基于深圳男男同性性行为人群的HIV-1广谱中和抗体筛选分析

目的:筛选基于深圳本地男男同性性行为者(Men who have sex with men,MSM)人群队列中HIV-1流行毒株的广谱中和抗体(Broadly neutralizing antibodies,bn Abs),为下一步机制和应用研究奠定基础。方法:建立小型MSM队列,按计划分别定期随访、留样,测序分析人群中HIV-1病毒流行亚型。选取将骨架质粒与系列标准HIV-1 env质粒12款,分别共转染293细胞制备单次感染能力假病毒。建立TZM-bl细胞实验测定并计算中和活性(ID50Titers)技术平台,用于选定病毒亚型样品的筛选。最后取所获具有一定广谱中和抗性的代表性血样,通过抗体竞争实验初步分析其结合位点。结果:近年来,深圳MSM的HIV-1流行亚型分布中,CRF07_BC(43.4%)和CRF55_01B(15.4%)占比快速增长。选择来自该人群队列CRF07_BC感染者34人88份血样进行广谱中和抗性检测,筛选出具有一定广谱中和抗性的血样10份(ID50 Titer≧25),其中2例显示了较佳中和宽度,可作用于全部12种假病毒中的7种(58.3%)。初步分析其结合机制均非靶向gp120。结论:本研究成功建立小型MSM队列和TZM-bl检测分析技术并应用于实践,初步筛选结果提示部分具有中和活性的患者血清内存在bn Abs。     

The role of conventional antibodies targeting the CD4 binding site and CD4-induced epitopes in the control of HIV-1 CRF01_AE viruses

HIV-1 CRF01_AE viruses are highly prevalent in Southeast Asia. However, vulnerability sites in Env of CRF01_AE viruses have not been investigated sufficiently. We examined the sensitivity of CRF01_AE viruses from Japan and Vietnam, together with subtype B viruses from Japan, to neutralization and Fc-mediated signaling. Neutralization coverage of broadly neutralizing antibodies (bnAbs), 2G12 and b12, was significantly low against CRF01_AE viruses, compared with subtype B viruses. In contrast, the conventional antibody targeting the CD4 binding site (CD4bs), 49G2, showed better neutralization and Fc-mediated signaling activities against CRF01_AE viruses than subtype B viruses. Fc-mediated signaling activity of anti-CD4 induced (CD4i) antibody, 4E9C, was also detected against CRF01_AE viruses more than subtype B viruses. These results suggest that conventional antibodies against CD4bs and CD4i may play an important role in the control of CRF01_AE viruses.     

Glycans Flanking the Hypervariable Connecting Peptide between the A and B Strands of the V1/V2 Domain of HIV-1 gp120 Confer Resistance to Antibodies That Neutralize CRF01_AE Viruses

Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.     

Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.     

Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120(Ds2)), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B'/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.     

Characterization of human immunodeficiency virus type 1 CRF01_AE env genes derived from recently infected Thai individuals

Transmitted/founder virus is responsible for the establishment of human immunodeficiency virus type 1 (HIV-1) infection and induces primary anti-HIV-1 immune responses; therefore, it is important to study the viral population to understand the early events of HIV-1 infection. We amplified HIV-1 env genes from sera derived from recently infected Thai individuals, and established envelope glycoproteins (Env)-recombinant viruses. Generated Env-recombinant viruses were tested for their neutralization susceptibility to neutralizing human monoclonal antibodies (NHMAbs) and entry inhibitors, as well as being subjected to genotypic analysis. Most recombinant viruses were susceptible to neutralization by NHMAbs to Env gp41, whereas approximately one-third of the recombinant viruses were susceptible to a NHMAb against the CD4 binding site of gp120. In addition, all env genes were classified into CRF01_AE genes and showed low genetic divergence. Taken together with our previous studies on CRF01_AE env genes derived from chronically infected Thai individuals, these results suggested that the immunological and genetic characteristics of CRF01_AE Env derived from recently infected Thai individuals were different from those derived from chronically infected individuals.     

