Fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the CD4 binding site of human immunodeficiency virus type 1 gp120

Alanine scanning mutagenesis was performed on monomeric gp120 of human immunodeficiency virus type 1 to systematically identify residues important for gp120 recognition by neutralizing and nonneutralizing monoclonal antibodies (MAbs) to the CD4 binding site (CD4bs). Substitutions that affected the binding of broadly neutralizing antibody b12 were compared to substitutions that affected the binding of CD4 and of two nonneutralizing anti-CD4bs antibodies (b3 and b6) with affinities for monomeric gp120 comparable to that of b12. Not surprisingly, the sensitivities to a number of amino acid changes were similar for the MAbs and for CD4. However, in contrast to what was seen for the MAbs, no enhancing mutations were observed for CD4, suggesting that the virus has evolved toward an optimal gp120-CD4 interaction. Although the epitope maps of the MAbs overlapped, a number of key differences between b12 and the other two antibodies were observed. These differences may explain why b12, in contrast to nonneutralizing antibodies, is able to interact not only with monomeric gp120 but also with functional oligomeric gp120 at the virion surface. Neutralization assays performed with pseudovirions bearing envelopes from a selection of alanine mutants mostly showed a reasonable correlation between the effects of the mutations on b12 binding to monomeric gp120 and neutralization efficacy. However, some mutations produced an effect on b12 neutralization counter to that predicted from gp120 binding data. It appears that these mutations have different effects on the b12 epitope on monomeric gp120 and functional oligomeric gp120. To determine whether monomeric gp120 can be engineered to preferentially bind MAb b12, recombinant gp120s were generated containing combinations of alanine substitutions shown to uniquely enhance b12 binding. Whereas b12 binding was maintained or increased, binding by five nonneutralizing anti-CD4bs MAbs (b3, b6, F105, 15e, and F91) was reduced or completely abolished. These reengineered gp120s are prospective immunogens that may prove capable of eliciting broadly neutralizing antibodies.     

PGV04, an HIV-1 gp120 CD4 binding site antibody, is broad and potent in neutralization but does not induce conformational changes characteristic of CD4

Recently, several broadly neutralizing monoclonal antibodies (bnMAbs) directed to the CD4-binding site (CD4bs) of gp120 have been isolated from HIV-1-positive donors. These include VRC01, 3BNC117, and NIH45-46, all of which are capable of neutralizing about 90% of circulating HIV-1 isolates and all of which induce conformational changes in the HIV-1 gp120 monomer similar to those induced by the CD4 receptor. In this study, we characterize PGV04 (also known as VRC-PG04), a MAb with potency and breadth that rivals those of the prototypic VRC01 and 3BNC117. When screened on a large panel of viruses, the neutralizing profile of PGV04 was distinct from those of CD4, b12, and VRC01. Furthermore, the ability of PGV04 to neutralize pseudovirus containing single alanine substitutions exhibited a pattern distinct from those of the other CD4bs MAbs. In particular, substitutions D279A, I420A, and I423A were found to abrogate PGV04 neutralization. In contrast to VRC01, PGV04 did not enhance the binding of 17b or X5 to their epitopes (the CD4-induced [CD4i] site) in the coreceptor region on the gp120 monomer. Furthermore, in contrast to CD4, none of the anti-CD4bs MAbs induced the expression of the 17b epitope on cell surface-expressed cleaved Env trimers. We conclude that potent CD4bs bnMAbs can display differences in the way they recognize and access the CD4bs and that mimicry of CD4, as assessed by inducing conformational changes in monomeric gp120 that lead to enhanced exposure of the CD4i site, is not uniquely correlated with effective neutralization at the site of CD4 binding on HIV-1.     

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.     

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.     

Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.

