Cryptic nature of envelope V3 region epitopes protects primary monocytotropic human immunodeficiency virus type 1 from antibody neutralization.

Characterization of biological and immunological properties of human immunodeficiency virus type 1 (HIV-1) is critical to developing effective therapies and vaccines for AIDS. With the use of a novel CD4+ T-cell line (PM-1) permissive to infection by both monocytotropic (MT) and T-cell-tropic virus types, we present a comparative analysis of the immunological properties of a prototypic primary MT isolate of HIV-1 strain JR-CSF (MT-CSF) with those of a T-cell-tropic variant (T-CSF) of the same virus, which emerged spontaneously in vitro. The parental MT-CSF infected only PM-1 cells and was markedly resistant to neutralization by sera from HIV-1-infected individuals, rabbit antiserum to recombinant MT-CSF gp120, and anti-V3 monoclonal antibodies. The T-CSF variant infected a variety of CD4+ T-cell lines, contained positively charged amino acid substitutions in the gp120 V3 region, and was highly sensitive to antibody neutralization. Neutralization and antibody staining of T-CSF-expressing cells were significantly inhibited by HIV-1 V3 peptides; in contrast, the MT strain showed only weak V3-specific binding of polyclonal and monoclonal antibodies. Exposure of PM-1 cells to a mixture of both viruses in the presence of human anti-HIV-1 neutralizing antiserum resulted in infection with only MT-CSF. These results demonstrate that although the V3 region of MT viruses is immunogenic, the target epitopes in the V3 principal neutralizing domain on the membrane form of the MT envelope appear to be cryptic or hidden from blocking antibodies.     

Systemic and mucosal immunity in rhesus macaques immunized with HIV-1 peptide and gp120 conjugated to Brucella abortus

HIV vaccine testing in primates is an important method for determining the possibility of vaccine benefit in humans. Goals of HIV-1 vaccination include establishing neutralizing antibodies and a strong CD8(+) T-cell response. We tested a novel vaccine conjugate for its ability to elicit relevant immune responses to HIV proteins and peptides in rhesus macaques. A neutralizing epitope, V3 loop peptide from HIV-1 envelope, was coupled to heat-inactivated Brucella abortus (V3-HKBA). Rhesus macaques were immunized with this conjugate in the anterior thigh. After two immunizations V3-specific antibodies were found in the sera and at mucosal sites. Neutralizing activity of these antibodies was demonstrated by syncytia inhibition assays. Cellular immune recall responses were demonstrated by antigen-specific induction of interferon-gamma and Regulation on Activation Noraml T Cell Expressed and Secreted (RANTES) secretion in vitro. These results confirm and extend preliminary studies in mice that suggest HKBA is an effective carrier that promotes neutralizing antibody secretion at relevant mucosal sites, as well as cellular immune responses that are correlated with viral protection.     

HIV-1 neutralization coverage is improved by combining monoclonal antibodies that target independent epitopes

HIV-1 neutralizing monoclonal antibodies (MAbs) define key targets for vaccine development and are being considered for passive prevention of infection. We analyzed the interaction of MAbs to two independent epitopes on the viral envelope glycoprotein. Potently neutralizing MAbs to the CD4 binding site and V1V2 region displayed no in vitro cross-competition and displayed additive, though not synergistic, neutralization activity. Predicted neutralization coverage of a combination of two MAbs reached 97% on a 208-isolate panel.     

In vivo emergence of HIV-1 highly sensitive to neutralizing antibodies

Background

The rapid and continual viral escape from neutralizing antibodies is well documented in HIV-1 infection. Here we report in vivo emergence of viruses with heightened sensitivity to neutralizing antibodies, sometimes paralleling the development of neutralization escape.

Methodology/Principal Findings

Sequential viral envs were amplified from seven HIV-1 infected men monitored from seroconversion up to 5 years after infection. Env-recombinant infectious molecular clones were generated and tested for coreceptor use, macrophage tropism and neutralization sensitivity to homologous and heterologous serum, soluble CD4 and monoclonal antibodies IgG1b12, 2G12 and 17b. We found that HIV-1 evolves sensitivity to contemporaneous neutralizing antibodies during infection. Neutralization sensitive viruses grow out even when potent autologous neutralizing antibodies are present in patient serum. Increased sensitivity to neutralization was associated with susceptibility of the CD4 binding site or epitopes induced after CD4 binding, and mediated by complex envelope determinants including V3 and V4 residues. The development of neutralization sensitive viruses occurred without clinical progression, coreceptor switch or change in tropism for primary macrophages.

