One approach to the development of an HIV vaccine is to design a protein template which can present gp120 epitopes inducing cross-neutralizing antibodies. To select a V3 sequence for immunogen design, we compared the neutralizing activities of 18 anti-V3 monoclonal antibodies (mAbs) derived from Cameroonian and Indian individuals infected with clade AG and C, respectively. It was found that V3 mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India. Interestingly, superior neutralizing activity of Cameroonian mAbs was also observed among the nine VH5-51/VL lambda genes encoding V3 mAbs which mediate a similar mode of recognition. This correlated with higher relative binding affinity to a variety of gp120s and increased mutation rates in V3 mAbs from Cameroon. These results suggest that clade C V3 is probably weakly immunogenic and that the V3 sequence of CRF02_AG viruses can serve as a plausible template for vaccine immunogen design.
HIV-1; V3 region; Non-B clade HIV-1; Immunoglobulin gene usage; Human monoclonal antibodies; HIV neutralizing antibodies
Hematogenous dissemination of Mycobacterium tuberculosis (M. tb) occurs during both primary and reactivated tuberculosis (TB). Although hematogenous dissemination occurs in non-HIV TB patients, in ∼80% of these patients, TB manifests exclusively as pulmonary disease. In contrast, extrapulmonary, disseminated, and/or miliary TB is seen in 60–70% of HIV-infected TB patients, suggesting that hematogenous dissemination is likely more common in HIV+ patients. To understand M. tb adaptation to the blood environment during bacteremia, we have studied the transcriptome of M. tb replicating in human whole blood. To investigate if M. tb discriminates between the hematogenous environments of immunocompetent and immunodeficient individuals, we compared the M. tb transcriptional profiles during replication in blood from HIV- and HIV+ donors. Our results demonstrate that M. tb survives and replicates in blood from both HIV- and HIV+ donors and enhances its virulence/pathogenic potential in the hematogenous environment. The M. tb blood-specific transcriptome reflects suppression of dormancy, induction of cell-wall remodeling, alteration in mode of iron acquisition, potential evasion of immune surveillance, and enhanced expression of important virulence factors that drive active M. tb infection and dissemination. These changes are accentuated during bacterial replication in blood from HIV+ patients. Furthermore, the expression of ESAT-6, which participates in dissemination of M. tb from the lungs, is upregulated in M. tb growing in blood, especially during growth in blood from HIV+ patients. Preliminary experiments also demonstrate that ESAT-6 promotes HIV replication in U1 cells. These studies provide evidence, for the first time, that during bacteremia, M. tb can adapt to the blood environment by modifying its transcriptome in a manner indicative of an enhanced-virulence phenotype that favors active infection. Additionally, transcriptional modifications in HIV+ blood may further accentuate M. tb virulence and drive both M. tb and HIV infection.
Neutralizing antibody assays are widely used in research toward development of a preventive HIV-1 vaccine. Currently, the neutralization potency of an antibody is typically quantified by the inhibitory concentration (IC) values (e.g., IC50), and the neutralization breadth is estimated by the empirical method. In this paper, we propose the AUC and pAUC measures for summarizing the titration curve, which complement the commonly used IC measure. We present multiple advantages of AUC over IC50, which include no complications due to censoring, the capability to explore low-level neutralization, and improved coverage probabilities and efficiency of estimators. We also propose statistical methods for determining positive neutralization and for estimating the neutralization breadth. The simulation results suggest that the AUC measure is preferable in particular as IC50s get closer to the highest concentration of antibodies tested. For the majority of the assay data, the AUC method is more powerful than the IC50 method. However, since these methods test different hypotheses, it is not unexpected that some virus-antibody combinations are AUC positive but IC50 negative or vice versa.
