Neutralization of human immunodeficiency virus type 1 by sCD4-17b,a single-chain chimeric protein,based on sequential interaction of gp120 with CD4 and coreceptor

We designed a novel single-chain chimeric protein, designated sCD4-17b, for neutralization of human immunodeficiency virus type 1 (HIV-1). The recombinant protein contains domains 1 and 2 of soluble CD4 (sCD4), connected via a flexible polypeptide linker to a single-chain variable region construct of 17b, a human monoclonal antibody that targets a conserved CD4-induced epitope on gp120 overlapping the coreceptor binding region. We hypothesized that the sCD4 moiety would bind gp120 and expose the 17b epitope; the 17b moiety would then bind, thereby blocking coreceptor interaction and neutralizing infection. The sCD4-17b protein, expressed by a recombinant vaccinia virus, potently neutralized a prototypic R5 clade B primary isolate, with a 50% inhibitory concentration of 3.2 nM (0.16 microg/ml) and >95% neutralization at 32 nM (1.6 microg/ml). The individual components (sCD4 and 17b, singly or in combination) had minimal effects at these concentrations, demonstrating that the activity of sCD4-17b reflected the ability of a single chimeric molecule to bind gp120 simultaneously via two independent moieties. sCD4-17b was highly potent compared to the previously characterized broadly cross-reactive neutralizing monoclonal antibodies IgGb12, 2G12, and 2F5. Multiple primary isolates were neutralized, including two previously described as antibody resistant. Neutralization occurred for both R5 and X4 strains and was not restricted to clade B. However, several primary isolates were insensitive over the concentration range tested, despite the known presence of binding sites for both CD4 and 17b. sCD4-17b has potential utility for passive immunization against HIV-1 in several contexts, including maternal transmission, postexposure prophylaxis, and sexual transmission (topical microbicide).     

A novel human antibody against human immunodeficiency virus type 1 gp120 is V1, V2, and V3 loop dependent and helps delimit the epitope of the broadly neutralizing antibody immunoglobulin G1 b12

The V1/V2 and V3 loops are proximal to the CD4 binding site (CD4bs) of human immunodeficiency virus type 1 (HIV-1) gp120 and undergo conformational change upon CD4 receptor engagement by the HIV-1 envelope spike. Nearly all of the reported monoclonal antibodies (MAbs) against the CD4bs exhibit a very limited capacity to neutralize HIV-1. However, one such human MAb, immunoglobulin G1 (IgG1) b12, is uniquely able to neutralize primary isolates across subtypes with considerable potency. The molecular basis for the anti-HIV-1 activity of b12 is not fully understood but is relevant to vaccine design. Here we describe a novel human MAb, 4KG5, whose binding to monomeric gp120 is moderately enhanced by IgG1 b12. In sharp contrast, 4KG5 binding to gp120 is inhibited by soluble CD4 (sCD4) and by all other (n = 14) anti-CD4bs MAbs tested. 4KG5 is unable to recognize gp120 in which either V1, V2, or V3 has been deleted, and MAbs against the V2 or V3 loops inhibit the binding of 4KG5 to gp120. Moreover, 4KG5 is able to inhibit the binding of the CD4-induced MAbs 17b and X5 in the absence of sCD4, whereas 17b and X5 only weakly inhibit the binding of 4KG5 to gp120. Mutagenesis of gp120 provides further evidence of a discontinuous epitope of 4KG5 that is formed by the V1/V2 loop, the V3 loop, and a portion of the bridging sheet (C4). 4KG5 was isolated as a single-chain Fv from a phage display library constructed from the bone marrow of an HIV-1-seropositive subject (FDA2) whose serum neutralizes HIV-1 across subtypes. Despite its source, we observed no significant neutralization with 4KG5 against the autologous (R2) virus and several other strains of HIV-1. The results suggest a model in which antibody access to the CD4bs on the envelope spike of HIV-1 is restricted by the orientation and/or dynamics of the V1/V2 and V3 loops, and b12 avoids these restrictions.     

Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs) mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs) by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER) of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s) of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics.     

A Human Antibody to the CD4 Binding Site of gp120 Capable of Highly Potent but Sporadic Cross Clade Neutralization of Primary HIV-1

Primary isolates of HIV-1 resist neutralization by most antibodies to the CD4 binding site (CD4bs) on gp120 due to occlusion of this site on the trimeric spike. We describe 1F7, a human CD4bs monoclonal antibody that was found to be exceptionally potent against the HIV-1 primary isolate JR-FL. However, 1F7 failed to neutralize a patient-matched primary isolate, JR-CSF even though the two isolates differ by <10% in gp120 at the protein level. In an HIV-1 cross clade panel (n = 157), 1F7 exhibited moderate breadth, but occasionally achieved considerable potency. In binding experiments using monomeric gp120s of select resistant isolates and domain-swap chimeras between JR-FL and JR-CSF, recognition by 1F7 was limited by sequence polymorphisms involving at least the C2 region of Env. Putative N-linked glycosylation site (PNGS) mutations, notably at position 197, allowed 1F7 to neutralize JR-CSF potently without improving binding to the cognate, monomeric gp120. In contrast, flow cytometry experiments using the same PNGS mutants revealed that 1F7 binding is enhanced on cognate trimeric Env. BN-PAGE mobility shift experiments revealed that 1F7 is sensitive to the diagnostic mutation D368R in the CD4 binding loop of gp120. Our data on 1F7 reinforce how exquisitely targeted CD4bs antibodies must be to achieve cross neutralization of two closely related primary isolates. High-resolution analyses of trimeric Env that show the orientation of glycans and polymorphic elements of the CD4bs that affect binding to antibodies like 1F7 are desirable to understand how to promote immunogenicity of more conserved elements of the CD4bs.     

Anti-HIV-1 response elicited in rabbits by anti-idiotype monoclonal antibodies mimicking the CD4-binding site

Antibodies against conserved epitopes on HIV-1 envelope glycoproteins (Env), such as the gp120 CD4-binding site (CD4bs), could contribute to protection against HIV-1. Env-based immunogens inducing such a response could be a major component of future anti-HIV-1 strategies. In this proof-of-concept study we describe the generation of two anti-idiotype (AI) murine antibodies mimicking the CD4bs epitope. Sera were collected from long-term non-progressor patients to obtain CD4bs-directed IgG, through sequential purification steps. The purified IgG were then used as Fab fragments to immunize mice for hybridoma generation. Two hybridomas (P1 and P2), reacting only against the CD4bs-directed IgG, were identified and characterized. The P1 and P2 antibodies were shown to recognize the idiotype of the broadly neutralizing anti-CD4bs human mAb b12. Both P1 and P2 Fabs were able to induce a strong anti-gp120 response in rabbits. Moreover, the rabbits' sera were shown to neutralize two sensitive tier 1 strains of HIV-1 in an Env-pseudotype neutralization assay. In particular, 3/5 rabbits in the P1 group and 1/5 in the P2 group showed greater than 80% neutralizing activity against the HXB2 pseudovirus. Two rabbits also neutralized the pseudovirus HIV-MN. Overall, these data describe the first anti-idiotypic vaccine approach performed to generate antibodies to the CD4bs of the HIV-1 gp120. Although future studies will be necessary to improve strength and breadth of the elicited neutralizing response, this proof-of-concept study documents that immunogens designed on the idiotype of broadly neutralizing Abs are feasible and could help in the design of future anti-HIV strategies.     

