Division of Retrovirology, NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK1
School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK2
Division of Virology, University College of London Medical School, Windeyer Building, 46 Cleveland Street, London W1P 6DB, UK3
School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading, Berks RG6 2AJ, UK4
Author for correspondence: Simon Jeffs. Fax +44 1707 649865. e-mail sjeffs{at}nibsc.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite these caveats, at least three human monoclonal antibodies (mAb) capable of neutralizing divergent primary isolates have been isolated. Two of these (b12 and 2G12) interact with gp120 epitopes, while a third (2F5) recognizes a C-terminal epitope in gp41 (Roben et al., 1994 ; Trkola et al., 1996
; Muster et al., 1993
). Furthermore, passive transfer of combinations of these antibodies have proved effective at controlling infection in model systems (Baba et al., 2000
; Mascola et al., 1999
, 2000
).
Thus the challenge is to improve the immunogenicity of recombinant gp120/41 to generate potent neutralizing responses. There are a number of ways by which this may be achieved, but most involve alteration of the molecular conformation of the molecule in order to expose these cryptic epitopes. These include (a) the removal of selected N-linked glycosylation sites (Back et al., 1994 ; Chackerian et al., 1997
; Chakrabarti et al., 2002
; Reitter et al., 1998
); (b) the use of multivalent envelope glycoprotein (Montefiori & Evans, 1999
); (c) a consideration of the role of monomeric versus oligomeric recombinant glycoprotein (Burton & Moore, 1998
; Montefiori & Evans, 1999
); (d) the use of fusion-competent immunogens (LaCasse et al., 1999
); and (e) the production of novel stabilized trimers of envelope glycoprotein (Chakrabarti et al., 2002
; Srivastava et al., 2002
; Binley et al., 2000
; Yang et al., 2000
).
In previous studies, we (Jeffs et al., 1996 ) and others (Pollard et al., 1992
; Wyatt et al., 1993
) have reported that removal of the V1, V2 and V3 hypervariable loops results in a molecule which is capable of binding sCD4 with an affinity comparable to that of the full-length protein. Furthermore, removal of these loops increases accessibility of the C1 and C4 regions to mAbs (Jeffs et al., 1996
). We therefore investigated the immunogenicity of this truncated protein (PR12) with the intention of re-directing the immune response from the variable loops to the more conserved regions critical for gp120 function.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anti-FL/PR12 gp120 sera were raised in pairs of naïve CBH/Cbi rats by three immunizations, at fortnightly intervals, with 10 µg of CHO-expressed IIIBBH10 FL or PR12 gp120 emulsified in Freund's complete (first immunization) or incomplete (second and third immunizations) adjuvant (McKeating et al., 1996 ). Anti-PR12 mAbs were generated in rats using the method of Shotton et al. (1995)
.
Transient expression and quantification of gp120; antibody-binding ELISAs
VTF7.3-infected human embryonal kidney 293 cells were transfected, using lipofectamine (Gibco BRL), with pCDNA.3 plasmids encoding divergent gp120 proteins. Cell supernatants were collected after 72 h, clarified by centrifugation at 1500 g and assayed for gp120 levels using a quantitative gp120 capture ELISA. This assay involves coating plates with the ovine antisera D7324 (Aalto Bioreagents), raised against a peptide conforming to the 15 C-terminal amino acids of IIIBBH10 gp120, to bind HIV-1 gp120 present in the sample. The presence of gp120 is detected by a rabbit CHO-derived IIIBBH10 gp120 antisera (ARP421, CFAR). All of the divergent proteins possess the conserved C-terminal D7324 epitope encoded by oligonucleotide 4141 used for PCR amplification. Bound ligands were visualized with anti-rabbit IgGHRP. Recombinant IIIBBH10 gp120 derived from CHO cells (EVA657, CFAR) was used for calibration.
For antibody-binding ELISAs, gp120 was allowed to bind to D7324 at an input concentration of 500 ng/ml, followed by addition of serially diluted mAb solutions. Detection was by the appropriate anti-species IgGHRP and the titre of mAb giving half-maximal binding was determined graphically.
