Immunization with a soluble CD4-gp120 complex preferentially induces neutralizing anti-human immunodeficiency virus type 1 antibodies directed to conformation-dependent epitopes of gp120.

Preservation of the conformation of recombinant gp120 in an adjuvant, enabling it to elicit conformation-dependent, epitope-specific, broadly neutralizing antibodies, may be critical for the development of any gp120-based human immunodeficiency virus type 1 (HIV-1) vaccine. It was hypothesized that recombinant gp120 complexed with recombinant CD4 could stabilize the conformation-dependent neutralizing epitopes and effectively deliver them to the immune system. Therefore, a soluble CD4-gp120 complex in Syntex adjuvant formulation was tested with mice for its ability to induce neutralizing anti-gp120 antibody responses. Seventeen monoclonal antibodies (MAbs) were generated and characterized. Immunochemical studies, neutralization assays, and mapping studies with gp120 mutants indicated that the 17 MAbs fell into three groups. Four of them were directed to what is probably a conformational epitope involving the C1 domain and did not possess virus-neutralizing activities. Another four MAbs bound to V3 peptide 302-321 and exhibited cross-reactive gp120 binding and relatively weak virus-neutralizing activities. These MAbs were very sensitive to amino acid substitutions, not only in the V3 regions but also in the base of the V1/V2 loop, implying a conformational constraint on the epitope. The last group of nine MAbs recognized conformation-dependent epitopes near the CD4 binding site of gp120 and inhibited the gp120-soluble CD4 interaction. Four of these nine MAbs showed broadly neutralizing activities against multiple laboratory-adapted strains of HIV-1, three of them neutralized only HIVIIIB, and the two lower-affinity MAbs did not neutralize any strain tested. Collectively, the results from this study indicate that immunization with the CD4-gp120 complex can elicit antibodies to conformationally sensitive gp120 epitopes, with some of the antibodies having broadly neutralizing activities. We suggest that immunization with CD4-gp120 complexes may be worth evaluating further for the development of an AIDS vaccine.     

Synergistic neutralization of human immunodeficiency virus type 1 by combinations of human monoclonal antibodies.

The ability of antibodies to the V3 region and the CD4-binding domain (CD4bd) of human immunodeficiency virus type 1 (HIV-1) to act in synergy to neutralize HIV has been demonstrated previously. However, synergy between antibodies to other HIV-1 epitopes has not been studied. We have used 21 combinations of human monoclonal antibodies (MAbs) directed against different epitopes of the gp120 and gp41 proteins of HIV-1 to evaluate their ability to act in synergy to neutralize HIV-1. Combinations of anti-V3 and anti-CD4bd antibodies, anti-V3 and anti-gp120 C-terminus antibodies, anti-CD4bd and anti-C-terminus antibodies, anti-V3 and anti-gp41 antibodies, and anti-CD4bd and anti-gp41 antibodies were tested. Our results show that some, but not all anti-V3 antibodies can act in synergy with anti-CD4bd antibodies. In addition, for the first time, antibodies to the C-terminus region have been found to act in synergy with the anti-CD4bd antibodies. Various anti-CD4bd MAbs also act in synergy when used together. The use of such cocktails of human MAbs for passive immunization against HIV-1 may prove to be important for therapy in postexposure settings and for prevention of maternal-fetal transmission of the virus. The results also provide information on the types of antibodies that should be elicited by an effective vaccine.     

Conserved and exposed epitopes on intact, native, primary human immunodeficiency virus type 1 virions of group M

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

Synergistic inhibition of human immunodeficiency virus type 1 envelope glycoprotein-mediated cell fusion and infection by an antibody to CD4 domain 2 in combination with anti-gp120 antibodies.

