Miroslaw K Gorny, Ph.D., M.D.

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-V2 monoclonal antibodies neutralize tier 1 HIV-1 pseudoviruses
Gorny M.K.; Williams C.; O'Neal T.; Wang X.; Seaman M.S.; Zolla-Fazner S.
2011 ;27(10):A9-A9, AIDS research & human retroviruses
Background: Antibodies (Abs) to the V2 region of gp120 are present in a minority of HIV-infected subjects, and, to date, little has been published on their immunologic or functional properties. Recent analysis of plasma from the RV144 clinical vaccine trial, however, revealed that anti-V2 Abs were present in almost all plasma from vaccine recipients, and their contribution to protection is being extensively analyzed. Methods: A panel of seven human anti-V2 monoclonal Abs (mAbs), generated previously using cellular techniques from FBMCs of individuals infected with clade B HIV-1, was analyzed for their immunoglobulin variable gene usage, ELISA cross- reactivity to recombinant gp120 derived from viruses from clades A, B and C, and neutralizing activity against pseudoviruses using the TZM-bl cell assay. Results: All the anti-V2 mAbs are IgG1 except for one which is IgG3. Five mAbs were sequenced: four use the same IGHV1-69 gene segment, suggesting preferential gene usage by Abs targeting this region. The remaining mAb uses the IGHV4-34 gene and has very different immunologic characteristics, including interference with sCD4 binding. All seven V2 mAbs are highly cross-reactive with comparable relative affinities. They bind to 19 recombinant gp120s representing eight from clade B, ten from clade C and, one from clade A. Four anti-V2 mAbs were tested for their neutralizing activity. Tier 1 pseudoviruses were neutralized, including six from clade B and two from clades A and C. The 50% neutralizing values ranged between < 0. 8 and 88. 7ig/ml, with a mean of 16. 6 ig/ml. Conclusion: The results indicate that human anti-V2 mAbs display cross-clade neutralization against Tier 1 pseudoviruses in the TZM. bl assay
— id: 139489, year: 2011, vol: 27, page: A9, 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,

Longitudinal Study of Primary HIV-1 Isolates in Drug-Naive Individuals Reveals the Emergence of Variants Sensitive to Anti-HIV-1 Monoclonal Antibodies
Haldar, Bijayesh; Burda, Sherri; Williams, Constance; Heyndrickx, Leo; Vanham, Guido; Gorny, Miroslaw K; Nyambi, Phillipe
2011 ;6(2):e17253-e17253, PLoS ONE
To study how virus evolution affects neutralization sensitivity and to determine changes that occur in and around epitopes, we tested the ability of 13 anti-HIV-1 gp120 (anti-V2, anti-V3, anti-CD4bd and anti-carbohydrate) human monoclonal antibodies (mAbs) to neutralize sequential viruses obtained from five HIV-1 chronically infected drug naive individuals. Overall, primary viruses collected from patients at first visit were resistant to neutralization by all anti-HIV-1 mAbs with the exception of one virus sensitive to IgG1b12. Four of the five patients' viruses evolved increased sensitivity to neutralization by anti-V3 mAbs. Virus collected from a patient obtained 31 months later, evolved increased sensitivity to anti-V2, anti-V3, and anti-CD4bd mAbs. Furthermore, the anti-V2 and anti-CD4bd mAbs also exhibited increased neutralization capacities against virus collected from a patient 29 months later. Of the seven anti-V3 mAbs, five showed increased potency to neutralize the evolved virus from a patient collected after 11 months, and three exhibited increased potency against viruses from two patients collected 29 and 36 months later. Anti-V3 mAbs exhibited the most breadth and potency in neutralizing the evolving viruses. Sequence analysis of the envelope regions revealed amino acid conservation within the V3 loop, while most of the changes identified occurred outside the core epitopes and in particular within the C3 region; these may account for increased neutralization sensitivity. These studies demonstrate that in vivo, HIV-1 can evolve increased neutralization sensitivity to mAbs and that the spectrum of neutralization capacities by mAbs can be broader when studied in longitudinal analysis
— id: 126515, year: 2011, vol: 6, page: e17253, 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,

Bunga Pad-induced ankle dermatitis in a figure skater
Mandell, Jenny A; Tlougan, Brook E; Cohen, David E
2011 Feb;22(1):58-59, Dermatitis
— id: 126511, year: 2011, vol: 22, page: 58, 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,

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,

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,

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,

Primary HIV-1 isolates in infected drug naive individuals that evolve increase neutralization sensitivity to anti-gp120 monoclonal antibodies (mAbs)
Haldar, B.; Burda, S.; Williams, C.; Heyndrickx, L.; Vanham, G.; Gorny, M. K.; Nyambi, P. N.
2010 OCT ;26(10):A42-A43, AIDS research & human retroviruses
— id: 117313, year: 2010, vol: 26, page: A42, 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,

