The breadth and potency of passively acquired human immunodeficiency virus type 1-specific neutralizing antibodies do not correlate with the risk of infant infection

Although a major goal of human immunodeficiency virus type 1 (HIV-1) vaccine efforts is to elicit broad and potent neutralizing antibodies (NAbs), there are no data that directly demonstrate a role for such NAbs in protection from HIV-1 infection in exposed humans. The setting of mother-to-child transmission provides an opportunity to examine whether NAbs provide protection from HIV-1 infection because infants acquire passive antibodies from their mothers prior to exposure to HIV-1 through breastfeeding. We evaluated the characteristics of HIV-1-specific NAbs in 100 breast-fed infants of HIV-1-positive mothers who were HIV-1 negative at birth and monitored them until age 2. A panel of eight viruses that included variants representative of those in the study region as well as more diverse strains was used to determine the breadth of the infant NAbs. From their mothers, infants acquired broad and potent NAbs that were capable of recognizing heterologous circulating HIV-1 variants of diverse subtypes, but the presence of NAbs of broad HIV-1 specificity was not associated with transmission risk. There was also no correlation between responses to any particular virus tested, which included a range of diverse variants that demonstrated different neutralization profiles, including recognition by specific antibodies with known epitope targets. The eight viruses tested exhibited neutralization profiles to a variety of monoclonal antibodies (2F5, PG9, and VRC01) similar to those of viruses present in pregnant women in the cohort. These results suggest that the breadth and potency of the heterologous antibody response in exposed infants, measured against a virus panel comprised of variants typical of those circulating in the population, does not predict protection.     

Cross-clade HIV-1 neutralizing antibodies induced with V3-scaffold protein immunogens following priming with gp120 DNA

The V3 epitope is a known target for HIV-1 neutralizing antibodies (NAbs), and V3-scaffold fusion proteins used as boosting immunogens after gp120 DNA priming were previously shown to induce NAbs in rabbits. Here, we evaluated whether the breadth and potency of the NAb response could be improved when boosted with rationally designed V3-scaffold immunogens. Rabbits were primed with codon-optimized clade C gp120 DNA and boosted with one of five V3-cholera toxin B fusion proteins (V3-CTBs) or with double combinations of these. The inserts in these immunogens were designed to display V3 epitopes shared by the majority of global HIV-1 isolates. Double combinations of V3-CTB immunogens generally induced more broad and potent NAbs than did boosts with single V3-CTB immunogens, with the most potent and broad NAbs elicited with the V3-CTB carrying the consensus V3 of clade C (V3(C)-CTB), or with double combinations of V3-CTB immunogens that included V3(C)-CTB. Neutralization of tier 1 and 2 pseudoviruses from clades AG, B, and C and of peripheral blood mononuclear cell (PBMC)-grown primary viruses from clades A, AG, and B was achieved, demonstrating that priming with gp120 DNA followed by boosts with V3-scaffold immunogens effectively elicits cross-clade NAbs. Focusing on the V3 region is a first step in designing a vaccine targeting protective epitopes, a strategy with potential advantages over the use of Env, a molecule that evolved to protect the virus by poorly inducing NAbs and by shielding the epitopes that are most critical for infectivity.     

Early HLA-B*57-Restricted CD8+ T Lymphocyte Responses Predict HIV-1 Disease Progression

Although HLA-B*57 (B57) is associated with slow progression to disease following HIV-1 infection, B57 heterozygotes display a wide spectrum of outcomes, including rapid progression, viremic slow progression, and elite control. Efforts to identify differences between B57-positive (B57(+)) slow progressors and B57(+) rapid progressors have largely focused on cytotoxic T lymphocyte (CTL) phenotypes and specificities during chronic stages of infection. Although CTL responses in the early months of infection are likely to be the most important for the long-term rate of HIV-1 disease progression, few data on the early CTL responses of eventual slow progressors have been available. Utilizing the Multicenter AIDS Cohort Study (MACS), we retrospectively examined the early HIV-1-specific CTL responses of 14 B57(+) individuals whose time to development of disease ranged from 3.5 years to longer than 25 years after infection. In general, a greater breadth of targeting of epitopes from structural proteins, especially Gag, as well as of highly conserved epitopes from any HIV-1 protein, correlated with longer times until disease. The single elite controller in the cohort was an outlier on several correlations of CTL targeting and time until disease, consistent with reports that elite control is typically not achieved solely by protective HLA-mediated CTLs. When targeting of individual epitopes was analyzed, we found that early CTL responses to the IW9 (ISPRTLNAW) epitope of Gag, while generally subdominant, correlated with delayed progression to disease. This is the first study to identify early CTL responses to IW9 as a correlate of protection in persons with HLA-B*57.     