Antibodies to several epitopes of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41) can synergize in inhibiting HIV-1 infection. In the present study we tested the ability of a monoclonal antibody (MAb), 5A8, which interacts with CD4 domain 2, and other CD4-specific MAbs to synergize with antibodies against gp120. We have previously found that 5A8 inhibits HIV-1 entry without interfering with gp120 binding to CD4, presumably by affecting a postbinding membrane fusion event. Because antibodies to the gp120 V3 loop also affect post-CD4-gp120-binding events, 5A8 was first tested in combination with anti-V3 loop antibodies for possible synergy. The anti-V3 loop antibodies 0.5 beta, NEA-9205, and 110.5 acted synergistically with 5A8 in inhibiting syncytium formation between gp120-gp41- and CD4-expressing cells. A human MAb to an epitope of gp120 involved in CD4 binding, IAM 120-1B1, and another anti-CD4 binding site antibody, PC39.13, also exerted synergistic effects in combination with 5A8. Similarly, an antibody against the gp120 binding site on CD4, 6H10, acted synergistically with an anti-V3 loop antibody, NEA-9205. However, a control anti-CD4 antibody, OKT4, which does not significantly inhibit syncytium formation alone, produced only an additive effect when combined with NEA-9205. Serum from HIV-1-infected individuals, which presumably contains antibodies to the V3 loop and the CD4 binding site, exhibited a strong synergistic effect with 5A8 in inhibiting infection by a patient HIV-1 isolate (0104B) and in blocking syncytium formation. These results indicate that therapeutics based on antibodies affecting both non-gp120 binding and gp120 binding epitopes of the target receptor molecule, CD4, could be efficient in patients who already contain anti-gp120 antibodies and could also be used to enhance passive immunization against HIV-1 in combination with anti-gp120 antibodies.     

Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1.

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.     

Two major groups of neutralizing anti-gp120 antibodies exist in HIV-infected individuals. Evidence for epitope diversity around the CD4 attachment site.

The aim of this study was to dissect neutralizing anti-gp120 antibody populations in seropositive asymptomatic individuals. Murine anti-Id mAb were raised against polyclonal affinity-purified human anti-gp120 antibodies. These anti-Id mAb were used to fractionate anti-gp120 antibodies from a pool of HIV-positive sera into idiotypically distinct anti-gp120 antibody (Id+Ab) preparations. Immunochemical and neutralization studies indicated that all Id+Ab that neutralized HIV-1 in vitro interacted with either the V3 loop or the CD4 attachment site of gp120. The V3-specific Id+Ab neutralized HIV-1 in a strain-restricted manner. Id+Ab specific for the CD4 attachment site exhibited different spectra of neutralizing activities against multiple strains of HIV-1. This finding indicates that multiple, antigenically diverse epitopes reside around the CD4 attachment site of gp120. Significantly, depletion of the Id+Ab from affinity-purified total anti-gp120 antibodies abrogated most of the neutralizing activities of these antibodies, suggesting that neutralizing anti-gp120 antibodies consist of two major specificities, either to the V3 region or to the CD4 attachment site. The understanding of specificities and neutralizing activities of different anti-gp120 antibodies in seropositive healthy individuals will be helpful for designing effective vaccines and immunotherapeutic strategies for AIDS.     

N-linked glycosylation of the V3 loop and the immunologically silent face of gp120 protects human immunodeficiency virus type 1 SF162 from neutralization by anti-gp120 and anti-gp41 antibodies

We examined how asparagine-linked glycans within and adjacent to the V3 loop (C2 and C3 regions) and within the immunologically silent face (V4, C4, and V5 regions) of the human immunodeficiency virus (HIV) SF612 envelope affect the viral phenotype. Five of seven potential glycosylation sites are utilized when the virus is grown in human peripheral blood mononuclear cells, with the nonutilized sites lying within the V4 loop. Elimination of glycans within and adjacent to the V3 loop renders SF162 more susceptible to neutralization by polyclonal HIV(+)-positive and simian/human immunodeficiency virus-positive sera and by monoclonal antibodies (MAbs) recognizing the V3 loop, the CD4- and CCR5-binding sites, and the extracellular region of gp41. Importantly, our studies also indicate that glycans located within the immunologically silent face of gp120, specifically the C4 and V5 regions, also conferred on SF162 resistance to neutralization by anti-V3 loop, anti-CD4 binding site, and anti-gp41 MAbs but not by antibodies targeting the coreceptor binding site. We also observed that the amino acid composition of the V4 region contributes to the neutralization phenotype of SF162 by anti-V3 loop and anti-CD4 binding site MAbs. Collectively, our data support the proposal that the glycosylation and structure of the immunologically silent face of the HIV envelope plays an important role in defining the neutralization phenotype of HIV type 1.     

Replicative function and neutralization sensitivity of envelope glycoproteins from primary and T-cell line-passaged human immunodeficiency virus type 1 isolates.

