Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope

Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3_B-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.     

Analysis of HIV-1 subtype B third variable region peptide motifs for induction of neutralizing antibodies against HIV-1 primary isolates

The HIV-1 gp120 V3 loop is a potent inducer of neutralizing antibodies for T cell line adapted-HIV-1, but less so for primary isolates. We hypothesized that peptides representative of the diversity of natural HIV-1 V3 loop variants might capture elements of conserved higher order structures and so stimulate broadly reactive neutralizing antibodies. We designed a panel of 29 subtype B V3 sequences postulated to reflect the range of V3 diversity. These peptides were used to immunize guinea pigs. The most effective peptide (62.19) clustered around the subtype B consensus sequence and induced antibodies that reproducibly neutralized 31% of the subtype B HIV-1 primary isolates evaluated, but exhibited limited cross-neutralization of non-subtype B HIV-1 strains. Taken together, these data demonstrated that the limited neutralization profile of antibodies induced by optimal subtype B V3 motifs likely represents the maximum breadth of neutralization of subtype B HIV-1 primary isolates attainable by anti-V3 peptide antibodies.     

Improving on nature: focusing the immune response on the V3 loop

The conventional wisdom suggests that "constant" rather than "variable" regions of the HIV envelope (Env) glycoproteins would induce the most broadly reactive antibodies (Abs). However, of the several epitopes in the conserved regions of gp120 and gp41 that induce neutralizing Abs, all are well-protected by protein folding, glycosylation, and/or oligomerization of the Env proteins on the virus surface; most are only transiently exposed during the process of infection or are poorly immunogenic. In contrast, the third variable region (V3) of gp120 appears to be at least partially exposed during various stages of the infectious process, is immunogenic in essentially all HIV+ subjects, and is capable of inducing Abs able to neutralize a broad array of primary isolates. While these Abs were originally thought to be isolate-specific, a large body of data now shows that anti-V3 Abs from HIV-infected individuals indeed show intra- and inter-clade cross-reactivity with respect to both binding to diverse gp120 molecules and neutralization of many primary isolates. This cross-reactivity of anti-V3 Abs is counter-intuitive if one focuses on the sequence variability rather than on the conserved V3 structures which must be present in order to allow this region of the virus envelope to mediate selection of and interaction with chemokine receptors. Current data, summarized here, support the hypothesis that the V3 region of gp120 can induce broadly-reactive, cross-neutralizing Abs and as such should constitute a prominent target of the immune response induced with an HIV vaccine.     

Characterization of neutralizing sites in the second variable and fourth variable region in gp125 and a conserved region in gp36 of human immunodeficiency virus type 2.

Several determinants of human immunodeficiency virus (HIV) have been suggested to harbor sites important for neutralization. The third variable region (V3) of the envelope glycoprotein (gp) is an important neutralizing determinant for both serotypes of HIV. The localization of additional neutralizing regions is an urgent task because the virus appears to mutate to phenotypes that escape neutralizing antibodies. Therefore, we have focused on the possibility of finding other immunodominant regions in the envelope glycoproteins of human immunodeficiency virus type 2 (HIV-2). By immunization of guinea pigs with peptides corresponding to different selected regions of gp125 and gp36, we have found three antigenic determinants located in the V2 and V4 regions of the envelope protein gp125, and one region in the glycoprotein gp36, which are important for human antibody binding and also as targets for neutralization. The peptide representing the V2 region had the most pronounced capacity to induce neutralizing anti-HIV-2 antibodies in guinea pigs. Neutralizing activity was also detected in an antipeptide guinea pig sera representing a linear site in gp36, amino acids 644-658. A substitution set of peptides representing the conserved antigenic site in the central part of gp36 was used to identify the role of individual amino acids important for human antibody binding.     