Conclusions

We propose that an interplay of selective forces for greater virus replication efficiency without the need to resist neutralizing antibodies in a compartment protected from immune surveillance may explain the temporal course described here for the in vivo emergence of HIV-1 isolates with high sensitivity to neutralizing antibodies.     

Analysis of Neutralization Specificities in Polyclonal Sera Derived from Human Immunodeficiency Virus Type 1-Infected Individuals

During human immunodeficiency virus type 1 (HIV-1) infection, patients develop various levels of neutralizing antibody (NAb) responses. In some cases, patient sera can potently neutralize diverse strains of HIV-1, but the antibody specificities that mediate this broad neutralization are not known, and their elucidation remains a formidable challenge. Due to variable and nonneutralizing determinants on the exterior envelope glycoprotein (Env), nonnative Env protein released from cells, and the glycan shielding that assembles in the context of the quaternary structure of the functional spike, HIV-1 Env elicits a myriad of binding antibodies. However, few of these antibodies can neutralize circulating viruses. We present a systematic analysis of the NAb specificities of a panel of HIV-1-positive sera, using methodologies that identify both conformational and continuous neutralization determinants on the HIV-1 Env protein. Characterization of sera included selective adsorption with native gp120 and specific point mutant variants, chimeric virus analysis, and peptide inhibition of viral neutralization. The gp120 protein was the major neutralizing determinant for most sera, although not all neutralization activity against all viruses could be identified. In some broadly neutralizing sera, the gp120-directed neutralization mapped to the CD4 binding region of gp120. In addition, we found evidence that regions of the gp120 coreceptor binding site may also be a target of neutralizing activity. Sera displaying limited neutralization breadth were mapped to the immunogenic V3 region of gp120. In a subset of sera, we also identified NAbs directed against the conserved, membrane-proximal external region of gp41. These data allow a more detailed understanding of the humoral responses to the HIV-1 Env protein and provide insights regarding the most relevant targets for HIV-1 vaccine design.     

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.     

Time frames for neutralization during the human immunodeficiency virus type 1 entry phase, as monitored in synchronously infected cell cultures

Characterization of the neutralizing interaction between antibody and virus is hindered by the nonsynchronized progression of infection in cell cultures. Discrete steps of the viral entry sequence cannot be discerned, and thus, the mode of antibody-mediated interference with virus infectivity remains undefined. Here, we magnetically synchronize the motion and cell attachment of human immunodeficiency virus type 1 (HIV-1) to monitor the progression of neutralization, both in solution and following virus attachment to the cell. By simultaneous transfer of all viral particles from reaction solution with antibody to the cell-bound state, the precise rate of neutralization of cell-free virus could be determined for each antibody. HIV-1 neutralization by both monoclonal and polyclonal antibody preparations followed distinct pseudo-first-order kinetics. For all antibodies, cell types, and HIV-1 strains examined, postattachment interference served a major role in the neutralizing effect. To monitor the progression of postattachment interference, we synchronized the entry process at initiation and measured the escape of cell-bound virus from antibody. We found that different antibodies neutralized the virus over different time frames during the entry phase. Virus was observed to progress through a sequence of shifting sensitivities to different antibodies during entry, suggested here to correlate with the exposure time of the target epitope on receptor-activated viral envelope proteins. Thus, by monitoring the progression of HIV-1 entry under synchronized conditions, we identify a new and significant determinant of antibody neutralization capacity, namely, the time frames for neutralization during the course of the viral entry phase.     

＂Unconventional＂ Neutralizing Activity of Antibodies Against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However,only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes,such as high sequence diversity,heavy glycosylation,and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohe-magglutinin (PHA)-stimulated human PBMCs as target cells. Thus,in essence the assay evaluates HIV-1 replication in CD4 T cells. Recently,several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera,although they do not have neutralizing activity when tested by the "conventional" neutralization assay,do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications,through opsonization of virus particles into macrophages and immature dendritic cells (iDCs),or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.     

