Recognition properties of V3-specific antibodies to V3 loop peptides derived from HIV-1 gp120 presented in multiple conformations

To identify structural constraints and amino acid sequences important for antibody recognition of the third variable domain (V3) of HIV-1 gp120, we have studied the solution conformation of three 35-mer circular V3 loop peptides derived from HIV-1 strains which differ in syncytium- (SI) and non-syncytium-inducing (NSI) capacity. In addition to 2D NMR and CD analyses, fluid- and solid-phase immunoassays were performed using V3-specific antibodies to V3 peptides and gp120 derived from different strains of HIV-1. NMR and CD spectroscopy indicated that circular and linear V3 loops exist in water as a dynamic ensemble of multiple conformations. Amino acid substitutions and biochemical modifications of the V3 loop were found to affect antibody binding depending on the presentation of the antigens. From NMR observations and immunological experiments, we provide evidence for a V3 loop specific monoclonal antibody interaction which is directed predominantly against linear epitopes rather than against discontinuous epitopes. The absence of a single defined solution conformation of 35-mer circular V3 peptides should be taken into account when using V3-related peptides to investigate structural elements in the V3 domain of the gp120 envelope protein of HIV-1 involved in biological processes of the virus.     

Antibodies to discontinuous or conformationally sensitive epitopes on the gp120 glycoprotein of human immunodeficiency virus type 1 are highly prevalent in sera of infected humans.

We have used an indirect-capture enzyme-linked immunosorbent assay to quantitate the reactivity of sera from human immunodeficiency virus type 1 (HIV-1)-infected humans with native recombinant gp120 (HIV-1 IIIB or SF-2) or with the gp120 molecule (IIIB or SF-2) denatured by being boiled in the presence of dithiothreitol with or without sodium dodecyl sulfate. Denaturation of IIIB gp120 reduced the titers of sera from randomly selected donors by at least 100-fold, suggesting that the majority of cross-reactive anti-gp120 antibodies present are directed against discontinuous or otherwise conformationally sensitive epitopes. When SF-2 gp120 was used, four of eight serum samples reacted significantly with the denatured protein, albeit with ca. 3- to 50-fold reductions in titer. Only those sera reacting with denatured SF-2 gp120 bound significantly to solid-phase-adsorbed SF-2 V3 loop peptide, and none bound to IIIB V3 loop peptide. Almost all antibody binding to reduced SF-2 gp120 was blocked by preincubation with the SF-2 V3 loop peptide, as was about 50% of the binding to native SF-2 gp120. When sera from a laboratory worker or a chimpanzee infected with IIIB were tested, the pattern of reactivity was reversed, i.e., there was significant binding to reduced IIIB gp120, but not to reduced SF-2 gp120. Binding of these sera to reduced IIIB gp120 was 1 to 10% that to native IIIB gp120 and was substantially decreased by preincubation with IIIB (but not SF-2) V3 loop peptide. To analyze which discontinuous or conformational epitopes were predominant in HIV-1-positive sera, we prebound monoclonal antibodies (MAbs) to IIIB gp120 and then added alkaline phosphatase-labelled HIV-1-positive sera. MAbs (such as 15e) that recognize discontinuous epitopes and compete directly with CD4 reduced HIV-1-positive sera binding by about 50%, whereas neutralizing MAbs to the C4, V2, and V3 domains of gp120 were either not inhibitory or only weakly so. Thus, antibodies to the discontinuous CD4-binding site on gp120 are prevalent in HIV-1-positive sera, antibodies to linear epitopes are less common, most of the antibodies to linear epitopes are directed against the V3 region, and most cross-reactive antibodies are directed against discontinuous epitopes, including regions involved in CD4 binding.     

Probing the structure of the human immunodeficiency virus surface glycoprotein gp120 with a panel of monoclonal antibodies.

