HIV-1 in Ethiopia: Phylogenetic divergence from other HIV-1 strains

Phylogenetic tree analysis was performed on selected polymerase chain reaction (PCR)-amplified and sequenced regions of the gag and env reading frames of several Ethiopian and Swedish human immunodeficiency virus type 1 (HIV-1) strains. These regions are considered to be conserved parts of the HIV-1 genome and correspond to the p7 of the core (gag) and part of the carboxy terminal of the gp41 protein of env respectively. Comparisons were made with all available HIV-1 sequences.The tree analysis showed that gag sequences from nine and env sequences from four Ethiopian strains all grouped together in separate branches distinct from all other sequenced European, North American, and African HIV-1 isolates. Thus, the Ethiopian strains seem to represent a highly divergent group of HIV-1, which might have developed during a relatively early stage of HIV-1 evolution.     

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.     

Comparing modified and plain peptide linked enzyme immunosorbent assay (ELISA) for detection of human immunodeficiency virus type-1 (HIV-1) and type-2 (HIV-2) antibodies

Serological diagnosis of human immunodeficiency virus (HIV) based on detection of HIV antibodies is one of the easiest, cheapest and simplest assay. Synthetic peptides corresponding to immunodominant regions of envelope glycoprotein (gp41, V3 loop for HIV-1 and gp36 for HIV-2) were used in the present study, to detect the anti-HIV antibodies in sera of Sexually Transmitted Diseases (STD), Tuberculosis (TB), Anti-Natal Care (ANC) patients. About 550 serum samples were tested using Enzyme Linked Immunosorbent Assay (ELISA) technique. The human sera positive for antibody to HIV-1 and HIV-2, reacted to different degrees with these peptides when used as a plain peptide with or without CGG motif/biotin motif at the amino terminus. The selected sequences are of Indian strain with 'C' serotype. The results showed a 100% sensitivity and specificity for V3 loop peptide and 98% sensitivity and specificity for gp41 peptide containing CGG moiety while the plain peptides showed similar sensitivities but low specificity's, i.e. 98% for V3 loop peptide and 42% for gp41 peptide when reacted with HIV-1 positive sera. The presence of biotin at the amino terminus did not provide any beneficial effect in increasing the sensitivity although the specificity was enhanced for both the peptide sequences, i.e. gp41 and V3 loop peptide. Furthermore, the gp36 peptide containing CGG moiety detected the HIV-2 sera with 100% sensitivity and 98% specificity while the sensitivity and specificity of gp36 plain peptide was reduced to 98 and 90%. Thus the study overall highlighted the importance of synthetic peptides containing CGG moiety as a capture antigen in detecting both HIV-1 & 2 sera using an indigenously built ELISA system which is simple, cheap, sensitive and cost effective for rural areas.     

Prolonged Infection in Rhesus Macaques with Simian Immunodeficiency Virus (SIVmac239) Results in Animal-Specific and Rarely Tissue-Specific Selection ofnefVariants

We analyzed the sequence ofnefgenes from different tissues of three rhesus macaques that had been infected with molecularly cloned SIV_mac239 for 88 to 92 weeks. Comparison of the predicted amino acid sequences revealed that each macaque had selected out specific amino acid substitutions and that most of this variation (70%) was confined to four regions, amino acids 39 to 75, 90 to 105, 153 to 167, and 191 to 217, comprising 36% of the protein. Thenefgenes in these animals underwent extensive genetic variation with average nucleotide and amino acid substitution rates varying from 0.86 to 2.84% and 2.47 to 6.27%, respectively, although tissue-specific selection ofnefvariants occurred in only 1 of 14 tissues examined in this study. Comparison of the rate of nucleotide and amino acid substitutions in thenefgenes to those previously reported in theenvin the central nervous system (CNS) and lymph node (LN) revealed that the predicted amino acid substitution rates for Nef were much higher than for the gp120 region ofenvin the CNS and LN tissues for one macaque. In the two other macaques, the predicted amino acid substitution rates were similar between these two proteins in LN tissues, but the amino acid substitution rates in Nef were significantly higher than in the gp120 from the CNS. Comparison of the nucleotide substitutions in the region of overlap between theenvand thenefrevealed that approximately 83% of the nucleotide substitutions in this area resulted in a Nef amino acid sequence change, 26% of the nucleotide substitutions resulted in a gp41 amino acid change, and 9.5% of nucleotide substitutions resulted in amino acid sequence changes in both proteins, suggesting a preference for the selection of amino acid substitutions in the Nef in these animals. Our results indicate that in animals infected with SIV_mac239 for prolonged periods, variation in thenefoccurs at rates similar to or exceeding that observed for theenvgene.     