PCR amplification of gp120 sequences both for expression of soluble proteins and for generation of chimeric constructs.
The PCR followed the protocols previously reported (McKeating et al., 1993 ). The gp120-encoding region was amplified from a panel of pBluescript (Stratagene) clones containing diverse envelope genes (the kind gift of F. McCutchan, Henry Jackson Foundation, Rockville, MD, USA) using primers 626 (sense, restriction enzyme site underlined) 5' GTG GGT CAC CGT CTA TTA TTG GG and 524 (antisense) 5' CAC CAC GCG TCT CTT TGC CTT GGT GGG. PCR products were purified using Geneclean (Bio 101), restriction enzyme digested with either BstEII or MluI, ligated into pHXB2-MCS
env (McKeating et al., 1993
) and transformed into competent TG2 E. coli. Individual colonies were screened for inserts by PCR using primers 626 and 524. Plasmid preparations of clones containing inserts were prepared using Wizard miniprep kits (Promega).
The gp120 open reading frame was also amplified using primers 4142 and 4141, uracilated derivatives of the primers 626 and 524 described above, with 100 ng of plasmid template and using the conditions described above. The PCR product was gel-purified and 100 ng annealed to 25 ng of the vector pCDNA3.tpa, which contains complementary uracilated sequences, in the presence of 0·25 U of uracil deglycosylase (CloneAmp, Gibco BRL). Colonies resulting from transformation with the annealed vector were screened for the presence of insert and plasmid DNA prepared from them.
Transfection of HXB2 chimeric clones and neutralization assays.
Plasmid DNAs (5 µg) were transfected into HeLa cells using lipofectamine. At 72 h post-transfection the cells were co-cultured with 2x106 PHA-stimulated peripheral blood mononuclear cells (PBMC) for 24 h. PBMC were recovered from the HeLa monolayer, washed and cultured in RPMI10% FCSIL-2 (5 U/ml). The extracellular fluid was tested for the presence of soluble p24 antigen as described previously (McKeating et al., 1993 ). Cell-free supernatants were collected from PBMC cultures and assessed for the levels of infectious virus by determining the median infectious dose on human phytohaemagglutinin (PHA) stimulation. Infection was measured by the detection of p24 antigen and the TCID50 values determined by the Karber formula (Lewis et al., 1998
).
Neutralization was assessed using PHA-stimulated PBMC as target cells and virus replication was assessed by measuring p24 antigen production as reported previously (Von Gegerfelt et al., 1991 ; Scarlatti et al., 1993
; Albert et al., 1990
). A 75 µl sample from each diluted rat antisera was incubated for 1 h at 37 °C with an equal volume of virus (in triplicate, at dilutions of 1:5, 1:25 and 1:125). Following virusantibody incubation, 1x105 PBMC in a final volume of 75 µl RPMI10% FCSIL-2 (5 U/ml) were added. PBMC were washed on days 1 and 3 by centrifugation and the medium changed (Albert et al., 1993
). The TCID50 of the virus stock was determined in parallel and the neutralization assay was evaluated for virus dilutions containing 1075 TCID50. The neutralizing titre of a serum is defined as the highest serum dilution which completely inhibits virus replication as assessed by p24 antigen production, that is an absorbance value below the cutoff value for the antigen ELISA of 3 pg (the mean+2 SD of negative controls containing cells only). Controls included in each assay were virus alone and cells alone. Positive virus replication controls contained either IL-2 medium or rat pre-bleed sera diluted 1:10. Positive anti-HIV-1 serum control utilized serum from an asymptomatic homosexual man with a high-titre of neutralizing antibodies against HIV-1 IIIB diluted 1:10 in medium.
Phylogenetic analysis of divergent envelope sequences.