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,

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,

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,

Molecular design of a mimotope that preserves conserved structural elements of the HIV-1 V3 crown
Jiang, X; Totrov, M; Sampson, J; Williams, C; Gorny, MK; Zollla-Pazner, S; Kong, X
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105702, 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,

Sequential HIV-1 isolates from infected individuals reveals emergence of neutralization sensitive HIV-1 strains in the course of infection
Nyambi, PN; Burda, S; Williams, C; Heyndrickx, L; Vanham, G; Gorny, MK
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105701, 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 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,

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,

Evolution of HIV-1 Subtype B Viruses and Neutralization Patterns with Heterologous Anti-HIV-1 Antibodies
Burda, S; Urbanski, M; Williams, C; Heyndrickx, L; Janssens, W; Vanham, G; Gorny, M; Nyambi, PN
2008 OCT ;24(1):75-76, AIDS research & human retroviruses
— id: 91416, year: 2008, vol: 24, page: 75, 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,

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,

In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120
Visciano, Maria Luisa; Tuen, Michael; Gorny, Miroslaw K; Hioe, Catarina E
2008 Mar 15;372(2):409-420, Virology
The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus. Moreover, mice immunized with the gp120/anti-CD4bs mAb complexes produced higher titers of gp120-specific serum IgG and IgA than mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced antibody production was directed against V3 and correlated with better exposure of V3 on the gp120/anti-CD4bs mAb complexes. The higher antibody reactivity was evident against the homologous V3(LAI) peptide, but not against heterologous V3 peptides. Potent neutralization activity against HIV-1(LAI) was also observed in the sera from mice immunized with gp120/anti-CD4bs mAb complexes, although the sera exhibited poor neutralizing activities against other viruses tested. These results indicate that the anti-CD4bs antibodies alter the antigenicity and immunogenicity of gp120, leading to enhanced production of anti-gp120 antibodies directed particularly against the V3 region
— id: 78633, year: 2008, vol: 372, page: 409, 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,

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,

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,

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,

Human Antibodies: Editorial
Gorny MK
2005 ;14(3-4):57-58, Human antibodies
— id: 64451, year: 2005, vol: 14, page: 57, 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,

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,

Characterization of antibodies that inhibit HIV gp120 antigen processing and presentation
Tuen, Michael; Visciano, Maria Luisa; Chien, Peter C Jr; Cohen, Sandra; Chen, Pei-de; Robinson, James; He, Yuxian; Pinter, Abraham; Gorny, Miroslaw K; Hioe, Catarina E
2005 Sep;35(9):2541-2551, European journal of immunology
Antibodies to the CD4-binding site (CD4bs) of HIV-1 envelope gp120 have been shown to inhibit MHC class II presentation of this antigen, but the mechanism is not fully understood. To define the key determinants contributing to the inhibitory activity of these antibodies, a panel of anti-CD4bs monoclonal antibodies with different affinities was studied and compared to antibodies specific for the chemokine receptor-binding site or other gp120 regions. Anti-CD4bs antibodies that completely obstruct gp120 presentation exhibit three common properties: relatively high affinity for gp120, acid-stable interaction with gp120, and the capacity to slow the kinetics of gp120 proteolytic processing. None of these antibodies prevents gp120 internalization into APC. Notably, the broadly virus-neutralizing anti-CD4bs IgG1b12 does not block gp120 presentation as strongly, because although IgG1b12 has a relatively high affinity, it dissociates from gp120 more readily at acidic pH and only moderately retards gp120 proteolysis. Other anti-gp120 antibodies, regardless of their affinities, do not affect gp120 presentation. Hence, high-affinity anti-CD4bs antibodies that do not dissociate from gp120 at endolysosomal pH obstruct gp120 processing and prevent MHC class II presentation of this antigen. The presence of such antibodies could contribute to the dearth of anti-gp120 T helper responses in chronically HIV-1-infected patients
— id: 58742, year: 2005, vol: 35, page: 2541, 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,

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,

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,

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,

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,

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,

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,

Gamma heavy chain disease in man: independent structural abnormalities and reduced transcription of a functionally rearranged lambda L-chain gene result in the absence of L-chains
Teng MH; Rosen S; Gorny MK; Alexander A; Buxbaum J
2000 Jun;26(3):177-185, Blood cells, molecules, & diseases
Human Ig heavy chain diseases of the alpha and gamma classes are characterized by the absence of light chain production as well as the disease-defining abnormalities in the heavy chain protein. Prior studies have suggested concomitant structural defects in productively rearranged L-chain genes as the reason for the absent L-chain proteins. We have found that the single rearranged lambda L-chain gene in the OMM heavy chain disease (HCD) cell line has a mutation in the splice donor site at the 3' end of the J exon, resulting in direct splicing of the 3' end of the leader to the acceptor site of the constant region. The cells contain an mRNA consisting of the leader-coding region joined directly to the constant region. The V-region exon is skipped and the shortened mRNA is translated into a truncated protein containing no V-region amino acids. We have also noted that, in contrast to most normal and neoplastic Ig-producing cells, the OMM cells produce an excess of heavy to light chain mRNA as well as protein. The excess is independent of the structural gene abnormality and is due to a low level of L-chain transcription, which can be increased by fusing the HCD cell to the murine myeloma cell line NS-1 or transfecting the defective OMM L-chain gene into a murine plasma cell. The latter data suggest that the OMM cells either lack a transcription factor present in mature plasma cells or have a functional repressor of L-chain transcription
— id: 18580, year: 2000, vol: 26, page: 177, 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,