We have probed the structures of monomeric and oligomeric gp120 glycoproteins from the LAI isolate of human immunodeficiency virus type 1 (HIV-1) with a panel of monoclonal antibodies (MAbs); most of these MAbs are directed against continuous epitopes. On native monomeric gp120, most of the first conserved (C1) domain is accessible to MAbs, although some regions of C1 are relatively inaccessible. All of the MAbs directed against the C2, C3, and C5 domains bind preferentially to denatured monomeric gp120, indicating that these regions of gp120 are poorly accessible on the native monomer, although the extreme C terminus in C5 is well exposed. Segments of the V1, V2, and V3 loops are exposed on the surface of monomeric gp120, although the base of the V3 loop is inaccessible. A portion of C4 is also available for MAb binding on monomeric gp120, as is the extreme C terminus in C5. However, on oligomeric gp120-gp41 complexes, only the V2 and V3 loops (and perhaps V1) are well exposed and a segment of the C4 region is partially exposed; continuous epitopes in C1 and C5 that are accessible to antibodies on monomeric gp120 are occluded on the oligomer. Although deletion of the V1, V2, and V3 loops resulted in increased exposure of several discontinuous epitopes overlapping the CD4-binding site, the exposure of most continuous epitopes on the monomeric gp120 glycoprotein was not affected. These results imply a HIV-1 gp120 structure in which the conserved continuous determinants are inaccessible; in some cases, this inaccessibility is due to intramolecular interactions between conserved regions, and in other cases, it is due to intermolecular interactions with other components of the glycoprotein spike. These findings have implications for the design of subunit vaccines based on gp120.     

Characterization of Simian-Human Immunodeficiency Virus Envelope Glycoprotein Epitopes Recognized by Neutralizing Antibodies from Infected Monkeys

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.     

Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains.

The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the human serum was only partially blocked by V3 peptide, indicating that this infected individual produced antibodies against epitopes in V3 that were expressed on the fusion glycoprotein but not by synthetic peptides. These data demonstrated that the chimeric glycoproteins described here effectively present native epitopes present in the V1/V2 and V3 domains of gp120 and provide efficient methods for detection of antibodies directed against native epitopes in these regions and for characterization of such epitopes.     

Comparative Immunogenicity of Evolved V1V2-Deleted HIV-1 Envelope Glycoprotein Trimers

Despite almost 30 years of research, no effective vaccine has yet been developed against HIV-1. Probably such a vaccine would need to induce both an effective T cell and antibody response. Any vaccine component focused on inducing humoral immunity requires the HIV-1 envelope (Env) glycoprotein complex as it is the only viral protein exposed on the virion surface. HIV-1 has evolved several mechanisms to evade broadly reactive neutralizing antibodies. One such a mechanism involves variable loop domains, which are highly flexible structures that shield the underlying conserved epitopes. We hypothesized that removal of such loops would increase the exposure and immunogenicity of these conserved regions. Env variable loop deletion however often leads to protein misfolding and aggregation because hydrophobic patches becoming solvent accessible. We have therefore previously used virus evolution to acquire functional Env proteins lacking the V1V2 loop. We then expressed them in soluble (uncleaved) gp140 forms. Three mutants were found to perform optimally in terms of protein expression, stability, trimerization and folding. In this study, we characterized the immune responses to these antigens in rabbits. The V1V2 deletion mutant ΔV1V2.9.VK induced a prominent response directed to epitopes that are not fully available on the other Env proteins tested but that effectively bound and neutralized the ΔV1V2 Env virus. This Env variant also induced more efficient neutralization of the tier 1 virus SF162. The immune refocusing effect was lost after booster immunization with a full-length gp140 protein with intact V1V2 loops. Collectively, this result suggests that deletion of variable domains could alter the specificity of the humoral immune response, but did not result in broad neutralization of neutralization-resistant virus isolates.     

Cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates.

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-Z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.     

Probing the structure of the V2 domain of human immunodeficiency virus type 1 surface glycoprotein gp120 with a panel of eight monoclonal antibodies: human immune response to the V1 and V2 domains.