Forty-six monoclonal antibodies (MAbs) able to bind to the native, monomeric gp120 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) LAI (HXBc2) strain were used to generate a competition matrix. The data suggest the existence of two faces of the gp120 glycoprotein. The binding sites for the viral receptor, CD4, and neutralizing MAbs appear to cluster on one face, which is presumably exposed on the assembled, oligomeric envelope glycoprotein complex. A second gp120 face, which is presumably inaccessible on the envelope glycoprotein complex, contains a number of epitopes for nonneutralizing antibodies. This analysis should be useful for understanding both the interaction of antibodies with the HIV-1 gp120 glycoprotein and neutralization of HIV-1.     

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.     

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

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

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.     

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.     

Nature of nonfunctional envelope proteins on the surface of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies are thought be distinguished from nonneutralizing antibodies by their ability to recognize functional gp120/gp41 envelope glycoprotein (Env) trimers. The antibody responses induced by natural HIV-1 infection or by vaccine candidates tested to date consist largely of nonneutralizing antibodies. One might have expected a more vigorous neutralizing response, particularly against virus particles that bear functional trimers. The recent surprising observation that nonneutralizing antibodies can specifically capture HIV-1 may provide a clue relating to this paradox. Specifically, it was suggested that forms of Env, to which nonneutralizing antibodies can bind, exist on virus surfaces. Here, we present evidence that HIV-1 particles bear nonfunctional gp120/gp41 monomers and gp120-depleted gp41 stumps. Using a native electrophoresis band shift assay, we show that antibody-trimer binding predicts neutralization and that the nonfunctional forms of Env may account for virus capture by nonneutralizing antibodies. We hypothesize that these nonfunctional forms of Env on particle surfaces serve to divert the antibody response, helping the virus to evade neutralization.     

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.     

HIV-1 virus-like particles bearing pure env trimers expose neutralizing epitopes but occlude nonneutralizing epitopes

Hypothetically, since native HIV-1 Env trimers are exclusively recognized by neutralizing antibodies, they might induce the neutralizing antibodies in a vaccine setting. This idea has not been evaluated due to the difficulty of separating trimers from nonfunctional Env (uncleaved gp160 and gp41 stumps). The latter are immunodominant and induce nonneutralizing antibodies. We previously showed that nonfunctional Env can be selectively cleared from virus-like particle (VLP) surfaces by enzyme digests (E. T. Crooks, T. Tong(,) K. Osawa, and J. M. Binley, J.Virol. 85:5825, 2011). Here, we investigated the effects of these digests on the antigenicity of VLPs and their sensitivity to neutralization. Before digestion, WT VLPs (bearing wild-type Env) and UNC VLPs (bearing uncleaved gp160) were recognized by various Env-specific monoclonal antibodies (MAbs), irrespective of their neutralizing activity, a result which is consistent with the presence of nonfunctional Env. After digestion, only neutralizing MAbs recognized WT VLPs, consistent with selective removal of nonfunctional Env (i.e., "trimer VLPs"). Digests eliminated the binding of all MAbs to UNC VLPs, again consistent with removal of nonfunctional Env. An exception was MAb 2F5, which weakly bound to digested UNC VLPs and bald VLPs (bearing no Env), perhaps due to lipid cross-reactivity. Trimer VLPs were infectious, and their neutralization sensitivity was largely comparable to that of undigested WT VLPs. However, they were ～100-fold more sensitive to the MAbs 4E10 and Z13e1, suggesting increased exposure of the gp41 base. Importantly, a scatterplot analysis revealed a strong correlation between MAb binding and neutralization of trimer VLPs. This suggests that trimer VLPs bear essentially pure native trimer that should allow its unfettered evaluation in a vaccine setting.     

