Susan B Zolla-Pazner, Ph.D.

Quantitative assessment of masking of neutralization epitopes in HIV-1
Agarwal, Alpna; Hioe, Catarina E; Swetnam, James; Zolla-Pazner, Susan; Cardozo, Timothy
2011 Sep 9;29(39):6736-6741, Vaccine
Despite the frequent observation of masking of HIV-1 neutralization epitopes, its extent has not been previously systematically assessed either for multiple epitopes presented by individual viruses or for individual epitopes across multiple viral strains. Using a recently developed method to identify amino acid sequence motifs required for recognition by HIV-1-neutralizing monoclonal antibodies (mAbs), we visualized the patterns of masking of specific epitopes targeted by mAbs in a diverse panel of HIV-1 isolates. We also calculated a specific masking intensity score for each virus based on the observed neutralization activity of mAbs against the epitopes in the virus. Finally, we combined these data with estimates of the conservation of each mAb-targeted epitope in circulating HIV-1 strains to estimate the effective neutralization potential (E(N)) for each mAb. Focusing on the V3 loop of gp120 as a prototype neutralization domain, we found that the V3 loop epitope targeted by mAb 2219 is one of the least masked mAbs and it has the highest E(N). Interestingly, although the V3 loop epitope targeted by mAb 3074 is present in over 87% of all viruses, it is 82.2% masked, so its E(N) is lower than that for mAb 2219. Notably, 50% of the viruses that mAb 3074 is able to neutralize are classified as subtype C viruses, while 70% or more of the viruses neutralized by mAbs 2219, 2557 or 447-52D are classified as subtype B. Thus, neutralization epitopes (in this case, in the V3 loop) have differential patterns of masking and also display distinct patterns of distribution among circulating HIV-1 viruses. Both factors combine to contribute to the practical vaccine value of any single epitope/mAb. Here we have developed a quantitative score for this value. These results have important implications for rational design of vaccines designed to induce neutralizing Abs by revealing epitopes that are minimally masked and maximally reactive with neutralizing Abs
— id: 137058, year: 2011, vol: 29, page: 6736, stat: Journal Article,

Crystal structure of human antibody 2909 reveals conserved features of quaternary structure-specific antibodies that potently neutralize HIV-1
Changela, Anita; Wu, Xueling; Yang, Yongping; Zhang, Baoshan; Zhu, Jiang; Nardone, Glenn A; O'Dell, Sijy; Pancera, Marie; Gorny, Miroslaw K; Phogat, Sanjay; Robinson, James E; Stamatatos, Leonidas; Zolla-Pazner, Susan; Mascola, John R; Kwong, Peter D
2011 Mar;85(6):2524-2535, Journal of virology
Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain specific, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120(3)/gp41(3)). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a beta-hairpin club extending approximately 20 A beyond the rest of the antibody. Sequence analysis and mass spectrometry identified sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reflecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability
— id: 133342, year: 2011, vol: 85, page: 2524, stat: Journal Article,

Nonneutralizing HIV-1 gp41 envelope cluster II human monoclonal antibodies show polyreactivity for binding to phospholipids and protein autoantigens
Dennison, S Moses; Anasti, Kara; Scearce, Richard M; Sutherland, Laura; Parks, Robert; Xia, Shi-Mao; Liao, Hua-Xin; Gorny, Miroslaw K; Zolla-Pazner, Susan; Haynes, Barton F; Alam, S Munir
2011 Feb;85(3):1340-1347, Journal of virology
HIV-1 gp41 envelope antibodies, which are frequently induced in HIV-1-infected individuals, are predominantly nonneutralizing. The rare and difficult-to-induce neutralizing antibodies (2F5 and 4E10) that target gp41 membrane-proximal epitopes (MPER) are polyspecific and require lipid binding for HIV-1 neutralization. These results raise the questions of how prevalent polyreactivity is among gp41 antibodies and how the binding properties of gp41-nonneutralizing antibodies differ from those of antibodies that are broadly neutralizing. In this study, we have characterized a panel of human gp41 antibodies with binding specificities within the immunodominant cluster I (gp41 amino acids [aa] 579 to 613) or cluster II (gp41 aa 644 to 667) for reactivity to autoantigens, to the gp140 protein, and with MPER peptide-lipid conjugates. We report that while none of the gp41 cluster I antibodies studied were polyspecific, all three gp41 cluster II antibodies bound either to lipids or autoantigens, thus showing the propensity of cluster II antibodies to manifest polyreactivity. All cluster II gp41 monoclonal antibodies (MAbs), including those that were lipid reactive, failed to bind to gp41 MPER peptide-lipid complexes. Cluster II antibodies bound strongly with nanomolar binding affinity (dissociation constant [K(d)]) to oligomeric gp140 proteins, and thus, they recognize conformational epitopes on gp41 that are distinct from those of neutralizing gp41 antibodies. These results demonstrate that lipid-reactive gp41 cluster II antibodies are nonneutralizing due to their inability to bind to the relevant neutralizing epitopes on gp41
— id: 133202, year: 2011, vol: 85, page: 1340, stat: Journal Article,

Human Anti-V3 HIV-1 Monoclonal Antibodies Encoded by the VH5-51/VL Lambda Genes Define a Conserved Antigenic Structure
Gorny, Miroslaw K; Sampson, Jared; Li, Huiguang; Jiang, Xunqing; Totrov, Maxim; Wang, Xiao-Hong; Williams, Constance; O'Neal, Timothy; Volsky, Barbara; Li, Liuzhe; Cardozo, Timothy; Nyambi, Phillipe; Zolla-Pazner, Susan; Kong, Xiang-Peng
2011 ;6(12):e27780-e27780, PLoS ONE
Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs
— id: 146267, year: 2011, vol: 6, page: e27780, stat: Journal Article,

Characterization of structural features and diversity of variable-region determinants of related quaternary epitopes recognized by human and rhesus macaque monoclonal antibodies possessing unusually potent neutralizing activities
Krachmarov, Chavdar; Lai, Zhong; Honnen, William J; Salomon, Aidy; Gorny, Miroslaw K; Zolla-Pazner, Susan; Robinson, James; Pinter, Abraham
2011 Oct;85(20):10730-10740, Journal of virology
A series of potently neutralizing monoclonal antibodies (MAbs) that target quaternary epitopes on the native Env trimer have recently been described. A common feature shared by these antibodies is the critical involvement of sites in both the V2 and V3 variable domains in antibody recognition. In this study the gp120 variable-region determinants were mapped for eight rhesus macaque monoclonal antibodies (RhMAbs) possessing potently neutralizing activity specific for a quaternary target in SF162 Env and compared to those originally identified for human MAb 2909. These studies showed that determinants for the epitopes defined by the RhMAbs differed in both the V2 (positions 160, 167, and 169) and V3 (positions 313 and 315) regions from 2909, and in a number of cases, from each other. Attempts to reconstitute expression of these epitopes on the cell surface by cotransfecting Envs containing either the V2 or the V3 determinant of the epitope were not successful, suggesting that these epitopes were expressed on individual protomers in a trimer-dependent manner. Several of the V2 positions found to be critical for expression of these quaternary epitopes also significantly affected exposure and neutralization sensitivity of targets in the V3 and CD4-binding domains. These results demonstrated a considerable diversity in the fine structure of this class of epitopes and further suggested a potentially important relationship between the expression of such quaternary epitopes and V1/V2-mediated masking of immunodominant epitopes
— id: 141138, year: 2011, vol: 85, page: 10730, stat: Journal Article,

A conserved determinant in the V1 loop of HIV-1 modulates the V3 loop to prime low CD4 use and macrophage infection
Musich, Thomas; Peters, Paul J; Duenas-Decamp, Maria Jose; Gonzalez-Perez, Maria Paz; Robinson, James; Zolla-Pazner, Susan; Ball, Jonathan K; Luzuriaga, Katherine; Clapham, Paul R
2011 Mar;85(5):2397-2405, Journal of virology
The CD4 binding site (CD4bs) on the HIV-1 envelope plays a major role in determining the capacity of R5 viruses to infect primary macrophages. Thus, envelope determinants within or proximal to the CD4bs have been shown to control the use of low CD4 levels on macrophages for infection. These residues affect the affinity for CD4 either directly or indirectly by altering the exposure of CD4 contact residues. Here, we describe a single amino acid determinant in the V1 loop that also modulates macrophage tropism. Thus, we identified an E153G substitution that conferred high levels of macrophage infectivity for several heterologous R5 envelopes, while the reciprocal G153E substitution abrogated infection. Shifts in macrophage tropism were associated with dramatic shifts in sensitivity to the V3 loop monoclonal antibody (MAb), 447-52D and soluble CD4, as well as more modest changes in sensitivity to the CD4bs MAb, b12. These observations are consistent with an altered conformation or exposure of the V3 loop that enables the envelope to use low CD4 levels for infection. The modest shifts in b12 sensitivity suggest that residue 153 impacts on the exposure of the CD4bs. However, the more intense shifts in sCD4 sensitivity suggest additional mechanisms that likely include an increased ability of the envelope to undergo conformational changes following binding to suboptimal levels of cell surface CD4. In summary, we show that a conserved determinant in the V1 loop modulates the V3 loop to prime low CD4 use and macrophage infection
— id: 134134, year: 2011, vol: 85, page: 2397, stat: Journal Article,

Crystal structures of human anti-V2 mAbs 697-30D and 8. 9D and what we can learn from their antigen-binding sites
Pan R.; Sampson J.M.; Spurrier B.; Totrov M.; O'Neal T.; Williams C.; Boliar S.; Allen S.; Mulenga J.; Robinson J.; Derdeyn C.A.; Gorny M.K.; Zolla-Pazner S.; Kong X.
2011 ;27(10):A120-A121, AIDS research & human retroviruses
Background: The immunogenic V2 region of HIV-1 gp120 has been largely overlooked as a target for AIDS vaccine discovery. However, recent studies of sera from vaccinees in RV144 trial suggested that anti-V2 Abs were elicited and possibly contributed to protection. Structural understanding of human anti-V2 mAbs and their epitopes can facilitate design of immunogens. Methods: We determined crystal structures of Fab fragments of two human anti-V2 mAbs 697-30D and 8. 9D, both at a resolution of 2. 5 A, and analyzed their antigen-binding sites (ABS) and possible modes of interactions with V1V2 of gp120. Results: MAb 697-30D, from a subtype B virus infected subject and encoded by VH1-69 gene, is a broadly cross-reactive anti-V2 mAb able to neutralize Tier 1 pseudoviruses; its epitope was mapped to conserved residues in V2. Structural analysis of Fab 697-30D revealed that its ABS consists of two distinct regions: (1) A surface pocket is located at the center of CDR loops formed by large aromatic residues, and it can accommodate residues with large side chains in the epitope identified by functional studies. (2) A convex hydrophobic surface is comprised of a cluster of CDR H2/H3 residues. Comparison with structures of other VH1- 69 mAbs suggests that mAb 697-30D likely binds to the region of a short helix or a relatively flat surface of V2. Autologous neutralizing mAb 8. 9D was isolated from a subtype C infected subject, and its epitope was mapped to the stem of V1V2. Its ABS is split by the upward positioned Tyr100b of CDR H3 into a positively-charged side and a negatively-charged side. Surface pockets in these regions can bind side chains of charged residues of V1V2, facilitating escape by mutations. Conclusion: Crystal structures of human anti-V2 mAbs provide structure-function insights of their epitopes. This information may contribute to rational design of immunogens targeting V2 region of gp120
— id: 139487, year: 2011, vol: 27, page: A120, stat: Journal Article,

A single amino acid substitution in the C4 region in gp120 confers enhanced neutralization of HIV-1 by modulating CD4 binding sites and V3 loop
Ringe R.; Sharma D.; Zolla-Pazner S.; Phogat S.; Risbud A.; Thakar M.; Paranjape R.; Bhattacharya J.
2011 ;418(2):123-132, Virology
Identification of vulnerability in the HIV-1 envelope (Env) will aid in Env-based vaccine design. We recently found an HIV-1 clade C Env clone (4-2.J45) amplified from a recently infected Indian patient showing exceptional neutralization sensitivity to autologous plasma in contrast to other autologous Envs obtained at the same time point. By constructing chimeric Envs and fine mapping between sensitive and resistant Env clones, we found that substitution of highly conserved isoleucine (I) with methionine (M) (ATA to ATG) at position 424 in the C4 domain conferred enhanced neutralization sensitivity of Env-pseudotyped viruses to autologous and heterologous plasma antibodies. When tested against monoclonal antibodies targeting different sites in gp120 and gp41, Envs expressing M424 showed significant sensitivity to anti-V3 monoclonal antibodies and modestly to sCD4 and b12. Substitution of I424M in unrelated Envs also showed similar neutralization phenotype, indicating that M424 in C4 region induces exposure of neutralizing epitopes particularly in CD4 binding sites and V3 loop. 2011 Elsevier Inc
— id: 137086, year: 2011, vol: 418, page: 123, stat: Journal Article,

Indirect Detection of an Epitope-Specific Response to HIV-1 gp120 Immunization in Human Subjects
Shmelkov, Evgeny; Nadas, Arthur; Swetnam, James; Zolla-Pazner, Susan; Cardozo, Timothy
2011 ;6(11):e27279-e27279, PLoS ONE
A specific response of human serum neutralizing antibodies (nAb) to a conformational epitope as a result of vaccination of human subjects with the surface envelope glycoprotein (gp120) of HIV-1 has not previously been documented. Here, we used computational analysis to assess the epitope-specific responses of human subjects, which were immunized with recombinant gp120 immunogens in the VAX003 and VAX004 clinical trials. Our computational methodology-a variation of sieve analysis-compares the occurrence of specific nAb targeted conformational 3D epitopes on viruses from infected individuals who received vaccination to the occurrence of matched epitopes in the viruses infecting placebo subjects. We specifically studied seven crystallographically defined nAb targeted conformational epitopes in the V3 loop, an immunogenic region of gp120. Of the six epitopes present in the immunogens and targeted by known monoclonal neutralizing antibodies, only the one targeted by the anti-V3 nAb 2219 exhibited a significant reduction in occurrence in vaccinated subjects compared to the placebo group. This difference occurred only in the VAX003 Thailand cohort. No difference was seen between vaccinated and placebo groups for the occurrence of an epitope that was not present in the immunogen. Thus, it can be theorized that a specific 2219-like human neutralizing antibody immune response to AIDSVAX immunization occurred in the VAX003 cohort, and that this response protected subjects from a narrow subset of HIV-1 viruses circulating in Thailand in the 1990s and bearing the conformational epitope targeted by the neutralizing antibody 2219
— id: 141491, year: 2011, vol: 6, page: e27279, stat: Journal Article,

Structural Analysis of Human and Macaque mAbs 2909 and 2.5B: Implications for the Configuration of the Quaternary Neutralizing Epitope of HIV-1 gp120
Spurrier, Brett; Sampson, Jared M; Totrov, Maxim; Li, Huiguang; O'Neal, Timothy; Williams, Constance; Robinson, James; Gorny, Miroslaw K; Zolla-Pazner, Susan; Kong, Xiang-Peng
2011 May 11;19(5):691-699, Structure
The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 A in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs
— id: 132586, year: 2011, vol: 19, page: 691, stat: Journal Article,

"Corrigendum to Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold"" [Virology 351 (2010) 513-523]"
Totrov M.; Jiang X.; Kong X.-P.; Cohen S.; Krachmarov C.; Salomon A.; Williams C.; Seaman M.S.; Abagyan R.; Cardozo T.; Gorny M.K.; Wang S.; Lu S.; Pinter A.; Zolla-Pazner S.
2011 ;409(2):360-360, Virology
— id: 119242, year: 2011, vol: 409, page: 360, stat: Journal Article,

Immunotypes of a Quaternary Site of HIV-1 Vulnerability and Their Recognition by Antibodies
Wu, Xueling; Changela, Anita; O'Dell, Sijy; Schmidt, Stephen D; Pancera, Marie; Yang, Yongping; Zhang, Baoshan; Gorny, Miroslaw K; Phogat, Sanjay; Robinson, James E; Stamatatos, Leonidas; Zolla-Pazner, Susan; Kwong, Peter D; Mascola, John R
2011 May;85(9):4578-4585, Journal of virology
HIV-1 is neutralized by a class of antibodies that preferentially recognize a site formed on the assembled viral spike. Such quaternary structure-specific antibodies have diverse neutralization breadths, with antibodies PG16 and PG9 able to neutralize 70 to 80% of circulating HIV-1 isolates while antibody 2909 is specific for strain SF162. We show that alteration between a rare lysine and a common N-linked glycan at position 160 of HIV-1 gp120 is primarily responsible for toggling between 2909 and PG16/PG9 neutralization sensitivity. Quaternary structure-specific antibodies appear to target antigenic variants of the same epitope, with neutralization breadth determined by the prevalence of recognized variants among circulating isolates
— id: 132734, year: 2011, vol: 85, page: 4578, stat: Journal Article,

Cross-Clade HIV-1 Neutralizing Antibodies Induced with V3-Scaffold Protein Immunogens following Priming with gp120 DNA
Zolla-Pazner, Susan; Kong, X-P; Jiang, Xunqing; Cardozo, Timothy; Nadas, Arthur; Cohen, Sandra; Totrov, Maxim; Seaman, Michael S; Wang, Shixia; Lu, Shan
2011 Oct;85(19):9887-9898, Journal of virology
The V3 epitope is a known target for HIV-1 neutralizing antibodies (NAbs), and V3-scaffold fusion proteins used as boosting immunogens after gp120 DNA priming were previously shown to induce NAbs in rabbits. Here, we evaluated whether the breadth and potency of the NAb response could be improved when boosted with rationally designed V3-scaffold immunogens. Rabbits were primed with codon-optimized clade C gp120 DNA and boosted with one of five V3-cholera toxin B fusion proteins (V3-CTBs) or with double combinations of these. The inserts in these immunogens were designed to display V3 epitopes shared by the majority of global HIV-1 isolates. Double combinations of V3-CTB immunogens generally induced more broad and potent NAbs than did boosts with single V3-CTB immunogens, with the most potent and broad NAbs elicited with the V3-CTB carrying the consensus V3 of clade C (V3(C)-CTB), or with double combinations of V3-CTB immunogens that included V3(C)-CTB. Neutralization of tier 1 and 2 pseudoviruses from clades AG, B, and C and of peripheral blood mononuclear cell (PBMC)-grown primary viruses from clades A, AG, and B was achieved, demonstrating that priming with gp120 DNA followed by boosts with V3-scaffold immunogens effectively elicits cross-clade NAbs. Focusing on the V3 region is a first step in designing a vaccine targeting protective epitopes, a strategy with potential advantages over the use of Env, a molecule that evolved to protect the virus by poorly inducing NAbs and by shielding the epitopes that are most critical for infectivity
— id: 137442, year: 2011, vol: 85, page: 9887, stat: Journal Article,

Quantitative Assessment of HIV-1 Neutralization Epitope Masking
Agarwal, A.; Swetnam, J.; Zolla-Pazner, S.; Cardozo, T.
2010 OCT ;26(10):A74-A74, AIDS research & human retroviruses
— id: 117318, year: 2010, vol: 26, page: A74, stat: Journal Article,

Map of broad and narrow neutralization in the V3 loop crown
Almond D.; Kong X.; Zolla-Pazner S.; Cardozo T.
2010 ;26(10):A27-A28, AIDS research & human retroviruses
Background: Sequence variability of the V3 loop crown has often been considered an impediment to eliciting broadly neutralization antibodies targeted to this region. Our work maps contacts between human anti-V3 monoclonal antibodies (mAbs) and the V3 crown in an attempt to make a structural distinction between the sites targeted by broadly as opposed to narrowly neutralizing mAbs. Methods: The 3D structure of the 16-residue V3 crown bound to anti-V3 mAb 2219 was divided into 4 regions (stem, hydrophilic patch, hydrophobic patch and turn) based on previously published analyses (Almond et. al., 2010). Known mAb/V3 contacts from Jiang et al., 2010 and others were then mapped onto this structure and compared to measurements of neutralization breadth by the respective mAbs in infectivity assays. Results: mAbs that entirely engage the less sequence variable zones of the V3 loop (the stem, turn and hydrophobic zones) tend to be more broadly neutralizing than mAbs that contact at least one side-chain in the most variable region of V3 (the hydrophilic zone). The contact in the highly variable region appears to narrow the Ab reactivity regardless of how many other contacts are formed with V3 backbone atoms or with side-chains in the conserved regions. Conclusion: Broadly neutralizing mAbs are targeted to the structurally conserved region, and they avoid contact with the highly sequence-variable zone. More narrowly neutralizing Abs make at least one side-chain contact within the highly-sequence variable zone: these latter mAbs are vulnerable to a single escape mutation at that side-chain/amino acid location, and indeed these side-chains are frequently mutated in circulating viruses. This knowledge could be exploited for HIV vaccine design by designing immunogens that immunologically silence the variable zone in the V3 loop
— id: 114523, year: 2010, vol: 26, page: A27, stat: Journal Article,

Structural conservation predominates over sequence variability in the crown of HIV type 1's V3 loop
Almond, David; Kimura, Tetsuya; Kong, XiangPeng; Swetnam, James; Zolla-Pazner, Susan; Cardozo, Timothy
2010 Jun;26(6):717-723, AIDS research & human retroviruses
The diversity of HIV-1 is a confounding problem for vaccine design, as the human immune response appears to favor poor or strain-specific responses to any given HIV-1 virus strain. A significant portion of this diversity is manifested as sequence variability in the loops of HIV-1's surface envelope glycoprotein. Here we show that the most variable sequence positions in the third variable (V3) loop crown cluster to a small zone on the surface of one face of the V3 loop ss-hairpin conformation. These results provide a novel visualization of the gp120 V3 loop, specifically demonstrating a surprising preponderance of conserved three-dimensional structure in a highly sequence-variable region. From a structural point of view, there appears to be less diversity in this region of the HIV-1 'principle neutralizing domain' than previously appreciated
— id: 110086, year: 2010, vol: 26, page: 717, stat: Journal Article,

Engineered immunogen presenting an epitope recognized by a neutralizing mAb elicits mammalian serum that recapitulate the mAb's specificity
Cardozo T.; Kong X.; Totrov M.; Wang S.; Lu S.; Gorny M.; Pinter A.; Seaman M.; Zolla-Pazner S.
2010 ;26(10):A26-A27, AIDS research & human retroviruses
Background: To exploit the promising properties of cross-strain neutralizing monoclonal antibodies (mAbs), immunogens should elicit polyclonal Ab responses in mammals that mimic the reactivity of these mAbs. To demonstrate the feasibility of this approach, the epitope recognized by the anti-V3 loop mAb 3074- which is present in approximately 90% of circulating HIV-1 viruses-was use as a template for immunogen design. Methods: A V3 loop-cholera toxin B fusion protein (V3₃₀₇₄-CTB) was designed to eliminate the epitopes targeted by several anti-V3 loop mAbs while preserving the epitope targeted by mAb 3074. New Zealand White rabbits were primed with codon-optimized clade C gp120 DNA and then boosted with V3₃₀₇₄-CTB. Anti-V3 mAbs and rabbit immune sera were assessed for neutralizing activity against (a) V3 chimeric psVs infecting U87 CD4 + CCR5 + cells, (b) primary isolates infecting TZM-bl cells, (c) Tier 1 and (d) Tier 2 psVs infecting TZM.bl cells. Results: V33074-CTB bound specifically to mAb 3074 but not to several other anti-V3 mAbs. A psV bearing the V3₃₀₇₄-designed sequence was neutralized only by mAb 3074. Immune sera elicited in rabbits using V3₃₀₇₄-CTB demonstrated 50% neutralization of (a) 7/7 V3 chimeric psVs carrying the V3 loops of clades A1, AG, AE, B, C, F, and H, (b) 3/10 primary isolates from clades A, AG and B, (c) 4/4 Tier 1 viruses, and (d) 4/14 Tier 2 viruses from clades B and C. Little neutralizing activity was seen in the immune sera against a V3 chimeric psV lacking the 3074 epitope, and the only three Tier 2 clade C viruses neutralized by mAb 3074 were also the only three Tier 2 Clade C viruses neutralized by the serum of the best responder. Conclusion: Our results demonstrate that V3₃₀₇₄-CTB elicited a polyclonal, cross-strain neutralizing Ab response in mammalian serum mirroring the specificity of 3074-the mAb used as a template for immunogen design
— id: 114522, year: 2010, vol: 26, page: A26, stat: Journal Article,

Distinct conformational states of HIV-1 gp41 are recognized by neutralizing and non-neutralizing antibodies
Frey, Gary; Chen, Jia; Rits-Volloch, Sophia; Freeman, Michael M; Zolla-Pazner, Susan; Chen, Bing
2010 Dec;17(12):1486-1491, Nature structural & molecular biology
HIV-1 envelope glycoprotein gp41 undergoes large conformational changes to drive fusion of viral and target cell membranes, adopting at least three distinct conformations during the viral entry process. Neutralizing antibodies against gp41 block HIV-1 infection by targeting gp41's membrane-proximal external region in a fusion-intermediate state. Here we report biochemical and structural evidence that non-neutralizing antibodies, capable of binding with high affinity to an immunodominant segment adjacent to the neutralizing epitopes in the membrane-proximal region, recognize a gp41 conformation that exists only when membrane fusion is complete. We propose that these non-neutralizing antibodies are induced in HIV-1-infected individuals by gp41 in a triggered, postfusion form and contribute to production of ineffective humoral responses. These results have important implications for gp41-based vaccine design
— id: 134407, year: 2010, vol: 17, page: 1486, stat: Journal Article,

Germline variable genes code for contact residues maintained during affinity maturation of human anti-V3 monoclonal antibodies encoded by VH5-51
Gorny M.K.; Sampson J.; Li H.; Jiang X.; Totrov M.; Wang X.; Li L.; Williams C.; Luthra K.; Nyambi P.; Zolla-Pazner S.; Kong X.
2010 ;26(10):A25-A25, AIDS research & human retroviruses
Background: Immunogenetic studies have revealed the importance of certain immunoglobulin (Ig) genes encoding monoclonal antibodies (mAbs) against various epitopes in the envelope of HIV-1. We have demonstrated recently that VH5-51 gene segment is preferentially utilized by 18 (35%) of 51 human anti-V3 mAbs. Methods: In this study, a panel of 18 anti-V3 mAbs were examined which were derived from individuals infected with clade B and non-clade B HIV-1; all were encoded by the VH5-51 gene segment and neutralized Tier 1 and Tier 2 viruses. Immunochemical and crys-tallographic methods were used to study their function and the structure of the antigen-combining site. Results: The VH5-51 gene used by all 18 mAbs paired only with lambda light chain genes, mainly with VL1-47 and VL3-1 genes. This restricted pairing of Ig genes resulted in the formation of a conserved antigen combining site, as documented by crystallo-graphic studies of five of these anti-V3 Fabs in complex with V3 peptides. The VH5-51-encoded V3mAbs recognize slight variations of an epitope which contains conserved residues in the N- and C-terminal b-strands of the V3 crown. This finding was confirmed by the binding of the mAbs to a peptide which mimics this region of V3 but lacks the tip of the V3 loop. Crystallographic analysis of the Fab/peptide complex showed that all contact residues in the CDR domains, except CDR H3 and L3, are germline-encoded and thus determine the conserved character of the binding site. The data indicate that affinity maturation of these mAbs has preserved the germline-encoded interaction with the antigen. Conclusion: The immunogenetic studies of anti-V3 neutralizing mAbs revealed that certain germline variable genes encode the contact residues which are maintained during antibody evolution; targeting epitopes preferentially recognized by such Ig genes with vaccines should induce broadly reactive neutralizing Ab responses
— id: 114521, year: 2010, vol: 26, page: A25, stat: Journal Article,

Characterization of a discontinuous epitope of the HIV envelope protein gp120 recognized by a human monoclonal antibody using chemical modification and mass spectrometric analysis
Hager-Braun, Christine; Hochleitner, Elisabeth O; Gorny, Miroslaw K; Zolla-Pazner, Susan; Bienstock, Rachelle J; Tomer, Kenneth B
2010 Oct;21(10):1687-1698, Journal of the American Society for Mass Spectrometry
A subset of the neutralizing anti-HIV antibodies recognize epitopes on the envelope protein gp120 of the human immunodeficiency virus. These epitopes are exposed during conformational changes when gp120 binds to its primary receptor CD4. Based on chemical modification of lysine and arginine residues followed by mass spectrometric analysis, we determined the epitope on gp120 recognized by the human monoclonal antibody 559/64-D, which was previously found to be specific for the CD4 binding domain. Twenty-four lysine and arginine residues in recombinant full-length glycosylated gp120 were characterized; the relative reactivities of two lysine residues and five arginine residues were affected by the binding of 559/64-D. The data show that the epitope is discontinuous and is located in the proximity of the CD4-binding site. Additionally, the reactivities of a residue that is located in the secondary receptor binding region and several residues distant from the CD4 binding site were also altered by Ab binding. These data suggest that binding of 559/64-D induced conformational changes which result in altered surface exposure of specific amino acids distant from the CD4-binding site. Consequently, binding of 559/64-D to gp120 affects not only the CD4-binding site, which is recognized as the epitope, but appears to have a global effect on surface exposed residues of the full-length glycosylated gp120
— id: 133796, year: 2010, vol: 21, page: 1687, stat: Journal Article,

Anti-V3 monoclonal antibodies display broad neutralizing activities against multiple HIV-1 subtypes
Hioe, Catarina E; Wrin, Terri; Seaman, Michael S; Yu, Xuesong; Wood, Blake; Self, Steve; Williams, Constance; Gorny, Miroslaw K; Zolla-Pazner, Susan
2010 ;5(4):e10254-e10254, PLoS ONE
BACKGROUND: The V3 loop of the HIV-1 envelope (Env) glycoprotein gp120 was identified as the 'principal neutralizing domain' of HIV-1, but has been considered too variable to serve as a neutralizing antibody (Ab) target. Structural and immunochemical data suggest, however, that V3 contains conserved elements which explain its role in binding to virus co-receptors despite its sequence variability. Despite this evidence of V3 conservation, the ability of anti-V3 Abs to neutralize a significant proportion of HIV-1 isolates from different subtypes (clades) has remained controversial. METHODS: HIV-1 neutralization experiments were conducted in two independent laboratories to test human anti-V3 monoclonal Abs (mAbs) against pseudoviruses (psVs) expressing Envs of diverse HIV-1 subtypes from subjects with acute and chronic infections. Neutralization was defined by 50% inhibitory concentrations (IC(50)), and was statistically assessed based on the area under the neutralization titration curves (AUC). RESULTS: Using AUC analyses, statistically significant neutralization was observed by >or=1 anti-V3 mAbs against 56/98 (57%) psVs expressing Envs of diverse subtypes, including subtypes A, AG, B, C and D. Even when the 10 Tier 1 psVs tested were excluded from the analysis, significant neutralization was detected by >or=1 anti-V3 mAbs against 46/88 (52%) psVs from diverse HIV-1 subtypes. Furthermore, 9/24 (37.5%) Tier 2 viruses from the clade B and C standard reference panels were neutralized by >or=1 anti-V3 mAbs. Each anti-V3 mAb tested was able to neutralize 28-42% of the psVs tested. By IC(50) criteria, 40/98 (41%) psVs were neutralized by >or=1 anti-V3 mAbs. CONCLUSIONS: Using standard and new statistical methods of data analysis, 6/7 anti-V3 human mAbs displayed cross-clade neutralizing activity and revealed that a significant proportion of viruses can be neutralized by anti-V3 Abs. The new statistical method for analysis of neutralization data provides many advantages to previously used analyses
— id: 109524, year: 2010, vol: 5, page: e10254, stat: Journal Article,

Conserved structural elements in the V3 crown of HIV-1 gp120
Jiang, Xunqing; Burke, Valicia; Totrov, Maxim; Williams, Constance; Cardozo, Timothy; Gorny, Miroslaw K; Zolla-Pazner, Susan; Kong, Xiang-Peng
2010 Aug;17(8):955-961, Nature structural & molecular biology
Binding of the third variable region (V3) of the HIV-1 envelope glycoprotein gp120 to the cell-surface coreceptors CCR5 or CXCR4 during viral entry suggests that there are conserved structural elements in this sequence-variable region. These conserved elements could serve as epitopes to be targeted by a vaccine against HIV-1. Here we perform a systematic structural analysis of representative human anti-V3 monoclonal antibodies in complex with V3 peptides, revealing that the crown of V3 has four conserved structural elements: an arch, a band, a hydrophobic core and the peptide backbone. These are either unaffected by or are subject to minimal sequence variation. As these regions are targeted by cross-clade neutralizing human antibodies, they provide a blueprint for the design of vaccine immunogens that could elicit broadly cross-reactive protective antibodies
— id: 111542, year: 2010, vol: 17, page: 955, stat: Journal Article,

Structures of human mAb 2909 and rhesus mAb 2.5B that target quaternary structure-dependent neutralizing epitopes of HIV-1 gp120
Kong, X.; Spurrier, B.; Sampson, J.; Totrov, M.; Williams, C.; Robinson, J.; Gorny, M. K.; Zolla-Pazner, S.
2010 OCT ;26(10):A56-A57, AIDS research & human retroviruses
— id: 117317, year: 2010, vol: 26, page: A56, stat: Journal Article,

Quaternary Epitope Specificities of Anti-HIV-1 Neutralizing Antibodies Generated in Rhesus Macaques Infected by the Simian/Human Immunodeficiency Virus SHIVSF162P4
Robinson, JE; Franco, K; Elliott, DH; Maher, MJ; Reyna, A; Montefiori, DC; Zolla-Pazner, S; Gorny, MK; Kraft, Z; Stamatatos, L
2010 APR ;84(7):3443-3453, Journal of virology
Monoclonal antibodies (MAbs) that neutralize human immunodeficiency virus type 1 (HIV-1) have been isolated from HIV-1-infected individuals or animals immunized with recombinant HIV-1 envelope (Env) glycoprotein constructs. The epitopes of these neutralizing antibodies (NAbs) were shown to be located on either the variable or conserved regions of the HIV-1 Env and to be linear or conformational. However, one neutralizing MAb, 2909, which was isolated from an HIV-1-infected subject, recognizes a more complex, quaternary epitope that is present on the virion-associated functional trimeric Env spike of the SF162 HIV-1 isolate. Here, we discuss the isolation of 11 anti-HIV NAbs that were isolated from three rhesus macaques infected with the simian/human immunodeficiency virus SHIVSF162P4 and that also recognize quaternary epitopes. A detailed epitope mapping analysis of three of these rhesus antibodies revealed that their epitopes overlap that of the human MAb 2909. Despite this overall similarity in binding, however, differences in specific amino acid and glycosylation pattern requirements for MAb 2909 and the rhesus MAbs were identified. These results highlight similarities in the B-cell responses of humans and macaques to structurally complex neutralization epitopes on related viruses, HIV-1 and SHIV
— id: 108315, year: 2010, vol: 84, page: 3443, stat: Journal Article,

Computational profiling the epitope-specific human neutralizing antibodies elicited in the AIDSVAX clinical trials
Shmelkov, E.; Nadas, A.; Swetnam, J.; Zolla-Pazner, S.; Cardozo, T.
2010 OCT ;26(10):A44-A44, AIDS research & human retroviruses
— id: 117314, year: 2010, vol: 26, page: A44, stat: Journal Article,

Comparative Magnitude of Cross-Strain Conservation of HIV Variable Loop Neutralization Epitopes
Swetnam, James; Shmelkov, Evgeny; Zolla-Pazner, Susan; Cardozo, Timothy
2010 ;5(12):e15994-e15994, PLoS ONE
Although the sequence variable loops of the human immunodeficiency virus' (HIV-1) surface envelope glycoprotein (gp120) can exhibit good immunogenicity, characterizing conserved (invariant) cross-strain neutralization epitopes within these loops has proven difficult. We recently developed a method to derive sensitive and specific signature motifs for the three-dimensional (3D) shapes of the HIV-1 neutralization epitopes in the third variable (V3) loop of gp120 that are recognized by human monoclonal antibodies (mAbs). We used the signature motif method to estimate the conservation of these epitopes across circulating worldwide HIV-1 strains. The epitope targeted by the anti-V3 loop neutralizing mAb 3074 is present in 87% of circulating strains, distributed nearly evenly among all subtypes. The results for other anti-V3 Abs are: 3791, present in 63% of primarily non-B subtypes; 2219, present in 56% of strains across all subtypes; 2557, present in 52% across all subtypes; 447-52D, present in 11% of primarily subtype B strains; 537-10D, present in 9% of primarily subtype B strains; and 268-D, present in 5% of primarily subtype B strains. The estimates correlate with in vitro tests of these mAbs against diverse viral panels. The mAb 3074 thus targets an epitope that is nearly completely conserved among circulating HIV-1 strains, demonstrating the presence of an invariant structure hidden in the dynamic and sequence-variable V3 loop in gp120. Since some variable loop regions are naturally immunogenic, designing immunogens to mimic their conserved epitopes may be a promising vaccine discovery approach. Our results suggest one way to quantify and compare the magnitude of the conservation
— id: 117360, year: 2010, vol: 5, page: e15994, stat: Journal Article,

Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold
Totrov, Maxim; Jiang, Xunqing; Kong, Xiang-Peng; Cohen, Sandra; Krachmarov, Chavdar; Salomon, Aidy; Williams, Constance; Seaman, Michael S; Abagyan, Ruben; Cardozo, Timothy; Gorny, Miroslaw K; Wang, Shixia; Lu, Shan; Pinter, Abraham; Zolla-Pazner, Susan
2010 Sep 30;405(2):513-523, Virology
V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boosting with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response
— id: 133786, year: 2010, vol: 405, page: 513, stat: Journal Article,

Analysis of neutralizing antibody responses induced by gp120 DNA prime followed by monovalent or polyvalent V3 epitope boost
Wang, S.; Lu, S.; Kong, X.; Cardozo, T.; Cohen, S.; Jiang, X.; Totrov, M.; Pinter, A.; Krachmarov, C.; Seaman, M.; Zolla-Pazner, S.
2010 OCT ;26(10):A54-A54, AIDS research & human retroviruses
— id: 117315, year: 2010, vol: 26, page: A54, stat: Journal Article,

Statistical approaches to analyzing HIV-1 neutralizing antibody assay data
Yu, X.; Gilbert, P. B.; Hioe, C. E.; Zolla-Pazner, S.; Self, S. G.
2010 OCT ;26(10):A55-A55, AIDS research & human retroviruses
— id: 117316, year: 2010, vol: 26, page: A55, stat: Journal Article,

Structure-function relationships of HIV-1 envelope sequence-variable regions refocus vaccine design
Zolla-Pazner, Susan; Cardozo, Timothy
2010 Jul;10(7):527-535, Nature reviews. Immunology
One of the main challenges of developing an HIV-1 vaccine lies in eliciting immune responses that can overcome the antigenic variability exhibited by HIV. Most HIV-1 vaccine development has focused on inducing immunity to conserved regions of the HIV-1 envelope. However, new studies of the sequence-variable regions of the HIV-1 gp120 envelope glycoprotein have shown that there are conserved immunological and structural features in these regions. Recombinant immunogens that include these features may provide the means to address the antigenic diversity of HIV-1 and induce protective antibodies that can prevent infection with HIV-1
— id: 110664, year: 2010, vol: 10, page: 527, stat: Journal Article,

Sequence variability in the crown of the V3 loop of the HIV-1 envelope is clustered within a small 3D structural zone
Almond, D; Kimura, T; Kong, X; Swetnam, J; Zolla-Pazner, S; Cardozo, T
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105709, year: 2009, vol: 6, page: 115, stat: Journal Article,

Structural basis of the cross-reactivity of genetically related human anti-HIV-1 mAbs: implications for design of V3-based immunogens
Burke, Valicia; Williams, Constance; Sukumaran, Madhav; Kim, Seung-Sup; Li, Huiguang; Wang, Xiao-Hong; Gorny, Miroslaw K; Zolla-Pazner, Susan; Kong, Xiang-Peng
2009 Nov 11;17(11):1538-1546, Structure
Human monoclonal antibodies 447-52D and 537-10D, both coded by the VH3 gene and specific for the third variable region (V3) of the HIV-1 gp120, were found to share antigen-binding structural elements including an elongated CDR H3 forming main-chain interactions with the N terminus of the V3 crown. However, water-mediated hydrogen bonds and a unique cation-pi sandwich stacking allow 447-52D to be broadly reactive with V3 containing both the GPGR and GPGQ crown motifs, while the deeper binding pocket and a buried Glu in the binding site of 537-10D limit its reactivity to only V3 containing the GPGR motif. Our results suggest that the design of immunogens for anti-V3 antibodies should avoid the Arg at the V3 crown, as GPGR-containing epitopes appear to select for B cells making antibodies of narrower specificity than V3 that carry Gln at this position
— id: 105343, year: 2009, vol: 17, page: 1538, stat: Journal Article,

Worldwide distribution of HIV type 1 epitopes recognized by human anti-V3 monoclonal antibodies
Cardozo, Timothy; Swetnam, James; Pinter, Abraham; Krachmarov, Chavdar; Nadas, Arthur; Almond, David; Zolla-Pazner, Susan
2009 Apr;25(4):441-450, AIDS research & human retroviruses
Epitopes, also known as antigenic determinants, are small clusters of specific atoms within macromolecules that are recognized by the immune system. Such epitopes can be targeted with vaccines designed to protect against specific pathogens. The third variable loop (V3 loop) of the HIV-1 pathogen's gp120 surface envelope glycoprotein can be a highly sensitive neutralization target. We derived sequence motifs for the V3 loop epitopes recognized by the human monoclonal antibodies (mAbs) 447-52D and 2219. Searching the HIV database for the occurrence of each epitope motif in worldwide viruses and correcting the results based on published WHO epidemiology reveal that the 447-52D epitope we defined occurs in 13% of viruses infecting patients worldwide: 79% of subtype B viruses, 1% of subtype C viruses, and 7% of subtype A/AG sequences. In contrast, the epitope we characterized for human anti-V3 mAb 2219 is present in 30% of worldwide isolates but is evenly distributed across the known HIV-1 subtypes: 48% of subtype B strains, 40% of subtype C, and 18% of subtype A/AG. Various assays confirmed that the epitopes corresponding to these motifs, when expressed in the SF162 Env backbone, were sensitively and specifically neutralized by the respective mAbs. The method described here is capable of accurately determining the worldwide occurrence and subtype distribution of any crystallographically resolved HIV-1 epitope recognized by a neutralizing antibody, which could be useful for multivalent vaccine design. More importantly, these calculations demonstrate that globally relevant, structurally conserved epitopes are present in the sequence variable V3 loop
— id: 99293, year: 2009, vol: 25, page: 441, stat: Journal Article,