We have analyzed a panel of eight murine monoclonal antibodies (MAbs) that depend on the V2 domain for binding to human immunodeficiency virus type 1 (HIV-1) gp120. Each MAb is sensitive to amino acid changes within V2, and some are affected by substitutions elsewhere. With one exception, the MAbs were not reactive with peptides from the V2 region, or only poorly so. Hence their ability to bind recombinant strain IIIB gp120 depended on the preservation of native structure. Three MAbs cross-reacted with strain RF gp120, but only one cross-reacted with MN gp120, and none bound SF-2 gp120. Four MAbs neutralized HIV-1 IIIB with various potencies, and the one able to bind MN gp120 neutralized that virus. Peptide serology indicated that antibodies cross-reactive with the HxB2 V1 and V2 regions are rarely present in HIV-1-positive sera, but the relatively conserved segment between the V1 and V2 loops was recognized by antibodies in a significant fraction of sera. Antibodies able to block the binding of V2 MAbs to IIIB or MN gp120 rarely exist in sera from HIV-1-infected humans; more common in these sera are antibodies that enhance the binding of V2 MAbs to gp120. This enhancement effect of HIV-1-positive sera can be mimicked by several human MAbs to different discontinuous gp120 epitopes. Soluble CD4 enhanced binding of one V2 MAb to oligomeric gp120 but not to monomeric gp120, perhaps by inducing conformational changes in the oligomer.     

Study of the V3 Loop as a Target Epitope for Antibodies Involved in the Neutralization of Primary Isolates versus T-Cell-Line-Adapted Strains of Human Immunodeficiency Virus Type 1

Previous studies characterized the third variable (V3) loop of the envelope gp120 as the principal neutralizing determinant for laboratory T-cell-line-adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1). However, primary viruses isolated from infected individuals are more refractory to neutralization than TCLA strains, suggesting that qualitatively different neutralizing antibodies may be involved. In this study, we investigated whether the V3 loop constitutes a linear target epitope for antibodies neutralizing primary isolates. By using peptides representative of the V3 regions of various primary isolates, an early, relatively specific and persistent antibody response was detected in sera from HIV-infected patients. To assess the relationship between these antibodies and neutralization, the same peptides were used in competition and depletion experiments. Addition of homologous V3 peptides led to a competitive inhibition in the neutralization of the TCLA strain HIV_MN/MT-4 but had no effect on the neutralization of the autologous primary isolate. Similarly, the removal of antibodies that bind to linear V3 epitopes resulted in a loss of HIV_MN/MT-4 neutralization, whereas no decrease in the autologous neutralization was measured. The different roles of V3-specific antibodies according to the virus considered were thereby brought to light. This confirmed the involvement of V3 antibodies in the neutralization of a TCLA strain but emphasized a more pronounced contribution of either conformational epitopes or epitopes outside the V3 loop as targets for antibodies neutralizing primary HIV-1 isolates. This result underlines the need to focus on new vaccinal immunogens with epitopes able to induce broadly reactive and efficient antibodies that neutralize a wide range of primary HIV-1 isolates.     

Rationally Targeted Mutations at the V1V2 Domain of the HIV-1 Envelope to Augment Virus Neutralization by Anti-V1V2 Monoclonal Antibodies

HIV-1 envelope glycoproteins (Env) are the only viral antigens present on the virus surface and serve as the key targets for virus-neutralizing antibodies. However, HIV-1 deploys multiple strategies to shield the vulnerable sites on its Env from neutralizing antibodies. The V1V2 domain located at the apex of the HIV-1 Env spike is known to encompass highly variable loops, but V1V2 also contains immunogenic conserved elements recognized by cross-reactive antibodies. This study evaluates human monoclonal antibodies (mAbs) against V2 epitopes which overlap with the conserved integrin α4β7-binding LDV/I motif, designated as the V2i (integrin) epitopes. We postulate that the V2i Abs have weak or no neutralizing activities because the V2i epitopes are often occluded from antibody recognition. To gain insights into the mechanisms of the V2i occlusion, we evaluated three elements at the distal end of the V1V2 domain shown in the structure of V2i epitope complexed with mAb 830A to be important for antibody recognition of the V2i epitope. Amino-acid substitutions at position 179 that restore the LDV/I motif had minimal effects on virus sensitivity to neutralization by most V2i mAbs. However, a charge change at position 153 in the V1 region significantly increased sensitivity of subtype C virus ZM109 to most V2i mAbs. Separately, a disulfide bond introduced to stabilize the hypervariable region of V2 loop also enhanced virus neutralization by some V2i mAbs, but the effects varied depending on the virus. These data demonstrate that multiple elements within the V1V2 domain act independently and in a virus-dependent fashion to govern the antibody recognition and accessibility of V2i epitopes, suggesting the need for multi-pronged strategies to counter the escape and the shielding mechanisms obstructing the V2i Abs from neutralizing HIV-1.     