AUC; breadth; HIV-1; neutralization assay; polynomial model; titration curve
In the RV144 HIV-1 vaccine efficacy trial, IgG antibody (Ab) binding levels to variable regions 1 and 2 (V1V2) of the HIV-1 envelope glycoprotein gp120 were an inverse correlate of risk of HIV-1 infection. To determine if V1V2-specific Abs cross-react with V1V2 from different HIV-1 subtypes, if the nature of the V1V2 antigen used to asses cross-reactivity influenced infection risk, and to identify immune assays for upcoming HIV-1 vaccine efficacy trials, new V1V2-scaffold antigens were designed and tested. Protein scaffold antigens carrying the V1V2 regions from HIV-1 subtypes A, B, C, D or CRF01_AE were assayed in pilot studies, and six were selected to assess cross-reactive Abs in the plasma from the original RV144 case-control cohort (41 infected vaccinees, 205 frequency-matched uninfected vaccinees, and 40 placebo recipients) using ELISA and a binding Ab multiplex assay. IgG levels to these antigens were assessed as correlates of risk in vaccine recipients using weighted logistic regression models. Levels of Abs reactive with subtype A, B, C and CRF01_AE V1V2-scaffold antigens were all significant inverse correlates of risk (p-values of 0.0008–0.05; estimated odds ratios of 0.53–0.68 per 1 standard deviation increase). Thus, levels of vaccine-induced IgG Abs recognizing V1V2 regions from multiple HIV-1 subtypes, and presented on different scaffolds, constitute inverse correlates of risk for HIV-1 infection in the RV144 vaccine trial. The V1V2 antigens provide a link between RV144 and upcoming HIV-1 vaccine trials, and identify reagents and methods for evaluating V1V2 Abs as possible correlates of protection against HIV-1 infection.
A human anti-HIV monoclonal antibody (mAb), 2909, selected on the basis of its potent neutralizing activity against HIV-1SF162, recognizes a complex epitope V2/V3 present on intact virions but not on soluble gp120. To confirm the quaternary nature of the epitope, 2909 binding was tested against the pseudovirus SF162 wild type (WT) expressing trimers and/or an SF162 mutant expressing monomeric envelope proteins. The construction of the SF162 mutant was made by an alanine substitution of nine hydrophobic residues in the N-terminal heptad repeat region of gp41 molecules that failed to form trimers on the virus surface. Monoclonal Ab 2909 bound only to SF162 WT virions and transfected cells as determined by mmunoprecipitation and flow cytometry, respectively, but showed no reactivity to the SF162 mutant expressing monomeric gp120. The data provide further evidence for the existence of a unique quaternary epitope V2/V3 on the surface of unliganded virus.
HIV-1; neutralizing antibody; V2/V3 regions
The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169–184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100–800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100–3200) and 3570 (range: 200–12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.
We previously showed that expression of human FcγRI on TZM-bl cells potentiates neutralization by gp41 membrane-proximal external region (MPER)-specific antibodies. Here we show that lysosomotropic reagents known to block phagocytosis do not diminish this effect. We also show that FcγRI occasionally potentiates neutralization by antibodies against the V3 loop of gp120 and cluster I of gp41. We conclude that FcγRI provides a kinetic advantage for neutralizing antibodies against partially cryptic epitopes independent of phagocytosis.
Neutralizing and non-neutralizing antibodies to linear epitopes on HIV-1 envelope glycoproteins have potential to mediate antiviral effector functions that could be beneficial to vaccine-induced protection. Here, plasma IgG responses were assessed in three HIV-1 gp120 vaccine efficacy trials (RV144, Vax003, Vax004) and in HIV-1-infected individuals by using arrays of overlapping peptides spanning the entire consensus gp160 of all major genetic subtypes and circulating recombinant forms (CRFs) of the virus. In RV144, where 31.2% efficacy against HIV-1 infection was seen, dominant responses targeted the C1, V2, V3 and C5 regions of gp120. An analysis of RV144 case-control samples showed that IgG to V2 CRF01_AE significantly inversely correlated with infection risk (OR= 0.54, p=0.0042), as did the response to other V2 subtypes (OR=0.60-0.63, p=0.016-0.025). The response to V3 CRF01_AE also inversely correlated with infection risk but only in vaccine recipients who had lower levels of other antibodies, especially Env-specific plasma IgA (OR=0.49, p=0.007) and neutralizing antibodies (OR=0.5, p=0.008). Responses to C1 and C5 showed no significant correlation with infection risk. In Vax003 and Vax004, where no significant protection was seen, serum IgG responses targeted the same epitopes as in RV144 with the exception of an additional C1 reactivity in Vax003 and infrequent V2 reactivity in Vax004. In HIV-1 infected subjects, dominant responses targeted the V3 and C5 regions of gp120, as well as the immunodominant domain, heptad repeat 1 (HR-1) and membrane proximal external region (MPER) of gp41. These results highlight the presence of several dominant linear B cell epitopes on the HIV-1 envelope glycoproteins. They also generate the hypothesis that IgG to linear epitopes in the V2 and V3 regions of gp120 are part of a complex interplay of immune responses that contributed to protection in RV144.