Neutralization efficiency and presence of anti-V3 antibodies in plasma of HIV-1 infected Northern Indians
Choudhary, Alok K.; Dutta, Subhashree; Wig, Naveet; Biswas, A.; Andrabi, Raiees; Kalra, Rajesh; Bhasin, Rama; Pazner, Susan Zolla; Laal, Suman; Luthra, Kalpana
2009 JUN ;51(10):160-160, Journal of acquired immune deficiency syndromes. JAIDS
— id: 113759, year: 2009, vol: 51, page: 160, stat: Journal Article,

International network for comparison of HIV neutralization assays: the NeutNet report
Fenyo, Eva Maria; Heath, Alan; Dispinseri, Stefania; Holmes, Harvey; Lusso, Paolo; Zolla-Pazner, Susan; Donners, Helen; Heyndrickx, Leo; Alcami, Jose; Bongertz, Vera; Jassoy, Christian; Malnati, Mauro; Montefiori, David; Moog, Christiane; Morris, Lynn; Osmanov, Saladin; Polonis, Victoria; Sattentau, Quentin; Schuitemaker, Hanneke; Sutthent, Ruengpung; Wrin, Terri; Scarlatti, Gabriella
2009 ;4(2):e4505-e4505, PLoS ONE
BACKGROUND: Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. METHODS: Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. FINDINGS: PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. CONCLUSIONS: The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation
— id: 98996, year: 2009, vol: 4, page: e4505, stat: Journal Article,

Preferential use of the VH5-51 gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1
Gorny, Miroslaw K; Wang, Xiao-Hong; Williams, Constance; Volsky, Barbara; Revesz, Kathy; Witover, Bradley; Burda, Sherri; Urbanski, Mateusz; Nyambi, Phillipe; Krachmarov, Chavdar; Pinter, Abraham; Zolla-Pazner, Susan; Nadas, Arthur
2009 Feb;46(5):917-926, Molecular immunology
Human anti-V3 monoclonal antibodies (mAbs) generated from HIV-1 infected individuals display diversity in the range of their cross-neutralization that may be related to their immunogenetic background. The study of the immunoglobulin (Ig) variable region gene usage of heavy chains have shown a preferential usage of the VH5-51 gene segment which was detected in 35% of 51 human anti-V3 mAbs. In contrast, human mAbs against other envelope regions of HIV-1 (anti-Env), including the CD4-binding domain, the CD4-induced epitope, and gp41 preferentially used the VH1-69 gene segment, and none of them used the VH5-51 gene. Furthermore, the usage of the VH4 family by anti-V3 mAbs was restricted to only one gene segment, VH4-59, while the VH3 gene family was used at a significantly lower frequency by all of the analyzed anti-HIV-1 mAbs. Multivariate analysis showed that usage of VH gene segments was significantly different between anti-V3 and anti-Env mAbs, and compared to antibodies from healthy subjects. In addition, the anti-V3 mAbs preferentially used the JH3 and D2-15 gene segments. The preferential usage of selected Ig gene segments and the characteristic pattern of Ig gene usage by anti-V3 mAbs can be related to the conserved structure of the V3 region
— id: 93792, year: 2009, vol: 46, page: 917, stat: Journal Article,

Neutralization of Tier 1 and Tier 2 pseudoviruses by human anti-V3 monoclonal antibodies
Gorny, MK; Williams, C; O'Neal, T; Choudhary, AK; Luthra, K; Wood, B; Seaman, MS; Nyambi, P; Zolla-Pazner, S
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105700, year: 2009, vol: 6, page: 115, stat: Journal Article,

Human monoclonal antibody 2909 binds to pseudovirions expressing trimers but not monomeric HIV-1 envelope proteins
Kimura, Tetsuya; Wang, Xiao-Hong; Williams, Constance; Zolla-Pazner, Susan; Gorny, Miroslaw K
2009 ;18(1):35-40, Human antibodies
A human anti-HIV monoclonal antibody (mAb), 2909, selected on the basis of its potent neutralizing activity against HIV-1<formula>_{SF162}</formula>, recognizes a complex epitope V2/V3 present on intact virions but not on soluble gp120. To confirm the quaternary nature of the epitope, 2909 binding was tested against the pseudovirus SF162 wild type (WT) expressing trimers and/or an SF162 mutant expressing monomeric envelope proteins. The construction of the SF162 mutant was made by an alanine substitution of nine hydrophobic residues in the N-terminal heptad repeat region of gp41 molecules that failed to form trimers on the virus surface. Monoclonal Ab 2909 bound only to SF162 WT virions and transfected cells as determined by immunoprecipitation and flow cytometry, respectively, but showed no reactivity to the SF162 mutant expressing monomeric gp120. The data provide further evidence for the existence of a unique quaternary epitope V2/V3 on the surface of unliganded virus
— id: 99240, year: 2009, vol: 18, page: 35, stat: Journal Article,

Worldwide epitope prevalence of crystallographically resolved anti-V3 antibodies
Swetnam, J; Zolla-Pazner, S; Cardozo, TJ
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105710, year: 2009, vol: 6, page: 115, stat: Journal Article,

Structure-guided design and immunological characterization of immunogen constructs presenting the HIV-1 gp120 V3 loop on a CTB scaffold
Totrov, M; Jiang, X; Kong, X; Cohen, S; Krachmarov, C; Williams, C; Cardozo, T; Gorny, M; Wang, S; Lu, S; Pinter, A; Zolla-Pazner, S
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105707, year: 2009, vol: 6, page: 115, stat: Journal Article,

Oligomer-specific conformations of the human immunodeficiency virus (HIV-1) gp41 envelope glycoprotein ectodomain recognized by human monoclonal antibodies
Yuan, Wen; Li, Xing; Kasterka, Marta; Gorny, Miroslaw K; Zolla-Pazner, Susan; Sodroski, Joseph
2009 Mar;25(3):319-328, AIDS research & human retroviruses
Trimerization of the human immunodeficiency virus (HIV-1) envelope glycoproteins is mediated by the ectodomain of the gp41 transmembrane glycoprotein. Here we investigate oligomer-specific conformations of gp41 by using monoclonal antibodies (MAbs) from HIV-1-infected humans. Human MAbs directed against the cluster I region of gp41 recognized trimeric, dimeric, and monomeric forms of soluble envelope glycoproteins; thus, the integrity of the cluster I epitopes is minimally affected by the oligomeric state. In contrast, human MAbs to the cluster II region were all oligomers specific. One cluster II MAb, 126-6, recognized exclusively the trimeric form of envelope glycoproteins, whereas the others recognized both trimeric and dimeric forms. Thus, a distinct trimer-specific conformation exists in the cluster II region of gp41. Analysis of soluble envelope glycoprotein mutants revealed that gp41 sequences immediately N-terminal to isoleucine 646 contribute to the formation of both the trimer and the trimer-specific conformational epitope
— id: 135248, year: 2009, vol: 25, page: 319, stat: Journal Article,

Induction of Cross-clade Neutralizing Antibodies in Rabbits Using a DNA Prime/Protein Boost Immunization Regimen
Zolla-Pazner, S.; Cohen, S.; Krachmarov, C.; Pinter, A.; Wang, S.; Lu, S.
2009 JUN ;51(10):95-95, Journal of acquired immune deficiency syndromes. JAIDS
— id: 113758, year: 2009, vol: 51, page: 95, stat: Journal Article,

Induction of cross-clade neutralizing antibodies with a prime/boost vaccine strategy focused on a neutralizing epitope
Zolla-Pazner, S; Kong, X; Cardozo, T; Hioe, C; Cohen, S; Jiang, X; Gorny, MK; Totrov, M; Pinter, A; Krachmarov, C; Seaman, MS; Wang, S; Lu, S
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105699, year: 2009, vol: 6, page: 115, stat: Journal Article,

Anti-V3 monoclonal antibodies display broad neutralizing activities against multiple HIV-1 subtypes
Zolla-Pazner, S; Wrin, T; Seaman, MS; Yu, X; Wood, B; Self, S; Hioe, CE
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105711, year: 2009, vol: 6, page: 115, stat: Journal Article,

Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope
Zolla-Pazner, Susan; Cohen, Sandra; Pinter, Abraham; Krachmarov, Chavdar; Wrin, Terri; Wang, Shixia; Lu, Shan
2009 Sep 15;392(1):82-93, Virology
Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3(B)-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity
— id: 101957, year: 2009, vol: 392, page: 82, stat: Journal Article,

Human immunodeficiency virus type 1 gp41 antibodies that mask membrane proximal region epitopes: antibody binding kinetics, induction, and potential for regulation in acute infection
Alam, S Munir; Scearce, Richard M; Parks, Robert J; Plonk, Kelly; Plonk, Steven G; Sutherland, Laura L; Gorny, Miroslaw K; Zolla-Pazner, Susan; Vanleeuwen, Stacie; Moody, M Anthony; Xia, Shi-Mao; Montefiori, David C; Tomaras, Georgia D; Weinhold, Kent J; Karim, Salim Abdool; Hicks, Charles B; Liao, Hua-Xin; Robinson, James; Shaw, George M; Haynes, Barton F
2008 Jan;82(1):115-125, Journal of virology
Two human monoclonal antibodies (MAbs) (2F5 and 4E10) against the human immunodeficiency virus type 1 (HIV-1) envelope g41 cluster II membrane proximal external region (MPER) broadly neutralize HIV-1 primary isolates. However, these antibody specificities are rare, are not induced by Env immunization or HIV-1 infection, and are polyspecific and also react with lipids such as cardiolipin or phosphatidylserine. To probe MPER anti-gp41 antibodies that are produced in HIV-1 infection, we have made two novel murine MAbs, 5A9 and 13H11, against HIV-1 gp41 envelope that partially cross-blocked 2F5 MAb binding to Env but did not neutralize HIV-1 primary isolates or bind host lipids. Competitive inhibition assays using labeled 13H11 MAb and HIV-1-positive patient plasma samples demonstrated that cluster II 13H11-blocking plasma antibodies were made in 83% of chronically HIV-1 infected patients and were acquired between 5 to 10 weeks after acute HIV-1 infection. Both the mouse 13H11 MAb and the three prototypic cluster II human MAbs (98-6, 126-6, and 167-D) blocked 2F5 binding to gp41 epitopes to variable degrees; the combination of 98-6 and 13H11 completely blocked 2F5 binding. These data provide support for the hypothesis that in some patients, B cells make nonneutralizing cluster II antibodies that may mask or otherwise down-modulate B-cell responses to immunogenic regions of gp41 that could be recognized by B cells capable of producing antibodies like 2F5
— id: 78808, year: 2008, vol: 82, page: 115, stat: Journal Article,

Structural Characterization of Neutralizing Human Anti-V3 Monoclonal Antibodies 3074 and 268-D
Burke, VJ; Kim, S; Williams, C; Gorny, MK; Zolla-Pazner, S; Kong, X
2008 OCT ;24(1):46-46, AIDS research & human retroviruses
— id: 91411, year: 2008, vol: 24, page: 46, stat: Journal Article,

Structure determination of an anti-HIV-1 Fab 447-52D-peptide complex from an epitaxially twinned data set
Dhillon, Amandeep K; Stanfield, Robyn L; Gorny, Miroslaw K; Williams, Constance; Zolla-Pazner, Susan; Wilson, Ian A
2008 Jul;64(Pt 7):792-802, Acta crystallographica. Section D, Biological crystallography
Although antibodies against the third variable loop (V3) of the HIV-1 viral envelope glycoprotein are among the first neutralizing antibodies to be detected in infected individuals, they are normally restricted in their specificity. X-ray crystallographic studies of V3-specific antibodies have contributed to a more thorough understanding of recognition of this epitope and of conserved features in the V3 loop that could potentially aid in the design of a multi-component vaccine. The human antibody 447-52D exhibits relatively broad neutralization of primary viral isolates compared with other V3-loop antibodies. A crystal structure of Fab 447-52D in complex with a V3 peptide (UG1033) was determined at 2.1 angstroms resolution. The structure was determined using an epitaxially twinned data set and in-house programs to detect and remove overlapping reflections. Although the processed data have lower than desired completeness and slightly higher than normal R values for the resolution, good-quality electron-density maps were obtained that enabled structure determination. The structure revealed an extended CDR H3 loop that forms a beta-sheet with the peptide, with the predominant contacts being main-chain hydrogen bonds. The V3 peptide and Fab show high structural homology with the previously reported structures of other Fab 447-52D complexes, reinforcing the idea that the V3 loop may adopt a small set of conserved structures, particularly around the crown of the beta-hairpin
— id: 81168, year: 2008, vol: 64, page: 792, stat: Journal Article,

Immunoglobulin Gene Usage by Neutralizing Human Anti-V3 HIV-1 Monoclonal Antibodies Derived from Clade B and Non-B HIV-1 Infected Individuals
Gorny, MK; Wang, X; Jiang, X; Williams, C; Volsky, B; Revesz, K; Witover, B; Krachmarov, C; Pinter, A; Nadas, A; Zolla-Pazner, S; Kong, X
2008 OCT ;24(1):46-46, AIDS research & human retroviruses
— id: 91412, year: 2008, vol: 24, page: 46, stat: Journal Article,

Structural Basis of the Antibody-Antigen Interaction in Human Anti-V3 HIV-1 Monoclonal Antibodies
Jiang, X; Burke, V; Williams, C; Gorny, MK; Zolla-Pazner, S; Kong, X
2008 OCT ;24(1):11-11, AIDS research & human retroviruses
— id: 91408, year: 2008, vol: 24, page: 11, stat: Journal Article,

Conservation of a Hydrophobic Residue in the Envelope V3 Domain could Contribute to Immune Avoidance in Subtype C HIV-1
Lynch, RM; Rong, R; Shen, T; Honnen, W; Mulenga, J; Allen, S; Blackwell, JL; Zolla-Pazner, S; Pinter, A; Gnanakaran, S; Hunter, E; Derdeyn, CA
2008 OCT ;24(1):51-51, AIDS research & human retroviruses
— id: 91414, year: 2008, vol: 24, page: 51, stat: Journal Article,

Recent advances in the characterization of HIV-1 neutralization assays for standardized evaluation of the antibody response to infection and vaccination
Polonis, Victoria R; Brown, Bruce K; Rosa Borges, Andrew; Zolla-Pazner, Susan; Dimitrov, Dimiter S; Zhang, Mei-Yun; Barnett, Susan W; Ruprecht, Ruth M; Scarlatti, Gabriella; Fenyo, Eva-Maria; Montefiori, David C; McCutchan, Francine E; Michael, Nelson L
2008 Jun 5;375(2):315-320, Virology
In AIDS vaccine development the pendulum has swung towards a renewed emphasis on the potential role for neutralizing antibodies in a successful global vaccine. It is recognized that vaccine-induced antibody performance, as assessed in the available neutralization assays, may well serve as a 'gatekeeper' for HIV-1 subunit vaccine prioritization and advancement. As a result, development of a standardized platform for reproducible measurement of neutralizing antibodies has received considerable attention. Here we review current advancements in our knowledge of the performance of different types of antibodies in a traditional primary cell neutralization assay and the newer, more standardized TZM-bl reporter cell line assay. In light of recently revealed differences (see accompanying article) in the results obtained in these two neutralization formats, parallel evaluation with both platforms should be contemplated as an interim solution until a better understanding of immune correlates of protection is achieved
— id: 78807, year: 2008, vol: 375, page: 315, stat: Journal Article,

Human and Rhesus MAbs Recognizing Distinct Groups of Epitopes that Elicit Neutralizing Antibodies against Autologous HIV-1 Strains
Robinson, JE; Franco, K; Elliott, D; Derdeyn, C; Montefiori, D; Tang, H; Gorny, M; Zolla-Pazner, S; Kraft, Z; Stamatatos, L
2008 OCT ;24(1):48-48, AIDS research & human retroviruses
— id: 91413, year: 2008, vol: 24, page: 48, stat: Journal Article,

Standardization of HIV Neutralization Assays for Use in Vaccine Research and Clinical Trials: Results from the NeutNet Working Group
Scarlatti, G; Alcami, J; Bongertz, V; Dispinseri, S; Donners, H; Fenyo, E; Heath, A; Heyndrickx, L; Holmes, H; Jassoy, C; Lusso, P; Montefiori, D; Moog, C; Morris, L; Osmanov, S; Polonis, VR; Sattentau, Q; Schuitemaker, H; Sutthent, R; Wrin, T; Zolla-Pazner, S
2008 OCT ;24(1):44-44, AIDS research & human retroviruses
— id: 91410, year: 2008, vol: 24, page: 44, stat: Journal Article,

Soluble CD4 broadens neutralization of V3-directed monoclonal antibodies and guinea pig vaccine sera against HIV-1 subtype B and C reference viruses
Wu, XL; Sambor, A; Nason, MC; Yang, ZY; Wu, L; Zolla-Pazner, S; Nabel, GJ; Mascola, JR
2008 OCT 25 ;380(2):285-295, Virology
To better understand the limits of antigenic reactivity and epitope accessibility of the V3 domain of primary HIV-1 isolates, we evaluated three human anti-V3 monoclonal antibodies (mAbs) and selected guinea pig vaccine sera for neutralization against reference panels of subtype B and C pseudoviruses derived from early stage infections. The mAbs and vaccine sera potently neutralized several prototype viruses, but displayed substantially less neutralization of most reference strains. In the presence of soluble CD4 (sCD4), the breadth of V3-mediated neutralization was increased; up to 80% and 77% of the subtype B and C viruses respectively were sensitive to V3-mediated neutralization. Unlike sCD4, the reaction of CD4-binding site mAbs b12 and F105 with native virus did not lead to full exposure of the V3 domain. These findings confirm that V3 antibodies recognize most primary viral strains, but that the epitope often has limited accessibility in the context of native envelope spike. Published by Elsevier Inc
— id: 90064, year: 2008, vol: 380, page: 285, stat: Journal Article,

Induction of Cross-clade, Anti-V3 Neutralizing Antibodies in Rabbits Using a DNA Prime/Protein Boost Immunization Regimen
Zolla-Pazner, S; Cohen, S; Krachmarov, C; Pinter, A; Wang, S; Lu, S
2008 OCT ;24(1):58-58, AIDS research & human retroviruses
— id: 91415, year: 2008, vol: 24, page: 58, stat: Journal Article,

Focusing the immune response on the V3 loop, a neutralizing epitope of the HIV-1 gp120 envelope
Zolla-Pazner, Susan; Cohen, Sandra Sharpe; Krachmarov, Chavdar; Wang, Shixia; Pinter, Abraham; Lu, Shan
2008 Mar 15;372(2):233-246, Virology
Rabbits were immunized with a novel regimen designed to focus the immune response on a single neutralizing epitope of HIV-1 gp120 and thereby preferentially induce neutralizing antibodies (Abs). Animals were primed with gp120 DNA from a clade A Env bearing the GPGR V3 motif and/or a clade C Env bearing the GPGQ V3 motif, and boosted with one or more fusion proteins containing V3 sequences from clades A, B and/or C. Immune sera neutralized three of four Tier 1 primary isolates, including strains heterologous to the immunizing strains, and potent cross-clade-neutralizing activity was demonstrated against V3 chimeric pseudoviruses carrying in a Tier 1 Env, the consensus V3 sequences from clades A1, AG, B, AE, or F. The broadest and most potent neutralizing responses were elicited with the clade C gp120 DNA and a combination of V3-fusion proteins from clades A, B and C. Neutralizing activity was primarily due to V3-specific Abs. The results demonstrate that the immune response can be focused on a neutralizing epitope and show that the anti-V3 Abs induced recognize a diverse set of V3 loops
— id: 78634, year: 2008, vol: 372, page: 233, stat: Journal Article,

Structural basis for coreceptor selectivity by the HIV type 1 V3 loop
Cardozo, Timothy; Kimura, Tetsuya; Philpott, Sean; Weiser, Barbara; Burger, Harold; Zolla-Pazner, Susan
2007 Mar;23(3):415-426, AIDS research & human retroviruses
The third variable region (V3) of the HIV-1 surface glycoprotein, gp120, plays a central role in the interaction of the virus envelope with the cell surface chemokine receptors, triggering membrane fusion and virus entry into human lymphocytes and macrophages. The CXCR4 and CCR5 chemokine receptors are used by 'X4-tropic' and 'R5-tropic' viruses, respectively. Recently, the crown of the V3 loop was shown to bear a close structural homology to the beta2-beta3 loop in the CXC and CC chemokines, the natural ligands of CXCR4 and CCR5, respectively. This homology can serve as the foundation for 3D molecular modeling of the V3 loops from primary isolates whose coreceptor usage was experimentally defined. The modeling revealed a charged 'patch' on the surface of V3 that correlates with coreceptor usage. This V3 surface patch is positively charged in X4-tropic viruses and negatively charged or neutral in R5-tropic viruses, and is formed by two amino acids, at position 11 and at position 24 or 25; amino acids 11 and 24 or 11 and 25 contact each other in 3D space. Residues at positions 11 and 25 were known previously to influence coreceptor usage, and the charge of the residues at these two positions is often used to predict viral tropism. However, we found that the predictive value of using the charge of residues 11, 24, and 25 to identify X4 or R5 tropism was improved over using only the charge of residues 11 and 25. Thus, the data suggest a new ' 11/24/25 rule' : a positively charged amino acid at position 11, 24, or 25 defines X4; otherwise R5. This rule gave an overall predictive value of 94% for 217 viruses whose tropism had been determined experimentally as either X4 or R5. The results have additional implications for the design of HIV therapeutics, vaccines, and strategies for monitoring disease progression
— id: 72077, year: 2007, vol: 23, page: 415, stat: Journal Article,

Type-specific epitopes targeted by monoclonal antibodies with exceptionally potent neutralizing activities for selected strains of human immunodeficiency virus type 1 map to a common region of the V2 domain of gp120 and differ only at single positions from the clade B consensus sequence
Honnen, W J; Krachmarov, C; Kayman, S C; Gorny, M K; Zolla-Pazner, S; Pinter, A
2007 Feb;81(3):1424-1432, Journal of virology
Only a few monoclonal antibodies (MAbs) have been isolated that recognize conserved sites in human immunodeficiency virus type 1 (HIV-1) Env proteins and possess broad neutralizing activities. Other MAbs directed against targets in various domains of Env have been described that are strongly neutralizing, but they possess limited breadth. One such MAb, 2909, possesses a uniquely potent neutralizing activity specific for a quaternary epitope on SF162 Env that requires the presence of both the V2 and the V3 domains. We now show that replacement of the SF162 V3 sequence with consensus V3 sequences of multiple subtypes led to attenuated but still potent neutralization by 2909 and that the main determinants for the type specificity of 2909 reside in the V2 domain. A substitution at position 160 completely eliminated 2909 reactivity, and mutations at position 167 either attenuated or potentiated neutralization by this antibody. Different substitutions at the same positions in V2 were previously shown to introduce epitopes recognized by MAbs 10/76b and C108g and to allow potent neutralization by these MAbs. Two substitutions at key positions in the V2 domain of JR-FL Env also allowed potent expression of the 2909 epitope, and single substitutions in YU2 V2 were sufficient for expression of the 2909, C108g, and 10/76b epitopes. These results demonstrate that the minimal epitopes for 2909, C108g, and 10/76b differed from that of the clade B consensus sequence only at single positions and suggest that all three MAbs recognize distinct variants of a relatively conserved sequence in V2 that is a particularly sensitive mediator of HIV-1 neutralization
— id: 93793, year: 2007, vol: 81, page: 1424, stat: Journal Article,

Targeted killing of virally infected cells by radiolabeled antibodies to viral proteins
Dadachova, Ekaterina; Patel, Mahesh C; Toussi, Sima; Apostolidis, Christos; Morgenstern, Alfred; Brechbiel, Martin W; Gorny, Miroslaw K; Zolla-Pazner, Susan; Casadevall, Arturo; Goldstein, Harris
2006 Nov;3(11):e427-e427, PLoS medicine
BACKGROUND: The HIV epidemic is a major threat to health in the developing and western worlds. A modality that targets and kills HIV-1-infected cells could have a major impact on the treatment of acute exposure and the elimination of persistent reservoirs of infected cells. The aim of this proof-of-principle study was to demonstrate the efficacy of a therapeutic strategy of targeting and eliminating HIV-1-infected cells with radiolabeled antibodies specific to viral proteins in vitro and in vivo. METHODS AND FINDINGS: Antibodies to HIV-1 envelope glycoproteins gp120 and gp41 labeled with radioisotopes bismuth 213 ((213)Bi) and rhenium 188 ((188)Re) selectively killed chronically HIV-1-infected human T cells and acutely HIV-1-infected human peripheral blood mononuclear cells (hPBMCs) in vitro. Treatment of severe combined immunodeficiency (SCID) mice harboring HIV-1-infected hPBMCs in their spleens with a (213)Bi- or (188)Re-labeled monoclonal antibody (mAb) to gp41 resulted in a 57% injected dose per gram uptake of radiolabeled mAb in the infected spleens and in a greater than 99% elimination of HIV-1-infected cells in a dose-dependent manner. The number of HIV-1-infected thymocytes decreased 2.5-fold in the human thymic implant grafts of SCID mice treated with the (188)Re-labeled antibody to gp41 compared with those treated with the (188)Re-control mAb. The treatment did not cause acute hematologic toxicity in the treated mice. CONCLUSIONS: The current study demonstrates the effectiveness of HIV-targeted radioimmunotherapy and may provide a novel treatment option in combination with highly active antiretroviral therapy for the eradication of HIV
— id: 78809, year: 2006, vol: 3, page: e427, stat: Journal Article,

Distinct sequence patterns characterize the V3 region of HIV type 1 gp120 from subtypes A and C
Felsovalyi, Klara; Nadas, Arthur; Zolla-Pazner, Susan; Cardozo, Timothy
2006 Jul;22(7):703-708, AIDS research & human retroviruses
The known sequences of HIV-1 viruses have been categorized into subtypes based on the phylogenetic partitioning of their env and gag gene sequences. The env gene encodes the protein gp120, which contains five sequence- variable regions (V1 to V5), of which the V3 loop is of central importance to viral infectivity. The V3 loop consensus sequences of HIV-1 subtype A and C viruses are similar, and more similar to one another than the V3 consensus sequences of any other two HIV-1 subtypes. However, using a position-specific statistical comparison, we found that the V3 region of these two subtypes is statistically distinct (p = approximately 0.0). (The p-value calculated to the lowest limit of representation on the computer used to run the calculation. This lowest limit was 10(16). Although theoretically a p-value cannot be equal to 0.0, the p-value for the comparisons in question can be intuitively considered to be extremely small, or approximately 0.0.)
— id: 67537, year: 2006, vol: 22, page: 703, stat: Journal Article,

Cross-clade neutralizing activity of human anti-V3 monoclonal antibodies derived from the cells of individuals infected with non-B clades of human immunodeficiency virus type 1
Gorny, Miroslaw K; Williams, Constance; Volsky, Barbara; Revesz, Kathy; Wang, Xiao-Hong; Burda, Sherri; Kimura, Tetsuya; Konings, Frank A J; Nadas, Arthur; Anyangwe, Christopher A; Nyambi, Phillipe; Krachmarov, Chavdar; Pinter, Abraham; Zolla-Pazner, Susan
2006 Jul;80(14):6865-6872, Journal of virology
The majority of global human immunodeficiency virus infections are caused by viruses characterized by a GPGQ motif at the tip of the V3 loop. Characterization of anti-V3 monoclonal antibodies (MAbs) that neutralize isolates with the GPGQ V3 motif is an important step in designing vaccines that will induce such Abs. Consequently, seven human anti-V3 MAbs derived from the cells of individuals infected with non-B-subtype viruses (anti-V3(non-B) MAbs) were generated from the cells of individuals from Africa infected with circulating recombinant forms CRF02_AG, CRF09_cpx, and CRF13_cpx, each of which contains a subtype A env gene. Sequence analysis of plasma viruses revealed a GPGQ motif at the apex of the V3 loop from six of the seven subjects and a GPGR motif from one subject. The MAbs were selected with fusion proteins (FP) containing V3(92UG037.8) or V3(JR-CSF) from subtype A or B, respectively. In virus binding assays, five of the seven (71%) anti-V3(non-B) MAbs bound to V3-FPs from both subtype A and subtype B, while only four of the nine (44%) anti-V3(B) MAbs recognized both V3-FPs. Using two neutralization assays, both the anti-V3(non-B) and the anti-V3(B) MAbs neutralized subtype B viruses with similar activities, while the anti-V3(non-B) MAbs exhibited a tendency toward both increased potency and breadth of neutralization against non-B viruses compared to anti-V3(B) MAbs. Statistical significance was not achieved, due in large measure to the sizes of the MAb panels, but the overall pattern of data strongly suggests that viruses with the GPGQ motif at the tip of the V3 loop induce anti-V3 Abs with broader cross-neutralizing activity than do viruses with the GPGR motif
— id: 67852, year: 2006, vol: 80, page: 6865, stat: Journal Article,

Immunoprophylaxis against mother-to-child transmission of HIV-1
Gorny, Miroslaw K; Zolla-Pazner, Susan
2006 Jul;3(7):e259-e259, PLoS medicine
— id: 74576, year: 2006, vol: 3, page: e259, stat: Journal Article,

Nonneutralizing antibodies are able to inhibit human immunodeficiency virus type 1 replication in macrophages and immature dendritic cells
Holl, Vincent; Peressin, Maryse; Decoville, Thomas; Schmidt, Sylvie; Zolla-Pazner, Susan; Aubertin, Anne-Marie; Moog, Christiane
2006 Jun;80(12):6177-6181, Journal of virology
Only five monoclonal antibodies (MAbs) neutralizing a broad range of primary isolates (PI) have been identified up to now. We have found that some MAbs with no neutralizing activities according to the 'conventional' neutralization assay, involving phytohemagglutinin-stimulated peripheral blood mononuclear cells as targets, efficiently inhibit the replication of human immunodeficiency virus type 1 (HIV-1) PI in macrophages and immature dendritic cells (iDC). The mechanism of inhibition is distinct from the neutralization of infectivity occurring via Fab fragments and involves the interaction of the F portion with the FcgammaRs present on macrophages and iDC. We propose that, if such nonneutralizing inhibitory antibodies limit mucosal HIV transmission, they should be induced by vaccination
— id: 78812, year: 2006, vol: 80, page: 6177, stat: Journal Article,

Efficient inhibition of HIV-1 replication in human immature monocyte-derived dendritic cells by purified anti-HIV-1 IgG without induction of maturation
Holl, Vincent; Peressin, Maryse; Schmidt, Sylvie; Decoville, Thomas; Zolla-Pazner, Susan; Aubertin, Anne-Marie; Moog, Christiane
2006 Jun 1;107(11):4466-4474, Blood
During mucosal HIV transmission, immature dendritic cells (DCs) present in the mucosa are among the first cellular targets of the virus. Previous studies have analyzed the inhibition of HIV-1 transfer from human mature DCs to T lymphocytes by neutralizing IgG, but so far no in vitro data regarding the capacity of antibodies to inhibit HIV-1 infection of immature DCs have been reported. Here, we found an increased HIV-inhibitory activity of monoclonal IgG and purified polyclonal IgG when immature monocyte-derived dendritic cells (iMDDCs) were used as target cells instead of autologous blood lymphocytes. We showed that FcgammaRII is involved in the mechanism for inhibiting HIV-1 infection of iMDDCs by IgG, whereas no induction of maturation was detected at concentrations of IgG that result in a 90% reduction of HIV replication. After induction of FcgammaRI expression on iMDDCs by IFN-gamma, an augmentation of the HIV-inhibitory activity of IgG, related to the expression of FcgammaRI, was observed. Taken together, our results demonstrate the participation of FcgammaRs in HIV-1 inhibition by IgG when iMDDCs are the targets. We propose that IgG is able to efficiently inhibit HIV-1 replication in iMDDCs and should be one of the components to be induced by vaccination
— id: 78814, year: 2006, vol: 107, page: 4466, stat: Journal Article,

Novel approach for differential diagnosis of HIV infections in the face of vaccine-generated antibodies: utility for detection of diverse HIV-1 subtypes
Khurana, Surender; Needham, James; Park, Susan; Mathieson, Bonnie; Busch, Michael P; Nemo, George; Nyambi, Phillipe; Zolla-Pazner, Susan; Laal, Suman; Mulenga, Joseph; Chomba, Elwyn; Hunter, Eric; Allen, Susan; McIntyre, James; Hewlett, Indira; Lee, Sherwin; Tang, Shixing; Cowan, Elliot; Beyrer, Chris; Altfeld, Marcus; Yu, Xu G; Tounkara, Anatole; Koita, Ousmane; Kamali, Anatoli; Nguyen, Nga; Graham, Barney S; Todd, Deborah; Mugenyi, Peter; Anzala, Omu; Sanders, Eduard; Ketter, Nzeera; Fast, Patricia; Golding, Hana
2006 Nov 1;43(3):304-312, Journal of acquired immune deficiency syndromes. JAIDS
Because increasing numbers of HIV vaccine candidates are being tested globally, it is essential to differentiate vaccine- from virus-induced antibodies. Most of the currently tested vaccines contain multiple viral components. As a result, many vaccine recipients give positive results in FDA-licensed HIV serodetection tests. We have identified conserved sequences in Env-gp41 and Gag-p6, which are recognized soon after infection but are not included in most HIV vaccine candidates. A new HIV serodetection assay, the HIV-SELECTEST, was established that distinguishes between vaccine-induced antibodies and seroconversion due to true HIV infections. It is important to make this assay globally relevant, because many clinical trials are conducted around the world where most HIV infections are due to non-B subtype HIV-1. Therefore, the current study examined the reactivity of plasma samples from >3,000 infections with diverse HIV subtypes worldwide. The HIV-SELECTEST performed at >99% specificity and sensitivity. Both recent and established infections with clades A, B, C, D, E, F, G, J, and CRFs were detected. Antibodies elicited by other vaccinations or infections endemic to the clinical trial sites did not react in this assay. Therefore, HIV-SELECTEST could be an important differential diagnostic tool for HIV vaccine trials, blood banks, and population screening worldwide
— id: 78810, year: 2006, vol: 43, page: 304, stat: Journal Article,

Factors determining the breadth and potency of neutralization by V3-specific human monoclonal antibodies derived from subjects infected with clade A or clade B strains of human immunodeficiency virus type 1
Krachmarov, C P; Honnen, W J; Kayman, S C; Gorny, M K; Zolla-Pazner, S; Pinter, Abraham
2006 Jul;80(14):7127-7135, Journal of virology
The neutralizing activities of anti-V3 antibodies for HIV-1 isolates is affected both by sequence variation within V3 and by epitope masking by the V1/V2 domain. To analyze the relative contribution of V3 sequence variation, chimeric Env genes that contained consensus V3 sequences from seven HIV-1 subtypes in the neutralization-sensitive SF162 Env backbone were constructed. Resulting viral pseudotypes were tested for neutralization by 15 anti-V3 MAbs isolated from humans infected with viruses of either subtype B (anti-V3(B) MAbs) or subtype A (anti-V3(A) MAbs). Pseudovirions with the subtype B consensus V3 sequence were potently neutralized (IC(50) < 0.006 microg/ml) by all but one of these MAbs, while pseudovirions with V3 subtypes A, C, F, H, AG, and AE were generally neutralized more effectively by anti-V3(A) MAbs than by anti-V3(B) MAbs. A V1/V2-masked Env version of SF162 Env with the consensus B V3 sequence was also neutralized by these MAbs, although with considerably lower potency, while similarly masked chimeras with V3 sequences of subtype A, C, or AG were weakly neutralized by anti-V3(A) MAbs but not by anti-V3(B) MAbs. Mutations in the V1/V2 domain of YU-2 Env increased the sensitivity of this highly resistant Env to a pool of anti-V3(B) MAbs several thousand-fold. These results demonstrated (i) the exceptional sensitivity of representative V3 domains of multiple subtypes to neutralization in the absence of epitope masking, (ii) the broader neutralizing activity of anti-V3(A) MAbs for viruses containing diverse V3 sequences, and (iii) the generality and dominant effect of V1/V2 masking on restriction of V3-mediated neutralization
— id: 93794, year: 2006, vol: 80, page: 7127, stat: Journal Article,

HIV-1 coreceptor selectivity: Structural analogy between HIV-1V3 regions and chemokine beta-hairpins is not the explanation - Response to matters arising
Rosen, O; Samson, AO; Sharon, M; Zolla-Pazner, S; Anglister, J
2006 APR ;14(4):649-651, Structure
— id: 63854, year: 2006, vol: 14, page: 649, stat: Journal Article,

Crystal structures of human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 2219 in complex with three different V3 peptides reveal a new binding mode for HIV-1 cross-reactivity
Stanfield, Robyn L; Gorny, Miroslaw K; Zolla-Pazner, Susan; Wilson, Ian A
2006 Jun;80(12):6093-6105, Journal of virology
Human monoclonal antibody 2219 is a neutralizing antibody isolated from a human immunodeficiency virus type 1-infected individual. 2219 was originally selected for binding to a V3 fusion protein and can neutralize primary isolates from subtypes B, A, and F. Thus, 2219 represents a cross-reactive, human anti-V3 antibody. Fab 2219 binds to one face of the variable V3 beta-hairpin, primarily contacting conserved residues on the N-terminal beta-strand of V3, leaving the V3 crown or tip largely accessible. Three V3/2219 complexes reveal the antibody-bound conformations for both the N- and C-terminal regions that flank the V3 crown and illustrate how twisting of the V3 loop alters the relative dispositions and pairing of the amino acids in the adjacent V3 beta-strands and how the antibody can accommodate V3 loops with different sequences
— id: 78813, year: 2006, vol: 80, page: 6093, stat: Journal Article,

The antigenic determinants on HIV p24 for CD4+ T cell inhibiting antibodies as determined by limited proteolysis, chemical modification, and mass spectrometry
Williams, Jason G; Tomer, Kenneth B; Hioe, Catarina E; Zolla-Pazner, Susan; Norris, Philip J
2006 Nov;17(11):1560-1569, Journal of the American Society for Mass Spectrometry
In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein-protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in protein-protein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. In this report, both limited proteolysis and chemical modification were combined with several mass spectrometric techniques in efforts to define the protein surface on the HIV core protein, p24, recognized by two different monoclonal human antibodies that were isolated from HIV+ patients. One of these antibodies, 1571, strongly inhibits the CD4+ T cell proliferative response to a known epitope (PEVIPMFSALSEGATP), while the other antibody, 241-D, does not inhibit as strongly. The epitopes for both of these antibodies were determined to be discontinuous and localized to the N-terminus of p24. Interestingly, the epitope recognized by the strongly inhibiting antibody, 1571, completely overlaps the T cell epitope PEVIPMFSALSEGATP, while the antibody 241-D binds to a region adjacent to the region of p24 recognized by the antibody 1571. These results suggest that, possibly due to epitope competition, antibodies produced during HIV infection can negatively affect CD4+ T cell-mediated immunity against the virus
— id: 78811, year: 2006, vol: 17, page: 1560, stat: Journal Article,

A highly conserved arginine in gp120 governs HIV-1 binding to both syndecans and CCR5 via sulfated motifs
de Parseval, Aymeric; Bobardt, Michael D; Chatterji, Anju; Chatterji, Udayan; Elder, John H; David, Guido; Zolla-Pazner, Susan; Farzan, Michael; Lee, Tun-Hou; Gallay, Philippe A
2005 Nov 25;280(47):39493-39504, Journal of biological chemistry
HIV-1 has maximized its utilization of syndecans. It uses them as in cis receptors to infect macrophages and as in trans receptors to infect T-lymphocytes. In this study, we investigated at a molecular level the mechanisms that control HIV-1-syndecan interactions. We found that a single conserved arginine (Arg-298) in the V3 region of gp120 governs HIV-1 binding to syndecans. We found that an amine group on the side chain of this residue is necessary for syndecan utilization by HIV-1. Furthermore, we showed that HIV-1 binds syndecans via a 6-O sulfation, demonstrating that this binding is not the result of random interactions between basic residues and negative charges, but the result of specific contacts between gp120 and a well defined sulfation in syndecans. Surprisingly, we found that Arg-298, which mediates HIV-1 binding to syndecans, also mediates HIV-1 binding to CCR5. We postulated that HIV-1 recognizes similar motifs on syndecans and CCR5. Supporting this hypothesis, we obtained several lines of evidence that suggest that the 6-O sulfation recognized by HIV-1 on syndecans mimics the sulfated tyrosines recognized by HIV-1 in the N terminus of CCR5. Our finding that CCR5 and syndecans are exploited by HIV-1 via a single determinant echoes the mechanisms by which chemokines utilize these two disparate receptors and suggests that the gp120/chemokine mimicry may represent a common strategy in microbial pathogenesis
— id: 78815, year: 2005, vol: 280, page: 39493, stat: Journal Article,

Identification of a new quaternary neutralizing epitope on human immunodeficiency virus type 1 virus particles
Gorny, Miroslaw K; Stamatatos, Leonidas; Volsky, Barbara; Revesz, Kathy; Williams, Constance; Wang, Xiao-Hong; Cohen, Sandra; Staudinger, Robert; Zolla-Pazner, Susan
2005 Apr;79(8):5232-5237, Journal of virology
The selection of human monoclonal antibodies (MAbs) specific for human immunodeficiency virus (HIV) type 1 by binding assays may fail to identify Abs to quaternary epitopes on the intact virions. The HIV neutralization assay was used for the selection of human MAb 2909, which potently neutralizes SF162 and recognizes an epitope on the virus surface but not on soluble proteins. Three regions of gp120, the V2 and V3 loops and the CD4 binding domain, contribute to the epitope recognized by MAb 2909. The existence of such a unique MAb, which defines a complex epitope formed by a quaternary structure, suggests that there may be other new neutralizing HIV epitopes to target with vaccines
— id: 54109, year: 2005, vol: 79, page: 5232, stat: Journal Article,

Antibodies that are cross-reactive for human immunodeficiency virus type 1 clade a and clade B v3 domains are common in patient sera from Cameroon, but their neutralization activity is usually restricted by epitope masking
Krachmarov, Chavdar; Pinter, Abraham; Honnen, William J; Gorny, Miroslaw K; Nyambi, Phillipe N; Zolla-Pazner, Susan; Kayman, Samuel C
2005 Jan;79(2):780-790, Journal of virology
Sera from human immunodeficiency virus type 1 (HIV-1)-infected North American patients recognized a fusion protein expressing a V3 loop from a clade B primary isolate virus (JR-CSF) but not from a clade A primary isolate virus (92UG037.8), while most sera from Cameroonian patients recognized both fusion proteins. Competition studies of consensus V3 peptides demonstrated that the majority of the cross-reactive Cameroonian sera contained cross-reactive antibodies that reacted strongly with both V3 sequences. V3-specific antibodies purified from all six cross-reactive sera examined had potent neutralizing activity for virus pseudotyped with envelope proteins (Env) from SF162, a neutralization-sensitive clade B primary isolate. For four of these samples, neutralization of SF162 pseudotypes was blocked by both the clade A and clade B V3 fusion proteins, indicating that this activity was mediated by cross-reactive antibodies. In contrast, the V3-reactive antibodies from only one of these six sera had significant neutralizing activity against viruses pseudotyped with Envs from typically resistant clade B (JR-FL) or clade A (92UG037.8) primary isolates. However, the V3-reactive antibodies from these cross-reactive Cameroonian sera did neutralize virus pseudotyped with chimeric Envs containing the 92UG037.8 or JR-FL V3 sequence in Env backbones that did not express V1/V2 domain masking of V3 epitopes. These data indicated that Cameroonian sera frequently contain cross-clade reactive V3-directed antibodies and indicated that the typical inability of such antibodies to neutralize typical, resistant primary isolate Env pseudotypes was primarily due to indirect masking effects rather than to the absence of the target epitopes
— id: 78821, year: 2005, vol: 79, page: 780, stat: Journal Article,