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   

Antibody 17b binding at the coreceptor site weakens the kinetics of the interaction of envelope glycoprotein gp120 with CD4

HIV-1 utilizes CD4 and the chemokine coreceptor for viral entry. The coreceptor CCR5 binding site on gp120 partially overlaps with the binding epitope of 17b, a neutralizing antibody of HIV-1. We designed a multicomponent biosensor assay to investigate the kinetic mechanism of interaction between gp120 and its receptors and the cooperative effect of the CCR5 binding site on the CD4 binding site, using 17b as a surrogate of CCR5. The Env gp120 proteins from four viral strains (JRFL, YU2, 89.6, and HXB2) and their corresponding C1-, V1/V2-, C5-deleted mutants (DeltaJRFL, DeltaYU2, Delta89.6, and DeltaHXB2) were tested in this study. We found that, across the primary and lab-adapted virus strains, 17b reduced the affinity of all four full-length Env gp120s for sCD4 by decreasing the on-rate and increasing the off-rate. This effect of 17b on full-length gp120 binding to sCD4 contrasts with the enhancing effect of sCD4 on gp120-17b interaction. For the corresponding loop-deleted mutants of Env gp120, the off-rates of the gp120-sCD4 interaction were greatly reduced in the presence of 17b, resulting in higher affinities (except for that of DeltaHXB2). The results suggest that, when 17b is prebound to full-length gp120, the V1/V2 loops may be relocated to a position that partially blocks the CD4-binding site, leading to weakening of the CD4 interaction. Given the fact that the 17b binding epitope partially overlaps with the binding site of CCR5, the kinetic results suggest that coreceptor CCR5 binding could have a similar "release" effect on the gp120-CD4 interaction by increasing the off-rate of the latter. The results also suggest that the neutralizing effect of 17b may arise not only from partially blocking the CCR5 binding site but also from reducing the CD4 binding affinity of gp120. This negative cooperative effect of 17b may provide insight into approaches to designing antagonists for viral entry.     

The C108g epitope in the V2 domain of gp120 functions as a potent neutralization target when introduced into envelope proteins derived from human immunodeficiency virus type 1 primary isolates

Monoclonal antibodies (MAbs) directed against epitopes in the V2 domain of human immunodeficiency virus type 1 gp120 often possess neutralizing activity, but these generally are highly type specific, neutralize only laboratory isolates, or have low potency. The most potent of these is C108g, directed against a type-specific epitope in HXB2 and BaL gp120s, which is glycan dependent and, in contrast to previous reports, dependent on intact disulfide bonds. This epitope was introduced into two primary Envs, derived from a neutralization-sensitive (SF162) and a neutralization-resistant (JR-FL) isolate, by substitution of two residues and, for SF162, addition of an N-linked glycosylation site. C108g effectively neutralized both variant Envs with considerably higher potency than standard MAbs against the V3 and CD4-binding domains and the broadly neutralizing MAbs 2G12 and 2F5. These amino acid substitutions also introduced the epitope recognized by a second V2-specific MAb, 10/76b, but this MAb possessed potent neutralizing activity only in the absence of the glycan required for C108g reactivity. In contrast to other gp120-specific neutralizing MAbs, C108g did not block binding of soluble Env proteins to either the CD4 or the CCR5 receptor, but studies with a fusion-arrested Env indicated that C108g neutralized at a step preceding the one blocked by the gp41-specific MAb, 2F5. These results indicate that the V1/V2 domain possesses targets that mediate potent neutralization of primary viral isolates via a novel mechanism and suggest that inclusion of carbohydrate determinants into these epitopes may help overcome the indirect masking effects that limit the neutralizing potency of antibodies commonly produced after infection.     

Exploration of antigenic variation in gp120 from clades A through F of human immunodeficiency virus type 1 by using monoclonal antibodies.