To better understand the limits of antigenic reactivity and epitope accessibility of the V3 domain of primary HIV-1 isolates, we evaluated three human anti-V3 monoclonal antibodies (mAbs) and selected guinea pig vaccine sera for neutralization against reference panels of subtype B and C pseudoviruses derived from early stage infections. The mAbs and vaccine sera potently neutralized several prototype viruses, but displayed substantially less neutralization of most reference strains. In the presence of soluble CD4 (sCD4), the breadth of V3-mediated neutralization was increased; up to 80% and 77% of the subtype B and C viruses respectively were sensitive to V3-mediated neutralization. Unlike sCD4, the reaction of CD4-binding site mAbs b12 and F105 with native virus did not lead to full exposure of the V3 domain. These findings confirm that V3 antibodies recognize most primary viral strains, but that the epitope often has limited accessibility in the context of native envelope spike.
An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.
In the case-control study of the RV144 vaccine trial, the levels of antibodies to the V1V2 region of the gp120 envelope glycoprotein were found to correlate inversely with risk of HIV infection. This recent demonstration of the potential role of V1V2 as a vaccine target has catapulted this region into the focus of HIV-1 research. We previously described seven human monoclonal antibodies (mAbs) derived from HIV-infected individuals that are directed against conformational epitopes in the V1V2 domain. In this study, using lysates of SF162 pseudoviruses carrying V1V2 mutations, we mapped the epitopes of these seven mAbs. All tested mAbs demonstrated a similar binding pattern in which three mutations (F176A, Y177T, and D180L) abrogated binding of at least six of the seven mAbs to ≤15% of SF162 wildtype binding. Binding of six or all of the mAbs was reduced to ≤50% of wildtype by single substitutions at seven positions (168, 180, 181, 183, 184, 191, and 193), while one change, V181I, increased the binding of all mAbs. When mapped onto a model of V2, our results suggest that the epitope of the conformational V2 mAbs is located mostly in the disordered region of the available crystal structure of V1V2, overlapping and surrounding the α4β7 binding site on V2.
The production of human monoclonal antibodies (mAbs) has been improved recently using the single B cell and PCR technology. A number of new anti-HIV-1 mAbs directed to various epitopes were produced by selecting single B cells from HIV positive individuals using the HIV-1 envelope (Env) proteins, and we tested whether the peptide can select B cells specific to a particular Env epitope. Using the fluorescently-labeled peptide tetramer representative of the V3 loop of HIV-1 Env gp120 for staining the B cells derived from one HIV-1 infected donor, four clonal human mAbs were produced with specificity to the V3 region. The clonality of the four V3 mAbs was based on the usage of the same immunoglobulin genes and almost identical sequence of CDRs. The amino acid changes were present only in the framework and, possibly, they could be related to the differences observed in the relative affinity binding of these four mAbs to V3 antigen. One representative V3 mAb displayed very potent neutralizing activity to one of two viruses tested. This study shows the feasibility of utilizing a peptide tetramer to select epitope-specific B cells and produce mAbs.