HIV-1 Autologous Antibody Neutralization Associates with Mother to Child Transmission

The HIV-1 characteristics associated with mother to child transmission (MTCT) are still poorly understood and if known would indicate where intervention strategies should be targeted. In contrast to horizontally infected individuals, exposed infants possess inherited antibodies (Abs) from their mother with the potential to protect against infection. We investigated the HIV-1 gp160 envelope proteins from seven transmitting mothers (TM) whose children were infected either during gestation or soon after delivery and from four non-transmitting mothers (NTM) with similar viral loads and CD4 counts. Using pseudo-typed viruses we tested gp160 envelope glycoproteins for TZM-bl infectivity, CD4 and CCR5 interactions, DC-SIGN capture and transfer and neutralization with an array of common neutralizing Abs (NAbs) (2F5, 2G12, 4E10 and b12) as well as mother and infant plasma. We found no viral correlates associated with HIV-1 MTCT nor did we find differences in neutralization with the panel of NAbs. We did, however, find that TM possessed significantly higher plasma neutralization capacities than NTM (P  = 0.002). Furthermore, we found that in utero (IU) TM had a higher neutralization capacity than mothers transmitting either peri - partum (PP) or via breastfeeding (BF) (P  = 0.002). Plasma from children infected IU neutralized viruses carrying autologous gp160 viral envelopes as well as those from their corresponding mothers whilst plasma from children infected PP and/or BF demonstrated poor neutralizing capacity. Our results demonstrate heightened autologous NAb responses against gp120/gp41 can associate with a greater risk of HIV-1 MTCT and more specifically in those infants infected IU. Although the number of HIV-1 transmitting pairs is low our results indicate that autologous NAb responses in mothers and infants do not protect against MTCT and may in fact be detrimental when considering IU HIV-1 transmissions.     

Infrequent detection of HIV-1-specific, but not cytomegalovirus-specific, CD8(+) T cell responses in young HIV-1-infected infants.

Early potent combination antiretroviral therapies (ART) for HIV-1 infection can preserve or restore immune function, but control of viral replication early in infection may interfere with the development of HIV-1-specific immune responses. Using an IFN-gamma ELISPOT assay, we evaluated the breadth and intensity of HIV-1-specific CD8(+) T cell responses in 17 vertically infected infants who began ART at 1-23 mo of age. CMV-specific responses were also characterized in three infants coinfected with HIV-1 and CMV. Before ART, HIV-1-specific CD8(+) T cell responses were detected in two of 13 (15%) infants <6 mo of age. HIV-1-specific CD8(+) T cells became undetectable in these two infants after the control of viral replication. Intermittent HIV-1-specific responses were noted in six infants who did not experience durable control of viral replication. In contrast, HIV-1-specific responses were detected before ART in four of four infants >6 mo of age and became persistently undetectable in only one child. CMV-specific CD8(+) T cell responses were persistently detected in all HIV-1 and CMV coinfected infants. In conclusion, HIV-1-specific CD8(+) T cell responses were less commonly detected before therapy in young infants than in older infants. Suppression of viral replication appeared to interfere with the development and maintenance of HIV-1-specific CD8(+) T cell responses. The detection of CMV-specific responses in HIV-1 and CMV coinfected infants suggests a selective defect in the generation or maintenance of HIV-1-specific CD8(+) T cell responses. Therapeutic HIV-1 vaccine strategies in young infants may prolong the clinical benefit of ART by expanding the HIV-1-specific CD8(+) T cell pool.     