Comparison and fine mapping of both high and low neutralizing monoclonal antibodies against the principal neutralization domain of HIV-1

Summary Monoclonal antibodies raised against viral lysate of HIV-1 (strain LAV-1) and against recombinant gp 160 of HIV-1 (strain HTLV IIIB) which neutralized HIV-1 in a type specific manner were mapped with the aid of peptides (Pepscan analysis). Each of these monoclonal antibodies bound to peptides located on the principal neutralizing domain (PND) of HIV-1. We found that the antigenic sites of the MAbs described in this paper are represented by linear peptides of at least 10 amino acids long. The affinity of the MAbs is high for these peptides and in the same order of magnitude as for native gp 160. The fine mapping of the epitopes may reflect structural features of the PND, for instance which amino acid side chains are exposed and which are buried in the protein. Furthermore the fine mapping of the epitopes explained the HIV type-specific neutralizing activity of the MAbs. Antibodies that bound to the tip of the loop (amino acids QRGPGRAF) have a higher neutralizing activity than antibodies that bound to amino acids towards the N-terminal side of the loop (amino acids KSIRI). Furthermore, MAbs that bound to virtually the same amino acids on the tip of the loop (amino acids IQRGPGRAF and RGPGRAFV) had different neutralizing activities due to different affinities for native gp 160. These data reveal that neutralizing activity not only is determined by the affinity of an antibody to the neutralizing site but also by its fine binding specificities to the V 3 loop of gp 120.     

5株不同亚型HIV-1毒株gp120序列特征分析及其蛋白的表达

目的 分析我国HIV-1主要亚型毒株的gp120区域的序列特征,并表达出gp120糖基化蛋白,为研究gp120的致病机制和设计疫苗奠定基础.方法 利用巢式PCR从来自于不同省份的5名HIV-1感染者外周血单个核细胞DNA中扩增全长gp120基因并测序,对序列特征进行分析,并将获得的gp120基因克隆入真核表达载体,体外表达获得gp120糖基化蛋白.结果 序列分析显示,此5条gp120序列分属HIV-1 Thai-B、BC重组和AE重组哑型,虽然亚型不同,但这些gp120序列在相同的位置都存在着一些保守的糖基化位点,且都具备相同的Furin蛋白酶第一切割位点,与参考序列比较发现,不同的HIV亚型序列中,除V3序列长度较为保守外,V1、V2、V4、V5各区域都有不同程度的缺失现象,与BC重组和AE重组亚型相比,Thai-B亚型V3的顶端环呈现出多种组合.最终将这5条gpl20序列都克隆人真核表达载体并表达出糖基化的gp120蛋白.结论 在设计疫苗和检测试剂时应考虑到膜区的高变性,gp120糖蛋白的体外表达有利于进一步开展针对我国主要HIV流行毒株膜蛋白致病机制和疫苗学的研究.     