The structure, replicative properties, and sensitivity to neutralization by soluble CD4 and monoclonal antibodies were examined for molecularly cloned envelope glycoproteins derived from human immunodeficiency virus type 1 (HIV-1) viruses either isolated directly from patients or passaged in T-cell lines. Complementation of virus entry into peripheral blood mononuclear cell targets by primary patient envelope glycoproteins exhibited efficiencies ranging from that observed for the HXBc2 envelope glycoproteins, which are derived from a T-cell line-passaged virus, to approximately fivefold-lower values. The ability of the envelope glycoproteins to complement virus entry roughly correlated with sensitivity to neutralization by soluble CD4. Laboratory-adapted viruses were sensitive to neutralization by monoclonal antibodies directed against the CD4-binding site and the third variable (V3) loop of the gp120 glycoprotein. By comparison, viruses with envelope glycoproteins from primary patient isolates exhibited decreased sensitivity to neutralization by these monoclonal antibodies; for these viruses, neutralization sensitivity correlated with replicative ability. Subinhibitory concentrations of soluble CD4 and a CD4-binding site-directed antibody significantly enhanced the entry of viruses containing envelope glycoproteins from some primary patient isolates. The sensitivity of viruses containing the different envelope glycoproteins to neutralization by soluble CD4 or monoclonal antibodies could be predicted by assays dependent on the binding of the inhibitory molecule to the oligomeric envelope glycoprotein complex but less well by assays measuring binding to the monomeric gp120 glycoprotein. These results indicate that the intrinsic structure of the oligomeric envelope glycoprotein complex of primary HIV-1 isolates, while often less than optimal with respect to the mediation of early events in virus replication, allows a relative degree of resistance to neutralizing antibodies. The interplay of selective forces for higher virus replication efficiency and resistance to neutralizing antibodies could explain the temporal course described for the in vivo emergence of HIV-1 isolates with differing phenotypes.     

Comparison of the Antibody Repertoire Generated in Healthy Volunteers following Immunization with a Monomeric Recombinant gp120 Construct Derived from a CCR5/CXCR4-Using Human Immunodeficiency Virus Type 1 Isolate with Sera from Naturally Infected Individuals

We have characterized sera from healthy volunteers immunized with a monomeric recombinant gp120 (rgp120) derived from a CCR5/CXCR4 (R5X4)-using subtype B isolate of human immunodeficiency virus type (HIV-1), HIV-1_W61D, in comparison to sera from long-term HIV-1-infected individuals, using homologous reagents. Sera from vaccinees and HIV-1 positive subjects had similar binding titers to native monomeric rgp120_W61D and showed a similar titer of antibodies inhibiting the binding of soluble CD4 (sCD4) to rgp120_W61D. However, extensive peptide binding studies showed that the overall pattern of recognition of vaccinee and HIV-1-positive sera is different, with vaccinee sera displaying a wider and more potent recognition of linear V1/V2 and V3 domain epitopes. Neutralization of homologous HIV-1_W61D or heterologous HIV-1_M2424/4 peripheral blood mononuclear cell (PBMC)-derived virus lines by vaccinee sera could be achieved, but only after adaptation of the viruses to T-cell lines and was quickly lost on readaptation to growth in PBMC. Sera from HIV-positive individuals were able to neutralize both PBMC-grown and T-cell line-adapted viruses. Interestingly, rgp120_W61D was recognized by monoclonal antibodies previously shown to neutralize primary HIV-1 isolates. The use of very potent adjuvants and R5X4 rgp120 led to an antibody response equivalent in binding activity and inhibition of binding of sCD4 to gp120 to that of HIV-positive individuals but did not lead to the induction of antibodies capable of neutralizing PBMC-grown virus.     

Primary isolates of human immunodeficiency virus type 1 are relatively resistant to neutralization by monoclonal antibodies to gp120, and their neutralization is not predicted by studies with monomeric gp120.

