Assessment of the population coverage of an HIV-1 vaccine targeting sequences surrounding the viral protease cleavage sites in Gag,Pol, or all 12 protease cleavage sites

Development of an effective HIV-1 vaccine has been a great challenge faced by the research community. Recently a novel strategy targeting the viral protease cleavage sites (PCSs) has been tested and shown promising results. This T cell-based vaccine strategy depends on individuals expressing certain HLA class I molecules and since each population has unique distributions of HLA class I alleles, population coverage analysis is required to assess feasibility. Utilizing the validated CD8 T cell epitope data from previous studies we conducted coverage analysis of an HIV-1 vaccine targeting the sequences surrounding all 12-PCSs, Gag-PCSs, and Pol-PCSs. The population coverage, average epitope hit, and minimum number of epitopes recognized by 90% of the population (PC90) was compiled for 66 countries and 16 geographical regions using the web tool provided by “Immune Epitope Database and Analysis Resource”. Our analysis shows that the coverage for an HIV-1 vaccine targeting sequences surrounding all 12 PCSs, 5 PCSs in Gag or 6 PCSs in Pol can cover ~ 70% to ~ 100% of the populations analyzed. There was no statistical difference in population coverages for the majority of populations examined when comparing the CD8 T cell epitope sets (12-PCSs, Gag-PCSs, and Pol-PCSs). As expected, vaccines targeting more sequences will have more CD8 T cell epitopes, as the mean average epitope hit for the 12-PCSs, Gag-PCSs, and Pol-PCSs across all countries studied was 9.45, 4.76, and 4.74, respectively, and across all geographical regions was 9.76, 4.99, and 4.92, respectively. The average PC90 for the 12-PCSs, Gag-PCSs, and Pol-PCSs across all countries studied was 2.53, 1.31, and 1.41, respectively, and across all geographical regions was 2.24, 1.23, and 1.29, respectively. Thus, vaccines targeting sequences surrounding the HIV-1 PCSs can cover broad populations; however, whether targeting a subset of the PCSs is sufficient to prevent acquisition requires further preclinical investigation.     

HIV-1病毒储存库的特征及检测方法

HIV-1在未治疗的艾滋病患者体内,虽可以持续复制,但也可潜伏在细胞内以逃避宿主的免疫清除,这一现象即病毒潜伏.由于病毒潜伏对抗病毒治疗带来了挑战,也引起人们的高度关注.此文对体外研究HIV-1潜伏库的实验方法 进展进行综述.     

Immunization with an HIV-1 immunogen induces CD4+ and CD8+ HIV-1-specific polyfunctional responses in patients with chronic HIV-1 infection receiving antiretroviral therapy

Development of polyfunctional T lymphocyte responses is critical in the immunological response against HIV-1. Fifty-four HIV-1 infected patients receiving antiretroviral treatment (ART) and immunization with an HIV-1 immunogen or placebo, periodically every 3 months throughout a period of 36 months, were evaluated for the purposes of analysing the development of HIV-1-specific CD4⁺ and CD8⁺ responses. A significant increase of proliferating and IFN-γ producing CD8⁺ HIV-1-specific T cells, of HIV-1-specific precursor frequencies for CD8⁺ and for CD4⁺ T cells and of Gag/pol-specific memory CTL precursors (CTLp) was observed in the immunogen group in comparison to placebo. IL-2 intracellular expression and IFN-γ and TNF- co-expression in HIV-1-specific CD8⁺ T cells were also substantially increased in the immunized group. A negative correlation between viral load and CD3⁺CD4⁺CFSE^low HIV-1-specific lymphoproliferative response and frequency of Gag/pol-specific CTLp was solely observed in the HIV-1 immunogen group. Long-term immunization in patients receiving ART helps to develop HIV-1-specific polyfunctional T cell responses.     