Design of an Escherichia coli Expressed HIV-1 gp120 Fragment Immunogen That Binds to b12 and Induces Broad and Potent Neutralizing Antibodies

b12, one of the few broadly neutralizing antibodies against HIV-1, binds to the CD4 binding site (CD4bs) on the gp120 subunit of HIV-1 Env. Two small fragments of HIV-1 gp120, b121a and b122a, which display about 70% of the b12 epitope and include solubility-enhancing mutations, were designed. Bacterially expressed b121a/b122a were partially folded and could bind b12 but not the CD4bs-directed non-neutralizing antibody b6. Sera from rabbits primed with b121a or b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity in a TZM-bl assay against a 16-virus panel that included nine Tier 2 and 3 viruses as well as in a five-virus panel previously designed to screen for broad neutralization. Using a mean IC₅₀ cut-off of 50, sera from control rabbits immunized with gp120 alone neutralized only one virus of the 14 non-Tier 1 viruses tested (7%), whereas sera from b121a- and b122a-immunized rabbits neutralized seven (50%) and twelve (86%) viruses, respectively. Serum depletion studies confirmed that neutralization was gp120-directed and that sera from animals immunized with gp120 contained lower amounts of CD4bs-directed antibodies than corresponding sera from animals immunized with b121a/b122a. Competition binding assays with b12 also showed that b121a/2a sera contained significantly higher amounts of antibodies directed toward the CD4 binding site than the gp120 sera. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals.     

Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.

Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently.     

Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG.

We have tested three human monoclonal antibodies (MAbs) IgG1b12, 2G12, and 2F5) to the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1), and a tetrameric CD4-IgG molecule (CD4-IgG2), for the ability to neutralize primary HIV-1 isolates from the genetic clades A through F and from group O. Each of the reagents broadly and potently neutralized B-clade isolates. The 2F5 MAb and the CD4-IgG2 molecule also neutralized strains from outside the B clade, with the same breadth and potency that they showed against B-clade strains. The other two MAbs were able to neutralize a significant proportion of strains from outside the B clade, although there was a reduction in their efficacy compared with their activity against B-clade isolates. Neutralization of isolates by 2F5 correlated with their possession of the LDKW motif in a segment of gp41 near the membrane-spanning domain. The other two MAbs and CD4-IgG2 recognize discontinuous binding sites on gp120, and so no comparison between genetic sequence and virus neutralization was possible. Our data show that a vaccine based on the induction of humoral immunity that is broadly active across the genetic clades is not impossible if immunogens that express the epitopes for MAbs such as 2F5, 2G12, and IgG1b12 in immunogenic configurations can be created. Furthermore, if the three MAbs and CD4-IgG2 produce clinical benefit in immunotherapeutic trials in the United States or Europe, they may also do so elsewhere in the world.     

The W100 pocket on HIV-1 gp120 penetrated by b12 is not a target for other CD4bs monoclonal antibodies

Background

The conserved CD4 binding site (CD4bs) on HIV-1 gp120 is a major target for vaccines. It is a priority to determine sites and structures within the CD4bs that are important for inclusion in vaccines. We studied a gp120 pocket penetrated by W100 of the potent CD4bs monoclonal antibody (mab), b12. We compared HIV-1 envelopes and corresponding mutants that carried blocked W100 pockets to evaluate whether other CD4bs mabs target this site.

Findings

All CD4bs mabs tested blocked soluble CD4 binding to gp120 consistent with their designation as CD4bs directed antibodies. All CD4bs mabs tested neutralized pseudovirions carrying NL4.3 wild type (wt) envelope. However, only b12 failed to neutralize pseudoviruses carrying mutant envelopes with a blocked W100 pocket. In addition, for CD4bs mabs that neutralized pseudovirions carrying primary envelopes, mutation of the W100 pocket had little or no effect on neutralization sensitivity.

Conclusions

Our data indicate that the b12 W100 pocket on gp120 is infrequently targeted by CD4bs mabs. This site is therefore not a priority for preservation in vaccines aiming to elicit antibodies targeting the CD4bs.     

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.     