A panel of anti-gp120 human monoclonal antibodies (HuMAbs), CD4-IgG, and sera from people infected with human immunodeficiency virus type 1 (HIV-1) was tested for neutralization of nine primary HIV-1 isolates, one molecularly cloned primary strain (JR-CSF), and two strains (IIIB and MN) adapted for growth in transformed T-cell lines. All the viruses were grown in mitogen-stimulated peripheral blood mononuclear cells and were tested for their ability to infect these cells in the presence and absence of the reagents mentioned above. In general, the primary isolates were relatively resistant to neutralization by the MAbs tested, compared with the T-cell line-adapted strains. However, one HuMAb, IgG1b12, was able to neutralize most of the primary isolates at concentrations of < or = 1 microgram/ml. Usually, the inability of a HuMAb to neutralize a primary isolate was not due merely to the absence of the antibody epitope from the virus; the majority of the HuMAbs bound with high affinity to monomeric gp120 molecules derived from various strains but neutralized the viruses inefficiently. We infer therefore that the mechanism of resistance of primary isolates to most neutralizing antibodies is complex, and we suggest that it involves an inaccessibility of antibody binding sites in the context of the native glycoprotein complex on the virion. Such a mechanism would parallel that which was previously postulated for soluble CD4 resistance. We conclude that studies of HIV-1 neutralization that rely on strains adapted to growth in transformed T-cell lines yield the misleading impression that HIV-1 is readily neutralized. The more relevant primary HIV-1 isolates are relatively resistant to neutralization, although these isolates can be potently neutralized by a subset of human polyclonal or monoclonal antibodies.     

Identification and characterization of a neutralization site within the second variable region of human immunodeficiency virus type 1 gp120.

Two monoclonal antibodies designated BAT085 and G3-136 were raised by immunizing BALB/c mice with gp120 purified from human immunodeficiency virus type 1 (HIV-1) IIIB-infected H9 cell extracts. Among three HIV-1 laboratory isolates (IIIB, MN, and RF), BAT085 neutralized only IIIB infection of CEM-SS cells, whereas G3-136 neutralized both IIIB and RF. These antibodies also neutralized a few primary HIV-1 isolates in the infection of activated human peripheral blood mononuclear cells. In indirect immunofluorescence assays, BAT085 bound to H9 cells infected with IIIB or MN, while G3-136 bound to H9 cells infected with IIIB or RF, but not MN. Using sequence-overlapping synthetic peptides of HIV-1 IIIB gp120, the binding site of BAT085 and G3-136 was mapped to a peptidic segment in the V2 region (amino acid residues 169 to 183). The binding of these antibodies to immobilized gp120 was not inhibited by the antibodies directed to the principal neutralization determinant in the V3 region or to the CD4-binding domain of gp120. In a competition enzyme-linked immunosorbent assay, soluble CD4 inhibited G3-136 but not BAT085 from binding to gp120. Deglycosylation of gp120 by endo-beta-N-acetylglucosaminidase H or reduction of gp120 by dithiothreitol diminished its reactivity with G3-136 but not with BAT085. These results indicate that the V2 region of gp120 contains multiple neutralization determinants recognized by antibodies in both a conformation-dependent and -independent manner.     

Structural modulations of the envelope gp120 glycoprotein of human immunodeficiency virus type 1 upon oligomerization and differential V3 loop epitope exposure of isolates displaying distinct tropism upon virion-soluble receptor binding.

We investigated the binding of conformation-dependent anti-V2, anti-V3, and anti-CD4-binding site monoclonal antibodies to monomeric and virion-associated gp120 from human immunodeficiency virus type 1 isolates displaying marked differences in cell tropism. For all viruses examined, we found that the half-maximal binding values of the anti-V2 and anti-CD4-binding site antibodies with virion-associated gp120 were higher than those with monomeric gp120, but the maximum amount of antibodies bound was diminished only for one of the anti-V2 antibodies tested. These observations suggest that upon gp120 oligomerization, the V2 loop and CD4-binding site undergo conformational changes and that particular epitopes within these domains are occluded in the oligomeric gp120. In contrast, although the overall binding patterns and half-maximal binding values of the anti-V3 loop antibodies tested were similar with monomeric and oligomeric gp120, all the V3 loop epitopes examined were less accessible to antibody binding on the virion surface. This masking of the V3 loop is more pronounced for the primary-like macrophage-tropic isolates examined. Lastly, we observe that upon soluble receptor-virion binding, specific V3 loop epitopes that differ for viruses displaying different tropisms are exposed.     

Antibody responses elicited in macaques immunized with human immunodeficiency virus type 1 (HIV-1) SF162-derived gp140 envelope immunogens: comparison with those elicited during homologous simian/human immunodeficiency virus SHIVSF162P4 and heterologous HIV-1 infection

The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIV(SF162P4), and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, DeltaV2gp140 (lacking the crown of the V2 loop), DeltaV3gp140 (lacking the crown of the V3 loop), and DeltaV2DeltaV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and DeltaV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and DeltaV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by DeltaV3gp140 or DeltaV2DeltaV3gp140. In contrast, the SHIV(SF162P4)-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.     

