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.     

Comprehensive Antigenic Map of a Cleaved Soluble HIV-1 Envelope Trimer

The trimeric envelope (Env) spike is the focus of vaccine design efforts aimed at generating broadly neutralizing antibodies (bNAbs) to protect against HIV-1 infection. Three recent developments have facilitated a thorough investigation of the antigenic structure of the Env trimer: 1) the isolation of many bNAbs against multiple different epitopes; 2) the generation of a soluble trimer mimic, BG505 SOSIP.664 gp140, that expresses most bNAb epitopes; 3) facile binding assays involving the oriented immobilization of tagged trimers. Using these tools, we generated an antigenic map of the trimer by antibody cross-competition. Our analysis delineates three well-defined epitope clusters (CD4 binding site, quaternary V1V2 and Asn332-centered oligomannose patch) and new epitopes at the gp120-gp41 interface. It also identifies the relationships among these clusters. In addition to epitope overlap, we defined three more ways in which antibodies can cross-compete: steric competition from binding to proximal but non-overlapping epitopes (e.g., PGT151 inhibition of 8ANC195 binding); allosteric inhibition (e.g., PGT145 inhibition of 1NC9, 8ANC195, PGT151 and CD4 binding); and competition by reorientation of glycans (e.g., PGT135 inhibition of CD4bs bNAbs, and CD4bs bNAb inhibition of 8ANC195). We further demonstrate that bNAb binding can be complex, often affecting several other areas of the trimer surface beyond the epitope. This extensive analysis of the antigenic structure and the epitope interrelationships of the Env trimer should aid in design of both bNAb-based therapies and vaccines intended to induce bNAbs.     

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.     

Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.     

Conformational and structural features of HIV-1 gp120 underlying the dual receptor antagonism by cross-reactive neutralizing antibody m18

We investigated the interaction between cross-reactive HIV-1 neutralizing human monoclonal antibody m18 and HIV-1YU-2 gp120 in an effort to understand how this antibody inhibits the entry of virus into cells. m18 binds to gp120 with high affinity (KD≈5 nM) as measured by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR analysis further showed that m18 inhibits interactions of gp120 with both soluble CD4 and CD4-induced antibodies that have epitopes overlapping the coreceptor binding site. This dual receptor site antagonism, which occurs with equal potency for both inhibition effects, argues that m18 is not functioning as a mimic of CD4, in spite of the presence of a putative CD4-like loop formed by HCDR3 in the antibody. Consistent with this view, m18 was found to interact with gp120 in the presence of saturating concentrations of a CD4-mimicking small molecule gp120 inhibitor, suggesting that m18 does not require unoccupied CD4 Phe43 binding cavity residues of gp120. Thermodynamic analysis of the m18-gp120 interaction suggests that m18 stabilizes a conformation of gp120 that is unique from and less structured than the CD4-stabilized conformation. Conformational mutants of gp120 were studied for their impact on m18 interaction. Mutations known to disrupt the coreceptor binding region and to lead to complete suppression of 17b binding had minimal effects on m18 binding. This argues that energetically important epitopes for m18 binding lie outside the disrupted bridging sheet region used for 17b and coreceptor binding. In contrast, mutations in the CD4 region strongly affected m18 binding. Overall, the results obtained in this work argue that m18, rather than mimicking CD4 directly, suppresses both receptor binding site functions of HIV-1 gp120 by stabilizing a nonproductive conformation of the envelope protein. These results can be related to prior findings about the importance of conformational entrapment as a common mode of action for neutralizing CD4bs antibodies, with differences mainly in epitope utilization and the extent of gp120 structuring.     

Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody

The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine-scanning mutagenesis overlaps that of b12. These results suggest that m14 is a novel CD4bs cross-reactive HIV-1-neutralizing antibody that exhibits a different inhibitory profile compared to the only known potent broadly neutralizing CD4bs human antibody, b12, and may have implications for our understanding of the mechanisms of immune evasion and for the development of inhibitors and vaccines.     

Antigenic properties of the human immunodeficiency virus envelope during cell-cell fusion

