Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

ABSTRACT: BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20--57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.     

Longitudinal study of primary HIV-1 isolates in drug-naïve individuals reveals the emergence of variants sensitive to anti-HIV-1 monoclonal antibodies

To study how virus evolution affects neutralization sensitivity and to determine changes that occur in and around epitopes, we tested the ability of 13 anti-HIV-1 gp120 (anti-V2, anti-V3, anti-CD4bd and anti-carbohydrate) human monoclonal antibodies (mAbs) to neutralize sequential viruses obtained from five HIV-1 chronically infected drug naïve individuals. Overall, primary viruses collected from patients at first visit were resistant to neutralization by all anti-HIV-1 mAbs with the exception of one virus sensitive to IgG1b12. Four of the five patients'' viruses evolved increased sensitivity to neutralization by anti-V3 mAbs. Virus collected from a patient obtained 31 months later, evolved increased sensitivity to anti-V2, anti-V3, and anti-CD4bd mAbs. Furthermore, the anti-V2 and anti-CD4bd mAbs also exhibited increased neutralization capacities against virus collected from a patient 29 months later. Of the seven anti-V3 mAbs, five showed increased potency to neutralize the evolved virus from a patient collected after 11 months, and three exhibited increased potency against viruses from two patients collected 29 and 36 months later. Anti-V3 mAbs exhibited the most breadth and potency in neutralizing the evolving viruses. Sequence analysis of the envelope regions revealed amino acid conservation within the V3 loop, while most of the changes identified occurred outside the core epitopes and in particular within the C3 region; these may account for increased neutralization sensitivity. These studies demonstrate that in vivo, HIV-1 can evolve increased neutralization sensitivity to mAbs and that the spectrum of neutralization capacities by mAbs can be broader when studied in longitudinal analysis.     

The v3 loop is accessible on the surface of most human immunodeficiency virus type 1 primary isolates and serves as a neutralization epitope

Antibodies (Abs) against the V3 loop of the human immunodeficiency virus type 1 gp120 envelope glycoprotein were initially considered to mediate only type-specific neutralization of T-cell-line-adapted viruses. However, recent data show that cross-neutralizing V3 Abs also exist, and primary isolates can be efficiently neutralized with anti-V3 monoclonal Abs (MAbs). The neutralizing activities of anti-V3 polyclonal Abs and MAbs may, however, be limited due to antigenic variations of the V3 region, a lack of V3 exposure on the surface of intact virions, or Ab specificity. For clarification of this issue, a panel of 32 human anti-V3 MAbs were screened for neutralization of an SF162-pseudotyped virus in a luciferase assay. MAbs selected with a V3 fusion protein whose V3 region mimics the conformation of the native virus were significantly more potent than MAbs selected with V3 peptides. Seven MAbs were further tested for neutralizing activity against 13 clade B viruses in a single-round peripheral blood mononuclear cell assay. While there was a spectrum of virus sensitivities to the anti-V3 MAbs observed, 12 of the 13 viruses were neutralized by one or more of the anti-V3 MAbs. MAb binding to intact virions correlated significantly with binding to solubilized gp120s and with the potency of neutralization. These results demonstrate that the V3 loop is accessible on the native virus envelope, that the strength of binding of anti-V3 Abs correlates with the potency of neutralization, that V3 epitopes may be shared rather than type specific, and that Abs against the V3 loop, particularly those targeting conformational epitopes, can mediate the neutralization of primary isolates.     

Anti-V3 Monoclonal Antibodies Display Broad Neutralizing Activities against Multiple HIV-1 Subtypes

Background

The V3 loop of the HIV-1 envelope (Env) glycoprotein gp120 was identified as the “principal neutralizing domain” of HIV-1, but has been considered too variable to serve as a neutralizing antibody (Ab) target. Structural and immunochemical data suggest, however, that V3 contains conserved elements which explain its role in binding to virus co-receptors despite its sequence variability. Despite this evidence of V3 conservation, the ability of anti-V3 Abs to neutralize a significant proportion of HIV-1 isolates from different subtypes (clades) has remained controversial.

Methods

HIV-1 neutralization experiments were conducted in two independent laboratories to test human anti-V3 monoclonal Abs (mAbs) against pseudoviruses (psVs) expressing Envs of diverse HIV-1 subtypes from subjects with acute and chronic infections. Neutralization was defined by 50% inhibitory concentrations (IC₅₀), and was statistically assessed based on the area under the neutralization titration curves (AUC).

