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Broadly neutralizing antibodies (bNAbs) are rarely elicited by current human immunodeficiency virus type 1 (HIV-1) vaccine designs, but the presence of bNAbs in naturally infected individuals may be associated with high plasma viral loads, suggesting that the magnitude, duration, and diversity of viral exposure may contribute to the development of bNAbs. Here, we report the isolation and characterization of a panel of human monoclonal antibodies (mAbs) from two subjects who developed broadly neutralizing autologous antibody responses during HIV-1 infection. In both subjects, we identified collections of mAbs that exhibited specificity only to a few autologous envelopes (Envs), with some mAbs exhibiting specificity only to a subset of Envs within the quasispecies of a particular sample at one time point. Neutralizing antibodies (NAbs) isolated from these subjects mapped mostly to epitopes in the Env V3 loop region and the CD4 binding site. None of the individual neutralizing mAbs recovered exhibited the cumulative breadth of neutralization present in the serum of the subjects. Surprisingly, however, the activity of polyclonal mixtures comprising individual mAbs that each possessed limited neutralizing activity, could achieve increased breadth of neutralizing activity against autologous isolates. While a single broadly neutralizing antibody targeting one epitope can mediate neutralization breadth, the findings presented here suggest that a cooperative polyclonal process mediated by diverse antibodies with more limited breadth targeting multiple epitopes also can achieve neutralization breadth against HIV-1.

Background The complex epidemic and significant diversity of HIV-1 strains in China pose serious challenges for surveillance and diagnostic assays, vaccine development and clinical management. There is a lack of HIV-1 isolates in current canonical HIV-1 subtype panels that can represent HIV-1 diversity in China; an HIV-1 subtype panel for China is urgently needed. Methods Blood samples were collected from HIV-1 infected patients participating in the drug-resistance surveillance program in China. The samples were isolated, cultured and stored as neat culture supernatant. The HIV-1 isolates were fully characterized. The panel was used to compare 2 viral load assays and 2 p24 assays as the examples of how this panel could be used. Results An HIV-1 subtype panel for China composed of 30 HIV-1 primary strains of four subtypes (B [including Thai-B], CRF01_AE, CRF07_BC and G) was established. The samples were isolated and cultured to a high-titer (106-109 copies/ml)/high-volume (40ml). The HIV-1 isolates were fully characterized by the final viral load, p24 concentration, gag-pol and envC2V3 sequencing, co-receptor prediction, determination of the four amino acids at the tip of the env V3-loop, glycosylation sites in the V3 loop and the drug-resistance mutations. The comparison of two p24 assays and two viral load assays on the isolates illustrated how this panel may be used for the evaluation of diagnostic assay performance. The Pearson value between p24 assays were 0.938. The viral load results showed excellent concordance and agreement for samples of Thai-B, but lower correlations for samples of CRF01_AE. Conclusion The current panel of 30 HIV-1 isolates served as a basis for the development of a comprehensive panel of fully characterized viral isolates, which could reflect the current dynamic and complex HIV-1 epidemic in China. This panel will be available to support HIV-1 research, assay evaluation, vaccine and drug development.

Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab) responses toward conserved regions of the viral Envelope (Env). However, the generation of neutralizing Abs (NAbs) targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER) of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.