HIV-1 Superinfection in Women Broadens and Strengthens the Neutralizing Antibody Response

Identifying naturally-occurring neutralizing antibodies (NAb) that are cross-reactive against all global subtypes of HIV-1 is an important step toward the development of a vaccine. Establishing the host and viral determinants for eliciting such broadly NAbs is also critical for immunogen design. NAb breadth has previously been shown to be positively associated with viral diversity. Therefore, we hypothesized that superinfected individuals develop a broad NAb response as a result of increased antigenic stimulation by two distinct viruses. To test this hypothesis, plasma samples from 12 superinfected women each assigned to three singly infected women were tested against a panel of eight viruses representing four different HIV-1 subtypes at matched time points post-superinfection (∼5 years post-initial infection). Here we show superinfected individuals develop significantly broader NAb responses post-superinfection when compared to singly infected individuals (RR = 1.68, CI: 1.23–2.30, p = 0.001). This was true even after controlling for NAb breadth developed prior to superinfection, contemporaneous CD4+ T cell count and viral load. Similarly, both unadjusted and adjusted analyses showed significantly greater potency in superinfected cases compared to controls. Notably, two superinfected individuals were able to neutralize variants from four different subtypes at plasma dilutions >1∶300, suggesting that their NAbs exhibit elite activity. Cross-subtype breadth was detected within a year of superinfection in both of these individuals, which was within 1.5 years of their initial infection. These data suggest that sequential infections lead to augmentation of the NAb response, a process that may provide insight into potential mechanisms that contribute to the development of antibody breadth. Therefore, a successful vaccination strategy that mimics superinfection may lead to the development of broad NAbs in immunized individuals.     

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.     

Neutralizing Antibody Escape during HIV-1 Mother-to-Child Transmission Involves Conformational Masking of Distal Epitopes in Envelope

HIV-1 variants transmitted to infants are often resistant to maternal neutralizing antibodies (NAbs), suggesting that they have escaped maternal NAb pressure. To define the molecular basis of NAb escape that contributes to selection of transmitted variants, we analyzed 5 viruses from 2 mother-to-child transmission pairs, in which the infant virus, but not the maternal virus, was resistant to neutralization by maternal plasma near transmission. We generated chimeric viruses between maternal and infant envelope clones obtained near transmission and examined neutralization by maternal plasma. The molecular determinants of NAb escape were distinct, even when comparing two maternal variants to the transmitted infant virus within one pair, in which insertions in V4 of gp120 and substitutions in HR2 of gp41 conferred neutralization resistance. In another pair, deletions and substitutions in V1 to V3 conferred resistance, but neither V1/V2 nor V3 alone was sufficient. Although the sequence determinants of escape were distinct, all of them involved modifications of potential N-linked glycosylation sites. None of the regions that mediated escape were major linear targets of maternal NAbs because corresponding peptides failed to compete for neutralization. Instead, these regions disrupted multiple distal epitopes targeted by HIV-1-specific monoclonal antibodies, suggesting that escape from maternal NAbs occurred through conformational masking of distal epitopes. This strategy likely allows HIV-1 to utilize relatively limited changes in the envelope to preserve the ability to infect a new host while simultaneously evading multiple NAb specificities present in maternal plasma.     

B cell depletion in HIV-1 subtype A infected Ugandan adults: relationship to CD4 T cell count, viral load and humoral immune responses

To better understand the nature of B cell dysfunctions in subjects infected with HIV-1 subtype A, a rural cohort of 50 treatment-naïve Ugandan patients chronically infected with HIV-1 subtype A was studied, and the relationship between B cell depletion and HIV disease was assessed. B cell absolute counts were found to be significantly lower in HIV-1+ patients, when compared to community matched negative controls (p<0.0001). HIV-1-infected patients displayed variable functional and binding antibody titers that showed no correlation with viral load or CD4+ T cell count. However, B cell absolute counts were found to correlate inversely with neutralizing antibody (NAb) titers against subtype A (p = 0.05) and subtype CRF02_AG (p = 0.02) viruses. A positive correlation was observed between subtype A gp120 binding antibody titers and NAb breadth (p = 0.02) and mean titer against the 10 viruses (p = 0.0002). In addition, HIV-1 subtype A sera showed preferential neutralization of the 5 subtype A or CRF02_AG pseudoviruses, as compared with 5 pseudoviruses from subtypes B, C or D (p<0.001). These data demonstrate that in patients with chronic HIV-1 subtype A infection, significant B cell depletion can be observed, the degree of which does not appear to be associated with a decrease in functional antibodies. These findings also highlight the potential importance of subtype in the specificity of cross-clade neutralization in HIV-1 infection.     