In order to assess the genetic variation present in the epitopes of the PR12 construct, we constructed an alignment of representative sequences from the major subtypes of HIV-1 (AF). The sequence for PR12 was added to this alignment and the deleted regions removed from all sequences (assigned weights of zero). Maximum-likelihood trees were constructed from the reduced data set and a tree constructed (using the PHYLIP suite of programs, version 3.5c, J. Felsenstein, University of Washington). Pairwise distances were calculated from the alignment (Kimura 2-parameter method) and comparisons made of intra-subtype and inter-subtype distances. For complete sequences, such comparisons show that members of a subtype are more closely related to one another than to unrelated subtypes (analysis not shown). Finally, bootstrap resampling of the data set by neighbour joining was used to assess the robustness of the tree obtained.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Immunogenicity of the truncated PR12 gp120 protein
Analysis of the antibody response from rats immunized with PR12 demonstrated that serum antibodies reacted preferentially with PR12 compared to FL gp120 (Fig. 2A). Similarly, serum from FL gp120-immunized rats bound with higher affinity to FL gp120 than to PR12 (Fig. 2B
). However, none of the sera were able to inhibit sCD4/FL or PR12 gp120 interactions by competition ELISA (data not shown), suggesting that this region remains immunosilent in PR12 immunized rats.
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Removal of the V1V3 loops may influence glycosylation and processing of the truncated molecule, such that increased mAb recognition may be due to an indirect effect of altered processing rather than a direct effect on the disruption of inter-domain interactions. Wyatt et al. (1993) reported that processing and transport to the cell surface of a V1V3-deleted gp160 protein was increased relative to the full-length molecule. It is therefore possible that cell type-dependent glycosylation patterns may explain the different binding affinities of some mAbs for gp120 expressed in 293 and CHO cells (Table 1
). Generally, only mAbs specific for epitopes within C1, C4 and the discontinuous CD4 binding site bound with greater affinity to FL gp120 expressed in 293 cells compared to that from CHO cells (Table 1
). These mAbs were also able to bind gp120 proteins of diverse clades, expressed from 293 cells, suggesting that their ability to bind full-length envelope glycoprotein is not specific for the immunizing strain (data not shown). Interestingly, exposure of the same gp120 regions, as assessed by mAb binding, was increased on the PR12 molecule (Table 1
and Fig. 1A
).
We were interested to know which regions, if any, were immunodominant on the PR12 molecule. Several authors have suggested that the enhanced exposure of epitopes within C4 and CD4bd may correlate with enhanced immunogenicity of these regions (Pollard et al., 1992 ; Wyatt et al., 1993
; Moore et al., 1994
; Jeffs et al., 1996
). Initially, we investigated the reactivity of the immune sera for a series of overlapping gp120 peptides. FL gp120 immune sera reacted with epitopes in the C1, V1, V2 and V3 regions, whereas PR12 immune sera failed to react to any of the tested gp120 peptides (data not shown). These data demonstrate that antibodies raised to PR12 were specific for conformation-dependent epitopes and that the immune response was not directed to new linear epitope(s). Neither PR12 nor FL gp120 immune sera competed with human mAb 697, specific for the CD4 binding site, for binding to FL gp120 (data not shown). Furthermore, we were unable to detect antibodies capable of inhibiting the envelope gpsCD4 interaction in either FL or PR12 gp120 immune sera (data not shown). Overall, these data suggest that CD4bd is not immunogenic in the PR12 molecule and that antibodies to this region do not contribute to the neutralizing activity of the immune sera. Moore et al. (1994)
reported a lack of association between the ability of antibodies to inhibit the sCD4gp120 interaction and their ability to neutralize, suggesting differential CD4bd exposure on soluble monomeric and viral oligomeric gp120. However, all neutralizing mAbs shown to recognize epitopes overlapping CD4bd have been reported to inhibit the in vitro sCD4gp120 interaction (Cordell et al., 1991
; McKeating et al., 1992
).