The reactivities of a panel of 14 monoclonal antibodies (MAbs) with monomeric gp120 derived from 67 isolates of human immunodeficiency virus type 1 of clades A through F were assessed by using an antigen-capture enzyme-linked immunosorbent assay. The MAbs used were all raised against gp120 or gp120 peptides from clade B viruses and were directed at a range of epitopes relevant to human immunodeficiency virus type 1 neutralization: the V2 and V3 loops, discontinuous epitopes overlapping the CD4-binding site, and two other discontinuous epitopes. Four of the five V3 MAbs showed modest cross-reactivity within clade B but very limited reactivity with gp120s from other clades. These reactivity patterns are consistent with the known primary sequence requirements for the binding of these MAbs. One V3 human MAb (19b), however, was much more broadly reactive than the others, binding to 19 of 29 clade B and 10 of 12 clade E gp120s. The 19b epitope is confined to the flanks of the V3 loop, and these sequences are relatively conserved in clade B and E viruses. In contrast to the limited reactivity of V3 MAbs, CD4-binding site MAbs were much more broadly reactive across clades, two of these MAbs (205-46-9 and 21h) being virtually pan-reactive across clades A through F. Another human MAb (A-32) to a discontinuous epitope was also pan-reactive. The CD4-binding site is strongly conserved between clades; but when considering the epitopes near the CD4-binding site, clade D gp120 appears to be the most closely related to clade B and clade E appears to be the least related. A tentative rank order for these epitopes is B/D-A/C-E/F. V2 MAbs reacted sporadically within and between clades, and no clear pattern was observable. While results from binding assays do not predict neutralization serotypes, they suggest that there may be antigenic subtypes related, but not identical, to the genetic subtypes.     

Effects of anti-gp120 monoclonal antibodies on CD4 receptor binding by the env protein of human immunodeficiency virus type 1.

Monoclonal antibodies (MAbs) to defined peptide epitopes on gp120 from human immunodeficiency virus type 1 were used to investigate the involvement of their epitopes in gp120 binding to the CD4 receptor. Recombinant vaccinia viruses were constructed that expressed either full-length gp120 (v-ED6), or a truncated gp120 lacking 44 amino acids at the carboxyl terminus (v-ED4). Binding of these glycoproteins to the CD4 receptor was detected directly with metabolically labeled gp120 or indirectly with the gp120 MAbs. Truncated gp120 from v-ED4 bound to CD4-positive cells less than 1/12 as well as gp120 from v-ED6, indicating that the C-terminal region of gp120, which is conserved in numerous isolates of human immunodeficiency virus type 1, is critical for CD4 binding. However, MAb 110-1, which recognizes a peptide contained in the region deleted from v-ED4 (amino acids 489 through 511), did not inhibit binding of gp120 to CD4. MAb 110-1 also reacted with gp120 bound to the CD4 receptor, indicating that the epitope for this antibody does not directly interact with CD4. A second MAb, 110-4, which recognizes a peptide epitope located between amino acids 303 and 323 and has potent viral neutralizing activity, also bound to gp120 on the CD4 receptor. Furthermore, pretreatment of gp120 with MAb 110-4 at concentrations approximately 1,000-fold higher than those required for complete virus neutralization inhibited subsequent CD4 binding by only about 65%. Taken together, these data suggest that neutralization mediated by antibody 110-4 does not result from binding of this MAb to the CD4-binding site of gp120.     

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.     

Neutralization of the human immunodeficiency virus type 1 primary isolate JR-FL by human monoclonal antibodies correlates with antibody binding to the oligomeric form of the envelope glycoprotein complex.

To test whether antibodies that are neutralizing or nonneutralizing for human immunodeficiency virus type 1 (HIV-1) primary isolates can be distinguished by their affinities for the oligomeric envelope glycoproteins, we selected HIV-1(JR-FL) as a model primary virus and a panel of 13 human monoclonal antibodies (MAbs) and evaluated three parameters: (i) half-maximal binding to recombinant monomeric envelope, gp120(JR-FL); (ii) half-maximal binding to oligomeric envelope of HIV-1(JR-FL) expressed on the surface of transfected 293 cells; and (iii) neutralization of HIV-1(JR-FL) in a peripheral blood mononuclear cell-based neutralization assay. Two conclusions can be drawn from these experiments. First, we confirm that antibody interactions with monomeric gp120 do not predict primary virus neutralization. Second, we show that neutralization correlates qualitatively with the relative affinity of an antibody for the oligomeric envelope glycoproteins, at least for HIV-1(JR-FL).