Longitudinal assessment of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon responses during the first year of life in HIV-1-infected infants

Human immunodeficiency virus type 1 (HIV-1) infection results in different patterns of viral replication in pediatric compared to adult populations. The role of early HIV-1-specific responses in viral control has not been well defined, because most studies of HIV-1-infected infants have been retrospective or cross-sectional. We evaluated the association between HIV-1-specific gamma interferon (IFN-gamma) release from the cells of infants of 1 to 3 months of age and peak viral loads and mortality in the first year of life among 61 Kenyan HIV-1-infected infants. At 1 month, responses were detected in 7/12 (58%) and 6/21 (29%) of infants infected in utero and peripartum, respectively (P = 0.09), and in approximately 50% of infants thereafter. Peaks of HIV-specific spot-forming units (SFU) increased significantly with age in all infants, from 251/10(6) peripheral blood mononuclear cells (PBMC) at 1 month of age to 501/10(6) PBMC at 12 months of age (P = 0.03), although when limited to infants who survived to 1 year, the increase in peak HIV-specific SFU was no longer significant (P = 0.18). Over the first year of life, infants with IFN-gamma responses at 1 month had peak plasma viral loads, rates of decline of viral load, and mortality risk similar to those of infants who lacked responses at 1 month. The strength and breadth of IFN-gamma responses at 1 month were not significantly associated with viral containment or mortality. These results suggest that, in contrast to HIV-1-infected adults, in whom strong cytotoxic T lymphocyte responses in primary infection are associated with reductions in viremia, HIV-1-infected neonates generate HIV-1-specific CD8+-T-cell responses early in life that are not clearly associated with improved clinical outcomes.     

Antibody responses elicited in macaques immunized with human immunodeficiency virus type 1 (HIV-1) SF162-derived gp140 envelope immunogens: comparison with those elicited during homologous simian/human immunodeficiency virus SHIVSF162P4 and heterologous HIV-1 infection

The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIV(SF162P4), and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, DeltaV2gp140 (lacking the crown of the V2 loop), DeltaV3gp140 (lacking the crown of the V3 loop), and DeltaV2DeltaV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and DeltaV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and DeltaV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by DeltaV3gp140 or DeltaV2DeltaV3gp140. In contrast, the SHIV(SF162P4)-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.     

Comprehensive analysis of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-secreting CD8+ T cells in primary HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) T cells provide an important defense in controlling HIV-1 replication, particularly following acquisition of infection. To delineate the breadth and potency of these responses in patients upon initial presentation and before treatment, we determined the fine specificities and frequencies of gamma interferon (IFN-gamma)-secreting CD8(+) T cells recognizing all HIV-1 proteins in patients with primary infection. In these subjects, the earliest detected responses were directed predominantly against Nef, Tat, Vpr, and Env. Tat- and Vpr-specific CD8(+) T cells accounted for the greatest frequencies of mean IFN-gamma spot-forming cells (SFC). Nef-specific responses (10 of 21) were more commonly detected. A mean of 2.3 epitopes were recognized with various avidities per subject, and the number increased with the duration of infection (R = 0.47, P = 0.031). The mean frequency of CD8(+) T cells (985 SFC/10(6) peripheral blood mononuclear cells) correlated with the number of epitopes recognized (R = 0.84, P < 0.0001) and the number of HLA-restricting alleles (R = 0.79, P < 0.0001). Neither the total SFC frequencies nor the number of epitopes recognized correlated with the concurrent plasma viral load. Seventeen novel epitopes were identified, four of which were restricted to HLA alleles (A23 and B72) that are common among African descendents. Thus, primary HIV-1 infection induces strong CD8(+)-T-cell immunity whose specificities broaden over time, but their frequencies and breadth do not correlate with HIV-1 containment when examined concurrently. Many novel epitopes, particularly directed to Nef, Tat, and Env, and frequently with unique HLA restrictions, merit further consideration in vaccine design.     