Neutralization of tier-2 viruses and epitope profiling of plasma antibodies from human immunodeficiency virus type 1 infected donors from India

Broadly cross neutralizing antibodies (NAbs) are generated in a group of HIV-1 infected individuals during the natural infection, but little is known about their prevalence in patients infected with viral subtypes from different geographical regions. We tested here the neutralizing efficiency of plasma antibodies from 80 HIV-1 infected antiretroviral drug naive patients against a panel of subtype-B and C tier 2 viruses. We detected cross-neutralizing antibodies in approximately 19–27% of the plasma, however the subtype-C specific neutralization efficiency predominated (p = 0.004). The neutralizing activity was shown to be exclusively mediated by the immunoglobulin G (IgG) fraction in the representative plasma samples. Epitope mapping of three, the most cross-neutralizing plasma (CNP) AIIMS206, AIIMS239 and AIIMS249 with consensus-C overlapping envelope peptides revealed ten different binding specificities with only V3 and IDR being common. The V3 and IDR were highly antigenic regions but no correlation between their reciprocal Max50 binding titers and neutralization was observed. In addition, the neutralizing activity of CNP was not substantially reduced by V3 and gp41 peptides except a modest contribution of MPER peptide. The MPER was rarely recognized by plasma antibodies though antibody depletion and competition experiments demonstrated MPER dependent neutralization in two out of three CNP. Interestingly, the binding specificity of one of the CNP (AIIMS206) overlapped with broadly neutralizing mAb 2F5 epitope. Overall, the data suggest that, despite the low immunogenicity of HIV-1 MPER, the antibodies directed to this region may serve as crucial reagents for HIV-1 vaccine design.     

Stoichiometry of antibody neutralization of human immunodeficiency virus type 1

The human immunodeficiency virus envelope glycoproteins function as trimers on the viral surface, where they are targeted by neutralizing antibodies. Different monoclonal antibodies neutralize human immunodeficiency virus type 1 (HIV-1) infectivity by binding to structurally and functionally distinct moieties on the envelope glycoprotein trimer. By measuring antibody neutralization of viruses with mixtures of neutralization-sensitive and neutralization-resistant envelope glycoproteins, we demonstrate that the HIV-1 envelope glycoprotein trimer is inactivated by the binding of a single antibody molecule. Virus neutralization requires essentially all of the functional trimers to be occupied by at least one antibody. This model applies to antibodies differing in neutralizing potency and to virus isolates with various neutralization sensitivities. Understanding these requirements for HIV-1 neutralization by antibodies will assist in establishing goals for an effective AIDS vaccine.     

"Unconventional" Neutralizing Activity of Antibodies Against HIV

Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.     

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.     

Antibody neutralization escape mediated by point mutations in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41

The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.     

Targeted Isolation of Antibodies Directed against Major Sites of SIV Env Vulnerability

The simian immunodeficiency virus (SIV) challenge model of lentiviral infection is often used as a model to human immunodeficiency virus type 1 (HIV-1) for studying vaccine mediated and immune correlates of protection. However, knowledge of the structure of the SIV envelope (Env) glycoprotein is limited, as is knowledge of binding specificity, function and potential efficacy of SIV antibody responses. In this study we describe the use of a competitive probe binding sort strategy as well as scaffolded probes for targeted isolation of SIV Env-specific monoclonal antibodies (mAbs). We isolated nearly 70 SIV-specific mAbs directed against major sites of SIV Env vulnerability analogous to broadly neutralizing antibody (bnAb) targets of HIV-1, namely, the CD4 binding site (CD4bs), CD4-induced (CD4i)-site, peptide epitopes in variable loops 1, 2 and 3 (V1, V2, V3) and potentially glycan targets of SIV Env. The range of SIV mAbs isolated includes those exhibiting varying degrees of neutralization breadth and potency as well as others that demonstrated binding but not neutralization. Several SIV mAbs displayed broad and potent neutralization of a diverse panel of 20 SIV viral isolates with some also neutralizing HIV-2_7312A. This extensive panel of SIV mAbs will facilitate more effective use of the SIV non-human primate (NHP) model for understanding the variables in development of a HIV vaccine or immunotherapy.     

Formalin inactivation of the lactate dehydrogenase-elevating virus reveals a major neutralizing epitope not recognized during natural infection.

