Conserved neutralizing epitopes of HIV type 1 CRF01_AE against primary isolates in long-term nonprogressors

Linear conserved B cell epitopes in envelope glycoprotein of long-term nonprogressors (LTNPs) HIV-1 CRF01_AE were determined. The envelope sequences of HIV-1 subtype E from Thailand were aligned to define consensus sequences. Then the peptides corresponding to these predicted regions were synthesized as peptides represent C1, C2, C3, C5, V2, V3, and gp41 regions. After that, the neutralizing B cell epitopes were determined by neutralized competitive assay with pool sera of typical progressor and LTNP HIV-1 CRF01_AE patients against HIV-1 CRF01_AE 24 primary isolates (PI) and laboratory strains (TCLA). We found that the strength and breadth of neutralization were greater for sera from LTNPs compared with sera from typical progressors. Peptides C1E and C2E could inhibit primary isolates but not the TCLA strain in LTNP sera. The new B cell epitopes, which were located in the C1 and C2 regions of CRF01_AE against primary HIV-1 isolates, were identified in HIV-1 CRF01_AE LTNPs. This may be important in HIV-1 vaccine development and trial.     

Monoclonal antibodies to the extracellular domain of HIV-1IIIB gp160 that neutralize infectivity, block binding to CD4, and react with diverse isolates.

Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.     

Identification of a novel HIV type 1 CRF01_AE cytotoxic T lymphocyte (CTL) epitope restricted by an HLA-Cw0602 allele and a novel HLA-A0206/peptide restriction

This report describes specific T cell responses to HIV-1 CRF01_AE Env and A Gag peptides in 20 HIV-1 CRF01_AE-infected Thai individuals using an interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISpot) assay. Twenty-six potentially novel HLA class I-restricted CD8+ T cell epitopes were identified in 14/20 subjects. Fine mapping analysis using the chromium release cytotoxic T lymphocyte (CTL) assay revealed a novel HLA-Cw0602 restricted epitope of HIV-1 CRF01_AE Env (NAKTIIVHL) and a previously identified HIV-1 A Gag epitope (ATLEEMMTA) with a novel HLA-A0206 restriction.     

Binding antibody to neutralizing epitope gp41 in HIV-1 subtype CRF 01_AE infection related to stage of disease

The responsiveness of gp41 antibody against epitope ELDKWA in HIV-1 infected subjects is of importance in neutralizing viral infectivity and for being related to disease progression. In this study, antibody titers to this neutralizing epitope from HIV-1 infected subjects at asymptomatic and AIDS stages in Thailand were investigated by peptide ELISA. The results showed that the frequency of antibody production against this neutralizing epitope was low (15-35%). Moreover, antibody titers to this epitope in sera from AIDS patients were significantly lower than those in sera from asymptomatic subjects which were collected in the same year (p=0.001). Comparison between the past (1992-1994) and present (2002) sera from asymptomatic infected individuals revealed that the earlier panel contained lower antibody titers than the later panel did (p = 0.05). In addition, random sera for HIV-1 infected subjects who were infected by diverse genetic subtypes, (A through G) including CRF 01_AE, had low titers of antibody to this region as well. It is assumed that antibody production to this epitope is low and related to the stage of HIV-1 infection.     

Search for epitope-specific antibody responses to the human immunodeficiency virus (HIV-1) envelope glycoproteins signifying resistance to disease development.

It is essential for the development of strategies for prevention and therapy of human immunodeficiency virus (HIV-1) infections to define host factors playing a dominant role in determining the clinical outcome of infection. Antibodies directed against restricted regions of the HIV-1 glycoproteins gp120 and gp41 are likely to represent important factors involved in host defense against HIV-1. Definition of qualitative and quantitative differences in the spectrum of anti-gp120 and anti-gp41 antibodies between two vastly different groups of HIV-1-infected individuals, long-term asymptomatic carriers, and individuals with acquired immunodeficiency syndrome (AIDS) who died, might reveal the epitope specificity of antibodies contributing to prevention of clinical disease. To accomplish this goal, sera from both groups were assayed for antibodies recognizing synthetic peptides from gp120/gp41 which were shown in earlier experiments to mimic epitopes on the two HIV-1 glycoproteins. None of the sera recognized all of the distinct 27 peptides from gp120 and gp41. The spectrum of antibodies was distinct for each of the sera from both groups of HIV-1-infected individuals. Nevertheless, antibody responses distinguishing the two groups from each other were discerned. In particular, it was possible to predict the unfavorable outcome of disease by comparative measurements of levels of antibodies to a peptide (303-338), corresponding to the entire V3 hypervariable loop of gp120 and/or by providing evidence for declining levels of these antibodies during the course of infection. Antibodies recognizing additional peptides [(219-245), (280-306), (425-452), (658-682), (729-758), (808-845), and (845-862)] were significantly less prevalent in AIDS patients than in asymptomatic carriers. It appears possible that maintenance of high levels of the respective antibodies would contribute to preventing AIDS in HIV-1-infected individuals. Active immunization with antigens containing epitopes defined by the respective peptides and/or administration of the corresponding antibodies may be considered as a modality for therapy of HIV-1 infections.     

