中国HIV-1 B/C重组毒株不同阶段感染者多聚酶蛋白特异性T淋巴细胞反应的研究

目的 研究中国主要流行的HIV-1 B/C重组毒株不同时期感染者多聚酶蛋白(Pol)特异性T淋巴细胞反应特征并确定主要识别的免疫优势区域.方法 本研究以11例感染时间<18个月和25例感染时间>3年的HIV-1 B/C重组毒株感染者为研究对象,以10例HIV-1阴性健康人作为对照,应用酶联免疫斑点检测技术综合测定了针对覆盖HIV-1 pol基因的249条重叠多肽产生IFN-γ的特异性T淋巴细胞免疫反应.结果 感染时间<18个月感染者中有8(72.73%)名检测到了分泌IUN-γ的HIV-1特异性T淋巴细胞免疫反应,主要识别位于逆转录酶区、氨基酸位置为Pol 481～631内的Pol5581、Pol5582、Pol5587、Pol5609、Pol5610和Pol5615六条多肽,分泌IFN-γ的特异性T淋巴细胞反应广度与外周血CD4+T细胞数呈现明显负相关(P=0.0212,r=-0.762);感染时间>3年感染者中有15(60%)名检测到了分泌IFN-γ的HIV-1特异性T淋巴细胞免疫反应,主要识别位于逆转录酶区、氨基酸位置为Pol241～295内的Pol5521、Pol5525、Pol5526、Pol5531四条多肽和Pol 708～722内的Pol5638多肽,分泌IFN-γ的特异性T淋巴细胞反应强度与病毒载量呈现明显正相关(P=0.006 95,r=0.660);健康人对照组无阳性反应.结论 中国HIV-1 B/C重组毒株不同阶段感染者主要识别多聚酶蛋白的不同区域.     

HIV-1B’/C重组病毒感染者Vif、Vpr和Vpu特异性细胞免疫应答研究

目的 探讨我国HIV-1 B'/C重组病毒感染者针对HIV-1调节蛋白的细胞免疫反应特征及其与病毒复制控制的关系.方法 以覆盖HIV-1 C亚型Vpr、Vpu和Vif蛋白全长的重叠肽段作为刺激抗原,利用ELISPOT方法检测新疆HIV-1 B'/C重组病毒感染者的特异性细胞免疫反应.使用SIGMAPLOT 10.0和SIGMASTAT 3.5进行统计分析,用双尾t检验比较组间差异,用Spearmam秩相关分析免疫反应与病毒载量及CD4细胞计数的关系.结果 在检测的60名HIV-1 B'/C重组病毒感染者中,能够识别Vif、Vpr和Vpu蛋白产生CIL应答者分别为68%、52%和8%,Vpr和Vif蛋白存在多个强CIL反应的免疫优势区域.研究中还发现针对Vpr、Vif和Vpu蛋白的CTL反应强度和广度与HIV感染者的病毒载量及CD4细胞数量无明显的相关性.结论 HIV-1 Vpr和Vif蛋白包含多个可被机体免疫系统特异性T细胞识别的免疫优势区域.对这些免疫优势区所包含的CIL表位进行鉴定并探讨其在自然感染过程中的作用,对新一代的HIV疫苗设计有重要的参考意义.     

中国人群HIV-1B亚型Nef蛋白特异性细胞毒性T细胞反应的研究

目的探讨中国人群HIV-1B亚型Nef蛋白特异性细胞毒性T细胞(CTL)反应特征及其与病毒载量以及CD4细胞数量间的关系。方法选取33例HIV-1B亚型感染者。用合成的HIV-1B亚型Nef全基因序列肽库作为抗原,ELISPOT方法检测HIV-1B亚型Nef蛋白特异性CTL反应,同时测定病毒载量及CD4细胞数量。结果70％的感染者对Nef产生特异性CTL反应,单一肽段能够被识别的频率不超过40％,应答强度为(1102±2136)SFC(斑点形成细胞数)／106 PBMC。HIV-1B亚型Nef特异性CTL应答的强度和频率之间没有显著的相关性。HIV-1 Neff特异性CTL反应强度与病毒载量间存在显著负相关,与CD4细胞数量间存在显著正相关。结论初步确定了Nef蛋白CTL应答的优势区域。这些区域主要集中在一些高度保守的氨基酸序列。提示HIV-1B亚型Nef特异性CTL应答在疾病进展中对机体具有保护性作用。     