Human immunodeficiency virus (HIV) fusion and entry involves sequential interactions between the viral envelope protein, gp120, cell surface CD4, and a G-protein-coupled coreceptor. Each interaction creates an intermediate gp120 structure predicted to display distinct antigenic features, including key functional domains for viral entry. In this study, we examined the disposition of these features during the fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various times and then arrested. The cells were then examined for reactivity with antibodies directed against receptor-induced epitopes on gp120. Analyses of cells arrested by cooling to 4( degrees )C revealed that antibodies against the CD4-induced coreceptor-binding domain, i.e., 17b, 48d, and CG10, faintly react with Env cells even in the absence of target cell or soluble CD4 (sCD4) interactions. Such reactivity increased after exposure to sCD4 but remained unchanged during fusion with target cells and was not intensified at the Env-target cell interface. Notably, the antibodies did not react with Env cells when treated with a covalent cross-linker either alone or during fusion with target cells. Immunoreactivity could not be promoted or otherwise altered on either temperature arrested or cross-linked cells by preventing coreceptor interactions or by using a 17b Fab. In comparison, two other gp120-CD4 complex-dependent antibodies against epitopes outside the coreceptor domain, 8F101 and A32, exhibited a different pattern of reactivity. These antibodies reacted with the Env-target cell interface only after 30 min of cocultivation, concurrent with the first visible transfer of cytoplasmic dye from Env to target cells. At later times, the staining surrounded entire syncytia. Such binding was entirely dependent on the formation of gp120-CD4-CXCR4 tricomplexes since staining was absent with SDF-treated or coreceptor-negative target cells. Overall, these studies show that access to the CD4-induced coreceptor-binding domain on gp120 is largely blocked at the fusing cell interface and is unlikely to represent a target for neutralizing antibodies. However, new epitopes are presented on intermediate gp120 structures formed as a result of coreceptor interactions. Such findings have important implications for HIV vaccine approaches based on conformational alterations in envelope structures.     

Selection pressure on HIV-1 envelope by broadly neutralizing antibodies to the conserved CD4-binding site

The monoclonal antibody (MAb) VRC01 was isolated from a slowly progressing HIV-1-infected donor and was shown to neutralize diverse HIV-1 strains by binding to the conserved CD4 binding site (CD4bs) of gp120. To better understand the virologic factors associated with such antibody development, we characterized HIV-1 envelope (Env) variants from this donor and five other donors who developed broadly neutralizing antibodies. A total of 473 env sequences were obtained by single-genome amplification, and 100 representative env clones were expressed and tested for entry and neutralization sensitivity. While VRC01 neutralizes about 90% of the genetically diverse heterologous HIV-1 strains tested, only selective archival Env variants from the VRC01 donor were sensitive to VRC01 and all of the Env variants derived from the donor plasma were resistant, indicating strong antibody-based selection pressure. Despite their resistance to this broadly reactive MAb that partially mimics CD4, all Env variants required CD4 for entry. Three other CD4bs MAbs from the same donor were able to neutralize some VRC01 escape variants, suggesting that CD4bs antibodies continued to evolve in response to viral escape. We also observed a relatively high percentage of VRC01-resistant Env clones in the plasma of four of five additional broadly neutralizing donors, suggesting the presence of CD4bs-directed neutralizing antibodies in these donors. In total, these data indicate that the CD4bs-directed neutralizing antibodies exert ongoing selection pressure on the conserved CD4bs epitope of HIV-1 Env.     

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.     

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.     

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.     

Biochemically defined HIV-1 envelope glycoprotein variant immunogens display differential binding and neutralizing specificities to the CD4-binding site

HIV-1 gp120 binds the primary receptor CD4. Recently, a plethora of broadly neutralizing antibodies to the gp120 CD4-binding site (CD4bs) validated this region as a target for immunogen design. Here, we asked if modified HIV-1 envelope glycoproteins (Env) designed to increase CD4 recognition might improve recognition by CD4bs neutralizing antibodies and more efficiently elicit such reactivities. We also asked if CD4bs stabilization, coupled with altering the Env format (monomer to trimer or cross-clade), might better elicit neutralizing antibodies by focusing the immune response on the functionally conserved CD4bs. We produced monomeric and trimeric Envs stabilized by mutations within the gp120 CD4bs cavity (pocket-filling; PF2) or by appending heterologous trimerization motifs to soluble Env ectodomains (gp120/gp140). Recombinant glycoproteins were purified to relative homogeneity, and ligand binding properties were analyzed by ELISA, surface plasmon resonance, and isothermal titration microcalorimetry. In some formats, the PF2 substitutions increased CD4 affinity, and importantly, PF2-containing proteins were better recognized by the broadly neutralizing CD4bs mAbs, VRC01 and VRC-PG04. Based on this analysis, we immunized selected Env variants into rabbits using heterologous or homologous regimens. Analysis of the sera revealed that homologous inoculation of the PF2-containing, variable region-deleted YU2 gp120 trimers (ΔV123/PF2-GCN4) more rapidly elicited CD4bs-directed neutralizing antibodies compared with other regimens, whereas homologous trimers elicited increased neutralization potency, mapping predominantly to the gp120 third major variable region (V3). These results suggest that some engineered Env proteins may more efficiently direct responses toward the conserved CD4bs and be valuable to elicit antibodies of greater neutralizing capacity.     

Antigenic properties of the human immunodeficiency virus transmembrane glycoprotein during cell-cell fusion

Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19 degrees C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.     