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。     

Human immunodeficiency virus type 1 superinfection occurs despite relatively robust neutralizing antibody responses

Superinfection by a second human immunodeficiency virus type 1 (HIV-1) strain indicates that gaps in protective immunity occur during natural infection. To define the role of HIV-1-specific neutralizing antibodies (NAbs) in this setting, we examined NAb responses in 6 women who became superinfected between ~1 to 5 years following initial infection compared to 18 women with similar risk factors who did not. Although superinfected individuals had less NAb breadth than matched controls at ~1 year postinfection, no significant differences in the breadth or potency of NAb responses were observed just prior to the second infection. In fact, four of the six subjects had relatively broad and potent NAb responses prior to infection by the second strain. To more specifically examine the specificity of the NAbs against the superinfecting virus, these variants were cloned from five of the six individuals. The superinfecting variants did not appear to be inherently neutralization resistant, as measured against a pool of plasma from unrelated HIV-infected individuals. Moreover, the superinfected individuals were able to mount autologous NAb responses to these variants following reinfection. In addition, most superinfected individuals had NAbs that could neutralize their second viral strains prior to their reinfection, suggesting that the level of NAbs elicited during natural infection was not sufficient to block infection. These data indicate that preventing infection by vaccination will likely require broader and more potent NAb responses than those found in HIV-1-infected individuals.     

Antibody responses induced by SHIV infection are more focused than those induced by soluble native HIV-1 envelope trimers in non-human primates

The development of an effective human immunodeficiency virus (HIV-1) vaccine is a high global health priority. Soluble native-like HIV-1 envelope glycoprotein trimers (Env), including those based on the SOSIP design, have shown promise as vaccine candidates by inducing neutralizing antibody responses against the autologous virus in animal models. However, to overcome HIV-1’s extreme diversity a vaccine needs to induce broadly neutralizing antibodies (bNAbs). Such bNAbs can protect non-human primates (NHPs) and humans from infection. The prototypic BG505 SOSIP.664 immunogen is based on the BG505 env sequence isolated from an HIV-1-infected infant from Kenya who developed a bNAb response. Studying bNAb development during natural HIV-1 infection can inform vaccine design, however, it is unclear to what extent vaccine-induced antibody responses to Env are comparable to those induced by natural infection. Here, we compared Env antibody responses in BG505 SOSIP-immunized NHPs with those in BG505 SHIV-infected NHPs, by analyzing monoclonal antibodies (mAbs). We observed three major differences between BG505 SOSIP immunization and BG505 SHIV infection. First, SHIV infection resulted in more clonal expansion and less antibody diversity compared to SOSIP immunization, likely because of higher and/or prolonged antigenic stimulation and increased antigen diversity during infection. Second, while we retrieved comparatively fewer neutralizing mAbs (NAbs) from SOSIP-immunized animals, these NAbs targeted more diverse epitopes compared to NAbs from SHIV-infected animals. However, none of the NAbs, either elicited by vaccination or infection, showed any breadth. Finally, SOSIP immunization elicited antibodies against the base of the trimer, while infection did not, consistent with the base being placed onto the virus membrane in the latter setting. Together these data provide new insights into the antibody response against BG505 Env during infection and immunization and limitations that need to be overcome to induce better responses after vaccination.     

HIV-1 Genetic Diversity and Its Impact on Baseline CD4+T Cells and Viral Loads among Recently Infected Men Who Have Sex with Men in Shanghai,China