Susceptibility of recently transmitted subtype B human immunodeficiency virus type 1 variants to broadly neutralizing antibodies

The ability of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) specific human monoclonal antibodies (MAbs) b12, 2G12, 2F5, and 4E10 to neutralize recently transmitted viruses has not yet been explored in detail. We investigated the neutralization sensitivity of subtype B HIV-1 variants obtained from four primary HIV infection cases and six transmission couples (four homosexual and two parenteral) to these MAbs. Sexually transmitted HIV-1 variants isolated within the first 2 months after seroconversion were generally sensitive to 2F5, moderately resistant to 4E10 and b12, and initially resistant but later more sensitive to 2G12 neutralization. In the four homosexual transmission couples, MAb neutralization sensitivity of HIV in recipients did not correlate with the MAb neutralization sensitivity of HIV from their source partners, whereas the neutralization sensitivity of donor and recipient viruses involved in parenteral transmission was more similar. For a fraction (11%) of the HIV-1 variants analyzed here, neutralization by 2G12 could not be predicted by the presence of N-linked glycosylation sites previously described to be involved in 2G12 binding. Resistance to 2F5 and 4E10 neutralization did also not correlate with mutations in the respective core epitopes. Overall, we observed that the neutralization resistance of recently transmitted subtype B HIV-1 variants was relatively high. Although 8 of 10 patients had viruses that were sensitive to neutralization by at least one of the four broadly neutralizing antibodies studied, 4 of 10 patients harbored at least one virus variant that seemed resistant to all four antibodies. Our results suggest that vaccine antigens that only elicit antibodies equivalent to b12, 2G12, 2F5, and 4E10 may not be sufficient to protect against all contemporary HIV-1 variants and that additional cross-neutralizing specificities need to be sought.     

Limited Neutralizing Antibody Specificities Drive Neutralization Escape in Early HIV-1 Subtype C Infection

We previously showed that HIV-1 subtype C viruses elicit potent but highly type-specific neutralizing antibodies (nAb) within the first year of infection. In order to determine the specificity and evolution of these autologous nAbs, we examined neutralization escape in four individuals whose responses against the earliest envelope differed in magnitude and potency. Neutralization escape occurred in all participants, with later viruses showing decreased sensitivity to contemporaneous sera, although they retained sensitivity to new nAb responses. Early nAb responses were very restricted, occurring sequentially and targeting only two regions of the envelope. In V1V2, limited amino acid changes often involving indels or glycans, mediated partial or complete escape, with nAbs targeting the V1V2 region directly in 2 cases. The alpha-2 helix of C3 was also a nAb target, with neutralization escape associated with changes to positively charged residues. In one individual, relatively high titers of anti-C3 nAbs were required to drive genetic escape, taking up to 7 weeks for the resistant variant to predominate. Thereafter titers waned but were still measurable. Development of this single anti-C3 nAb specificity was associated with a 7-fold drop in HIV-1 viral load and a 4-fold rebound as the escape mutation emerged. Overall, our data suggest the development of a very limited number of neutralizing antibody specificities during the early stages of HIV-1 subtype C infection, with temporal fluctuations in specificities as escape occurs. While the mechanism of neutralization escape appears to vary between individuals, the involvement of limited regions suggests there might be common vulnerabilities in the HIV-1 subtype C transmitted envelope.     

Human immunodeficiency virus type 1 primary isolate neutralization resistance is associated with the syncytium-inducing phenotype and lower CD4 cell counts in subtype CRF01_AE-infected patients

A number of human immunodeficiency virus type 1 (HIV-1) non-B-subtype products have been developed for present or future vaccine trials; in Thailand, several studies using subtype B and/or CRF01_AE vaccines have been conducted. To better characterize the biologic properties of these subtypes, 70 HIV-1 subtype B and E isolates were phenotyped as syncytium-inducing (SI) or non-syncytium-inducing (NSI) isolates and assessed for sensitivity to neutralizing antibody (NAb). A significantly higher number of NSI subtype E viruses were neutralization sensitive than SI subtype E viruses (P = 0.009), while no association between viral phenotype and sensitivity to NAb was observed for subtype B (P = 0.856), suggesting a difference in the neutralization patterns of subtypes B and E. Strikingly, concurrent CD4 T-cell numbers were significantly lower for subtype E-infected patients whose isolates were more resistant to NAb, both for the overall study group (P < 0.001) as well as for the 22 patients with NSI isolates (P = 0.013). Characterization of the evolution of biologic properties of both B and non-B HIV-1 subtypes will provide a clearer understanding of the repertoire of antibodies that must be elicited for a vaccine to be effective against all phenotypes and subtypes.     