Comparing antigenicity and immunogenicity of engineered gp120

We have engineered monomeric gp120 in such a way as to favorably present the conserved epitope for the broadly neutralizing antibody b12 while lowering the exposure of epitopes recognized by some weakly neutralizing and nonneutralizing antibodies. The work presented here describes the immune response in rabbits immunized with two prototype, engineered gp120s to explore the relationship between antigenicity and immunogenicity for these mutants. The GDMR gp120 mutant (residues 473 to 476 on gp120 altered from GDMR to AAAA) has a series of substitutions on the edge of the CD4 binding site (CD4bs), and the mCHO gp120 mutant has seven extra glycans relative to the wild-type protein. Importantly, serum mapping showed that both mutants did not elicit antibodies against a number of epitopes that had been targeted for dampening. The sera from rabbits immunized with the GDMR gp120 mutant neutralized some primary viruses at levels somewhat better than the wild-type gp120 immune sera as a result of an increased elicitation of anti-V3 antibodies. Unlike wild-type gp120 immune sera, GDMR gp120 immune sera failed to neutralize HXBc2, a T-cell line adapted (TCLA) virus. This was associated with loss of CD4bs/CD4-induced antibodies that neutralize TCLA but not primary viruses. The mCHO gp120 immune sera did not neutralize primary viruses to any significant degree, reflecting the masking of epitopes of even weakly neutralizing antibodies without eliciting b12-like antibodies. These results show that antibody responses to multiple epitopes on gp120 can be dampened. More precise focusing to a neutralizing epitope will likely require several iterations comparing antigenicity and immunogenicity of engineered proteins.     

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.     

Gp120/CD4 Blocking Antibodies Are Frequently Elicited in ART-Na?ve Chronically HIV-1 Infected Individuals

Antibodies with the ability to block the interaction of HIV-1 envelope glycoprotein (Env) gp120 with CD4, including those overlapping the CD4 binding site (CD4bs antibodies), can protect from infection by HIV-1, and their elicitation may be an interesting goal for any vaccination strategy. To identify gp120/CD4 blocking antibodies in plasma samples from HIV-1 infected individuals we have developed a competitive flow cytometry-based functional assay. In a cohort of treatment-naïve chronically infected patients, we showed that gp120/CD4 blocking antibodies were frequently elicited (detected in 97% plasma samples) and correlated with binding to trimeric HIV-1 envelope glycoproteins. However, no correlation was observed between functional CD4 binding blockade data and titer of CD4bs antibodies determined by ELISA using resurfaced gp120 proteins. Consistently, plasma samples lacking CD4bs antibodies were able to block the interaction between gp120 and its receptor, indicating that antibodies recognizing other epitopes, such as PGT126 and PG16, can also play the same role. Antibodies blocking CD4 binding increased over time and correlated positively with the capacity of plasma samples to neutralize the laboratory-adapted NL4.3 and BaL virus isolates, suggesting their potential contribution to the neutralizing workforce of plasma in vivo. Determining whether this response can be boosted to achieve broadly neutralizing antibodies may provide valuable information for the design of new strategies aimed to improve the anti-HIV-1 humoral response and to develop a successful HIV-1 vaccine.     

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.     

Binding interactions between soluble HIV envelope glycoproteins and quaternary-structure-specific monoclonal antibodies PG9 and PG16

PG9 and PG16 are antibodies isolated from a subject infected with HIV-1 and display broad anti-HIV neutralizing activities. They recognize overlapping epitopes, which are preferentially expressed on the membrane-anchored trimeric form of the HIV envelope glycoprotein (Env). PG9 and PG16 were reported not to bind to soluble mimetics of Env. The engineering of soluble Env proteins on which the PG9 and PG16 epitopes are optimally exposed will support efforts to elicit broad anti-HIV neutralizing antibodies by immunization. Here, we identified several soluble gp140 Env proteins that are recognized by PG9 and PG16, and we investigated the molecular details of those binding interactions. The IgG versions of PG9 and PG16 recognize the soluble trimeric gp140 form less efficiently than the corresponding monomeric gp140 form. In contrast, the Fab versions of PG9 and PG16 recognized the monomeric and trimeric gp140 forms with identical binding kinetics and with binding affinities similar to the high binding affinity of the anti-V3 antibody 447D to its epitope. Our data also indicate that, depending on the Env backbone, the interactions of PG9 and PG16 with gp140 may be facilitated by the presence of the gp41 ectodomain and are independent of the proper enzymatic cleavage of gp140 into gp120 and gp41. The identification of soluble Env proteins that express the PG9 and PG16 epitopes and the detailed characterization of the molecular interactions between these two antibodies and their ligands provide important and novel information that will assist in improving the engineering of future Env immunogens.     

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4⁺ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.