CD4 epitope masking by gp120/anti-gp120 antibody complexes. A potential mechanism for CD4+ cell function down-regulation in AIDS patients.

The in vitro suppressive effect of gp120 and gp120/anti-gp120 antibody is well known but not yet proven to operate in vivo. We report findings consistent with the presence of gp120/anti-gp120 antibody complexes on CD4+ lymphocytes from HIV-infected patients with advanced disease. PBMC from most AIDS patients showed selective masking of the CD4 epitope associated with the gp120 binding site; immunoprecipitation of PBMC with anti-CD4 mAb disclosed high amounts of IgG bound to CD4 receptors. Antibodies against HIV env proteins, but not other HIV products or CD4 Ag, were detected in purified CD4+ cell culture supernatants; in vitro culture was associated with normalization of both CD4 expression in PBMC and the lymphocyte proliferative response to anti-CD3. gp120 presence could not be directly demonstrated, but findings strongly suggested that CD4+ lymphocytes from most HIV-infected patients with advanced disease were covered with gp120/anti-gp120 antibody complexes, which are responsible for down-regulation of surface CD4 expression as well as functional lymphocyte impairment; this event may represent an important mechanism in the pathogenesis of HIV-associated immunodeficiency.     

Human immunodeficiency virus type 1 neutralization by a single molecule of V3-targeted antibody

Human immunodeficiency virus type-1 (HIV-1) infection generally provokes antibody responses to the viral envelope glycoprotein. Two major regions of gp120, the third variable (V3) domain and the CD4-binding site, have been identified as neutralization targets. The precise mechanism of HIV-1 neutralization by antibodies against the V3 domain is still unknown. It is shown that by kinetic neutralization studies, one molecule of V3-targeted monoclonal antibody (0.5beta) is enough to neutralize one virion. This antibody, which neutralized more than 99% of the virus, inhibited the binding of the virus to cells by 42%. HIV-1 pseudotyped with G glycoprotein from vesicular stomatitis virus was also neutralized by 0.5beta, suggesting that the antibody did not inhibit the viral attachment but caused some alteration in the envelope. These results indicate that the antibody plays an additional role on steric change of the envelope involved in inhibition of viral entry.     

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.     

Additive effects characterize the interaction of antibodies involved in neutralization of the primary dualtropic human immunodeficiency virus type 1 isolate 89.6

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

Analysis of the cross-reactive anti-gp120 antibody population in human immunodeficiency virus-infected asymptomatic individuals.

This study was undertaken to analyze the specificity and neutralizing properties of cross-reactive anti-gp120 antibodies (Abs) in the sera of two human immunodeficiency virus (HIV)-infected asymptomatic individuals. Two panels of murine monoclonal anti-idiotype Abs (anti-id MAbs) were established against cross-reactive polyclonal anti-gp120 Abs purified from HIV+ sera by sequential affinity chromatography using gp120SF2- and gp120IIIB-Sepharose columns. These panels of anti-id MAbs were then used to affinity purify idiotype-positive (Id+) anti-gp120 Abs from HIV+ sera. The recovery of each of these Id+ Abs by purification indicated that several idiotypically distinct cross-reactive anti-gp120 Abs are present in sera over a wide range of concentrations. Immunological and biological studies showed that although all of the Id+ Abs were reactive against gp120SF2 and gp120IIIB, they exhibited unique epitope specificities and distinct neutralizing activities. Most of the Id+ Abs were directed against epitopes in the CD4 attachment site (CD4 site epitopes) of gp120 and exhibited a spectrum of broadly neutralizing activities. On the other hand, a minor population of Id+ Abs showed specificity for the V3 region of gp120 and exhibited limited cross-neutralizing activities. Together, these studies indicate that the CD4 site epitope-specific Abs are heterogeneous with respect to their clonality, neutralizing activity, and concentration in sera. This heterogeneity suggests that anti-gp120 Abs to the CD4 attachment site are developed in response to multiple overlapping epitopes present on the original virus isolate and/or epitopes on mutated variants which emerged over time.     

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.     

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.