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,

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,

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,

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,

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,

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,

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,

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,

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,

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,

Role of the V2, V3, and CD4-binding domains of GP120 in curdlan sulfate neutralization sensitivity of HIV-1 during infection of T lymphocytes
Jagodzinski, P P; Wustner, J; Kmieciak, D; Wasik, T J; Fertala, A; Sieron, A L; Takahashi, M; Tsuji, T; Mimura, T; Fung, M S; Gorny, M K; Kloczewiak, M; Kaneko, Y; Kozbor, D
1996 Dec 15;226(2):217-227, Virology
A sulfated polysaccharide, curdlan sulfate (CRDS) with 1,3-beta-D-glucan as a main chain, inhibits HIV-1 infection of human peripheral blood lymphocytes (PBLs) by binding to the V3 region of gp 120. We previously showed that T cell (T)-tropic HIV-1 isolates are over 10-fold more sensitive to neutralization by CRDS than macrophage (MT)-tropic viruses, which possesses a relatively less charged amino acid composition in the V3 sequence. To analyze the interaction of CRDS with V3 and its association with neutralization sensitivity of HIV-1 isolates, we examined the effect of CRDS on the binding of neutralizing antibodies to monomeric and oligomeric gp 120 mutants of T- and MT-tropic HIV-1 clones in which the V3 loop was either deleted or substituted by V3 of another isolate. Our results showed that the presence and the amino acid composition of the V3 loop appears to determine the extent of interaction of CRDS with the V2 and CD4-binding regions on native gp 120 monomers; however, the positive charge of V3 has less effect on this interaction on oligomeric gp 120. Furthermore, our results established that only the CRDS-induced masking of V3 on oligomeric gp120 appears to be associated with the anti-HIV-1 activity of CRDS in vitro. Our findings underline the usefulness of CRDS for understanding the structural constraints on gp 120 that drive the transition from MT- to T-tropic isolates in vivo and enable the virus to use multiple fusion cofactors
— id: 93796, year: 1996, vol: 226, page: 217, 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,

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,

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,

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,

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,

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,

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,

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,

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,

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,

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,

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,

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,

Immunoregulatory effect of T cell subsets on PWM-induced IgG synthesis by blood lymphocytes in lung cancer
Gorny, M K; Jezewska, E; Skrzypczak, A; Slowik-Gabryelska, A; Zeromski, J
1991 ;39(3):243-252, Archivum immunologii & therapiae experimentalis
The mechanism of pokeweed mitogen (PWM) dependent decreased IgG production by blood lymphocytes from lung cancer patients was studied in comparison to control patients and blood donors. It has been shown that the depletion of monocytes has some influence on IgG synthesis but is not a decisive factor. Also, quantitative alterations in the CD4 and CD8 lymphocyte subsets do not significantly influence the PWM stimulation index for IgG synthesis. The assessment of T lymphocyte suppressor activity in lung cancer patients was performed by means of a co-culture with blood mononuclear cells, while helper activity was evaluated through co-culture with donor B lymphocytes. It has been found that lung cancer patient T lymphocytes have no increased suppressor activity, however, especially in the CD4 subset, display the decrease of helper function for B lymphocytes in PWM-induced IgG synthesis. The weakened helper function of CD4 lymphocytes may explain the suppression of specific antibody synthesis do novo which is evident in patients with lung cancer
— id: 93797, year: 1991, vol: 39, page: 243, 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,

Model-based optimization of infectivity parameters: a study of the early epidemic in San Francisco
Ahlgren, D J; Gorny, M K; Stein, A C
1990 ;3(6):631-643, Journal of acquired immune deficiency syndrome
By combining dynamic modeling of human immunodeficiency virus (HIV) transmission with mathematical optimization techniques, we calculate values of epidemiological parameters characterizing the early epidemic (1978-1986) among homosexual and bisexual men in San Francisco. The seroconversion fraction data reported by the San Francisco hepatitis B vaccine trials cohort study for this period is accurately simulated by a model assuming varying infectivity among three stages of HIV infection (early antigen stage, HIV antibody-positive stage, and AIDS stage). Using optimization techniques, we generate curves of the annual number of new partners and the annual number of risk contacts as functions of time. We project future case rates using optimized parameter values, and we study the sensitivity of these projections to variations in parameters, including the population size and the incubation period
— id: 93799, year: 1990, vol: 3, page: 631, stat: Journal Article,