HIV-1; V3 region; immunoglobulin gene usage; human monoclonal antibodies; HIV neutralizing antibodies
HIV-1 R5 viruses vary extensively in their capacity to infect macrophages. R5 viruses that confer efficient infection of macrophages are able to exploit low levels of CD4 for infection and predominate in brain tissue, where macrophages are a major target for infection. HIV-1 R5 founder viruses that are transmitted were reported to be non-macrophage-tropic. Here, we investigated the sensitivities of macrophage-tropic and non-macrophage-tropic R5 envelopes to neutralizing antibodies. We observed striking differences in the sensitivities of Env+ pseudovirions to soluble CD4 (sCD4) and to neutralizing monoclonal antibodies (MAbs) that target the CD4 binding site. Macrophage-tropic R5 Envs were sensitive to sCD4, while non-macrophage-tropic Envs were significantly more resistant. In contrast, all Envs were sensitive to VRC01 regardless of tropism, while MAb b12 conferred an intermediate neutralization pattern where all the macrophage-tropic and about half of the non-macrophage-tropic Envs were sensitive. CD4, b12, and VRC01 share binding specificities on the outer domain of gp120. However, these antibodies differ in their ability to induce conformational changes on the trimeric envelope and in specificity for residues on the V1V2 loop stem and β20-21 junction that are targets for CD4 in recruiting the bridging sheet. These distinct specificities of CD4, b12, and VRC01 likely explain the observed differences in Env sensitivity to inhibition by these reagents and provide an insight into the envelope mechanisms that control macrophage tropism. We present a model where the efficiency of bridging-sheet recruitment by CD4 is a major determinant of HIV-1 R5 envelope sensitivity to soluble CD4 and macrophage tropism.
Human monoclonal antibodies (mAbs) 447-52D and 537-10D, both coded by the VH3 gene and specific for the third variable region (V3) of the HIV-1 gp120, were found to share antigen binding structural elements including an elongated CDR H3 forming main-chain interactions with the N-terminus of the V3 crown. However, water-mediated hydrogen bonds and a unique cation-π sandwich stacking allow 447-52D to be broadly reactive with V3 containing both the GPGR and GPGQ crown motifs, while the deeper binding pocket, and a buried Glu in the binding site of 537-10D limit its reactivity to only V3 containing the GPGR motif. Our results suggest that the design of immunogens for anti-V3 antibodies should avoid the Arg at the V3 crown, as GPGR-containing epitopes appear to select for B cells making antibodies of narrower specificity than V3 that carry Gln at this position.
HIV-1; gp120; V3; monoclonal antibody; antibody-antigen interactions
The recent analysis of the first successful RV144 vaccine trial revealed that a high titer of plasma anti-V2 antibodies (Abs) correlated with a decreased risk of HIV-1 infection in vaccine recipients. To understand the mechanism of immune correlates, we studied seven anti-V2 monoclonal Abs (mAbs) developed from HIV-1 infected individuals. The V2 mAbs target conserved epitopes, including the binding site for α4β7 integrin, and are broadly cross-reactive with various gp120 proteins. Preferential usage of the VH1-69 gene by V2 mAbs may depend on selection by the same antigenic structure. Six of seven V2 mAbs weakly neutralized four to eight of the 41 pseudoviruses tested and resistance to neutralization was correlated with longer V2 domains. The data suggest the presence of shared, conserved structural elements in the V2 loop, and these can be used in the design of vaccine immunogens inducing broadly reactive Abs with anti-viral activities.
HIV-1; V2 domain; Envelope proteins; Human monoclonal antibodies; HIV neutralizing antibodies; Glycosylation
The RV144 clinical trial of a prime/boost immunizing regimen using recombinant canary pox (ALVAC-HIV) and two gp120 proteins (AIDSVAX B and E) was previously shown to have a 31.2% efficacy rate. Plasma specimens from vaccine and placebo recipients were used in an extensive set of assays to identify correlates of HIV-1 infection risk. Of six primary variables that were studied, only one displayed a significant inverse correlation with risk of infection: the antibody (Ab) response to a fusion protein containing the V1 and V2 regions of gp120 (gp70-V1V2). This finding prompted a thorough examination of the results generated with the complete panel of 13 assays measuring various V2 Abs in the stored plasma used in the initial pilot studies and those used in the subsequent case-control study. The studies revealed that the ALVAC-HIV/AIDSVAX vaccine induced V2-specific Abs that cross-react with multiple HIV-1 subgroups and recognize both conformational and linear epitopes. The conformational epitope was present on gp70-V1V2, while the predominant linear V2 epitope mapped to residues 165–178, immediately N-terminal to the putative α4β7 binding motif in the mid-loop region of V2. Odds ratios (ORs) were calculated to compare the risk of infection with data from 12 V2 assays, and in 11 of these, the ORs were ≤1, reaching statistical significance for two of the variables: Ab responses to gp70-V1V2 and to overlapping V2 linear peptides. It remains to be determined whether anti-V2 Ab responses were directly responsible for the reduced infection rate in RV144 and whether anti-V2 Abs will prove to be important with other candidate HIV vaccines that show efficacy, however, the results support continued dissection of Ab responses to the V2 region which may illuminate mechanisms of protection from HIV-1 infection and may facilitate the development of an effective HIV-1 vaccine.