HIV-1B/C重组病毒感染者Gag特异性T淋巴细胞免疫应答的研究

目的 研究中国主要流行的HIV-1 B/C重组毒株感染者Gag特异性T淋巴细胞反应特征.方法 本研究以10例感染时间＜1年和25例感染时间＞3年未接受抗病毒治疗的HIV-1 B/C重组毒株感染者为研究对象,以10例HIV-1阴性健康人作为对照,用Elispot方法检测其针对HIV-1B/C同义B Gag重叠多肽产生γ干扰素的特异性T淋巴细胞反应.结果 8例(8/10)感染时间＜1年的HIV-1 B/C重组毒株感染者产生Gag特异性分泌γ干扰素的T淋巴细胞反应,主要识别散在分布的五条多肽;17例(68％)感染时间＞3年的感染者产生反应,主要识别p17区域内的一条和p24区域内六条多肽.感染时间＞3年组产生IFN-γ的特异性T淋巴细胞反应强度与病毒载量呈明显正相关(P =0.0318,r=0.519),感染时间＜1年的感染者反应强度明显高于感染时间＞3年的感染者(P=0.021).健康人对照组无阳性反应.结论 HIV-1 B/C重组病毒感染者在疾病进程不同阶段识别Gag的不同区域.     

HIV-1 Gag、Tat、Rev和Nef蛋白特异性的免疫应答

目的：探讨中国HIV／AIDS患者HIV—1 Gag、Tat、Rev和Nef蛋白特异性CTL应答的特征。方法：应用覆盖HIV-1 B、C亚型Gag、Tat、Rev和Nef蛋白的220个肽段作为抗原，通过ELISPOT方法俭测HIV／AIDS患者HIV特异性CTL应答。结果：无沦HIV—1 B亚型还是HIV-1C亚型所构建肽库的应答强度和频率，主要集中在Gag和Nef蛋白，Tat和Rev蛋白也有不同程度的应答。HIV—1 B、C亚型间应答比较，整体应答强度大致相同，但免疫优势区间存在着一定的差异，B亚型Gagp24亚蛋白的288～313氨基酸区应答最强，而C亚型Gagp24亚蛋白的155～181氨基酸区应答最强；两个亚型免疫优势区应答频率最高的都是Nef蛋白106～143氨基酸区(48．1％)。结论：中国人群CTL应答多集中在Gag和Nef蛋白，B、C业型间略有差异且存在交叉识别，这对设计针对中国人群的HIV疫苗是有重要的意义。     

HIV-1B／C重组病毒感染者Gag特异性T淋巴细胞免疫应答的研究

目的研究中国主要流行的HIV-1B／C重组毒株感染者Gag特异性T淋巴细胞反应特征。方法本研究以10例感染时间〈1年和25例感染时间〉3年未接受抗病毒治疗的HIV-1B／C重组毒株感染者为研究对象,以10例HIV．1阴性健康人作为对照,用Elispot方法检测其针对HIV-1B／C同义BGag重叠多肽产生1干扰素的特异性T淋巴细胞反应。结果8例（8／10）感染时间〈1年的HIV-1B／C重组毒株感染者产生Gag特异性分泌Y干扰素的T淋巴细胞反应,主要识别散在分布的五条多肽;17例（68％）感染时间〉3年的感染者产生反应,主要识别p17区域内的-条和p24区域内六条多肽。感染时间〉3年组产生IFN-吖的特异性T淋巴细胞反应强度与病毒载量呈明显正相关（P=0．0318,r=0．519）,感染时间〈1年的感染者反应强度明显高于感染时间〉3年的感染者（P=0．021）。健康人对照组无阳性反应。结论HIV-1B／C重组病毒感染者在疾病进程不同阶段识别Gag的不同区域。     