Neutralization of HIV-1 primary isolates by polyclonal and monoclonal human antibodies

To examine antibody-mediated neutralization of HIV-1 primary isolates in vitro, we tested sera and plasma from infected individuals against four clade B primary isolates. These isolates were analyzed further for neutralization by a panel of several human anti-HIV-1 mAb in order to identify the neutralizing epitopes of these viruses. Each of the HIV-1+ serum and plasma specimens tested had neutralizing activities against one or more of the four primary isolates. Of the three individual sera, one (FDA-2) neutralized all of the four isolates, while the other two sera were effective against only one virus. The pooled plasma and serum samples reacted broadly with these isolates. Based on the neutralizing activities of the mAb panel, each virus isolate exhibited a distinct pattern of reactivity, suggesting antigenic diversity among clade B viruses. Neutralizing epitopes were found in the V3 loop and CD4- binding domain of gp120, as well as near the transmembrane region (cluster II epitope) of gp41. A mAb directed to the cluster I epitope of gp41 near the immunodominant disulfide loop weakly neutralized one primary isolate. None of the mAb in the panel affected one primary isolate, US4, although this virus was sensitive to neutralization by some of the polyclonal antibody specimens. This isolate was also resistant to neutralization by a cocktail of 10 mAb, most of which individually inhibited at least one of the other three viruses tested. These results suggest that neutralizing activity for this latter virus is present in certain HIV-1+ sera/plasma, but is not exhibited by the mAb in the panel. Thus, effective neutralizing antibodies against primary isolates can be generated by humans upon exposure to HIV-1, but not all of these antigenic specificities are represented in a large panel of human anti-HIV-1 mAb.      

Focusing the immune response on the V3 loop, a neutralizing epitope of the HIV-1 gp120 envelope

Rabbits were immunized with a novel regimen designed to focus the immune response on a single neutralizing epitope of HIV-1 gp120 and thereby preferentially induce neutralizing antibodies (Abs). Animals were primed with gp120 DNA from a clade A Env bearing the GPGR V3 motif and/or a clade C Env bearing the GPGQ V3 motif, and boosted with one or more fusion proteins containing V3 sequences from clades A, B and/or C. Immune sera neutralized three of four Tier 1 primary isolates, including strains heterologous to the immunizing strains, and potent cross-clade-neutralizing activity was demonstrated against V3 chimeric pseudoviruses carrying in a Tier 1 Env, the consensus V3 sequences from clades A1, AG, B, AE, or F. The broadest and most potent neutralizing responses were elicited with the clade C gp120 DNA and a combination of V3-fusion proteins from clades A, B and C. Neutralizing activity was primarily due to V3-specific Abs. The results demonstrate that the immune response can be focused on a neutralizing epitope and show that the anti-V3 Abs induced recognize a diverse set of V3 loops.     

A broad range of mutations in HIV-1 neutralizing human monoclonal antibodies specific for V2, V3, and the CD4 binding site

The HIV vaccine-induced neutralizing antibodies (Abs) display low rates of mutation in their variable regions. To determine the range of neutralization mediated by similar human monoclonal Abs (mAbs) but derived from unselected chronically HIV-1 infected subjects, we tested a panel of 66 mAbs specific to V3, CD4 binding site (CD4bs) and V2 regions. The mAbs were tested against 41 pseudoviruses, including 15 tier 1 and 26 tier 2, 3 viruses, showing that the neutralization potency and breadth of anti-V3 mAbs were significantly higher than those of the anti-CD4bs and anti-V2 mAbs, and only anti-V3 mAbs were able to neutralize some tier 2, 3 viruses. The percentage of mutations in the variable regions of the heavy (VH) and light (VL) chains varied broadly in a range from 2% to 18% and correlated moderately with the neutralization breadth of tier 2, 3 viruses. There was no correlation with neutralization of tier 1 viruses as some mAbs with low and high percentages of mutations neutralized the same number of viruses. The electrostatic interactions between anti-V3 mAbs and the charged V3 region may contribute to their neutralization because the isoelectric points of the VH CDR3 of 48 anti-V3 mAbs were inversely correlated with the neutralization breadth of tier 2, 3 viruses. The results demonstrate that infection-induced antibodies to CD4bs, V3 and V2 regions can mediate cross-clade neutralization despite low levels of mutations which can be achieved by HIV-1 vaccine-induced antibodies.     