HIV-1 Neutralizing Antibodies Display Dual Recognition of the Primary and Coreceptor Binding Sites and Preferential Binding to Fully Cleaved Envelope Glycoproteins

The gp120 CD4 binding site (CD4bs) and coreceptor binding site (CoRbs) are two functionally conserved elements of the HIV-1 envelope glycoproteins (Env). We previously defined the presence of CD4bs-neutralizing antibodies in the serum of an HIV-1-infected individual and subsequently isolated the CD4bs-specific monoclonal antibodies (MAbs) VRC01 and VRC03 from the memory B cell population. Since this donor''s serum also appeared to contain neutralizing antibodies to the CoRbs, we employed a differential fluorescence-activated cell sorter (FACS)-based sorting strategy using an Env trimer possessing a CoRbs knockout mutation (I420R) to isolate specific B cells. The MAb VRC06 was recovered from these cells, and its genetic sequence allowed us to identify a clonal relative termed VRC06b, which was isolated from a prior cell sort using a resurfaced core gp120 probe and its cognate CD4bs knockout mutant. VRC06 and VRC06b neutralized 22% and 44% of viruses tested, respectively. Epitope mapping studies revealed that the two MAbs were sensitive to mutations in both the gp120 CoRbs and the CD4bs and could cross-block binding of both CD4bs and CoRbs MAbs to gp120. Fine mapping indicated contacts within the gp120 bridging sheet and the base of the third major variable region (V3), which are elements of the CoRbs. Cell surface binding assays demonstrated preferential recognition of fully cleaved Env trimers over uncleaved trimers. Thus, VRC06 and VRC06b are Env trimer precursor cleavage-sensitive neutralizing MAbs that bind to a region of gp120 that overlaps both the primary and the secondary HIV-1 receptor binding sites.     

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.     

Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies

Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV(+) plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (相似文献   

Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates.

Primary isolates of human immunodeficiency virus type 1 (HIV-1) are much less sensitive to neutralization by soluble CD4 (sCD4) and sCD4-immunoglobulin (Ig) chimeras (CD4-IgG) than are HIV-1 strains adapted to growth in cell culture. We demonstrated that there are significant reductions (10- to 30-fold) in the binding of sCD4 and CD4-IgG to intact virions of five primary isolates compared with sCD4-sensitive, cell culture-adapted isolates RF and IIIB. However, soluble envelope glycoproteins (gp120) derived from the primary isolate virions, directly by detergent solubilization or indirectly by recombinant DNA technology, differed in affinity from RF and IIIB gp120 by only one- to threefold. The reduced binding of sCD4 to these primary isolate virions must therefore be a consequence of the tertiary or quaternary structure of the envelope glycoproteins in their native, oligomeric form on the viral surface. In addition, the rate and extent of sCD4-induced gp120 shedding from these primary isolates was lower than that from RF. We suggest that reduced sCD4 binding and increased gp120 retention together account for the relative resistance of these primary isolates to neutralization by sCD4 and CD4-IgG and that virions of different HIV-1 isolates vary both in the mechanism of sCD4 binding and in subsequent conformational changes in their envelope glycoproteins.     

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.     

Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein.

The humoral immune response to human immunodeficiency virus type 1 (HIV-1) is often studied by using monomeric or denatured envelope proteins (Env). However, native HIV-1 Env complexes that maintain quaternary structure elicit immune responses that are qualitatively distinct from those seen with monomeric or denatured Env. To more accurately assess the levels and types of antibodies elicited by HIV-1 infection, we developed an antigen capture enzyme-linked immunosorbent assay using a soluble, oligomeric form of HIV-1IIIB Env (gp140) that contains gp120 and the gp41 ectodomain. The gp140, captured by various monoclonal antibodies (MAbs), retained its native oligomeric structure: it bound CD4 and was recognized by MAbs to conformational epitopes in gp120 and gp41, including oligomer-specific epitopes in gp41. We compared the reactivities of clade B and clade E serum samples to captured Env preparations and found that while both reacted equally well with oligomeric gp140, clade B seras reacted more strongly with monomeric gp120 than did clade E samples. However, these differences were minimized when gp120 was captured by a V3 loop MAb, which may lead to increased exposure of the CD4 binding site. We also measured the ability of serum samples to block binding of MAbs to epitopes in gp120 and gp41. Clade B serum samples consistently blocked binding of oligomer-dependent MAbs to gp41 and, to a slightly lesser extent, MAbs to the CD4 binding site in gp120. Clade E serum samples showed equivalent or greater blocking of oligomer-dependent gp41 antibodies and considerably less blocking of CD4-binding-site MAbs. Finally, we found that < 5% of the antibodies in clade B sera bound to epitopes present only in monomeric gp120, 30% bound to epitopes present in both monomeric gp120 and oligomeric gp140, and 70% bound to epitopes present in oligomeric gp140, which includes gp41. Thus, captured oligomeric Env closely reflects the antigenic characteristics of Env protein on the surface of virions and infected cells, retains highly conserved epitopes that are recognized by antibodies raised against different clades, and makes it possible to detect a much greater fraction of total anti-HIV-1 Env activity in sera than does native monomeric gp120.     