Human monoclonal antibodies to the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein gp41 enhance HIV-1 infection in vitro
Robinson, W E Jr; Kawamura, T; Gorny, M K; Lake, D; Xu, J Y; Matsumoto, Y; Sugano, T; Masuho, Y; Mitchell, W M; Hersh, E
1990 Apr;87(8):3185-3189, Proceedings of the National Academy of Sciences of the United States of America
Three of 16 human monoclonal antibodies (hu-mAbs) enhanced human immunodeficiency virus type 1 (HIV-1) infection of MT-2 target cells by means of a mechanism that is dependent on complement. Enhanced infections are characterized by an increase in cytopathic effects and antigen synthesis as well as an increase in the production of progeny virus as detected by release of reverse transcriptase activity and infectious virus into the culture medium. Analyses by radioimmunoprecipitation, Western blot, and ELISA using the pENV9 envelope fragment localize the antigenic specificities of these three hu-mAbs to the N-terminal two-thirds of the transmembrane protein gp41. Competitive binding experiments indicate that the hu-mAbs are reactive with immunodominant epitopes of gp41 recognized by sera from essentially all HIV-1-infected subjects. Combination dose-effect experiments demonstrate that these hu-mAbs can act synergistically in vitro to enhance HIV-1 infection. These data demonstrate that hu-mAbs directed against the HIV-1 transmembrane glycoprotein gp41 can enhance HIV-1 infection in vitro. The availability of these reagents allows for the mapping of enhancing epitopes on HIV-1 and provides a means for studying whether deletion of such enhancing epitopes from candidate HIV-1 vaccines might improve the protective immune response to HIV-1 in immunized humans and chimpanzees
— id: 93798, year: 1990, vol: 87, page: 3185, 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,

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,

Assessment of phenotype of blood lymphocyte subsets prior and after PWM stimulation in patients with lung carcinoma
Jezewska, E; Gorny, M K; Skrzypczak, A; Slowik-Gabryelska, A; Zeromski, J
1989 ;59(1):31-36, Archiv fur Geschwulstforschung
Surface phenotypes of peripheral blood lymphocytes of lung cancer patients and those of two control groups were assessed by means of indirect immunofluorescence with monoclonal antibodies, prior and after 10 day pokeweed mitogen (PWM) in vitro stimulation. There was no significant alteration in pan T cell per cent values prior and after mitogen stimulation in all groups tested. CD4+ cells in lung cancer group were however significantly decreased as compared to blood donor group (37.3% vs 44.9%, p less than 0.05). This decline was even more pronounced in III/IVo stage of tumour progression according to TNM classification (36.8%, p less than 0.05). These changes, however were not cancer specific, because similar decrease of CD4+ cells was seen in a group of patients with nonneoplastic lung conditions (35.7%, p less than 0.01). Following 10 day PWM culture per cent values of CD4+ cells did not change significantly. The assessment of CD8+ lymphocytes has shown marked differences in two subgroups of lung cancer, namely in II (17.4%) and III/IV (26.2%) of tumour progression (p less than 0.05), which returned to normal values following PWM culture. CD4/CD8 ratio was distinctly depressed in cancer patients in relation to donors. The evaluation of surface markers of B lymphocytes activated cells and monocytes did not show significant alterations in all groups examined. Per cent of HNK1+ cells was heightened in cancer group, especially in III/IV stage of tumour progression in relation to donors (21.7% and 22.8% vs 17.3%, p less than 0.05 respectively). PWM stimulation resulted in marked fall of HNK1+ cells to values corresponding to those in donor group. This study indicates some alterations in per cent values of blood lymphocytes subpopulations belonging mainly to T cell lineage in lung cancer patients linked to tumour staging which only partially return to normal following PWM stimulation
— id: 93800, year: 1989, vol: 59, page: 31, 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,

Alloantibodies, autoantibodies, and immune complexes in patients with lung cancer
Gorny, M K; Lawniczak, M; Jenek, R; Slowik-Gabryelska, A; Kaczmarek, E; Zeromski, J
1988 ;166(2):97-105, Lung
The sera of patients with lung cancer, nonmalignant lung disease, and blood donors were subjected to various immunologic assays. Nine assays, based on immunoradiometric (IRMA) and immunoenzymatic (ELISA) principles, included 3 types of fetal cell antibodies, 2 established lung cancer cell antibodies, anti-DNA, anti-IgG autoantibodies, and immune complex assays based on C1q binding and anti-C3 activity. Antitumor cell antibody level was significantly lower in patients with lung cancer compared to blood donors. In the remaining 7 assays, the lung cancer patients tended towards higher median values compared to both control patients and blood donors, but without statistical significance, with the exception of anti-DNA antibodies. Statistical analysis of all 9 assays taken together has shown significant differences between the 3 groups. When only 5 assays were used to assess 3 types of fetal cell antibodies, anti-DNA antibodies, and immune complexes by means of ELISA anti-C3, the margins between groups increased. A range of values for the selected assays was established that may discriminate 70% of tested individuals of the 3 groups. These results suggest the existence of a characteristic profile of deranged humoral immunity in lung cancer patients
— id: 93802, year: 1988, vol: 166, page: 97, stat: Journal Article,