Antibody-dependent cell-mediated cytotoxicity directed by a human monoclonal antibody reactive with gp120 of HIV-1.

We used a human monoclonal antibody (MAb; 15e) to identify an antibody-dependent cell-mediated cytotoxicity (ADCC) epitope on HIV-1 gp120. 15e has been shown to recognize a conformation-dependent epitope on gp120 which is important in both CD4 binding and neutralizing of HIV-1 infection. 15e binds to gp120 of HIV-1IIIB but not HIV-1RF. Using a standard ADCC assay, 15e was found to mediate ADCC against cells infected with HIV-1IIIB but not HIV-1RF. 15e did not mediate ADCC against cells with recombinant gp120 bound to surface CD4, indicating that 15e does not mediate innocent bystander ADCC against uninfected CD4 cells. To better define the 15e epitope, we performed ADCC against target cells infected with a vaccinia vector which expresses processed HIV-1IIIB gp160 from which the third variable region was deleted (amino acids, 312-328). MAb 15e efficiently mediated ADCC against cells expressing this altered form of gp120, indicating that this region is not contributing to the conformational epitope defined by 15e. 15e defines an important epitope in the human immune response to HIV-1 infection. Antibodies with 15e-like activity may be useful in immunoprophylaxis or immunotherapy of HIV-1 infection.     

Maternal antibodies to gp120 V3 sequence do not correlate with protection against vertical transmission of human immunodeficiency virus.

A retrospective study of sera from mothers infected with human immunodeficiency virus (HIV-1) was undertaken to investigate whether the titers or affinities of antibodies against the third hypervariable region (V3 loop) of gp120 correlated with transmission of the virus from mother to child. The cohort comprised 7 mothers who transmitted HIV-1 to their children and 20 who did not. Sera were screened for reactivity against two synthetic peptides, one encompassing the entire V3 loop of gp120 (amino acids 297-330) and the other containing an immunodominant epitope from gp41 (amino acids 596-614). Doubling dilutions of sera were tested to obtain antibody titers against both peptides: Anti-gp41 titers were used to normalize the anti-V3 titers. Maternal sera were also screened for the presence of high-affinity antibodies against the V3 peptide. No differences were observed in either titers or affinities of maternal antibodies to the V3 sequence from transmitters and nontransmitters.     

Immunocytochemical determination of antigen and epitope specificity of HIV-1-specific B cells in lymph-node biopsies from HIV-1-infected individuals.

Knowledge about B-cell dysfunction and HIV-specific antibody production is necessary for the understanding of both HIV-1-related immunopathology and the (vaccine-induced) humoral immunity involved in protection against AIDS. This paper describes the application of recently developed methods to detect epitope specificity of B cells in lymph-node biopsies with antigen-enzyme conjugates. Cryosections of five lymph-node biopsies from HIV-1-infected individuals and four control tissues were stained with a panel of HIV-1 antigen-enzyme conjugates: recombinant HIV-1 proteins (gp 160, gp 120 and p24), labelled with peroxidase, and synthetic peptides representing neutralizing epitopes from gp120 and gp41, labelled with alkaline phosphatase. Antibody-forming cells (AFCs) were detected in all the HIV-1-infected biopsies with gp160, gp120 and/or p24, in numbers up to 350 per section. AFCs producing specific antibodies against peptide 101 (SP 101), representing the neutralizing epitope 586-608 of gp41, were detected in one patient. These techniques allow correlation of in vivo function of B cells with lymph-node pathology, clinical stage of the disease and serological data. Their potential for the elucidation of HIV-related immunopathogenesis and the development of vaccines is discussed.     