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HIV-1 subtype C (C-HIV) is responsible for most HIV-1 cases worldwide. Although the pathogenesis of C-HIV is thought to predominantly involve CCR5-restricted (R5) strains, we do not have a firm understanding of how frequently CXCR4-using (X4 and R5X4) variants emerge in subjects with progressive C-HIV infection. Nor do we completely understand the molecular determinants of coreceptor switching by C-HIV variants. Here, we characterized a panel of HIV-1 envelope glycoproteins (Envs) (n = 300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naive subjects who experienced progression from chronic to advanced stages of C-HIV infection, and show that CXCR4-using C-HIV variants emerged in only one individual. Mutagenesis studies and structural models suggest that the evolution of R5 to X4 variants in this subject principally involved acquisition of an "Ile-Gly" insertion in the gp120 V3 loop and replacement of the V3 "Gly-Pro-Gly" crown with a "Gly-Arg-Gly" motif, but that the accumulation of additional gp120 "scaffold" mutations was required for these V3 loop changes to confer functional effects. In this context, either of the V3 loop changes could confer possible transitional R5X4 phenotypes, but when present together they completely abolished CCR5 usage and conferred the X4 phenotype. Our results show that the emergence of CXCR4-using strains is rare in this cohort of untreated individuals with advanced C-HIV infection. In the subject where X4 variants did emerge, alterations in the gp120 V3 loop were necessary but not sufficient to confer CXCR4 usage.

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4(+) cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1(V3-M5) derived from HIV-1(JR-FLan) is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1(JR-FLan) to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.

BackgroundHuman Immunodeficiency virus type-1 (HIV) entry into target cells involves binding of the viral envelope (Env) to CD4 and a coreceptor, mainly CCR5 or CXCR4. The only currently licensed HIV entry inhibitor, maraviroc, targets CCR5, and the presence of CXCX4-using strains must be excluded prior to treatment. Co-receptor usage can be assessed by phenotypic assays or through genotypic prediction. Here we compared the performance of a phenotypic Env-Recombinant Viral Assay (RVA) to the two most widely used genotypic prediction algorithms, Geno2Pheno[coreceptor] and webPSSM.MethodsCo-receptor tropism of samples from 73 subtype B and 219 non-B infections was measured phenotypically using a luciferase-tagged, NL4-3-based, RVA targeting Env. In parallel, tropism was inferred genotypically from the corresponding V3-loop sequences using Geno2Pheno[coreceptor] (5-20% FPR) and webPSSM-R5X4. For discordant samples, phenotypic outcome was retested using co-receptor antagonists or the validated Trofile® Enhanced-Sensitivity-Tropism-Assay.ResultsThe lower detection limit of the RVA was 2.5% and 5% for X4 and R5 minority variants respectively. A phenotype/genotype result was obtained for 210 samples. Overall, concordance of phenotypic results with Geno2Pheno[coreceptor] was 85.2% and concordance with webPSSM was 79.5%. For subtype B, concordance with Geno2pheno[coreceptor] was 94.4% and concordance with webPSSM was 79.6%. High concordance of genotypic tools with phenotypic outcome was seen for subtype C (90% for both tools). Main discordances involved CRF01_AE and CRF02_AG for both algorithms (CRF01_AE: 35.9% discordances with Geno2Pheno[coreceptor] and 28.2% with webPSSM; CRF02_AG: 20.7% for both algorithms). Genotypic prediction overestimated CXCR4-usage for both CRFs. For webPSSM, 40% discordance was observed for subtype A.ConclusionsPhenotypic assays remain the most accurate for most non-B subtypes and new subtype-specific rules should be developed for non-B subtypes, as research studies more and more draw conclusions from genotypically-inferred tropism, and to avoid unnecessarily precluding patients with limited treatment options from receiving maraviroc or other entry inhibitors.

BACKGROUND: The net charge of the hypervariable V3 loop on the HIV-1 envelope gp120 outer domain plays a key role in modulating viral phenotype. However, the molecular mechanisms underlying the modulation remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: By combining computational and experimental approaches, we examined how V3 net charge could influence the phenotype of the gp120 interaction surface. Molecular dynamics simulations of the identical gp120 outer domain, carrying a V3 loop with net charge of +3 or +7, showed that the V3 change alone could induce global changes in fluctuation and conformation of the loops involved in binding to CD4, coreceptor and antibodies. A neutralization study using the V3 recombinant HIV-1 infectious clones showed that the virus carrying the gp120 with +3 V3, but not with +7 V3, was resistant to neutralization by anti-CD4 binding site monoclonal antibodies. An information entropy study shows that otherwise variable surface of the gp120 outer domain, such as V3 and a region around the CD4 binding loop, are less heterogeneous in the gp120 subpopulation with +3 V3. CONCLUSIONS/SIGNIFICANCE: These results suggest that the HIV-1 gp120 V3 loop acts as an electrostatic modulator that influences the global structure and diversity of the interaction surface of the gp120 outer domain. Our findings will provide a novel structural basis to understand how HIV-1 adjusts relative replication fitness by V3 mutations.