The HIV-1 epidemic among men who have sex with men (MSM) has been spreading throughout China. Shanghai, a central gathering place for MSM, is facing a continuously increasing incidence of HIV-1 infection. In order to better understand the dynamics of HIV-1 diversity and its influence on patient’s immune status at baseline on diagnosis, 1265 newly HIV-1-infected MSM collected from January 2009 to December 2013 in Shanghai were retrospectively analyzed for genetic subtyping, CD4+T cell counts, and viral loads. HIV-1 phylogenetic analysis revealed a broad viral diversity including CRF01_AE (62.13%), CRF07_BC (24.51%), subtype B (8.06%), CRF55_01B (3.24%), CER67_01B (0.95%), CRF68_01B (0.4%), CRF08_BC (0.08%) and CRF59_01B (0.08%). Twenty-four unique recombination forms (URFs) (1.98%) were identified as well. Bayesian inference analysis indicated that the introduction of CRF01_AE strain (1997) was earlier than CRF07_BC strain (2001) into MSM population in Shanghai based on the time of the most recent common ancestor (tMRCA). Three epidemic clusters and five sub-clusters were found in CRF01_AE. Significantly lower CD4+T cell count was found in individuals infected with CRF01_AE than in those infected with CRF07_BC infection (P<0.01), whereas viral load was significantly higher those infected with CRF01_AE than with CRF07_BC (P<0.01). In addition, the patients with >45 years of age were found to have lower CD4+T cell counts and higher viral loads than the patients with <25 years of age (P<0.05). This study reveals the presence of HIV-1 subtype diversity in Shanghai and its remarkable influence on clinical outcome. A real-time surveillance of HIV-1 viral diversity and phylodynamics of epidemic cluster, patient’s baseline CD4+T cell count and viral load would be of great value to monitoring of disease progression, intervention for transmission, improvement of antiretroviral therapy strategy and design of vaccines.     

CRF01_AE-Specific Neutralizing Activity Observed in Plasma Derived from HIV-1-Infected Thai Patients Residing in Northern Thailand: Comparison of Neutralizing Breadth and Potency between Plasma Derived from Rapid and Slow Progressors

Background

Development of a protective vaccine against human immunodeficiency virus type 1 (HIV-1) is an important subject in the field of medical sciences; however, it has not yet been achieved. Potent and broadly neutralizing antibodies are found in the plasma of some HIV-1-infected patients, whereas such antibody responses have failed to be induced by currently used vaccine antigens. In order to develop effective vaccine antigens, it is important to reveal the molecular mechanism of how strong humoral immune responses are induced in infected patients. As part of such studies, we examined the correlation between the anti-HIV-1 neutralizing antibody response and disease progression.

Methodology/Principal Findings

We evaluated the anti-HIV-1 neutralizing activity of plasma derived from 33 rapid and 34 slow progressors residing in northern Thailand. The level of neutralizing activity varied considerably among plasmas, and no statistically significant differences in the potency and breadth of neutralizing activities were observed overall between plasma derived from rapid and slow progressors; however, plasma of 4 slow progressors showed neutralizing activity against all target viruses, whereas none of the plasma of rapid progressors showed such neutralizing activity. In addition, 21% and 9% of plasmas derived from slow and rapid progressors inhibited the replication of more than 80% of CRF01_AE Env-recombinant viruses tested, respectively. Neutralization of subtype B and C Env-recombinant viruses by the selected plasma was also examined; however, these plasma samples inhibited the replication of only a few viruses tested.

Conclusions/Significance

Although no statistically significant differences were observed in the potency and breadth of anti-HIV-1 neutralizing activities between plasma derived from rapid and slow progressors, several plasma samples derived from slow progressors neutralized CRF01_AE Env-recombinant viruses more frequently than those from rapid progressors. In addition, plasma derived from HIV-1-infected Thai patients showed CRF01_AE-specific neutralizing activity.     