HIV-1 envelope pseudotyped viral vectors and infectious molecular clones expressing the same envelope glycoprotein have a similar neutralization phenotype, but culture in peripheral blood mononuclear cells is associated with decreased neutralization sensitivity
Louder, Mark K; Sambor, Anna; Chertova, Elena; Hunte, Tai; Barrett, Sarah; Ojong, Fallon; Sanders-Buell, Eric; Zolla-Pazner, Susan; McCutchan, Francine E; Roser, James D; Gabuzda, Dana; Lifson, Jeffrey D; Mascola, John R
2005 Sep 1;339(2):226-238, Virology
Recombinant lentiviral vectors pseudotyped with heterologous HIV-1 envelope glycoproteins allow rapid and accurate measurement of antibody-mediated HIV-1 neutralization. However, the neutralization phenotypes of envelope pseudoviruses have not been directly compared to isogenic replication competent HIV-1. We produced pseudoviruses expressing three different HIV-1 envelope glycoproteins and subcloned the same three env genes into a replication competent NL4-3 molecular clone. For each of the antibodies tested, the neutralization dose-response curves of pseudoviruses and corresponding replication competent viruses were similar. Thus, envelope pseudoviruses can be used to study the anti-HIV-1 neutralizing antibody response. A single passage of replication competent virus derived from 293T cells through peripheral blood mononuclear cells (PBMC) caused a substantial decrease in sensitivity to neutralizing antibodies. This was associated with an increase in average virion envelope glycoprotein content of the PBMC-derived virus. Replication competent HIV-1 and isogenic envelope pseudoviruses have similar neutralization characteristics, but passage into PBMC is associated with decreased sensitivity to neutralization
— id: 78816, year: 2005, vol: 339, page: 226, stat: Journal Article,

The C108g epitope in the V2 domain of gp120 functions as a potent neutralization target when introduced into envelope proteins derived from human immunodeficiency virus type 1 primary isolates
Pinter, Abraham; Honnen, William J; D'Agostino, Paul; Gorny, Miroslaw K; Zolla-Pazner, Susan; Kayman, Samuel C
2005 Jun;79(11):6909-6917, Journal of virology
Monoclonal antibodies (MAbs) directed against epitopes in the V2 domain of human immunodeficiency virus type 1 gp120 often possess neutralizing activity, but these generally are highly type specific, neutralize only laboratory isolates, or have low potency. The most potent of these is C108g, directed against a type-specific epitope in HXB2 and BaL gp120s, which is glycan dependent and, in contrast to previous reports, dependent on intact disulfide bonds. This epitope was introduced into two primary Envs, derived from a neutralization-sensitive (SF162) and a neutralization-resistant (JR-FL) isolate, by substitution of two residues and, for SF162, addition of an N-linked glycosylation site. C108g effectively neutralized both variant Envs with considerably higher potency than standard MAbs against the V3 and CD4-binding domains and the broadly neutralizing MAbs 2G12 and 2F5. These amino acid substitutions also introduced the epitope recognized by a second V2-specific MAb, 10/76b, but this MAb possessed potent neutralizing activity only in the absence of the glycan required for C108g reactivity. In contrast to other gp120-specific neutralizing MAbs, C108g did not block binding of soluble Env proteins to either the CD4 or the CCR5 receptor, but studies with a fusion-arrested Env indicated that C108g neutralized at a step preceding the one blocked by the gp41-specific MAb, 2F5. These results indicate that the V1/V2 domain possesses targets that mediate potent neutralization of primary viral isolates via a novel mechanism and suggest that inclusion of carbohydrate determinants into these epitopes may help overcome the indirect masking effects that limit the neutralizing potency of antibodies commonly produced after infection
— id: 78817, year: 2005, vol: 79, page: 6909, stat: Journal Article,

Induced fit in HIV-neutralizing antibody complexes: evidence for alternative conformations of the gp120 V3 loop and the molecular basis for broad neutralization
Rosen, Osnat; Chill, Jordan; Sharon, Michal; Kessler, Naama; Mester, Brenda; Zolla-Pazner, Susan; Anglister, Jacob
2005 May 17;44(19):7250-7258, Biochemistry
Human monoclonal antibody (mAb) 447-52D neutralizes a broad spectrum of HIV-1 isolates, whereas murine mAb 0.5beta, raised against gp120 of the X4 isolate HIV-1(IIIB), neutralizes this strain specifically. Two distinct gp120 V3 peptides, V3(MN) and V3(IIIB), adopt alternative beta-hairpin conformations when bound to 447-52D and 0.5beta, respectively, suggesting that the alternative conformations of this loop play a key role in determining the coreceptor specificity of HIV-1. To test this hypothesis and to better understand the molecular basis underlying an antibody's breadth of neutralization, the solution structure of the V3(IIIB) peptide bound to 447-52D was determined by NMR. V3(IIIB) and V3(MN) peptides bound to 447-52D exhibited the same N-terminal strand conformation, while the V3(IIIB) peptide revealed alternative N-terminal conformations when bound to 447-52D and 0.5beta. Comparison of the three known V3 structures leads to a model in which a 180 degrees change in the orientation of the side chains and the resulting one-residue shift in hydrogen bonding patterns in the N-terminal strand of the beta-hairpins markedly alter the topology of the surface that interacts with antibodies and that can potentially interact with the HIV-1 coreceptors. Predominant interactions of 447-52D with three conserved residues of the N-terminal side of the V3 loop, K312, I314, and I316, can account for its broad cross reactivity, whereas the predominant interactions of 0.5beta with variable residues underlie its strain specificity
— id: 78818, year: 2005, vol: 44, page: 7250, stat: Journal Article,

Vaccination of rhesus macaques with recombinant Mycobacterium bovis bacillus Calmette-Guerin Env V3 elicits neutralizing antibody-mediated protection against simian-human immunodeficiency virus with a homologous but not a heterologous V3 motif
Someya, Kenji; Cecilia, Dayaraj; Ami, Yasushi; Nakasone, Tadashi; Matsuo, Kazuhiro; Burda, Sherri; Yamamoto, Hiroshi; Yoshino, Naoto; Kaizu, Masahiko; Ando, Shuji; Okuda, Kenji; Zolla-Pazner, Susan; Yamazaki, Shudo; Yamamoto, Naoki; Honda, Mitsuo
2005 Feb;79(3):1452-1462, Journal of virology
Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guerin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations
— id: 78820, year: 2005, vol: 79, page: 1452, stat: Journal Article,

Improving on nature: focusing the immune response on the V3 loop
Zolla-Pazner, Susan
2005 ;14(3-4):69-72, Human antibodies
The conventional wisdom suggests that 'constant' rather than 'variable' regions of the HIV envelope (Env) glycoproteins would induce the most broadly reactive antibodies (Abs). However, of the several epitopes in the conserved regions of gp120 and gp41 that induce neutralizing Abs, all are well-protected by protein folding, glycosylation, and/or oligomerization of the Env proteins on the virus surface; most are only transiently exposed during the process of infection or are poorly immunogenic. In contrast, the third variable region (V3) of gp120 appears to be at least partially exposed during various stages of the infectious process, is immunogenic in essentially all HIV+ subjects, and is capable of inducing Abs able to neutralize a broad array of primary isolates. While these Abs were originally thought to be isolate-specific, a large body of data now shows that anti-V3 Abs from HIV-infected individuals indeed show intra- and inter-clade cross-reactivity with respect to both binding to diverse gp120 molecules and neutralization of many primary isolates. This cross-reactivity of anti-V3 Abs is counter-intuitive if one focuses on the sequence variability rather than on the conserved V3 structures which must be present in order to allow this region of the virus envelope to mediate selection of and interaction with chemokine receptors. Current data, summarized here, support the hypothesis that the V3 region of gp120 can induce broadly-reactive, cross-neutralizing Abs and as such should constitute a prominent target of the immune response induced with an HIV vaccine
— id: 66460, year: 2005, vol: 14, page: 69, stat: Journal Article,

Infection With HIV Type 1 Group M Non-B Subtypes in Individuals Living in New York City
Achkar, Jacqueline M; Burda, Sherri T; Konings, Frank A J; Urbanski, Mateusz M; Williams, Constance A U; Seifen, Dorothee; Kahirimbanyi, Martha N; Vogler, Mary; Parta, Mark; Lupatkin, Helene C; Zolla-Pazner, Susan; Nyambi, Phillipe N
2004 Jul 1;36(3):835-844, Journal of acquired immune deficiency syndromes. JAIDS
OBJECTIVE:: To document infection with HIV type 1 (HIV-1) group M non-B subtypes in individuals living in New York City. DESIGN:: From October 1999 through April 2003, HIV-1-seropositive individuals were selected from 3 clinics in New York City based on having risk factors for infection with HIV-1 non-B subtypes. METHODS:: HIV-1 RNA was extracted from plasma samples, and partial gag, pol, or env genes were amplified by PCR analysis. The infecting HIV-1 group M subtype was determined based on results of either heteroduplex mobility assay or sequencing and phylogenetic analysis. RESULTS:: Ninety-seven subjects were enrolled in the study. Of the 97 subjects, 91 (94%) were selected based on having emigrated from a non-European country, while 6 (6%) were native United States citizens. Subtypes were successfully determined in 53 (55%) of the 97 plasma samples tested. The subtypes in 2 plasma samples were unclassifiable. HIV-1 infections were classified as those due to the following group M subtypes: A (n = 4; 7%), B (n = 12; 22%), C (n = 8; 15%), F (n = 2; 4%), CRF01_AE-like (n = 7; 13%), CRF02_AG-like (n = 19; 34%), an intersubtype recombinant form G/A (n = 1; 2%), and unclassifiable viruses (n = 2; 4%). CONCLUSION:: This study reveals infection with a broad variety of HIV-1 group M subtypes mostly in the immigrant population of New York City as well as how several non-B subtypes are being introduced into the United States
— id: 56107, year: 2004, vol: 36, page: 835, stat: Journal Article,

Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies
Binley, James M; Wrin, Terri; Korber, Bette; Zwick, Michael B; Wang, Meng; Chappey, Colombe; Stiegler, Gabriela; Kunert, Renate; Zolla-Pazner, Susan; Katinger, Hermann; Petropoulos, Christos J; Burton, Dennis R
2004 Dec;78(23):13232-13252, Journal of virology
Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV(+) plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (</=7%). MAbs b6 (directed against the CD4 binding site) and X5 (directed against a CD4-induced epitope of gp120) neutralized only sensitive primary clade B viruses. The HIV(+) plasma neutralized 70% of the viruses, including some from all major clades. Further analysis revealed five neutralizing immunotypes that were somewhat associated with clades. As well as the significance for vaccine design, our data have implications for passive-immunization studies in countries where clade C viruses are common, given that only MAbs b12 and 4E10 were effective against viruses from this clade
— id: 78822, year: 2004, vol: 78, page: 13232, stat: Journal Article,

The v3 loop is accessible on the surface of most human immunodeficiency virus type 1 primary isolates and serves as a neutralization epitope
Gorny, Miroslaw K; Revesz, Kathy; Williams, Constance; Volsky, Barbara; Louder, Mark K; Anyangwe, Christopher A; Krachmarov, Chavdar; Kayman, Samuel C; Pinter, Abraham; Nadas, Arthur; Nyambi, Phillipe N; Mascola, John R; Zolla-Pazner, Susan
2004 Mar;78(5):2394-2404, Journal of virology
Antibodies (Abs) against the V3 loop of the human immunodeficiency virus type 1 gp120 envelope glycoprotein were initially considered to mediate only type-specific neutralization of T-cell-line-adapted viruses. However, recent data show that cross-neutralizing V3 Abs also exist, and primary isolates can be efficiently neutralized with anti-V3 monoclonal Abs (MAbs). The neutralizing activities of anti-V3 polyclonal Abs and MAbs may, however, be limited due to antigenic variations of the V3 region, a lack of V3 exposure on the surface of intact virions, or Ab specificity. For clarification of this issue, a panel of 32 human anti-V3 MAbs were screened for neutralization of an SF162-pseudotyped virus in a luciferase assay. MAbs selected with a V3 fusion protein whose V3 region mimics the conformation of the native virus were significantly more potent than MAbs selected with V3 peptides. Seven MAbs were further tested for neutralizing activity against 13 clade B viruses in a single-round peripheral blood mononuclear cell assay. While there was a spectrum of virus sensitivities to the anti-V3 MAbs observed, 12 of the 13 viruses were neutralized by one or more of the anti-V3 MAbs. MAb binding to intact virions correlated significantly with binding to solubilized gp120s and with the potency of neutralization. These results demonstrate that the V3 loop is accessible on the native virus envelope, that the strength of binding of anti-V3 Abs correlates with the potency of neutralization, that V3 epitopes may be shared rather than type specific, and that Abs against the V3 loop, particularly those targeting conformational epitopes, can mediate the neutralization of primary isolates
— id: 42250, year: 2004, vol: 78, page: 2394, stat: Journal Article,

Defining human immunodeficiency virus (HIV) type 1 immunotypes with six human monoclonal antibodies
Nadas, Arthur; Zhong, Ping; Burda, Sherri; Zekeng, Leopold; Urbanski, Mateusz; Gorny, Miroslaw K; Zolla-Pazner, Susan; Nyambi, Phillipe N
2004 Jan;20(1):55-65, AIDS research & human retroviruses
Studies of HIV-1 immunological relatedness have revealed that genetic diversity does not parallel antigenic diversity and have recently shown that HIV-1 strains from different geographic regions from around the world can be grouped into a small number of immunologically defined groups (immunotypes). Previously, the binding patterns of 28 monoclonal antibodies (mAbs) (specific for V3 and C5 of gp120 and cluster I of gp41) with 26 HIV-1 virions obtained globally were determined in a virus binding assay. Analysis of the binding patterns of these 728 mAb/virus combinations now reveals that a particular subset containing six of the 28 mAbs can correctly immunotype 24 of the 26 isolates (92%) into three immunotypes. Like the original panel of mAbs, the subset of six mAbs identified was directed against epitopes in the V3 and C5 regions of gp 120 as well as cluster I of gp41. The binding patterns ('profiles') of these six mAbs with 24 additional HIV-1 virions from Cameroon confirmed that epitopes in V3 and C5 of gp120 and cluster I of gp41 are well exposed on these viruses. Multivariate analysis of the binding patterns of these six mAbs with all 50 viruses (26 obtained globally and 24 obtained from Cameroon) indicates that the viruses from Cameroon have binding profiles similar to viruses from the rest of the world and can be classified into the same three immunotypes that were previously described. This study suggests that a vaccine against HIV-1 need not be based on geographic origin of the virus or on clade, but may better be based on antigenic properties that classify the plethora of different HIV-1 viruses into immunologically defined groups
— id: 42249, year: 2004, vol: 20, page: 55, stat: Journal Article,

The V1/V2 domain of gp120 is a global regulator of the sensitivity of primary human immunodeficiency virus type 1 isolates to neutralization by antibodies commonly induced upon infection
Pinter, Abraham; Honnen, William J; He, Yuxian; Gorny, Miroslaw K; Zolla-Pazner, Susan; Kayman, Samuel C
2004 May;78(10):5205-5215, Journal of virology
A major problem hampering the development of an effective vaccine against human immunodeficiency virus type 1 (HIV-1) is the resistance of many primary viral isolates to antibody-mediated neutralization. To identify factors responsible for this resistance, determinants of the large differences in neutralization sensitivities of HIV-1 pseudotyped with Env proteins derived from two prototypic clade B primary isolates were mapped. SF162 Env pseudotypes were neutralized very potently by a panel of sera from HIV-infected individuals, while JR-FL Env pseudotypes were neutralized by only a small fraction of these sera. This differential sensitivity to neutralization was also observed for a number of monoclonal antibodies (MAbs) directed against sites in the V2, V3, and CD4 binding domains, despite often similar binding affinities of these MAbs towards the two soluble rgp120s. The neutralization phenotypes were switched for chimeric Envs in which the V1/V2 domains of these two sequences were exchanged, indicating that the V1/V2 region regulated the overall neutralization sensitivity of these Envs. These results suggested that the inherent neutralization resistance of JR-FL, and presumably of related primary isolates, is to a great extent mediated by gp120 V1/V2 domain structure rather than by sequence variations at the target sites. Three MAbs (immunoglobulin G-b12, 2G12, and 2F5) previously reported to possess broad neutralizing activity for primary HIV-1 isolates neutralized JR-FL virus at least as well as SF162 virus and were not significantly affected by the V1/V2 domain exchanges. The rare antibodies capable of neutralizing a broad range of primary isolates thus appeared to be targeted to exceptional epitopes that are not sensitive to V1/V2 domain regulation of neutralization sensitivity
— id: 78824, year: 2004, vol: 78, page: 5205, stat: Journal Article,

Structural rationale for the broad neutralization of HIV-1 by human monoclonal antibody 447-52D
Stanfield, Robyn L; Gorny, Miroslaw K; Williams, Constance; Zolla-Pazner, Susan; Wilson, Ian A
2004 Feb;12(2):193-204, Structure
447-52D is a human monoclonal antibody isolated from a heterohybridoma derived from an HIV-1-infected individual. This antibody recognizes the hypervariable gp120 V3 loop, and neutralizes both X4 and R5 primary isolates, making it one of the most effective anti-V3 antibodies characterized to date. The crystal structure of the 447-52D Fab in complex with a 16-mer V3 peptide at 2.5 A resolution reveals that the peptide beta hairpin forms a three-stranded mixed beta sheet with complementarity determining region (CDR) H3, with most of the V3 side chains exposed to solvent. Sequence specificity is conferred through interaction of the type-II turn (residues GPGR) at the apex of the V3 hairpin with the base of CDR H3. This novel mode of peptide-antibody recognition enables the antibody to bind to many different V3 sequences where only the GPxR core epitope is absolutely required
— id: 42251, year: 2004, vol: 12, page: 193, stat: Journal Article,

Human parvovirus B19 transgenic mice become susceptible to polyarthritis
Takasawa, N; Munakata, Y; Ishii, KK; Takahashi, Y; Takahashi, M; Fu, Y; Ishii, T; Fujii, H; Saito, T; Takano, H; Noda, T; Suzuki, M; Nose, M; Zolla-Patzner, S; Sasaki, T
2004 OCT 1 ;173(7):4675-4683, Journal of immunology
Human parvovirus B19 (B19) often causes acute polyarthritis in adults. in this papers we analyzed nucleotide sequences of the B19 genome of patients with rheumatoid arthritis (RA), and then introduced the nonstructual protein 1 (NS1) gene of B19 into C57BL/6 mice that had a genetic origin not susceptible to arthritis. The transgenic mice developed no lesions spontaneously, but were susceptible to type H collagen (CH)-induced arthritis. B19 NS1 was expressed in synovial cells on the articular lesions that were histologically characteristic of granulomatous synovitis and pannus formation in cartilage and bone. Serum levels of anti-CII Abs and TNF-alpha increased in NS1 transgenic mice to the same levels as those of DBA/1 mice, which were susceptible to polyarthritis. Stimulation with CH increased secretion of Th1-type- and Th2-type cytokines in NS1 transgenic mice, indicating that a nonpermissive H-2(b) haplotype in the wild type of C57BL/6 mice can be made susceptible to polyarthritis through the expression of NS1. This study is the first to show that a viral agent from the joints in humans can cause CH-induced arthritis resembling RA
— id: 46489, year: 2004, vol: 173, page: 4675, stat: Journal Article,

Characterization of the outer domain of the gp120 glycoprotein from human immunodeficiency virus type 1
Yang, Xinzhen; Tomov, Vesko; Kurteva, Svetla; Wang, Liping; Ren, Xinping; Gorny, Miroslaw K; Zolla-Pazner, Susan; Sodroski, Joseph
2004 Dec;78(23):12975-12986, Journal of virology
The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1(YU2) gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine
— id: 78823, year: 2004, vol: 78, page: 12975, stat: Journal Article,

Identifying epitopes of HIV-1 that induce protective antibodies
Zolla-Pazner, Susan
2004 Mar;4(3):199-210, Nature reviews. Immunology
— id: 42248, year: 2004, vol: 4, page: 199, stat: Journal Article,

The cross-clade neutralizing activity of a human monoclonal antibody is determined by the GPGR V3 motif of HIV type 1
Zolla-Pazner, Susan; Zhong, Ping; Revesz, Kathy; Volsky, Barbara; Williams, Constance; Nyambi, Phillipe; Gorny, Miroslaw K
2004 Nov;20(11):1254-1258, AIDS research & human retroviruses
Both polyclonal and monoclonal human antibodies (Abs) to the V3 domain of HIV-1 gp120 display cross-clade neutralizing activity against primary isolates and T cell-adapted virus strains. The most broadly neutralizing of the human anti-V3 monoclonal Abs (mAbs), 447-52D, recognizes 14 amino acids, including the GPxR core epitope at the tip of the V3 loop. Monoclonal Ab 447-52D neutralized 92% of 38 primary isolates carrying the GPGR V3 motif regardless of whether the viruses belonged to clades A, B, F, or H; in contrast, none of 19 viruses with the GPGQ and other non-GPGR/Q sequences at the tip of the V3 loop was sensitive to mAb 447-52D. These data are consistent with the crystallographic resolution of a complex of the Fab fragment of mAb 447-52D with a V3 peptide that shows that the binding specificity of the mAb is due to recognition of the GPGR motif at the tip of the loop. The critical role of the Arg residue in this motif was determined using viruses pseudotyped with the envelope of primary isolate CA1 containing the GPGR motif or with a mutated envelope with a Gln (Q) replacing the Arg (R) at the tip of the loop. While the wild-type pseudovirus was neutralized by mAb 447-52D, the pseudovirus carrying the point mutation was resistant to neutralization. These data illuminate the structural basis for both the breadth and specificity of a broadly neutralizing human mAb and contribute to our understanding of the epitopes recognized by Abs that protect against infection with HIV-1
— id: 48046, year: 2004, vol: 20, page: 1254, stat: Journal Article,

Chronic lymphocytic leukemia with prostate infiltration mediated by specific clonal membrane-bound IgM
Bogdan, Carol A; Alexander, Alice A; Gorny, Miroslaw K; Matute, Reynaldo; Marjanovic, Nada; Zolla-Pazner, Susan; Walden, Paul D; Furneaux, Henry M; Sidhu, Gurdip S; Jacobson, Daniel R
2003 May 1;63(9):2067-2071, Cancer research
Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of monoclonal B lymphocytes in the hematopoietic organs. Rarely, CLL cells accumulate in a single atypical site. The mechanism underlying this unusual distribution of CLL cells has not been studied previously. We obtained peripheral blood from five patients having early stage CLL with heavy prostate infiltration. These patients' circulating CLL cells bound strongly in vitro to cultured prostate cell lines PC3, LNCaP, and DU145 and to short-term cultures of fresh prostate cells but not to colon, breast, or bladder cells. CLL cells from patients without prostate infiltration did not bind in vitro to any cell line. Peripheral blood CLL cells from one patient with CLL with heavy prostate infiltration were fused with a mouse-human heteromyeloma line to make hybridomas expressing the same monoclonal IgM as the patient's CLL cells. The hybridoma cells bound specifically to prostate cells. IgM secreted by the hybridoma blocked binding of the patient's CLL cells to prostate cells. Flow cytometry and immunohistochemistry demonstrated that the secreted IgM bound specifically to prostate cells. These results indicate that CLL with atypical prostate infiltration can be mediated by specific surface-bound IgM against an antigen expressed specifically by prostate cells and suggest that a similar mechanism might also apply to cases of CLL with atypical infiltration into other organs
— id: 42245, year: 2003, vol: 63, page: 2067, stat: Journal Article,

Expression, purification, and isotope labeling of the Fv of the human HIV-1 neutralizing antibody 447-52D for NMR studies
Kessler, Naama; Zvi, Anat; Ji, Min; Sharon, Michal; Rosen, Osnat; Levy, Rina; Gorny, Miroslaw; Zolla-Pazner, Suzan; Anglister, Jacob
2003 Jun;29(2):291-303, Protein expression & purification
The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, V(L) and V(H), respectively. The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy. The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates. This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. The V3 loop is an immunodominant neutralizing epitope of HIV-1. To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed V(H) and V(L) were constructed. A pelB signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm. For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon. A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers. The forward primers for the V(L) and V(H) were based on constant region sequences. The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli. High level of expression was obtained by individual expression of the V(H) and the V(L) chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment. The V(L) of mAb 447-52D was uniformly labeled with 13C and 15N nuclei (U-13C/15N). Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible
— id: 42244, year: 2003, vol: 29, page: 291, stat: Journal Article,

Alternative conformations of HIV-1 V3 loops mimic beta hairpins in chemokines, suggesting a mechanism for coreceptor selectivity
Sharon, Michal; Kessler, Naama; Levy, Rina; Zolla-Pazner, Susan; Gorlach, Matthias; Anglister, Jacob
2003 Feb;11(2):225-236, Structure
The V3 loop of the HIV-1 envelope glycoprotein gp120 is involved in binding to the CCR5 and CXCR4 coreceptors. The structure of an HIV-1(MN) V3 peptide bound to the Fv of the broadly neutralizing human monoclonal antibody 447-52D was solved by NMR and found to be a beta hairpin. This structure of V3(MN) was found to have conformation and sequence similarities to beta hairpins in CD8 and CCR5 ligands MIP-1alpha, MIP-1beta, and RANTES and differed from the beta hairpin of a V3(IIIB) peptide bound to the strain-specific murine anti-gp120(IIIB) antibody 0.5beta. In contrast to the structure of the bound V3(MN) peptide, the V3(IIIB) peptide resembles a beta hairpin in SDF-1, a CXCR4 ligand. These data suggest that the 447-52D-bound V3(MN) and the 0.5beta-bound V3(IIIB) structures represent alternative V3 conformations responsible for selective interactions with CCR5 and CXCR4, respectively
— id: 42246, year: 2003, vol: 11, page: 225, stat: Journal Article,

Combined use of serum and urinary antibody for diagnosis of tuberculosis
Singh, Krishna K; Dong, Yuxin; Hinds, Laura; Keen, Marc A; Belisle, John T; Zolla-Pazner, Susan; Achkar, Jacqueline M; Nadas, Arthur J; Arora, Vijay K; Laal, Suman
2003 Aug 1;188(3):371-377, Journal of infectious diseases
Efforts to devise immunoassays for tuberculosis (TB) that can be adapted to rapid formats are ongoing. The present study was aimed at determining whether urinary anti-Mycobacterium tuberculosis antibodies are present in patients with TB, to evaluate the feasibility of developing a urine antibody-based diagnostic test. Urinary antibodies directed against the culture filtrate proteins of M. tuberculosis, MPT 32, and the 81-kDa GlcB protein were detectable in patients with TB, although the sensitivity of antibody detection was lower (53%-64%), compared with serum antibodies (68%-77%). Surprisingly, with all 3 antigens, the use of paired serum and urine samples provided higher sensitivities of antibody detection than either single specimen, and anti-GlcB antibodies were present in the serum and/or urine of 39 (90%) of 43 smear-positive patients with TB. Although, with the current methods and antigens, the level of sensitivity is insufficient to design a urinary antibody diagnostic test, these studies provide the foundation for further studies on the development of a urine antibody-based immunoassay for TB
— id: 39136, year: 2003, vol: 188, page: 371, stat: Journal Article,

Evidence for CD4-enchanced signaling through the chemokine receptor CCR5
Staudinger, Robert; Phogat, Sanjay K; Xiao, Xiaodong; Wang, Xiahong; Dimitrov, Dimiter S; Zolla-Pazner, Susan
2003 Mar 21;278(12):10389-10392, Journal of biological chemistry
The chemokine receptor CCR5 is constitutively associated with the T cell co-receptor CD4 in plasma cell membranes, but the physiological role of this interaction has not been elucidated. Here we show that detergent-solubilized, purified CCR5 can directly associate with purified soluble fragments of the extracellular portion of CD4. We further demonstrate that the physical association of CCR5 and CD4 in membrane vesicles results in the formation of a receptor complex that exhibits macrophage inflammatory protein 1beta (MIP-1beta) binding properties that are distinct from CCR5. The affinity of the CD4-CCR5 complex for MIP-1beta was 3.5-fold lower than for CCR5, but the interaction of CD4 and CCR5 resulted in a receptor complex that exhibited enhanced G-protein signaling as compared with CCR5 alone. MIP-1beta-induced G-protein activation was further increased by simultaneous stimulation of CD4 with its natural agonist, interleukin-16. Thus, the physical association of CD4 and CCR5 results in receptor cross-talk with allosteric CD4-dependent regulation of the binding and signaling properties of CCR5. Although the precise physiological role of the CD4 effects on CCR5-mediated signaling remains unknown, one can speculate that the cross-talk is a component of mechanisms involved in the fine tuning of immune system cell responses
— id: 42247, year: 2003, vol: 278, page: 10389, stat: Journal Article,

Genetic and biological properties of HIV type 1 isolates prevalent in villagers of the Cameroon equatorial rain forests and grass fields: further evidence of broad HIV type 1 genetic diversity
Zhong P; BUrda S; Konings F; Urbanski M; Ma L; Zekeng L; Ewane L; Agyingi L; Agwara M; Saa; Afane ZE; Kinge T; Zolla-Pazner S; Nyambi P
2003 Dec;19(12):1167-1178, AIDS research & human retroviruses
To understand the evolution of HIV-1, the genetic and biological characteristics of viruses that infect persons living in regions in which the virus has been evolving for several decades must be studied. Thus, we investigated teh genetic subtypes, coreceptor usage, and syncytium-inducing ability of viruses in 47 HIV-1-infected blood samples from individuals living in rural villages in the equatorial rain forest and grass field regions in Cameroon. Heteroduplex mobility analysis (HMA) of gag (part of p24 and p7) and env (C2V5) or sequence and phylogenetic analysis of gag (part of p24 and p7), pol (protease), and env (C2V5), revealed a broad HIV-1 group M genetic diversity. Subtype analysis revealed genetic evidence of seven subtypes (A, C, D, F, G, H, and J) and three circulating recombinant froms (CRFs) (CRF01_AE, CRF02_AG, and CRF11_cpx). Only 15 (32%) of the 47 samples analyzed revealed a concordant subtype in all three genes (gag, pol, and env), while discordant subtypes and CRFs were identified for the remaining 32 (68%) samples. Two patterns of HIV-1 diversity could be discerned in two provinces. While more concordant subtypes in gag, pol, and env genes were identified in villages of South province (10 of 13, 77%), the HIV-1 diversity in the West province was characterized by intersubtype recombinants (63%). Five new intersubtype recombinants were identified including Agag Jpol Genv, Ggag Upol Aenv, AGgag Jpol Aenv, Agag AGpol Henv, and Cgag AGpol AGenv. All of the 40 viruses tested used the R5 coreceptor, of which four also used the X4 coreceptor. Four viruses were able to induce syncytia in MT-2 cells, however, syncytium induction did not correlate with coreceptor usage. This study further reveals the complexity of HIV-1 infection in rural Cameron and points to the future of the global epidemic, which may be characterized by more genetically diverse viruses
— id: 42243, year: 2003, vol: 19, page: 1167, stat: Journal Article,

Re-examining the V3 Loop and its Role in Inducing Protective Antibodies
Zolla-Pazner, Susan
[S.l.] : NIH, 2003,
— id: 1434, year: 2003, vol: , page: , stat: ,

High levels of antibodies to the CD4 binding domain of human immunodeficiency virus type 1 glycoprotein 120 are associated with faster disease progression
Chien, Peter C Jr; Cohen, Sandra; Kleeberger, Cynthia; Giorgi, Janis; Phair, John; Zolla-Pazner, Susan; Hioe, Catarina E
2002 Jul 15;186(2):205-213, Journal of infectious diseases
Human monoclonal antibodies (Abs) to the CD4 binding domain of human immunodeficiency virus (HIV) type 1 glycoprotein (gp) 120 (gp120(CD4bd)) inhibit gp120 presentation to gp120-specific T helper (Th) cells. Since Th responses are critical to control HIV, anti-gp120(CD4bd) Abs could be involved in HIV pathogenesis. Therefore, anti-gp120(CD4bd) Ab levels were compared in serum samples from matched pairs of HIV-positive rapid progressors (RPs) and slow progressors (SPs). Many RPs had higher levels of anti-gp120(CD4bd) Abs than their corresponding SPs. However, Ab levels to whole gp120 and to its C5 domain were similar. Hence, the higher levels of anti-gp120(CD4bd) Abs detected in the serum of RPs do not reflect generalized increases in Ab levels to whole gp120. Moreover, anti-gp120(CD4bd) Ab levels correlated with the amount of inhibition of gp120-specific Th proliferation in the presence of respective serum immunoglobulin G. These findings document a novel mechanism of HIV pathogenesis mediated by anti-gp120(CD4bd) Abs exhibiting suppressive activity on gp120 presentation
— id: 32469, year: 2002, vol: 186, page: 205, stat: Journal Article,

Truncation of the cytoplasmic domain induces exposure of conserved regions in the ectodomain of human immunodeficiency virus type 1 envelope protein
Edwards, Terri G; Wyss, Stephanie; Reeves, Jacqueline D; Zolla-Pazner, Susan; Hoxie, James A; Doms, Robert W; Baribaud, Frederic
2002 Mar;76(6):2683-2691, Journal of virology
We have described a CD4-independent variant of HXBc2, termed 8x, that binds directly to CXCR4 and mediates CD4-independent virus infection. Determinants for CD4 independence map to residues in the V3 and V4-C4 domains together with a single nucleotide deletion in the transmembrane domain which introduces a frameshift (FS) at position 706. This FS results in a truncated cytoplasmic domain of 27 amino acids. We demonstrate here that while introduction of the 8x FS mutation into heterologous R5, X4, or R5X4 Env proteins did not impart CD4 independence, it did affect the conformation of the gp120 surface subunit, exposing highly conserved domains involved in both coreceptor and CD4 binding. In addition, antigenic changes in the gp41 ectodomain were also observed, consistent with the idea that the effects of cytoplasmic domain truncation must in some way be transmitted to the external gp120 subunit. Truncation of gp41 also resulted in the marked neutralization sensitivity of all Env proteins tested to human immunodeficiency virus-positive human sera and monoclonal antibodies directed against the CD4 or coreceptor-binding sites. These results demonstrate a structural interdependence between the cytoplasmic domain of gp41 and the ectodomain of the Env protein. They also may help explain why the length of the gp41 cytoplasmic domain is retained in vivo and may provide a way to genetically trigger the exposure of neutralization determinants in heterologous Env proteins that may prove useful for vaccine development
— id: 78826, year: 2002, vol: 76, page: 2683, stat: Journal Article,

Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates
Golding, Hana; Zaitseva, Marina; de Rosny, Eve; King, Lisa R; Manischewitz, Jody; Sidorov, Igor; Gorny, Miroslaw K; Zolla-Pazner, Susan; Dimitrov, Dimiter S; Weiss, Carol D
2002 Jul;76(13):6780-6790, Journal of virology
Human immunodeficiency virus type 1 (HIV-1) entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1 entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Suboptimal temperature (31.5 degrees C) was used to prolong fusion intermediates as monitored by confocal microscopy. After transfer to 37 degrees C, these fusion intermediates progressed to syncytium formation with enhanced kinetics compared with effector-target (E/T) cell mixtures that were incubated only at 37 degrees C. gp41 peptides DP-178, DP-107, and IQN17 blocked fusion more efficiently (5- to 10-fold-lower 50% inhibitory dose values) when added to E/T cells at the suboptimal temperature prior to transfer to 37 degrees C. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37 degrees C only if preincubated with E/T cells at the suboptimal temperature. Similar fusion inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41 and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site
— id: 78825, year: 2002, vol: 76, page: 6780, stat: Journal Article,

Human monoclonal antibodies specific for conformation-sensitive epitopes of V3 neutralize human immunodeficiency virus type 1 primary isolates from various clades
Gorny, Miroslaw K; Williams, Constance; Volsky, Barbara; Revesz, Kathy; Cohen, Sandra; Polonis, Victoria R; Honnen, William J; Kayman, Samuel C; Krachmarov, Chavdar; Pinter, Abraham; Zolla-Pazner, Susan
2002 Sep;76(18):9035-9045, Journal of virology
The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3(JR-CSF) sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates
— id: 39604, year: 2002, vol: 76, page: 9035, stat: Journal Article,

Synergy determination issues - Authors' reply
Nadas, A; Zolla-Pazner, S
2002 OCT ;76(20):10578-10578, Journal of virology
— id: 32551, year: 2002, vol: 76, page: 10578, stat: Journal Article,

Authors' Reply
Nadas, Arthur; Zolla-Pazner, Susan
2002 ;76(20):886-892, Journal of virology
— id: 98806, year: 2002, vol: 76, page: 886, stat: Journal Article,

HIV infection in rural villages of Cameroon
Nyambi, Phillipe; Zekeng, Leopold; Kenfack, Henriette; Tongo, Marcel; Nanfack, Aubin; Nkombe, Innocent; Ndonko, Flavien; Shang, Judith; Burda, Sherri; Mbah, Henry; Agyingi, Lucy; Zhong, Ping; Nadas, Arthur; Zolla-Pazner, Susan; Marmor, Michael
2002 Dec 15;31(5):506-513, Journal of acquired immune deficiency syndromes. JAIDS
OBJECTIVE: To evaluate HIV-1 antibody seroprevalence and risk factors for HIV seropositivity in rural areas of Cameroon. METHOD: The prevalences of HIV antibodies in 53 villages in rural Cameroon visited during May-October 2000 were determined with an HIV1/2 rapid assay, standard ELISA, and western blot. Demographic data and risk factors were elicited via face-to-face interviews with a structured questionnaire. RESULTS: HIV seroprevalence was 5.8% (243/4156, 95% confidence interval [CI] = 5.1-6.6) overall, 6.3% (151/2394, 95% CI = 5.4-7.4) among females and 5.2% (92/1762, 95% CI = 4.3-6.4) among males. HIV seroprevalence among persons aged 15 - 70 years did not differ significantly by province (5.6% in Center, 4.5% in East, 6.9% in South, and 5.8% in South-West) ( =.10). Analysis of age- and gender-standardized prevalence by village across provinces indicated a near-significant difference (nonparametric Wilcoxon signed rank test, =.06), with highest prevalence in South-West, followed by South, Center, and East. Multivariate analysis revealed that single women were significantly more likely to be HIV seropositive than were married or widowed women. Women with a history of sexual relations while traveling were at significantly increased risk of HIV seropositivity (OR adjusted for age and marital status = 2.4, 95% CI = 1.4-9.7). Among men, those who reported ever having a sexually transmitted disease were at significantly increased risk of HIV-seropositivity (OR adjusted for age = 1.8, 95% CI = 1.1-2.8). CONCLUSION: We have documented a wide range of HIV prevalences among rural villages of Cameroon. Age, marital status (in women) and sexual risk factors appear to be associated with HIV infection in this setting
— id: 39352, year: 2002, vol: 31, page: 506, stat: Journal Article,

In memoriam: G. Jeanette Thorbecke 1929-2001
Tsiagbe, Vincent K; Coico, Richard; Ponzio, Nicholas M; Zolla-Pazner, Susan
2002 Apr 15;168(8):3695-3696, Journal of immunology
— id: 34673, year: 2002, vol: 168, page: 3695, stat: Journal Article,

Remembrance: G. Jeanette Thorbecke
Tsiagbe, Vincent K; Coico, Richard; Ponzio, Nicholas M; Zolla-Pazner, Susan
2002 Mar;35(2):75-78, Autoimmunity
— id: 78819, year: 2002, vol: 35, page: 75, stat: Journal Article,

In memorium G. Jeanette Thorbecke, 1929-2001
Tsiagbe, Vincent K; Zolla-Pazner, Susan; Coico, Richard; Ponzio, Nicholas M
2002 Jun;160(6):1917-1920, American journal of pathology
— id: 34672, year: 2002, vol: 160, page: 1917, stat: Journal Article,

A variable region 3 (V3) mutation determines a global neutralization phenotype and CD4-independent infectivity of a human immunodeficiency virus type 1 envelope associated with a broadly cross-reactive, primary virus-neutralizing antibody response
Zhang, Peng Fei; Bouma, Peter; Park, Eun Ju; Margolick, Joseph B; Robinson, James E; Zolla-Pazner, Susan; Flora, Michael N; Quinnan, Gerald V Jr
2002 Jan;76(2):644-655, Journal of virology
The human serum human immunodeficiency virus type 1 (HIV-1)-neutralizing serum 2 (HNS2) neutralizes many primary isolates of different clades of HIV-1, and virus expressing envelope from the same donor, clone R2, is neutralized cross-reactively by HIV-immune human sera. The basis for this cross-reactivity was investigated. It was found that a rare mutation in the proximal limb of variable region 3 (V3), 313-4 PM, caused virus pseudotyped with the R2 envelope to be highly sensitive to neutralization by monoclonal antibodies (MAbs) directed against conformation-sensitive epitopes at the tip of the V3 loop, such as 19b, and moderately sensitive to MAbs against CD4 binding site (CD4bs) and CD4-induced (CD4i) epitopes, soluble CD4 (sCD4), and HNS2. In addition, introduction of this sequence by mutagenesis caused enhanced sensitivity to neutralization by 19b, anti-CD4i MAb, and HNS2 in three other primary HIV-1 envelopes and by anti-CD4bs MAb and sCD4 in one of the three. The 313-4 PM sequence also conferred increased infectivity for CD4(+) CCR5(+) cells and the ability to infect CCR5(+) cells upon all of these four and two of these four HIV-1 envelopes, respectively. Neutralization of R2 by HNS2 was substantially inhibited by the cyclized R2 V3 35-mer synthetic peptide. Similarly, the peptide also had some lesser efficacy in blocking neutralization of R2 by other sera or of neutralization of other primary viruses by HNS2. Together, these results indicate that the unusual V3 mutation in the R2 clone accounts for its uncommon neutralization sensitivity phenotype and its capacity to mediate CD4-independent infection, both of which could relate to immunogenicity and the neutralizing activity of HNS2. This is also the first primary HIV-1 isolate envelope glycoprotein found to be competent for CD4-independent infection
— id: 78827, year: 2002, vol: 76, page: 644, stat: Journal Article,