Analysis of genetic variability within the immunodominant epitopes of envelope gp41 from human immunodeficiency virus type 1 (HIV-1) group M and its impact on HIV-1 antibody detection

The serodiagnosis of human immunodeficiency virus type 1 (HIV-1) infection primarily relies on the detection of antibodies, most of which are directed against the immunodominant regions (IDR) of HIV-1 structural proteins. Among these, the N-terminal region of gp41 contains cluster I (amino acids [aa] 580 to 623), comprising the cytotoxic T-lymphocyte epitope (AVERYLKDQQLL) and the cysteine loop (CSGKLIC), and cluster II (aa 646 to 682), comprising an ectodomain region (ELDKWA). To delineate the epitope diversity within clusters I and II and to determine whether the diversity affects serologic detection by U.S. Food and Drug Administration (FDA)-licensed enzyme immunoassay (EIA) kits, gp41 Env sequences from 247 seropositive persons infected with HIV-1 group M, subtypes A (n = 42), B (n = 62), B' (n = 13), C (n = 38), D (n = 41), E (n = 18), F (n = 27), and G (n = 6), and 6 HIV-1-infected but persistently seronegative (HIPS) persons were analyzed. While all IDR were highly conserved among both seropositive and HIPS persons, minor amino acid substitutions (<20% for any one residue, mostly conservative) were observed for all subtypes, except for B', in comparison with the consensus sequence for each subtype. Most importantly, none of the observed substitutions among the group M plasma specimens affected antibody detection, since all specimens (n = 152) tested positive with all five FDA-licensed EIA kits. Furthermore, all specimens reacted with a group M consensus gp41 peptide (WGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASW), and high degrees of cross-reactivity (>80%) were observed with an HIV-1 group N peptide, an HIV-1 group O peptide, and a peptide derived from the homologous region of gp41 from simian immunodeficiency virus from chimpanzee (SIVcpz). Taken together, these data indicate that the minor substitutions observed within the IDR of gp41 of HIV-1 group M subtypes do not affect antibody recognition and that all HIV-1-seropositive specimens containing the observed substitutions react with the FDA-licensed EIA kits regardless of viral genotype and geographic origin.     

Molecular analysis of the proviral DNA of equine infectious anemia virus in mules in Greece

Molecular analysis of the regulatory and structurally important genetic segments of equine infectious anemia virus (EIAV) in mules is presented. We have previously reported clinicopathological and laboratory findings in mules infected with EIAV, both naturally and after experimental inoculation. In this study the fragment coding for integrase, gp90, tat and the fusion domain of gp45 of the proviral genome from these animals was sequenced and compared with one another and with that of EIAV strains already published in the literature. Significant variations were observed mainly in the sequences of the gp90 surface protein. In the two wild type sequences, there were substitutions in the V5 hypervariable domain of this protein. In the sequences of the experimentally inoculated animals and the donor strain, variations were due to insertions/duplications in the V3 principal neutralizing domain (PND) and substitutions in the V5 hypervariable domain. Finally, when compared with the already published strains, the wild type sequences had single amino acid substitutions across the whole protein and multiple substitutions in the V4-V6 variable domains. In general, the two Greek wild type sequences were closer to two of the American strains (WSU5 and Massachusetts), than to the two Japanese (V26 and V70) or the third American strain (Wyoming_wi) used in this study.     

Selected amino acid changes in HIV-1 subtype-C gp41 are associated with specific gp120(V3) signatures in the regulation of co-receptor usage