中国HIV-1 B'亚型毒株nef基因多态性及其对疾病进展影响的研究

目的 研究我国北方地区B'亚型HIV-1感染者病毒基因组中nef基因多态性、重要功能区的保守程度,探索其与疾病进展的关系.方法 HIV-1 B'亚型感染长期不进展者(LTNP)30例,典型进展者(TP)42例,从全血标本中提取全前病毒DNA,经nested-PCR扩增nef基因全长,扩增产物纯化后直接测序,对测得的序列进行系统进化和氨基酸变异分析,并计算比较LTNP组与TP的HIV/AIDS组氨基酸突变位置和频率的差异.结果 nef氨基酸序列长度可变区R21K/E/H/I/Q取代,TP组突变频率(59.52%)低于LTNP组(93.33%,P<0.005,OR=0.11).功能区外S15R/K/N取代,TP组突变频率(64.29%)高于LTNP组(33.33%,P<0.01,OR=3.60);K39R/E/N取代,TP组突变频率高于LTNP组(P<0.005).其他功能区内有少数序列发生变异,但在两组间无差异.结论 未发现中国北方地区HIV-1 B'亚型nef基因与疾病长期不进展相关联的明显缺失或缺陷,但nef基因序列长度可变区R21位K/E/H/I/Q取代R可能与疾病缓慢进展有关,S15R/K/N取代、K39R/E/N取代可能与疾病进展相关.nfe氨基酸序列的重要功能区较为保守.     

中国HIV-1 B'/C亚型感染者对异体病毒中和作用与疾病进展关系研究

目的 探讨中国HIV-1 B'/C亚型感染者对异体病毒中和作用与疾病进展的关系.方法 根据CD4 T淋巴细胞数量和有尤临床症状将HIV-1 B'/C亚型感染者分为HIV慢性感染组和AIDS组.将HIV-1感染者血清稀释(1/10～1/320)后,与在基因结构特点上同源性很低的3株HIV-1作用,以检测其中和作用.同时以正常人血清加病毒悬液为对照孔,能够抑制对照孔50%病毒复制的血清为中和作用阳性.将某个HIV-1感染者血浆能够中和异体病毒的个数占3个异体病毒的百分率定义为HIV-1感染者中和异体病毒的宽度;将某个HIV-1感染者血浆中和3个异体病毒抗体滴度的几何平均滴度定义为HIV-1感染者中和异体病毒的强度.结果 HIV-1慢性感染组与AIDS组之间中和异体病毒的宽度和强度差异有统计学意义,HIV-1慢性感染组显著高于AIDS组.HIV-1慢性感染组中和异体病毒的宽度和强度与病毒载量呈正相关,而AIDS组巾和异体病毒的宽度和强度与病毒载量没有显著的相关性.HIV-1慢性感染组和AIDS组中和异体病毒的宽度和强度与CD4 T淋巴细胞数均没有显著的相关性.结论 中国HIV-1B'/C亚型感染者不同疾病进展阶段针对异体病毒中和作用能力不同,HIV慢性感染组显著高于AIDS组,当疾病进展到AIDS期时,失去对异体病毒的中和作用,提示针对异体病毒的中和抗体与疾病进程有关.     

北京市性传播HIV-1感染者流行毒株gag基因序列测定和亚型分析

目的 了解北京市性传播HIV-1感染者流行毒株亚型特点和流行规律.方法 随机采集北京市2008年新确证性传播HIV感染者的抗凝全血标本100份,分离血浆,提取病毒RNA,用套式聚合酶链反应扩增病毒gag基因,并进行序列测定和亚型分析.结果 系统进化分析确定北京市性传播HIV-1感染者流行毒株存在8个亚型或流行重组型,分别为B亚型22份,B'亚型8份,C亚型1份,CRF01_AE 38份,CRF02_AG 2份,CRF07 BC 9份,CRF08_BC 3份,疑似C/CRF01_AE重组型1份.结论 CRF01_AE和B亚型分别占45.2%和26.2%,为性传播感染者主要的亚型,应该加强我市HIV-1亚型流行情况的监测.