Genetic and neutralization properties of subtype C human immunodeficiency virus type 1 molecular env clones from acute and early heterosexually acquired infections in Southern Africa

A standard panel of subtype C human immunodeficiency virus type 1 (HIV-1) Env-pseudotyped viruses was created by cloning, sequencing, and characterizing functional gp160 genes from 18 acute and early heterosexually acquired infections in South Africa and Zambia. In general, the gp120 region of these clones was shorter (most evident in V1 and V4) and less glycosylated compared to newly transmitted subtype B viruses, and it was underglycosylated but no different in length compared to chronic subtype C viruses. The gp120s also exhibited low amino acid sequence variability (12%) in V3 and high variability (39%) immediately downstream of V3, a feature shared with newly transmitted subtype B viruses and chronic viruses of both subtypes. When tested as Env-pseudotyped viruses in a luciferase reporter gene assay, all clones possessed an R5 phenotype and resembled primary isolates in their sensitivity to neutralization by HIV-1-positive plasmas. Results obtained with a multisubtype plasma panel suggested partial subtype preference in the neutralizing antibody response to infection. The clones were typical of subtype C in that all were resistant to 2G12 (associated with loss of N-glycosylation at position 295) and most were resistant to 2F5, but all were sensitive to 4E10 and many were sensitive to immunoglobulin G1b12. Finally, conserved neutralization epitopes in the CD4-induced coreceptor binding domain of gp120 were poorly accessible and were difficult to induce and stabilize with soluble CD4 on Env-pseudotyped viruses. These results illustrate key genetic and antigenic properties of subtype C HIV-1 that might impact the design and testing of candidate vaccines. A subset of these gp160 clones are suitable for use as reference reagents to facilitate standardized assessments of vaccine-elicited neutralizing antibody responses.     

Activity of broadly neutralizing antibodies, including PG9, PG16, and VRC01, against recently transmitted subtype B HIV-1 variants from early and late in the epidemic

For the development of a neutralizing antibody-based human immunodeficiency virus type 1 (HIV-1) vaccine, it is important to characterize which antibody specificities are most effective against currently circulating HIV-1 variants. We recently reported that HIV-1 has become more resistant to antibody neutralization over the course of the epidemic, and we here explore whether this increased neutralization resistance is also observed for the newly identified broadly neutralizing antibodies (BrNAbs) PG9, PG16, and VRC01. Furthermore, we performed a comprehensive analysis of the neutralizing sensitivity of currently circulating recently transmitted subtype B viruses to the currently most known BrNAbs. Virus variants isolated less than 6 months after seroconversion from individuals who seroconverted between 2003 and 2006 (n = 21) were significantly more resistant to neutralization by VRC01 than viruses from individuals who seroconverted between 1985 and 1989 (n = 14). In addition, viruses from contemporary seroconverters tended to be more resistant to neutralization by PG16, which coincided with the presence of more mutations at positions in the viral envelope that may potentially influence neutralization by this antibody. Despite this increased neutralization resistance, all recently transmitted viruses from contemporary seroconverters were sensitive to at least one BrNAb at concentrations of ≤5 μg/ml, with PG9, PG16, and VRC01 showing the greatest breadth of neutralization at lower concentrations. These results suggest that a vaccine capable of eliciting multiple BrNAb specificities will be necessary for protection of the population against HIV-1 infection.     

Tiered Categorization of a Diverse Panel of HIV-1 Env Pseudoviruses for Assessment of Neutralizing Antibodies