The majority of studies have characterized the tropism of HIV-1 subtype-B isolates, but little is known about the determinants of tropism in other subtypes. So, the goal of the present study was to genetically characterize the envelope of viral proteins in terms of co-receptor usage by analyzing 356 full-length env sequences derived from HIV-1 subtype-C infected individuals. The co-receptor usage of V3 sequences was inferred by using the Geno2Pheno and PSSM algorithms, and also analyzed to the "11/25 rule". All reported env sequences were also analyzed with regard to N-linked glycosylation sites, net charge and hydrophilicity, as well as the binomial correlation phi coefficient to assess covariation among gp120(V3) and gp41 signatures and the average linkage hierarchical agglomerative clustering were also performed. Among env sequences present in Los Alamos Database, 255 and 101 sequences predicted as CCR5 and CXCR4 were selected, respectively. The classical V3 signatures at positions 11 and 25, and other specific V3 and gp41 amino acid changes were found statistically associated with different co-receptor usage. Furthermore, several statistically significant associations between V3 and gp41 signatures were also observed. The dendrogram topology showed a cluster associated with CCR5-usage composed by five gp41 mutated positions, A22V, R133M, E136G, N140L, and N166Q that clustered with T2V(V3) and G24T(V3) (bootstrap=1). Conversely, a heterogeneous cluster with CXCR4-usage, involving S11GR(V3), 13-14insIG/LG(V3), P16RQ(V3), Q18KR(V3), F20ILV(V3), D25KRQ(V3), Q32KR(V3) along with A30T(gp41), S107N(gp41), D148E(gp41), A189S(gp41) was identified (bootstrap=0.86). Our results show that as observed for HIV-1 subtype-B, also in subtype-C specific and different gp41 and gp120V3 amino acid changes are associated individually or together with CXCR4 and/or CCR5 usage. These findings strengthen previous observations that determinants of tropism may also reside in the gp41 protein.     

Molecular and structural characterization of HIV-1 subtype B Brazilian isolates with GWGR tetramer at the tip of the V3-loop

One of most intriguing features of the HIV-1 subtype B epidemic in Brazil is the high frequency of isolates exhibiting tryptophan (W) in the tetramer (GWGR) at the tip of the V3 loop. We observed that the frequencies of glutamic and aspartic acids at site 25 of the V3 loop are quite distinct in GWGR isolates compared with viruses with other tetramers. The basic amino acids at sites 11 and 25 of V3 are strongly linked with CCR5-to-CXCR4 coreceptor shift. We therefore predicted phenotype usage and found that GWGR isolates are exclusively CCR5-using. Further evidence of this came from intrahost sequences, where basic amino acid substitutions at sites 11 and 25 emerged only in isolates presenting a tryptophan-to-glycine replacement at the tetramer of the V3. In addition, modeled 3D-structures of the V3 loop of GWGR and GGGR in intrahost viruses differ essentially in the binding region of the coreceptor.     

Antibodies of HIV-1 positive subjects and experimentally immunized primates and rodents bind to sequence divergent regions of the third variable domain (V3) of gp120.

Several motifs have been found to be the target of the neutralizing antibody response to HIV, the human immunodeficiency virus. One of the well characterized motifs maps to a loop within the third hypervariable region (V3) of the exterior envelope glycoprotein gp120 at amino acid positions 308-331 and is referred to as the principal neutralizing determinant (PND). The sequence of this V3 loop raises the question of the immunogenicity and the degree of diversity of the antibody response to the PND. We show here that this neutralization-related motif is highly immunogenic in HIV-positive subjects and in experimentally immunized primates and rodents submitted to various anti-HIV immunization regimens. In probing the diversity of the antibody response to PNDs corresponding to 11 HIV sequence-divergent isolates in serum samples of 101 HIV-positive individuals we found that human antibodies exhibit binding affinity to up to nine PND synthetic peptides. This antibody binding was in all cases tested inhibitable by the homologous PND synthetic peptide. We additionally demonstrate that this antibody cross-reactivity towards sequence-divergent PNDs is detectable in the sera of mice and chimpanzees experimentally immunized against a single HIV-1 isolate. Finally, we noticed that there is a hierarchy of reactivity among the various PNDs wherein the synthetic peptide corresponding to the MN isolate was generally the most prominently recognized by antibodies of human, non-human primate, and rodent origins. Based on these findings and on features of the sequences analyzed we suggest that, despite its overall sequence variability, the PND encompasses conserved amino acid positions or epitopes that are the targets of antibodies recognizing sequence-divergent isolates. We also propose that the high positive charge density of the most frequently recognized PNDs and the high antigenicity value of some of their residues are critical to the broad immunoreactivity of this neutralization-related motif.     