Analysis of the neutralizing antibody response elicited in rabbits by repeated inoculation with trimeric HIV-1 envelope glycoproteins

The elicitation of broadly neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, gp120 and gp41, remains a major challenge. Attempts to utilize monomeric gp120 as an immunogen to elicit high titers of neutralizing antibodies have been disappointing. Envelope glycoprotein constructs that better reflect the trimeric structure of the functional envelope spike have exhibited improved immunogenicity compared with monomeric gp120. We have described soluble gp140 ectodomain constructs with a heterologous trimerization motif; these have previously been shown to elicit antibodies in mice that were able to neutralize a number of HIV-1 isolates, among them primary isolate viruses. Recently, solid-phase proteoliposomes retaining the envelope glycoproteins as trimeric spikes in a physiologic membrane setting have been described. Here, we compare the immunogenic properties of these two trimeric envelope glycoprotein formulations and monomeric gp120 in rabbits. Both trimeric envelope glycoprotein preparations generated neutralizing antibodies more effectively than gp120. In contrast to monomeric gp120, the trimeric envelope glycoproteins elicited neutralizing antibodies with some breadth of neutralization. Furthermore, repeated boosting with the soluble trimeric formulations resulted in an increase in potency that allowed neutralization of a subset of neutralization-resistant HIV-1 primary isolates. We demonstrate that the neutralization is concentration-dependent, is mediated by serum IgG and that the major portion of the neutralizing activity is not directed against the gp120 V3 loop. Thus, mimics of the trimeric envelope glycoprotein spike described here elicit HIV-1-neutralizing antibodies that could contribute to a protective immune response and provide platforms for further modifications to improve the efficiency of this process.     

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1).

Antibody mediated and cell mediated immune responses to the envelope glycoproteins gp120 and gp41 of the human immunodeficiency virus (HIV-1) are considered important for protection against infection and for attenuation of disease symptoms after infection. Virus neutralizing antibodies are mostly subtype specific and primarily directed against epitopes on a hypervariable loop from the V3 region of HIV-1 gp120. Such epitopes are recognized by helper and cytotoxic T-cells suggesting that all protective immune responses to HIV-1 are predominantly subtype specific. The extraordinary primary sequence variability of gp120 indicates that a combination of subtype specific components will be required to design a broadly effective protective immunogen against HIV-1. Peptides from hypervariable loops of the V3 region of 21 distinct HIV-1 isolates (clones) were synthesized and used to raise rabbit antisera. The antisera contained high levels of antibodies recognizing the homologous peptides and the parent gp120 sequence. The serological cross-reactivity between the distinct peptides was evaluated and related to amino acid divergence. The corresponding relationship approximated a linear regression with a correlation coefficient r = 0.718. The 21 peptides were combined into a single immunogen which elicited broadly reactive antibodies recognizing all 21 peptides as well as gp120 from the only isolate tested, HIV-1 IIIB. The results suggest the possibility of developing broadly protective HIV-1 immunogens by combining judiciously selected subtype specific peptides derived from envelope glycoproteins of divergent virus isolates.     

Structure-based,targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization.     

HIV-specific lymphoproliferative responses in asymptomatic HIV-infected individuals.

During HIV-1 infection, the current tenet is that only anti-gag antibodies decline, while those directed at env remain stable. Among the latter antibodies, those directed to the V3 domain of gp120 are assumed to play a role in the immune surveillance against HIV-1. We investigated the correlation between anti-V3 antibody levels and the clinical stage of infection and the ability to neutralize syncytium formation. Using a V3-specific antigen-limited ELISA, we analysed the antibody levels of a panel of 93 HIV-1+ sera to V3 peptides derived from different HIV-1 strains and from the North American/European consensus sequence V3(Cs). Sera preferentially recognized V3 peptides from the representative V3(MN) strain and V3(Cs). Antibody reactivity to V3(MN) or V3(Cs) actually declined in relation with progression to AIDS, while antibodies against whole recombinant gp160 or gp41 immunodominant epitope remained stable. There was a strong correlation (P < 0.0001) between anti-V3 (Cs)/V3(MN) antibody levels and serum titres that neutralized HIV-1MN-mediated syncytia. Serology based on V3-specific antigen-limited ELISA indicates that anti-V3 antibody reactivity may decline during the course of HIV infection.     