HIV type 1 group M clades infecting subjects from rural villages in equatorial rain forests of Cameroon
Zhong, Ping; Burda, Sherri; Urbanski, Mateusz; Kenfack, Henriette; Tongo, Marcel; Heyndrickx, Leo; Nanfack, Aubin; Shang, Judith; Agyingi, Lucy; Zolla-Pazner, Susan; Zekeng, Leopold; Nyambi, Phillipe
2002 Dec 15;31(5):495-505, Journal of acquired immune deficiency syndromes. JAIDS
Though the HIV-1 subtypes infecting patients living in urban and semi-urban areas in Cameroon have been reported, information on the subtypes infecting patients in rural villages is lacking. To begin to understand the diversity of the HIV-1 group M subtypes infecting persons living in rural villages in the equatorial rain forest regions of Cameroon, 49 plasma samples from 14 rural villages in four provinces of Cameroon were analyzed using heteroduplex mobility analysis (HMA), DNA sequencing, and phylogenetic tree analysis on the basis of env C2V5, gag, or pol regions. Sixty-one percent of the group M infections were clade A or CRF02_AG-like as subtyped by env and gag. Of the remaining group M infections, 12% were either A or CRF02_AG-like or CRF01_AE-like in recombination with other clades; 25% were infections that were entirely non-A or non-CRF02_AG-like; and 2% were CRF11_cpx. The HIV-1 group M clades identified included A, D, F (F2), G, and H. The CRF strains identified were CRF02_AG-like, CRF01_AE-like, and CRF11_cpx. Two new intersubtype recombinant infections, H/G and A/F2, were identified. This study suggests that the HIV-1 diversity in rural villages in the equatorial rain forest of Cameroon is at least as broad as has been observed in major cities of Cameroon and that multiple HIV-1 group M subtypes are infecting persons living in the countryside of Cameroon
— id: 39353, year: 2002, vol: 31, page: 495, stat: Journal Article,

Chronic lymphocytic leukemia with prostate or bladder infiltration mediated by specific clonal membrane-bound IgM
Bogdan, CA; Gorny, MK; Zolla-Pazner, S; Furneaux, HM; Jacobson, DR
2001 NOV 16 ;98(11):362A-363A, Blood
— id: 55370, year: 2001, vol: 98, page: 362A, stat: Journal Article,

Inhibition of human immunodeficiency virus type 1 gp120 presentation to CD4 T cells by antibodies specific for the CD4 binding domain of gp120
Hioe CE; Tuen M; Chien PC Jr; Jones G; Ratto-Kim S; Norris PJ; Moretto WJ; Nixon DF; Gorny MK; Zolla-Pazner S
2001 Nov;75(22):10950-10957, Journal of virology
Human immunodeficiency virus (HIV)-specific CD4 T-cell responses, particularly to the envelope glycoproteins of the virus, are weak or absent in most HIV-infected patients. Although these poor responses can be attributed simply to the destruction of the specific CD4 T cells by the virus, other factors also appear to contribute to the suppression of these virus-specific responses. We previously showed that human monoclonal antibodies (MAbs) specific for the CD4 binding domain of gp120 (gp120(CD4BD)), when complexed with gp120, inhibited the proliferative responses of gp120-specific CD4 T-cells. MAbs to other gp120 epitopes did not exhibit this activity. The present study investigated the inhibitory mechanisms of the anti-gp120(CD4BD) MAbs. The anti-gp120(CD4BD) MAbs complexed with gp120 suppressed gamma interferon production as well as proliferation of gp120-specific CD4 T cells. Notably, the T-cell responses to gp120 were inhibited only when the MAbs were added to antigen-presenting cells (APCs) during antigen pulse; the addition of the MAbs after pulsing caused no inhibition. However, the anti-gp120(CD4BD) MAbs by themselves, or as MAb/gp120 complexes, did not affect the presentation of gp120-derived peptides by the APCs to T cells. These MAb/gp120 complexes also did not inhibit the ability of APCs to process and present unrelated antigens. To test whether the suppressive effect of anti-gp120(CD4BD) antibodies is caused by the antibodies' ability to block gp120-CD4 interaction, APCs were treated during antigen pulse with anti-CD4 MAbs. These treated APCs remained capable of presenting gp120 to the T cells. These results suggest that anti-gp120(CD4BD) Abs inhibit gp120 presentation by altering the uptake and/or processing of gp120 by the APCs but their inhibitory activity is not due to blocking of gp120 attachment to CD4 on the surface of APCs
— id: 26598, year: 2001, vol: 75, page: 10950, stat: Journal Article,

Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120
Jeffs, S A; Gorny, M K; Williams, C; Revesz, K; Volsky, B; Burda, S; Wang, X H; Bandres, J; Zolla-Pazner, S; Holmes, H
2001 Dec 3;79(3):209-213, Immunology letters
Recombinant gp120 of the HIV-1(IIIB) isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120(LAI). Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A,1570C,1570D,1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V(H)3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies
— id: 93795, year: 2001, vol: 79, page: 209, stat: Journal Article,

Effect of soluble cd4 on exposure of epitopes on primary, intact, native human immunodeficiency virus type 1 virions of different genetic clades
Mbah HA; Burda S; Gorny MK; Williams C; Revesz K; Zolla-Pazner S; Nyambi PN
2001 Aug;75(16):7785-7788, Journal of virology
We have used a virus-binding assay to examine conformational changes that occur when soluble CD4 (sCD4) binds to the surface of intact, native, primary human immunodeficiency virus type 1 virions. The isolates examined belong to seven genetic clades (A to H) and are representative of syncytium-inducing and non-syncytium-inducing phenotypes. Conformational changes in epitopes in the C2, V2, V3, C5, and CD4 binding domain (CD4bd) of gp120 and the cluster I and II regions of gp41 of these viruses were examined using human monoclonal antibodies that are directed at these regions. The studies revealed that sCD4 binding causes a marked increase in exposure of epitopes in the V3 loop, irrespective of the clade or the phenotype of the virus. Sporadic increases in exposure were observed in some epitopes in the V2 region, while no changes were observed in the C2, C5, or CD4bd of gp120 or the cluster I and II regions of gp41
— id: 21111, year: 2001, vol: 75, page: 7785, stat: Journal Article,

Additive effects characterize the interaction of antibodies involved in neutralization of the primary dualtropic human immunodeficiency virus type 1 isolate 89.6
Verrier F; Nadas A; Gorny MK; Zolla-Pazner S
2001 Oct;75(19):9177-9186, Journal of virology
Human immunodeficiency virus-type I (HIV-1) infection elicits antibodies (Abs) directed against several regions of the gp120 and gp41 envelope glycoproteins. Many of these Abs are able to neutralize T-cell-line-adapted strains (TCLA) of HIV-1, but only a few effectively neutralize primary HIV-1 isolates. The nature of HIV-1 neutralization has been carefully studied using human monoclonal Abs (MAbs), and the ability of such MAbs to act in synergy to neutralize HIV-1 has also been extensively studied. However, most synergy studies have been conducted using TCLA strains. To determine the nature of Ab interaction in HIV-1 primary isolate neutralization, a panel of 12 anti-HIV-1 human immunoglobulin G (IgG) MAbs, specific for epitopes in gp120 and gp41, were used. Initial tests showed that six of these MAbs, as well as sCD4, used individually, were able to neutralize the dualtropic primary isolate HIV-1(89.6); MAbs giving significant neutralization at 2 to 10 microg/ml included 2F5 (anti-gp41), 50-69 (anti-gp41), IgG1b12 (anti-gp120(CD4bd)), 447-52D (anti-gp120(V3)), 2G12 (anti-gp120), and 670-D (anti-gp120(C5)). For studies of reagent interaction, 16 binary combinations of reagents were tested for their ability to neutralize HIV-1(89.6). Reagent combinations tested included one neutralizing MAb with sCD4, six pairs consisting of two neutralizing MAbs, and nine pairs consisting of one neutralizing MAb with another non-neutralizing MAb. To assess the interaction of the latter type of combination, a new mathematical treatment of reagent interaction was developed since previously used methods could be used only when both reagents neutralize. Synergy was noted between sCD4 and a neutralizing anti-gp120(V3) MAb. Antagonism was noted between two pairs of anti-gp41 MAbs (one neutralizing and one non-neutralizing). All of the other 13 pairs of MAbs tested displayed only additive effects. These studies suggest that Abs rarely act in synergy to neutralize primary isolate HIV-1(89.6); many anti-HIV-1 Abs act additively to mediate this biological function
— id: 26673, year: 2001, vol: 75, page: 9177, stat: Journal Article,

Antibody binding and neutralization of primary and T-cell line-adapted isolates of human immunodeficiency virus type 1
York J; Follis KE; Trahey M; Nyambi PN; Zolla-Pazner S; Nunberg JH
2001 Mar;75(6):2741-2752, Journal of virology
The relative resistance of human immunodeficiency virus type 1 (HIV-1) primary isolates (PIs) to neutralization by a wide range of antibodies remains a theoretical and practical barrier to the development of an effective HIV vaccine. One model to account for the differential neutralization sensitivity between Pls and laboratory (or T-cell line-adapted [TCLA]) strains of HIV suggests that the envelope protein (Env) complex is made more accessible to antibody binding as a consequence of adaptation to growth in established cell lines. Here, we revisit this question using genetically related PI and TCLA viruses and molecularly cloned env genes. By using complementary techniques of flow cytometry and virion binding assays, we show that monoclonal antibodies targeting the V3 loop, CD4-binding site, CD4-induced determinant of gp120, or the ectodomain of gp41 bind equally well to PI and TCLA Env complexes, despite large differences in neutralization outcome. The data suggest that the differential neutralization sensitivity of PI and TCLA viruses may derive not from differences in the initial antibody binding event but rather from differences in the subsequent functioning of the PI and TCLA Envs during virus entry. An understanding of these as yet undefined differences may enhance our ability to generate broadly neutralizing HIV vaccine immunogens
— id: 27422, year: 2001, vol: 75, page: 2741, stat: Journal Article,

Polymerase chain reaction-based assay for antibody-mediated neutralization of HIV-1 reveals a population of nonneutralized virus undetected by conventional p24 assay [In Process Citation]
Achkar JM; Wang XH; Nyambi P; Gorny MK; Zolla-Pazner S; Bandres JC
2000 Jul 1;24(3):203-210, Journal of acquired immune deficiency syndromes. JAIDS
To be successful with strategies involving passive immunization or the generation of neutralizing antibodies against HIV, it is crucial that we improve our understanding of the process of antibody-mediated HIV neutralization. We have used a neutralization assay based on polymerase chain reaction (PCR) that is more rapid and sensitive than the conventional p24 neutralization assay based on enzyme-linked immunosorbent assay (ELISA). PCR assays permit measurement of the number of infectious events and can detect small amounts of HIV-1 only a few days postinfection. In these studies, the human anti-V3 monoclonal antibody 694/98-D was used to neutralize the infectivity of the laboratory isolate HIVIIIB for CEM-SS cells. 8E5/LAV cells, which contain a single integrated copy of proviral DNA per cell, served as a standard to determine the amount of HIV-1 copies in infected CEM-SS cells. Evaluation of antibody-mediated neutralization was possible at 2 to 3 days postinfection, at a time when p24 readouts were not conclusive. We achieved >95% neutralization of HIVIIIB, and of its molecular clone HXB2, using the monoclonal antibody 694/98-D. This degree of neutralization is probably highly significant in vivo. Nevertheless, a small amount of both HIVIIIB and HXB2 ( approximately 5%) escapes neutralization and can consistently be detected after a few days by this sensitive assay. Experiments with different anti-HIV monoclonal antibodies and viruses showed that the assay could be applied to anti-V3 as well as anti-CD4 binding domain antibodies as well as HIV laboratory strains or primary isolates
— id: 9227, year: 2000, vol: 24, page: 203, stat: Journal Article,

HIV phenotype correlates with the relative amounts of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II in the virion envelope [In Process Citation]
Bastiani Lallos L; Cecilia D; Fenyo EM; Laal S; Zolla-Pazner S
2000 Jul 28;14(11):1523-1531, AIDS
OBJECTIVE: The biological phenotype of HIV-1 has been associated with various aspects of its infectivity, including syncytium formation and coreceptor usage. Adhesion molecules, present on both the target cell and the virus, have also been shown to play a role in the infectious process. A possible correlation between the presence of adhesion molecules in the envelope of HIV-1 with the biological phenotype of the virus is examined. DESIGN: The envelopes of 56 isolates of HIV-1 of known biological phenotype were analyzed for the presence of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II molecules. METHODS: The coreceptor usage of each isolate was determined in a GHOST cell or a U87.CD4 infectivity assay. The presence of LFA-1 and MHC class II in each virus envelope was then determined using a virus-binding enzyme-linked immunosorbent assay (ELISA). RESULTS: Viruses using the chemokine receptor CCR5 have relatively higher levels of MHC class II than LFA-1 in their envelopes compared with those using CXCR4. CONCLUSIONS: The finding that there is a differential incorporation of MHC class II and LFA-1 molecules by CXCR4- and CCR5-using viruses augments the list of properties contributing to the biological phenotype of HIV-1. This may explain, in part, how CXCR4-using viruses are able to bind to and infect a broader range of cell types than CCR5-using viruses, and why CXCR4-using viruses are associated with a more aggressive disease course
— id: 9226, year: 2000, vol: 14, page: 1523, stat: Journal Article,

Effects of oligomerization on the epitopes of the human immunodeficiency virus type 1 envelope glycoproteins
Gorny MK; VanCott TC; Williams C; Revesz K; Zolla-Pazner S
2000 Feb 15;267(2):220-228, Virology
To understand the differential expression of epitopes on monomeric and oligomeric forms of the envelope glycoproteins, nine human monoclonal antibodies (mAbs) were derived from the cells of human immunodeficiency virus-infected subjects by selection with soluble oligomeric gp140 (o.140). These nine mAbs and 12 human mAbs selected with V3 peptides, viral lysates, and rgp120, specific for the V2, V3, C5, CD4-binding domain (CD4bd), and gp41, were tested in a binding assay to compare the exposure of these regions on monomeric gp120 or gp41 and on o.140. None of the 21 mAbs were oligomer specific. However, mAbs to V3 and CD4bd were 'oligomer sensitive,' whereas mAbs to V2 and the distal epitope of C5 tended to be 'monomer sensitive' (i.e., to react better with the oligomer or monomer, respectively). The majority of anti-gp41 mAbs reacted similarly with monomer and oligomer. Although the uncleaved o.140 used in this study differs from the cleaved gp120/41 oligomer found on the native virus particle, these results suggest that new epitopes are not introduced by oligomerization of viral envelope proteins, that such oligomer-specific epitopes, if they exist, are not highly immunogenic, and/or that they are not efficiently selected using soluble o.140.
— id: 8547, year: 2000, vol: 267, page: 220, stat: Journal Article,

Recognition by human monoclonal antibodies of free and complexed peptides representing the prefusogenic and fusogenic forms of human immunodeficiency virus type 1 gp41
Gorny MK; Zolla-Pazner S
2000 Jul;74(13):6186-6192, Journal of virology
Human immunodeficiency virus type 1 (HIV-1) entry into target cells appears to be triggered when two heptad repeat regions in the ectodomain of gp41 associate, converting the prefusogenic form of gp41 to a fusogenic form. Peptides from these two heptad repeat regions, designated N51 and C43, form a coiled coil consisting of an alpha-helical trimer of heterodimers which approximates the core of the fusogenic form of gp41. To understand the antigenic structures of gp41 in these two configurations, and to examine the specificity of anti-gp41 antibodies produced by HIV-1-infected individuals, human anti-gp41 monoclonal antibodies (MAbs) were tested for their reactivity against N51, C43, and the complex formed by these peptides. Of 11 MAbs, 7 reacted with the complex but with neither of the parent peptides. These MAbs reacted optimally with the N51-C43 complex prepared at a 1:1 ratio and appeared to recognize the fusogenic form of gp41 in which the two heptad repeat regions are associated to form the coiled coil. The existence of antibodies from HIV-infected humans that exclusively recognize the N51-C43 complex constitutes the first proof that the coiled-coil conformation of gp41 exists in vivo and is immunogenic. Two of the 11 MAbs were specific for the hydrophilic loop region of gp41 and failed to react with either peptide alone or with the peptide complex, while the remaining 2 MAbs reacted with peptide C43. One of these two latter MAbs, 98-6, also reacted well with the equimolar N51-C43 complex, while reactivity with C43 by the other MAb, 2F5, was inhibited by even small amounts of N51, suggesting that the interaction of these peptides occludes or disrupts the epitope recognized by MAb 2F5. MAbs 98-6 and 2F5 are also unusual among the MAbs tested in their ability to neutralize multiple primary HIV isolates, although 2F5 displays more broad and potent activity. The data suggest that anti-gp41 neutralizing activity is associated with specificity for a region in C43 which participates in complex formation with N51
— id: 9231, year: 2000, vol: 74, page: 6186, stat: Journal Article,

Anti-CD4-binding domain antibodies complexed with HIV type 1 glycoprotein 120 inhibit CD4+ T cell-proliferative responses to glycoprotein 120
Hioe CE; Jones GJ; Rees AD; Ratto-Kim S; Birx D; Munz C; Gorny MK; Tuen M; Zolla-Pazner S
2000 Jun 10;16(9):893-905, AIDS research & human retroviruses
HIV-specific CD4+ helper T cell responses, particularly to the envelope glycoproteins, are usually weak or absent in the majority of HIV-seropositive individuals. Since antibodies, by their capacity to alter antigen uptake and processing, are known to have modulatory effects on CD4+ T cell responses, we investigated the effect of antibodies produced by HIV-infected individuals on the CD4+ T cell response to HIV-1 gp120. Proliferative responses of gp120-specific CD4+ T cells were inhibited in the presence of either serum immunoglobulin from HIV-infected individuals or human monoclonal antibodies specific for the CD4-binding domain (CD4bd) of gp120. Human monoclonal antibodies to other gp120 epitopes did not have the same effect. The anti-CD4bd antibodies complexed with gp120 suppressed T cell lines specific for varying gp120 epitopes but did not affect T cell proliferation to non-HIV antigens. Moreover, inhibition by the anti-CD4bd/gp120 complexes was observed regardless of the types of antigen-presenting cells used to stimulate the T cells. These results indicate that the presence of anti-CD4bd antibodies complexed with gp120 can strongly suppress CD4+ helper T responses to gp120
— id: 9230, year: 2000, vol: 16, page: 893, stat: Journal Article,

Suppression of HIV gp120-specific proliferative responses is mediated by immune complexes
Hioe, Catarina E; Jones, Gareth J; Rees, Ann D; Ratto-Kim, Silvia; Birx, Deborah; Munz, Christian; Gorny, Miroslaw K; Tuen, Michael; Zolla-Pazner, Susan
2000 Sept 10-15;3(5):252-252, Journal of human virology
— id: 15799, year: 2000, vol: 3, page: 252, stat: Journal Article,

Suppression of HIV env-specific proliferative responses is mediated by immune complexes
Hioe, CE; Jones, G; Rees, A; Ratto-Kim, S; Birx, D; Munz, C; Gorny, MK; Tuen, M; Zolla-Pazner, S
2000 APR 20 ;14(6):A934-A934, FASEB journal
— id: 54630, year: 2000, vol: 14, page: A934, stat: Journal Article,

Mass spectrometric characterization of a discontinuous epitope of the HIV envelope protein HIV-gp120 recognized by the human monoclonal antibody 1331A
Hochleitner EO; Gorny MK; Zolla-Pazner S; Tomer KB
2000 Apr 15;164(8):4156-4161, Journal of immunology
The characterization of a discontinuous epitope in the C5 region of the HIV envelope protein HIV-gp120, recognized by 1331A, a human mAb, is reported. Regions involved in affinity binding in the HIV-gp120 molecule were identified by epitope excision/extraction methods followed by matrix assisted laser desorption-time of flight mass spectrometry. In epitope excision, the protein is bound in its native conformation to an immobilized Ab and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the Ab. A series of proteolytic digestions of the 1331A/HIV-gp120 complex allowed the identification of protected amino acids in two noncontinuous regions of the C5 region of HIV-gp120. Interaction of the Ab with amino acids I487 and E507 of HIV-gp120 is essential for efficient binding. This is the first application of this approach for the identification and characterization of a discontinuous epitope. The results are consistent with molecular modeling results, indicating that these amino acids are located on opposite sides of a hydrophobic pocket. This pocket is thought to be of importance for the interaction of HIV-gp120 with the transmembrane protein HIV-gp41
— id: 9233, year: 2000, vol: 164, page: 4156, stat: Journal Article,

Conserved and exposed epitopes on intact, native, primary human immunodeficiency virus type 1 virions of group M
Nyambi PN; Mbah HA; Burda S; Williams C; Gorny MK; Nadas A; Zolla-Pazner S
2000 Aug;74(15):7096-7107, Journal of virology
We have examined the exposure and conservation of antigenic epitopes on the surface envelope glycoproteins (gp120 and gp41) of 26 intact, native, primary human immunodeficiency virus type 1 (HIV-1) group M virions of clades A to H. For this, 47 monoclonal antibodies (MAbs) derived from HIV-1-infected patients were used which were directed at epitopes of gp120 (specifically V2, C2, V3, the CD4-binding domain [CD4bd], and C5) and epitopes of gp41 (clusters I and II). Of the five regions within gp120 examined, MAbs bound best to epitopes in the V3 and C5 regions. Only moderate to weak binding was observed by most MAbs to epitopes in the V2, C2, and CD4bd regions. Two anti-gp41 cluster I MAbs targeted to a region near the tip of the hydrophilic immunodominant domain bound strongly to >90% of isolates tested. On the other hand, binding of anti-gp41 cluster II MAbs was poor to moderate at best. Binding was dependent on conformational as well as linear structures on the envelope proteins of the virions. Further studies of neutralization demonstrated that MAbs that bound to virions did not always neutralize but all MAbs that neutralized bound to the homologous virus. This study demonstrates that epitopes in the V3 and C5 regions of gp120 and in the cluster I region of gp41 are well exposed on the surface of intact, native, primary HIV-1 isolates and that cross-reactive epitopes in these regions are shared by many viruses from clades A to H. However, only a limited number of MAbs to these epitopes on the surface of HIV-1 isolates can neutralize primary isolates
— id: 9229, year: 2000, vol: 74, page: 7096, stat: Journal Article,

Immunoreactivity of intact virions of human immunodeficiency virus type 1 (HIV-1) reveals the existence of fewer HIV-1 immunotypes than genotypes [In Process Citation]
Nyambi PN; Nadas A; Mbah HA; Burda S; Williams C; Gorny MK; Zolla-Pazner S
2000 Nov;74(22):10670-10680, Journal of virology
In order to protect against organisms that exhibit significant genetic variation, polyvalent vaccines are needed. Given the extreme variability of human immunodeficiency virus type 1 (HIV-1), it is probable that a polyvalent vaccine will also be needed for protection from this virus. However, to understand how to construct a polyvalent vaccine, serotypes or immunotypes of HIV must be identified. In the present study, we have examined the immunologic relatedness of intact, native HIV-1 primary isolates of group M, clades A to H, with human monoclonal antibodies (MAbs) directed at epitopes in the V3, C5, and gp41 cluster I regions of the envelope glycoproteins, since these regions are well exposed on the virion surface. Multivariate analysis of the binding data revealed three immunotypes of HIV-1 and five MAb groups useful for immunotyping of the viruses. The analysis revealed that there are fewer immunotypes than genotypes of HIV and that clustering of the isolates did not correlate with either genotypes, coreceptor usage (CCR5 and CXCR4), or geographic origin of the isolates. Further analysis revealed distinct MAb groups that bound preferentially to HIV-1 isolates belonging to particular immunotypes or that bound to all three immunotypes; this demonstrates that viral immunotypes identified by mathematical analysis are indeed defined by their immunologic characteristics. In summary, these results indicate (i) that HIV-1 immunotypes can be defined, (ii) that constellations of epitopes that are conserved among isolates belonging to each individual HIV-1 immunotype exist and that these distinguish each of the immunotypes, and (iii) that there are also epitopes that are routinely shared by all immunotypes
— id: 15283, year: 2000, vol: 74, page: 10670, stat: Journal Article,

Defining immunotypes of HIV
Nyambi, P N; Nadas, A; Mbah, H A; Burda, S; Williams, C; Gorny, M K; Zolla-Pazner, S
2000 Sept 10-15;3(5):259-259, Journal of human virology
— id: 15798, year: 2000, vol: 3, page: 259, stat: Journal Article,

A global neutralization resistance phenotype of human immunodeficiency virus type 1 is determined by distinct mechanisms mediating enhanced infectivity and conformational change of the envelope complex
Park EJ; Gorny MK; Zolla-Pazner S; Quinnan GV Jr
2000 May;74(9):4183-4191, Journal of virology
We have described previously genetic characterization of neutralization-resistant, high-infectivity, and neutralization-sensitive, low-infectivity mutants of human immunodeficiency virus type 1 (HIV-1) MN envelope. The distinct phenotypes of these clones are attributable to six mutations affecting functional interactions between the gp120 C4-V5 regions and the gp41 leucine zipper. In the present study we examined mechanisms responsible for the phenotypic differences between these envelopes using neutralization and immunofluorescence assays (IFA). Most monoclonal antibodies (MAbs) tested against gp120 epitopes (V3, CD4 binding site, and CD4-induced) were 20 to 100 times more efficient at neutralizing pseudovirus expressing sensitive rather than resistant envelope. By IFA cells expressing neutralization sensitive envelope bound MAbs to gp120 epitopes more, but gp41 epitopes less, than neutralization-resistant envelope. This binding difference appeared to reflect conformational change, since it did not correlate with the level of protein expression or gp120-gp41 dissociation. This conformational change was mostly attributable to one mutation, L544P, which contributes to neutralization resistance but not to infectivity enhancement. The V420I mutation, which contributes a major effect to both high infectivity and neutralization resistance, had no apparent effect on conformation. Notably, a conformation-dependent V3 neutralization epitope remained sensitive to neutralization and accessible to binding by MAbs on neutralization-resistant HIV-1 envelope. Sensitivity to sCD4 did not distinguish the clones, suggesting that the phenotypes may be related to post-CD4-binding effects. The results demonstrate that neutralization resistance can be determined by distinguishable effects of mutations, which cause changes in envelope conformation and/or function(s) related to infectivity. A conformation-dependent V3 epitope may be an important target for neutralization of resistant strains of HIV-1
— id: 9232, year: 2000, vol: 74, page: 4183, stat: Journal Article,

Serodiagnostic potential of culture filtrate antigens of Mycobacterium tuberculosis
Samanich KM; Keen MA; Vissa VD; Harder JD; Spencer JS; Belisle JT; Zolla-Pazner S; Laal S
2000 Jul;7(4):662-668, Clinical & diagnostic laboratory immunology
Our studies of the humoral responses of tuberculosis (TB) patients have defined the repertoire of culture filtrate antigens of Mycobacterium tuberculosis that are recognized by antibodies from cavitary and noncavitary TB patients and demonstrated that the profile of antigens recognized changes with disease progression (K. Samanich et al., J. Infect. Dis. 178:1534-1538, 1998). We have identified several antigens with strong serodiagnostic potential. In the present study we have evaluated the reactivity of cohorts of human immunodeficiency virus (HIV)-negative, smear-positive; HIV-negative, smear-negative; and HIV-infected TB patients, with three of the candidate antigens, an 88-kDa protein, antigen (Ag) 85C, and MPT32, and compared the reactivity of the same patient cohort with the 38-kDa antigen and Ag 85A. We have also compared the reactivity of native Ag 85C and MPT32 with their recombinant counterparts. The evaluation of the reactivity was done by a modified enzyme-linked immunosorbent assay described earlier (S. Laal et al., Clin. Diag. Lab. Immunol. 4:49-56, 1997), in which all sera are preadsorbed against Escherichia coli lysates to reduce the levels of cross-reactive antibodies. Our results demonstrate that (i) antigens identified on the basis of their reactivity with TB patients' sera provide high sensitivities for serodiagnosis, (ii) recombinant Ag 85C and MPT32, expressed in E. coli, show reduced reactivity with human TB sera, and (iii) of the panel of antigens tested, the 88-kDa protein is the most promising candidate for serodiagnosis of TB in HIV-infected individuals. Moreover, these results reaffirm that both the extent of the disease and the bacterial load may play a role in determining the antigen profile recognized by antibodies
— id: 11616, year: 2000, vol: 7, page: 662, stat: Journal Article,

Human monoclonal antibody 98-6 reacts with the fusogenic form of gp41
Taniguchi Y; Zolla-Pazner S; Xu Y; Zhang X; Takeda S; Hattori T
2000 Aug 1;273(2):333-340, Virology
A mixture of two peptides from gp41 (N36 and C34) forms an alpha-helical structure that is thought to represent the fusogenic form of gp41. A human anti-gp41 monoclonal antibody (mAb 98-6), generated from the cells of an infected individual, reacted poorly with C34, but binding was strongly enhanced when N36 was added, indicating that the mAb reacts with a conformational epitope present in the fusogenic structure formed by the interaction of peptides N36 and C34. The epitope recognized by mAb 98-6 was found in lysates of virions on oligomeric forms of gp41 (dimers, trimers, and tetramers). On infected cells, the epitope was present as oligomers of gp41, as monomers of gp41, and as part of the envelope polyprotein gp160, obtained after biotinylation of intact cells, which were then lysed and immunoprecipitated with various mAbs. In lysates of infected cells, the epitope was present as part of both monomeric gp41 and gp160. These studies demonstrate that infected humans can respond to the fusogenic form of gp41 and that the anti-gp41 mAb studied here recognizes a conformational epitope formed by the interaction of two regions of gp41, which forms an alpha-helical bundle. This epitope is found on several forms of gp41 as it occurs in virions, on the surface of infected cells, and in infected cells.
— id: 9228, year: 2000, vol: 273, page: 333, stat: Journal Article,

A human immunodeficiency virus prime-boost immunization regimen in humans induces antibodies that show interclade cross-reactivity and neutralize several X4-, R5-, and dualtropic clade B and C primary isolates
Verrier F; Burda S; Belshe R; Duliege AM; Excler JL; Klein M; Zolla-Pazner S
2000 Nov;74(21):10025-10033, Journal of virology
A human immunodeficiency virus (HIV) vaccine that will be useful in diverse geographic regions will need to induce a broad immune response characterized by cross-clade immunity. To test whether a clade B-based HIV candidate vaccine could induce interclade humoral responses, including neutralizing activity against primary HIV-1 isolates, sera were tested from recipients of a vaccine consisting of recombinant canarypox virus vCP205 and recombinant gp120(SF2). Serum antibodies exhibited strong immunochemical cross-reactivity with V3 peptides from clades B, C, and F, with weaker activity for several V3 peptides from clades A, D, G, and H; essentially no reactivity could be demonstrated with V3 peptides from clades E and O. Extensive cross-clade reactivity was also documented by enzyme-linked immunosorbent assay with all nine recombinant HIV envelope glycoproteins tested from clades B, D, and E. In addition, vaccinees' sera displayed significant neutralizing activity against 5 of 14 primary isolates tested, including one X4 virus and two dualtropic viruses (from clade B) and two R5 viruses (from clades B and C). This is the first demonstration of the induction by a candidate HIV vaccine constructed from clade B laboratory strains of HIV of neutralizing activity against R5 and clade C primary isolates. The data suggest that, by virtue of their ability to induce cross-clade immune responses, appropriately formulated HIV vaccines based on a finite number of HIV isolates may ultimately be able to protect against the wide range of HIV isolates affecting the populations of many geographic regions
— id: 39544, year: 2000, vol: 74, page: 10025, stat: Journal Article,

A longitudinal study of neutralizing antibodies and disease progression in HIV-1-infected subjects
Cecilia D; Kleeberger C; Munoz A; Giorgi JV; Zolla-Pazner S
1999 Jun;179(6):1365-1374, Journal of infectious diseases
Sera from human immunodeficiency virus-1-infected participants in the Multicenter AIDS Cohort Study were tested to assess the association between serum neutralizing antibodies (NAbs) and disease progression. Each of 14 pairs, retrospectively matched for age, sex, race, and CD4+ lymphocyte numbers early in the study, consisted of a rapid progressor (RP) who developed AIDS and a long-term nonprogressor (LTNP) who remained asymptomatic. Serum samples were drawn early, when all participants were asymptomatic, and late, when the RPs had developed clinical AIDS. The LTNPs and RPs had similar levels of NAbs against primary isolates at the early time point, indicating that NAb levels are not predictive of disease progression; at the late time point, the LTNPs had significantly higher titers because of an increase in the level of serum NAbs in the LTNPs and/or a decrease in the NAbs in the RPs. The patterns of neutralizing activity over time suggest that changes in effective NAbs against different viruses do not occur in parallel
— id: 9234, year: 1999, vol: 179, page: 1365, stat: Journal Article,

Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins
Gigler A; Dorsch S; Hemauer A; Williams C; Kim S; Young NS; Zolla-Pazner S; Wolf H; Gorny MK; Modrow S
1999 Mar;73(3):1974-1979, Journal of virology
Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 microgram/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 micrograms/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development
— id: 57034, year: 1999, vol: 73, page: 1974, stat: Journal Article,

Enhanced HIV type 1 neutralization by human anti-glycoprotein 120 monoclonal antibodies in the presence of monoclonal antibodies to lymphocyte function-associated molecule 1
Hioe CE; Hildreth JE; Zolla-Pazner S
1999 Apr 10;15(6):523-531, AIDS research & human retroviruses
Cellular adhesion receptor LFA-1 and its ICAM ligands are known to play a role in HIV infection. The presence of these molecules on virions and target cells promotes virus infectivity and has previously been shown to hinder virus neutralization by anti-HIV antibodies. To delineate the effect of these molecules on neutralization of HIV-1, human monoclonal antibodies (MAbs) to V3 and the CD4-binding domain (CD4bd) of gp120 were examined in the presence of anti-LFA-1 MAbs. When either of two anti-LFA-1 MAbs was present, higher levels of virus neutralization were achieved by both anti-V3 and anti-CD4bd MAbs. This effect was observed with primary HIV-1 isolates as well as with a laboratory-adapted strain. However, this activity was seen only when an anti-LFA-1 MAb was combined with anti-gp120 MAbs that exhibited virus-specific neutralizing activities, demonstrating the specificity of both the anti-LFA-1 and anti-gp120 MAbs. Enhanced neutralization by anti-gp120 MAbs was observed if the anti-LFA-1 MAb was present during the initial 24 hr only, if added 24 hr after infection, or if present throughout the culture period. These data suggest that the anti-LFA-1 MAbs could act at different stages of HIV-1 infection, including the initial virus-cell interaction as well as during the amplification and spread of virus from cell to cell. These findings demonstrate the significant role of LFA-1 in HIV-1 infection and have important implications for evaluating the neutralizing activity of anti-HIV antibodies
— id: 6102, year: 1999, vol: 15, page: 523, stat: Journal Article,

Exclusion of HIV coreceptors CXCR4, CCR5, and CCR3 from the HIV envelope
Lallos LB; Laal S; Hoxie JA; Zolla-Pazner S; Bandres JC
1999 Jul 1;15(10):895-897, AIDS research & human retroviruses
Both HIV-1 primary isolates and laboratory strains incorporate cell-derived molecules into their envelopes depending on the host cell in which they are grown. This incorporation is not random and, specifically, HIV-1 has been shown to select against the incorporation into its surface of CD4, its main receptor. In this study, we have looked at the incorporation of HIV coreceptors CXCR4, CCR5, and CCR3 into the HIV envelope. For this purpose, we grew HIV-1 primary isolate BZ167 in several cell lines and PBMCs, and the envelope profiles of the resulting viruses were determined with a virus-binding ELISA. While the virus particle gained several molecules when passed through the different cell lines (e.g., ICAM-3, LFA-1, ICAM-1, or MHC class II), BZ167 never incorporated significant levels of CXCR4, CCR5, or CCR3 into its envelope even though some or all of the cell lines in which it was grown expressed them. These results show that HIV-1 selects against the incorporation of these chemokine receptors into its envelope molecule, as it does against the incorporation of CD4
— id: 6159, year: 1999, vol: 15, page: 895, stat: Journal Article,

The implications of antigenic diversity for vaccine development
Zolla-Pazner S; Gomy MK; Nyambi PN
1999 Mar;66(1-3):159-164, Immunology letters
The reactivity of human monoclonal and polyclonal anti-HIV-1 antibodies demonstrates that shared epitopes, including those that induce neutralizing antibodies, exist and are recognized by the human immune system. A priori, there is no reason why cross-clade neutralizing antibodies could not be induced by an appropriately constructed HIV vaccine. But to construct such a vaccine, it is critical to understand, as completely as possible, the antigenic structure of HIV, to establish and identify immunologic classifications for HIV, and to choose rationally the minimum number and types of viruses from these immunologic groupings that will induce the broadest protective responses
— id: 9235, year: 1999, vol: 66, page: 159, stat: Journal Article,

Immunotyping of human immunodeficiency virus type 1 (HIV): an approach to immunologic classification of HIV
Zolla-Pazner S; Gorny MK; Nyambi PN; VanCott TC; Nadas A
1999 May;73(5):4042-4051, Journal of virology
Because immunologic classification of human immunodeficiency virus type 1 (HIV) might be more relevant than genotypic classification for designing polyvalent vaccines, studies were undertaken to determine whether immunologically defined groups of HIV ('immunotypes') could be identified. For these experiments, the V3 region of the 120-kDa envelope glycoprotein (gp120) was chosen for study. Although antibodies (Abs) to V3 may not play a major protective role in preventing HIV infection, identification of a limited number of immunologically defined structures in this extremely variable region would set a precedent supporting the hypothesis that, despite its diversity, the HIV family, like the V3 region, might be divisible into immunotypes. Consequently, the immunochemical reactivities of 1,176 combinations of human anti-V3 monoclonal Abs (MAbs) and V3 peptides, derived from viruses of several clades, were studied. Extensive cross-clade reactivity was observed. The patterns of reactivities of 21 MAbs with 50 peptides from clades A through H were then analyzed by a multivariate statistical technique. To test the validity of the mathematical approach, a cluster analysis of the 21 MAbs was performed. Five groups were identified, and these MAb clusters corresponded to classifications of these same MAbs based on the epitopes which they recognize. The concordance between the MAb clusters identified by mathematical analysis and by their specificities supports the validity of the mathematical approach. Therefore, the same mathematical technique was used to identify clusters within the 50 peptides. Seven groups of peptides, each containing peptides from more than one clade, were defined. Inspection of the amino acid sequences of the peptides in each of the mathematically defined peptide clusters revealed unique 'signature sequences' that suggest structural motifs characteristic of each V3-based immunotype. The results suggest that cluster analysis of immunologic data can define immunotypes of HIV. These immunotypes are distinct from genotypic classifications. The methods described pave the way for identification of immunotypes defined by immunochemical and neutralization data generated with anti-HIV Env MAbs and intact, viable HIV virions
— id: 9236, year: 1999, vol: 73, page: 4042, stat: Journal Article,

Passive immunization with a human immunodeficiency virus type 1-neutralizing monoclonal antibody in Hu-PBL-SCID mice: isolation of a neutralization escape variant
Andrus L; Prince AM; Bernal I; McCormack P; Lee DH; Gorny MK; Zolla-Pazner S
1998 Apr;177(4):889-897, Journal of infectious diseases
A monoclonal antibody (694/98-D) directed toward the V3 loop of human immunodeficiency virus type 1 (HIV-1) was evaluated for pre- and postexposure prophylaxis in SCID mice reconstituted with human peripheral blood lymphocytes (Hu-PBL-SCID). Fifty percent protection against the HIV-1LAI strain was obtained by preexposure administration of 1.32 mg/kg antibody. However, virus isolated from 1 mouse 3 weeks after passive immunization with 13.2 mg/kg antibody proved resistant to subsequent in vitro neutralization by 694/98-D. V3 loop sequence analysis of cloned virus revealed amino acid changes within the linear core epitope recognized by 694/98-D and in one flanking amino acid. Further evaluation of 694/98-D for postexposure prophylaxis in mice revealed that 694/ 98-D was effective when given 15 min after virus. However, efficacy declined to 50% if treatment was delayed to 1 h after virus inoculation. These studies point out some potential drawbacks of passive immunization with monoclonal antibodies
— id: 7483, year: 1998, vol: 177, page: 889, stat: Journal Article,

Human immunodeficiency virus (HIV) envelope binds to CXCR4 independently of CD4, and binding can be enhanced by interaction with soluble CD4 or by HIV envelope deglycosylation
Bandres JC; Wang QF; O'Leary J; Baleaux F; Amara A; Hoxie JA; Zolla-Pazner S; Gorny MK
1998 Mar;72(3):2500-2504, Journal of virology
Chemokine receptor CXCR4 (also known as LESTR and fusin) has been shown to function as a coreceptor for T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have developed a binding assay to show that HIV envelope (Env) can interact with CXCR4 independently of CD4 but that this binding is markedly enhanced by the previous interaction of Env with soluble CD4. We also show that nonglycosylated HIV-1(SF-2) gp120 or sodium metaperiodate-treated oligomeric gp160 from HIV-1(451) bound much more readily to CXCR4 than their counterparts with intact carbohydrate residues did
— id: 57100, year: 1998, vol: 72, page: 2500, stat: Journal Article,

Flow cytometric analysis of the binding of HIV envelope to CXCR4, relevance of the interaction with soluble CD4 and HIV envelope glycosylation
Bandres JC; Wang QF; O'Leary J; Hoxie JA; Zolla-Pazner S; Gorny MK
1998 Feb 1-5;5:87-87, Conference on Retroviruses & Opportunistic Infections
Chemokine receptor CXCR4 has been shown to function as a coreceptor for T-cell tropic strains of HIV-1. We have developed a flow cytometric binding assay to study the interaction between HIV Env and CXCR4. Binding assay: ogp160 from HIV(451) was allowed to bind to CD4+, CXCR4+ cell line CEM-SS as well as to CD4-, CXCR4+ cell line J25. Binding was detected using a human monoclonal antibody against the C-terminal portion of gpl60 (1331-A) and a secondary PE-labeled goat anti-human IgG. Results: HIV Envelope was able to interact directly with CXCR4 independent of CD4 as evidenced by binding to J25 cells. We also show that this binding is greatly enhanced by the previous interaction of Env with soluble CD4. In addition, non-glycosylated HIV-1(SF-2) gp120 or sodium metaperiodate-treated oligomeric gpl60 from HIV-1(451) were also found to bind much more readily to CXCR4 than their counterparts with intact carbohydrate residues. Conclusions: our results demonstrate that there is a direct interaction between HIV Env and CXCR4, describe a flow cytometric assay to investigate this process and provide new insights into its function
— id: 6007, year: 1998, vol: 5, page: 87, stat: Journal Article,