In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case–control analysis to identify antibody and cellular immune correlates of infection risk.
In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up.
Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P = 0.02; q = 0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P = 0.03; q = 0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies.
This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.
Despite the frequent observation of masking of HIV-1 neutralization epitopes, its extent has not previously been systematically assessed either for multiple epitopes presented by individual viruses or for individual epitopes across multiple viral strains. Using a recently developed method to identify amino acid sequence motifs required for recognition by HIV-1-neutralizing monoclonal antibodies (mAbs), we visualized the patterns of masking of specific epitopes targeted by mAbs in a diverse panel of HIV-1 isolates. We also calculated a specific masking intensity score for each virus based on the observed neutralization activity of mAbs against the epitopes in the virus. Finally, we combined these data with estimates of the conservation of each mAb-targeted epitope in circulating HIV-1 strains to estimate the effective neutralization potential (EN) for each mAb. Focusing on the V3 loop of gp120 as a prototype neutralization domain, we found that the V3 loop epitope targeted by mAb 2219 is one of the least masked mAbs and it has the highest EN. Interestingly, although the V3 loop epitope targeted by mAb 3074 is present in over 87% of all viruses, it is 82.2% masked, so its EN is lower than that for mAb 2219. Notably, 50% of the viruses that mAb 3074 is able to neutralize are classified as subtype C viruses, while 70% or more of the viruses neutralized by mAbs 2219, 2557 or 447-52D are classified as subtype B. Thus, neutralization epitopes (in this case, in the V3 loop) have differential patterns of masking and also display distinct patterns of distribution among circulating HIV-1 viruses. Both factors combine to contribute to the practical vaccine value of any single epitope/mAb. Here we have developed a quantitative score for this value. These results have important implications for rational design of vaccines designed to induce neutralizing Abs by revealing epitopes that are minimally masked and maximally reactive with neutralizing Abs.
HIV-1; V3; variable loop; masking; epitope; neutralization
A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis.
The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20Å in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.
HIV/AIDS; gp120; quaternary neutralizing epitope (QNE); monoclonal antibody (mAb); crystal structure; immunogen design; vaccine
HIV-1's subtype C V3 loop consensus sequence exhibits increased resistance to anti-V3 antibody-mediated neutralization as compared to the subtype B consensus sequence. The dynamic 3D structure of the consensus C V3 loop crown, visualized by ab initio folding, suggested that the resistance derives from structural rigidity and non-β-strand secondary protein structure in the N-terminal strand of the β-hairpin of the V3 loop crown, which is where most known anti-V3 loop antibodies bind. The observation of either rigidity or non-β-strand structure in this region correlated with observed resistance to antibody-mediated neutralization in a series of chimeric pseudovirus (psV) mutants. The results suggest the presence of an epitope-independent, neutralization-relevant structural difference in the antibody-targeted region of the V3 loop crown between subtype C and subtype B, a difference that we hypothesize may contribute to the divergent pattern of global spread between these subtypes. As antibodies to a variable loop were recently identified as an inverse correlate of risk for HIV infection, the structure-function relationships discussed in this study may have relevance to HIV vaccine research.