Background α-defensin-5 (HD5) is a key effector of the innate immune system with broad anti-bacterial and anti-viral activities. Specialized epithelial cells secrete HD5 in the genital and gastrointestinal mucosae, two anatomical sites that are critically involved in HIV-1 transmission and pathogenesis. We previously found that human neutrophil defensins (HNP)-1 and -2 inhibit HIV-1 entry by specific bilateral interaction both with the viral envelope and with its primary cellular receptor, CD4. Despite low amino acid identity, human defensin-5 (HD5) shares with HNPs a high degree of structural homology. Methodology/Principal Findings Here, we demonstrate that HD5 inhibits HIV-1 infection of primary CD4+ T lymphocytes at low micromolar concentration under serum-free and low-ionic-strength conditions similar to those occurring in mucosal fluids. Blockade of HIV-1 infection was observed with both primary and laboratory-adapted strains and was independent of the viral coreceptor-usage phenotype. Similar to HNPs, HD5 inhibits HIV-1 entry into the target cell by interfering with the reciprocal interaction between the external envelope glycoprotein, gp120, and CD4. At high concentrations, HD5 was also found to downmodulate expression of the CXCR4 coreceptor, but not of CCR5. Consistent with its broad spectrum of activity, antibody competition studies showed that HD5 binds to a region overlapping with the CD4- and coreceptor-binding sites of gp120, but not to the V3 loop region, which contains the major determinants of coreceptor-usage specificity. Conclusion/Significance These findings provide new insights into the first line of immune defense against HIV-1 at the mucosal level and open new perspectives for the development of preventive and therapeutic strategies.

BackgroundThe HIV-1 subtype B epidemic in Brazil is peculiar because of the high frequency of isolates having the GWGR tetramer at V3 loop region. It has been suggested that GWGR is a distinct variant and less pathogenic than other subtype B isolates.Methodology/principal findingsNinety-four percent of the HIV-1 subtype B worldwide sequences (7689/8131) obtained from the Los Alamos HIV database contain proline at the tetramer of the V3 loop of the env gene (GPGR) and only 0.74% (60/8131) have tryptophan (GWGR). By contrast, 48.4% (161/333) of subtype B isolates from Brazil have proline, 30.6% (102/333) contain tryptophan and 10.5% (35/333) have phenylalanine (F) at the second position of the V3 loop tip. The proportion of tryptophan and phenylalanine in Brazilian isolates is much higher than in worldwide subtype B sequences (chi-square test, p = 0.0001). The combined proportion of proline, tryptophan and phenylalanine (GPGR+GWGR+GFGR) of Brazilian isolates corresponds to 89% of all amino acids in the V3 loop. Phylogenetic analysis revealed that almost all subtype B isolates in Brazil have a common origin regardless of their motif (GWGR, GPGR, GGGR, etc.) at the V3 tetramer. This shared ancestral origin was also observed in CRF28_BF and CRF29_BF in a genome region (free of recombination) derived from parental subtype B. These results imply that tryptophan substitution (e.g., GWGR-to-GxGR), which was previously associated with the change in the coreceptor usage within the host, also occurs at the population level.Conclusions/significanceBased on the current findings and previous study showing that tryptophan and phenylalanine in the V3 loop are related with coreceptor usage, we propose that tryptophan and phenylalanine in subtype B isolates in Brazil are kept by selective mechanisms due to the distinct coreceptor preferences in target cells of GWGR, GFGR and GFGR viruses.