Fine mapping of an immunodominant region of the transmembrane protein of the human immunodeficiency virus (HIV-1)

Peptides of HIV sequences are significant for antibody screening systems, and because of the limited number of amino acids they have to represent immunodominant regions of the virus proteins in order to maintain sensitivity. We detected, in a region of the outside loop of the transmembrane protein gp41 of the human immunodeficiency virus HIV-1 (amino acid 586-620), two immunodominant sequences which are distinct from each other. Whereas in the first immunodominant region the sensitivity and specificity of ELISA were inadequate, a neighbouring region is well suited for use as antigen for an anti-HIV screening ELISA.     

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.     

Amino Acid Substitutions in the V3 Loop Are Responsible for Adaptation to Growth in Transformed T-Cell Lines of a Primary Human Immunodeficiency Virus Type 1

A T-cell-line-tropic, syncytium-inducing, sCD4- and serum neutralization-sensitive variant (R3H) of the macrophage-tropic, non-syncytium-inducing, sCD4^- and serum neutralization-resistant molecular clone HIV-1_SF162 was obtained by passage through the T-cell line HUT 78. Sequence analyses of the V1-V5 regions of envelope gp120 of the variant R3H revealed amino acid substitutions in the V3 (amino acids 307, 312) and V4 (amino acid 390) domains. Site-directed mutagenesis of the HIV-1_SF162 genome showed that specific mutations in the V3 loop of this isolate can alter tropism and cytopathicity of the virus, but only moderately affect its sensitivity to sCD4 and serum neutralization. These results show that adaptation to growth in T-cell lines can render a primary-like virus sensitive to sCD4 and serum neutralization. However, the extent of T-cell line tropism does not correlate with the degree of susceptibility to sCD4 and serum neutralization. The latter appears to be dependent on the amino acid compositions of the V3 loop and other regions of envelope gp120, whereas the former is primarily determined by the V3 loop. Our findings further illustrate the importance of the V3 loop in influencing HIV-1 cell tropism and syncytium formation, and the interplay among V3 loop residues in maintaining a structure of the loop that influences biological phenotype of the virus.     

Sequence variation and consensus sequence of V3 loop on HIV-1 gp120

Mutation in the V3 loop of HIV-1 gp120 could affect syncytium formation, virus infectivity and neutralization. To acquire more information of the V3 loop mutation, we analyzed amino acid sequences of the V3 loop of 24504 isolates from most HIV-1 clades (including A, B, C, D, E, F, G and H clades). The consensus sequence of the V3 loop of each subtype with the highest frequency emerging on each position is constituted and the conservation of each amino acid in this region is also calculated. Exploring the restricted mutation of the V3 region could help to understand mechanism of HIV entry and to develop new strategy against HIV-1.     

Molecular subtyping of the HIV-1 V3 loop sequences detected in HIV-1-positive patients in southern Taiwan

Polymerase chain reaction and nucleotide sequence analysis were performed to amplify and determine the V3 loop sequences of human immunodeficiency virus type 1 (HIV-1) from ten seropositive patients at National Cheng Kung University Hospital, Tainan. The nucleotide sequences and the deduced amino acid (a. a.) sequences of these V3 regions were compared with those of known HIV-1 prototypes. The V3 loop a. a. sequences detected in eight individuals belong to subtype B which predominates in North America and Europe, whereas two individuals were infected with HIV-1 subtype E which is mainly found in the heterosexual populations of Thailand. Sequence analysis of these variant HIV-1 strains revealed a number of interesting features and a phylogenetic tree was also constructed according to the V3 loop nucleotide sequences of these variant strains and HIV-1 isolates from other parts of the world. Furthermore, our results suggest that the north vs south geographical separation in terms of HIV-1 epidemiology in Taiwan is insignificant.     

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.     

Genotypic changes in human immunodeficiency virus type 1 envelope glycoproteins on treatment with the fusion inhibitor enfuvirtide and their influence on changes in drug susceptibility in vitro.