We noted that the neutralizing human mAb 2G12, specific for a glycan-dependent epitope within the carboxyl region of gp120, bound with equivalent affinities to FL and PR12 gp120. Furthermore, PR12 immune sera were able to compete with 2G12 for binding to FL gp120, whereas FL gp120 immune sera were unable to compete (Fig. 4). It is interesting to note that 2G12 is one of the few mAbs where an association between affinity for soluble gp120 and neutralization efficiency has been reported (Moore et al., 1995
). These data suggest that 2G12-like antibodies may contribute to the neutralizing activity of the PR12 immune sera and this epitope may be immunogenic within PR12. In order to evaluate the immunogenicity of this region during natural infection, a panel of human HIV-positive sera were tested for their ability to compete with 2G12 binding to FL gp120. Our data are in agreement with those of Trkola et al. (1996)
in that none of the sera were able to compete with 2G12 (data not shown), suggesting that this epitope is poorly immunogenic in naturally infected individuals.
In order to further evaluate the PR12 immune response, we generated a number of mAbs from rats immunized with PR12. Five mAbs specific for conformation-dependent epitopes were obtained, three of which, 62/59b, 62/41 and 62/51, were only able to bind PR12 (Table 4). These mAbs failed to react with any of the gp120 proteins listed in Table 2
or to neutralize HXB2 (data not shown). The two additional mAbs, 58/1b/7i and 58/30, demonstrated broad cross-reactivity for gp120 proteins of different clades (data not shown), in that both mAbs were able to bind all proteins tested so far (total of 24 from clades AG). Both mAbs showed increased binding affinity for PR12 and were able to compete with the C4 mAb 55/45b/6e (data not shown), suggesting that C4-dependent epitopes are immunogenic on PR12. We therefore tested the ability of the polyclonal immune sera to compete with mAb 55/45b/6e for FL gp120 binding; however, no competition was observed with either FL gp120 or PR12 immune sera. Unfortunately, neither of these mAbs demonstrated neutralizing activity for HXB2 or any of the chimeric viruses (data not shown). In a parallel study, PR12 was used to select human mAbs from HIV-infected individuals (Jeffs et al., 2001
). Five mAbs were generated with specificities overlapping the CD4 binding domain, one of which, mAb 1570, recognized a conserved epitope in recombinant envelope proteins derived from clade A, B, C, D and E viruses. Initial results suggest that this mAb may show cross-clade neutralizing activity (Zolla-Pazner and Jeffs, unpublished results). Clearly, one potential disadvantage of immunizing with PR12 is the generation of PR12-specific antibodies. However, mAbs 58/1b, 58/30 (this study) and 1570 (Jeffs et al., 2001
) do appear to show broad cross-clade reactivity. Furthermore, the ability of PR12 serum antibodies to neutralize and bind divergent gp120 proteins suggests that PR12-specific antibodies may be a minor component of the polyclonal response (Table 2
). Clearly, it is important to attempt to identify the epitopes responsible for inducing this neutralizing activity; however, the polyclonal response may result from antibodies recognizing multiple epitopes and exerting synergistic effects.
In summary, we have shown that removal of the V1V3 loops from IIIB gp120 results in a protein, PR12, with altered immunogenicity compared to the full-length protein. Differences were found both in the ability of the immune sera to recognize glycoproteins of diverse origins and in their ability to neutralize viruses chimeric for primary gp120 proteins. These data lead us to conclude that the immune response to PR12 is directed toward conserved epitopes present on the majority of viral glycoproteins. None of the mAbs derived from the PR12-immunized rats were able to neutralize virus infectivity and hence the epitopes responsible for the induction of this cross-neutralizing activity remain to be elucidated. However, PR12 immune sera were able to compete with the human neutralizing mAb 2G12 for gp120 binding, implying that this epitope may be immunogenic within the truncated protein. In contrast, the epitopes recognized by PR12-selected human mAbs appear to be located within the CD4 binding domain and at least one of these shows cross-clade neutralizing activity (Jeffs et al., 2001 ). The challenge ahead is to induce potent cross-clade neutralizing antibody responses in experimental animals using glycoprotein immunogens based on primary isolates of HIV-1. To date, this has proved notoriously difficult to achieve. Indeed, one very promising approach using immunogens comprising formaldehyde-fixed co-cultures of cells expressing HIV-1 envelope glycoprotein, CD4 and CCR5 (LaCasse et al., 1999