The chemokine receptor CXCR4 is shared by primary and laboratory-adapted strains of feline immunodeficiency virus (FIV) for viral entry. Our previous studies implicated a contiguous nine-amino-acid region of the V3 loop of the FIV envelope surface as important in CXCR4 binding and virus entry. The binding is specific for CXCR4 since it can be inhibited by AMD3100, a selective CXCR4 inhibitor. Additional site-directed mutagenesis was used to further reveal the key residues. Binding studies indicated that basic residues R395, K397, R399 as well as N398 are critical for CXCR4 binding. The effect of other amino acid residues on receptor binding depends on the type of amino acid residue substituted. The binding study results were confirmed on human CXCR4-expressing SupT1 cells and correlated with entry efficiency using a virus entry assay. Amino acid residues critical for CXCR4 are not critical for interactions with the primary binding receptor CD134, which has an equivalent role as CD4 for HIV-1 binding. The ELISA results show that W394 and W400 are crucial for the recognition by neutralizing anti-V3 antibodies. Since certain strains of HIV-1 also use CXCR4 as the entry receptor, the findings make the feline model attractive for development of broad-based entry antagonists and for study of the molecular mechanism of receptor/virus interactions.

HIV-1 cell entry commonly uses, in addition to CD4, one of the chemokine receptors CCR5 or CXCR4 as coreceptor. Knowledge of coreceptor usage is critical for monitoring disease progression as well as for supporting therapy with the novel drug class of coreceptor antagonists. Predictive methods for inferring coreceptor usage based on the third hypervariable (V3) loop region of the viral gene coding for the envelope protein gp120 can provide us with these monitoring facilities while avoiding expensive phenotypic tests. All simple heuristics (such as the 11/25 rule) as well as statistical learning methods proposed to date predict coreceptor usage based on sequence features of the V3 loop exclusively. Here, we show, based on a recently resolved structure of gp120 with an untruncated V3 loop, that using structural information on the V3 loop in combination with sequence features of V3 variants improves prediction of coreceptor usage. In particular, we propose a distance-based descriptor of the spatial arrangement of physicochemical properties that increases discriminative performance. For a fixed specificity of 0.95, a sensitivity of 0.77 was achieved, improving further to 0.80 when combined with a sequence-based representation using amino acid indicators. This compares favorably with the sensitivities of 0.62 for the traditional 11/25 rule and 0.73 for a prediction based on sequence information as input to a support vector machine and constitutes a statistically significant improvement. A detailed analysis and interpretation of structural features important for classification shows the relevance of several specific hydrogen-bond donor sites and aliphatic side chains to coreceptor specificity towards CCR5 or CXCR4. Furthermore, an analysis of side chain orientation of the specificity-determining residues suggests a major role of one side of the V3 loop in the selection of the coreceptor. The proposed method constitutes the first approach to an improved prediction of coreceptor usage based on an original integration of structural bioinformatics methods with statistical learning., Author Summary HIV-1 cell entry requires a chemokine coreceptor in addition to the CD4 cell surface receptor. The two most common types of HIV coreceptors are called CCR5 and CXCR4. Whereas CCR5-using viral variants dominate directly after infection and during early stages of the disease, in about 50% of the patients, CXCR4-using variants appear in later stages of the disease, suggesting the coreceptor switch to be a determinant of disease progression. HIV coreceptors received substantial attention as antiviral drug targets, with CCR5 antagonists being currently tested in phase III clinical studies. Treatment with coreceptor antagonists requires continuous monitoring of coreceptor usage. The prominent role of coreceptors in disease progression and their potential as antiviral drug targets provides incentives for methodological improvements in coreceptor prediction and better understanding of the underlying determining factors regarding sequence and structural aspects. Our proposed method is the first approach to predict coreceptor usage based on structural information as opposed to established sequence-based methods. Including structural information improves predictive performance and is a first step towards a deeper understanding of the structural aspects of coreceptor usage.