Mechanism of neutralization by the broadly neutralizing HIV-1 monoclonal antibody VRC01

The structure of VRC01 in complex with the HIV-1 gp120 core reveals that this broadly neutralizing CD4 binding site (CD4bs) antibody partially mimics the interaction of the primary virus receptor, CD4, with gp120. Here, we extended the investigation of the VRC01-gp120 core interaction to the biologically relevant viral spike to better understand the mechanism of VRC01-mediated neutralization and to define viral elements associated with neutralization resistance. In contrast to the interaction of CD4 or the CD4bs monoclonal antibody (MAb) b12 with the HIV-1 envelope glycoprotein (Env), occlusion of the VRC01 epitope by quaternary constraints was not a major factor limiting neutralization. Mutagenesis studies indicated that VRC01 contacts within the gp120 loop D, the CD4 binding loop, and the V5 region were necessary for optimal VRC01 neutralization, as suggested by the crystal structure. In contrast to interactions with the soluble gp120 monomer, VRC01 interaction with the native viral spike did not occur in a CD4-like manner; VRC01 did not induce gp120 shedding from the Env spike or enhance gp41 membrane proximal external region (MPER)-directed antibody binding to the Env spike. Finally, VRC01 did not display significant reactivity with human antigens, boding well for potential in vivo applications. The data indicate that VRC01 interacts with gp120 in the context of the functional spike in a manner distinct from that of CD4. It achieves potent neutralization by precisely targeting the CD4bs without requiring alterations of Env spike configuration and by avoiding steric constraints imposed by the quaternary structure of the functional Env spike.     

Association of enhanced HIV-1 neutralization by a single Y681H substitution in gp41 with increased gp120-CD4 interaction and macrophage infectivity

HIV-1 variants that show unusual sensitivity to autologous antibodies due to presence of critical neutralization signatures would likely contribute towards rational envelope based HIV-1 vaccine design. In the present study, we found that presence of a naturally occurring H681 in gp41 membrane proximal external region (MPER) of a clade C envelope (Env) obtained from a recently infected Indian patient conferred increased sensitivity to autologous and heterologous plasma antibodies. Furthermore, Env-pseudotyped viruses expressing H681 showed increased sensitivity to soluble CD4, b12 and 4E10 monoclonal antibodies both in related and unrelated Envs and was corroborated with increased Env susceptibility and binding to cellular CD4 as well as with prolonged exposure of MPER epitopes. The increased gp120-CD4 interaction was further associated with relative exposure of CD4-induced epitopes and macrophage infectivity. In summary, our data indicate that Y681H substitution exposes neutralizing epitopes in CD4bs and MPER towards comprehensive interference in HIV-1 entry.     