BACKGROUND: Previous studies have established the importance of substitutions at amino acids 36-45 of HIV-1 gp41 in the development of viral resistance to the peptide fusion inhibitor enfuvirtide. However, the influence of other loci in the HIV-1 envelope is not well established. OBJECTIVE: To identify positions showing genotypic changes that are associated with particularly high levels of changes in enfuvirtide susceptibility. STUDY DESIGN: We examined full-length baseline and on treatment sequences of gp120 and gp41 for isolates from 369 patients in Phase III studies of enfuvirtide, including 281 patients receiving ENF+OB and 88 patients receiving OB alone. Individual changes in gp41 and gp120 were evaluated for correlations with on treatment phenotype changes by analysis of variance (ANOVA). This modeling was done with (two-way) and without (one-way) ANOVA adjusting for the effects of any changes in gp41 amino acids 36-45 modeled as a single variable (ANY(36-45)). Positions displaying significance levels of p<0.05 by either one- or two-way ANOVA were then studied by multi-way ANOVA (stepwise regression). RESULTS: In addition to changes at gp41 amino acids 36-45, changes at three positions in the HR2 domain (126, 129 and 133) occurred significantly more often in patients undergoing virologic failure on enfuvirtide. However, ANY(36-45) alone accounted for slightly more than 90% of the variation in phenotype explained by the ANOVA models. Relative to ANY(36-45) alone, significant increases in the geometric mean of the fold-change in inhibitory concentration (19.6-236.3-fold higher) were observed for amino acid changes at positions gp41: 18, 42,126, 247, 256 and 312; gp120: 330, 389 and 424 and significant reductions (18.8-29.7-fold lower) for gp41: 3, 46, 165, 232 and 324. CONCLUSIONS: This study represents a statistical approach to highlight positions in HIV envelope that undergo mutations in the presence of enfuvirtide. Several of the identified positions have been implicated in the viral fusion process by other studies. The specific impact of positions 330. Three hundred and eighty-nine and 424 on viral fusion kinetics remains to be studied further by site-directed mutagenesis experiments.     

Induction of V3-Specific Cytotoxic T Lymphocyte Responses by HIVgagParticles Carrying Multiple Immunodominant V3 Epitopes of gp120

Efforts to develop a vaccine to prevent infection of human immunodeficiency virus (HIV) have focused on the induction of neutralizing antibodies. In our previous study, we reported that chimericgag–envvirus-like particles (VLPs) induce neutralizing antibodies which block HIV infection. In addition to the neutralizing antibodies, the cytotoxic T-lymphocyte (CTL) response is considered to be another major immune defense mechanism required for recovery from many different viral infections. In the present study, we have constructed chimeric fusion proteins using HIV-2gagprecursor protein with (1) four neutralizing epitopes from HIV-1 gp160; (2) three tandem copies of consensus V3 domain, which have been derived from 245 different isolates of HIV-1 and carries both the principal neutralizing determinant (PND) and CTL epitopes; and (3) V3 domains from HIV-1_IIIB, HIV-1_MN, HIV-1_RF, and HIV-1_SF2. These chimeric fusion proteins were expressed in a large quantity within insect cells, and released as VLPs into the cell culture medium. The purifiedgag–envVLPs from all three constructs appear to be spherical particles similar to immature HIV but slightly larger than thegagVLPs. Immunoprecipitation analysis showed that the chimeric proteins were recognized not only by HIV-1 positive patient sera, but also by monoclonal and polyclonal antisera raised against V3 peptides of HIV-1_IIIB, HIV-1_MN, HIV-1_RF, and the gp120 antiserum against HIV-1_SF2. Balb/C mice immunized with these chimeric VLPs successfully induced CTL activity against V3 peptide-stimulated target cells. In addition, a high degree of cross-reactivity was observed among the four different strains of HIV-1 V3 domain, indicating that the tandem multiple consensus V3 peptide sequence carried by HIV-2gagcan be used as a potential HIV vaccine against various HIVs.     

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.