Results

Using AUC analyses, statistically significant neutralization was observed by ≥1 anti-V3 mAbs against 56/98 (57%) psVs expressing Envs of diverse subtypes, including subtypes A, AG, B, C and D. Even when the 10 Tier 1 psVs tested were excluded from the analysis, significant neutralization was detected by ≥1 anti-V3 mAbs against 46/88 (52%) psVs from diverse HIV-1 subtypes. Furthermore, 9/24 (37.5%) Tier 2 viruses from the clade B and C standard reference panels were neutralized by ≥1 anti-V3 mAbs. Each anti-V3 mAb tested was able to neutralize 28–42% of the psVs tested. By IC₅₀ criteria, 40/98 (41%) psVs were neutralized by ≥1 anti-V3 mAbs.

Conclusions

Using standard and new statistical methods of data analysis, 6/7 anti-V3 human mAbs displayed cross-clade neutralizing activity and revealed that a significant proportion of viruses can be neutralized by anti-V3 Abs. The new statistical method for analysis of neutralization data provides many advantages to previously used analyses.     

Characterization of neutralizing antibody responses elicited by clade A envelope immunogens derived from early transmitted viruses

The vast majority of studies with candidate immunogens based on the human immunodeficiency virus envelope (Env) have been conducted with Env proteins derived from clade B viruses isolated during chronic infection. Whether non-clade B Env protein immunogens will elicit antibodies with epitope specificities that are similar to those of antibodies elicited by clade B Envs and whether the antibodies elicited by Envs derived from early transmitted viruses will be similar to those elicited by Envs derived from viruses isolated during chronic infection are currently unknown. Here we performed immunizations with four clade A Envs, cloned directly from the peripheral blood of infected individuals during acute infection, which differed in lengths and extents of glycosylation. The antibody responses elicited by these four Envs were compared to each other and to those elicited by a well-characterized clade B Env immunogen derived from the SF162 virus, which was isolated during chronic infection. Only one clade A Env, the one with the fewer glycosylation sites, elicited homologous neutralizing antibodies (NAbs); these did not target the V1, V2, or V3 regions. In contrast, all four clade A Envs elicited anti-V3 NAbs against "easy-to-neutralize" clade B and clade A isolates, irrespective of the variable region length and extent of glycosylation of the Env used as an immunogen. These anti-V3 NAbs did not access their epitopes on homologous and heterologous clade A, or B, neutralization-resistant viruses. The length and extent of glycosylation of the variable regions on the clade A Env immunogens tested did not affect the breadth of the elicited NAbs. Our data also indicate that the development of cross-reactive NAbs against clade A viruses faces similar hurdles to the development of cross-reactive anti-clade B NAbs.     

Ability of the V3 loop of simian immunodeficiency virus to serve as a target for antibody-mediated neutralization: correlation of neutralization sensitivity, growth in macrophages, and decreased dependence on CD4

To better define the effects of sequence variation and tropism on the ability of the simian immunodeficiency virus SIVmac V3 loop to act as a target of antibody-mediated neutralization, a series of experiments were performed. Three SIV strains, SIVmac239, SIVmac316, and SIVmac155/T3, each with defined differences in env sequence and tropism, were used to construct a panel of viruses chimeric for a portion of envelope that includes the V2 and V3 regions. Peptides with sequences corresponding to the V3 loops of the parental viruses were used to immunize rabbits. The polyclonal rabbit antibodies and plasma from SIVmac239-infected animals were then used to assess the neutralization sensitivity of the parental and chimeric viruses. One of the parental viruses, SIVmac316, which is able to replicate to high titer in alveolar macrophages and can infect cells in a CD4-independent fashion, was highly sensitive to neutralization by plasma from SIVmac-infected rhesus macaques, with average 50% neutralization titers of 1:20,480; this same strain was also sensitive to neutralization by the anti-V3 loop peptide sera. Other parental and chimeric viruses were less sensitive to neutralization with this same panel of antibodies, but as seen with SIVmac316, those viruses that were able to productively replicate in alveolar macrophages were more sensitive to antibody-mediated neutralization. To further define the amino acids involved in increased sensitivity to neutralization, a panel of viruses was constructed by changing envelope residues in SIVmac316 to the corresponding SIVmac239 amino acids. The increased neutralization sensitivity observed for SIVmac316 was mapped principally to three amino acid changes spread throughout gp120. In addition, the increased sensitivity to neutralization by V3-directed antibodies correlated with the ability of the various viruses to replicate to high levels in alveolar macrophage cultures and a CD4-negative cell line, BC7/CCR5. These results demonstrate that the V3 loop of SIVmac Env can act as an efficient target of neutralizing antibodies in a fashion that is highly dependent on sequence context. In addition, these studies suggest a correlation between decreased dependence on CD4 and increased sensitivity to antibody-mediated neutralization.     