Maternal antibodies to HIV-1 envelope domains: No correlation with HIV-1 vertical transmission in patients from Argentina

The role of the maternal antibody response in relation to vertical human immunodeficiency virus type 1 (HIV-1) transmission was investigated in HIV-1-infected mothers from Argentina. Sera from 23 transmitting and 18 nontransmitting HIV-1-infected mothers were tested for the presence of antibodies to V3 loop gp120 peptides representing both Argentinian sequences and several well-characterized viral isolates from different geographic areas. Argentinian sera from transmitting mothers had significantly higher capacity to react with four of 14 V3 loop peptides tested than sera from nontransmitting mothers. Frequency of reactivity against the other peptides did not differ between the two maternal groups. Furthermore, no differences in antibody affinity were found between transmitting and nontransmitting mothers. Sera were also tested against overlapping peptides covering a neutralizing epitope of the HIV-1 MN gp41 (amino acids 648-677). Statistical analysis indicated that no correlation between anti-gp41 antibodies and vertical transmission exists. Although we used V3 loop peptides based on local HIV-1 sequences, our data showed that maternal antibodies to these peptides, as well as to gp41 peptides, are not correlated with protection against HIV-1 vertical transmission.     

Identification of amino acids in the V3 region of gp120 critical for virus neutralization by human HIV-1-specific antibodies.

The importance of the dependence on single amino acids in the V3 region of HIV-1 gp120 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-1-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81%) reacted with amino acids 304-318, compared with only one of eight sera (13%) negative in neutralization. When any of the four amino acids G, P, G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gp120 is critical for virus neutralization by human HIV-1-specific antibodies.     

Ex vivo neutralization of HIV-1 quasi-species by a broadly reactive humanized monoclonal antibody KD-247

By immunizing mice sequentially with six different V3 peptides we obtained a murine monoclonal antibody (MAb) C25, and its humanized counterpart KD-247. The MAb recognizes the sequence IGPGRA at the tip of the V3 loop and displays broad neutralizing activity against a variety of HIV-1 isolates. KD-247 was tested in an ex vivo neutralization assay to determine its capability to contain the spread of a quasi species population of clade B HIV-1 derived from two patients. The epitope of KD-247 was generally matching with the V3 sequences of various clones isolated from plasma and peripheral blood mononuclear cells (PBMC) of two patients. Complete or strong inhibition of viral replication was observed when the patients' PBMC were cultured with a high concentration of KD-247. Some neutralization escape variants, which had mutations in the V3 or outside of the V3 loop, emerged only at a low concentration of the MAb. These results suggest that KD-247 could be a good candidate for immunotherapy against HIV-1 in vivo.     

An optimally constrained V3 peptide is a better immunogen than its linear homolog or HIV-1 gp120

Synthetic peptides offer an attractive option for development of a V3-directed vaccine. However, immunization with flexible linear peptides may result in an immune response to multiple conformations, many of which differ from the native conformation of the corresponding region in the protein. Here we show that optimization of the location of a disulfide bond in peptides constrained to mimic the β-hairpin conformation of the V3, yields an immunogen that elicits a 30-fold stronger HIV-1 neutralizing response in rabbits compared with the homologous linear V3 peptide. The HIV-1 neutralizing response elicited by the optimally constrained peptide is also significantly stronger than that elicited by a gp120 construct in which the V3 is exposed. Neutralization of an HIV-1 strain that shares only 72% identity with the immunizing peptide was demonstrated. The most effective immunogen was also able to neutralize primary isolates that are more resistant to neutralization such as SS1196 and 6535.     

Genetic variability of the V1 and V2 env domains of SIVcpz-ant and neutralization pattern of plasma viruses in a chimpanzee infected naturally.