Five hybridomas that secrete monoclonal antibodies which neutralize the infectivity of lactate dehydrogenase-elevating virus (LDV) were isolated from BALB/c mice primed with Formalin-inactivated LDV. Competition analyses indicated that all five neutralizing monoclonal antibodies recognize contiguous, if not identical, epitopes on the envelope glycoprotein of LDV (VP-3) which are not recognized by nonneutralizing VP-3-specific monoclonal antibodies isolated from the same fusion. Despite the presence of neutralizing activity, polyclonal anti-LDV antibodies obtained from persistently infected mice did not compete for binding to LDV with four of the five neutralizing monoclonal antibodies tested. The results indicate that the envelope glycoprotein of LDV possesses a major neutralizing epitope which is poorly recognized, if at all, by mice during a natural infection but is rendered immunogenic by Formalin inactivation of the virus. The epitope was also not immunogenic in a rabbit, since its polyclonal LDV-neutralizing antibodies did not inhibit binding of the mouse monoclonal antibodies to LDV. Passive immunization with the neutralizing monoclonal antibodies did not protect mice from LDV infection and did not alter the course of infection. Neutralizing monoclonal antibodies have been used to select a neutralization escape variant by a novel combination of in vitro and in vivo isolation.     

Human Immunodeficiency Virus Type 2 (HIV-2)/HIV-1 Envelope Chimeras Detect High Titers of Broadly Reactive HIV-1 V3-Specific Antibodies in Human Plasma

Deciphering antibody specificities that constrain human immunodeficiency virus type 1 (HIV-1) envelope (Env) diversity, limit virus replication, and contribute to neutralization breadth and potency is an important goal of current HIV/AIDS vaccine research. Transplantation of discrete HIV-1 neutralizing epitopes into HIV-2 scaffolds may provide a sensitive, biologically functional context by which to quantify specific antibody reactivities even in complex sera. Here, we describe a novel HIV-2 proviral scaffold (pHIV-2_KR.X7) into which we substituted the complete variable region 3 (V3) of the env gene of HIV-1_YU2 or HIV-1_Ccon to yield the chimeric proviruses pHIV-2_KR.X7 YU2 V3 and pHIV-2_KR.X7 Ccon V3. These HIV-2/HIV-1 chimeras were replication competent and sensitive to selective pharmacological inhibitors of virus entry. V3 chimeric viruses were resistant to neutralization by HIV-1 monoclonal antibodies directed against the CD4 binding site, coreceptor binding site, and gp41 membrane proximal external region but exhibited striking sensitivity to HIV-1 V3-specific monoclonal antibodies, 447-52D and F425 B4e8 (50% inhibitory concentration of [IC₅₀] <0.005 μg/ml for each). Plasma specimens from 11 HIV-1 clade B- and 10 HIV-1 clade C-infected subjects showed no neutralizing activity against HIV-2 but exhibited high-titer V3-specific neutralization against both HIV-2/HIV-1 V3 chimeras with IC₅₀ measurements ranging from 1:50 to greater than 1:40,000. Neutralization titers of B clade plasmas were as much as 1,000-fold lower when tested against the primary HIV-1_YU2 virus than with the HIV-2_KR.X7 YU2 V3 chimera, demonstrating highly effective shielding of V3 epitopes in the native Env trimer. This finding was replicated using a second primary HIV-1 strain (HIV-1_BORI) and the corresponding HIV-2_KR.X7 BORI V3 chimera. We conclude that V3 is highly immunogenic in vivo, eliciting antibodies with substantial breadth of reactivity and neutralizing potential. These antibodies constrain HIV-1 Env to a structure(s) in which V3 epitopes are concealed prior to CD4 engagement but do not otherwise contribute to neutralization breadth and potency against most primary virus strains. Triggering of the viral spike to reveal V3 epitopes may be required if V3 immunogens are to be components of an effective HIV-1 vaccine.     

Immune Focusing and Enhanced Neutralization Induced by HIV-1 gp140 Chemical Cross-Linking

Experimental vaccine antigens based upon the HIV-1 envelope glycoproteins (Env) have failed to induce neutralizing antibodies (NAbs) against the majority of circulating viral strains as a result of antibody evasion mechanisms, including amino acid variability and conformational instability. A potential vaccine design strategy is to stabilize Env, thereby focusing antibody responses on constitutively exposed, conserved surfaces, such as the CD4 binding site (CD4bs). Here, we show that a largely trimeric form of soluble Env can be stably cross-linked with glutaraldehyde (GLA) without global modification of antigenicity. Cross-linking largely conserved binding of all potent broadly neutralizing antibodies (bNAbs) tested, including CD4bs-specific VRC01 and HJ16, but reduced binding of several non- or weakly neutralizing antibodies and soluble CD4 (sCD4). Adjuvanted administration of cross-linked or unmodified gp140 to rabbits generated indistinguishable total gp140-specific serum IgG binding titers. However, sera from animals receiving cross-linked gp140 showed significantly increased CD4bs-specific antibody binding compared to animals receiving unmodified gp140. Moreover, peptide mapping of sera from animals receiving cross-linked gp140 revealed increased binding to gp120 C1 and V1V2 regions. Finally, neutralization titers were significantly elevated in sera from animals receiving cross-linked gp140 rather than unmodified gp140. We conclude that cross-linking favors antigen stability, imparts antigenic modifications that selectively refocus antibody specificity and improves induction of NAbs, and might be a useful strategy for future vaccine design.     

Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate

Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines.     

Neutralization escape mutants define a dominant immunogenic neutralization site on hepatitis A virus.

Hepatitis A virus is an hepatotrophic human picornavirus which demonstrates little antigenic variability. To topologically map immunogenic sites on hepatitis A virus which elicit neutralizing antibodies, eight neutralizing monoclonal antibodies were evaluated in competition immunoassays employing radiolabeled monoclonal antibodies and HM-175 virus. Whereas two antibodies (K3-4C8 and K3-2F2) bound to intimately overlapping epitopes, the epitope bound by a third antibody (B5-B3) was distinctly different as evidenced by a lack of competition between antibodies for binding to the virus. The other five antibodies variably blocked the binding of both K3-4C8-K3-2F2 and B5-B3, suggesting that these epitopes are closely spaced and perhaps part of a single neutralization immunogenic site. Several combinations of monoclonal antibodies blocked the binding of polyclonal human convalescent antibody by greater than 96%, indicating that the neutralization epitopes bound by these antibodies are immunodominant in humans. Spontaneously arising HM-175 mutants were selected for resistance to monoclonal antibody-mediated neutralization. Fourteen clonally isolated mutants demonstrated substantial resistance to multiple monoclonal antibodies, including K3-4C8-K3-2F2 and B5-B3. In addition, 13 mutants demonstrated a 10-fold or greater reduction in neutraliztion mediated by polyclonal human antibody. Neutralization resistance was associated with reduced antibody binding. These results suggest that hepatitis A virus may differ from poliovirus in possessing a single, dominant neutralization immunogenic site and therefore may be a better candidate for synthetic peptide or antiidiotype vaccine development.     

Characterization of primary isolate-like variants of simian-human immunodeficiency virus

Several different strains of simian-human immunodeficiency virus (SHIV) that contain the envelope glycoproteins of either T-cell-line-adapted (TCLA) strains or primary isolates of human immunodeficiency virus type 1 (HIV-1) are now available. One of the advantages of these chimeric viruses is their application to studies of HIV-1-specific neutralizing antibodies in preclinical AIDS vaccine studies in nonhuman primates. In this regard, an important consideration is the spectrum of antigenic properties exhibited by the different envelope glycoproteins used for SHIV construction. The antigenic properties of six SHIV variants were characterized here in neutralization assays with recombinant soluble CD4 (rsCD4), monoclonal antibodies, and serum samples from SHIV-infected macaques and HIV-1-infected individuals. Neutralization of SHIV variants HXBc2, KU2, 89.6, and 89.6P by autologous and heterologous sera from SHIV-infected macaques was restricted to an extent that these viruses may be considered heterologous to one another in their major neutralization determinants. Little or no variation was seen in the neutralization determinants on SHIV variants 89.6P, 89.6PD, and SHIV-KB9. Neutralization of SHIV HXBc2 by sera from HXBc2-infected macaques could be blocked with autologous V3-loop peptide; this was less true in the case of SHIV 89.6 and sera from SHIV 89.6-infected macaques. The poorly immunogenic but highly conserved epitope for monoclonal antibody IgG1b12 was a target for neutralization on SHIV variants HXBc2, KU2, and 89.6 but not on 89.6P and KB9. The 2G12 epitope was a target for neutralization on all five SHIV variants. SHIV variants KU2, 89.6, 89.6P, 89.6PD, and KB9 exhibited antigenic properties characteristic of primary isolates by being relatively insensitive to neutralization in peripheral blood mononuclear cells with serum samples from HIV-1-infected individuals and 12-fold to 38-fold less sensitive to inhibition with recombinant soluble CD4 than TCLA strains of HIV-1. The utility of nonhuman primate models in AIDS vaccine development is strengthened by the availability of SHIV variants that are heterologous in their neutralization determinants and exhibit antigenic properties shared with primary isolates.