Quantitative assessment of masking of neutralization epitopes in HIV-1

Despite the frequent observation of masking of HIV-1 neutralization epitopes, its extent has not been previously systematically assessed either for multiple epitopes presented by individual viruses or for individual epitopes across multiple viral strains. Using a recently developed method to identify amino acid sequence motifs required for recognition by HIV-1-neutralizing monoclonal antibodies (mAbs), we visualized the patterns of masking of specific epitopes targeted by mAbs in a diverse panel of HIV-1 isolates. We also calculated a specific masking intensity score for each virus based on the observed neutralization activity of mAbs against the epitopes in the virus. Finally, we combined these data with estimates of the conservation of each mAb-targeted epitope in circulating HIV-1 strains to estimate the effective neutralization potential (E_N) for each mAb. Focusing on the V3 loop of gp120 as a prototype neutralization domain, we found that the V3 loop epitope targeted by mAb 2219 is one of the least masked mAbs and it has the highest E_N. Interestingly, although the V3 loop epitope targeted by mAb 3074 is present in over 87% of all viruses, it is 82.2% masked, so its E_N is lower than that for mAb 2219. Notably, 50% of the viruses that mAb 3074 is able to neutralize are classified as subtype C viruses, while 70% or more of the viruses neutralized by mAbs 2219, 2557 or 447-52D are classified as subtype B. Thus, neutralization epitopes (in this case, in the V3 loop) have differential patterns of masking and also display distinct patterns of distribution among circulating HIV-1 viruses. Both factors combine to contribute to the practical vaccine value of any single epitope/mAb. Here we have developed a quantitative score for this value. These results have important implications for rational design of vaccines designed to induce neutralizing Abs by revealing epitopes that are minimally masked and maximally reactive with neutralizing Abs.     

Cross-reactive potential of human T-lymphocyte responses in HIV-1 infection

An effective HIV-1 vaccine should elicit sufficient breadth of immune recognition to protect against the genetically diverse forms of the circulating virus. Evaluation of the breadth and magnitude of cellular immune responses to epitope variants is important for HIV-1 vaccine assessment. We compared HIV-1 Gag-specific T-lymphocyte responses in 20 HIV-1-infected individuals representing two different HIV-1 subtypes, B and C. By assessing T lymphocyte responses with peptides based on natural HIV-1 variants, we found evidence for limited cross-reactivity and significantly enhanced within-clade responses among clade B-infected subjects, and not among clade C-infected subjects.     

Packaging limits and stability of HIV-1 sequences in a coxsackievirus B vector

Enteroviruses elicit protective mucosal immune responses that could be harnessed as part of a strategy to prevent sexual transmission of the human immunodeficiency virus-1 (HIV-1). We report the construction of replication-competent recombinant vectors of coxsackievirus B3 (CVB3) that express one or more portions of the HIV-1 Gag protein. Vectors containing the capsid domain of Gag were initially genetically unstable with protein expression lost after brief passage in tissue culture. Codon modification to increase the G/C content of the HIV-1 capsid sequence resulted in enhanced genetic stability of CVB3 vectors during in vitro passage. Cells infected with a vector expressing the matrix (MA) subunit of the HIV-1 Gag protein were susceptible to lysis by CD8 T cell clones specific for the SL9 epitope found within MA. These studies suggest that CVB3 vectors may be useful as vaccine vector candidates, if hurdles in class I antigen presentation and stability can be overcome.     

Improving immunogenicity of HIV-1 envelope gp120 by glycan removal and immune complex formation

HIV-1 envelope (Env) gp120 is an important target for neutralizing antibody (Ab) responses against the virus; however, developing gp120 vaccines that elicit potent and broad neutralizing Abs has proven to be a formidable challenge. Previously, removal of an N-linked glycan at residue 448 by an N to Q mutation (N448Q) has been found to enhance the in vitro antigenicity of neutralizing epitopes in the V3 loop. In this study the mutated gp120 was first compared with wild type gp120 for immunogenicity in mice using a DNA prime and protein boost immunization regimen. The N448Q mutant did not elicit higher titers of anti-gp120 serum Abs and failed to generate anti-V3 Abs. The sera also had no virus-neutralizing activity, even though the mutant induced higher levels of lymphoproliferation and cytokine production. Subsequently, the N448Q mutant was used to construct an immune complex vaccine with the anti-CD4 binding site monoclonal antibody (mAb) 654. The N448Q/654 complex stimulated comparably high levels of serum Abs to gp120 and V3 as the wild type complex. However, Abs against the C1 and C2 regions in the gp120 core were more elevated. Importantly, the mutant complex also elicited higher titers of neutralizing Abs activity than the wild type counterpart. Similar results were achieved with a complex made with gp120 bearing an N448E mutation, confirming the importance of the N448-linked glycan in modulating gp120 immunogenicity. Neutralizing activity was directed to V3 and other undefined neutralizing epitopes. Improved immunogenicity of the immune complexes correlated with alterations in exposure of V3 and other Ab epitopes and their stability against proteases. These data demonstrate the advantage of combining site-specific N-glycan removal and immune complex formation as a novel vaccine strategy to improve immunogenicity of targeted Ab epitopes on critical regions of HIV-1 gp120.     