海南省及云南西双版纳州HIV-1的分子流行病学比较研究

目的调查比较HIV-1在海南省及云南西双版纳州的流行情况及分子流行病学分析高危因素传播的情况以及传播链的鉴定。方法我们与海南省CDC及西双版纳州CDC合作在其境内开展了HIV血清学调查,筛查了海南省（1991～2006）及西双版纳州（1996～2005）高危人群志愿者血清样本。在本次两地调查中,我们通过系统进化分析方法,对已诊断的HIV感染者进行分子流行病学的追踪,来分析其中的主要流行亚群以及结合个案追踪进行传播链的鉴定。结果我们共筛查了海南省499725人,共检出HIV阳性感染者523例（0.1%）,以注射器吸毒感染为主要传播途径（经共用注射器吸毒感染占69．2％、经性接触传播占19．3％、供血和使用血液／血液制品感染占3．3％、母婴传播占0．8％、不详占7．7％）。然而,在西双版纳州筛查中发现了较海南省高20倍的检出率：在25390受检人中,共检出HIV阳性感染者501例（2％）并以异性性传播为主（经异性性接触传播占77．3％、经共用注射器吸毒感染占21．1％、经同性性接触传播占0．4％、母婴传播占1．2％）。在海南省抽样的83人中,以CRF01_-AE重组亚型（70人,84．3％）为主要病毒亚型,其他病毒亚型包括B’亚型（8人）、C亚型（2人）、CRF08_BC重组亚型（1人）、B亚型（1人）和1个未报道过的CRF01～AE／B’重组亚型。同样,在版纳抽样的44人中,也以CRF01_AE重组亚型（27人,61．3％）为主要病毒亚型,其他病毒亚型包括CRF08_BC重组亚型（15人）、G亚型（1人）、和1个未报道过的B／C重组亚型。在海南省抽样中有66人（79．5％）分布于4个大小不同的传播群,传播群1（59人）较大（奠基效应）,属于CRF01_AE重组亚型,传播群2、3、4则较小,分别为3、2、2人,分别属于CRF01_AE重组亚型、C亚型和B’亚型。相反,在版纳抽样中有18人（40．9％）分布于8个较小的传播群（每群平均含2．25感染个体）。在海南省抽样中怀疑的6对异性性接触的传播链中,经系统进化分析方法确认了其中的4对,2对被拒绝。在版纳抽样中怀疑的8对异性性接触的传播链中,经系统进化分析方法确认了其中的5对,2对被拒绝,1对无法分析,此外还新发现了3对异性性接触传播群。结论HIV-1感染者的分子流行病学的追踪和分析对于追溯艾滋病流行的源头和地区性预防策略的制定有着重大的意义。     

Preservation of T cell proliferation restricted by protective HLA alleles is critical for immune control of HIV-1 infection

HIV-1-infected persons with HLA-B27 and -B57 alleles commonly remain healthy for decades without antiretroviral therapy. Properties of CD8+ T cells restricted by these alleles considered to confer disease protection in these individuals are elusive but important to understand and potentially elicit by vaccination. To address this, we compared CD8+ T cell function induced by HIV-1 immunogens and natural infection using polychromatic flow cytometry. HIV-1-specific CD8+ T cells from all four uninfected immunized and 21 infected subjects secreted IFN-gamma and TNF-alpha. However, CD8+ T cells induced by vaccination and primary infection, but not chronic infection, proliferated to their cognate epitopes. Notably, B27- and B57-restricted CD8+ T cells from nonprogressors exhibited greater expansion than those restricted by other alleles. Hence, CD8+ T cells restricted by certain protective alleles can resist replicative defects, which permits expansion and antiviral effector activities. Our findings suggest that the capacity to maintain CD8+ T cell proliferation, regardless of MHC-restriction, may serve as an important correlate of disease protection in the event of infection following vaccination.     

In vitro inhibition of HIV-1 replication in autologous CD4~+ T cells indicates viral containment by multifactorial mechanisms

HIV-1-specific cytotoxic T lymphocytes(CTLs) and neutralizing antibodies(NAbs) are present during chronic infection, but the relative contributions of these effector mechanisms to viral containment remain unclear. Here, using an in vitro model involving autologous CD4+ T cells,primary HIV-1 isolates, HIV-1-specific CTLs, and neutralizing monoclonal antibodies, we show that b12, a potent and broadly neutralizing monoclonal antibody to HIV-1, was able to block viral infection when preincubated with virus prior to infection, but was much less effective than CTLs at limiting virus replication when added to infected cell cultures. However, the same neutralizing antibody was able to contain viruses by antibody-dependent cell-mediated virus inhibition in vitro,which was mediated by natural killer cells(NKs) and dependent on an Fc-Fc receptor interaction.Meanwhile, bulk CTLs from HIV-1 controllers were more effective in suppression of virus replication than those from progressors. These findings indicate that control of HIV-1 replication in activated CD4~+ T cells is ineffectively mediated by neutralizing antibodies alone, but that both CTLs and antibody-dependent NK-mediated immune mechanisms contribute to viral containment. Our study systemically compared three major players in controlling HIV-1 infection, CTLs, NAbs, and NKs, in an autologous system and highlighted the multifactorial mechanisms for viral containment and vaccine success.