Nearly all persons newly infected with HIV-1 harbor exclusively CCR5-using virus. CXCR4-using variants eventually arise in up to 50% of patients infected with subtypes B or D. This transition to efficient CXCR4 utilization is often co-incident with progression to AIDS. The basis for HIV-1’s initial dependence on CCR5, the selective force(s) that drive CXCR4-utilization, and the evolutionary pathways by which it occurs are incompletely understood. Greater knowledge of these processes will inform interventions at all stages, from vaccination to cure. The determinants of co-receptor use map primarily, though not exclusively, to the V3 loop of gp120. In this study, we describe five clonal variants with identical V3 loops but divergent CXCR4 use. Mutagenesis revealed two residues controlling this phenotypic switch: a rare polymorphism in C1 and a highly conserved N-glycan in C2. To our knowledge, this is the first description of co-receptor usage regulated by the N-glycan at position 262.

The most predominant HIV-1 strains in China's current epidemic is the Circulating Recombinant Form 07_BC (CRF07_BC). CRF07_BC is mainly considered as a CCR5-tropic (R5) virus, since CXCR4-tropic (X4) viruses have thus far not been found in this subtype, and the molecular determinants of coreceptor switching remain unknown. To investigate the mechanisms underlying coreceptor requirement in CRF07_BC viruses, we characterized a panel of pNL4-3-based chimeric viruses with mutated V3 loop regions derived from an HIV-1 CRF07_BC infectious clone pXJDC13. Among 17 chimeric viruses, seven were dual-tropic and induced syncytium formation in MT-2 cells. Two amino acid insertions between positions 13 and 14, as well as arginine substitution at position 11 or 16 (IG insertion and P16R mutation or MG insertion and S11R mutation), conferred the chimeric viruses CXCR4-tropic features, which were same as subtype C X4 viruses. Next, to construct CRF07_BC X4 variants, mutated V3 loops were cloned into the CRF07_BC infectious clone pXJDC13. These V3 loops, which in the pNL4-3 backbone conferred chimeric viruses with CXCR4-using ability, abrogated infectivity completely in the CRF07_BC pXJDC13 genetic background. Similarly, IG insertion or MG insertion and S11R mutation dramatically diminished or completely abolished viral infectivity in other envelopes of subtype C or CRF07_BC. These results suggest that the effects of IG insertion and P16R mutation or MG insertion and S11R mutation on CXCR4 usage are context dependent, and additional mutations elsewhere in the envelope are needed to compensate for these fitness-reducing alterations.

A CXCR4 inhibitor-resistant HIV-1 was isolated from a dual-X4 HIV-1 in vitro. The resistant variant displayed competitive resistance to the CXCR4 inhibitor AMD3100, indicating that the resistant variant had a higher affinity for CXCR4 than that of the wild-type HIV-1. Amino acid sequence analyses revealed that the resistant variant harbored amino acid substitutions in the V2, C2, and C4 regions, but no remarkable changes in the V3 loop. Site-directed mutagenesis confirmed that the changes in the C2 and C4 regions were principally involved in the reduced sensitivity to AMD3100. Furthermore, the change in the C4 region was associated with increased sensitivity to soluble CD4, and profoundly enhanced the entry efficiency of the virus. Therefore, it is likely that the resistant variant acquired the higher affinity for CD4/CXCR4 by the changes in non-V3 regions. Taken together, a CXCR4 inhibitor-resistant HIV-1 can evolve using a non-V3 pathway.

Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.