The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1⁺) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1⁺ plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.The development of an HIV-1 vaccine that can elicit protective humoral and cellular immunity is one of the highest priorities in the global fight against HIV/AIDS (2, 44). Data from lentiviral animal models suggest that antibodies capable of neutralizing primary strains of HIV-1 may have the capacity to prevent HIV-1 infection (1, 28, 30, 35). However, the ability to design immunogens that can elicit such broadly reactive neutralizing antibodies (NAbs) has proven to be a formidable obstacle, due in part to the extensive genetic diversity of HIV-1 and the complex escape mechanisms employed by the envelope gp120 and gp41 glycoproteins that form the trimeric viral envelope spike (Env) (20, 34, 45). As improved vaccine immunogens enter the stage of detailed preclinical analysis, the in vitro assays used for evaluating vaccine sera will need to detect incremental advances in the magnitude, breadth, and durability of NAb responses (37). Such data can then be used to distinguish and prioritize among antibody-based vaccine immunogens. Furthermore, highly reproducible and quantitative data on vaccine-elicited NAbs can enhance our understanding of the relationship between Env immunogen design and the resulting antibody response generated.Current recommendations for evaluating candidate vaccine sera for NAb activity include the use of standard reference panels of molecularly cloned HIV-1 Env pseudoviruses and a tiered algorithm of testing (27). Reference virus panels should represent genetically and geographically diverse subsets of viruses with neutralization phenotypes that are generally representative of primary isolate strains that a vaccine would need to protect against. As such, standard reference panels for HIV-1 subtypes B and C have been described (22, 23), and efforts continue toward the creation of virus reference panels representing additional genetic subtypes. For tiered evaluation of NAb activity, vaccine sera are first tested against homologous Env pseudoviruses and/or a small number of isolates that are known to be highly sensitive to antibody-mediated neutralization (commonly referred to as tier 1 viruses). A more rigorous assessment of the potency and breadth of vaccine-induced NAbs entails testing against more resistant reference panel viruses (commonly referred to as tier 2 viruses) that are either matched or mismatched in genetic subtype to the vaccine immunogen (second and third tiers of testing, respectively). This tiered approach for testing candidate HIV-1 vaccine sera is advantageous in that it provides increasingly stringent levels for assessing the potency and breadth of NAbs, uses standardized panels of reference viruses for consistency and reproducibility, and allows for the generation of comparative data sets for evaluating different candidate vaccine regimens.While the tiered algorithm for evaluating vaccine sera has gained acceptance in the field, a major limitation has been the lack of objective data to characterize HIV-1 Env pseudoviruses according to their overall sensitivity or resistance to antibody-mediated neutralization. The category of sensitive, tier 1 viruses arose in part from the observation that HIV-1 isolates passaged through T-cell lines often become highly sensitive to antibody-mediated neutralization (33). Compared to these laboratory-adapted viruses, most primary isolate strains are moderately resistant to NAbs. Yet, even among recently isolated circulating viral Envs, there is a wide spectrum of neutralization sensitivity. Some HIV-1 isolates have a neutralization phenotype closer to that of tier 1 viruses, while others appear to be quite neutralization resistant (6, 19, 22, 23). Overall, there are few data from which to understand or categorize the viral neutralization phenotypes of HIV-1 strains. As a result, we have a limited ability to assess the potential potency of vaccine-elicited NAbs or to estimate the percentage of circulating HIV-1 isolates that would be neutralized. Further categorization of isolates into distinct subgroups based on sensitivity to NAbs may reveal patterns of neutralization that could provide a greater understanding of the NAb response generated by current and future vaccine immunogens. In addition, the structure-based design of novel immunogens may be facilitated by an ability to monitor the types of viruses neutralized and to specifically map the viral epitopes targeted by vaccine-elicited NAbs.In this study, we assembled a diverse panel of 109 HIV-1 Env pseudoviruses, including multiple representatives from clades A, B, and C and circulating recombinant forms (CRFs) CRF07_BC and CRF02_AG-related. These were tested for their sensitivities using HIV-1-positive (HIV-1⁺) plasma samples representative of clades A, B, and C and CRF01_AE and CRF02_AG. Clinical, demographic, and viral genetic sequence data were collected for each virus. The neutralization phenotype of each virus was assessed with a panel of seven clade-specific HIV-1⁺ plasma pools. Viruses were rank ordered according to average neutralization sensitivity, and k-means clustering was utilized to identify four subgroups of viruses with neutralization phenotypes ranging from highly sensitive to resistant. Together, these results will improve the ability to rigorously evaluate antibody-based HIV-1 vaccines and will facilitate the interpretation of assay results to identify immunogens with improved capacity to elicit broadly cross-reactive NAbs.     

Antibody binding is a dominant determinant of the efficiency of human immunodeficiency virus type 1 neutralization

Primary and laboratory-adapted variants of human immunodeficiency virus type 1 (HIV-1) exhibit a wide range of sensitivities to neutralization by antibodies directed against the viral envelope glycoproteins. An antibody directed against an artificial FLAG epitope inserted into the envelope glycoproteins of three HIV-1 isolates with vastly different neutralization sensitivities inhibited all three viruses equivalently. Thus, naturally occurring HIV-1 isolates that are neutralization resistant are not necessarily more impervious to the inhibitory consequences of bound antibody. Moreover, the binding affinity of the anti-FLAG antibody correlated with neutralizing potency, underscoring the dominant impact on neutralization of antibody binding to the envelope glycoproteins.     

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

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

Refined identification of neutralization-resistant HIV-1 CRF02_AG viruses

We studied neutralization of CRF02_AG HIV-1-infected plasma samples. In contrast to previous reports, these samples neutralized CRF02_AG viruses better than other viruses. This included six of eight CRF02_AG viruses previously designated resistant (tier 2/3 or 3). Only viruses 253-11 and 278-50 remained highly resistant, but they were sensitive to membrane-proximal external region (MPER)-specific monoclonal antibodies, suggesting neutralization targets for even these viruses. We propose using high-neutralizing-within-subtype samples for evaluation of neutralization resistance of viruses.     