Comparison of HIV Type 1 ADA gp120 monomers versus gp140 trimers as immunogens for the induction of neutralizing antibodies

Designing an immunogen for effective neutralizing antibody induction against diverse primary isolates of human immunodeficiency virus type 1 (HIV-1) is a high priority for HIV-1 vaccine development. Soluble gp120 envelope (Env) glycoprotein subunit vaccines elicit high titers of antibodies that neutralize T cell line-adapted (TCLA) strains but the antibodies possess poor neutralizing activity against many primary isolates. Previously, we generated soluble trimeric recombinant gp140 from the HIV-1 primary isolate ADA. Here we compared monomeric ADAgp120 and trimeric ADAgp140 as immunogens for neutralizing antibody responses in guinea pigs. Both immunogens generated a neutralizing antibody response that was detectable against the vaccine strain and several heterologous strains. The magnitude of this response was significantly greater in ADAgp140-immunized animals when measured against the TCLA strain, MN, and the R5 primary isolate, Bal. Two additional isolates (SS1196 and Bx08) were neutralized equally by sera from both groups of animals whereas other isolates were neutralized weakly or not at all. Despite equal titers of V3 loop specific binding antibodies in sera from both groups of animals, neutralization of ADA by sera from gp140-immunized animals was insensitive to the presence of ADA-V3 peptide, whereas addition of this peptide to sera from gp120- immunized animals blocked all detectable neutralizing activity against ADA. These results support the idea that trimeric gp140 is an improved immunogen compared to monomeric gp120 but that additional improvements are required to afford broad protection against a spectrum of heterologous primary HIV-1 isolates. This ADAgp140 immunogen may be considered a starting point from which to engineer additional improvements for cross-reactive neutralizing antibody induction.     

Importation of multiple HIV type 1 strains into West Papua, Indonesia (Irian Jaya).

HIV-1 from 16 sexually transmitted disease clinic patients in Timika, West Papua, Indonesia was amplified by RT-PCR and subtyped by a combination of envelope and gag region heteroduplex mobility analysis (HMA) and direct PCR DNA sequencing. HMA showed the presence of 14 subtype E (CRF01_AE) and 2 subtype B HIV-1. Phylogenetic analysis of a 540-bp V3-V4 region of gp120 showed that 9 of 10 CRF01_AE variants clustered tightly with a median distance of 1.3% (range, 0.5 to 2.2%) whereas 1 CRF01_AE variant diverged significantly from the others (median distance, 10.7%; range, 10.1 to 11.8%). One subtype B virus envelope was typical of United States/European strains whereas the other appeared to be related to Thai subtype B' variants. These results reflect the independent introduction of multiple HIV-1 strains into West Papua, with the rapid spread in the majority of infected patients tested of a single strain of HIV-1E (CRF01_AE).     

Identification of two neutralizing and 8 non-neutralizing epitopes on simian immunodeficiency virus envelope using monoclonal antibodies.

Ten new monoclonal antibodies (MAbs) to SIV envelope were produced and characterized. Using a panel of 28 MAbs, 10 antibody binding sites on SIV envelope protein were identified. Seven sites were located in gp120 and three in gp41. Five sites in gp120 and two in gp41 were defined by overlapping peptides. The remaining two sites on gp120 and one on gp41 were distinguished by competition binding assays but could not be defined by overlapping peptides, suggesting that they were discontinuous or conformational epitopes. Five of the 28 MAbs consistently and reliably neutralized the infectivity of SIVmac251. Two of these bound to a peptide (aa171-190) in the V2 region. The remaining three MAbs bound to a conformational epitope on gp120. These two neutralizing epitopes on SIV are analogous to similar epitopes recently described in HIV-1. In contrast, three MAbs binding to the V3 region of SIV failed to neutralize infectivity, suggesting that this region in SIV may by functionally different from the V3 loop in HIV-1.     