Evaluation of phenotype of mononuclear host cells isolated from primary tumour and peripheral blood of patients with laryngeal carcinoma
Zeromski, J; Pietrzak, J; Szmeja, Z; Jezewska, E; Gorny, M K; Kruk-Zagajewska, A
1988 Jan-Feb;105(1-2):149-154, Acta oto-laryngologica
Mononuclear host cells isolated from primary laryngeal carcinoma were assessed by means of indirect immunofluorescence with a panel of monoclonal antibodies against various lymphocyte subsets and macrophages. Tumours of various staging groups were examined in parallel with cells isolated from patient and donor peripheral blood (PBL). It was found that percentage values of cells bearing T3 and T4 phenotype were reduced both in tumour infiltrating cells (TIC) and in PBL population. The fall in T4+ cells in PBL from cancer patients in T3 and T4 staging groups was statistically significant (p less than 0.01) as compared with donor cells. Corresponding values for T8+ cells from TIC were increased in T1 and T2 staging groups of cancer, but showed a gradual fall in advanced stages. The T4+/T8+ cell ratio was decreased in both TIC and PBL cells. The HNK-1+ (NK) cell pattern in TIC was analogous to that for T8+ cells, i.e. the cell percentage decreased with advance in tumour growth. Corresponding values for OKM-1+ were increased in TIC and in patient blood, though in TIC they grew in proportion to tumour growth. Ia+ (HLA-DR+) cells in peripheral blood were significantly increased in patients versus those of donors (p less than 0.01), but only in T3 and T4 staging groups of examined cancer. These results show that subsets of tumour infiltrating cells in laryngeal carcinoma are a complex phenomenon, associated with growth and progression of tumour
— id: 93801, year: 1988, vol: 105, page: 149, stat: Journal Article,

Incidence of alloantibodies and immune complexes in bronchial secretions of lung cancer patients
Gorny, M K; Lawniczak, M; Skrzypczak, A; Zeromski, J
1987 ;34(2):189-198, Neoplasma
Bronchial secretions may accumulate immunoglobulins (Igs) produced by lymphocytes infiltrating tumor and its vicinity, and tumor associated antigens to higher degree than cancer patient serum. All three classes of Igs were increased in bronchial secretions from lung cancer patients as compared to control patients. Similar relationships were found for IgG and IgA/albumin ratios and IgG and IgA indices. The level of alloantibodies to fetal cells (measured by immunoradiometric--IRMA--assay) and immune complexes (IC, measured by IRMA--Clq and ELISA--anti C3) were significantly higher in lung cancer patients versus control patients in bronchial secretions. The differences were similar in sera but they were not significant. On the contrary, alloantibody binding to tumor cells was higher in control group. Absorption experiments with normal cells abolished antitumor and antifetal activity, what indicates natural character of these alloantibodies. Significant correlations between alloantibody to fetal cells and IC levels indicate that fetal antigens may contribute to antigenic component of IC. These findings suggest the link of alloantibodies and IC levels in bronchial secretions with neoplastic growth
— id: 93803, year: 1987, vol: 34, page: 189, stat: Journal Article,

[Immune complex levels in the serum of patients with ulcerative colitis and neoplasms of the digestive system]
Stachowiak, C; Zeromski, J; Gorny, M K; Lawniczak, M; Rzymski, K
1986 Mar 31;41(13):393-396, Polski tygodnik lekarski (Warsaw, Poland : 1960)
— id: 93804, year: 1986, vol: 41, page: 393, stat: Journal Article,