), has recently been retracted following reports that a major fraction of the reported neutralization was due to a specific cytotoxic effect of the antisera (Nunberg, 2002
). We remain convinced that envelope-based immunogens do induce protective humoral responses. However, considerable effort is required to identify in vitro assays that correlate with protection observed in either model systems or clinical trials in vivo. Certainly at the moment it is important that promising results are demonstrated to be reproducible and evaluated using a wide variety of neutralization assays and HIV-1 isolates. In addition, a wide variety of projects are currently in progress in our laboratory, designed to evaluate the comparative immunogenicity of monomeric and oligomeric glycoproteins with modifications such as hypervariable loop-deletion, specific N-linked glycan removal and the addition of potentially adjuvanting molecules to the polypeptide backbone. It is hoped that these modifications will provide information on the structural components that will be required for an effective HIV envelope-based AIDS vaccine.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albert, J., Bjorling, E., Vongegerfelt, A., Scarlatti, G., Zhang, Y. J., Fenyo, E-M. & Thorstensson, R. (1993). Antigen-detection is a reliable method for evaluating HIV/SIV neutralization assays. AIDS Research and Human Retroviruses 9, 501-504.[Medline]
Baba, T. W., Liska, V., Hofmann-Lehmann, R., Vlasak, J., Xu, W., Ayehunie, S., Cavacini, L. A., Posner, M. R., Katinger, H., Stiegler, G., Bernacky, B. J., Rizvi, T. A., Schmidt, R., Hill, L. R., Keeling, M. E., Lu, Y., Wright, J. E., Chou, T.-C. & Ruprecht, R. M. (2000). Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus interaction. Nature Medicine 6, 200-206.[Medline]
Back, N. K. T., Smith, L. & Dejog, J. J. (1994). An N-glycan within the human immunodeficiency virus type 1 gp120 V3 loop affects virus neutralization. Virology 199, 431-443.[Medline]
Berman, P. W., Matthews, T. J., Riddle, L., Champe, M., Hobbs, M. R., Nakamura, G. R., Mercer, J. & Eastman, D. J. (1992). Antisera raised against gp120 from the MN isolate of HIV-1. Journal of Virology 66, 4464-4469.[Abstract]
Binley, J. M., Sanders, R. W., Las, B., Schuelke, N., Master, A., Guo, Y., Fajumo, F., Anselma, D. J., Maddon, P. J., Olson, W. C. & Moore, J. P. (2000). A recombinant human immunodeficiency virus type I envelope glycoprotein complex stabilised by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. Journal of Virology 74, 627-643.
Buchacher, A., Predl, R., Strutzenberger, K., Steinfellner, W., Trkola, A., Purtscher, M., Gruber, G., Tauer, C., Steindl, F., Jungbauer, A. & Katinger, H. (1994). Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and EpsteinBarr virus transformation for peripheral blood lymphocyte immortalization. AIDS Research and Human Retroviruses 10, 359-369.[Medline]
Burton, D. R. & Montefiori, D. C. (1997). The antibody response in HIV-1 infection. AIDS 11, Supplement A, S87S98.
Burton, D. R. & Moore, J. P. (1998). Why do we not have an HIV vaccine and how can we make one? Nature Medicine 4, 495-498.[Medline]
Chackerian, B., Rudensey, L. M. & Overbaugh, J. (1997). Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of simian immunodeficiency virus that evolve in the host alter recognition by neutralizing antibodies. Journal of Virology 71, 7719-7727.[Abstract]
Chakrabarti, B. K., Kong, W.-P., Wu, B.-Y., Yang, Z.-Y., Friborg, J., Ling, X., King, S. R., Montefiori, D. C. & Nable, G. J. (2002). Modifications of the human immunodeficiency virus envelope glycoprotein enhance immunogenicity for genetic immunization. Journal of Virology 76, 5357-5368.