Safety and immunogenicity of combinations of recombinant subtype E and B human immunodeficiency virus type 1 envelope glycoprotein 120 vaccines in healthy Thai adults

Safety and immunogenicity of 2 recombinant human immunodeficiency virus (HIV) type 1 envelope glycoprotein (gp) 120 vaccines derived from SF2 (subtype B) and CM235 (CRF01_AE, Thai E) were evaluated in 370 Thai adults at low risk of HIV infection. Various doses of CM235 (25, 50, or 100 microg) and SF2 (0, 25, or 50 microg) gp120 were used. Eighty volunteers received placebo. There were no serious adverse events related to vaccination. Binding antibody developed in all vaccine recipients. There was no dose response to CM235 gp120, but a dose response to gp120 SF2 was present. Neutralizing antibodies to subtype E HIV-1 NPO3 and subtype B HIV-1 SF2 developed in 84% and 82% of vaccine recipients, respectively. Lymphoproliferative responses were detected in >95% of vaccine recipients. There was no evidence of antigenic interference in HIV-specific humoral or cellular responses. The gp120 Thai E and SF2 vaccines were safe and immunogenic in combination and could be advanced into phase 3 testing.     

Identification of human neutralization-inducing regions of the human immunodeficiency virus type 1 envelope glycoproteins.

Four major neutralizing regions of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein were identified and characterized with a panel of 80 HIV-1 antibody-positive human sera. Levels of neutralizing antibodies against the HIV-1 strains IIIB, SF2, and RF were compared with reactivity in ELISAs against peptides that correspond to certain regions of the HIV-1 envelope. A correlation between high neutralizing activity and strong seroreactivity against specific peptides suggested that the corresponding regions might be involved in neutralization. This was further substantiated by using peptides to inhibit neutralization by a panel of 10 HIV-1 antibody-positive sera. The positions of three neutralizing sites, defined earlier mostly by antisera from animals, were confirmed in the present study. Human sera thus recognize the strain-specific third variable region of gp120 (amino acids 304-318), the C-terminal end of gp120 (amino acids 489-508), and the conserved region in the intracellular part of gp41 (amino acids 732-746). It is likely that these different regions mediate help rather than self-sufficient neutralization. Furthermore, a human neutralizing region was detected in a conserved part of gp41 (amino acids 647-671). Accordingly, neutralizing antibodies directed to this region were found to be cross-reactive between HIV-1 strains. Peptides corresponding to these four regions were able to inhibit neutralization mediated by serum from HIV-1 antibody-positive individuals. These results indicate that this conserved B-cell epitope of the HIV-1 envelope elicits a virus-neutralizing antibody response during natural infection in humans and may therefore be considered for inclusion in a vaccine against HIV-1.     

Lack of correlation between maternal antibodies to V3 loop peptides of gp120 and perinatal HIV-1 transmission. The NYC Perinatal HIV Transmission Collaborative Study.

Recent reports have suggested that maternal antibodies to specific epitopes of the variable region 3 (V3 loop) of gp120 of HIV-1 might protect against perinatal transmission. In an attempt to confirm these findings, sera from 34 HIV-1-seropositive mothers, representing 13 episodes of mother-to-infant transmission and 23 episodes of non-transmission (two mothers had two pregnancies each during the study period), were tested for the presence of antibodies to various regions of the gp120 V3 loop. Synthetic peptides were generated from HIV-1MN. Of the four peptides tested by enzyme-linked immunosorbent assay (ELISA), only antibody to the C53 peptide (Env310-322, principal neutralizing determinant) was present in maternal sera. Antibody to the C53 sequence was present in 11 specimens from transmitting mothers and 21 from non-transmitting mothers (84.6 and 91.3%, respectively, P = 0.6). No reactivity was detected against the C51, C57, or C58 peptide sequences, located on the sides of the V3 loop. In an antigen-limited ELISA, only two specimens from transmitting mothers and two specimens from non-transmitting mothers had detectable 'high-affinity' antibodies to C53 at low antigen concentrations (15.4 and 8.7%, respectively; P = 0.6). Our results do not support previous reports that epitope-specific antibodies to the V3 loop peptides protect against perinatal transmission. Further research is required to determine whether any specific maternal humoral response might influence HIV-1 perinatal transmission.     