T-lymphocyte subsets and immunoglobulin concentrations in bronchoalveolar lavage of patients with sarcoidosis and high and low intensity alveolitis
Bauer, W; Gorny, M K; Baumann, H R; Morell, A
1985 Nov;132(5):1060-1065, American review of respiratory disease
Reproducible volumes of bronchoalveolar lavage (BAL) fluid were recovered from 19 patients with pulmonary sarcoidosis and from 6 control subjects using a standardized technique. Studies of cell surface markers showed that BAL from 9 patients contained more than 8 X 10(6) helper T-cells. These patients were classified as having high intensity alveolitis, whereas in 10 patients with lower cell counts, low intensity alveolitis was diagnosed. Activated helper T-cells coexpressing Leu-3a and Ia-antigens were significantly more numerous in BAL from patients with high intensity alveolitis than in BAL from the other patients and the control subjects. Concentrations of IgM, IgA, and IgG, but not of albumin were markedly increased in BAL from patients with high intensity alveolitis when compared with the other subjects. In BAL from both patient and control groups, numbers of helper T-cells and particularly of activated helper T-cells were correlated with immunoglobulin concentrations. In 4 patients with high intensity alveolitis, prednisone-induced and spontaneous clinical improvement were paralleled by a decrease in helper and activated helper T-cells and in immunoglobulin concentrations in BAL, indicating conversion of high to low intensity disease. No change was observed in 4 patients with low intensity alveolitis
— id: 93805, year: 1985, vol: 132, page: 1060, stat: Journal Article,

[Bronchoalveolar immunoglobulins in sarcoidosis]
Bauer, W; Gorny, M K; Morell, A; Baumann, H R
1985 Jan 19;115(3):103-105, Schweizerische medizinische wochenschrift = Journal suisse de medecine
In 9 patients with active and 10 patients with inactive sarcoidosis, and in 6 normal controls, the concentration of IgM, IgA and IgG broncho-alveolar lavage (BAL) fluid was measured. Patients with active sarcoidosis showed significantly higher values of these immunoglobulins than patients with inactive sarcoidosis or normals. In addition, a significant correlation between the immunoglobulin levels and the number of helper-T lymphocytes in the BAL fluid was demonstrated
— id: 93806, year: 1985, vol: 115, page: 103, stat: Journal Article,

Production of immunoglobulins and alloantibodies by lymphocytes from lung cancer patients
Gorny, M K; Morell, A
1985 ;33(5):677-684, Archivum immunologii & therapiae experimentalis
Spontaneous and PWM stimulated production of immunoglobulins (Igs) were measured in culture of lymphocytes isolated from peripheral blood (PBL) of lung cancer patients, blood donors and patients with benign lung diseases. Culture supernatants were tested for antibody activity against lung cancer cells and normal cells and some of them were absorbed with normal lung cells and fetal liver cells. Igs levels and antibody activity in culture supernatants were measured by radioimmunometric assay. Significantly increased level of Igs in unstimulated culture of PBL and low index of PWM-stimulated production of Igs was found in lung cancer patients in contrast to the controls. Supernatants of PBL cultures from lung cancer patients had significant low antibody activity to lung cancer cells and were effectively absorbed by normal lung cells and fetal liver cells. The results suggest in vivo polyclonal activation of B cells in patients with lung cancer and simultaneously diminished production of alloantibodies with some features of natural antibodies following polyclonal activation in vitro
— id: 93808, year: 1985, vol: 33, page: 677, stat: Journal Article,

Identification of a tumor-related protein antigen in immune complexes derived from pleural effusions of patients with bronchial carcinoma
Lawniczak, M; Jezewska, E; Gorny, M K; Zeromski, J
1985 ;32(1):9-20, Neoplasma
Pleural effusions from 15 patients with advanced primary bronchial carcinoma, from 2 patients with metastatic lung cancer and from 6 patients with nonmalignant disease were studied. Immune complexes were found in examined fluids in amounts corresponding to 2.5-210 mg/100 ml of aggregated IgG by means of ELISA solid phase anti C3 and 125ICIq binding radioimmunoassay. Following determination of protein content and salting out by ammonium sulfate of examined fluids, the sediments were subjected to subsequent chromatographic procedure including molecular sieving (Sephadex G-200, Sepharose 4B) and affinity chromatography on Protein A-Sepharose CL-4B. The yield--apparently pure immune complexes--was then split by means of chaotropic agent 2.5 M KSCN. It permitted to obtain 2 fractions: one contained IgG while the other was a non-Ig protein of m. w. = 150 000. The latter isolated from malignant effusions possessed antigenic activity in the leukocyte migration inhibition (LMI) assay. It resulted in inhibition of migration of allogenic peripheral blood leukocytes from lung cancer patients in 87% of cases. It had no activity against leukocytes from nonmalignant disease patients. LMI activity of the final second fraction derived from malignant effusion was significantly different from that of other fractions obtained both from malignant and nonmalignant fluids
— id: 93807, year: 1985, vol: 32, page: 9, stat: Journal Article,

[Antibodies against encephalitogenic protein and myelin glycoprotein in the cerebrospinal fluid of patients with multiple sclerosis. Effect of encorton treatment on the autoantibody and immune complex levels]
Waigt, A; Gorny, M K
1984 ;25(3-4):307-318, Folia medica Cracoviensia
— id: 93809, year: 1984, vol: 25, page: 307, stat: Journal Article,