Selective exclusion of HIV coreceptors from HIV virions
Bastiani L; Laal S; Hoxie JA; Zolla-Pazner S; Bandres JC
1998 Feb 1-5;5:85-85, Conference on Retroviruses & Opportunistic Infections
Both primary isolates and laboratory strains of HIV-1 incorporate different cell-derived molecules into their envelopes depending on the host cell in which they are grown. This incorporation in not random; for example, HIV-1 has been shown to exclude CD4 from its envelope. In this study we have examined the incorporation into the envelope of HIV-1 of three coreceptors -- CXCR4,CCR5, and CCR3-- as well as CD4. For this purpose, the HIV-1 SI primary isolate BZ167 was passaged into PHA-stimulated PBMC and CEM-SS cells and the incorporation of various cell-derived molecules into the virion envelope was determined with a virus binding ELISA. As previously shown, BZ167 grown in PHA-stimulated PBMC expressed most of the adhesion molecules tested (such as LFA-1, ICAM-1, and MHC class II), while CEM-SS-grown BZ167 did not express significant levels of any of these adhesion molecules. CEM-SS cells express both CD4 and CXCR4 whereas PHA-stimulated PBMCs express CD4, CXCR4, CCR5, and CCR3. In contrast, BZ167 grown in either cell type lacked CD4 as well as the coreceptors, CXCR4, CCR5, and CCR3
— id: 6008, year: 1998, vol: 5, page: 85, stat: Journal Article,

Neutralization profiles of primary human immunodeficiency virus type 1 isolates in the context of coreceptor usage
Cecilia D; KewalRamani VN; O'Leary J; Volsky B; Nyambi P; Burda S; Xu S; Littman DR; Zolla-Pazner S
1998 Sep;72(9):6988-6996, Journal of virology
Most strains of human immunodeficiency virus type 1 (HIV-1) which have only been carried in vitro in peripheral blood mononuclear cells (primary isolates) can be neutralized by antibodies, but their sensitivity to neutralization varies considerably. To study the parameters that contribute to the differential neutralization sensitivity of primary HIV-1 isolates, we developed a neutralization assay with a panel of genetically engineered cell lines (GHOST cells) that express CD4, one of eight chemokine receptors which function as HIV-1 coreceptors, and a Tat-dependent green fluorescent protein reporter cassette which permits the evaluation and quantitation of HIV-1 infection by flow cytometry. All 21 primary isolates from several clades could grow in the various GHOST cell lines, and their use of one or more coreceptors could easily be defined by flow cytometric analysis. Ten of these primary isolates, three that were CXCR4 (X4)-tropic, three that were CCR5 (R5)-tropic, and four that were dual- or polytropic were chosen for study of their sensitivity to neutralization by human monoclonal and polyclonal antibodies. Viruses from the X4-tropic category of viruses were first tested since they have generally been considered to be particularly neutralization sensitive. It was found that the X4-tropic virus group contained both neutralization-sensitive and neutralization-resistant viruses. Similar results were obtained with R5-tropic viruses and with dual- or polytropic viruses. Within each category of viruses, neutralization sensitivity and resistance could be observed. Therefore, sensitivity to neutralization appears to be the consequence of factors that influence the antibody-virus interaction and its sequelae rather than coreceptor usage. Neutralization of various viruses by the V3-specific monoclonal antibody, 447-52D, was shown to be dependent not only on the presence of the relevant epitope but also on its presentation. An epitope within the envelope of a particular virus is not sufficient to render a virus sensitive to neutralization by an antibody that recognizes that epitope. Moreover, conformation-dependent factors may overcome the need for absolute fidelity in the match between an antibody and its core epitope, permitting sufficient affinity between the viral envelope protein and the antibody to neutralize the virus. The studies indicate that the neutralization sensitivity of HIV-1 primary isolates is a consequence of the complex interaction between virus, antibody, and target cell
— id: 7527, year: 1998, vol: 72, page: 6988, stat: Journal Article,

Immune responses to human immunodeficiency virus (HIV) type 1 induced by canarypox expressing HIV-1MN gp120, HIV-1SF2 recombinant gp120, or both vaccines in seronegative adults. NIAID AIDS Vaccine Evaluation Group
Clements-Mann ML; Weinhold K; Matthews TJ; Graham BS; Gorse GJ; Keefer MC; McElrath MJ; Hsieh RH; Mestecky J; Zolla-Pazner S; Mascola J; Schwartz D; Siliciano R; Corey L; Wright PF; Belshe R; Dolin R; Jackson S; Xu S; Fast P; Walker MC; Stablein D; Excler JL; Tartaglia J; Paoletti E
1998 May;177(5):1230-1246, Journal of infectious diseases
A safety and immunogenicity trial was conducted in vaccinia-immune and vaccinia-naive human immunodeficiency virus (HIV)-uninfected adults who were randomized to receive 10(6) or 10(7) TCID50 of canarypox (ALVAC) vector expressing HIV-1MN gp160 or 10(5.5) TCID50 of ALVAC-rabies virus glycoprotein control at 0 and 1 or 2 months and ALVAC-gp160 or 50 microg of HIV-1SF2 recombinant (r) gp120 in microfluidized emulsion at 9 and 12 months; others received rgp120 at 0, 1, 6, and 12 months. All vaccines were well-tolerated. Neither vaccinia-immune status before vaccination nor ALVAC dose affected HIV immune responses. HIV-1MN and HIV-1SF2 neutralizing antibodies were detected more often (100%) in ALVAC-gp160/rgp120 recipients than in recipients of ALVAC-gp160 (<65%) or rgp120 (89%) alone. ALVAC-gp160/rgp120 also elicited more frequent HIV V3-specific and fusion-inhibition antibodies, antibody-dependent cellular cytotoxicity, lymphoproliferation, and cytotoxic CD8+ T cell activity than did either vaccine alone. Trials with ALVAC expressing additional HIV components and rgp120 are underway
— id: 7533, year: 1998, vol: 177, page: 1230, stat: Journal Article,

A human monoclonal antibody specific for the V3 loop of HIV type 1 clade E cross-reacts with other HIV type 1 clades
Gorny MK; Mascola JR; Israel ZR; VanCott TC; Williams C; Balfe P; Hioe C; Brodine S; Burda S; Zolla-Pazner S
1998 Feb 10;14(3):213-221, AIDS research & human retroviruses
To ascertain the antigenic relationship between HIV-1 viruses belonging to various genetically defined subgroups (clades), shared epitopes need to be defined. Human monoclonal antibodies (MAbs) are particularly useful for this purpose because they can detect complex regions of viral proteins that may be missed by sequence analysis and because, by definition, they react with epitopes that stimulate the human immune system. Monoclonal antibodies derived from the cells of HIV-1 clade B-infected subjects have been used extensively for this purpose. Here we describe the first human MAb derived from a clade E-infected individual; the MAb is specific for the V3 loop, recognizing a core epitope represented by the amino acids TRTSVR on the N-terminal side of the crown of the V3 loop. The IgG1(kappa) MAb, designated 1324E, binds to the clade E consensus V3 loop, to rgp120 proteins from clade E and to peripheral blood mononuclear cells infected in vitro with the virus that infected the subject from whose cells the MAb-producing heterohybridoma was derived. Strong cross-reactivity of the MAb to the V3 peptides, rgp120 proteins, and native monomeric gp120s representing clades A and C, as well as to cells infected with a clade C primary isolate, revealed a shared V3 epitope between these clades. When tested for its neutralizing ability, MAb 1324E neutralized a clade E isolate that had been adapted for growth in H9 cells but failed to neutralize five clade E primary isolates
— id: 7582, year: 1998, vol: 14, page: 213, stat: Journal Article,

Humoral immunity to HIV, SIV, and SHIV
Haigwood NL; Zolla-Pazner S
1998 ;12 Suppl A:S121-S132, AIDS
— id: 9238, year: 1998, vol: 12 Suppl A, page: S121, stat: Journal Article,

Role of cellular adhesion molecules in HIV type 1 infection and their impact on virus neutralization
Hioe CE; Bastiani L; Hildreth JE; Zolla-Pazner S
1998 Oct;14 Suppl 3:S247-S254, AIDS research & human retroviruses
While CD4 and several chemokine receptors are the principal receptors for human immunodeficiency virus type 1 (HIV-1) viruses, other cell membrane proteins also play a role in HIV-1 infection. A large array of host cell-derived membrane proteins, including adhesion molecules, are incorporated into the envelope of HIV-1 virions, and the profile of host cell proteins acquired by the virus depends on the cells used to propagate the virus. The major leukocyte adhesion molecules, such as leukocyte-function associated antigen-1 (LFA-1), intercellular adhesion molecule-1 (ICAM-1), and CD44, retain their biological functions when expressed on the virion surface, and have been shown to increase virus-cell interaction, enhance virus infectivity, and extend the host cell range of the virus. LFA-1 and its ICAM ligands are also necessary for syncytium formation and cell-to-cell transmission of HIV-1. Furthermore, several studies demonstrate that the presence and level of cell-derived adhesion molecules on the surface of HIV-1 virions affect the process by which antibody-mediated virus neutralization occurs and is measured: the level of virus neutralization is influenced by the host cell-derived adhesion molecules present on the virus, and thus, by the type of host cells in which the virus was produced. Adhesion molecules expressed on the target cells used in neutralization assays similarly affect HIV-1 neutralization by virus-specific antibodies. Consistent with these observations is the finding that neutralizing activities of both HIV+ plasma and human anti-gp120 monoclonal antibodies (Mabs) are enhanced by an anti-LFA-1 Mab capable of blocking LFA-1 functions. Hence, LFA-1, ICAM-1, and other cellular adhesion molecules are involved in different stages of HIV-1 infection and profoundly affect HIV-1 neutralization by virus-specific antibodies. These findings illuminate the biology of virus-cell interactions and have significant implications for evaluating candidate HIV vaccines
— id: 7346, year: 1998, vol: 14 Suppl 3, page: S247, stat: Journal Article,

Neutralizing antibodies directed against the V3 loop select for different escape variants in a virus with mutated reverse transcriptase (M184V) than in wild-type human immunodeficiency virus type 1
Inouye P; Cherry E; Hsu M; Zolla-Pazner S; Wainberg MA
1998 Jun 10;14(9):735-740, AIDS research & human retroviruses
The M184V substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) encodes high-level resistance to the (-)-enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC) and low-level resistance to each of 2',3'-dideoxycytidine (ddC) and 2',3'-dideoxyinosine (ddI). This mutation also results in decreased HIV replication fitness in primary cells, diminished RT processivity, and increased RT fidelity. To assess the effect of this substitution on genetic variation in the HIV env region, we cultured both M184V-containing and wild-type BH10 in MT-4 cells in the presence of the neutralizing monoclonal antibody 447-52D, targeted to the GPGR epitope within the V3 loop of gp120. Outgrowth of viruses resistant to neutralization was followed by sequence analysis of the V3 loop by standard methodology. Wild-type HIV first showed escape after 15-22 days in culture. Sequence analysis revealed an arginine-to-lysine change within the GPGR epitope in the V3 loop (R20K, AGA --> AAA) in six of six clones sequenced after day 36. In contrast, M184V-containing HIV first showed escape between days 25 and 32 and sequence analysis revealed an aspartate-to-tyrosine change at amino acid 5 in V3 (N5Y; AAC --> TAC) in two of six clones at day 36 and in five of five clones at day 55. Similar results were obtained in two independent antibody selection protocols. The escape mutation in the wild type is consistent with the G --> A hypermutation observed in wild-type HIV-1, recently shown to cause an initial M184I change (before M184V) in 3TC-treated patients. In contrast, the N5Y substitution seen with M184V-containing HIV-1 is an A --> T transversion in V3
— id: 8080, year: 1998, vol: 14, page: 735, stat: Journal Article,

Coreceptor utilization by human immunodeficiency virus type 1 is not a primary determinant of neutralization sensitivity
LaCasse RA; Follis KE; Moudgil T; Trahey M; Binley JM; Planelles V; Zolla-Pazner S; Nunberg JH
1998 Mar;72(3):2491-2495, Journal of virology
We have examined the relationship between coreceptor utilization and sensitivity to neutralization in a primary isolate of human immunodeficiency virus type 1 and its T-cell line-adapted (TCLA) derivative. We determined that adaptation of the primary-isolate (PI) virus 168P results in the loss of the unique capacity of PI viruses to utilize the CCR5 coreceptor and in the acquisition by the TCLA 168C virus of sensitivity to neutralization by V3-directed monoclonal antibodies (MAbs). In experiments wherein infection by 168P is directed via either the CCR5 or the CXCR4 pathway, we demonstrate that the virus, as well as pseudotyped virions bearing a molecularly cloned 168P envelope protein, remains refractory to neutralization by MAbs 257-D, 268-D, and 50.1 regardless of the coreceptor utilized. This study suggests that coreceptor utilization is not a primary determinant of differential neutralization sensitivity in PI and TCLA viruses
— id: 57188, year: 1998, vol: 72, page: 2491, stat: Journal Article,

Synergistic neutralization of simian-human immunodeficiency virus SHIV-vpu+ by triple and quadruple combinations of human monoclonal antibodies and high-titer anti-human immunodeficiency virus type 1 immunoglobulins
Li A; Katinger H; Posner MR; Cavacini L; Zolla-Pazner S; Gorny MK; Sodroski J; Chou TC; Baba TW; Ruprecht RM
1998 Apr;72(4):3235-3240, Journal of virology
We have tested triple and quadruple combinations of human monoclonal antibodies (MAbs), which are directed against various epitopes on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, and a high-titer anti-HIV-1 human immunoglobulin (HIVIG) preparation for their abilities to neutralize a chimeric simian-human immunodeficiency virus (SHIV-vpu+). This virus encodes the HIV-1 strain IIIB env, tat, rev, and vpu genes. The quantitative nature of the Chou-Talalay method (Adv. Enzyme Regul. 22:27-55, 1984) allows ranking of various combinations under identical experimental conditions. Of all triple combinations tested, the most potent neutralization was seen with MAbs 694/98D plus 2F5 plus 2G12 (directed against domains on V3, gp41, and gp120, respectively) as measured by the total MAb concentration required to reach 90% neutralization (90% effective concentration [EC90], 2.0 microg/ml). All triple combinations involving MAbs and/or HIVIG that were tested yielded synergy with combination index values of < 1; the dose reduction indices (DRIs) ranged from 3.1 to 26.2 at 90% neutralization. When four MAbs (the previous three plus MAb F105, directed against the CD4 binding site) were combined, higher neutralization potency (EC90 1.8 microg/ml) and a higher degree of synergy compared to any triple combination were seen. The mean DRIs of the quadruple combination were approximately twice that of the most synergistic triple combination. We conclude that human MAbs targeting different HIV-1 envelope glycoprotein epitopes exhibit strong synergy when used in combination, a fact that could be exploited clinically for passive immunoprophylaxis against HIV-1
— id: 9239, year: 1998, vol: 72, page: 3235, stat: Journal Article,

Early-stage HIV infection and hepatitis C virus infection are associated with elevated serum porphyrin levels
Lim HW; Pereira A; Sassa S; Kim M; Zolla-Pazner S
1998 Dec;39(6):956-959, Journal of the American Academy of Dermatology
BACKGROUND: Porphyria cutanea tarda is known to be associated with HIV infection and hepatitis C virus (HCV). OBJECTIVE: Our purpose was to evaluate whether early infection with HIV, with or without HCV infection, is associated with elevated serum porphyrin levels. METHODS: Serum porphyrin levels were measured in samples obtained from 103 patients with early HIV infection. The results were compared with those of 89 late-stage HIV-positive patients and 78 HIV-negative patients. RESULTS: The highest median porphyrin level was in early-stage HIV-positive/HCV-positive samples, followed in decreasing order by those in early-stage HIV-positive/HCV-negative, late-stage HIV-positive/HCV-positive, late-stage HIV-positive/HCV-negative, HIV-negative/HCV-positive, and HIV-negative/HCV-negative groups. Elevated porphyrin levels were independently associated with early-stage HIV infection (P < .0001) and HCV infection (P = .03). CONCLUSION: This finding suggests abnormal porphyrin metabolism is most noticeable in early-stage HIV infection; it becomes less severe with the progression of HIV disease
— id: 57195, year: 1998, vol: 39, page: 956, stat: Journal Article,

[Increase of circulating CD3+CD4-CD8-CD19+ cells in the latent phase of HIV-1 infection]
Nozaki-Renard J; O'Leary J; Zolla-Pazner S; Tada T
1998 ;192(5):1007-1015, Comptes rendus des seances de la Societe de Biologie & des ses Filiales
Having reported that HIV-1-infected T cell lines are rescued as CD4- from cytolysis by human complement factor B, we now show the presence of an in vivo counterpart of such CD4- T cells by demonstrating the circulating CD3+ CD4- CD8- CD29+ cells in the blood of seropositive subjects (n = 91, classified by the immunologic scale scores 0, 1, 2 and 3). The cell population was found to be significantly increased in the early phase of infection in score 0: 195/mm3 (p < 0.005) and in score 1:376/mm3 (p = 0.001). With the infection progressing to score 2, the cells decreased to 220/mm3 (p < 0.001) and finally to the same range: 101/mm3, as that of uninfected subjects. Further elucidation of the mechanism of the appearance and disappearance of that population in vivo could help to elucidate protective immunologic processes
— id: 9237, year: 1998, vol: 192, page: 1007, stat: Journal Article,

Mapping of epitopes exposed on intact human immunodeficiency virus type 1 (HIV-1) virions: a new strategy for studying the immunologic relatedness of HIV-1
Nyambi PN; Gorny MK; Bastiani L; van der Groen G; Williams C; Zolla-Pazner S
1998 Nov;72(11):9384-9391, Journal of virology
To study the antigenic conservation of epitopes of human immunodeficiency virus type 1 (HIV-1) isolates of different clades, the abilities of human anti-HIV-1 gp120 and gp41 monoclonal antibodies (MAbs) to bind to intact HIV-1 virions were determined by a newly developed virus-binding assay. Eighteen human anti-HIV MAbs, which were directed at the V2, V3 loop, CD4-binding domain (CD4bd), C5, or gp41 regions, were used. Nine HIV-1 isolates from clades A, B, D, F, G, and H were used. Microtiter wells were coated with the MAbs, after which virus was added. Bound virus was detected after lysis by testing for p24 antigen with a noncommercial p24 enzyme-linked immunosorbent assay. The anti-V3 MAbs strongly bound the four clade B viruses and viruses from the non-B clades, although binding was weaker and more sporadic with the latter. The degrees of binding by the anti-V3 MAbs to CXCR4- and CCR5-tropic viruses were similar, suggesting that the V3 loops of these two categories of viruses are similarly exposed. The anti-C5 MAbs bound isolates of clades A, B, and D. Only weak and sporadic binding of all the viruses tested with anti-CD4bd, anti-V2, and anti-gp41 MAbs was detected. These results suggest that V3 and C5 structures are shared and well exposed on intact virions of different clades compared to the CD4bd, V2, and gp41 regions
— id: 7969, year: 1998, vol: 72, page: 9384, stat: Journal Article,

Binding of monoclonal antibodies (mAb) to intact HIV-1 virions: a new strategy for mapping conserved HIV-1 epitopes
Nyambi PN; Gorny MK; Williams C; Zolla-Pazner S
1998 Feb 1-5;5:96-96, Conference on Retroviruses & Opportunistic Infections
Identification of conserved antigenic epitopes on intact HIV virions will facilitate the design of a vaccine against HIV. Previous attempts to map conserved epitopes on different HIV strains used peptides or recombinant proteins in binding assays which do not mimic the intact virion structure. The best case scenario is to use intact virions for mapping conserved epitopes. In this study, we determined the ability of mAbs to bind to intact virions using a simple binding assay in microtiter wells. Bound virus was detected after lysis by testing for p24 antigen in a non-commercial p24 ELISA. To determine binding, five anti-HIV-1 V(3) loop, two anti-CD4 binding domain (CD4bd) mAbs derived from HIV-infected persons and anti-HIV-1 pooled immunoglobulin (HIVIG) from infected persons were tested with PBMC culture supernatants of four HIV-1 isolates of subtype B (two primary and two laboratory strains). All five V(3) mAbs bound to two viruses (one primary [CA5] and one lab [MN] strain). Three of four and one of five V(3) mAbs bound to IIIB (lab strain) and BZ167 (primary strain), respectively. Interestingly, mAb 447-52D bound to all the four viruses suggesting that its epitope is highly conserved amongst the isolates tested. Binding of the two CD4bd mAbs to the four isolates was weak and sporadic. HIVIG bound to all the four isolates when used at concentrations five fold higher than the mAbs. These results suggest that both the lab and primary isolates tested share conserved antigenic epitopes. Testing isolates of different clades with this strategy will facilitate the identification of conserved epitopes and antigenic relationships
— id: 6011, year: 1998, vol: 5, page: 96, stat: Journal Article,

Vaccine protection against a heterologous, non-syncytium-inducing, primary human immunodeficiency virus
Robert-Guroff M; Kaur H; Patterson LJ; Leno M; Conley AJ; McKenna PM; Markham PD; Richardson E; Aldrich K; Arora K; Murty L; Carter L; Zolla-Pazner S; Sinangil F
1998 Dec;72(12):10275-10280, Journal of virology
Vaccine-induced protection of chimpanzees against laboratory-adapted and syncytium-inducing, multiply passaged primary human immunodeficiency virus type 1 (HIV-1) isolates, but not against non-syncytium-inducing, minimally passaged ones, has been demonstrated. Following challenge with such an isolate, HIV-15016, we obtained complete protection in one of three chimpanzees previously protected against low- and high-dose HIV-1SF2 exposures after immunization with an adenovirus-HIV-1MN gp160 priming-HIV-1SF2 gp120 boosting regimen. At challenge, the protected chimpanzee exhibited broad humoral immunity, including neutralizing antibody activity. These results demonstrate the potential of this combination vaccine strategy and suggest that vaccine protection against an HIV isolate relevant to infection of people is feasible
— id: 57234, year: 1998, vol: 72, page: 10275, stat: Journal Article,

Delineation of human antibody responses to culture filtrate antigens of Mycobacterium tuberculosis
Samanich KM; Belisle JT; Sonnenberg MG; Keen MA; Zolla-Pazner S; Laal S
1998 Nov;178(5):1534-1538, Journal of infectious diseases
This study was undertaken to define the antigens in culture filtrates of actively replicating Mycobacterium tuberculosis that are recognized by antibodies from tuberculosis (TB) patients. Two-dimensional Western blots were probed with sera from healthy controls and TB patients that were preabsorbed with Escherichia coli lysates to deplete cross-reactive antibodies. Antibodies from TB patients recognized 26 of the >100 culture filtrate proteins, and the repertoire changed with disease progression. Only 12 of 26 antigens, including 3 proteins implicated in colonization and invasion by mycobacteria (MPT51, MPT32, and 85C), and 9 (as yet undefined proteins) were reactive with sera from TB patients with early noncavitary or cavitary disease. Eight additional antigens, including 4 undefined proteins, were recognized only by sera from a subset of patients with advanced cavitary disease. Studies suggest that 3 of the antigens recognized by sera from patients with early TB (85C, MPT32, and a 88-kDa protein) have strong serodiagnostic potential
— id: 7777, year: 1998, vol: 178, page: 1534, stat: Journal Article,

Induction of neutralizing antibodies to T-cell line-adapted and primary human immunodeficiency virus type 1 isolates with a prime-boost vaccine regimen in chimpanzees
Zolla-Pazner S; Lubeck M; Xu S; Burda S; Natuk RJ; Sinangil F; Steimer K; Gallo RC; Eichberg JW; Matthews T; Robert-Guroff M
1998 Feb;72(2):1052-1059, Journal of virology
Five chimpanzees were immunized by administration of one or more intranasal priming doses of one to three recombinant adenoviruses containing a gp160 insert from human immunodeficiency virus type 1 (HIV-1) MN (HIV-1MN) followed by one or more boosts of recombinant HIV-1SF2 gp120 delivered intramuscularly with MF59 adjuvant. This regimen resulted in humoral immune responses in three of five animals. Humoral responses included immunochemically active anti-H1V-1 antibodies (Abs) directed to recombinant gp120 and neutralizing Abs reactive with T-cell-line-adapted HIV-1MN and HIV-1SF2. In addition, neutralizing activity was detected to the two homologous primary isolates and to two of three heterologous primary isolates which, like the immunizing strains, can use CXCR4 as a coreceptor for infection. The three animals with detectable neutralizing Abs and a fourth exhibiting the best cytotoxic T-lymphocyte response were protected from a low-dose intravenous challenge with a cell-free HIV-1SF2 primary isolate administered 4 weeks after the last boost. Animals were rested for 46 weeks and then rechallenged, without a boost, with an eightfold-higher challenge dose of HIV-1SF2. The three animals with persistent neutralizing Abs were again protected. These data show that a strong, long-lived protective Ab response can be induced with a prime-boost regimen in chimpanzees. The data suggest that in chimpanzees, the presence of neutralizing Abs correlates with protection for animals challenged intravenously with a high dose of a homologous strain of HIV-1, and they demonstrate for the first time the induction of neutralizing Abs to homologous and heterologous primary isolates
— id: 57296, year: 1998, vol: 72, page: 1052, stat: Journal Article,

Neutralization of syncytium-inducing primary isolates by sera from human immunodeficiency virus (HIV)-uninfected recipients of candidate HIV vaccines
Zolla-Pazner S; Xu S; Burda S; Duliege AM; Excler JL; Clements-Mann ML
1998 Nov;178(5):1502-1506, Journal of infectious diseases
Most candidate human immunodeficiency virus (HIV)-1 vaccines induce antibodies that neutralize T cell line-adapted HIV-1 strains. Until recently, however, no neutralizing activity against primary HIV-1 isolates had been demonstrated in sera from human vaccinees. Since most candidate HIV-1 vaccines have been constructed from T cell line-adapted syncytium-inducing (SI) strains, experiments were done to test whether sera from recipients of SI-based vaccines could preferentially neutralize SI primary HIV-1 isolates. Various neutralization assays were performed with sera from volunteers receiving ALVACgp160MN and/or rgp120SF2. Neutralizing activity was detected against 4 of 8 SI primary isolates but against none of 5 non-SI primary isolates. The data suggest that, for the induction of neutralizing antibodies to a broad array of HIV-1 primary isolates, a polyvalent vaccine will be needed containing representatives of more than a single category of viruses
— id: 7874, year: 1998, vol: 178, page: 1502, stat: Journal Article,

Host cell-dependent alterations in envelope components of human immunodeficiency virus type 1 virions
Bastiani L; Laal S; Kim M; Zolla-Pazner S
1997 May;71(5):3444-3450, Journal of virology
In addition to gp41 and gp120, an array of cell adhesion molecules is present on the envelope of human immunodeficiency virus type 1 (HIV-1). To examine the role of the host cell in the acquisition of these molecules by virions, both laboratory-adapted and primary isolates were sequentially passaged into different host cells. Viruses obtained from the various host cells were examined for the presence of 10 different cell-derived molecules by a virus binding enzyme-linked immunosorbent assay. Virus progeny raised in peripheral blood mononuclear cells expressed most of the adhesion molecules tested, with the level of LFA-1 being the highest. When viruses were passaged into CEM-SS or SupT1 cells, the expression of most of the adhesion molecules on the virus envelope was lost. In contrast, when viruses were passaged into MT2 cells, the virus progeny bore high levels of LFA-3, ICAM-1, and major histocompatibility complex classes I and II. These studies demonstrate for the first time the host cell dependence of the adhesion molecule profile present on the envelope of primary isolates of HIV-1. The presence of several adhesion molecules that have not previously been identified as components of the envelope of either laboratory or primary isolates is also described. In addition, we show that the adhesion molecule profile of the virions is acquired, or lost, within one passage and is maintained with subsequent passages in the same cell type
— id: 9243, year: 1997, vol: 71, page: 3444, stat: Journal Article,

Host cell-dependent alterations in HIV envelope components
Bastiani L; Laal S; Zolla-Pazner S
1997 Jan 22-26;4:148-148, Conference on Retroviruses & Opportunistic Infections
We have recently demonstrated that HIV(IIIB), when grown in different host cells, carried different adhesion molecules in its envelope. In the current study, we have examined the acquisition of adhesion molecules by primary isolates of HIV-1 under similar circumstances. Five PBMC-grown primary isolates of HIV-1 were passaged through CEM-SS, MT2, or SupT1 cells and back passaged in PBMC. The adhesion molecule profile on the progeny virions from each host cell was determined with a virus binding ELISA. The parental and back passaged primary isolates expressed high levels of LFA-1, ICAM-1, ICAM-3, and MHC class II. The profile on the envelope of the MT2-grown virions was altered in that they now bear high levels of LFA-3, ICAM-1, and MHC class I and II. In contrast, few of the adhesion molecules were detected on virions from CEM-SS or SupT1 cells. These alterations in the envelope profile of adhesion molecules were detected after a single passage and were maintained with additional passages in the same cell line. Since adhesion molecules play a critical role in virus-cell interactions, these studies suggest that the tropism of HIV-1 for various cells may be critically dependent on the host cell in which the virion was produced
— id: 6002, year: 1997, vol: 4, page: 148, stat: Journal Article,

Host cell-dependent alterations in cell-derived proteins carried by HIV virions
Bastiani, L; Laal, S; ZollaPazner, S
1997 JAN ;99(1):204-204, Journal of allergy & clinical immunology
— id: 53279, year: 1997, vol: 99, page: 204, stat: Journal Article,

Anti-human immunodeficiency virus type 1 human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries
Boots LJ; McKenna PM; Arnold BA; Keller PM; Gorny MK; Zolla-Pazner S; Robinson JE; Conley AJ
1997 Dec 10;13(18):1549-1559, AIDS research & human retroviruses
A phage display library screening approach was used to identify peptide sequences that could bind to anti-HIV-1 MAbs whose binding specificities are complex. Most of the antibodies used recognize discontinuous epitopes in gp120 and one recognizes gp41. Both a 15-mer and a 21-mer display library (each with a complexity of greater than 60 x 10[6]) and two constrained, V3 region-biased libraries, all expressed as recombinant pIII protein of filamentous phage, were used. The unmapped anti-gp120 human MAb A32 recognized a set of related linear sequences and repeatedly identified a single phage sequence that could form a cyclic disulfide structure. Selection methods were also developed so that phage could be obtained by competition selection in the presence of antibody bound to native, monomeric gp120 antigen (used with MAb IgG1b12 and the anti-gp120 V3 region MAb 447-52D) or gp120 variable region 3 synthetic peptides (used with anti-gp120 V3 region MAb 19b). The potent, virus-neutralizing MAb IgG1b12 recognized numerous sequences and, when used in competition with gp120, recognized only one sequence. These studies extend the range of antibody determinant studies that can be performed with display phage libraries, demonstrate a workable experimental strategy for use of competition ligands to discriminate among phage mimotopes, and provide a large number of mimotopes that bind potent virus-neutralizing MAbs for HIV-1 vaccine studies
— id: 7510, year: 1997, vol: 13, page: 1549, stat: Journal Article,

A human monoclonal antibody directed to the V3 loop of clade E cross-reacts with other HIV-1 subtypes
Gorny MK; Mascola JR; Israel ZR; Williams C; Balfe P; Vancott TC; Hioe C; Brodine S; Burda S; Zollapazner S
1997 Jan 22-26;4:77-77, Conference on Retroviruses & Opportunistic Infections
To ascertain the antigenic relationship between HIV-1 viruses belonging to various genetically defined subgroups (clades), shared epitopes need to be defined. Human monoclonal antibodies (mAbs) are particularly useful for this purpose because they can detect complex regions of viral proteins which may be missed by sequence analysis. Here we describe the first human mAb derived from a clade E-infected individual; the mAb is specific for the V3 loop, recognizing a core epitope represented by the amino acids TRTSVR on the Nterminal side of the crown of the V3 loop. The IgG1 kappa mAb, designated 1324E, binds to the clade E consensus V3 loop, to rgp120 proteins from clade E and to peripheral blood mononuclear cells infected in vitro with the virus which infected the subject from whose cells the mAb-producing heterohybridoma was derived. Strong cross-reactivity was revealed by the ability of the mAb to bind to the V3 consensus peptide representing clades A and C and to rgp120 proteins from clade A and C, revealing a shared V3 epitope between these three clades. The mAb neutralized a clade E isolate which had been adapted for growth in H9 cells but failed to neutralize five clade E primary isolates
— id: 6003, year: 1997, vol: 4, page: 77, stat: Journal Article,

Human monoclonal antibodies to the V3 loop of HIV-1 with intra- and interclade cross-reactivity
Gorny MK; VanCott TC; Hioe C; Israel ZR; Michael NL; Conley AJ; Williams C; Kessler JA 2nd; Chigurupati P; Burda S; Zolla-Pazner S
1997 Nov 15;159(10):5114-5122, Journal of immunology
Five human anti-V3 mAbs were generated from Ab-producing cells derived from the blood of HIV-1-infected individuals from North America and selected using the V3 peptide of a divergent clade B isolate, HIV(RF). The anti-V3(RF) mAbs were mapped to a cluster of three overlapping epitopes present in the KSITKGP sequence located in the hypervariable region on the N-terminal side of the V3 loop. Broad immunochemical cross-reactivity was noted when the mAbs were tested for binding to V3 peptides derived from four clade A viruses, nine clade B viruses, and two clade C viruses. These results demonstrate antigenic relatedness in the V3 regions of these three HIV-1 clades. Affinities determined by surface plasmon resonance were higher for recombinant gp120 than for V3 peptides, suggesting that these mAbs recognize both linear and conformationally dependent epitopes of the V3 loop. Two of the mAbs neutralized four clade B T cell line-adapted and primary isolates with varying degrees of potency. The two neutralizing mAbs were the most cross-reactive with V3 peptides from several clades, had the highest affinity for V3(RF) and V3(MN), and stained HIV-infected cells. The data suggest that cross-reactivity, affinity, cell surface staining, and neutralizing activity are characteristics that describe an optimal fit between Ag and Ab. The results also demonstrate that the V3 peptides representing the sequence of several clade A, B, and C viruses share antigenic features that are recognized by the human immune response, a finding that suggests that cross-clade immunity to HIV-1 may be inducible by HIV-1 vaccines
— id: 7581, year: 1997, vol: 159, page: 5114, stat: Journal Article,

Immune correlates of protection from HIV infection and AIDS
Heeney JL; Bruck C; Goudsmit J; Montagnier L; Schultz A; Tyrrell D; Zolla-Pazner S
1997 Jan;18(1):4-8, Immunology today
— id: 9246, year: 1997, vol: 18, page: 4, stat: Journal Article,

Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor
Hill CM; Deng H; Unutmaz D; Kewalramani VN; Bastiani L; Gorny MK; Zolla-Pazner S; Littman DR
1997 Sep;71(9):6296-6304, Journal of virology
Several members of the chemokine receptor family have recently been identified as coreceptors, with CD4, for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. In this report, we show that the envelope glycoproteins of several strains of HIV-2 and simian immunodeficiency virus (SIV) employ the same chemokine receptors for infection. Envelope glycoproteins from HIV-2 use CCR5 or CXCR4, while those from several strains of SIV use CCR5. Our data indicate also that some viral envelopes can use more than one coreceptor for entry and suggest that some of these coreceptors remain to be identified. To further understand how different envelope molecules use CCR5 as an entry cofactor, we show that soluble purified envelope glycoproteins (SU component) from CCR5-tropic HIV-1, HIV-2, and SIV can compete for binding of iodinated chemokine to CCR5. The competition is dependent on binding of the SU glycoprotein to cell surface CD4 and implies a direct interaction between envelope glycoproteins and CCR5. This interaction is specific since it is not observed with SU glycoprotein from a CXCR4-tropic virus or with a chemokine receptor that is not competent for viral entry (CCR1). For HIV-1, the interaction can be inhibited by antibodies specific for the V3 loop of SU. Soluble CD4 was found to potentiate binding of the HIV-2 ST and SIVmac239 envelope glycoproteins to CCR5, suggesting that a CD4-induced conformational change in SU is required for subsequent binding to CCR5. These data suggest a common fundamental mechanism by which structurally diverse HIV-1, HIV-2, and SIV envelope glycoproteins interact with CD4 and CCR5 to mediate viral entry
— id: 57413, year: 1997, vol: 71, page: 6296, stat: Journal Article,

Resting cell neutralization assay for HIV-1 primary isolates
Hioe C; Burda S; Chigurupati P; Xu S; Zolla-Pazner S
1997 Aug;12(4):300-305, Methods
A technique is described for detecting the activity of neutralizing polyclonal or monoclonal antibodies against HIV-1 primary isolates. Most commonly, neutralizing antibody activity for HIV-1 is assessed by quantifying the ability of antibodies to inhibit virus infection in mitogen-activated peripheral blood mononuclear cells or transformed lymphocytes. Because the target of HIV infection in vivo is neither a mitogen-activated nor a transformed cell, an assay using unstimulated peripheral blood mononuclear cells as a more physiologic target cell was developed. This 'resting cell assay' mainly utilizes primary HIV-1 isolates that have been carried for only a few passages in vitro. The result is an assay that is more efficient to perform and that detects neutralizing activity with comparable or greater sensitivity than that previously described for assays of primary HIV-1 isolates
— id: 7163, year: 1997, vol: 12, page: 300, stat: Journal Article,

Neutralization of HIV-1 primary isolates by polyclonal and monoclonal human antibodies
Hioe CE; Xu S; Chigurupati P; Burda S; Williams C; Gorny MK; Zolla-Pazner S
1997 Sep;9(9):1281-1290, International immunology
To examine antibody-mediated neutralization of HIV-1 primary isolates in vitro, we tested sera and plasma from infected individuals against four clade B primary isolates. These isolates were analyzed further for neutralization by a panel of several human anti-HIV-1 mAb in order to identify the neutralizing epitopes of these viruses. Each of the HIV-1+ serum and plasma specimens tested had neutralizing activities against one or more of the four primary isolates. Of the three individual sera, one (FDA-2) neutralized all of the four isolates, while the other two sera were effective against only one virus. The pooled plasma and serum samples reacted broadly with these isolates. Based on the neutralizing activities of the mAb panel, each virus isolate exhibited a distinct pattern of reactivity, suggesting antigenic diversity among clade B viruses. Neutralizing epitopes were found in the V3 loop and CD4-binding domain of gp120, as well as near the transmembrane region (cluster II epitope) of gp41. A mAb directed to the cluster I epitope of gp41 near the immunodominant disulfide loop weakly neutralized one primary isolate. None of the mAb in the panel affected one primary isolate, US4, although this virus was sensitive to neutralization by some of the polyclonal antibody specimens. This isolate was also resistant to neutralization by a cocktail of 10 mAb, most of which individually inhibited at least one of the other three viruses tested. These results suggest that neutralizing activity for this latter virus is present in certain HIV-1+ sera/plasma, but is not exhibited by the mAb in the panel. Thus, effective neutralizing antibodies against primary isolates can be generated by humans upon exposure to HIV-1, but not all of these antigenic specificities are represented in a large panel of human anti-HIV-1 mAb
— id: 9240, year: 1997, vol: 9, page: 1281, stat: Journal Article,

Prevalence of a V2 epitope in clade B primary isolates and its recognition by sera from HIV-1-infected individuals
Israel ZR; Gorny MK; Palmer C; McKeating JA; Zolla-Pazner S
1997 Jan;11(1):128-130, AIDS
— id: 7074, year: 1997, vol: 11, page: 128, stat: Journal Article,

Surrogate marker of preclinical tuberculosis in human immunodeficiency virus infection: antibodies to an 88-kDa secreted antigen of Mycobacterium tuberculosis
Laal S; Samanich KM; Sonnenberg MG; Belisle JT; O'Leary J; Simberkoff MS; Zolla-Pazner S
1997 Jul;176(1):133-143, Journal of infectious diseases
Antibodies to purified, size-fractionated secreted proteins of Mycobacterium tuberculosis in sera from patients with human immunodeficiency virus (HIV) infection and active tuberculosis (HIV/TB patients), and in stored sera obtained from the same patients prior to clinical manifestation of TB, were evaluated by ELISA, and the repertoire of antigens recognized was analyzed by immunoblotting. Compared with non-HIV/TB patients, HIV/TB patients had lower levels of anti-mycobacterial antibodies, and these were directed toward a restricted set of antigens. Antibodies to an 88-kDa secreted antigen were present in the sera of 74% of HIV/TB patients during the years (1.5-6) prior to manifestation of active, clinical tuberculosis, although only 66% were positive by the time tuberculosis was diagnosed. The presence of antibodies to the 88-kDa antigen can serve as a surrogate marker for identifying HIV-infected persons with active, subclinical disease who are at a high risk of developing clinical tuberculosis
— id: 7953, year: 1997, vol: 176, page: 133, stat: Journal Article,

Human humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-molecular-mass antigens
Laal S; Samanich KM; Sonnenberg MG; Zolla-Pazner S; Phadtare JM; Belisle JT
1997 Jan;4(1):49-56, Clinical & diagnostic laboratory immunology
The selection of antigens of Mycobacterium tuberculosis for most studies of humoral responses in tuberculosis patients has been restricted to molecules that were either immunodominant in immunized animals or amenable to biochemical purification rather than those that were reactive with the human immune system. Delineation of antigens that elicit humoral responses during the natural course of disease progression in humans has been hindered by the presence of cross-reactive antibodies to conserved regions on ubiquitous prokaryotic antigens in sera from healthy individuals and tuberculosis patients. The levels of cross-reactive antibodies in the sera were reduced by preadsorption with Escherichia coli lysates, prior to studying their reactivity against a large panel of M. tuberculosis antigens to which the human immune system may be exposed during natural infection and disease. Thus, reactivity against pools of secreted, cellular, and cell wall-associated antigens of M. tuberculosis was assessed by an enzyme-linked immunosorbent assay (ELISA). Initial results suggested that the secreted protein preparation contained antigens most frequently recognized by the humoral responses of pulmonary tuberculosis patients. The culture filtrate proteins were subsequently size fractionated by preparative polyacrylamide gel electrophoresis, characterized by reaction with murine monoclonal antibodies to known antigens of M. tuberculosis by an ELISA, and assessed for reactivity with tuberculous and nontuberculous sera. Results show that a secreted antigen of 88 kDa elicits a strong antibody response in a high percentage of patients with pulmonary tuberculosis. This and other antigens identified on the basis of their reactivity with patient sera may prove useful for developing serodiagnosis for tuberculosis
— id: 9247, year: 1997, vol: 4, page: 49, stat: Journal Article,