Specific neutralizing epitope changes have been observed in a chimpanzee infected naturally with SIVcpz, which differ from HIV-1 infecting humans. To characterize further these changes, a longitudinal study of env genomic sequence variation of SIVcpz-ant isolates was undertaken in this animal. The V1 and V2 regions of the env were determined to arise from specific recombination events. To determine whether recombination of the V1 and V2 domains was possibly associated with the emergence of neutralization escape viruses, envelope sequences and gene length polymorphisms from PBMC and plasma viral variants were studied over a 7-year period. PBMCs and plasma-associated infectious virus titers as well as plasma RNA viral loads were monitored longitudinally. The first 5 viruses isolated from the plasma were found to be neutralization escape variants. Sequence analysis of their V1 and the V2 regions indicated that a 20 amino acid stretch of the V1 region had undergone recombination and was also associated with the emergence of isolates eliciting strong neutralization responses. These findings support the hypothesis that recombination of the V1 and V2 regions of the envelope play a role in neutralization escape of SIVcpz in chimpanzees infected naturally. Furthermore, the data confirm that the neutralizing antibody response plays an important role in the decline of plasma infectious virus titers in HIV-1 related SIVcpz nonpathogenic infection.     

Functional and immunochemical cross-reactivity of V2-specific monoclonal antibodies from HIV-1-infected individuals

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.     

Comparative studies on neutralisation of primary HIV-1 isolates by human sera and rabbit anti-V3 peptide sera.

IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.     

Antigenicity and Predefined Specificities of the Multi-Epitope Vaccine in Candidate Consisting of Neutralizing Epitope and Mutated Epitopes Suggested a New Way against HIV-1 Mutation

A seven-amino acid epitope GPGRAFY located inside the V3 loop on envelope protein gp120 of HIV-1 is the principal neutralizing epitope (PNE), and a subset of anti-V3 antibodies specific for this epitope show a broad range of neutralizing activity. But this epitope undergoes restricted mutation. In this study, three epitope peptides [C-(GPGRAFY)2, C-(GPGQTFY)2 and C-(GPGQAWY)2] that contain neutralizing epitope GPGRAFY and its two mutated epitope GPGQTFY and GPGQAWY, were synthesized and then conjugated to carrier protein KLH (keyhole limpet hemocyanin). the epitope-vaccines C-(GPGRAFY)2-KLH, C-(GPGQTFY)2-KLH and C-(GPGQAWY)2-KLH induced high levels of antibodies to three V3 loop peptides that contain these epitopes respectively, and the antibody response induced by each epitope-vaccine showed predefined epitope-specific. When these three epitope-peptides mixed together and conjugated to carrier protein, or conjugated to carrier protein separately and then mixed together, high levels of epitope-specific antibodies which respectively recognized these epitopes on V3 loop peptide and both mutated peptides all can be induced by both of them. In blotting assay, these epitope-specific antibodies all recognized the neutralizing epitope and mutated epitopes on peptides respectively. In addition, the reactivity of the antibodies with whole gp120 molecule which contained the epitope GPGRAFY was tested. Only the GPGRAFY-epitope-specific antibodies but not the other antibodies recognized the gp120 molecule. These results provide experimental evidence that the candidate multi-epitope-vaccine containing neutralizing epitope and mutated epitopes may bring new hope against viral mutation resulting in HIV-1 immune evasion and may be developed as an effective vaccine with a broad neutralizing activity against HIV-1 infection.     

A single amino acid substitution in the C4 region in gp120 confers enhanced neutralization of HIV-1 by modulating CD4 binding sites and V3 loop

Identification of vulnerability in the HIV-1 envelope (Env) will aid in Env-based vaccine design. We recently found an HIV-1 clade C Env clone (4-2.J45) amplified from a recently infected Indian patient showing exceptional neutralization sensitivity to autologous plasma in contrast to other autologous Envs obtained at the same time point. By constructing chimeric Envs and fine mapping between sensitive and resistant Env clones, we found that substitution of highly conserved isoleucine (I) with methionine (M) (ATA to ATG) at position 424 in the C4 domain conferred enhanced neutralization sensitivity of Env-pseudotyped viruses to autologous and heterologous plasma antibodies. When tested against monoclonal antibodies targeting different sites in gp120 and gp41, Envs expressing M424 showed significant sensitivity to anti-V3 monoclonal antibodies and modestly to sCD4 and b12. Substitution of I424M in unrelated Envs also showed similar neutralization phenotype, indicating that M424 in C4 region induces exposure of neutralizing epitopes particularly in CD4 binding sites and V3 loop.