Cross-clade neutralizing activity of human anti-V3 monoclonal antibodies derived from the cells of individuals infected with non-B clades of human immunodeficiency virus type 1

The majority of global human immunodeficiency virus infections are caused by viruses characterized by a GPGQ motif at the tip of the V3 loop. Characterization of anti-V3 monoclonal antibodies (MAbs) that neutralize isolates with the GPGQ V3 motif is an important step in designing vaccines that will induce such Abs. Consequently, seven human anti-V3 MAbs derived from the cells of individuals infected with non-B-subtype viruses (anti-V3(non-B) MAbs) were generated from the cells of individuals from Africa infected with circulating recombinant forms CRF02_AG, CRF09_cpx, and CRF13_cpx, each of which contains a subtype A env gene. Sequence analysis of plasma viruses revealed a GPGQ motif at the apex of the V3 loop from six of the seven subjects and a GPGR motif from one subject. The MAbs were selected with fusion proteins (FP) containing V3(92UG037.8) or V3(JR-CSF) from subtype A or B, respectively. In virus binding assays, five of the seven (71%) anti-V3(non-B) MAbs bound to V3-FPs from both subtype A and subtype B, while only four of the nine (44%) anti-V3(B) MAbs recognized both V3-FPs. Using two neutralization assays, both the anti-V3(non-B) and the anti-V3(B) MAbs neutralized subtype B viruses with similar activities, while the anti-V3(non-B) MAbs exhibited a tendency toward both increased potency and breadth of neutralization against non-B viruses compared to anti-V3(B) MAbs. Statistical significance was not achieved, due in large measure to the sizes of the MAb panels, but the overall pattern of data strongly suggests that viruses with the GPGQ motif at the tip of the V3 loop induce anti-V3 Abs with broader cross-neutralizing activity than do viruses with the GPGR motif.     

Human monoclonal antibodies specific for conformation-sensitive epitopes of V3 neutralize human immunodeficiency virus type 1 primary isolates from various clades

The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3(JR-CSF) sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.     

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

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

The first hypervariable region of the gp120 Env glycoprotein defines the neutralizing susceptibility of heterologous human immunodeficiency virus type 1 isolates to neutralizing antibodies elicited by the SF162gp140 immunogen

Current vaccine efforts to elicit cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus (HIV) focus on the engineering of soluble mimetics of the trimeric HIV Env glycoprotein (commonly termed gp140 immunogens). Such immunogens are thought to be more effective than previously tested monomeric gp120 immunogens at eliciting cross-reactive NAbs. Still, the breadth of neutralizing antibody responses elicited by gp140 immunogens is narrow. Understanding why antibodies elicited by gp140 immunogens fail to neutralize a wide range of heterologous primary HIV isolates is necessary for improving the design of such immunogens. We previously reported that antibodies elicited in macaques by SF162 Env-derived gp140 immunogens fail to neutralize several heterologous “neutralization-resistant” primary HIV type 1 isolates, such as JRFL, ADA, and YU2. Here we show that by replacing the V1 region of Env on these heterologous viruses with that of SF162, we render them highly susceptible to neutralization by the SF162gp140-elicited antibodies. We observed that viral neutralization was mediated not only by vaccine-elicited anti-V1 but also by anti-V3 antibodies and antibodies directed against as yet unidentified Env regions, depending on the heterologous Env background. Our study indicates that common neutralization epitopes are differentially exposed on diverse primary HIV isolates and that the V1 loop contributes to this differential exposure. Therefore, the antibody responses elicited by soluble gp140 immunogens will have to overcome several distinct obstacles in order to neutralize diverse primary HIV isolates.     