Multibranched V3 peptides inhibit human immunodeficiency virus infection in human lymphocytes and macrophages.

Synthetic polymeric constructions (SPCs) including the consensus sequence of the human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein gp120 V3 loop (GPGRAF) blocked the fusion between HIV-1- and HIV-2-infected cells and CD4+ uninfected cells. A structure-activity relationship study using V3 SPC analogs showed that the most efficient inhibitor of cell fusion was an eight-branched SPC with the hexapeptide motif GPGRAF (i.e., [GPGRAF]8-SPC). N-terminal acetylation or incorporation of D-amino acids in the GPGRAF sequence of this SPC resulted in significant loss of activity. Analogs with fewer than six residues in the motif (i.e., GPGRA or GPGR), as well as SPCs with a nonrelevant sequence, did not inhibit cell fusion, demonstrating the high specificity of the antifusion activity. [GPGRAF]8-SPC, which was not toxic to CEM cells at concentrations of up to 50 microM, inhibited 50% of HIV-1(LAI) replication in these cells at a concentration of 0.07 microM. Moreover, [GPGRAF]8-SPC inhibited the infection of human peripheral blood mononuclear cells by several HIV-1 and HIV-2 isolates, including laboratory strains [HIV-1(LAI), HIV-1(NDK), and HIV-2(ROD)], and fresh primary isolates, including two zidovudine-resistant HIV-1 isolates and two HIV-2 isolates obtained from infected individuals. The multibranched peptide also inhibited infection of human primary macrophages by the highly cytopathic macrophage-tropic isolate HIV-1(89.6). The antiviral activity of [GPGRAF]8-SPC was not related to a virucidal effect, since preincubation of HIV-1 with the peptide did not affect its infectious titer. This result is in agreement with the concept that the multibranched peptide mimics a part of the V3 loop and thus interacts with the host cell. The therapeutic properties of synthetic multibranched peptides based on the V3 loop consensus motif should be evaluated in HIV-infected patients.     

Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1-infected individuals

A small proportion of HIV-infected individuals generate a neutralizing antibody (NAb) response of exceptional magnitude and breadth. A detailed analysis of the critical epitopes targeted by broadly neutralizing antibodies should help to define optimal targets for vaccine design. HIV-1-infected subjects with potent cross-reactive serum neutralizing antibodies were identified by assaying sera from 308 subjects against a multiclade panel of 12 "tier 2" viruses (4 each of subtypes A, B, and C). Various neutralizing epitope specificities were determined for the top 9 neutralizers, including clade A-, clade B-, clade C-, and clade A/C-infected donors, by using a comprehensive set of assays. In some subjects, neutralization breadth was mediated by two or more antibody specificities. Although antibodies to the gp41 membrane-proximal external region (MPER) were identified in some subjects, the subjects with the greatest neutralization breadth targeted gp120 epitopes, including the CD4 binding site, a glycan-containing quaternary epitope formed by the V2 and V3 loops, or an outer domain epitope containing a glycan at residue N332. The broadly reactive HIV-1 neutralization observed in some subjects is mediated by antibodies targeting several conserved regions on the HIV-1 envelope glycoprotein.     

epitopes immediately below the base of the V3 loop of gp120 as targets for the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals

Epitopes that drive the initial autologous neutralizing antibody response in HIV-1-infected individuals could provide insights for vaccine design. Although highly strain specific, these epitopes are immunogenic, vulnerable to antibody attack on infectious virus, and could be involved in the ontogeny of broadly neutralizing antibody responses. To delineate such epitopes, we used site-directed mutagenesis, autologous plasma samples, and autologous monoclonal antibodies to map the amino acid changes that led to escape from the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals. Additional mapping of the epitopes was accomplished by using alanine scanning mutagenesis. Escape in the two individuals occurred by different pathways, but the responses in both cases appeared to be directed against the same region of gp120. In total, three amino acid positions were identified that were independently associated with autologous neutralization. Positions 295 and 332 are located immediately before and after the N- and C-terminal cysteines of the V3 loop, respectively, the latter of which affected an N-linked glycan that was critical to the neutralization epitope. Position 415 affected an N-linked glycan at position 413 in the C terminus of V4 that might mask epitopes near the base of V3. All three sites lie in close proximity on a four-stranded antiparallel sheet on the outer domain of gp120. We conclude that a region just below the base of the V3 loop, near the coreceptor binding domain of gp120, can be a target for autologous neutralization.     