Antibody recognition of SIVmac envelope peptides in plasma from macaques experimentally infected with SIV/Mne

Four stretches of amino acid sequences encoded in conserved HIV-1 env domains and four parallel regions of the SIVmac env (two from gp120 and two from gp41/p32E) were fused to the NH2 terminus of beta-galactosidase by recombinant DNA techniques and used to analyze sera from three macaque species experimentally infected with SIV/Mne. All SIVmac env sequences were recognized by sera from the SIV/Mne-inoculated macaques. Western blot analysis performed with whole SIV/Mne, SIVmac, SIVagm, and HIV-1 antigens and sera from SIV/Mne-infected macaques also demonstrates that SIV/Mne is immunologically more closely related to SIVmac than to SIVagm or to HIV-1. Antibody levels to the gp120 NH2-terminal SIV-88 epitope appear to decrease in the infected Macaca nemestrina with progression of disease, as was also reported for the parallel HIV-1 epitope in HIV-1-infected individuals. Sera from all infected macaques reacted with the p32E-SIV-582 epitope (EKYLEDQAQLNAWGCAFRQVC). High titers to this immunodominant epitope could be detected at least 9 weeks postinfection and at a time when primarily the p28 and p32E antibodies were detectable in Western blots performed with whole disrupted SIV/Mne virus. In the majority of animals, antibody titers of 1:100,000 to SIV-582 develop during the infection and persist until death. Antibody responses to the SIV env epitopes in SIV/Mne-infected macaques thus resemble in many aspects (prevalence and immunogenicity) those observed previously for the corresponding HIV-1 env epitopes in HIV-1-infected humans.     

The conserved carboxy terminal region of HIV-1 gp120 is recognized by seronegative HIV-exposed people

OBJECTIVE: To screen HIV-positive, long-term exposed seronegative and low-risk individuals for the presence of antibodies against regions of HIV-1 gp120 that share some degree of homology with HLA. METHODS: Sera were obtained from 63 HIV-1-infected subjects [52 Centers for Disease Control and Prevention (CDC) stage 2 and 11 stages 3/4], 32 HIV-exposed uninfected (HEU) subjects and from 24 low-risk HIV-1 seronegative individuals. They were tested by a peptide-based enzyme-linked immunosorbent assay (ELISA) for reactivity against peptides derived from the HIV-1 gp120 C-terminal region that contain regions of MHC sequence/structural similarity. Ten randomly selected sera from each group were also screened for anti-class I antibodies. RESULTS: Thirty per cent of the long-term HIV-1-exposed seronegative individuals had antibodies against the conserved C-terminal region (C5) of HIV-1 gp120. However, sera from HEU individuals showed no reactivity against other peptides derived from the C2 region of gp120, also an HLA homologous region. Anti-C terminal gp120 antibodies were mainly of IgM subclass, although IgG-specific antibodies were also present. In addition, 70% of HEU individuals had antibodies to HLA class I molecules compared with 15% of HIV-positive patients (restricted to only those HIV-positive patients with anti C-terminal antibodies). CONCLUSION: Our results suggest that antibody responses against the C-terminal region of HIV gp120 and HLA class I may represent markers of apparent natural protection against HIV-1 infection.     

Antibody responses of chimpanzees immunized with synthetic peptides corresponding to full-length V3 hypervariable loops of HIV-1 envelope glycoproteins.

Immunization of primates or humans with human immunodeficiency virus type 1 (HIV-1) glycoproteins usually elicited moderate immune responses to the principal neutralizing determinant (PND) located within the V3 hypervariable loop of gp120. Since an antibody response to the PND appears to be protective, experiments were carried out to determine the responsiveness of chimpanzees to immunization with synthetic peptides corresponding to the full-length V3 loop. Seven chimpanzees (4 preimmunized with gp160, 2 preimmunized with HIV-1 antigens unrelated to gp160, and 1 unimmunized) were vaccinated with a mixture of full-length V3 loop peptides from 21 distinct HIV-1 isolates (clones) either in unconjugated form or linked to carrier proteins from HIV-1 nef and gag P18, respectively. Six chimpanzees developed high levels of antibodies to the peptides (dilution endpoints 1: greater than 25,000), and 5 had high levels of antibodies to gp120 from HIV-1IIIB (endpoint titers 1: greater than 500,000). Chimpanzees immunized with peptide-carrier conjugates (4) had antibodies to the carrier proteins nef and gag P18, respectively (endpoint titers 1: greater than or equal to 35,000). Virus-neutralizing (VN) antibodies were detected in sera of 5 of 7 chimpanzees, but were present at titers of 1: greater than or equal to 400 only in sera of 2 chimpanzees. One of these was challenged with HIV-1 and was protected against infection, as reported elsewhere. The antibodies were primarily specific for the HIV-1 isolate used for primary immunization before boosting with peptides. The relatively low dilution endpoints of VN antibodies as compared with endpoints determined by site-specific immunoassays probably can be ascribed to imperfect mimicry of conformational epitopes by synthetic peptides. Nevertheless, sequential or simultaneous immunization with recombinant envelope glycoproteins of HIV-1 and selected synthetic peptides offers an approach for eliciting protective immunity against HIV-1.     