The c3-v4 region is a major target of autologous neutralizing antibodies in human immunodeficiency virus type 1 subtype C infection

The early autologous neutralizing antibody response in human immunodeficiency virus type 1 (HIV-1) subtype C infections is often characterized by high titers, but the response is type specific with little to no cross-neutralizing activity. The specificities of these early neutralizing antibodies are not known; however, the type specificity suggests that they may target the variable regions of the envelope. Here, we show that cross-reactive anti-V3 antibodies developed within 3 to 12 weeks in six individuals but did not mediate autologous neutralization. Using a series of chimeric viruses, we found that antibodies directed at the V1V2, V4, and V5 regions contributed to autologous neutralization in some individuals, with V1V2 playing a more substantial role. However, these antibodies did not account for the total neutralizing capacity of these sera against the early autologous virus. Antibodies directed against the C3-V4 region were involved in autologous neutralization in all four sera studied. In two sera, transfer of the C3-V4 region rendered the chimera as sensitive to antibody neutralization as the parental virus. Although the C3 region, which contains the highly variable α2-helix was not a direct target in most cases, it contributed to the formation of neutralization epitopes as substitution of this region resulted in neutralization resistance. These data suggest that the C3 and V4 regions combine to form important structural motifs and that epitopes in this region are major targets of the early autologous neutralizing response in HIV-1 subtype C infection.     

Induction of antibodies in guinea pigs and rhesus monkeys against the human immunodeficiency virus type 1 envelope: neutralization of nonpathogenic and pathogenic primary isolate simian/human immunodeficiency virus strains

We have compared the abilities of human immunodeficiency virus type 1 (HIV-1) envelope V3 peptides and recombinant gp120 to induce antibodies that neutralize simian/human immunodeficiency viruses (SHIVs). SHIV-89.6 is a nonpathogenic SHIV that expresses the envelope protein of primary HIV-1 isolate 89.6. SHIV-89.6P, clone KB9, is a pathogenic SHIV variant derived from SHIV-89.6. Infection of rhesus monkeys with these SHIVs rarely induces anti-V3 region antibodies. To determine the availability of the gp120 V3 loop for neutralizing antibody binding on SHIV-89.6 and KB9 virions, we have constructed immunogenic C4-V3 peptides from these SHIVs and induced anti-V3 antibodies in guinea pigs and rhesus monkeys. We found that both SHIV-89.6 and KB9 C4-V3 peptides induced antibodies that neutralized SHIV-89.6 but that only SHIV-KB9 C4-V3 peptide induced antibodies that neutralized SHIV-KB9. Immunoprecipitation assays demonstrated that SHIV-KB9 C4-V3 peptide-induced antibodies had a greater ability to bind SHIV-KB9 envelope proteins than did antibodies raised against SHIV-89.6 C4-V3 peptide. We have used a series of mutant HIV-1 envelope constructs to map the gp120 determinants that affect neutralization by anti-V3 antibodies. The residue change at position 305 of arginine (in SHIV-89.6) to glutamic acid (in SHIV-KB9) played a central role in determining the ability of peptide-induced anti-V3 antiserum to neutralize primary isolate SHIVs. Moreover, residue changes in the SHIV-89.6 V1/V2 loops also played roles in regulating the availability of the V3 neutralizing epitope on SHIV-89.6 and -KB9. Thus, SHIV-89.6 and -KB9 V3 region peptides are capable of inducing neutralizing antibodies against these primary isolate SHIVs, although the pathogenic SHIV-KB9 is less easily neutralized than its nonpathogenic variant SHIV-89.6. In contrast to natural infection with SHIV-89.6, in which few animals make anti-V3 antibodies, C4-V3 peptides frequently induced anti-V3 antibodies that neutralized primary isolate SHIV strains.     

Synergistic neutralization of human immunodeficiency virus type 1 by a chimpanzee monoclonal antibody against the V2 domain of gp120 in combination with monoclonal antibodies against the V3 loop and the CD4-binding site.