Some secondary metabolites from plants show to have potent inhibitory activities against microbial pathogens, such as human immunodeficiency virus (HIV), herpes simplex virus (HSV), Treponema pallidum, Neisseria gonorrhoeae, etc. Here we report that lignosulfonic acid (LSA), a polymeric lignin derivative, exhibits potent and broad activity against HIV-1 isolates of diverse subtypes including two North America strains and a number of Chinese clinical isolates values ranging from 21.4 to 633 nM. Distinct from other polyanions, LSA functions as an entry inhibitor with multiple targets on viral gp120 as well as on host receptor CD4 and co-receptors CCR5/CXCR4. LSA blocks viral entry as determined by time-of-drug addiction and cell-cell fusion assays. Moreover, LSA inhibits CD4-gp120 interaction by blocking the binding of antibodies specific for CD4-binding sites (CD4bs) and for the V3 loop of gp120. Similarly, LSA interacts with CCR5 and CXCR4 via its inhibition of specific anti-CCR5 and anti-CXCR4 antibodies, respectively. Interestingly, the combination of LSA with AZT and Nevirapine exhibits synergism in viral inhibition. For the purpose of microbicide development, LSA displays low in vitro cytotoxicity to human genital tract epithelial cells, does not stimulate NF-kappaB activation and has no significant up-regulation of IL-1alpha/beta and IL-8 as compared with N-9. Lastly, LSA shows no adverse effect on the epithelial integrity and the junctional protein expression. Taken together, our findings suggest that LSA can be a potential candidate for tropical microbicide., published_or_final_version

A specific response of human serum neutralizing antibodies (nAb) to a conformational epitope as a result of vaccination of human subjects with the surface envelope glycoprotein (gp120) of HIV-1 has not previously been documented. Here, we used computational analysis to assess the epitope-specific responses of human subjects, which were immunized with recombinant gp120 immunogens in the VAX003 and VAX004 clinical trials. Our computational methodology--a variation of sieve analysis--compares the occurrence of specific nAb targeted conformational 3D epitopes on viruses from infected individuals who received vaccination to the occurrence of matched epitopes in the viruses infecting placebo subjects. We specifically studied seven crystallographically defined nAb targeted conformational epitopes in the V3 loop, an immunogenic region of gp120. Of the six epitopes present in the immunogens and targeted by known monoclonal neutralizing antibodies, only the one targeted by the anti-V3 nAb 2219 exhibited a significant reduction in occurrence in vaccinated subjects compared to the placebo group. This difference occurred only in the VAX003 Thailand cohort. No difference was seen between vaccinated and placebo groups for the occurrence of an epitope that was not present in the immunogen. Thus, it can be theorized that a specific 2219-like human neutralizing antibody immune response to AIDSVAX immunization occurred in the VAX003 cohort, and that this response protected subjects from a narrow subset of HIV-1 viruses circulating in Thailand in the 1990s and bearing the conformational epitope targeted by the neutralizing antibody 2219.

Background The B′, CRF07_BC and CRF01_AE are the predominant HIV-1 subtypes in China. It is essential to determine their baseline susceptibility to HIV entry inhibitors before these drugs are used in China. Methodology/Principal Findings The baseline susceptibility of 14 representative HIV-1 isolates (5 CRF07_BC, 4 CRF01_AE, and 5 B′), most of which were R5 viruses, obtained from drug-naive patients to HIV entry inhibitors, including two fusion inhibitors (enfuvirtide and C34), two CCR5 antagonists (maraviroc and TAK779) and one CXCR4 antagonist (AMD3100), were determined by virus inhibition assay. The sequences of their env genes were amplified and analyzed. These isolates possessed similar susceptibility to C34, but they exhibited different sensitivity to enfuvirtide, maraviroc or TAK779. CRF07_BC isolates, which carried polymorphisms of A578T and V583I in the N-terminal heptad repeat and E630Q, E662A, K665S, A667K and S668N in the C-terminal heptad repeat of gp41, were about 5-fold less sensitive than B′ and CRF01_AE isolates to enfuvirtide. Subtype B′ isolates with a unique polymorphism site of F317W in V3 loop, were about 4- to 5-fold more sensitive than CRF07_BC and CRF01_AE isolates to maraviroc and TAK779. AMD3100 at the concentration as high as 5 µM exhibited no significant inhibitory activity against any of the isolates tested. Conclusion Our results suggest that there are significant differences in baseline susceptibility to HIV entry inhibitors among the predominant HIV-1 subtypes in China and the differences may partly result from the naturally occurring polymorphisms in these subtypes. This study provides useful information for rational design of optimal therapeutic regimens for HIV-1-infected patients in China.