Serum Neutralizing Activities from a Beijing Homosexual Male Cohort Infected with Different Subtypes of HIV-1 in China

Protective antibodies play a critical role in an effective HIV vaccine; however, eliciting antibodies to block infection by viruses from diverse genetic subtypes remains a major challenge. As the world’s most populous country, China has been under the threat of at least three major subtypes of circulating HIV-1 viruses. Understanding the cross reactivity and specificities of serum antibody responses that mediate broad neutralization of the virus in HIV-1 infected Chinese patients will provide valuable information for the design of vaccines to prevent HIV-1 transmission in China. Sera from a cohort of homosexual men, who have been managed by a major HIV clinical center in Beijing, China, were analyzed for cross-sectional neutralizing activities against pseudotyped viruses expressing Env antigens of the major subtype viruses (AE, BC and B subtypes) circulating in China. Neutralizing activities in infected patients’ blood were most capable of neutralizing viruses in the homologous subtype; however, a subset of blood samples was able to achieve broad neutralizing activities across different subtypes. Such cross neutralizing activity took 1–2 years to develop and CD4 binding site antibodies were critical components in these blood samples. Our study confirmed the presence of broadly neutralizing sera in China’s HIV-1 patient population. Understanding the specificity and breadth of these neutralizing activities can guide efforts for the development of HIV vaccines against major HIV-1 viruses in China.     

Phenotypic studies on recombinant human immunodeficiency virus type 1 (HIV-1) containing CRF01_AE env gene derived from HIV-1-infected patient,residing in central Thailand

Human immunodeficiency virus type 1 (HIV-1) env genes were cloned from blood samples of HIV-1-infected Thai patients, and 35 infectious CRF01_AE envelope glycoprotein (Env)-recombinant viruses were established. In this report, we examined the neutralization susceptibility of these viruses to human monoclonal antibodies, 2G12, IgG1 b12, 2F5 and 4E10, pooled patient plasma, coreceptor antagonists and fusion inhibitor, T-20. The neutralization susceptibility of CRF01_AE Env-recombinant viruses to 2F5, 4E10, patient plasma, coreceptor antagonists and T-20 varied, while most viruses showed low susceptibility to 2G12 and IgG1 b12. Several dual-tropic viruses showed lower susceptibility to 2F5 and 4E10 than CXCR4- or CCR5-tropic viruses. Neutralization susceptibility of the CRF01_AE Env-recombinant virus to pooled patient plasma was negatively correlated with the length of the V1/V2 region or the number of potential N-linked glycosylation sites in conserved regions of gp120. No correlation was found between the coreceptor usage and neutralization susceptibility of the virus to T-20, whereas several dual-tropic viruses showed higher susceptibility to coreceptor antagonists than CXCR4- or CCR5-tropic viruses. We propose that these CRF01_AE Env-recombinant viruses are useful to further study the molecular mechanism of the susceptibility of CRF01_AE Env to neutralizing antibodies and viral entry inhibitors.     

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

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

Temporal and spatial dynamics of human immunodeficiency virus type 1 circulating recombinant forms 08_BC and 07_BC in Asia

Human immunodeficiency virus type 1 (HIV-1) CRF08_BC and CRF07_BC are two major recombinants descended from subtypes B' and C. Despite their massive epidemic impact in China, their migration patterns and divergence times remain unknown. Phylogenetic and population genetic analyses were performed on 228 HIV-1 sequences representing CRF08_BC, CRF07_BC, and subtype C strains from different locations across China, India, and Myanmar. Genome-specific rates of evolution and divergence times were estimated using a Bayesian Markov chain Monte Carlo framework under various evolutionary models. CRF08_BC originated in 1990.3 (95% credible region [CR], 1988.6 to 1991.9) in Yunnan province before spreading to Guangxi (south) and Liaoning (northeast) around 1995. Inside Guangxi region, the eastward expansion of CRF08_BC continued from Baise city (west) to Binyang (central) between 1997 and 1998 and later spread into Pingxiang around 1999 in the south, mainly through injecting drug users. Additionally, CRF07_BC diverged from its common ancestor in 1993.3 (95% CR, 1991.2 to 1995.2) before crossing the border into southern Taiwan in late 1990s. Phylogenetic analysis indicates that both CRF08_BC and CRF07_BC can trace their origins to Yunnan. The parental Indian subtype C lineage likely entered China around 1981.2 (95% CR, 1976.7 to 1985.9). Using a multiple unlinked locus model, we also showed that the dates of divergence calculated in this study may not be significantly affected by intrasubtype recombination among different lineages. This is the first phylodynamic study depicting the spatiotemporal dynamics of HIV/AIDS in East Asia.     

Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.