Synergistic neutralization of human immunodeficiency virus type 1 (HIV-1) was observed in studies using a chimpanzee anti-V2 monoclonal antibody (MAb), C108G, in combination with anti-V3 loop and anti-CD4 binding-site (bs) MAbs of different epitope specificities. C108G paired with either of two anti-V3 loop MAbs or either of two anti-CD4 bs MAbs synergistically neutralized both the uncloned IIIB and clonal HXB2 strains of virus in H9 target cells. Synergism was quantitated by calculation of combination indices. Significant synergy with a given MAb pair was seen over a range of MAb ratios, with the optimal effect centering around the ratio at which the MAbs were equipotent for a given HIV-1 strain (on the basis of the 50% neutralization titer). In preliminary experiments with monocytotropic strains of HIV-1 in peripheral blood mononuclear cell targets, significant synergism was also observed between anti-V2-anti-V3 and anti-V2-anti-CD4 bs MAb pairs. Synergism by all MAb pairs tested was greater against heterogeneous isolates of HIV-1 (IIIB and Ba-L) than against clonal isolates (HXB2 and NLHXADA), suggesting that strain broadening may be a component of the synergism observed against the heterogeneous isolates. In addition, conformational changes in gp120 upon binding of one or both MAbs may result in increased affinity or exposure of the epitope of one or both MAbs. Finally, a three-MAb combination of C108G, an anti-V3 MAb, and an anti-CD4 bs MAb was more effective in neutralizing the HXB2 strain of HIV-1 than any of the three two-MAb combinations within this trio, as determined by the dose reduction indices of each MAb required to achieve a given level of neutralization. This is the first report of synergistic neutralization of HIV-1 by a three-MAb combination composed of MAbs directed against the three major neutralization epitope clusters in gp120. Implications for vaccine design and for immunoprophylaxis and immunotherapy with a combination of MAbs are discussed.     

Factors determining the breadth and potency of neutralization by V3-specific human monoclonal antibodies derived from subjects infected with clade A or clade B strains of human immunodeficiency virus type 1

The neutralizing activities of anti-V3 antibodies for HIV-1 isolates is affected both by sequence variation within V3 and by epitope masking by the V1/V2 domain. To analyze the relative contribution of V3 sequence variation, chimeric Env genes that contained consensus V3 sequences from seven HIV-1 subtypes in the neutralization-sensitive SF162 Env backbone were constructed. Resulting viral pseudotypes were tested for neutralization by 15 anti-V3 MAbs isolated from humans infected with viruses of either subtype B (anti-V3(B) MAbs) or subtype A (anti-V3(A) MAbs). Pseudovirions with the subtype B consensus V3 sequence were potently neutralized (IC(50) < 0.006 microg/ml) by all but one of these MAbs, while pseudovirions with V3 subtypes A, C, F, H, AG, and AE were generally neutralized more effectively by anti-V3(A) MAbs than by anti-V3(B) MAbs. A V1/V2-masked Env version of SF162 Env with the consensus B V3 sequence was also neutralized by these MAbs, although with considerably lower potency, while similarly masked chimeras with V3 sequences of subtype A, C, or AG were weakly neutralized by anti-V3(A) MAbs but not by anti-V3(B) MAbs. Mutations in the V1/V2 domain of YU-2 Env increased the sensitivity of this highly resistant Env to a pool of anti-V3(B) MAbs several thousand-fold. These results demonstrated (i) the exceptional sensitivity of representative V3 domains of multiple subtypes to neutralization in the absence of epitope masking, (ii) the broader neutralizing activity of anti-V3(A) MAbs for viruses containing diverse V3 sequences, and (iii) the generality and dominant effect of V1/V2 masking on restriction of V3-mediated neutralization.     

Conformational changes affecting the V3 and CD4-binding domains of human immunodeficiency virus type 1 gp120 associated with env processing and with binding of ligands to these sites.

Two neutralizing human monoclonal antibodies (HuMAbs) directed against epitopes located near the tip of the V3 loop of human immunodeficiency virus type 1 env protein recognized solubilized gPr160, but not gp120, in radioimmunoprecipitation assays. Efficient immunoprecipitation of solubilized gp120 by these antibodies did occur in the presence of HuMAb 1125H, directed against a conformational epitope overlapping the CD4-binding site, or its F(ab')2 fragment. In contrast to the inability of the anti-V3 antibodies to immunoprecipitate solubilized gp120, these HuMAbs did bind to gp120 in intact virions; this level of binding increased severalfold in the presence of the F(ab')2 fragment of 1125H. These results demonstrate that neutralization epitopes in the V3 loop are sequestered in soluble gp120 but partly exposed in gPr160 and in virion-associated gp120 and that binding of antibodies to the discontinuous CD4-binding site leads to conformational changes that result in the exposure of V3 epitopes in soluble gp120 and their enhanced accessibility in gPr160 and in virion-associated gp120. Enhanced binding of suboptimal concentrations of 1125H to soluble gp120 was also induced by the presence of an anti-V3 HuMAb, indicating the occurrence of reciprocal allosteric interactions between the V3 loop and the CD4-binding site. It is likely that these effects contribute to the synergistic neutralization of human immunodeficiency virus type 1 previously reported for antibodies directed against these two regions.     