Molecular epidemiology of HIV type 1 in Singapore and identification of novel CRF01_AE/B recombinant forms

To investigate HIV-1 molecular epidemiology in Singapore, we sequenced portions of three regions of the HIV-1 genome (protease HXB2: 2163 to 2620, gp120 HXB2: 6904 to 7628, and gp41 HXB2: 7817 to 8264) from 212 plasma samples collected between February 2008 and August 2009. From these samples, 109 (51.4%) generated interpretable data in all regions. Sixty-one (56.0%) were identified as CRF01_AE, 26 (23.9%) as subtype B and 14 (12.8%) as possible novel recombinant forms. The main novel recombinant pattern, detected in 13 sequences, had subtype B in protease and gp41 and CRF01_AE in gp120. There was intermixing of subtypes within transmission risk groups. However, 85% of subjects infected with the novel recombinant forms self-identified as men who have sex with men or bisexuals compared with only 41% of individuals infected with CRF01_AE and 62% infected with subtype B (p = 0.001).     

Antibodies to conserved epitopes of the HIV-1 envelope in sera from long-term non-progressors: prevalence and association with neutralizing activity

OBJECTIVE: Previous studies have shown that broadly neutralizing antibodies (NAb) are more frequent in long-term non-progressors (LTNP) than in other HIV-1 infected patients, but nothing is known about the envelope regions targeted by these broadly NAb. We investigated whether the breadth of neutralizing activity of sera was associated with the presence of specific antibodies (2F5- and/or 4E10-like, b12-like or 2G12-like antibodies) directed against conserved epitopes known to be involved in broad neutralization. METHODS: We assessed the ability of sera from 67 LTNP of the French ANRS cohort (ANRS CO15) to neutralize four heterologous primary isolates of four various clades. Competitive and non-competitive ELISA were developed for the specific comparison of levels of antibodies against these specific epitopes in neutralizing and non-neutralizing sera from LTNP. RESULTS: We found that higher 2G12-like antibody levels were significantly associated with the broadest neutralizing activity in sera from LTNP. Levels of 2G12-like antibodies were higher in the sera that neutralized the four isolates than in the others, with a median of 5.7 microg/ml [interquartile range (IQR), 2.7-9.3 microg/ml] versus 2.3 microg/ml (IQR, 1.1-3.9 microg/ml) (Mann-Whitney test, P = 0.03). Levels of antibodies against the other targeted envelope epitopes did not differ significantly between broadly and non-broadly neutralizing sera. CONCLUSION: These results suggest that the antigenicity of the "silent face" of gp120 that exposes the 2G12 epitope should be analysed in more detail, to find ways to induce broadly neutralizing antibodies.     

Characterization of recombinant gp120 and gp160 from HIV-1: binding to monoclonal antibodies and soluble CD4

We compared four preparations of recombinant HIV-1 envelope glycoprotein: mammalian (Chinese hamster ovary cells) gp120 (Celltech); baculovirus gp120 from American Biotechnologies Inc. (ABT) and from MicroGeneSys (MGS); and baculovirus gp160 (Institute of Virology, Oxford, UK). Each envelope glycoprotein binds to a neutralizing monoclonal antibody (MAb) directed against the V3 loop, confirming the integrity of this type-specific neutralization epitope. MGS gp120 binds abnormally well to a MAb which recognizes an epitope preferentially exposed on denatured gp120. Consistent with this finding, MGS gp120 binds to soluble CD4 (sCD4) with an affinity 50-100-fold lower than that of Celltech gp120. The affinity of Celltech gp120 from sCD4 is 2.3 nM, indistinguishable from that of gp120 extracted from HIV-1 virions. Baculovirus gp120 (ABT) and gp160 also have a high affinity for sCD4. A significant proportion of anti-gp120 antibodies in HIV-positive human sera recognize epitopes that are dependent on the mammalian glycosylation pattern, and a human HIV-positive serum inhibits the binding of mammalian gp120 to sCD4 five- to 10-fold more potently than it inhibits baculovirus gp120 binding to sCD4.