CSF antibodies to myelin basic protein and oligodendrocytes in multiple sclerosis and other neurological diseases
Gorny, M K; Wroblewska, Z; Pleasure, D; Miller, S L; Wajgt, A; Koprowski, H
1983 Jun;67(6):338-347, Acta neurologica Scandinavica
Cerebrospinal fluid (CSF) from 18 multiple sclerosis (MS) patients, 13 subacute sclerosing panencephalitis (SSPE) patients, 22 other neurological disease (OND) patients, and 7 neurotic patients as controls were tested in an 125I-labeled anti-human F(ab')2 binding assay for the presence of antibodies to normal human brain cells from tissue culture, human fibroblasts, plasma membranes of MS and normal human brain, myelin basic protein (MBP) and bovine oligodendrocytes. Antibodies to MBP and to oligodendrocytes were found in the CSF of MS, SSPE and OND patients. Absorption of CSF with bovine CNS myelin significantly diminished binding activity to oligodendrocytes. Antibodies in the CSF against MBP and oligodendrocytes, on which some myelin determinants are expressed, seem to be a common feature of diseases in which demyelination is a component
— id: 93811, year: 1983, vol: 67, page: 338, stat: Journal Article,

The influence of high-dose prednisone medication on autoantibody specific activity and on circulating immune complex level in cerebrospinal fluid of multiple sclerosis patients
Wajgt, A; Gorny, M K; Jenek, R
1983 Dec;68(6):378-385, Acta neurologica Scandinavica
In agreement with the close correlation between intrathecal IgG production and anti-MBP (myelin basic protein) and anti-MAG (myelin-associated glycoprotein) antibody activity in the CSF of active MS cases, and parallel to the reduction of intrathecal IgG synthesis resulting from corticosteroids medication, we have found a significant reduction of anti-MBP and anti-MAG antibody activity expressed per 0.5 micrograms of CSF IgG in the same group of 40 MS patients subjected to high-dose prednisone therapy. Every patient received 3980 mg of prednisone over 54 days. In native CSF of 30% (21/70) of active MS cases, circulating immune complexes (CIC) were detected by C1q binding solid-phase RIA. There was no correlation between CIC level in the CSF or MS patients and 1. IgG index which was used as an indicator of intrathecal IgG synthesis, or 2. CSF anti-MBP specific antibody activity, or 3. CSF anti-MAG specific antibody activity. High-dose prednisone therapy resulted in a highly significant reduction of the CSF CIC level. CIC were also found in the CSF of patients affected with various chronic diseases of the CNS
— id: 93810, year: 1983, vol: 68, page: 378, stat: Journal Article,

Radioimmunometric demonstration of immunoglobulin G on cancer cells derived from malignant pleural effusions
Gorny, M K; Micksche, M; Wolf, A
1982 ;39(1):6-12, Oncology (New York)
Well-preserved viable cancer cells, lymphocytes and mesothelial cells were prepared from various human malignant and non-malignant pleural effusions by a Ficoll-Hypaque discontinuous gradient. Using 125I-labelled protein A from Staphylococcus aureus as tracer, IgG was found on all cancer cells and also on apparently non-malignant mesothelial cells. Loss of cell surface IgG by overnight incubation, and fluctuation of surface IgG levels in the same patient occurred with cancer cells as well as with mesothelial cells. The IgG in vivo coat of cancer cells reflected the IgG levels of the sera rather than those of the pleural effusions. Binding of IgG from autologous and allogeneic sera and from supernatants of acid and mild overnight elution of cancer cells and mesothelial cells did not indicate a cancer-specific uptake of IgG. Various unspecific modes of IgG uptake by cancer cells are discussed including the possibility of a non-immune mechanism. Modification and loss of antigens by metastatic cells have also been considered
— id: 93813, year: 1982, vol: 39, page: 6, stat: Journal Article,

Assessment of specific anti-tumour immunity in lung cancer patients by means of leukocyte migration inhibition test. The role of antigenic preparations
Zeromski, J; Jezewska, E; Lawniczak, M; Gorny, M K
1982 Jan-Dec;14(1-2):68-73, Materia medica Polona = Polish journal of medicine & pharmacy
— id: 93812, year: 1982, vol: 14, page: 68, stat: Journal Article,

Autoantibodies in lung cancer patients demonstrated on fixed tissue culture cells. An immunofluorescent study
Gorny, M K; Jezewska, E; Zeromski, J
1981 ;51(5):418-423, Archiv fur Geschwulstforschung
A panel of sera derived from 138 patients with primary bronchogenic carcinoma, non-neoplastic lung conditions and from blood donors was tested for presence of autoantibodies by indirect immunofluorescence on fixed cells of established lung cancer cell line and lung fibroblasts as a substrate. Autoantibodies were detected in 87% and 64% out of patient sera respectively and in 9% of donor sera. Immunofluorescence patterns permitted to distinguish 3 antibody specificities: anti-nucleolar, anti-cytoplasmic and anti-nuclear ones. The major differences were noted in incidence of anti-nucleolar antibodies, which were present in 77% of lung cancer patients and only in 14% of patients with non-neoplastic lung conditions. The autoantibodies in question belonged to IgG and to lesser degree IgA class of immunoglobulin and were not apparently cancer specific because absorption with normal tissue homogenates removed their activity
— id: 93814, year: 1981, vol: 51, page: 418, stat: Journal Article,