Broad and potent neutralizing antibody responses elicited in natural HIV-2 infection

Compared with human immunodeficiency virus type 1 (HIV-1), little is known about the susceptibility of HIV-2 to antibody neutralization. We characterized the potency and breadth of neutralizing antibody (NAb) responses in 64 subjects chronically infected with HIV-2 against three primary HIV-2 strains: HIV-2(7312A), HIV-2(ST), and HIV-2(UC1). Surprisingly, we observed in a single-cycle JC53bl-13/TZM-bl virus entry assay median reciprocal 50% inhibitory concentration (IC(50)) NAb titers of 1.7 × 10(5), 2.8 × 10(4), and 3.3 × 10(4), respectively. A subset of 5 patient plasma samples tested against a larger panel of 17 HIV-2 strains where the extracellular gp160 domain was substituted into the HIV-2(7312A) proviral backbone showed potent neutralization of all but 4 viruses. The specificity of antibody neutralization was confirmed using IgG purified from patient plasma, HIV-2 Envs cloned by single-genome amplification, viruses grown in human CD4(+) T cells and tested for neutralization sensitivity on human CD4(+) T target cells, and, as negative controls, env-minus viruses pseudotyped with HIV-1, vesicular stomatitis virus, or murine leukemia virus Env glycoproteins. Human monoclonal antibodies (MAbs) specific for HIV-2 V3 (6.10F), V4 (1.7A), CD4 binding site (CD4bs; 6.10B), CD4 induced (CD4i; 1.4H), and membrane-proximal external region (MPER; 4E10) epitopes potently neutralized the majority of 32 HIV-2 strains bearing Envs from 13 subjects. Patient antibodies competed with V3, V4, and CD4bs MAbs for binding to monomeric HIV-2 gp120 at titers that correlated significantly with NAb titers. HIV-2 MPER antibodies did not contribute to neutralization breadth or potency. These findings indicate that HIV-2 Env is highly immunogenic in natural infection, that high-titer broadly neutralizing antibodies are commonly elicited, and that unlike HIV-1, native HIV-2 Env trimers expose multiple broadly cross-reactive epitopes readily accessible to NAbs.     

Regional clustering of shared neutralization determinants on primary isolates of clade C human immunodeficiency virus type 1 from South Africa

Clade C is one of the most prevalent genetic subtypes of human immunodeficiency virus type 1 (HIV-1) in the world today and one of the least studied with respect to neutralizing antibodies. Most information on HIV-1 serology as it relates to neutralization is derived from clade B. Clade C primary isolates of HIV-1 from South Africa and Malawi were shown here to resemble clade B isolates in their resistance to inhibition by soluble CD4 and their sensitivity to neutralization by human monoclonal antibody immunoglobulin G1b12 and, to a lesser extent, 2F5. Unlike clade B isolates, however, all 16 clade C isolates examined resisted neutralization by 2G12. Infection with clade C HIV-1 in a cohort of female sex workers in South Africa generated antibodies that neutralized the autologous clade C isolate and T-cell-line-adapted (TCLA) strains of clade B. Neutralization of clade B TCLA strains was much more sensitive to the presence of autologous gp120 V3 loop peptides compared to the neutralization of clade C isolates in most cases. Thus, the native structure of gp120 on primary isolates of clade C will likely pose a challenge for neutralizing antibody induction by candidate HIV-1 vaccines much the same as it has for clade B. The autologous neutralizing antibody response following primary infection with clade C HIV-1 in South Africa matured slowly, requiring at least 4 to 5 months to become detectable. Once detectable, extensive cross-neutralization of heterologous clade C isolates from South Africa was observed, suggesting an unusual degree of shared neutralization determinants at a regional level. This high frequency of cross-neutralization differed significantly from the ability of South African clade C serum samples to neutralize clade B isolates but did not differ significantly from results of other combinations of clade B and C reagents tested in checkerboard assays. Notably, two clade C serum samples obtained after less than 2 years of infection neutralized a broad spectrum of clade B and C isolates. Other individual serum samples showed a significant clade preference in their neutralizing activity. Our results suggest that clades B and C are each comprised of multiple neutralization serotypes, some of which are more clade specific than others. The clustering of shared neutralization determinants on clade C primary HIV-1 isolates from South Africa suggests that neutralizing antibodies induced by vaccines will have less epitope diversity to overcome at a regional level.