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.

Although the sequence variable loops of the human immunodeficiency virus' (HIV-1) surface envelope glycoprotein (gp120) can exhibit good immunogenicity, characterizing conserved (invariant) cross-strain neutralization epitopes within these loops has proven difficult. We recently developed a method to derive sensitive and specific signature motifs for the three-dimensional (3D) shapes of the HIV-1 neutralization epitopes in the third variable (V3) loop of gp120 that are recognized by human monoclonal antibodies (mAbs). We used the signature motif method to estimate the conservation of these epitopes across circulating worldwide HIV-1 strains. The epitope targeted by the anti-V3 loop neutralizing mAb 3074 is present in 87% of circulating strains, distributed nearly evenly among all subtypes. The results for other anti-V3 Abs are: 3791, present in 63% of primarily non-B subtypes; 2219, present in 56% of strains across all subtypes; 2557, present in 52% across all subtypes; 447-52D, present in 11% of primarily subtype B strains; 537-10D, present in 9% of primarily subtype B strains; and 268-D, present in 5% of primarily subtype B strains. The estimates correlate with in vitro tests of these mAbs against diverse viral panels. The mAb 3074 thus targets an epitope that is nearly completely conserved among circulating HIV-1 strains, demonstrating the presence of an invariant structure hidden in the dynamic and sequence-variable V3 loop in gp120. Since some variable loop regions are naturally immunogenic, designing immunogens to mimic their conserved epitopes may be a promising vaccine discovery approach. Our results suggest one way to quantify and compare the magnitude of the conservation.

The V3 loop of the HIV-1 Env protein is the primary determinant of viral coreceptor usage, whereas the V1V2 loop region is thought to influence coreceptor binding and participate in shielding of neutralization-sensitive regions of the Env glycoprotein gp120 from antibody responses. The functional properties and antigenicity of V1V2 are influenced by changes in amino acid sequence, sequence length and patterns of N-linked glycosylation. However, how these polymorphisms relate to HIV pathogenesis is not fully understood. We examined 5185 HIV-1 gp120 nucleotide sequence fragments and clinical data from 154 individuals (152 were infected with HIV-1 Subtype B). Sequences were aligned, translated, manually edited and separated into V1V2, C2, V3, C3, V4, C4 and V5 subregions. V1-V5 and subregion lengths were calculated, and potential N-linked glycosylation sites (PNLGS) counted. Loop lengths and PNLGS were examined as a function of time since infection, CD4 count, viral load, and calendar year in cross-sectional and longitudinal analyses. V1V2 length and PNLGS increased significantly through chronic infection before declining in late-stage infection. In cross-sectional analyses, V1V2 length also increased by calendar year between 1984 and 2004 in subjects with early and mid-stage illness. Our observations suggest that there is little selection for loop length at the time of transmission; following infection, HIV-1 adapts to host immune responses through increased V1V2 length and/or addition of carbohydrate moieties at N-linked glycosylation sites. V1V2 shortening during early and late-stage infection may reflect ineffective host immunity. Transmission from donors with chronic illness may have caused the modest increase in V1V2 length observed during the course of the pandemic., Author Summary The HIV envelope gene (env) encodes viral surface proteins (Env) that are vital to the basic processes used by the virus to infect and cause disease in humans. Adaptations in env determine which cells the virus can infect, and permit the virus to avoid elimination by the immune system. Env is one of the most variable genes known, and it can change dramatically over time in a single individual. However, Env-host cell interactions are complex and incompletely understood, and changes in this viral protein during infection have not yet been systematically described. We examined a large number of env sequences from 154 individuals at various stages of HIV infection but who had never received antiretroviral treatment. We found that the env V1V2 region lengthens during chronic infection and becomes more heavily glycosylated. However, these changes partially reverse during late-stage illness, possibly in response to a weakening host immune system. V1V2 lengths are also increasing over time in the epidemic at large, possibly related to the epidemiology of HIV transmission within the subtype B epidemic. These results provide fundamental insights into the biology of HIV.