Synergistic neutralization of a chimeric SIV/HIV type 1 virus with combinations of human anti-HIV type 1 envelope monoclonal antibodies or hyperimmune globulins
Li A; Baba TW; Sodroski J; Zolla-Pazner S; Gorny MK; Robinson J; Posner MR; Katinger H; Barbas CF 3rd; Burton DR; Chou TC; Ruprecht RM
1997 May 20;13(8):647-656, AIDS research & human retroviruses
A panel of 14 human IgG monoclonal antibodies (MAbs) specific for envelope antigens of the human immunodeficiency virus type 1 (HIV-1), 2 high-titer human anti-HIV-1 immunoglobulin (HIVIG) preparations, and 15 combinations of MAbs or MAb/HIVIG were tested for their ability to neutralize infection of cultured human T cells (MT-2) with a chimeric simian immunodeficiency virus (SHIV-vpu+), which expressed HIV-1 IIIB envelope antigens. Eleven MAbs and both HIVIGs were neutralizing. When used alone, the anti-CD4-binding site MAb b12, the anti-gp41 MAb 2F5, and the anti-gp120 MAb 2G12 were the most potent. When combination regimens involving two MAbs targeting different epitopes were tested, synergy was seen in all paired MAbs, except for one combination that revealed additive effects. The lowest effective antibody concentration for 50% viral neutralization (EC50) and EC90 were achieved with combinations of MAbs b12, 2F5, 2G12, and the anti-V3 MAb 694/98D. Depending on the combination regimen, the concentration of MAbs required to reach 90% virus neutralization was reduced approximately 2- to 25-fold as compared to the dose requirement of individual MAbs to produce the same effect. Synergy of the combination regimens implies that combinations of antibodies may have a role in passive immunoprophylaxis against HIV-1. The ability of SHIV to replicate in rhesus macaques will allow us to test such approaches in vivo
— id: 9242, year: 1997, vol: 13, page: 647, stat: Journal Article,

Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization
Lubeck MD; Natuk R; Myagkikh M; Kalyan N; Aldrich K; Sinangil F; Alipanah S; Murthy SC; Chanda PK; Nigida SM Jr; Markham PD; Zolla-Pazner S; Steimer K; Wade M; Reitz MS Jr; Arthur LO; Mizutani S; Davis A; Hung PP; Gallo RC; Eichberg J; Robert-Guroff M
1997 Jun;3(6):651-658, Nature medicine
A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans
— id: 9241, year: 1997, vol: 3, page: 651, stat: Journal Article,

Binding of antibodies to virion-associated gp120 molecules of primary-like human immunodeficiency virus type 1 (HIV-1) isolates: effect on HIV-1 infection of macrophages and peripheral blood mononuclear cells
Stamatatos L; Zolla-Pazner S; Gorny MK; Cheng-Mayer C
1997 Mar 17;229(2):360-369, Virology
Using immunobiochemical approaches we previously studied the conformation and surface exposure of different immunodominant regions within the oligomeric, virion-associated form of the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) (L Stamatatos and C. Cheng-Mayer (1995) J. Virol. 69, 6191-6198). These studies allowed us to determine to what extent epitopes within these immunodominant regions of the oligomeric gp120 are occluded or accessible to antibody binding on the virion surface of two primary-like (HIV-1SF162 and HIV-1SF128A) and one. T-cell-line-adapted (HIV-1SF2) isolates. Here, we investigate whether binding of anti-gp120 monoclonal antibodies (MAbs) to exposed epitopes of the immunodominant regions of oligomeric gp120 results in neutralization of HIV-1 infection and whether certain exposed sites are better targets for neutralization than others. We also test whether proposed mechanisms for antibody-mediated neutralization of T-cell-line-adapted HIV-1 isolates, e.g., antibody-mediated gp120-virion dissociation, are applicable to primary-like HIV-1 isolates. We assess the neutralization potential of anti-V2 loop, anti-V3 loop, and anti-CD4 binding site MAbs using human primary macrophages or peripheral blood mononuclear cells (PBMC) as target cells and HIV-1SF162 and HIV-1SF128A as infecting isolates. Our data indicate that: (i) not every exposed epitope of the immunodominant regions of gp120 oligomers on the virion surface is a target for neutralization; (ii) during virus-cell fusion specific exposure of previously occluded V3 loop epitopes within gp120 oligomers occurs, which may become targets for neutralization; (iii) antibody-mediated gp120-virion dissociation does not appear to be a major mechanism of neutralization for the primary-like HIV-1 isolates tested here; and (iv) infection of macrophages is more sensitive to neutralization by anti-gp120 monoclonal antibodies than PBMC. We also report that binding of one of the two anti-CD4 binding site MAbs tested mediated enhancement of macrophage cell infection in a concentration-dependent fashion, while binding of the other anti-CD4 binding site MAb resulted in efficient neutralization of infection of both macrophages and PBMC
— id: 9245, year: 1997, vol: 229, page: 360, stat: Journal Article,

Neutralization of a clade B primary isolate by sera from human immunodeficiency virus-uninfected recipients of candidate AIDS vaccines [see comments]
Zolla-Pazner S; Alving C; Belshe R; Berman P; Burda S; Chigurupati P; Clements ML; Duliege AM; Excler JL; Hioe C; Kahn J; McElrath MJ; Sharpe S; Sinangil F; Steimer K; Walker MC; Wassef N; Xu S
1997 Apr;175(4):764-774, Journal of infectious diseases
The inability of antibodies induced by experimental human immunodeficiency virus type 1 (HIV-1) vaccines to neutralize HIV-1 primary isolates may be due to a failure to elicit such antibodies, antigenic differences between the vaccine and the strains tested, insensitivity of the assays used, or to a combination of factors. New neutralization assays were used to determine the ability of candidate AIDS vaccines to generate neutralizing antibodies for clade B primary isolate BZ167, which is closely related in portions of its envelope to the immunizing strains. Sera from HIV-uninfected volunteers in vaccine trials were tested, and neutralizing activity was found in recipients of recombinant (r) gp120MN or of rgp160MN-containing canarypox boosted with rgp120SF-2. Detection of antibodies that neutralize primary isolate BZ167 correlated with neutralizing activity for homologous vaccine strains. These data demonstrate that certain candidate AIDS vaccines can elicit antibodies that neutralize a primary isolate of HIV-1
— id: 9244, year: 1997, vol: 175, page: 764, stat: Journal Article,

Neutralization assays using the BZ167 strain of human immunodeficiency virus type 1 - Reply
ZollaPazner, S
1997 NOV ;176(5):1411-1412, Journal of infectious diseases
— id: 98353, year: 1997, vol: 176, page: 1411, stat: Journal Article,

Functional studies of bispecific antibodies directed against HIV-1 and the Fc gamma I receptor type I
Gorny MK; Keler T; Burda S; Williams C; Gabriel JL; Mitchell WM; Deo YM; Zolla-Pazner S
1996 ;48:173-183, Antibiotics & chemotherapy
— id: 9251, year: 1996, vol: 48, page: 173, stat: Journal Article,

Abnormal serum porphyrin levels in patients with the acquired immunodeficiency syndrome with or without hepatitis C virus infection
Nomura N; Zolla-Pazner S; Simberkoff M; Kim M; Sassa S; Lim HW
1996 Aug;132(8):906-910, Archives of dermatology
OBJECTIVE: To define the contributions of human immunodeficiency virus (HIV) and hepatitis C virus infections to the development of porphyria cutanea tarda. DESIGN: Analysis of serum porphyrin levels in a cohort of 167 subjects. Serum samples were divided into 4 groups corresponding to the status of HIV and hepatitis C virus infections: positive-positive, positive-negative, negative-positive, and negative-negative. SETTING: Serum samples positive for HIV were obtained from the serum bank of an acquired immunodeficiency syndrome-HIV research center, and HIV-negative samples were obtained from a regional blood center. MAIN OUTCOME MEASURES: Spectrofluorometric measurement of serum porphyrin levels. RESULTS: The median values of porphyrin were 2.31 nmol/L (interquartile range [difference between the 25th and 75th percentiles]: 4.55) in the positive-positive group, 1.99 nmol/L (interquartile range: 1.63) in the positive-negative group, 1.31 nmol/L (interquartile range: 1.58) in the negative-positive group, and 1.14 nmol/L (interquartile range: 0.92) in the negative-negative group. The fluorescence emission spectra of samples with elevated porphyrin levels were identical with that reported for porphyria cutanea tarda. Elevated porphyrin levels were significantly associated with HIV infection (P < .001) and were observed in patients with an elevated level of alanine aminotransferase (P = .03). Infection with hepatitis C virus was also associated with an elevation in porphyrin levels, although the increase was not statistically significant (P = .16). Porphyrin levels in patients positive for HIV were not correlated with serum urea nitrogen or creatinine levels. None of the patients had symptomatic porphyria cutanea tarda. CONCLUSIONS: Factors associated with increased serum porphyrin levels included HIV infection, elevated alanine aminotransferase levels, and, to a lesser extent, hepatitis C virus infection. These findings suggest that patients with the above risk factors are potentially predisposed to the development of symptomatic porphyria cutanea tarda on further exposure to hepatotoxic agents
— id: 9248, year: 1996, vol: 132, page: 906, stat: Journal Article,

Characterization by serial deletion competition ELISAs of HIV-1 V3 loop epitopes recognized by monoclonal antibodies
Seligman SJ; Binley JM; Gorny MK; Burton DR; Zolla-Pazner S; Sokolowski KA
1996 Jun;33(9):737-745, Molecular immunology
Characterization of the sites recognized by antibody on the V3 loop of the envelope glycoprotein gp120 of HIV-1 was done by competition ELISAs on a series of four mouse mAbs, a human mAb and a human Fab. The solid-phase antigen consisted of biotin-YNKRK-RIHIGPGRAFYTTKN, a sequence from the center of the V3 loop of gp120MN, applied to streptavidin-coated wells. Competing antigens were two series of peptides with the HIV-1MN sequence each serially deleted at either the N or C terminus but kept constant at the other terminus. For each series, the amino acid at the deleting end needed to give a minimum KD was identified. The epitope was defined as the sequence including both of the identified amino acids as terminal amino acids. For the six antibodies reported, the epitope length ranged from seven to 14 amino acids. Use of a cyclic peptide as competing fluid-phase antigen suggested the influence of conformational constraints on presumed 'linear' epitopes. The operationally-defined epitope was longer than the contact residues in one of two instances in which the X-ray crystallographic structure had been determined. The longer estimates of epitope length in the current study based on competition ELISAs with serial deletions suggest that non-contact residues are significant both in epitope definition and in functional applications including immunogen design
— id: 9250, year: 1996, vol: 33, page: 737, stat: Journal Article,

Mechanisms contributing to the neutralization of HIV-1
Zolla-Pazner S
1996 Jun;51(1-2):89-93, Immunology letters
The phenomenon of virus neutralization is a function of three variables: the antibody (Ab), the virus and the target cell. Variation in any one of these parameters may drastically affect the results of assays for neutralization. In focusing on the virus as a variable in assays for the neutralization of HIV-1, it has been shown that the range of sensitivity of primary HIV-1 isolates is quite large. This may be due to the structure and biology of the virus particle, its density of envelope proteins, its ability to retain or shed these proteins, and its phenotype, determining the type of cells it will infect. The Ab used for neutralization also contributes to the efficiency of neutralization. Thus, the 'match' between the specificity of the Ab and the structure and availability of the epitope on the virus will affect the interaction and contribute to the resultant reduction in virus infectivity. Similarly, the strength of interaction between the virus and the neutralizing Ab, dependent on the affinity of the Ab for the virus epitope, will also be a determining factor. However, other factors contribute to the neutralization sensitivity of primary isolates of HIV-1. One factor that has been almost completely overlooked in the recent literature is the role that the target cell plays in revealing reduced virus infectivity. The facility and mechanism through which different cell types bind a virion and are infected by it will contribute profoundly to the efficiency with which Ab-mediated neutralization can be detected. These factors are discussed below with particular reference to interpreting (and re-interpreting) the current literature on HIV-1 neutralization
— id: 9249, year: 1996, vol: 51, page: 89, stat: Journal Article,

NEUTRALIZATION OF PRIMARY HIV-1 ISOLATES BY ANTI-ENVELOPE MONOCLONAL-ANTIBODIES
DSOUZA, MP; MILMAN, G; BRADAC, JA; MCPHEE, D; HANSON, CV; HENDRY, RM; CORCORAN, T; STOTT, J; FUNG, M; HANSON, C; LAMAN, J; MASCOLA, J; MCPHEE, D; RASHEED, S; RICHMAN, D; SCHUITEMAKER, H; THIRIART, C; WAINBERG, M; WEBER, J; BEDDOWS, S; TILLEY, S; ROBINSON, J; ZOLLAPAZNER, S; KATINGER, H; CUMMINS, L
1995 AUG ;9(8):867-874, AIDS
Objective: To evaluate human monoclonal antibodies (MAb) for neutralizing activity against primary HIV-1 isolates in peripheral blood mononuclear cells. Design: Neutralization activity data were obtained from 11 laboratories on a coded panel consisting of six human MAb to HIV envelope V3, CD4-binding region or gp41. Hyperimmune globulin against HIV-1 and normal human immunoglobulin G were supplied as controls. Each laboratory received pre-titered virus for use in the studies. Methods: Each laboratory measured neutralization of the MAb against laboratory strain HIVMN, genomic clone HIVJR-CSF, two subtype B and one subtype D primary isolates. Results: The titers of the centrally supplied virus stocks as determined by re-titration or back-titration varied among laboratories and were generally 10-100-fold less than provided. The neutralizing activity of each MAb varied by as much as a 1000-fold among laboratories. These differences may result from varying sensitivity in neutralization assay protocols and the differing susceptibility of primary cells to infection with HIV-1. Conclusions: To consolidate the data from multiple laboratories, the neutralization titers were compared by classifying antibodies as neutralizing if the antibody concentration for 50% virus inhibition was less than or equal to 10 mu g/ml. By this criterion, the CD4-binding region and gp41 MAb neutralized all four subtype B viruses and the subtype D isolate in a few of the laboratories. The V3 MAb neutralized only HIVMN and the closely related HIVJR-CSF viruses
— id: 87238, year: 1995, vol: 9, page: 867, stat: Journal Article,

Presentation of HIV V3 loop epitopes for enhanced antigenicity, immunogenicity and diagnostic potential
Fontenot JD; VanCott TC; Parekh BS; Pau CP; George JR; Birx DL; Zolla-Pazner S; Gorny MK; Gatewood JM
1995 Oct;9(10):1121-1129, AIDS
OBJECTIVE: To evaluate the immunological properties of a panel of human mucin MUC1/HIV V3 loop chimeras. DESIGN: The immunodominant epitope of MUC1 (APDTR) was found to be structurally isomorphous with the tip of the principle neutralizing determinant (PND) of HIV-1 (MN) (GPGRA). A panel of 120 residue, six tandem repeat (TR) and 60 residue, three TR chimeric antigens were constructed in which the repeating MUC1 epitope is replaced by HIV-1 PND. Each 20 residue TR contains one PND epitope. The PND of HIV-1 is presented in the native beta-turn conformation at the crest of each repeating knob structure of the mucin-like protein. METHODS: The antigenicity of the chimeric antigens were compared using enzyme-linked immunosorbent assay (ELISA) and HIV-infected patient sera. Structural effects of antibody-antigen interactions were determined using surface plasmon resonance, with human monoclonal antibodies, chimeric antigens and the cyclic and linear V3 loops. Immunogenicity of three versus six TR was measured in mice. RESULTS: Nine residues of the HIV PND substituted into the mucin backbone were equivalent to the 36 residue cyclic V3 loop in ELISA. The 120 residue antigens induced high titer, immunoglobulin (Ig) M and IgG, and HIV-specific antibodies in mice. CONCLUSIONS: MUC1/V3 chimeras efficiently detect HIV-specific antibodies in patient sera. Multivalent presentation of the PND is advantageous for higher affinity antibody-antigen interactions and for inducing HIV-specific IgM and IgG antibodies
— id: 9254, year: 1995, vol: 9, page: 1121, stat: Journal Article,

Functional activities of 20 human immunodeficiency virus type 1 (HIV-1)-specific human monoclonal antibodies
Forthal DN; Landucci G; Gorny MK; Zolla-Pazner S; Robinson WE Jr
1995 Sep;11(9):1095-1099, AIDS research & human retroviruses
Antibodies that are useful in the treatment of HIV infection should result in virus neutralization or lysis of infected cells but should not enhance infection. In this study, the potential clinical use of 20 HIV-1-specific human monoclonal antibodies (HuMAbs) was determined by measuring their enhancing (C-ADE) activities using HIVLAI as the target virus. Two HuMAbs mediated both C-ADE and ADCC, two exclusively neutralized, and five exclusively mediated ADCC. Ten HuMAbs demonstrated no activity in any of the three assays. Three antibodies that neutralized HIVLAI were tested against HIVSF2; all three also neutralized HIVSF2. Four of five HuMAbs mediating ADCC against HIVLAI that were also tested against HIVSF2 had ADCC activity against HIVSF2. These results demonstrate that many HuMAbs have unique functions, allowing the separation of potentially beneficial and harmful activities. Combinations of HuMAbs with ADCC and neutralizing functions may have therapeutic utility
— id: 9255, year: 1995, vol: 11, page: 1095, stat: Journal Article,

SERUM PORPHYRIN PROFILE IN PATIENTS WITH HIV-INFECTION
NOMURA, N; FOTIADES, J; LIM, HW; SASSA, S; ZOLLAPAZNER, S; SIMBERKOFF, M
1995 APR ;104(4):654-654, Journal of investigative dermatology
— id: 87384, year: 1995, vol: 104, page: 654, stat: Journal Article,

Induction of HIV type 1 neutralizing and env-CD4 blocking antibodies by immunization with genetically engineered HIV type 1-like particles containing unprocessed gp160 glycoproteins
Rovinski B; Rodrigues L; Cao SX; Yao FL; McGuinness U; Sia C; Cates G; Zolla-Pazner S; Karwowska S; Matthews TJ; et al
1995 Oct;11(10):1187-1195, AIDS research & human retroviruses
Genetically engineered, noninfectious HIV-1-like particles containing processed envelope glycoproteins represent potential candidate immunogens for a vaccine against HIV-1. However, since the gp120 glycoprotein is known to be rapidly lost from the surface of infected cells and purified virions as a result of its low-affinity interaction with gp41, shedding of this extracellular subunit could compromise the immunogenic potential of particle-based HIV-1 vaccine candidates. In this study, we demonstrate for the first time the feasibility of producing fully assembled HIV-1-like particles containing only unprocessed gp160 glycoproteins. Monkey kidney Vero cells were transfected with an inducible, human metallothionein-based expression vector containing most of the HIV-1LAI coding sequences that were genetically modified to introduce safety mutations and destroy the major cleavage site of the HIV-1 envelope glycoprotein. A stably-transfected cell line was isolated and shown to secrete HIV-1-like particles containing unprocessed gp160. Immunization with these particles induced HIV-1 cross-neutralizing, syncytium-inhibiting and env-CD4 blocking antibodies. Thus, these novel HIV-1-like particles represent alternative candidate immunogens for the development of a particle-based AIDS vaccine
— id: 9253, year: 1995, vol: 11, page: 1187, stat: Journal Article,

Role of virion-associated glycosylphosphatidylinositol-linked proteins CD55 and CD59 in complement resistance of cell line-derived and primary isolates of HIV-1
Saifuddin M; Parker CJ; Peeples ME; Gorny MK; Zolla-Pazner S; Ghassemi M; Rooney IA; Atkinson JP; Spear GT
1995 Aug 1;182(2):501-509, Journal of experimental medicine
This study investigates whether cell-derived glycosylphosphatidylinositol-linked complement control proteins CD55 and CD59 can be incorporated into HIV-1 virions and contribute to complement resistance. Virus was prepared by transfection of cell lines with pNL4-3, and primary isolates of HIV-1 were derived from patients' PBMCs. Virus was tested for sensitivity to complement-mediated virolysis in the presence of anti-gp160 antibody. Viral preparations from JY33 cells, which lack CD55 and CD59, were highly sensitive to complement. HIV-1 preparations from H9 and U937 cells, which express low levels of CD55 and CD59, had intermediate to high sensitivity while other cell line-derived viruses and primary isolates of HIV-1 were resistant to complement-mediated virolysis. Although the primary isolates were not lysed, they activated complement as measured by binding to a complement receptor positive cell line. While the primary isolates were resistant to lysis in the presence of HIV-specific antibody, antibody to CD59 induced lysis. Likewise, antibody to CD55 and CD59 induced lysis of cell line-derived virus. Western blot analysis of purified virus showed bands corresponding to CD55 and CD59. Phosphatidylinositol-specific phospholipase C treatment of either cell line-derived or primary isolates of HIV-1 increased sensitivity to complement while incubation of sensitive virus with purified CD55 and CD59 increased resistance to complement. These results show that CD55 and CD59 are incorporated into HIV-1 particles and function to protect virions from complement-mediated destruction, and they are the first report of host cell proteins functioning in protection of HIV-1 from immune effector mechanisms
— id: 9256, year: 1995, vol: 182, page: 501, stat: Journal Article,

Epitope exposure on functional, oligomeric HIV-1 gp41 molecules
Sattentau QJ; Zolla-Pazner S; Poignard P
1995 Jan 10;206(1):713-717, Virology
We have used cells infected with the HIV-1 molecular clone HX10 to study the binding of monoclonal antibodies (mAbs) to different epitopes within the extracellular domain of the HIV-1 transmembrane glycoprotein gp41. Gp41 mAb binding to the infected cells at 4 degrees was variable but weaker than the binding of an anti-gp120/V3 loop mAb and increased substantially for three of the gp41 antibodies at 37 degrees. Treatment of the cells with soluble CD4 (sCD4) at 37 degrees increased gp41 mAb binding to epitopes spanning residues 521-663, implying that these regions had probably been masked by gp120, which following interaction with sCD4 had subsequently dissociated from gp41. By contrast, the binding of a mAb to residues 662-667 which form a neutralization epitope was reduced by sCD4 binding. Another region which has been described as containing a neutralization epitope spans residues 725-750. MAbs to this region bound equally well to noninfected and HIV-infected cells, and binding was not increased in the presence of sCD4. These data strongly imply that this epitope is not exposed on the external surface of the membrane, a finding in accord with the proposed cytoplasmic localization of this region
— id: 9259, year: 1995, vol: 206, page: 713, stat: Journal Article,

CHARACTERIZATION BY COMPETITION ELISAS OF V3 LOOP-MAB 391/95-D BINDING
SELIGMAN, SJ; GORNY, MK; ZOLLAPAZNER, S
1995 APR 2 ;54(3):226-226, Journal of cellular biochemistry
— id: 87319, year: 1995, vol: 54, page: 226, stat: Journal Article,

Summary of the workshop on passive immunotherapy in the prevention and treatment of HIV infection. The Passive Antibody Workshop Participants
Stiehm ER; Mofenson L; Zolla-Pazner S; Jackson B; Martin NL; Ammann AJ
1995 Apr;75(1):84-93, Clinical immunology & immunopathology
— id: 9258, year: 1995, vol: 75, page: 84, stat: Journal Article,

Serotyping of primary human immunodeficiency virus type 1 isolates from diverse geographic locations by flow cytometry
Zolla-Pazner S; O'Leary J; Burda S; Gorny MK; Kim M; Mascola J; McCutchan F
1995 Jun;69(6):3807-3815, Journal of virology
The immunologic relatedness of the various human immunodeficiency virus type 1 (HIV-1) clades was determined with 13 human anti-HIV-1 monoclonal antibodies (MAbs) to six immunogenic regions of the HIV-1 structural proteins. The immunoreactivity of the native, oligomeric viral envelope glycoproteins expressed on the surfaces of human peripheral blood mononuclear cells infected in vitro with primary isolates from clades A through E was determined by flow cytometry. Some epitopes in the immunodominant region of gp41 and the C terminus of gp120 appear to be HIV-1 group specific in that they are expressed on the surfaces of cells in cultures infected with the majority of viruses tested from clades A to E. Epitopes within the V3 region appear to be clade restricted. Surprisingly, one MAb to an epitope in the C terminus of gp120 was entirely clade B specific. Staining with anti-V2 and anti-CD4 binding domain (CD4bd) reagents was infrequently detected. Anti-CD4bd MAbs stained only CD4-negative T cells because the CD4bd of gp120 appeared to be complexed with membrane CD4. When present, the epitopes of V2 and the CD4bd appeared to be expressed on cells infected with various clades. Thus, the results suggest that MAbs to gp41, the C terminus, and the V3 loop of gp120 are most useful in serotyping primary isolates of HIV-1, providing group-specific, clade-restricted, and clade-specific reagents. The use of the immunofluorescent method with the reagents described herein distinguishes infection with clade B from that with all other HIV-1 clades. With additional MAbs, this technique will allow a broadly applicable, reproducible, and practical method for serotyping HIV-1
— id: 9257, year: 1995, vol: 69, page: 3807, stat: Journal Article,

A resting cell assay for improved detection of antibody-mediated neutralization of HIV type 1 primary isolates
Zolla-Pazner S; Sharpe S
1995 Dec;11(12):1449-1458, AIDS research & human retroviruses
The sensitivity with which antibody-mediated neutralization is detected in vitro is dependent on the virus, the antibody, the target cells, and the culture conditions used in the assay. Using activated and transformed target cells, the ability of various culture-adapted and primary strains of HIV-1 to be neutralized by different polyclonal and monoclonal antibody preparations has been thoroughly studied. However, the vast majority of HIV-1-susceptible CD4+ cells in vivo are not activated or transformed, but are quiescent. Because resting lymphocytes can be infected with HIV-1, we initiated studies to determine (1) if the use of resting lymphocytes as target cells would result in a neutralization assay with increased sensitivity, (2) if the degree of target cell activation had a measurable effect on the sensitivity with which antibody-mediated neutralization could be detected, and (3) whether, using a more sensitive assay, neutralizing antibodies in patients' sera might be detectable that had been below the threshold of detection when using 'conventional' assays. The experiments described in the studies below reveal that an inverse relationship exists between the level of target cell activation and the sensitivity with which neutralization can be detected. Moreover, using an assay in which unstimulated peripheral blood mononuclear cells serve as target cells, experiments show that antibody-mediated neutralization of primary and prototype laboratory isolates of HIV-1 can be detected with 10- to 100-fold greater sensitivity than when stimulated peripheral blood mononuclear cells are used as target cells. With this resting cell assay, neutralizing activity can be detected in the sera of HIV-positive subjects that, by previously used 'conventional' neutralization assays, was undetectable
— id: 9252, year: 1995, vol: 11, page: 1449, stat: Journal Article,

Improved neutralization of HIV-1 primary isolates
Zolla-Pazner S; Sharpe S
1995 Jan 29-Feb 2;2:169-169, National Conference on Human Retroviruses & Related Infections
No single factor has slowed the development of an HIV vaccine as much as the discovery that sera from vaccines can neutralize lab strains of HIV-1 but not primary isolates. The role of the many differences between culture-adapted lab strains and primary isolates in this phenomenon has been extensively studied as has the immunogenicity of the various vaccine candidates. However, the influence of the target cell on the neutralization assay has not been extensively studied as a factor contributing to the poor detection of neutralizing antibodies (Abs) for primary isolates. This led us to investigate the role of activated and transformed lymphocytes as target cells in neutralization assays and to question whether it would be more relevant, and more sensitive, to test for neutralization of Ab-treated primary isolates on quiescent lymphocytes. Hence, neutralization experiments were performed holding all variables constant save the activation state of the peripheral blood mononuclear cells (PBMCs). For these experiments, one of three primary isolates was exposed to increasing amounts of polyclonal or monoclonal Abs. The virus/Ab mixture was added to either resting or PHA-stimulated PBMCs. At 24 hr, PHA and IL-2 were added to all cultures and p24 was measured on day 6 of culture. When a pool of HIV+ sera was tested on resting cells, the 50% neutralizing dose (ND50) was 1:400; the same serum pool tested for neutralization on PHA-blasts showed no significant neutralizing activity. Similarly, when the serum of a long-term survivor was tested on resting and stimulated PBMCs, the ND50 were 1:200 and undetectable, respectively. When the human anti-V3 mAb 447-52D was tested on resting and stimulated cells, the ND50 were 0.5 and 10 ug mAb/ml. Of 26 sera from HIV+ subjects, 16 (62%) were found capable of neutralizing greater than or equal to 50% of a clade B primary isolate with a titre of greater than or equal to 1:50; thus, using the resting cell assay, detection of neutralizing Ab for primary isolates in patients' sera is considerably increased over that reported by most labs. Conclusions: Using quiescent lymphocytes as targets for infection in neutralization assays increases the sensitivity of the assay significantly; frequently, sera without apparent neutralizing activity (when tested by conventional neutralization assays) can be shown, with the resting cell assay, to possess significant neutralizing activity
— id: 5999, year: 1995, vol: 2, page: 169, stat: Journal Article,

THE ROLE OF PROTECTIVE ANTIBODIES LN THE PREVENTION AND CONTROL OF HIV-INFECTION
ZOLLAPAZNER, S; SHARPE, S; XU, S; BURDA, S; ANDRUS, L; GIORGI, J
1995 JUL-AUG ;11(4):S134-S134, AIDS research & human retroviruses
— id: 87232, year: 1995, vol: 11, page: S134, stat: Journal Article,

Neutralization of primary human immunodeficiency virus type 1 isolates by the broadly reactive anti-V3 monoclonal antibody, 447-52D
Conley AJ; Gorny MK; Kessler JA 2nd; Boots LJ; Ossorio-Castro M; Koenig S; Lineberger DW; Emini EA; Williams C; Zolla-Pazner S
1994 Nov;68(11):6994-7000, Journal of virology
Human monoclonal antibody 447-52D binds to the V3 determinant of the human immunodeficiency virus type 1 (HIV-1) gp120 external glycoprotein. Its binding requires the expression of the GPxR sequence at the center of the V3 domain. HIV-1 variants that are adapted to replication in T-lymphoid cell lines and express this sequence motif are efficiently neutralized by the antibody (M. K. Gorny, A. J. Conley, S. Karwowska, A. Buchbinder, J.-Y. Xu, E. A. Emini, S. Koenig, and S. Zolla-Pazner, J. Virol. 66:7538-7542, 1992). In the present study, the antiviral activity of 447-52D was further defined with regard to its ability to mediate neutralization of primary HIV-1 clinical isolates. Again, the antibody was found to potently neutralize those isolates that expressed the binding sequence. We confirmed that this determinant is commonly expressed by virus isolates belonging to the subtype (clade) B sequence classification. As such, 447-52D may be useful for prophylactic and immunotherapeutic intervention. In addition, the study demonstrated that neutralization of primary HIV-1 isolates is possible if mediated by an appropriate antibody
— id: 9261, year: 1994, vol: 68, page: 6994, stat: Journal Article,

Delayed-type hypersensitivity skin tests are an independent predictor of human immunodeficiency virus disease progression. Department of Veterans Affairs Cooperative Study Group
Gordin FM; Hartigan PM; Klimas NG; Zolla-Pazner SB; Simberkoff MS; Hamilton JD
1994 Apr;169(4):893-897, Journal of infectious diseases
Delayed-type hypersensitivity (DTH) testing was evaluated as a predictor of human immunodeficiency virus (HIV) disease progression in 336 symptomatic patients with baseline CD4 cell counts of 200-500/mm3 who were participating in a randomized trial of early versus late therapy with zidovudine. Patients with a response of > 2 mm to any of seven antigens were categorized as reactive; those without were anergic. Anergic patients were significantly more likely than reactive patients to have HIV disease progression as evidenced by decrease in CD4 cell count (52% vs. 27%), development of AIDS (33% vs. 17%), or death (18% vs. 9%) (P < or = .02), irrespective of time of zidovudine initiation. By multivariate analysis, DTH results were an independent predictor of HIV progression separate from CD4 cell count, p24 antigen positivity, or level of beta 2-microglobulin. DTH skin tests are an independent predictor of HIV disease progression and may be of value in the evaluation of a patient's immune status
— id: 9264, year: 1994, vol: 169, page: 893, stat: Journal Article,

Human anti-V2 monoclonal antibody that neutralizes primary but not laboratory isolates of human immunodeficiency virus type 1
Gorny MK; Moore JP; Conley AJ; Karwowska S; Sodroski J; Williams C; Burda S; Boots LJ; Zolla-Pazner S
1994 Dec;68(12):8312-8320, Journal of virology
A human immunoglobulin G1 lambda monoclonal antibody (MAb), 697-D, was developed that recognizes the V2 region of human immunodeficiency virus type 1 (HIV-1) gp120. Substitutions at amino acid positions 176/177, 179/180, 183/184, and 192 to 194 in the V2 loop of gp120 each completely abolished the binding capacity of 697-D in an enzyme-linked immunosorbent assay format. Competition analysis with three different neutralizing murine anti-V2 MAbs confirmed the specificity of 697-D. The 697-D epitope is primarily conformation dependent, although there was weak reactivity of the MAb with a V2 peptide spanning residues 161 to 180. Treatment of recombinant gp120 HIVIIIB with sodium metaperiodate, which oxidizes carbohydrates, abolished the binding of the MAb, showing the dependence of the epitope on intact carbohydrates. The broad reactivity of 697-D was displayed by its binding to the gp120 molecules from four of four laboratory isolates and five of five primary isolates. The MAb 697-D neutralized three out of four primary isolates but failed to neutralize any of four laboratory strains of HIV-1. 697-D and a human anti-V3 MAb, 447-52-D, displayed similar potency in neutralizing primary isolates, indicating that the V2 region of gp120, like the V3 region and the CD4-binding domain, can induce potent neutralizing antibodies against HIV-1 in humans
— id: 57488, year: 1994, vol: 68, page: 8312, stat: Journal Article,

Synergistic neutralization of human immunodeficiency virus type 1 by combinations of human monoclonal antibodies
Laal S; Burda S; Gorny MK; Karwowska S; Buchbinder A; Zolla-Pazner S
1994 Jun;68(6):4001-4008, Journal of virology
The ability of antibodies to the V3 region and the CD4-binding domain (CD4bd) of human immunodeficiency virus type 1 (HIV-1) to act in synergy to neutralize HIV has been demonstrated previously. However, synergy between antibodies to other HIV-1 epitopes has not been studied. We have used 21 combinations of human monoclonal antibodies (MAbs) directed against different epitopes of the gp120 and gp41 proteins of HIV-1 to evaluate their ability to act in synergy to neutralize HIV-1. Combinations of anti-V3 and anti-CD4bd antibodies, anti-V3 and anti-gp120 C-terminus antibodies, anti-CD4bd and anti-C-terminus antibodies, anti-V3 and anti-gp41 antibodies, and anti-CD4bd and anti-gp41 antibodies were tested. Our results show that some, but not all anti-V3 antibodies can act in synergy with anti-CD4bd antibodies. In addition, for the first time, antibodies to the C-terminus region have been found to act in synergy with the anti-CD4bd antibodies. Various anti-CD4bd MAbs also act in synergy when used together. The use of such cocktails of human MAbs for passive immunization against HIV-1 may prove to be important for therapy in postexposure settings and for prevention of maternal-fetal transmission of the virus. The results also provide information on the types of antibodies that should be elicited by an effective vaccine
— id: 9263, year: 1994, vol: 68, page: 4001, stat: Journal Article,

Studies with monoclonal antibodies to the V3 region of HIV-1 gp120 reveal limitations to the utility of solid-phase peptide binding assays
Moore JP; Cao Y; Conley AJ; Wyatt R; Robinson J; Gorny MK; Zolla-Pazner S; Ho DD; Koup RA
1994 Apr;7(4):332-339, Journal of acquired immune deficiency syndrome
Using human monoclonal antibodies (HuMAbs) r(1)-447 (L-736,523) and 19b to the V3 region of HIV-1 gp120, we have explored epitope presentation on V3-peptides and on the corresponding gp120 proteins. HuMAb r(1)-447 binds strongly to the MN and SF-2 peptides and gp120 proteins. In contrast, while this HuMAb binds equally avidly to both the HxB2 and the BRU/BH10 peptides, it binds but weakly to the HxB2 V3 loop on gp120 and fails to bind at all to BH10 gp120. Thus, the solid-phase peptide binding assay can falsely predict reactivity of an MAb with a gp120 protein. Conversely, HuMAb 19b fails to bind to a peptide from the V3 loop of HIV-1 AD-6 in solid-phase assays, but binds to the same peptide in solution and also to AD-6 gp120. Thus, the solid-phase peptide binding assay can fail to predict reactivity of an MAb with a gp120 protein. Furthermore, serum antibodies from individual AD-6 do not react well with the AD-6 V3-peptide in a solid-phase assay, but react strongly with the corresponding MN V3-peptide. On the basis of peptide binding assays, we had assumed that the AD-6 virus was 'MN-like' with a prototypic North American/European subtype B GPGR motif at the crown of the V3 loop. However, direct sequencing demonstrates that the AD-6 V3 loop contains a variant GPGK motif. This highlights a limitation of V3-peptide-based assays for serotyping viruses
— id: 56567, year: 1994, vol: 7, page: 332, stat: Journal Article,

Antibodies to the HIV-1 V3 loop in serum from infected persons contribute a major proportion of immune effector functions including complement activation, antibody binding, and neutralization
Spear GT; Takefman DM; Sharpe S; Ghassemi M; Zolla-Pazner S
1994 Nov 1;204(2):609-615, Virology
Previous studies have shown that the V3 region of the HIV envelope is both critical to viral functions and immunogenic. However, the relative contribution of anti-V3 antibodies in the sera of infected individuals in mediating immune effector functions directed at whole intact virus and infected cells has not been determined. This study used peptides corresponding to several regions of the HIV envelope as inhibitors of antibody binding and antibody effector functions directed at virions and virus-infected cells in order to assess the relative importance of V3-specific antibodies in sera from infected persons. Approximately 40% of the antibody in serum which could bind to native viral proteins on HIVMN-infected cells was blocked by a peptide corresponding to the central 15 amino acids of the V3 loop. In contrast, little if any blocking of serum antibody binding was observed with peptides corresponding to flanking regions of HIVMN V3 or three regions of gp41. Since antiviral antibody can also activate immune effector functions, we determined whether peptides could block antibody-dependent activation of the complement system by HIV-infected cells or free virus. Surprisingly, the V3 loop peptide blocked 75-95% of complement activation on HIV-infected cells. While the V3 loop peptide also blocked a substantial portion of the neutralizing activity in serum from infected persons for free virus it was again more effective in inhibiting complement-mediated effects on free virus. Accordingly, antibody-dependent, complement-mediated virolysis was inhibited by 61-79%. The results of these experiments indicate that (1) a substantial portion (30-40%) of the antibody in sera from infected persons that is capable of binding to HIV-infected cells and HIV virions is V3-specific, and (2) these V3-specific antibodies are particularly important for complement activation on infected cells and virions. This indicates that the central portion of the V3 loop, while constituting less than 3% of the amino acid sequence of the HIV envelope, apparently provides a major gp160 site for immune effector functions, especially complement activation
— id: 9260, year: 1994, vol: 204, page: 609, stat: Journal Article,

Enhanced in vitro human immunodeficiency virus type 1 replication in B cells expressing surface antibody to the TM Env protein
Tani Y; Donoghue E; Sharpe S; Boone E; Lane HC; Zolla-Pazner S; Cohen DI
1994 Mar;68(3):1942-1950, Journal of virology
The human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 tightly binds CD4 as its principal cellular receptor, explaining the tropism of HIV-1 for CD4+ cells. Nevertheless, reports documenting HIV infection or HIV binding in cells lacking CD4 surface expression have raised the possibility that cellular receptors in addition to CD4 may interact with HIV envelope. Moreover, the lymphocyte adhesion molecule LFA-1 appears to play an important role in augmenting HIV-1 viral spread and cytopathicity in vitro, although the mechanism of this function is still not completely defined. In the course of characterizing a human anti-HIV gp41 monoclonal antibody, we transfected a CD4-negative, LFA-1-negative B-cell line to express an anti-gp41 immunoglobulin receptor (surface immunoglobulin [sIg]/gp41). Despite acquiring the ability to bind HIV envelope, such transfected B cells could not be infected by HIV-1. These cells were not intrinsically defective for supporting HIV-1 infection, because when directed to produce surface CD4 by using retroviral constructs, they acquired the ability to replicate HIV-1. Interestingly, transfected cells expressing both surface CD4 and sIg/gp41 receptors replicated HIV much better than cells expressing only CD4. The enhancement resided specifically in sIg/gp41, because isotype-specific, anti-IgG1 antibodies directed against sIg/gp41 blocked the enhancement. These data directly establish the ability of a cell surface anti-gp41 receptor to enhance HIV-1 replication
— id: 9265, year: 1994, vol: 68, page: 1942, stat: Journal Article,

Dissociation rate of antibody-gp120 binding interactions is predictive of V3-mediated neutralization of HIV-1
VanCott TC; Bethke FR; Polonis VR; Gorny MK; Zolla-Pazner S; Redfield RR; Birx DL
1994 Jul 1;153(1):449-459, Journal of immunology
mAbs specific for the V3 loop of HIV-1 are capable of neutralizing laboratory strains of HIV-1 in vitro. In this report we use surface plasmon resonance and biosensor technology to demonstrate that the ability of V3-specific mAbs to neutralize HIV-1(MN) correlated with the dissociation rate constant of the homologous mAb-gp120 binding interaction. mAbs capable of binding diverse strains of gp120 with similar association rate constants exhibited marked differences in the dissociation rate. The dissociation rate, and not the association rate, was found to be predictive of the neutralization capacity of the mAb. Furthermore, synthetic peptides corresponding to the V3 loop of HIV-1(IIIB, MN) yielded quantitatively similar binding kinetic profiles abrogating the need for purified recombinant gp120 protein and potentially facilitating the screening of more diverse isolates. Biosensor immobilized V3 peptides were found to mimic their conformational structure in solution. This offers advantages to peptides studied by ELISA where some degree of denaturation and masking of epitopes can occur upon adsorption of peptides to plastic surfaces. The impact of amino acid substitutions within epitopes on subsequent mAb binding was dissected by observing alterations in dissociation rates. The technique provides rapid kinetic analyses of V3 Ab binding interactions with diverse V3 sequences, facilitating the efficient screening and selection of those most likely to possess the broadest and most potent HIV-1 neutralizing potentials
— id: 9262, year: 1994, vol: 153, page: 449, stat: Journal Article,

Human monoclonal antibodies to HIV-1 define synergistic activities leading to enhanced neutralization
Zolla-Pazner S; Laal S; Burda S; Buchbinder A
1994 ;46:38-47, Antibiotics & chemotherapy
— id: 9266, year: 1994, vol: 46, page: 38, stat: Journal Article,

THE PROFESSOR, THE UNIVERSITY AND INDUSTRY
ZOLLAPAZNER, S
1994 MAR ;270(3):120-120, Scientific american
— id: 52565, year: 1994, vol: 270, page: 120, stat: Journal Article,

Generation of neutralizing anti-B19 parvovirus human monoclonal antibodies from patients infected with human immunodeficiency virus
Arakelov S; Gorny MK; Williams C; Riggin CH; Brady F; Collett MS; Zolla-Pazner S
1993 Sep;168(3):580-585, Journal of infectious diseases
The prevalence of IgG antibodies to human B19 parvovirus (anti-B19) is elevated in individuals infected with human immunodeficiency virus (HIV), especially during the later stages of HIV infection. In subjects with high titers of IgG anti-B19, 86% (19 of 22) had circulating B cells producing anti-B19. Immortalization of these cells with Epstein-Barr virus and generation of heterohybridomas by fusion with a mouse X human heteromyeloma resulted in the production of two cell lines producing IgG1 kappa monoclonal antibodies (MAbs). Both of these MAbs were specific for conformational epitopes on the VP2 capsid protein of B19 parvovirus and both were capable of neutralizing 50% of the viral infectivity in a human erythroid colony-forming unit assay at < or = 1 micrograms of MAb/mL. These human MAbs are potentially useful in the treatment of acute B19 parvovirus infection
— id: 9268, year: 1993, vol: 168, page: 580, stat: Journal Article,