Antibodies that are cross-reactive for human immunodeficiency virus type 1 clade a and clade B v3 domains are common in patient sera from Cameroon, but their neutralization activity is usually restricted by epitope masking

Sera from human immunodeficiency virus type 1 (HIV-1)-infected North American patients recognized a fusion protein expressing a V3 loop from a clade B primary isolate virus (JR-CSF) but not from a clade A primary isolate virus (92UG037.8), while most sera from Cameroonian patients recognized both fusion proteins. Competition studies of consensus V3 peptides demonstrated that the majority of the cross-reactive Cameroonian sera contained cross-reactive antibodies that reacted strongly with both V3 sequences. V3-specific antibodies purified from all six cross-reactive sera examined had potent neutralizing activity for virus pseudotyped with envelope proteins (Env) from SF162, a neutralization-sensitive clade B primary isolate. For four of these samples, neutralization of SF162 pseudotypes was blocked by both the clade A and clade B V3 fusion proteins, indicating that this activity was mediated by cross-reactive antibodies. In contrast, the V3-reactive antibodies from only one of these six sera had significant neutralizing activity against viruses pseudotyped with Envs from typically resistant clade B (JR-FL) or clade A (92UG037.8) primary isolates. However, the V3-reactive antibodies from these cross-reactive Cameroonian sera did neutralize virus pseudotyped with chimeric Envs containing the 92UG037.8 or JR-FL V3 sequence in Env backbones that did not express V1/V2 domain masking of V3 epitopes. These data indicated that Cameroonian sera frequently contain cross-clade reactive V3-directed antibodies and indicated that the typical inability of such antibodies to neutralize typical, resistant primary isolate Env pseudotypes was primarily due to indirect masking effects rather than to the absence of the target epitopes.     

Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization.     

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

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

Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1-infected individuals

A small proportion of HIV-infected individuals generate a neutralizing antibody (NAb) response of exceptional magnitude and breadth. A detailed analysis of the critical epitopes targeted by broadly neutralizing antibodies should help to define optimal targets for vaccine design. HIV-1-infected subjects with potent cross-reactive serum neutralizing antibodies were identified by assaying sera from 308 subjects against a multiclade panel of 12 "tier 2" viruses (4 each of subtypes A, B, and C). Various neutralizing epitope specificities were determined for the top 9 neutralizers, including clade A-, clade B-, clade C-, and clade A/C-infected donors, by using a comprehensive set of assays. In some subjects, neutralization breadth was mediated by two or more antibody specificities. Although antibodies to the gp41 membrane-proximal external region (MPER) were identified in some subjects, the subjects with the greatest neutralization breadth targeted gp120 epitopes, including the CD4 binding site, a glycan-containing quaternary epitope formed by the V2 and V3 loops, or an outer domain epitope containing a glycan at residue N332. The broadly reactive HIV-1 neutralization observed in some subjects is mediated by antibodies targeting several conserved regions on the HIV-1 envelope glycoprotein.     

Human immunodeficiency virus type 1 neutralization by plasma from B or F genotype infected individuals

Anti-human immunodeficiency virus type 1 (HIV-1) "binding antibodies" (antibodies capable of binding to synthetic peptides or proteins) occur throughout HIV-1 infection, are high-titered and highly cross-reactive, as confirmed in this study by analyzing plasma from B and F genotype HIV-1 infected individuals. Plasma from individuals infected with clade F HIV-1 displayed the most frequent cross-reactivity, in high titers, while Bbr plasma showed much higher specificity. Similarly, neutralization of a reference HIV-1 isolate (HIV-1 MN) was more frequently observed by plasma from F than B genotype infected individuals. No significant difference was seen in neutralization susceptibility of primary B, Bbr or F clade HIV-1 by plasma from individuals infected with the classical B (GPGR) or F HIV-1, but Bbr (GWGR) plasma were less likely to neutralize the F genotype primary HIV-1 isolates. The data indicate that both B and F genotype derived vaccines would be equally effective against B and F HIV-1 infection, with a slightly more probable effectiveness for F than B genotype. Although the Bbr variant appears to induce a much more specific humoral immune response, the susceptibility in neutralizing the Brazilian HIV-1 B genotype Bbr variant is similar to that observed with the classical B genotype HIV-1.