[Clinical evaluation of quantitative determination of immunoglobulins and complement in ulcerative colitis]
Stachowiak, C; Gorny, M K; Zeromski, J
1980 May 26;35(21):773-776, Polski tygodnik lekarski (Warsaw, Poland : 1960)
— id: 93815, year: 1980, vol: 35, page: 773, stat: Journal Article,

Anti-tumor antibodies in lung cancer patients. Immunofluorescence study using various indicator cells
Gorny, M K; Jezewska, E; Krzysko, R; Stawarz, M; Zeromski, J
1979 ;26(6):729-736, Neoplasma
By means of indirect immunofluorescence a number of primary lung cancer patient sera and control sera were tested for anti-tumor antibody activity on living tumor cells as a substrate. Antibodies against surface antigens were the most frequently detected in autologous system (in 65%) on cells derived from fresh surgical material of lung cancer. They were also found in 50% of cases using tumor cells from primary short-term culture. When established cell line of lung cancer was used (E-14) in allogeneic system, the antibodies were detected in only 22% of examined lung cancer sera. Absorption of positive sera with homogenates of normal tissues did not abolish their specific activity. Positive reactions were confined to squamous cell type of bronchogenic carcinoma
— id: 93816, year: 1979, vol: 26, page: 729, stat: Journal Article,

Local monoclonal immunoglobulin production in cancer patient
Gorny, M K; Zeromski, J
1975 Feb 15;31(2):238-239, Experientia
— id: 93818, year: 1975, vol: 31, page: 238, stat: Journal Article,

Comparative studies on cell-mediated immunity in experimental trichinnellosis. Macrophage migration inhibition and cell adherence around larva in vitro
Gustowska, L; Rauhut, W; Gorny, M K; Zeromski, J
1975 ;21(4-5):639-648, Wiadomosci parazytologiczne
— id: 93819, year: 1975, vol: 21, page: 639, stat: Journal Article,

Letter: Cytotoxicity in normal and multiple-sclerosis sera against mouse cells expressing theta-antigen
Tachovsky, T G; Palmer, J C; Gorny, M K; Koprovski, H
1975 Dec 13;2(7946):1204-1205, Lancet
— id: 93817, year: 1975, vol: 2, page: 1204, stat: Journal Article,

Behaviour of local and systemic immunoglobulins in patients with lung cancer
Zeromski, J; Gorny, M K; Wruk, M; Sapula, J
1975 ;49(4):548-563, International archives of allergy & applied immunology
Tissue blocks from lung parenchyma in the vicinity of primary lung cancer and regional lymph nodes were collected during surgery from 22 patients. Cryostat sections were assessed for the presence of five five immunoglobulin classes by means of immunofluorescence. Imprints made from surfaces of freshly resected tumours were used for indirect immunofluorescent reaction to detect anti-tumour antibodies in the autologous system sera of patients after surgery. Also, sera of 60 lung cancer patients were assessed for IgG, A, M levels. It has been found that the direct vicinity of primary lung cancer and regional lymph nodes was a site of intensive immunoglobulin synthesis is plasma cells, with a predominance of the IgA and IgG classes. Serum anti-tumour antibody was found in 3 cases out of 22. There were significantly elevated serum immunoglobulin levels with predominance of IgA and IgG. Inoperable patients had higher immunoglobulin levels then those who underwent surgery
— id: 93820, year: 1975, vol: 49, page: 548, stat: Journal Article,

[Letter: IgM immunocytes and gastrointestinal cancers]
Gorny, M K; Drews, M; Zeromski, J
1974 Apr 6;3(14):893-893, La nouvelle presse medicale
— id: 93822, year: 1974, vol: 3, page: 893, stat: Journal Article,

[Immune response of rabbit lymph nodes following implantation of xenogenous cartilage and bone into soft tissue]
Gorny, M K; Krajnik, J; Zeromska, B; Jachimowicz, B
1974 Jul;46(7):965-972, Polski przeglad chirurgiczny
— id: 93821, year: 1974, vol: 46, page: 965, stat: Journal Article,

Malignancy associated with antinuclear antibodies
Zeromski, J O; Gorny, M K; Jarczewska, K
1972 Nov 11;2(7785):1035-1036, Lancet
— id: 93823, year: 1972, vol: 2, page: 1035, stat: Journal Article,