EFFECT OF HIV-NEUTRALIZING ANTIBODIES ON INFECTION OF TROPHOBLASTS
BOURINBAIAR, AS; BORKOWSKY, W; KRASINSKI, K; ZOLLAPAZNER, S
1993 MAR 29 ;43(4):88-88, Journal of cellular biochemistry
— id: 54216, year: 1993, vol: 43, page: 88, stat: Journal Article,

Human monoclonal antibodies to the V3 loop of HIV-1 gp120 mediate variable and distinct effects on binding and viral neutralization by a human monoclonal antibody to the CD4 binding site
Cavacini LA; Emes CL; Power J; Buchbinder A; Zolla-Pazner S; Posner MR
1993 Apr;6(4):353-358, Journal of acquired immune deficiency syndrome
Interactive effects between human monoclonal antibodies specific for the V3 loop (257-D and 447-D) and an epitope within the CD4 binding site (F105) of HIV-1 gp120 were evaluated for neutralization of viral cytopathogenicity and binding to HIV-infected cells. Regardless of antibody pair, only additive effects were observed in neutralization of MN and SF2 virus though each antibody alone had potent neutralizing activity on these strains. Significant cooperativity was observed between F105 and 447-D in neutralization of RF. Relatively high concentrations (> 100 micrograms/ml) of each individual antibody are required for partial neutralization (25--40%) of RF. Coincubation with 10 micrograms/ml of each antibody increased neutralization activity 3--4-fold more than predicted for additive effects alone. No enhancement was seen upon coincubation of F105 with 257-D which does not neutralize RF. Antibody interactions with native antigen on HIV-infected cells was measured by flow cytometry. Results were consistent with neutralization results in the majority of flow cytometry experiments; however, enhanced binding did not necessarily predict enhanced neutralization. These data support the notion that either a conformational change occurs with binding of V3 loop antibodies which enhances the binding and neutralizing activity of antibodies directed to the CD4 binding site of gp120 or vice versa, or new antigenic sites are exposed by the V3 loop antibodies on cell surfaces and virions. Of importance, cooperativity is observed even at very low antibody concentrations
— id: 9272, year: 1993, vol: 6, page: 353, stat: Journal Article,

Molecular mimicry accompanying HIV-1 infection: human monoclonal antibodies that bind to gp41 and to astrocytes
Eddleston M; de La Torre JC; Xu JY; Dorfman N; Notkins A; Zolla-Pazner S; Oldstone MB
1993 Oct;9(10):939-944, AIDS research & human retroviruses
Monoclonal antibodies that bound to HIV gp41 and cross-reacted with astrocytes were recovered from the blood of three patients infected with HIV-1. Mapping of the specificity of these monoclonal antibodies, using synthetic gp41 peptides, located their epitope to amino acids 644-663 and established their conformation dependence. Six other human monoclonal anti-HIV antibodies were found to bind to HIV gp41 or gp120 but not to reactive astrocytes in brain tissue. Sharing of linear or conformational protein determinants between disparate viral and host proteins is termed molecular mimicry. The consequences of such mimicry by anti-viral antibodies interacting with astrocytes may play a role in the dementia of AIDS patients since a major function of astrocytes is to maintain the appropriate milieu for neuronal function. The finding of such cross-reactive antibodies adds to the evidence for a possible autoimmune pathogenesis in some of the disease manifestations accompanying HIV infection
— id: 9267, year: 1993, vol: 9, page: 939, stat: Journal Article,

Repertoire of neutralizing human monoclonal antibodies specific for the V3 domain of HIV-1 gp120
Gorny MK; Xu JY; Karwowska S; Buchbinder A; Zolla-Pazner S
1993 Jan 15;150(2):635-643, Journal of immunology
HIV infection induces antibodies that mediate neutralization of cell-free virus and may protect against viral infection. Neutralizing human mAb that bind to the third hypervariable domain (V3) of gp120 have been generated from PBL of HIV-seropositive subjects. Twelve such mAb recognize 9 different epitopes spanning an 11 amino acid stretch at the tip of the V3MN loop. The epitopes of an additional two mAb have not been precisely determined but occur within a 15-mer peptide around the tip of the V3 loop. Eleven of 13 mAb are reactive with at least one other V3 peptide besides MN, indicating that cross-reactivity is a common phenomenon amongst anti-V3 antibodies. All the mAb achieved 50% neutralization of HIVMN at antibody concentrations of 12 to 4700 ng/ml. Two mAb, which recognized epitopes at the top of the V3 loop, GPGR and GRAF, neutralized strains as divergent as MN and IIIB. The affinities of all mAb tested were shown to be substantially higher for the rgp120 than for a 23-mer peptide from the V3 loop of the homologous strain. A significant inverse correlation was demonstrated between affinities and the 50% neutralizing doses for HIVMN
— id: 8427, year: 1993, vol: 150, page: 635, stat: Journal Article,

ROLE OF CARBOHYDRATE (CHO) RESIDUES OF HIV-1 GP120 IN ITS BINDING TO ANTIBODY
KARWOWSKA, S; GORNY, MK; ZOLLAPAZNER, S
1993 APR 15 ;150(8):A316-A316, Journal of immunology
— id: 54233, year: 1993, vol: 150, page: A316, stat: Journal Article,

Identification of HIV vaccine candidate peptides by screening random phage epitope libraries
Keller PM; Arnold BA; Shaw AR; Tolman RL; Van Middlesworth F; Bondy S; Rusiecki VK; Koenig S; Zolla-Pazner S; Conard P; et al
1993 Apr;193(2):709-716, Virology
Most synthetic HIV-1 gp120 V3 loop peptides that are used as immunogens in experimental HIV-1 vaccine studies are modeled from the naturally occurring viral gp120 V3 loops. In experimental animals these immunogens generally elicit type (or variant)-specific neutralizing antibodies that are not broadly reactive among HIV-1 variants. In an attempt to find a more general structure for the V3 loop, we have obtained candidates that mimic V3 loop sequences by screening random epitopes displayed in a fusion phage 15-residue epitope library. Human monoclonal antibody 447-52D, a highly potent and broadly reactive virus-neutralizing antibody that recognizes the conserved V3 loop tip motif GPXR, was the probe. By using a screening method that was designed specifically for this work, we identified hundreds of reactive phage clones, 70 of which were sequenced. Over 98% of the epitopes contain the motif GPXR, yet none of the 70 are an identical match to any V3 variant loop described to date. One of these sequences was synthesized as the beta-maleimidopropionyl 15-mer peptide, covalently conjugated to a carrier and used to immunize rabbits. High anti-peptide titers were obtained in all animals with three of four individual responses also binding to a peptide that is representative of the 'North American consensus' V3 loop. The sera from these three positive rabbits neutralized HIV-1 variant SF-2 in vitro. In addition, one of them was capable of neutralizing variant AL-1. Both of these variants are considered to have V3 loops of the North American consensus type. Thus, neutralizing responses were obtained by use of an immunogen that was selected for its ability to bind a broadly reactive human monoclonal antibody rather than modeled from an HIV-1 gp120 V3 loop sequence
— id: 9273, year: 1993, vol: 193, page: 709, stat: Journal Article,

Synergistic interaction of various pairs of human monoclonal antibodies in the neutralization of HIV-1
Laal S; Burda S; Buchbinder A; Zolla-Pazner S
1993 Dec 12-16;1:67-67, National Conference on Human Retroviruses & Related Infections
Studies have shown that combinations of anti-V3 region and anti-CD4 binding domain (CD4bd) monoclonal antibodies are synergistic for neutralization of HIV-1. We have used a panel of human monoclonal antibodies (humAb) directed against various immunogenic regions on the HIV envelope, to perform a systematic analysis of the epitopes that must be targeted simultaneously to achieve enhanced neutralization. The panel included 2 humAbs to the gp120-V3 region, 3 to the gp120-CD4bd, 2 to the gp120-C-terminal region, 1 each to gp41 cluster I (aa571-641) and II (aa644-663), and 1 (2F5 from H. Kattinger) to a gp41 region adjacent to cluster II (aa662-667). The assay used was the modified syncytium formation microassay (Nara et al., Laal et al.) that measures cell free virus neutralization. The results show that a) not all anti-V3 and anti-CD4bd humAb pairs show synergy; b) anti-CD4bd humAbs can act in synergy both with anti-V3 and anti-C-terminus humAbs; c) anti-V3 humAb 447-D is able to act in synergy with 5 different anti-CD4bd humAbs; d)none of the anti-gp41 humAbs is able to act in synergy with either anti-V3 or anti-CD4bd humAbs. These results have important implications for rational design of regimens for active and passive immunization against HIV
— id: 6012, year: 1993, vol: 1, page: 67, stat: Journal Article,

A rapid, automated microtiter assay for measuring neutralization of HIV-1
Laal S; Burda S; Sharpe S; Zolla-Pazner S
1993 Aug;9(8):781-785, AIDS research & human retroviruses
A sensitive, rapid, and quantitative ELISA for p24 is described, which can be used as the read-out to test for HIV-1 neutralization in the syncytium-forming microassay and can replace the counting of syncytia under the microscope. This assay can be used reliably for divergent strains of HIV-1, including those that do not induce syncytia. The new read-out permits evaluation of neutralization at 3 days rather than 5 days. Using this assay, the neutralizing activity of several new and previously described human monoclonal antibodies against HIV-1 was characterized
— id: 9271, year: 1993, vol: 9, page: 781, stat: Journal Article,

Epitopes of HIV-1 glycoproteins recognized by the human immune system
Laal S; Zolla-Pazner S
1993 ;56:91-111, Chemical immunology
— id: 9274, year: 1993, vol: 56, page: 91, stat: Journal Article,

Non-covalent complexes of HIV gp120 with CD4 and/or mAbs enhance activation of gp120-specific T clones and provide intermolecular help for anti-CD4 antibody production
Manca F; Seravalli E; Valle MT; Fenoglio D; Kunkl A; Li Pira G; Zolla-Pazner S; Celada F
1993 Sep;5(9):1109-1117, International immunology
The 'dangerous liaison' between CD4 and gp120 that offers the first entry opportunity to HIV may also provoke perturbations of the immune control of the host with far-reaching immunopathological consequences. We wondered whether a mechanism of intermolecular help (T help across the gap of a non-covalent bond, in contrast to the intramolecular help of carrier to hapten) could break self-tolerance and be the cause of the frequent anti-CD4 autoantibodies found in AIDS patients. To determine whether this hypothesis deserves further testing, we designed a series of in vitro and in vivo experiments of increasing complexity, focused on the presentation of gp120 to specific T cells by antigen presenting cells (APC) exposed to the envelope protein in the form of non-covalent complexes. Bi-molecular complexes were constructed by allowing gp120 or gp160 to bind specific human mAbs. Tri-molecular complexes were constructed by introducing CD4 as an intermediate ligand between gp120 and mouse mAbs specific for CD4. In all cases the use of complexes did enhance the immunogenic capacity of substimulatory doses of gp120 or gp160 by facilitating uptake by APC via Fc receptor and consequent presentation to specific human T cell clones. Finally, help for the production in vivo of anti-CD4 antibodies was obtained from T lymphocytes specific for gp120 when CD4-primed memory B cells were pulsed with CD4 complexed with gp120, thus demonstrating in the mouse the entire cycle of intermolecular help via non-covalent interaction, and setting the stage for future experiments on self-tolerance breakage in a human molecular context
— id: 9269, year: 1993, vol: 5, page: 1109, stat: Journal Article,

Resistance of a human serum-selected human immunodeficiency virus type 1 escape mutant to neutralization by CD4 binding site monoclonal antibodies is conferred by a single amino acid change in gp120
McKeating JA; Bennett J; Zolla-Pazner S; Schutten M; Ashelford S; Brown AL; Balfe P
1993 Sep;67(9):5216-5225, Journal of virology
We have selected an HXB2 variant which can replicate in the presence of a neutralizing human serum. Sequencing of the gp120 region of the env gene from the variant and parental viruses identified a single amino acid substitution in the third conserved region of gp120 at residue 375 (AGT-->AAT, Ser-->Asn; designated 375 S/N). The escape mutant was found to be resistant to neutralization by soluble CD4 (sCD4) and four monoclonal antibodies (MAbs), 39.13g, 1.5e, G13, and 448, binding to epitopes overlapping that of the CD4 binding site (CD4 b.s.). Introduction of the 375 S/N mutation into HXB2 by site-directed mutagenesis confirmed that this mutation is responsible for the neutralization-resistant phenotype. Both sCD4 and three of the CD4 b.s. MAbs (39.13g, 1.5e, and G13) demonstrated reduced binding to the native 375 S/N mutant gp120. The ability to select for an escape variant resistant to multiple independent CD4 b.s. MAbs by a human serum confirms the reports that antibodies to the discontinuous CD4 b.s. are a major component of the group-specific neutralizing activity in human sera
— id: 9270, year: 1993, vol: 67, page: 5216, stat: Journal Article,

Complement activation by human monoclonal antibodies to human immunodeficiency virus
Spear GT; Takefman DM; Sullivan BL; Landay AL; Zolla-Pazner S
1993 Jan;67(1):53-59, Journal of virology
It has been shown that the incubation of human immunodeficiency virus (HIV) with polyclonal antibodies from HIV-infected persons and complement results in complement-mediated neutralization due, at least in part, to virolysis. The current study was performed to determine whether any of a panel of 16 human monoclonal antibodies to HIV could activate complement and, if so, which determinants of the HIV envelope could serve as targets for antibody-dependent complement-mediated effects. Human monoclonal antibodies directed to the third variable region (V3 region) of HIVMN gp120 induced C3 deposition on infected cells and virolysis of free virus. Antibodies to two other sites on HIVMN gp120 and two sites on gp41 induced few or no complement-mediated effects. Similarly, only anti-V3 antibodies efficiently caused complement-mediated effects on the HIVIIIB isolate. In general, the level of C3 deposition on infected cells paralleled the relative level of bound monoclonal antibodies. As expected, pooled polyclonal antibodies from infected persons were much more efficient than monoclonal antibodies inducing C3 deposition per unit of bound immunoglobulin. Treatment of virus or infected cells with soluble CD4 resulted in increases in anti-gp41 antibody-mediated virolysis and C3 deposition but decreases in anti-V3 antibody-mediated virolysis and C3 deposition. In general, virolysis of HIV was more sensitive as an indicator of complement-mediated effects than infected-cell surface C3 deposition, suggesting the absence of or reduced expression of functional complement control proteins on the surface of free virus. Thus, this study shows that human monoclonal antibodies to the V3 region of gp120 are most efficient in causing virolysis of free virus and C3 deposition on infected cells. Elution of gp120 with soluble CD4 exposes epitopes on gp41 that can also bind antibody, resulting in virolysis and C3 deposition. These findings establish a serologically defined model system for the further study of the interaction of complement and HIV
— id: 9275, year: 1993, vol: 67, page: 53, stat: Journal Article,

EXPRESSION OF AN ANTI-GP41 RECEPTOR ON THE SURFACE OF A B-CELL ASSOCIATED WITH ENHANCED REPLICATION OF HIV=1
TANI, Y; BOONE, E; DONOHUE, E; ZOLLAPAZNER, S; LANE, HC; COHEN, DI
1993 JUN ;6(6):704-704, Journal of acquired immune deficiency syndromes & human retrovirology
— id: 54143, year: 1993, vol: 6, page: 704, stat: Journal Article,

Augmented neutralization demonstrated by combining HIVIG and human monoclonal antibodies
Zolla-Pazner S; Burda S; Andrus L; Prince A
1993 Dec 12-16;1:73-73, National Conference on Human Retroviruses & Related Infections
Preparations containing pooled human immunoglobulin derived from HIV-infected individuals (HIVIG) have been reported to possess the ability to neutralize HIV-1. Human monoclonal antibodies (humAbs) to HIV-1 derived from the cells of HIV-seropositive individuals also have neutralizing capability. We have attempted to enhance the neutralizing ability of HIVIG by combining it with humAbs targeted towards different epitopes on the HIV envelope. The humAbs used included: 694/98-D which recognizes the GRAF sequence of the gp120-V3 region, 447-52-D, specific for the GPGR sequence of the same region, and 729-D, which is directed against the CD4 binding domain of gp120. The assay used was the modified syncytium formation assay (Nara et al., Laal et al.) that measures the neutralization of cell-free virus. The results show that the addition of 0.2 to 1.3% of these humAbs to HIVIG (on a weight-to-weight basis) reduces by 2.5- to 5-fold the amount of HIVIG required to achieve 50% neutralization. The combinations tested demonstrated an additive rather than synergistic interaction (CI50=0.7-1.3). These results suggest that the amount of HIVIG required for passive immunization could be greatly decreased by the addition of small amounts of humAbs
— id: 5998, year: 1993, vol: 1, page: 73, stat: Journal Article,

HUMAN MONOCLONAL-ANTIBODIES TO GP120 - BIOLOGIC FUNCTION, IMMUNOCHEMICAL CHARACTERISTICS AND CLINICAL POTENTIAL
ZOLLAPAZNER, S; GORNY, MK; BUCHBINDER, A; KARWOWSKA, S; XU, JY
1993 JUN ;6(6):677-677, Journal of acquired immune deficiency syndromes & human retrovirology
— id: 54141, year: 1993, vol: 6, page: 677, stat: Journal Article,

Synergy between human monoclonal antibodies to HIV extends their effective biologic activity against homologous and divergent strains
Buchbinder A; Karwowska S; Gorny MK; Burda ST; Zolla-Pazner S
1992 Apr;8(4):425-427, AIDS research & human retroviruses
— id: 9284, year: 1992, vol: 8, page: 425, stat: Journal Article,

Synergy between human monoclonal antibodies to HIV extends their effective biologic activity against homologous and divergent strains
Buchbinder A; Zolla-Pazner S; Karwowska S; Gorny MK; Burda ST
1992 Aug;8(8):1395-1395, AIDS research & human retroviruses
— id: 9279, year: 1992, vol: 8, page: 1395, stat: Journal Article,

Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody
Gorny MK; Conley AJ; Karwowska S; Buchbinder A; Xu JY; Emini EA; Koenig S; Zolla-Pazner S
1992 Dec;66(12):7538-7542, Journal of virology
The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is thought to induce potent neutralizing antibodies which are generally defined as type specific and reactive with individual viral isolates. In contrast, the CD4-binding domain is thought to induce neutralizing antibodies that are group specific and capable of neutralizing all isolates of HIV-1. However, in this study, we used a panel of human monoclonal antibodies to these regions of gp120 which displays specificities and neutralizing activities that challenge these tenets. In particular, we used a human monoclonal antibody to the V3 domain with exceptionally potent and broad neutralizing activity against many diverse HIV-1 isolates. The anti-CD4-binding domain antibodies, on the other hand, showed a more restricted pattern of activity
— id: 9276, year: 1992, vol: 66, page: 7538, stat: Journal Article,

Production of human monoclonal antibodies specific for conformational and linear non-V3 epitopes of gp120
Karwowska S; Gorny MK; Buchbinder A; Gianakakos V; Williams C; Fuerst T; Zolla-Pazner S
1992 Jun;8(6):1099-1106, AIDS research & human retroviruses
Three IgG1 human monoclonal antibodies (MAbs) directed against conformational epitopes of the gp120 envelope protein of HIV-1 were produced, as was a single human MAb to a linear epitope spanning amino acids 487-509 in the C-terminal portion of gp120. All three conformation-dependent MAbs reacted optimally with recombinant gp120 (rgp120) captured on plastic via its carbohydrate moieties with Concanavalin A. These MAbs were able to block the interaction between recombinant CD4 (rCD4) and rgp120; they were also able to achieve 50% neutralization of HTLV-IIIB and MN strains of HIV-1 in a concentration range of 0.5-12.8 micrograms/mL. The MAb to the linear determinant is the first reported human MAb specific for the immunodominant portion of gp120; this MAb was most reactive with rgp120 when it was coated directly on plastic. It could neither inhibit rCD4-rgp120 binding nor neutralize either HTLV-IIIB or MN. The binding affinities of the four human MAbs for rgp120 in solution, reflected by their dissociation constants (Kd), ranged from 0.5 x 10(-8) to 7.5 x 10(-8) M
— id: 9283, year: 1992, vol: 8, page: 1099, stat: Journal Article,

Amino acid residues of the human immunodeficiency virus type I gp120 critical for the binding of rat and human neutralizing antibodies that block the gp120-sCD4 interaction
McKeating JA; Thali M; Furman C; Karwowska S; Gorny MK; Cordell J; Zolla-Pazner S; Sodroski J; Weiss RA
1992 Sep;190(1):134-142, Virology
We have characterized the discontinuous epitopes recognized by two rat and three human neutralizing monoclonal antibodies (mAb) by examining the effect of single amino acid changes in conserved residues of gp120 on mAb recognition. A human mAb derived from an infected individual, 448D, and two rat mAbs, 39.13g and 39.3b, respectively, derived by immunization with native recombinant gp120, recognize similar epitopes. Recognition of the envelope glycoproteins by these mAbs was affected by changes in gp120 amino acid residues 88, 113, 117, 257, 368, or 370. The gp120 amino acids 257, 368, and 370 have previously been reported to be important for CD4 binding, which is consistent with the ability of these mAbs to block the gp120-CD4 interaction. Residues 88, 113, and 117 are not thought to be important for CD4 binding, suggesting that the antibody epitopes overlap, but are distinct from, the CD4 binding region. We also found that some alterations in gp120 residues 88, 117, 368, or 421 reduced the ability of polyclonal sera from HIV-1-infected individuals to inhibit the interaction of the mutant gp120 glycoproteins with soluble CD4. Thus, changes in the HIV-1 gp120 glycoprotein that minimally affect the receptor binding may allow escape from neutralizing antibodies directed against the CD4 binding region
— id: 9278, year: 1992, vol: 190, page: 134, stat: Journal Article,

Expression and characterization of genetically engineered human immunodeficiency virus-like particles containing modified envelope glycoproteins: implications for development of a cross-protective AIDS vaccine
Rovinski B; Haynes JR; Cao SX; James O; Sia C; Zolla-Pazner S; Matthews TJ; Klein MH
1992 Jul;66(7):4003-4012, Journal of virology
Noninfectious human immunodeficiency virus type 1 (HIV-1) viruslike particles containing chimeric envelope glycoproteins were expressed in mammalian cells by using inducible promoters. We engineered four expression vectors in which a synthetic oligomer encoding gp120 residues 306 to 328 (amino acids YNKRKRIHIGP GRAFYTTKNIIG) from the V3 loop of the MN viral isolate was inserted at various positions within the endogenous HIV-1LAI env gene. Expression studies revealed that insertion of the heterologous V3(MN) loop segment at two different locations within the conserved region 2 (C2) of gp120, either 173 or 242 residues away from the N terminus of the mature subunit, resulted in the secretion of fully assembled HIV-like particles containing chimeric LAI/MN envelope glycoproteins. Both V3 loop epitopes were recognized by loop-specific neutralizing antibodies. However, insertion of the V3(MN) loop segment into other regions of gp120 led to the production of envelope-deficient viruslike particles. Immunization with HIV-like particles containing chimeric envelope proteins induced specific antibody responses against both the autologous and heterologous V3 loop epitopes, including cross-neutralizing antibodies against the HIV-1LAI and HIV-1MN isolates. This study, therefore, demonstrates the feasibility of genetically engineering optimized HIV-like particles capable of eliciting cross-neutralizing antibodies
— id: 9281, year: 1992, vol: 66, page: 4003, stat: Journal Article,

p24 antibody production in p24 antibody-negative HIV-infected subjects
Stickler MC; Sharpe S; Zolla-Pazner S
1992 Summer;5(2):123-132, Viral immunology
To determine if HIV-infected patients with no detectable serum antibodies to p24 are producing antibodies to p24 (anti-p24), blood was obtained from 49 HIV-infected patients at various stages of infection. Serum p24 antigen levels were measured and peripheral blood mononuclear cells were cultured for 1 week without mitogenic stimulation. The presence of anti-p24 in culture supernatants and sera was determined by radioimmunoprecipitation assays. Cells from 89% of the patients who had anti-p24 in their sera spontaneously synthesized anti-p24 in vitro. Similarly, cells from 83% of the HIV-infected patients who had no detectable anti-p24 in their sera spontaneously produced anti-p24 in vitro. Thus the absence of anti-p24 in serum did not reflect suppression in the ability of patients' cells to synthesize and secrete antibodies to p24. However, cells from patients whose sera contained anti-p24 spontaneously synthesized more anti-p24 than did cells from patients whose sera lacked anti-p24, suggesting that these two groups of patients may represent individuals with inherently high or low responses to p24 epitopes, respectively
— id: 9285, year: 1992, vol: 5, page: 123, stat: Journal Article,

Human monoclonal and polyclonal anti-human immunodeficiency virus-1 antibodies share a common clonotypic specificity
Wang H; Muller S; Zolla-Pazner S; Kohler H
1992 Jul;22(7):1749-1755, European journal of immunology
Human monoclonal and purified polyclonal anti-human immunodeficiency virus (HIV)-1 antibodies were tested for binding to a murine monoclonal anti-idiotypic antibody (1F7, IgM, kappa). Four human monoclonal anti-p24 and three human monoclonal anti-gp120 antibodies express the 1F7 clonotype, while one human monoclonal anti-gp41 antibody does not bind to 1F7. Affinity-purified anti-p24 and anti-gp120 antibodies from HIV-1-infected individuals also react with 1F7. Western blot analysis and enzyme-linked immunosorbent assay confirmed that 1F7 reacts with human antibodies of different HIV-1 antigen specificities. A survey of sera from 329 HIV-1-infected individuals showed binding to 1F7 in 239 sera (72.6%) while 1F7 was not reacting with 109 HIV-1-negative sera. These results show that 1F7 idiotype is an HIV-1 infection-associated clonotypic marker shared by anti-HIV-1 antibodies with different epitope specificites
— id: 9282, year: 1992, vol: 22, page: 1749, stat: Journal Article,

Passive immunization for the prevention and treatment of HIV infection
Zolla-Pazner S; Gorny MK
1992 Nov;6(11):1235-1247, AIDS
— id: 9277, year: 1992, vol: 6, page: 1235, stat: Journal Article,

Summary: Clinical Immunology Working Group
Zolla-Pazner S; Mathieson BJ; Walker MC; Walker B
1992 Aug;8(8):1441-1443, AIDS research & human retroviruses
— id: 9280, year: 1992, vol: 8, page: 1441, stat: Journal Article,

B-CELLS FROM HIV-INFECTED INDIVIDUALS ARE FUNCTIONALLY NORMAL BUT SUBJECT TO PROFOUND ABNORMAL IMMUNOREGULATION
ZOLLAPAZNER, S; EDELMAN, A
1992 MAY ;8(5):892-892, AIDS research & human retroviruses
— id: 51933, year: 1992, vol: 8, page: 892, stat: Journal Article,

Molecular characterization of five human anti-human immunodeficiency virus type 1 antibody heavy chains reveals extensive somatic mutation typical of an antigen-driven immune response
Andris JS; Johnson S; Zolla-Pazner S; Capra JD
1991 Sep 1;88(17):7783-7787, Proceedings of the National Academy of Sciences of the United States of America
We report the heavy chain variable region sequences from the cDNAs of five previously described monoclonal cell lines producing human antibodies specific for the human immunodeficiency virus type 1 and detail the molecular characteristics, germ-line origins, and extent of somatic mutation among these antibodies. Three of the five heavy chain variable regions derive from the VHIV gene family, but each has arisen from a different heavy chain variable region (VH) gene segment within the VHIV family. In addition, one is derived from a VHI gene segment, and one is derived from a VHV gene segment. Since four of the five antibodies arise from known germ-line VH elements, a precise determination of the extent of somatic variation is possible. The amount of variation from the closest germ-line sequence ranges from 4.5% to 14.8% among these antibodies, most of which is concentrated in the complementarity-determining regions. In general, the diversity (D) segments are long, characteristic of D-D fusions and/or extensive terminal deoxynucleotidyltransferase activity; however, definitive homologies cannot be found with the known germ-line D segments. Joining (JH) gene segment utilization appears random. The use of five different germ-line VH gene segments and extensive somatic mutation provides evidence that a polyclonal, antigen-driven immune response occurs during the natural infection with human immunodeficiency virus
— id: 9287, year: 1991, vol: 88, page: 7783, stat: Journal Article,

Proliferative response of mononuclear cells from HIV-infected patients to B-cell mitogens: effects of lymphocyte subset frequency, T-cell defects and prostaglandins
Edelman AS; Zolla-Pazner S
1991 Nov;7(11):953-961, AIDS research & human retroviruses
Proliferative responses of mononuclear cells from patients seropositive for human immunodeficiency virus to B-cell mitogens are severely depressed compared with those of controls. The role of several immunoregulatory phenomena was analyzed. Experimental results show that addition of exogenous lymphokines to cultures increases responses to anti-mu and SAC. Addition of indomethacin to cultures greatly increases the SAC response and causes a smaller increase in the pokeweed mitogen (PWM) response. When both exogenous lymphokines and indomethacin are present in cultures, responses of patients' cells to all three mitogens are positively correlated with the percentage of CD4+ T cells and negatively correlated with the percentage of CD8+ T cells. Responses to anti-mu and SAC are also positively correlated with the percentage of B cells in these cultures. On the basis of these correlations between B-cell responses and lymphocyte subset frequency, patients' B-cell responses can be mathematically corrected to estimate the responsiveness of the B cells in the presence of normal numbers of CD4+ and CD8+ cells. These corrected responses for all three mitogens are virtually identical to control responses. Furthermore, responses of enriched B-cell populations from HIV+ subjects and normal controls to SAC were not significantly different when assays were performed in the presence of indomethacin and exogenous lymphokines. These results suggest that B cells from HIV+ patients are inherently normal in their responsiveness to B-cell mitogens. The depressed function is imposed upon them as a result of the abnormal frequency of lymphocyte subsets in the blood, by increased prostaglandin production, and deficient lymphokine production
— id: 9286, year: 1991, vol: 7, page: 953, stat: Journal Article,

Production of site-selected neutralizing human monoclonal antibodies against the third variable domain of the human immunodeficiency virus type 1 envelope glycoprotein
Gorny MK; Xu JY; Gianakakos V; Karwowska S; Williams C; Sheppard HW; Hanson CV; Zolla-Pazner S
1991 Apr 15;88(8):3238-3242, Proceedings of the National Academy of Sciences of the United States of America
Cell lines secreting IgG1 human monoclonal antibodies (mAb) to the envelope glycoprotein, gp120, of human immunodeficiency virus (HIV) have been produced by transformation of peripheral blood cells from HIV-infected individuals and by fusion of transformed cells to a human-mouse heteromyeloma cell line (SHM-D33). Two human mAbs were site-selected by means of a 23-mer synthetic peptide spanning a portion of the third variable domain of gp120 from the MN strain of HIV. The two heterohybridomas produce three times more IgG than do their parent lymphoblastoid cell lines. The specificities of these mAbs have been mapped to sequences near the tip of the disulfide loop of the gp120 third variable domain, Lys-Arg-Ile-His-Ile and His-Ile-Gly-Pro-Gly-Arg, respectively. The mAbs have dissociation constants of 3.7 x 10(-6) M and 8.3 x 10(-7) M, neutralize HIVMN in vitro at nanogram levels, and bear the characteristics of antibodies associated with protective immunity in vivo
— id: 9290, year: 1991, vol: 88, page: 3238, stat: Journal Article,

Passive immunization for the treatment and prevention of HIV infection
Karwowska S; Zolla-Pazner S
1991 ;2(1-2):31-48, Biotechnology therapeutics
Passive immunization using serum or immunoglobulin preparations has been used in the prophylaxis and treatment of many bacterial and viral diseases. Preliminary attempts to use these methods to prevent HIV infection in chimpanzees have been promising. With the identification of polyclonal and monoclonal antibodies with protective activity against HIV in in vitro systems, the possibility of using these reagents in vivo takes on new relevance. The potential and problems of using passively administered anti-HIV antibodies for HIV prophylaxis and treatment are discussed, as well as the relative merits of polyclonal versus monoclonal reagents
— id: 9291, year: 1991, vol: 2, page: 31, stat: Journal Article,

Two immunodominant domains of gp41 bind antibodies which enhance human immunodeficiency virus type 1 infection in vitro
Robinson WE Jr; Gorny MK; Xu JY; Mitchell WM; Zolla-Pazner S
1991 Aug;65(8):4169-4176, Journal of virology
Four of eight human monoclonal antibodies (huMAbs) to gp41 were identified which could enhance human immunodeficiency virus type 1 (HIV-1) infection in vitro by complement-mediated antibody-dependent enhancement (C'-ADE). These enhancing huMAbs were mapped to two distinct domains on the HIV-1 gp41 transmembrane glycoprotein by using synthetic peptides. The first domain, amino acids 579 to 613 (peptide AA579-613), was recognized by three of the four enhancing huMAbs. The AA579-613 peptide blocked C'-ADE of HIV-1 infection in vitro whether it was mediated by these three huMAbs or by human polyclonal anti-HIV serum. The second domain, amino acids 644 to 663, bound the remaining enhancing huMAb. This peptide weakly blocked C'-ADE mediated by the huMAb and by an HIV immune globulin fraction but did not block C'-ADE mediated by a patient's serum. The patient's serum did react with the peptide in an enzyme immunoassay. The huMAbs to the two domains could interact in vitro to enhance HIV-1 infection in a synergistic manner. These two domains, which bind enhancing antibodies, are conserved between HIV-1 isolates as well as between HIV-2 and simian immunodeficiency virus isolates. These data demonstrate the existence of two conserved regions within the HIV-1 gp41 which bind enhancing antibodies; these two domains, amino acids 579 to 613 and 644 to 663, may prove important in HIV-1 vaccine development and in immunopathogenesis of HIV-1 infection
— id: 9289, year: 1991, vol: 65, page: 4169, stat: Journal Article,

Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies
Xu JY; Gorny MK; Palker T; Karwowska S; Zolla-Pazner S
1991 Sep;65(9):4832-4838, Journal of virology
Immunogenic regions of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) were previously mapped by examining polyclonal sera from HIV-infected patients and rodent polyclonal and monoclonal antibodies (MAbs) to peptides of gp41. To define the epitopes within these regions to which infected humans respond during the course of infection, the specificity of human MAbs to these regions had to be studied. Using 10 human MAbs identified initially by their reactivity to whole gp41 in HIV-1 lysates, the epitopes within the immunodominant region of gp41 and within a second immunogenic region of gp41 have been mapped. Thus, five MAbs (from five different patients) to the immunodominant domain of gp41 in the vicinity of the cysteines at positions 598 and 604 (hereinafter designated cluster I) reacted with a stretch of 11 amino acids from positions 590 to 600. Four of these five MAbs were reactive with linear epitopes, while one MAb required the conformation conferred by the disulfide bridge between the aforementioned cysteines. Three MAbs to cluster I revealed dissociation constants ranging from 10(-6) to 10(-8) M, depending on the MAb tested and the size of the synthetic or recombinant peptide used in the assay. Five additional MAbs reacted with a second immunogenic region between positions 644 and 663 (designated cluster II). Four of these five MAbs were specific for conformational determinants. Titration of sera from HIV-infected patients showed that there was about 100-fold more antibody to cluster I than to cluster II in patients' sera, confirming the immunodominance of cluster I
— id: 9288, year: 1991, vol: 65, page: 4832, stat: Journal Article,

Response of mononuclear cells from HIV-infected patients to B-cell mitogens: correlation with immunological and clinical features of disease progression
Edelman AS; Zolla-Pazner S
1990 Sep;4(9):859-864, AIDS
Proliferation of mononuclear cells from HIV-seropositive patients to B-cell mitogens was studied in the absence and presence of mixed lymphocyte culture supernatants (MLC-sup). The results show: (1) patients' responses to B-cell mitogens overlap with normal responses but are, on average, consistently lower than normal; (2) the addition of MLC-sup increases the proliferative responses to T-cell-independent mitogens, but does not bring patient's responses up to control levels; (3) HIV-positive patients in all clinical categories have decreased responses to B-cell mitogens. Although some patients with AIDS Centers for Disease Control (CDC) group IVC and IVD have the lowest responses, asymptomatic (CDC group II) and AIDS-related complex (ARC; CDC groups III/IVA and IVB) patients also show significant defects. (4) The same patients were recategorized using an immunological staging system. Those patients with more normal immunohematological parameters have significantly greater responses to mitogens compared with patients with more abnormal immunological parameters. The data suggest that immunological staging could provide more information than clinical classification with respect to the underlying immunopathogenic events occurring in HIV infection
— id: 9293, year: 1990, vol: 4, page: 859, stat: Journal Article,

Immunoconjugates containing ricin A chain and either human anti-gp41 or CD4 kill H9 cells infected with different isolates of HIV, but do not inhibit normal T or B cell function
Till MA; Ghetie V; May RD; Auerbach PC; Zolla-Pazner S; Gorny MK; Gregory T; Uhr JW; Vitetta ES
1990 ;3(6):609-614, Journal of acquired immune deficiency syndrome
We have previously reported that immunoconjugates composed of deglycosylated ricin A chain coupled to either recombinant (r) CD4 or two different monoclonal human anti-gp41 antibodies (rCD4-dgA and anti-gp41-dgA, respectively) are specifically toxic to HIV-infected lines of human T cells (H9) and monocytes (U937). In order to further evaluate these immunoconjugates as potential therapeutic reagents for killing HIV-infected cells, H9 cells infected with five different isolates of HIV were used as target cells in vitro. All three HIV-specific immunoconjugates were toxic to H9 cells infected with each HIV isolate, but were virtually nontoxic to uninfected cells. Chloroquine markedly potentiated the specific toxicity of all three conjugates, particularly the anti-gp41-dgAs. None of the conjugates affected the ability of normal peripheral blood B cells to respond to mitogen or the ability of normal T cells to respond to alloantigens
— id: 9294, year: 1990, vol: 3, page: 609, stat: Journal Article,

Identification of sites within gp41 that serve as targets for antibody-dependent cellular cytotoxicity by using human monoclonal antibodies
Tyler DS; Stanley SD; Zolla-Pazner S; Gorny MK; Shadduck PP; Langlois AJ; Matthews TJ; Bolognesi DP; Palker TJ; Weinhold KJ
1990 Nov 15;145(10):3276-3282, Journal of immunology
In an effort to determine the functional activity of anti-HIV-1 human mAb and to define the epitopes against which they are directed, supernatants from 10 EBV-transformed lymphoblastoid cell lines producing mAb to HIV were tested. Five clones producing mAb to gp41 and five producing mAb to p24 were identified. The anti-HIV-1 human mAb were tested in neutralization and cell fusion assays in the form of cell culture supernatants at concentrations ranging from 1.7 to 22.0 micrograms/ml. None of the human mAb were found either to inhibit HIV-1-(IIIB or RF) associated cell fusion or to neutralize HIV-1 (IIIB) infection of AA5 cells. All human mAb were additionally tested in 6 h 51Cr release assays for their ability to direct HIV-1 specific antibody-dependent cellular cytotoxicity (ADCC). For ADCC assays, PBMC were isolated from healthy seronegative donors and used as effector cells. HIV-1 infected (IIIB, RF, and MN) CEM.NKR cells as well as CEM.NKR cells with purified gp120 adsorbed onto their surface served as targets. None of the anti-p24 mAb mediated ADCC. In contrast, three of the anti-gp41 mAb were able to direct a significant level of ADCC against each of the infected targets, but as expected, failed to lyse gp120 adsorbed cells. To define the specific epitopes against which the anti-gp41 mAb were directed, seven small peptides homologous to regions within the extracellular domain of gp41 were synthesized. Using RIA, two of the mAb could be mapped. The most effective ADCC-directing human mAb bound to a peptide comprising amino acids 644-663, whereas the least effective ADCC directing anti-gp41 human mAb bound to a region within the immunodominant portion of gp41 outlined by amino acids 579-604. Together, these results for the first time assign a functional activity to human mAb directed at specific regions within gp41 by demonstrating that areas within this molecule can serve as targets for ADCC
— id: 9292, year: 1990, vol: 145, page: 3276, stat: Journal Article,

AIDS: a syndrome of immune dysregulation, dysfunction, and deficiency
Edelman AS; Zolla-Pazner S
1989 Jan;3(1):22-30, FASEB journal
Acquired immune deficiency syndrome (AIDS) is a disease caused by the human immunodeficiency virus (HIV) in which cellular immune functions are severely impaired. Acute infection and subsequent destruction of helper T cells, although occurring readily in cell cultures, do not appear to be the only mechanisms mediating helper T cell loss. Other mechanisms that may account for the loss of helper T cells include: T cell syncytia formation, decreased T cell production, and autoimmune-related destruction of helper T cells. Immune abnormalities seen early in the course of HIV infection include immune hyperactivation and autoimmune phenomena suggestive of immune dysregulation rather than immune deficiency. Many changes in immune function are, in fact, seen in HIV-seropositive patients who possess a normal number of helper T cells. Mechanisms (other than the loss of helper T cells) that may contribute to the immune abnormalities seen in these patients include noninfectious effects of HIV and HIV proteins, effects of HIV on non-T cells, autoimmune-related manifestations of HIV infection, and HIV-induced activation of normal immunosuppressive circuits
— id: 9300, year: 1989, vol: 3, page: 22, stat: Journal Article,

Generation of human monoclonal antibodies to human immunodeficiency virus
Gorny MK; Gianakakos V; Sharpe S; Zolla-Pazner S
1989 Mar;86(5):1624-1628, Proceedings of the National Academy of Sciences of the United States of America
Based on the finding that cells producing antibodies to human immunodeficiency virus (HIV) circulate in the peripheral blood of HIV-infected individuals, attempts were made to immortalize such B cells with Epstein-Barr virus. Mononuclear cells from 58 HIV-seropositive subjects at various stages of HIV infection were transformed, and anti-HIV cell lines were derived from 4 subjects, all of whom were in early stages of infection. Seven of these cell lines have been stable with respect to antibody production for up to 15 months. Three lines are producing IgG antibody to the 41-kDa HIV transmembrane glycoprotein gp41 and 4 produce IgG antibodies to the 24-kDa HIV core protein p24, its precursors and a breakdown product. The antibodies are reactive by ELISA, by radioimmunoprecipitation, and by Western blot, demonstrating the feasibility of producing multiple stable cell lines synthesizing human monoclonal antibodies to HIV by immortalization of peripheral blood cells with Epstein-Barr virus
— id: 9298, year: 1989, vol: 86, page: 1624, stat: Journal Article,

Specific immunity to HIV and other retroviral infections
Gorny MK; Pinter A; Zolla-Pazner S
1989 ;1:181-199, Progress in AIDS pathology
— id: 9301, year: 1989, vol: 1, page: 181, stat: Journal Article,