A series of potently neutralizing monoclonal antibodies (MAbs) that target quaternary epitopes on the native Env trimer have recently been described. A common feature shared by these antibodies is the critical involvement of sites in both the V2 and V3 variable domains in antibody recognition. In this study the gp120 variable-region determinants were mapped for eight rhesus macaque monoclonal antibodies (RhMAbs) possessing potently neutralizing activity specific for a quaternary target in SF162 Env and compared to those originally identified for human MAb 2909. These studies showed that determinants for the epitopes defined by the RhMAbs differed in both the V2 (positions 160, 167, and 169) and V3 (positions 313 and 315) regions from 2909, and in a number of cases, from each other. Attempts to reconstitute expression of these epitopes on the cell surface by cotransfecting Envs containing either the V2 or the V3 determinant of the epitope were not successful, suggesting that these epitopes were expressed on individual protomers in a trimer-dependent manner. Several of the V2 positions found to be critical for expression of these quaternary epitopes also significantly affected exposure and neutralization sensitivity of targets in the V3 and CD4-binding domains. These results demonstrated a considerable diversity in the fine structure of this class of epitopes and further suggested a potentially important relationship between the expression of such quaternary epitopes and V1/V2-mediated masking of immunodominant epitopes.
The V3 epitope is a known target for HIV-1 neutralizing antibodies (NAbs), and V3-scaffold fusion proteins used as boosting immunogens after gp120 DNA priming were previously shown to induce NAbs in rabbits. Here, we evaluated whether the breadth and potency of the NAb response could be improved when boosted with rationally designed V3-scaffold immunogens. Rabbits were primed with codon-optimized clade C gp120 DNA and boosted with one of five V3-cholera toxin B fusion proteins (V3-CTBs) or with double combinations of these. The inserts in these immunogens were designed to display V3 epitopes shared by the majority of global HIV-1 isolates. Double combinations of V3-CTB immunogens generally induced more broad and potent NAbs than did boosts with single V3-CTB immunogens, with the most potent and broad NAbs elicited with the V3-CTB carrying the consensus V3 of clade C (V3C-CTB), or with double combinations of V3-CTB immunogens that included V3C-CTB. Neutralization of tier 1 and 2 pseudoviruses from clades AG, B, and C and of peripheral blood mononuclear cell (PBMC)-grown primary viruses from clades A, AG, and B was achieved, demonstrating that priming with gp120 DNA followed by boosts with V3-scaffold immunogens effectively elicits cross-clade NAbs. Focusing on the V3 region is a first step in designing a vaccine targeting protective epitopes, a strategy with potential advantages over the use of Env, a molecule that evolved to protect the virus by poorly inducing NAbs and by shielding the epitopes that are most critical for infectivity.
Any strategy for curing HIV infection must include a method to eliminate viral-infected cells. Based on our earlier proof-of-principle results targeting HIV-1 infected cells with radiolabeled antibody (mAb) to gp41 viral antigen, we embarked on identifying a suitable candidate mAb for preclinical development.
Among the several human mAbs to gp41 tested, mAb 2556 was found to have high affinity, reactivity with multimeric forms of gp41 present on both the surface of virus particles and cells expressing HIV-1 Env, and recognition of a highly conserved epitope of gp41 shared by all HIV-1 subtypes. Also, mAb 2556 was the best in competition with HIV-1+ serum antibodies, which is an extremely important consideration for efficacy in the treatment of HIV patients. When radiolabeled with alpha-emitting radionuclide 213-Bismuth (213Bi) - 213Bi-2556 efficiently and specifically killed ACH-2 human lymphocytes chronically infected with HIV-1, and HIV-1 infected human peripheral blood mononuclear cells (hPBMCs). The number of binding sites for 213Bi-2556 on the surface of the infected cells was >106. The in vivo experiments were performed in two HIV-1 mouse models – splenic and intraperitoneal. In both models, the decrease in HIV-1 infected hPBMCs from the spleens and peritoneum, respectively, was dose-dependent with the most pronounced killing of hPBMCs observed in the 100 µCi 213Bi-2556 group (P = 0.01). Measurement of the blood platelet counts and gross pathology of the treated mice demonstrated the lack of toxicity for 213Bi-2556.
We describe the preclinical development of a novel radiolabeled mAb reagent that could potentially be part of an HIV eradication strategy that is ready for translation into the clinic as the next step in its development. As viral antigens are very different from “self” human antigens - this approach promises high selectivity, increased efficacy and low toxicity, especially in comparison to immunotoxins.