Examination of sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals for antibodies reactive with peptides corresponding to the principal neutralizing determinant of HIV-1 gp120 and for in vitro neutralizing activity.

Sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals from the United States and Tanzania were examined for antibody reactivity to four synthetic peptides which corresponded to the principal neutralizing determinant from the V3 region of HIV-1 gp120. We observed that the majority of sera from both countries contained antibodies reactive with a V3 peptide whose sequence is based on that of the HIV-1 MN isolate. We were unable to establish a relationship between the presence of V3-reactive antibodies, as measured by enzyme-linked immunosorbent assay and neutralization of homologous HIV-1 isolates, in sera from either the United States or Tanzania. We observed that some sera which contained high antibody titers to the V3 peptides failed to neutralize HIV-1, while others with no antibody reactivity to the panel of V3 peptides exhibited in vitro neutralizing activity. These results suggest that neutralizing epitopes exist outside the V3 loop and that the presence of V3-reactive antibodies in sera does not imply in vitro neutralization of the homologous HIV-1 isolate. In addition, it appears that the V3 loop may consist of both neutralizing and nonneutralizing epitopes. The identification of neutralizing as well as nonneutralizing epitopes will be important for the design of potential HIV-1 vaccines.     

Relationship of the human immunodeficiency virus type 1 gp120 third variable loop to a component of the CD4 binding site in the fourth conserved region.

Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein.

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.     

Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding.

The binding of human immunodeficiency virus type 1 (HIV-1) to the cellular receptor CD4 has been suggested to induce conformational changes in the viral envelope glycoproteins that promote virus entry. Conserved, discontinuous epitopes on the HIV-1 gp120 glycoprotein recognized by the 17b, 48d, and A32 antibodies are preferentially exposed upon the binding of soluble CD4 (sCD4). The binding of the 17b and 48d antibodies to the gp120 glycoprotein can also be enhanced by the binding of the A32 antibody. Here we constructed HIV-1 gp120 mutants in which the variable segments of the V1/V2 and V3 structures were deleted, individually or in combination, while the 17b, 48d, and A32 epitopes were retained. The effects of the variable loop deletions on the function of the HIV-1 envelope glycoproteins and on the exposure of epitopes induced by sCD4 or A32 binding to the monomeric gp120 glycoprotein were examined. The variable-loop-deleted envelope glycoproteins were able to mediate virus entry, albeit at lower efficiencies than those of the wild-type glycoproteins. Thus, the V1/V2 and V3 variable sequences contribute to the efficiency of HIV-1 entry but are not absolutely required for the process. Neither the V1/V2 nor V3 loops were necessary for the increase in exposure of the 17b/48d epitopes induced by binding of the A32 monoclonal antibody. By contrast, induction of the 17b, 48d, and A32 epitopes by sCD4 binding apparently involves a movement of the V1/V2 loops, which in the absence of CD4 partially mask these epitopes on the native gp120 monomer. The results obtained with a mutant glycoprotein containing a deletion of the V1 loop alone indicated that the contribution of the V2 loop to these phenomena was more significant than that of the V1 sequences. These results suggest that the V1/V2 loops, which have been previously implicated in CD4-modulated, postattachment steps in HIV-1 entry, contribute to CD4-induced gp120 conformational changes detected by the 17b, 48d, and A32 antibodies.     