Connor, R. I., Korber, B. T. M., Graham, B. S., Hahn, B. H., Ho, D. D., Walker, B. D., Neuman, A. U., Vermund, S. H., Mestecky, J., Jackson, S., Fenamore, E., Cao, Y., Gao, F., Kalams, S., Kuntsman, K. J., McDonald, D., McWilliams, N., Trkola, A., Moore, J. P. & Wolinksky, S. M. (1998). Immunological and virological analyses of persons infected by human immunodeficiency virus type I while participating in trials of recombinant gp120 subunit vaccines. Journal of Virology 72, 1552-1576.
Cordell, J., Moore, J. P., Dean, C. J., Klasse, P. J., Weiss, R. A. & McKeating, J. A. (1991). Rat monoclonal antibodies to nonoverlapping epitopes of human immunodeficiency virus type 1 gp120 block CD4 binding in vitro. Virology 185, 72-79.[Medline]
Fenouillet, E., Blanes, N., Benjouad, A. & Gluckman, J. C. (1995). Anti-V3 antibody reactivity correlates with clinical stage of HIV-1 infection and with serum neutralizing activity. Clinical and Experimental Immunology 99, 419-424.[Medline]
Jeffs, S. A., McKeating, J., Lewis, S., Craft, H., Biram, D., Stephens, P. E. & Brady, R. L. (1996). Antigenicity of truncated forms of the human immunodeficiency virus type 1 envelope glycoprotein. Journal of General Virology 77, 1403-1410.[Abstract]
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). Characterisation of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1IIIB gp120. Immunology Letters 79, 209-213.[Medline]
LaCasse, R. A., Follis, K. E., Trahey, M., Scarborough, J. D., Littman, D. R. & Nunberg, J. H. (1999). Fusion-competent vaccines: broad neutralisation of primary isolates of HIV. Science 283, 357-362.
Lewis, J., Balfe, P., Arnold, C., Kaye, S., Tedder, R. S. & McKeating, J. M. (1998). Development of a neutralizing antibody response during acute primary human immunodeficiency virus type 1 infection and the emergence of antigenic variants. Journal of Virology 72, 8943-8951.
McKeating, J. A., Cordell, J., Dean, C. J. & Balfe, P. (1992). Synergistic interaction between ligands binding to the CD4 binding site and V3 domain of human immunodeficiency virus type 1 gp120. Virology 191, 732-742.[Medline]
McKeating, J. A., Shotton, C., Cordell, J., Graham, S., Balfe, P., Sullivan, N., Charles, M., Page, M., Bolmstedt, A., Olofsson, S., Wu, Z., Pinter, A., Dean, C., Sodroski, J. & Weiss, R. A. (1993). Characterization of neutralizing monoclonal antibodies to linear and conformation-dependent epitopes within the first and second variable domains of human immunodeficiency virus type 1 gp120. Journal of Virology 67, 4932-4944.[Abstract]
McKeating, J. A., Shotton, C., Jeffs, S., Palmer, C., Hammond, A., Lewis, J., Oliver, K., May, J & Balfe, P. (1996). Immunogenicity of full-length and truncated forms of the human immunodeficiency virus type I envelope glycoprotein. Immunology Letters 51, 101-105.[Medline]
Mascola, J. R., Lewis, M. G., Stiegler, G., Harris, D., VanCott, T. C., Hayes, D., Louder, M. L., Brown, C. R., Sapan, C. V., Frankel., S. S., Lu, Y., Robb, M. L., Katinger, H. & Birx, D. L. (1999). Protection of macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. Journal of Virology 73, 4009-4018.