Oligomeric structure of gp41, the transmembrane protein of human immunodeficiency virus type 1
Pinter A; Honnen WJ; Tilley SA; Bona C; Zaghouani H; Gorny MK; Zolla-Pazner S
1989 Jun;63(6):2674-2679, Journal of virology
We characterized the structural forms of the human immunodeficiency virus env-encoded proteins with a panel of monoclonal and polyclonal antibodies. Western blot (immunoblot) assays with antibodies specific for gp41 invariably recognized a major component of 160 kilodaltons and a less intense component of 120 kilodaltons in viral lysates. We demonstrated that these species are noncovalently associated tetramers and trimers of gp41 which represent the native form of this protein in virions. These complexes were stable when boiled in the presence of low concentrations of sodium dodecyl sulfate but were dissociated to gp41 monomers at high sodium dodecyl sulfate concentrations. Moreover, two human monoclonal antibodies preferentially recognized the oligomeric complexes over monomeric gp41 in Western blots, indicating the presence of epitopes recognized by the human immune system on the gp41 multimers which are not efficiently expressed by the dissociated monomers. The demonstration of the existence of multimeric env complexes and the enhanced and altered antigenicity of such multimers may be relevant to the design of subunit and recombinant human immunodeficiency virus env vaccines
— id: 9296, year: 1989, vol: 63, page: 2674, stat: Journal Article,

Human immunodeficiency virus-infected T cells and monocytes are killed by monoclonal human anti-gp41 antibodies coupled to ricin A chain
Till MA; Zolla-Pazner S; Gorny MK; Patton JS; Uhr JW; Vitetta ES
1989 Mar;86(6):1987-1991, Proceedings of the National Academy of Sciences of the United States of America
Two human monoclonal antibodies specific for the envelope glycoprotein (gp), gp41, of the human immunodeficiency virus were conjugated to deglycosylated ricin A chain. These immunotoxins killed human immunodeficiency virus-infected H9 (T cell) and U937 (monocyte) cell lines but were nontoxic to the uninfected cell lines or to class II-positive Daudi cells. Specific killing of infected H9 cells could be completely blocked by recombinant gp160 and partially blocked by unconjugated anti-gp41 antibody but was not blocked by recombinant gp120 or human IgG demonstrating specificity for gp41. The specific toxicity of the immunotoxins for infected U937 cells was markedly potentiated by chloroquine
— id: 9299, year: 1989, vol: 86, page: 1987, stat: Journal Article,

An immuno-dot blot assay for the detection of antibody to HIV
Xu JY; Gorny MK; Zolla-Pazner S
1989 Jun 21;120(2):179-183, Journal of immunological methods
This paper describes a simple and rapid immuno-dot blot assay for the detection of antibody to HIV. It utilizes nanogram quantities of HIV lysate, immobilized on nitrocellulose paper, and microliter quantities of serum or culture supernatants for the detection of antibody. The test is highly sensitive and specific. It offers the opportunity to perform multiple assays at a minimal cost and provides the additional advantage of not requiring any sophisticated or expensive equipment to perform the test or interpret the results
— id: 9295, year: 1989, vol: 120, page: 179, stat: Journal Article,

Reinterpretation of human immunodeficiency virus western blot patterns
Zolla-Pazner S; Gorny MK; Honnen WJ; Pinter A
1989 May 11;320(19):1280-1281, New England journal of medicine
— id: 9297, year: 1989, vol: 320, page: 1280, stat: Journal Article,

ROLE OF MACROPHAGES FROM PLASMACYTOMA-BEARING MICE IN SUPPRESSION OF B-CELL PROLIFERATION
KLEIN, I; ZOLLAPAZNER, S
1988 MAR 25 ;2(6):A1667-A1667, FASEB journal
— id: 41800, year: 1988, vol: 2, page: A1667, stat: Journal Article,

Association of human immunodeficiency virus infection and autoimmune phenomena
Kopelman RG; Zolla-Pazner S
1988 Jan;84(1):82-88, American journal of medicine
Patients infected with human immunodeficiency virus have a variety of presentations including fevers, lymphadenopathy, rash, renal dysfunction, and neurologic and hematologic disorders. Many of these features are also seen in patients with systemic lupus erythematosus (SLE). Herein are described five patients ultimately diagnosed as having acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) in whom the differential diagnosis included SLE because of multi-system disease and autoimmune phenomena, especially positive antinuclear antibodies. Serum samples from 151 consecutive patients with AIDS or ARC were examined and 19 with low titer-positive antinuclear antibodies were found (17 at 1:20 and two at 1:160). These observations suggest that SLE and human immunodeficiency virus infection may share clinical and serologic features
— id: 9303, year: 1988, vol: 84, page: 82, stat: Journal Article,

B-cell activation in HIV infection: relationship of spontaneous immunoglobulin secretion to various immunological parameters
Mizuma H; Litwin S; Zolla-Pazner S
1988 Mar;71(3):410-416, Clinical & experimental immunology
Peripheral blood mononuclear cells from HIV-infected individuals spontaneously secrete elevated levels of IgG, IgM and IgD. This increased level of synthesis and secretion is similar in HIV-infected subjects with no or few symptoms, in ARC patients and in AIDS patients. Thus, abnormal B-cell activation is characteristic of patients with mild as well as severe manifestations of HIV infection. The level of spontaneous cellular secretion of IgG, IgM and IgD correlates with serum levels of these isotypes. Levels of spontaneous cellular secretion of IgG and IgM correlate negatively with the percentage but not with the absolute number of T4-positive cells and correlate positively with the percentage but not with the absolute number of T8-positive cells. The data suggest that the proportional distribution of these T-cell subsets is a critical factor in the B-cell dysregulation leading to overproduction of IgG and IgM. On the other hand, spontaneous IgD secretion correlates with neither the percent nor the absolute number of T4 or T8 cells suggesting that the increase of IgD-secretion by B cells is independent of the T-cell defects. The data imply that more than one mechanism underlies the B-cell activation in HIV-infected individuals
— id: 9302, year: 1988, vol: 71, page: 410, stat: Journal Article,

Control of B cell proliferation: arrest of B cells in late G1 underlies immunosuppression induced by plasma cell tumors
Berman JE; Zolla-Pazner S
1987 May 1;138(9):2805-2812, Journal of immunology
Immunosuppression in mice bearing plasma cell tumors (PC-mice) provides a model system for the study of negative B cell regulation. Our previous studies demonstrated that B cell proliferation is suppressed in these mice by a cascade of interactions involving macrophages and soluble factors. The present report pinpoints the G1 phase of the cell cycle as the stage of B cell proliferation inhibited in PC-mice. Modulation of surface immunoglobulin (sIg) with anti-mu, an early membrane activation event, occurred normally on B cells from the spleens of PC-mice. However, examination of the size profile and the expression of sIgD and sIgM on B cells from the spleens of PC-mice showed an accumulation of large-sized, low intensity sIgD+ cells, suggesting a block in B cell activation in the late G1 phase of the cell cycle. This was confirmed by experiments in vitro that demonstrated that although LPS-stimulated B cells from the spleens of PC-mice enlarged to a size characteristic of G1 phase, most did not additionally enlarge into S phase even after 3 days of culture, nor did they incorporate significant amounts of [3H]thymidine. Additional confirmation of a block in late G1 was obtained by using analysis of [3H]thymidine incorporation, cell size, and cell cycle after normal cells were cultured in supernatants from cloned PC lines containing the factor(s) that initiates the cascade of events leading to suppression of B cell proliferation. The relevance of these findings to PC-induced immunosuppression and to the regulation of normal B cell proliferation during the G1 phase of the cell cycle is discussed.
— id: 9307, year: 1987, vol: 138, page: 2805, stat: Journal Article,

Four-year prospective study of homosexual men: correlation of immunologic abnormalities, clinical status, and serology to human immunodeficiency virus
el-Sadr W; Marmor M; Zolla-Pazner S; Stahl RE; Lyden R; William D; D'Onofrio S; Weiss SH; Saxinger WC
1987 Apr;155(4):789-793, Journal of infectious diseases
— id: 9120, year: 1987, vol: 155, page: 789, stat: Journal Article,

Detection of HTLV-III antibody in cerebrospinal fluid from patients with AIDS and pre-AIDS with the use of a commercial test system
Johnson JE; Zolla-Pazner S
1987 Sep;88(3):351-353, American journal of clinical pathology
Sixty-one paired sera and cerebrospinal fluid (CSF) specimens were tested for the presence of human T-cell lymphotropic virus/lymphadenopathy-associated virus (HTLV-III) antibodies. Of 16 sera negative for HTLV-III antibodies by enzyme-linked immunosorbent assay (ELISA), the corresponding CSF results were also negative. Forty-two of 45 (93%) CSF specimens tested (in which the corresponding sera were HTLV-III antibody positive) were positive by ELISA. Western blot analysis (WB) of 24 paired specimens, as well as retrospectively acquired case histories, confirmed the results obtained by ELISA.
— id: 9304, year: 1987, vol: 88, page: 351, stat: Journal Article,

Serum IgD elevation is an early marker of B cell activation during infection with the human immunodeficiency viruses
Mizuma H; Zolla-Pazner S; Litwin S; el-Sadr W; Sharpe S; Zehr B; Weiss S; Saxinger WC; Marmor M
1987 Apr;68(1):5-14, Clinical & experimental immunology
Serum IgD levels in individuals infected with the human immunodeficiency viruses (HIV) were studied as a means of monitoring the character and timing of B cell activation in individuals with this infection. Significantly increased levels of IgD were characteristic of homosexual men who were HIV seropositive but asymptomatic or mildly symptomatic. The hyper IgD globulinaemia became progressively more pronounced in patients with increasingly severe infection and reached its most marked level in patients with AIDS-related complex (ARC). In ARC patients, IgD levels were increased 8.8-fold above normal which was disproportionately greater than the 2.4-fold increase in IgG, the 1.8-fold increase in IgA and the 1.6-fold increase in IgM. IgD levels declined in AIDS patients (although remained elevated compared to controls). The data suggest that an unusual type of B cell activation is responsible for the unique pattern of hypergammaglobulinaemia seen in this disease and that the B cell activation occurs early in the pathogenesis of HIV infection, often before development of symptoms, and continues throughout the course of infection
— id: 9121, year: 1987, vol: 68, page: 5, stat: Journal Article,

Nonrandom development of immunologic abnormalities after infection with human immunodeficiency virus: implications for immunologic classification of the disease
Zolla-Pazner S; Des Jarlais DC; Friedman SR; Spira TJ; Marmor M; Holzman R; Mildvan D; Yancovitz S; Mathur-Wagh U; Garber J; et al
1987 Aug;84(15):5404-5408, Proceedings of the National Academy of Sciences of the United States of America
Blood specimens from 165 intravenous drug users who were seropositive for the human immunodeficiency virus (HIV), from 158 seropositive homosexual men with lymphadenopathy, and from 77 patients with acquired immunodeficiency syndrome (AIDS) were assessed immunologically. Immunologic parameters were analyzed by the Guttman scalogram technique to determine if immunologic abnormalities occurred in a nonrandom pattern. The following four patterns emerged: (i) seropositivity for HIV with no immunologic abnormalities; (ii) seropositivity for HIV with a depressed T4/T8 cell ratio; (iii) seropositivity with a depressed T4/T8 cell ratio and T4-cell depletion; and (iv) seropositivity with a depressed T4/T8 cell ratio, T4-cell depletion, and lymphopenia. Ninety-two to 100% of subjects in each of the three groups of patients were found \'to scale\' because the abnormalities occurred in the cumulative, ordered fashion described. This nonrandom occurrence of abnormalities indicates an ordered progression of immunologic abnormalities in individuals infected with HIV, a finding useful in the staging of both symptomatic and asymptomatic HIV-seropositive subjects.
— id: 9305, year: 1987, vol: 84, page: 5404, stat: Journal Article,

Potential use of serotherapy in the prevention and treatment of infection with the human immunodeficiency virus
Zolla-Pazner S; Pinter A; Mizuma H
1987 Aug;17(1-2):45-53, Journal of virological methods
While prevention of infection with the human immunodeficiency virus (HIV) using prophylactic immunization and treatment with anti-viral drugs would appear to be the methods of choice for the prevention and treatment of this infection, neither safe and effective vaccines nor anti-viral agents have yet been developed. A third approach should thus be considered which could be employed both for prophylaxis and treatment of this disease. This approach utilizes specific, anti-HIV antibodies, passively administered, to prevent and/or slow the infectious process. The disadvantages of using xenogeneic antibodies and the advantages of using human antibodies are discussed. The need for large quantities of human antibodies to HIV necessitates the production of cell lines producing these antibodies. The various techniques of producing these lines are summarized. Finally, preliminary data supporting the feasibility of producing human cell lines producing antibody to HIV are presented
— id: 9306, year: 1987, vol: 17, page: 45, stat: Journal Article,

A stage model of HTLV-III LAV infection in intravenous drug users
Des Jarlais DC; Friedman SR; Spira TJ; Zolla-Pazner S; Marmor M; Holzman R; Mildvan D; Yancovitz S; Mathur-Wagh U; Garber J; et al
1986 ;67:328-334, NIDA research monograph series
— id: 9123, year: 1986, vol: 67, page: 328, stat: Journal Article,

Immunosuppression and infection in multiple myeloma
Jacobson DR; Zolla-Pazner S
1986 Sep;13(3):282-290, Seminars in oncology
Patients with multiple myeloma are at increased risk of severe bacterial infection. A variety of immune deficits has been described in such patients, including a decreased primary antibody response and defects in complement and granulocyte function. The depressed humoral response appears to result primarily from the activity of suppressor monocytes. Pneumovax (Merck Sharp & Dohme, West Point, Penn) should be administered to patients with myeloma, although its effectiveness in this population has not been proven. The role of other potential modalities of treatment and prophylaxis, such as IV gamma globulin, requires further study
— id: 9308, year: 1986, vol: 13, page: 282, stat: Journal Article,

IGD SECRETION AS A MARKER OF B-CELL ACTIVATION IN AIDS
ZOLLAPAZNER, S; MIZUMA, H; ELSADR, W; SHARPE, S; LITWIN, S
1986 JAN ;4(6):107-107, Journal of cellular biochemistry
— id: 41515, year: 1986, vol: 4, page: 107, stat: Journal Article,

Control of B cell proliferation: inhibition of responses to B cell mitogens induced by plasma cell tumors
Berman JE; Zolla-Pazner S
1985 May;134(5):2872-2878, Journal of immunology
A multitude of factors has been described that positively and negatively regulate B cell proliferation. A model system for the study of negative control of B cell function is provided by mice bearing plasmacytomas (PC-mice). In PC-mice, the primary immune response, as measured by development of antibody-forming cells (AFC), is severely suppressed. The present report specifically identifies a block in B cell proliferation as the apparent cause of this reduction in AFC production. Thus, the proliferative response of B cells from the spleens of PC-mice (PC-spleens) was significantly impaired when stimulated with four different B cell mitogens (lipopolysaccharide, Salmonella typhimurium mitogen, anti-mu conjugated to Sepharose, and 8-mercaptoguanosine). Nevertheless, the mitogen-responsiveness of these B cells was recovered when they were segregated by various methods from macrophages. These data suggest that the proliferative ability of the B cells in PC-spleens is inherently normal. In concordance with this conclusion, it was shown that suppressor cells from PC-spleens can block the proliferation of normal B cells derived from nontumor-bearing mice. This inhibition does not require direct cell contact and is mediated via soluble factors. The relevance of these results to previous studies of PC-induced immunosuppression and to the control of normal B cell proliferation is discussed.
— id: 9309, year: 1985, vol: 134, page: 2872, stat: Journal Article,

The acquired immunodeficiency syndrome: an ultrastructural study
Sidhu GS; Stahl RE; el-Sadr W; Cassai ND; Forrester EM; Zolla-Pazner S
1985 Apr;16(4):377-386, Human pathology
Blood and a variety of tissues from 97 patients with the acquired immunodeficiency syndrome (AIDS) and 25 with the AIDS prodrome were studied ultrastructurally. Tubuloreticular structures (TRS) were found in 85 per cent of the patients with AIDS and in 92 per cent of those with the prodrome. Test tube and ring-shaped forms (TRF), found in 41 per cent of the patients with AIDS and in 8 per cent of those with the prodrome, increased with disease progression. Among the patients with AIDS, as the number of sites examined per case increased, the incidence of TRS and TRF tended to approach 100 per cent, suggesting that they are present in all patients with AIDS. Other changes seen frequently were immunologic capping of blood lymphocytes, intramitochondrial iron in blood reticulocytes and marrow normoblasts, megakaryocytic immaturity and platelet phagocytosis, collections of membranous rings in hepatocytic cytoplasm, suggestive of non-A, non-B hepatitis, and proliferations and engorgement of hepatic Ito cells with lipid. The data suggest that TRS and TRF can be used as diagnostic and prognostic markers.
— id: 9310, year: 1985, vol: 16, page: 377, stat: Journal Article,

BETA-2-MICROGLOBULIN AND THE ACQUIRED IMMUNODEFICIENCY SYNDROME IN A LOW-INCIDENCE AREA - REPLY
Zollapazner, S
1985 ;253(1):44-44, JAMA
— id: 31003, year: 1985, vol: 253, page: 44, stat: Journal Article,

SYMPOSIUM HELD IN THE HONOR OF DR ZOLTAN OVARY AT NEW-YORK- UNIVERSITY-MEDICAL-CENTER ON NOVEMBER 26, 1984
Zollapazner, S; Thorbecke, GJ
1985 ;94(1):254-264, Cellular immunology
— id: 30872, year: 1985, vol: 94, page: 254, stat: Journal Article,

Changes in splenic histology and cytology in mice bearing plasmacytomas
Berman JE; Zolla-Pazner S
1984 Aug;87(1):137-146, Cellular immunology
Studies of immunosuppression in plasmacytoma-bearing mice (PC-mice) yield important information for understanding a variety of immune phenomena. Most investigations of this model system have utilized splenic cells; thus, valid interpretation of much of this data rests on knowledge of the nature of the cells present in the spleens of PC-mice (PC-spleen). Nevertheless, no comprehensive description of PC-spleens has ever been made and is therefore the subject of this report. Major differences exist between normal and PC-spleens. PC-spleens are enlarged and contain an increased number of cells, the greater proportion of which are large in size. On the basis of morphology and expression of cell surface markers the absolute number of B cells and T cells per spleen was found to be normal or somewhat increased in PC-mice. However, the percentage of these cells was decreased due to an increase in other cell types causing changes predominantly in the red pulp of the spleen. The major populations expanded are polymorphonuclear leukocytes and macrophages. The numbers of megakaryocytes, immature precursor cells, and metastatic tumor cells are also increased to a smaller degree. The implications and relevance of these data to studies of PC-induced immunosuppression are discussed.
— id: 9311, year: 1984, vol: 87, page: 137, stat: Journal Article,

Kaposi's sarcoma among four different AIDS risk groups
De Jarlais DC; Marmor M; Thomas P; Chamberland M; Zolla-Pazner S; Sencer DJ
1984 Apr 26;310(17):1119-1119, New England journal of medicine
— id: 9127, year: 1984, vol: 310, page: 1119, stat: Journal Article,

The acquired immune deficiency syndrome: laboratory findings, clinical features, and leading hypotheses
El-Sadr W; Stahl R; Sidhu G; Zolla-Pazner S
1984 ;2(2):73-85, Diagnostic immunology
— id: 9313, year: 1984, vol: 2, page: 73, stat: Journal Article,

Culture and recovery of macrophages and cell lines from tissue culture-treated and -untreated plastic dishes
Fleit SA; Fleit HB; Zolla-Pazner S
1984 Mar 30;68(1-2):119-129, Journal of immunological methods
Macrophages can be separated from other cell types by their ability to readily attach and spread on glass or on plastic surfaces which are treated for optimal growth of cultured cells (tissue culture-treated plastic). To detach macrophages from these surfaces, techniques must be used which require prior preparation of special flasks or vessels, utilize expensive equipment, are time-consuming and almost uniformly require that the macrophages be exposed to various chemicals. We now report that macrophages can be enriched and recovered efficiently after attachment to disposable polystyrene bacteriologic petri dishes simply by gentle scraping with a rubber policeman. In this paper we compare this method to others currently in use in which resident peritoneal cells, peritoneal exudate cells or cells from bone marrow-derived cultures are detached from treated dishes using cold shock, chelating agents and lidocaine. In all studies, advantages were noted when cells were incubated in untreated dishes and detached by gentle scraping. In addition, untreated dishes supported the growth of adherent cell lines IC-21 and L929B and yielded large numbers of cells, with high viability, which were easily harvested.
— id: 9312, year: 1984, vol: 68, page: 119, stat: Journal Article,

Immunologic abnormalities among male homosexuals in New York City: changes over time
Marmor M; el-Sadr W; Zolla-Pazner S; Lazaro C; Stahl RE; William D
1984 ;437:312-319, Annals of the New York Academy of Sciences
— id: 9129, year: 1984, vol: 437, page: 312, stat: Journal Article,

Kaposi\'s sarcoma in homosexual men. A seroepidemiologic case-control study
Marmor M; Friedman-Kien AE; Zolla-Pazner S; Stahl RE; Rubinstein P; Laubenstein L; William DC; Klein RJ; Spigland I
1984 Jun;100(6):809-815, Annals of internal medicine
The cases of 20 male homosexuals with Kaposi\'s sarcoma and the acquired immunodeficiency syndrome were compared with those of 40 age- and race-matched male homosexual controls. Patients with Kaposi\'s sarcoma had lower OKT4/OKT8 (T-helper/T-suppressor) ratios than controls, due to smaller numbers of OKT4 cells. Serum IgG concentrations and antibody titers to cytomegalovirus in patients exceeded those in controls, but patients had lower antibody titers to Epstein-Barr virus. Logistic regression analysis comparing patients with controls showed significant relative risks for Kaposi\'s sarcoma associated with the number of partners per month in receptive anal-genital intercourse, occasions per month of \' fisting ,\' and cytomegalovirus antibody titers. Cytomegalovirus titers also were inversely correlated with OKT4 cell concentrations in the control group. Significantly greater OKT4 cell concentrations were found at diagnosis in HLA-DR5-positive patients than in HLA-DR5-negative patients. Patients who have HLA-DR5 may express disease at lesser degrees of immunodeficiency than HLA-DR5-negative patients.
— id: 9126, year: 1984, vol: 100, page: 809, stat: Journal Article,

Osteonecrosis of the femoral head associated with pregnancy. Report of three cases
Pellicci PM; Zolla-Pazner S; Rabhan WN; Wilson PD Jr
1984 May;(185):59-63, Clinical orthopaedics & related research
Osteonecrosis of the femoral head developed in three patients during pregnancy. There were no demonstrable predisposing factors. The symptoms were first noted during the last trimester and persisted following delivery. Results of examination, radiography, and scanning were consistent with a diagnosis of osteonecrosis, and in one patient the diagnosis was substantiated by biopsy at the time of core decompression and bone grafting. An association between pregnancy and osteonecrosis, while established by case reports in the literature, does not seem to be recognized by obstetricians or orthopedic surgeons
— id: 47453, year: 1984, vol: , page: 59, stat: Journal Article,

ULTRASTRUCTURAL FEATURES OF ACQUIRED IMMUNE-DEFICIENCY SYNDROME
SIDHU, GS; STAHL, RE; ELSADR, W; ZOLLAPAZNER, S
1984 ;50(Suppl 1):A54-A54, Laboratory investigation
— id: 40861, year: 1984, vol: 50, page: A54, stat: Journal Article,

The use of beta-2 microglobulin in the diagnosis of AIDS, suspected AIDS, and preclinical AIDS
Zolla-Pazner S; el-Sadr W; William D; Stahl R; Marmor M
1984 ;437:526-529, Annals of the New York Academy of Sciences
— id: 9128, year: 1984, vol: 437, page: 526, stat: Journal Article,

Quantitation of beta 2-microglobulin and other immune characteristics in a prospective study of men at risk for acquired immune deficiency syndrome
Zolla-Pazner S; William D; El-Sadr W; Marmor M; Stahl R
1984 Jun 8;251(22):2951-2955, JAMA
Serum samples from 24 patients with acquired immune deficiency syndrome (AIDS) and from 15 patients with an early or milder form of this disease (\'suspected AIDS\') were found to contain elevated levels of beta 2-microglobulin (beta 2M). Therefore, prospective studies of 40 asymptomatic homosexual men from New York City were undertaken to determine whether quantitation of beta 2M and other immunologic variables was useful in recognizing those in populations at high risk for this disease who have a high probability for experiencing symptoms consistent with AIDS. After 20 to 26 months of follow-up, two of those persons now have AIDS and four have suspected AIDS. All six of these persons had elevated serum beta 2M levels and other immunologic abnormalities when they entered this study. Of those tested, only one other man had an increased level of beta 2M; neither he nor any of the remaining 33 persons in this group developed AIDS.
— id: 9125, year: 1984, vol: 251, page: 2951, stat: Journal Article,

B-CELLS IN THE PATHOGENESIS OF AIDS
ZOLLAPAZNER, S
1984 ;5(10):289-291, Immunology today
— id: 41040, year: 1984, vol: 5, page: 289, stat: Journal Article,

Identification of a new marker (Ly RL male 1) for cells of the T lineage by an auto-antithymocyte serum
Basch RS; Buxbaum JN; Zolla-Pazner S
1983 Oct 1;81(1):144-156, Cellular immunology
The sera of mice surviving challenge with a Thy-1-negative variant of the thymoma RL male 1 contain antibodies which identify a new cell surface antigen (Ly RL male 1) present on cells of the T lineage. This antigen appears early in the development of the lineage and it can be detected on most thymocyte precursors. Its presence on prothymocytes can serve to distinguish these cells from their multipotential precursors. The antigen is present on many thymocytes, and dividing thymocytes are more susceptible to its cytotoxic activity than is the total population. Ly RL male 1-antigen-positive cells can be detected in peripheral lymphoid tissues by both functional assays and absorption. Treatment of peripheral lymphoid cells with the antiserum leads to significant reduction in MLR and helper activity but does not alter mitogen reactivity or lymphocytotoxicity. Animals with significant serum levels of anti-RL male 1 are deficient in their ability to produce IgG antibody to sheep erythrocytes.
— id: 9316, year: 1983, vol: 81, page: 144, stat: Journal Article,

ALTERED B-CELL PROLIFERATION AND SURFACE IG EXPRESSION IN PLASMACYTOMA-BEARING MICE
BERMAN, J; ZOLLAPAZNER, S
1983 ;42(4):943-943, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 40714, year: 1983, vol: 42, page: 943, stat: Journal Article,

Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS)
Gallo RC; Sarin PS; Gelmann EP; Robert-Guroff M; Richardson E; Kalyanaraman VS; Mann D; Sidhu GD; Stahl RE; Zolla-Pazner S; Leibowitch J; Popovic M
1983 May 20;220(4599):865-867, Science
Several isolates of a human type-C retrovirus belonging to one group, known as human T-cell leukemia virus (HTLV), have previously been obtained from patients with adult T-cell leukemia or lymphoma. The T-cell tropism of HTLV and its prevalence in the Caribbean basin prompted a search for it in patients with the epidemic T-cell immune deficiency disorder known as AIDS. Peripheral blood lymphocytes from one patient in the United States and two in France were cultured with T-cell growth factor (TCGF) an shown to express HTLV antigens. Virus from the U.S. patient was isolated and characterized and shown to be related to HTLV subgroup I. The virus was also transmitted into normal human T cells from umbilical cord blood of a newborn. Whether or not HTLV-I or other retroviruses of this family with T-cell tropism cause AIDS, it is possible that patients from whom the virus can be isolated can also transmit it to others. If the target cell of AIDS is the mature T cell as suspected, the methods used in these studies may prove useful for the long-term growth of these cells and for the identification of antigens specific for the etiological agent of AIDS.
— id: 9317, year: 1983, vol: 220, page: 865, stat: Journal Article,

Ultrastructural markers of AIDS
Sidhu GS; Stahl RE; El-Sadr W; Zolla-Pazner S
1983 Apr 30;1(8331):990-991, Lancet
— id: 9318, year: 1983, vol: 1, page: 990, stat: Journal Article,

AIDS: a medical conundrum
Stahl RE; Friedman-Kien A; Zolla-Pazner S
1983 Dec;10(6):550-558, Journal of cutaneous pathology
— id: 9314, year: 1983, vol: 10, page: 550, stat: Journal Article,

Histiocytoid hemangioma with features of angiolymphoid hyperplasia and Kaposi's sarcoma. A study by light microscopy, electron microscopy, and immunologic techniques
Waldo E; Sidhu GS; Stahl R; Zolla-Pazner S
1983 Dec;5(6):525-538, American journal of dermatopathology
We examined by light and electron microscopy 99 vascular lesions removed on 17 occasions over a 2-year period from a 55-year-old black man. The lesions all showed histologic features of a vascular neoplasm composed of enlarged 'histiocytoid' endothelial cells and overlapping features of angiolymphoid hyperplasia with eosinophilia and Kaposi's sarcoma. An apparently unique feature was early loss of melanin from the overlying epidermis and a mononuclear inflammatory-cell infiltrate. The patient also had an abnormal immunologic state very similar to that seen in homosexuals with Kaposi's sarcoma.
— id: 9315, year: 1983, vol: 5, page: 525, stat: Journal Article,

Disseminated Kaposi\'s sarcoma in homosexual men
Friedman-Kien AE; Laubenstein LJ; Rubinstein P; Buimovici-Klein E; Marmor M; Stahl R; Spigland I; Kim KS; Zolla-Pazner S
1982 Jun;96(6 Pt 1):693-700, Annals of internal medicine
Nineteen cases from an epidemic of disseminated Kaposi\'s sarcoma in homosexual men were studied by clinical, virologic, immunologic, and genetic methods. The patients were all male homosexuals ranging in age from 29 to 52 years, with histories of multiple sexually transmitted diseases and exposure to both prescription and recreational drugs. Sites of disease included skin (16 of 19 patients), lymph nodes (13 patients), gastrointestinal tract (12 patients), spleen (three patients), and lung (one patient). Most patients had elevated levels of serum immunoglobins, positive antibody titers to hepatitis A and B virus, cytomegalovirus and Epstein-Barr virus, and impairment of cell-mediated immunologic reactions. The frequency of HLA-DR5 in these patients was significantly elevated. Two of the 19 patients died. Although the precise cause of this epidemic is unknown, it is likely that a genetic predisposition, an acquired immunoregulatory defect, and one or more infectious agents and drugs may be involved.
— id: 9135, year: 1982, vol: 96, page: 693, stat: Journal Article,

Mycobacterium avium-intracellulare: a cause of disseminated life-threatening infection in homosexuals and drug abusers
Greene JB; Sidhu GS; Lewin S; Levine JF; Masur H; Simberkoff MS; Nicholas P; Good RC; Zolla-Pazner SB; Pollock AA; Tapper ML; Holzman RS
1982 Oct;97(4):539-546, Annals of internal medicine
Five men developed disseminated infection with Mycobacterium avium-intracellulare. These patients all lived in the New York City area and presented with their illnesses between January 1981 and September 1981; four were homosexual and one was an intravenous drug abuser. Four patients died. All five patients had defects in the cell-mediated immune response. The infections were characterized histopathologically by poor or absent granulomatous tissue reaction. Clinical isolates of M. avium-intracellulare from all five patients agglutinated commonly used antimycobacterial drugs. The spectrum of opportunistic infections among populations of homosexuals and drug abusers should be expanded to include disseminated disease due to M. avium-intracellulare.
— id: 9319, year: 1982, vol: 97, page: 539, stat: Journal Article,

Immunologic abnormalities in homosexual men. Relationship to Kaposi\'s sarcoma
Stahl RE; Friedman-Kien A; Dubin R; Marmor M; Zolla-Pazner S
1982 Aug;73(2):171-178, American journal of medicine
Studies were performed to define the immunologic status of various groups of homosexual men including homosexual men with Kaposi\'s sarcoma, healthy homosexual men who were of similar ages to the homosexual patients with Kaposi\'s sarcoma and homosexual men with hyperplastic lymphadenopathy. Heterosexual men with Kaposi\'s sarcoma were also studied. Immunologic parameters which were examined included serum immunoglobulin levels, enumeration of B cells, T cells, and T-cell subsets, and quantitation of lymphocyte responsive to phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Significant immunologic abnormalities were observed in all three groups of homosexuals studied. These were most severe in the homosexuals with Kaposi\'s sarcoma, somewhat less severe in homosexual men with lymphadenopathy, and least marked but still significant in healthy homosexual men. Heterosexual men with Kaposi\'s sarcoma displayed essentially normal immunologic profiles. The possible etiologic factors underlying the immunologic abnormalities in the male homosexual population studied and the role of an altered immune system in the development of and the fulminant course of Kaposi\'s sarcoma in these patients are discussed.
— id: 9134, year: 1982, vol: 73, page: 171, stat: Journal Article,

IMMUNE ABNORMALITIES IN HOMOSEXUAL MEN WITH KAPOSIS SARCOMA
Stahl, R; Friedmankien, A; Dubin, R; Marmor, M; Zollapazner, S
1982 ;41(4):955-955, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 30458, year: 1982, vol: 41, page: 955, stat: Journal Article,

Immunoregulatory circuits in myeloma
Ullrich S; Zolla-Pazner S
1982 Feb;11(1):87-111, Clinics in haematology
— id: 9320, year: 1982, vol: 11, page: 87, stat: Journal Article,

PURIFICATION OF AN IMMUNOREGULATORY FACTOR RELEASED BY MALIGNANT PLASMA-CELLS
Ullrich, S; Zollapazner, S
1982 ;41(3):480-480, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 30482, year: 1982, vol: 41, page: 480, stat: Journal Article,

PURIFICATION OF A SOLUBLE IMMUNOREGULATORY SUBSTANCE RELEASED BY PLASMACYTOMA CELLS
ULLRICH, SE; ZOLLAPAZNER, S
1982 ;24(10):293-299, UCLA symposia on molecular & cellular biology
— id: 40380, year: 1982, vol: 24, page: 293, stat: Journal Article,

Markers of macrophage heterogeneity: altered frequency of macrophage subpopulations after various pathologic stimuli
Roubin R; Kennard J; Foley D; Zolla-Pazner S
1981 Jun;29(6):423-432, Journal of the Reticuloendothelial Society
— id: 9322, year: 1981, vol: 29, page: 423, stat: Journal Article,

Studies of antibody affinity in plasmacytoma-bearing mice: evidence for a maturational defect of B lymphocytes
Zolla-Pazner S; Gilbert M; Fleit SA
1981 Jul 15;62(1):149-155, Cellular immunology
— id: 9321, year: 1981, vol: 62, page: 149, stat: Journal Article,

CHARACTERIZATION OF THE IGM RECEPTOR ON BONE-MARROW MACROPHAGES
ZOLLAPAZNER, S; FUHRER, E; DECKER, M
1981 ;40(3):364-364, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 40247, year: 1981, vol: 40, page: 364, stat: Journal Article,

Origin and function of suppressor macrophages in myeloma
Kennard J; Zolla-Pazner S
1980 Jan;124(1):268-273, Journal of immunology
— id: 9325, year: 1980, vol: 124, page: 268, stat: Journal Article,

A disposable diffusion chamber system used to study immunoregulation by soluble factors
Ullrich SE; Zolla-Pazner S
1980 ;39(1-2):147-154, Journal of immunological methods
A disposable two-chamber culture system is described which is useful for studying various soluble factors which regulate the immune response. This method requires small numbers of cells and less than 2 ml of medium per culture. The vessel is compact, relatively inexpensive and all component parts are readily available from commercial suppliers. This culture system circumvents the major problems associated with dual chamber methods previously developed for the study of immunoregulatory molecules affecting antibody production.
— id: 9324, year: 1980, vol: 39, page: 147, stat: Journal Article,

Osteonecrosis of the femoral head during pregnancy
Zolla-Pazner S; Pazner SS; Lanyi V; Meltzer M
1980 Aug 15;244(7):689-690, JAMA
— id: 9323, year: 1980, vol: 244, page: 689, stat: Journal Article,

MEDIATION OF PLASMACYTOMA-INDUCED IMMUNOSUPPRESSION BY 2 SOLUBLE FACTORS
Kennard, J; Zollapazner, S
1979 ;38(3):1426-1426, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 30150, year: 1979, vol: 38, page: 1426, stat: Journal Article,

Markers of macrophage heterogeneity. I. Studies of macrophages from various organs of normal mice
Roubin R; Zolla-Pazner S
1979 Dec;9(12):972-978, European journal of immunology
A unique subpopulation of macrophages (M phi) was identified among the spleen and bone marrow M phi of normal mice. After 24 h of culture, approximately 2.5% of the adherent cells cluster into 'foci' of 10-30 cells. On the basis of their phagocytic and morphologic characteristics, these focus-forming M phi (FF-M phi) appeared to be highly activated. Uncoated sheep erythrocytes (E) were ingested by FF-m phi indicating that opsonization was not a prerequisite for phagocytosis. However, IgM-coated E (EIgM) were more readily phagocytosed by FF-M phi than were E suggesting that IgM is recognized as an effective opsonin by these cells. EIgM and E coated with IgM and complement (C) (EIgMC) were ingested by approximately the same percentage of FF-M phi; thus, if these cells possess complement receptors in addition to structures which bind EIgM, the C receptors do not enhance the ability of FF-M phi to ingest opsonized particles. The non-focus-forming M phi, e.g. individual M phi (I-M phi), in the spleen and bone marrow can, themselves, be divided into various subpopulations distinguished by their ability to bind and ingest E, EIgM ana EIgMC. These may represent various subpopulations of M phi or M phi at various stages of activation or differentiation. While spleen and bone marrow M phi contained FF-M phi and I-M phi which vary in their ability to ingest E, EIgM and EIgMC, theM phi of the peritoneum and blood of normal mice were far more homogeneous. Peritoneal and blood M phi did not form foci, ad did not ingest E or EIgM in significant amounts although a small percentage were able to ingest EIgMC. These data suggest that the population of M phi in the spleen and bone marrow are far more heterogenous than those found in the peritoneum or blood and that binding and phagocytosis of various coated and uncoated erythrocytes can be studied to elucidate this heterogeneity.
— id: 9326, year: 1979, vol: 9, page: 972, stat: Journal Article,

Humoral mediator of antigenic competition demonstrated in vivo
Zolla-Pazner S; Koehne C; Oratz R
1979 ;59(3):349-356, International archives of allergy & immunology
To determine if a soluble mediator of antigenic competition could be demonstrated in vivo, various groups of mice received implants of intraperitoneal Millipore diffusion chambers containing normal spleen cells and sheep erythrocytes (SRC). Priming and boosting the chamber hosts so that a vigorous secondary immune response to horse erythrocytes (HoRC) coincided with chamber implantation resulted in the suppression of the anti-SRC response of the chamber-enclosed cells. Similarly, passive immunization of the chamber hosts with SRC-absorbed anti-HoRC hyperimmune serum suppressed the response of the chamber-enclosed cells to SRC. Thus, serum from hyperimmune mice contains a humoral suppressor substance which mediates antigenic competition.
— id: 9327, year: 1979, vol: 59, page: 349, stat: Journal Article,

SUPPRESSION OF INVITRO ANTIBODY-RESPONSE BY A SOLUBLE FACTOR FROM SPLEEN-CELLS OF PLASMACYTOMA-BEARING MICE
Kennard, J; Cooper, NS; Zollapazner, S
1978 ;37(6):1664-1664, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 29806, year: 1978, vol: 37, page: 1664, stat: Journal Article,

HIGHLY ACTIVATED MACROPHAGES IN NORMAL MURINE SPLEEN AND BONE- MARROW
Roubin, R; Kennard, J; Zollapazner, S
1978 ;37(6):1655-1655, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 29805, year: 1978, vol: 37, page: 1655, stat: Journal Article,

Suppression of the humoral immune response by plasmacytomas: mediation by adherent mononuclear cells
Kolb JP; Arrian S; Zolla-Pazner S
1977 Feb;118(2):702-709, Journal of immunology
Mice bearing plasmacytomas have a severely impaired ability to mount a primary immune response; T cells from these mice, however, appear by both in vivo and in vitro criteria to function normally. This unusual pattern of immunodeficiency appears to be mediated by a regulatory cell found in the spleens and peritoneal cavities but not in the lymph nodes or thymuses of mice bearing plasmacytomas. The number of cells with suppressor activity in the spleens of plasmacytoma-bearing mice is directly proportional to the size of the subcutaneous tumor borne by the host. These cells are capable of suppressing antibody production in in vitro cultures of normal spleen cells but have no demonstrable effect on the ability of normal spleen cells to proliferate in vitro in response to phytohemagglutinin or 8-Br-guanosine-3', 5'-monophosphate (T and B cell mitogens, respectively). Characterization of the suppressor cell population on the basis of its cell surface properties, its radioresistance, its morphology, and its ability to adhere to various solid matrices suggest that these cells are adherent mononuclear cells. These data support the concept that plasma cell tumors indirectly induce an impairment in the humoral immune response of their hosts by stimulating the expression of regulatory functions in a population of splenic and peritoneal macrophages.
— id: 9328, year: 1977, vol: 118, page: 702, stat: Journal Article,

Cellular specificity of plasmacytoma-induced immunosuppression
Zolla-Pazner S; Sullivan B; Richardson D
1976 Aug;117(2):563-568, Journal of immunology
The pattern of immunodeficiency in plasmacytoma-bearing mice appears to be unique. Mice bearing these tumors exhibit a severe impairment in their ability to mount a primary immune response to thymus-dependent and -independent antigens. However, cell-mediated immune functions in these mice apparently remain intact. Thus, when T cell activity of lymph node cells from plasmacytoma-bearing mice was tested in vivo by sensitization with dinitrofluorobenzene and in vitro by responsiveness to phytohemmagglutinin, allogeneic cells, and dinitrobenzene sulfonate, cell-mediated immunity was found to be normal.
— id: 9329, year: 1976, vol: 117, page: 563, stat: Journal Article,

The synthesis and assembly of immunoglobulins by malignant human plasmacytes. 3. Heterogeneity in IgA polymer assembly
Buxbaum, J N; Zolla, S; Scharff, M D; Franklin, E C
1974 May;4(5):367-369, European journal of immunology
— id: 99124, year: 1974, vol: 4, page: 367, stat: Journal Article,

RESTORATION OF IMMUNE COMPETENCE IN TOLERANT MICE BY PARABIOSIS TO NORMAL MICE
ZOLLA, S; NAOR, D
1974 ;140(5):1421-1426, Journal of experimental medicine
— id: 98741, year: 1974, vol: 140, page: 1421, stat: Journal Article,

CELLULAR BASIS OF IMMUNODEPRESSION IN MICE WITH PLASMACYTOMAS
ZOLLA, S; NAOR, D; TANAPATC.P
1974 ;112(6):2068-2076, Journal of immunology
— id: 98748, year: 1974, vol: 112, page: 2068, stat: Journal Article,