宁夏HIV-1感染者原发性耐药及基因变异型研究

目的 掌握本地区HIV-1 病毒耐药毒株的流行与传播情况,为有效的抗病毒治疗提供参考依据。方法 2018年3月1日-6月30日在宁夏开展HIV/AIDS抗病毒治疗前HIV-1耐药的横断面调查。提取获得HIV-1 pol基因区序列,根据2015年WHO耐药监测指南的推荐标准,采用Shafer 的耐药性突变积分表耐药评分,分值≥15判为耐药。结果 调查68例HIV/AIDS,58例巢式PCR检测获得HIV-1病毒pol区基因序列。年龄中位数为36.5岁（IQR:30～50）;男性55例(94.8%),女性3例(5.2%), Shafer 的耐药性突变积分表耐药评分,7例判为耐药,耐药率12.1%。存在4种基因亚型,分别是B亚型、CRF01_AE 亚型、CRF07_BC亚型和CRF55_01B亚型。结论 宁夏HIV-1原发性耐药率处于较高水平,应继续加强艾滋病抗病毒治疗前耐药监测。     

Phylogeographic analysis on the travel-related introduction of HIV-1 non-B subtypes to Northern Poland

Phylodynamic, sequence data based reconstructions for the surveillance of the geographic spatial spread are a powerful tool in molecular epidemiology. In this study region of origin for the set of 57 partial pol sequences derived from the patients the history of travel-related HIV transmission was analyzed using phylogeographic approach. Maximum likelihood trees based on the sets of country-annotated reference sequences were inferred for identified non-B variants. Region of sequence import was assigned using on the highest approximate likelihood ratios. Import of the A1 clades was traced to the Eastern Europe and associated with immigration from this region. Subtype C infections clustered most frequently with sequences of the South African origin while majority of subtype Ds were similar to the European clades. Subtype G sequences clustered with Portuguese lineage, CRF01_AE with Eastern or South-Eastern Asian. Eastern European, Middle African or Western African lineage was assigned for the CFR02_AG. Rare circulating recombinants originated either from Central Africa (CRF11_cpx – Democratic Republic of Congo, CRF13_cpx – Central African Republic, CRF37_cpx – Cameroon) or South America (CRF28_BF and CRF46_BF - Brazil). Import of the HIV-1 non-B variants, including recombinant forms previously rarely found in Poland and Europe is frequent among travelers. Observed founder events result in the heterosexually-driven introduction of the novel HIV-1 variants into the population.     

同性和异性传播感染人群的HIV-1亚型分布和特征分析

目的了解河北省男男同性传播和异性传播人群HIV-1亚型分布和特征。方法从感染者的血浆样品中提取病毒RNA,逆转录后采用套式PCR扩增HIV-1 gag和env基因的部分片段,对PCR产物直接进行核苷酸序列测定,所获序列与各亚型国际参考株序列比对,确定基因型并进行系统进化树分析。结果同性传播的52例HIV-1感染者中,发现了3种HIV-1亚型和重组型,以CRF01-AE为主,占65.4%(34/52),其次为B亚型占30.8%(16/52);异性传播的60例HIV-1感染者中,存在6种亚型和重组型,以B亚型为主占43.3%(26/60),其次是CRF01-AE占30.0%(18/60),CRF07-BC占15%(9/60);两类人群的亚型分布差异有统计学意义(P<0.001)。异性感染人群的各亚型组内基因距离均大于同性感染人群。结论在河北省同性传播人群和异性传播人群中HIV-1亚型分布不均衡。     

A universal anti-HIV-1 Tat epitope vaccine that is fully synthetic and self-adjuvanting

Circulating HIV-1 Tat protein is essential for maintenance of the chronic HIV replication that predicates both HIV transmission and clinical progression to AIDS/death. A synthetic anti-Tat epitope vaccine (TUTI-16) was designed to induce neutralizing antibodies to Tat and, hopefully, provide immunological control of HIV replication. TUTI-16 is composed of (1) a conserved Tat B cell epitope (Tat 4–12), rendered universal by introducing known variant amino acids at variable positions 7, 9 and 12 during solid phase synthesis, (2) a promiscuous T helper sequence and (3) a lipopeptide toll-like receptor 2 (TLR2) agonist. TUTI-16 induced high titer antibodies against all 8 known variants of the Tat epitope.     