Mascola, J. R., Stiegler, G, VanCott, T. C., Katinger, H., Carpenter, C. B., Hanson, C. E., Beary, H., Hayes, D., Frankel, S. S., Birx, D. L. & Lewis, M. G. (2000). Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nature Medicine 6, 207-210.[Medline]
Montefiori, D. C. & Evans, T. G. (1999). Towards an HIV type1 vaccine that generates potent, broadly cross-reactive neutralizing antibodies. AIDS Research and Human Retroviruses 15, 689-698.[Medline]
Moore, J. P., Sattentau, Q., Wyatt, R. & Sodroski, J. (1994). Probing the structure of the human immunodeficiency virus surface glycoprotein gp120 with a panel of monoclonal antibodies. Journal of Virology 68, 469-484.[Abstract]
Moore, J. P., Trkola, A., Korber, B., Boots, L. J., Kessler, J., McCutchan, F. E., Mascola, J., Ho, D. D., Robinson, J. & Conley, A. J. (1995). A human monoclonal antibody to a complex epitope in the V3 region of gp120 of human immunodeficiency virus type 1 has broad reactivity within and outside clade B. Journal of Virology 69, 122-30.[Abstract]
Muster, T., Steindl, F., Purtscher, M., Trkola, A., Klima, A., Himmler, G., Ruker, F. & Katinger, H. (1993). A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. Journal of Virology 67, 6642-6647.[Abstract]
Nunberg, J. (2002). Retraction (letter). Science 296, 1025.
Nyambi, P. N., Gorny, M. K., Bastiani, L., Van Der Groen, G., Williams, C. & Zolla-Pazner, S. (1998). 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. Journal of Virology 72, 9384-9391.
Pollard, S. R., Rosa, M. D., Rosa, J. J. & Wiley, D. C. (1992). Truncated variants of gp120 bind CD4 with high affinity and suggest a minimum CD4 binding region. EMBO Journal 11, 585-591.[Abstract]
Reitter, J. N., Means, R. E. & Desrosiers, R. C. (1998). A role for carbohydrates in immune evasion in AIDS. Nature Medicine 4, 679-684.[Medline]
Roben, P., Moore, J. P., Thali, M., Sodroski, J. & Barbas, C. F.III (1994). Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. Journal of Virology 68, 4821-4828.[Abstract]
Scarlatti, G. E., Albert, J., Rossi, P., Hodara, V., Biraghi, P., Muggiasca, L. & Fenyo, E.-M. (1993). Mother-to-child transmission of human immunodeficiency virus type 1 correlation with neutralising antibodies against primary isolates. Journal of Infectious Diseases 168, 207-210.[Medline]
Shotton, C., Arnold, C., Sattentau, Q., Sodroski, J. & McKeating, J. M. (1995). Identification and characterisation of monoclonal antibodies specific for polymorphic antigenic determinants within the V2 region of the HIV-1 envelope glycoprotein. Journal of Virology 69, 222-230.[Abstract]
Srivastava, I. K., Stamatatos, L., Legg, H., Kan, E., Fong, A., Coates, S. R., Leung, L., Wininger, M., Donnolly, J. L., Ulmer, J. B. & Barnett, S. W. (2002). Purification and characterization of oligomeric envelope glycoprotein from a primary R5 subtype B human immunodeficiency virus. Journal of Virology 76, 2835-2847.
Trkola, A., Purtscher, M., Muster, T., Ballaun, C., Buchacher, A., Sullivan, N., Srinivasan, K., Sodroski, J., Moore, J. P. & Katinger, H. (1996). Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. Journal of Virology 70, 1100-1108.[Abstract]
Von Gegerfelt, A., Albert, J., Morfeldt-Manson, L., Broliden, K. & Fenyo, E.-M. (1991). Isolate-specific neutralizing antibodies in patients with progressive HIV-1-related disease. Virology 185, 162-168.[Medline]
Wyatt, R., Sullivan, N., Thali, M., Repke, H., Ho, D., Robinson, J., Posner, M. & Sodroski, J. (1993). Functional and immunologic characterization of human immunodeficiency virus type 1 envelope glycoproteins containing deletions of the major variable regions. Journal of Virology 67, 4557-4565.[Abstract]
Yang, X., Florin, L., Farzan, M., Kolchinsky, P., Kwong, P. D., Sodroski, J. & Wyatt, R. (2000). Modifications that stabilize human immunodeficiency virus envelope glycoprotein trimers in solution. Journal of Virology 74, 4746-4754.
Received 30 April 2002;
accepted 5 July 2002.