Dissecting the neutralizing antibody specificities of broadly neutralizing sera from human immunodeficiency virus type 1-infected donors

Attempts to elicit broadly neutralizing antibody responses by human immunodeficiency virus type 1 (HIV-1) vaccine antigens have been met with limited success. To better understand the requirements for cross-neutralization of HIV-1, we have characterized the neutralizing antibody specificities present in the sera of three asymptomatic individuals exhibiting broad neutralization. Two individuals were infected with clade B viruses and the third with a clade A virus. The broadly neutralizing activity could be exclusively assigned to the protein A-reactive immunoglobulin G (IgG) fraction of all three donor sera. Neutralization inhibition assays performed with a panel of linear peptides corresponding to the third hypervariable (V3) loop of gp120 failed to inhibit serum neutralization of a panel of HIV-1 viruses. The sera also failed to neutralize chimeric simian immunodeficiency virus (SIV) and HIV-2 viruses displaying highly conserved gp41-neutralizing epitopes, suggesting that antibodies directed against these epitopes likely do not account for the broad neutralizing activity observed. Polyclonal IgG was fractionated on recombinant monomeric clade B gp120, and the neutralization capacities of the gp120-depleted samples were compared to that of the original polyclonal IgG. We found that the gp120-binding antibody population mediated neutralization of some isolates, but not all. Overall, the data suggest that broad neutralization results from more than one specificity in the sera but that the number of these specificities is likely small. The most likely epitope recognized by the monomeric gp120 binding neutralizing fraction is the CD4 binding site, although other epitopes, such as the glycan shield, cannot be excluded.     

Conformational changes affecting the V3 and CD4-binding domains of human immunodeficiency virus type 1 gp120 associated with env processing and with binding of ligands to these sites.

Two neutralizing human monoclonal antibodies (HuMAbs) directed against epitopes located near the tip of the V3 loop of human immunodeficiency virus type 1 env protein recognized solubilized gPr160, but not gp120, in radioimmunoprecipitation assays. Efficient immunoprecipitation of solubilized gp120 by these antibodies did occur in the presence of HuMAb 1125H, directed against a conformational epitope overlapping the CD4-binding site, or its F(ab')2 fragment. In contrast to the inability of the anti-V3 antibodies to immunoprecipitate solubilized gp120, these HuMAbs did bind to gp120 in intact virions; this level of binding increased severalfold in the presence of the F(ab')2 fragment of 1125H. These results demonstrate that neutralization epitopes in the V3 loop are sequestered in soluble gp120 but partly exposed in gPr160 and in virion-associated gp120 and that binding of antibodies to the discontinuous CD4-binding site leads to conformational changes that result in the exposure of V3 epitopes in soluble gp120 and their enhanced accessibility in gPr160 and in virion-associated gp120. Enhanced binding of suboptimal concentrations of 1125H to soluble gp120 was also induced by the presence of an anti-V3 HuMAb, indicating the occurrence of reciprocal allosteric interactions between the V3 loop and the CD4-binding site. It is likely that these effects contribute to the synergistic neutralization of human immunodeficiency virus type 1 previously reported for antibodies directed against these two regions.     

Longer V1V2 region with increased number of potential N-linked glycosylation sites in the HIV-1 envelope glycoprotein protects against HIV-specific neutralizing antibodies

Human immunodeficiency virus type 1 (HIV-1) has the ability to adapt to the host environment by escaping from host immune responses. We previously observed that escape from humoral immunity, both at the individual and at a population level, coincided with longer variable loops and an increased number of potential N-linked glycosylation sites (PNGS) in the viral envelope glycoprotein (Env) and, in particular, in variable regions 1 and 2 (V1V2). Here, we provide several lines of evidence for the role of V1V2 in the resistance of HIV-1 to neutralizing antibodies. First, we determined that the increasing neutralization resistance of a reference panel of tier-categorized neutralization-sensitive and -resistant HIV-1 variants coincided with a longer V1V2 loop containing more PNGS. Second, an exchange of the different variable regions of Env from a neutralization-sensitive HIV-1 variant into a neutralization-resistant escape variant from the same individual revealed that the V1V2 loop is a strong determinant for sensitivity to autologous-serum neutralization. Third, exchange of the V1V2 loop of neutralization-sensitive HIV-1 variants from historical seroconverters with the V1V2 loop of neutralization-resistant HIV-1 variants from contemporary seroconverters decreased the neutralization sensitivity to CD4-binding site-directed antibodies. Overall, we demonstrate that an increase in the length of the V1V2 loop and/or the number of PNGS in that same region of the HIV-1 envelope glycoprotein is directly involved in the protection of HIV-1 against HIV-specific neutralizing antibodies, possibly by shielding underlying epitopes in the envelope glycoprotein from antibody recognition.