Mucosal antibodies induced by tandem repeat of 2F5 epitope block transcytosis of HIV-1

Induction of mucosal antibodies to prevent HIV infection is an important strategy for the HIV-1 prophylaxis. Here we report an epitope-vaccine based antigen that was able to elicit mucosal antibodies capable of blocking HIV-1 transcytosis. Because the ELDKWA epitope of neutralizing antibody 2F5 plays a crucial role in transcytosis, a series of immunogens that contain tandem copies of ELDKWA were prepared. Mice were immunized with these immunogens intranasally, and received intraperitoneal + intranasal boosters. The immunogens that contained more ELDKWA epitopes elicited higher level of mucosal ELDKWA-epitope specific IgAs and systemic IgGs. Although the antisera from the immunized mice exhibited mild neutralizing potency to HIV-1 isolates HXB2 and JRFL, the affinity purified mucosal ELDKWA-epitope specific antibodies could block the transcytosis of cell-free CNE3 (a primary isolate of subtype CRF01_AE) in human tight epithelial models.     

HIV-1 CRF55_01B毒株流行特征的研究进展

CRF55_01B是我国报道的HIV-1主要流行毒株之一,具有较为独特的流行病学和基因进化特征。近几年HIV-1分子流行病学监测发现CRF55_01B已经成为我国第五个主要流行HIV-1毒株。本研究从他的发现起源、流行情况、致病性和耐药性及参与的重组等方面进行综述,并讨论了与其流行密切相关的社会和生物学因素。     

无锡市2013-2016年HIV-1亚型及进化分析

目的了解无锡市HIV-1亚型流行及进化特征,为预测本地HIV-1疫情变化提供参考依据。方法样本来源于2013年4月至2016年7月无锡市部分CD4+T淋巴细胞监测队列,进行HIV-1基因的扩增和测序,采用ChromasPro 1.6和MEGA 7.0软件构建HIV-1序列数据库;采用FastTree 2.1.10和BEAST 1.7.2软件和贝叶斯系统进化推断法重构HIV-1历史传播情况,采用SPSS 22.0软件进行统计学分析。结果有205例HIV-1感染者,其中≥50岁占32.68%(67/205)。共检测出CRF01_AE、CRF07_BC、CRF67_01B、B、CRF08_BC、CRF68_0B、CRF78_cpx 7种HIV-1基因型及1例独特重组型。流行亚型以CRF01_AE(51.67%,93/180)及CRF07_BC(17.22%,31/180)为主,不同亚型之间传播方式的差异有统计学意义(χ2=16.99,P≤0.05)。CRF67_01B型(12.78%,23/180)所占比例较高。贝叶斯系统进化推断法分析结果显示,无锡市CRF67_01B型进化率为2.29×10-3,最近共同祖先时间约为2003.10年,与江苏省及安徽省来源的参考株可能存在亲缘关系,CRF67_01B型于2003年开始在无锡市出现传播。结论2013-2016年无锡市HIV-1亚型复杂多样,CRF67_01B型已经开始在无锡市流行,应持续监测HIV-1亚型变化,从分子角度为疫情预测提供参考依据。     

2007-2010年浙江省HIV双阳夫妻对流行病学特征分析

目的了解2007-2010年浙江省双方HIV-1抗体阳性夫妻流行病学特征,为预防艾滋病在夫妻间传播提供依据。方法通过浙江省艾滋病疫情报告资料和个案调查建立艾滋病阳性夫妻数据库,并进行流行病学特征分析。结果 2007-2010年全省共报告421对(842例)双方HIV-1抗体阳性的夫妻。男性和女性中本省户籍分别占62.00%、51.31%;夫妻主要危险行为是婚外异性性接触和婚内性接触。仅男方有危险行为、仅女方有危险行为、双方均有危险行为者分别占51.00%、22.09%、24.94%。80.52%感染夫妻通过临床可疑和医院体检中发现。80.8%的夫妻检测发现时间间隔在一个月以内。共发现17例经母婴传播感染儿童。结论本省内及外省的农村男性流动人群、外省的女性流动人群是造成夫妻间传播的重要人群。感染危险行为主要为婚外异性性接触,以夫妻一方有危险行为为主,夫妻双方有危险行为比例在增加。加强农村流动人群的高危性行为干预,促进夫妻双方共同定期检测、夫妻间行为干预可能有利于预防艾滋病在家庭内传播。     