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 coreceptors 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.

High-throughput sequencing platforms provide an approach for detecting rare HIV-1 variants and documenting more fully quasispecies diversity. We applied this technology to the V3 loop-coding region of env in samples collected from 4 chronically HIV-infected subjects in whom CCR5 antagonist (vicriviroc [VVC]) therapy failed. Between 25,000-140,000 amplified sequences were obtained per sample. Profound baseline V3 loop sequence heterogeneity existed; predicted CXCR4-using populations were identified in a largely CCR5-using population. The V3 loop forms associated with subsequent virologic failure, either through CXCR4 use or the emergence of high-level VVC resistance, were present as minor variants at 0.8-2.8% of baseline samples. Extreme, rapid shifts in population frequencies toward these forms occurred, and deep sequencing provided a detailed view of the rapid evolutionary impact of VVC selection. Greater V3 diversity was observed post-selection. This previously unreported degree of V3 loop sequence diversity has implications for viral pathogenesis, vaccine design, and the optimal use of HIV-1 CCR5 antagonists.

The third variable loop (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope is a principal determinant of antibody neutralization and progression to AIDS. Although it is undoubtedly an important target for vaccine research, extensive genetic variation in V3 remains an obstacle to the development of an effective vaccine. Comparative methods that exploit the abundance of sequence data can detect interactions between residues of rapidly evolving proteins such as the HIV-1 envelope, revealing biological constraints on their variability. However, previous studies have relied implicitly on two biologically unrealistic assumptions: (1) that founder effects in the evolutionary history of the sequences can be ignored, and; (2) that statistical associations between residues occur exclusively in pairs. We show that comparative methods that neglect the evolutionary history of extant sequences are susceptible to a high rate of false positives (20%–40%). Therefore, we propose a new method to detect interactions that relaxes both of these assumptions. First, we reconstruct the evolutionary history of extant sequences by maximum likelihood, shifting focus from extant sequence variation to the underlying substitution events. Second, we analyze the joint distribution of substitution events among positions in the sequence as a Bayesian graphical model, in which each branch in the phylogeny is a unit of observation. We perform extensive validation of our models using both simulations and a control case of known interactions in HIV-1 protease, and apply this method to detect interactions within V3 from a sample of 1,154 HIV-1 envelope sequences. Our method greatly reduces the number of false positives due to founder effects, while capturing several higher-order interactions among V3 residues. By mapping these interactions to a structural model of the V3 loop, we find that the loop is stratified into distinct evolutionary clusters. We extend our model to detect interactions between the V3 and C4 domains of the HIV-1 envelope, and account for the uncertainty in mapping substitutions to the tree with a parametric bootstrap., Author Summary The third variable loop (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope is a principal determinant of viral growth characteristics and an important target for the immune system. Interactions between residues of V3 allow the virus to shift between combinations of residues to escape the immune system while retaining its structure and functions. Comparative study of HIV-1 V3 sequences can detect such interactions by the covariation of sites in the sequence, which can then be used to inform vaccine development, but current methods for detecting such associations rely on biologically unrealistic assumptions. We demonstrate that these assumptions cause an excessive number of spurious associations, and present a new approach that couples phylogenetic and Bayesian network models, and greatly reduces this number while retaining the ability to detect real associations. Our analysis reveals that the V3 loop is stratified into discrete layers of interacting residues, suggesting a partition of functions along this viral structure with implications for vaccine development.