重组沙门菌载体HIV-12F5表位疫苗毒力及分布

目的研究表达Ⅰ型人类免疫缺陷病毒(HIV-1)2F5表位的重组沙门菌(SA-2F5)对BALB/c小鼠的半数致死量(LD₅₀),并观察其在小鼠体内的生物学分布。方法BALB/c小鼠随机分8组,以0,104~1010菌落形成单位(CFU)重组菌灌胃,30d后用改良寇氏法测定LD₅₀;BALB/c小鼠随机分2组,灌胃给予107CFU重组沙门菌或磷酸盐缓冲液(PBS),分别于感染后第3,9,16,30 d,无菌操作取各组肝、脾、肠系膜淋巴结,检测重组沙门菌在小鼠体内的分布。结果SA-2F5对BALB/c小鼠的LD₅₀为1.45×10⁹CFU;感染后第3d,肝、脾、肠系膜淋巴结活菌数依次为5.38×10⁴,4.87×10⁴,9.08×10³CFU。第9d各脏器活菌数分别降至7.35×10²,7.50×10²及3.00×10²CFU。第16d各脏器活菌数基本与第9d相当。之后各脏器中活菌数持续下降,至第30d肝、脾内检测不到活菌,肠系膜淋巴结内活菌为2.1×10²CFU。结论重组沙门菌SA-2F5毒力显著降低,在小鼠体内具有良好的生物学分布。     

The use of immune complex vaccines to enhance antibody responses against neutralizing epitopes on HIV-1 envelope gp120

The capacity of immune complexes to augment antibody (Ab) responses is well established. The enhancing effects of immune complexes have been attributed mainly to Fc-mediated adjuvant activity, while the ability of Abs to induce antigenic alterations of specific epitopes as a result of immune complex formation has been less well studied. Previously we have shown that the interaction of anti-CD4-binding site (CD4bs) Abs with HIV-1 gp120 induces conformation changes that lead to enhanced antigenicity and immunogenicity of neutralizing epitopes in the V3 loop. The present study shows that significant increases in the antigenicity of the V3 and C1 regions of gp120 were attained for several subtype B gp120s and a subtype C gp120 upon immune complex formation with the anti-CD4bs monoclonal Ab (mAb) 654-D. Such enhancement was observed with immune complexes made with other anti-CD4bs mAbs and anti-V2 mAbs, but not with anti-C2 mAbs, indicating this activity is determined by antigen specificity of the mAb that formed the immune complex. When immune complexes of gp120_LAI/654-D and gp120_JRFL/654-D were tested as immunogens in mice, serum Abs to gp120 and V3 were generated at significantly higher titers than those induced by the respective uncomplexed gp120s. Notably, the anti-V3 Ab responses had distinct fine specificities; gp120_JRFL/654-D stimulated more cross-reactive anti-V3 Abs than gp120_LAI/654-D. Neutralizing activities against viruses with heterologous envelope were also detected in sera of mice immunized with gp120_JRFL/654-D, although the neutralization breadth was still limited. Overall this study shows the potential use of gp120/Ab complexes to augment the immunogenicity of HIV-1 envelope gp120, but further improvements are needed to elicit virus-neutralizing Ab responses with higher potency and breadth.     

HIV-1 Tat-specific IgG antibodies in high-responders target a B-cell epitope in the cysteine-rich domain and block extracellular Tat efficiently

Tat, an important regulatory protein of HIV-1, has been implicated in HIV-related pathogenesis. Immune responses to Tat, although underrepresented, confer protection against disease progression, in natural infection and experimental immunization, making Tat an attractive vaccine candidate. Information on immune responses to Tat from India which has the second largest HIV incidence has been lacking. Here we report a cross-sectional study evaluating the humoral response to Tat from a large number of samples from two southern states of India. 14% of the seropositive (63/447) and 4.6% of seronegative samples (7/150) harbored Tat-reactive antibodies. A significant number of the seropositive samples contained high levels of anti-Tat antibodies (31/447) which demonstrated class-switch to IgG1 and bound to Tat with high avidity. Cross-reactivity analysis showed that these antibodies interacted with Tat from different clades with variable degree withthe highest interaction with subtype-AE and the least with subtype-B Tat. Importantly, a B-cell epitope in the cysteine-rich domain was found to be the most immunodominant one and antibodies interacting with this epitope blocked extracellular Tat efficiently. To the best of our knowledge this is the first report on immune responses to Tat from Indian populations and the data presented here could significantly contribute to HIV Tat vaccine design.