Abstract：

Objective To investigate the subtype distribution and sequence characteristics of HIV-1 strains prevalent among sexual infectors in Beijing. Methods We collected the blood samples from 100HIV sexual infectors in Beijing during 2008 and separated plasma specimens. RNA was extracted from the plasma and the gag gene was amplified by RT-PCR and nest-PCR. The PCR products were sequenced directly and phylogenetic analyses of gag gene was performed using the MEGA4 software. Results Among 100 HIV-1 plasma samples,84 gag gene fragments were amplified and analyzed. Eight HIV subtypes including B(22 strains), B'(8 strains),C( 1 strain) ,CRF01_AE (38 strains) ,CRF02_AG (2 strains) ,CRF07_BC(9 strains) ,CRF08_BC(3 strains) and C/CRF01_AE recombinant like strain( 1 strain) were identified circulating in Beijing. Conclusion CRF01 _AE and subtype B were predominant in Beijing account for 45.2% and 26.2% and the surveillance of HIV gene variation should be paid more attention.     

河北省HIV-1流行株基因序列测定及亚型分析

目的 了解河北省HIV-1流行株的亚型分布和流行趋势.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 对154份HIV-1感染者的样品进行扩增,得到了148份样品的HIV-1基因片段.发现6种HIV-1亚型和重组型,以及2例未定型.其中B'亚型61例(41.2%)、CRF01_AE 59例(39.9%)、CRF07_BC 16例(10.8%)、CRF08_BC 6例(4.1%)、C亚和B01亚型各2例(1.4%).在河北省首次发现了B01亚型.结论 2009年河北省存在多种HIV-1亚型和流行重组型,主要是B'亚型和CRF01_AE重组型,应加强对HIV-1毒株亚型变异的监测,及时调整防治策略.     

Characterization of Gag and Nef-specific ELISpot-based CTL responses in HIV-1 infected Indian individuals

Cytotoxic T lymphocyte (CTL) responses to Gag have been most frequently linked to control of viremia whereas CTL responses to Nef have direct relationship with viral load. IFN-γ ELISpot assay was used to screen CTL responses at single peptide level directed at HIV-1 subtype C Gag and Nef proteins in 30 antiretroviral therapy naive HIV-1 infected Indian individuals. PBMCs from 73.3% and 90% of the study population showed response to Gag and Nef antigens, respectively. The magnitude of Gag-specific CTL responses was inversely correlated with plasma viral load (r = −0.45, P = 0.001), whereas magnitude of Nef-specific responses was directly correlated (r = 0.115). Thirteen immunodominant regions (6 in Gag, 7 in Nef) were identified in the current study. The identification of Gag and Nef-specific responses across HIV-1 infected Indian population and targeting epitopes from multiple immunodominant regions may provide useful insight into the designing of new immunotherapy and vaccines.     

三个不同亚型HIV-1调节/辅助基因DNA疫苗的构建和免疫原性研究

目的 构建表达中国主要流行亚型HIV-1 tat-rev-integrase(c-half)-vif-nef(TRIVN)融合基因的DNA疫苗,并比较其免疫原性.方法 按人源密码子使用频率对HIV-1 CN54(B'/C重组亚型)与RL42(B'亚型)的tat、rev、integrase(C端144个氨基酸)、vif和nef基因序列进行优化,构建DNA疫苗.通过Western blot测定上述DNA疫苗与HIV-1 AE2f株来源的tat-rev-integrase(c-half)-vifnef融合基因DNA疫苗的体外表达效率;利用小鼠模型比较3个DNA疫苗单独免疫与混合免疫的免疫原性特征.结果 限制性酶切及DNA测序结果表明两个融合基因重组质粒构建正确;Western blot 检测结果显示:3个DNA疫苗的体外表达效率基本相当.小鼠免疫后ELISPOT检测结果显示:在总T细胞反应强度方面AE2f-TRIVN最强[(948.0±330.0)SFCs/106脾细胞],次之为混合DNA免疫组(500.0±155.0 SFCs/106脾细胞),再者为RL42-TRIVN[(195.1±44.0)SFCs/106脾细胞],CN54-TRIVN最弱[(89.5±17.0)SFCs/106脾细胞].T细胞反应分布情况显示:3个DNA疫苗单独免疫时,T细胞反应主要集中在Integrase和Vif蛋白上;而混合免疫可以部分改善针对Nef蛋白的免疫识别.结论 3个亚型TRIVN DNA疫苗中以AE2f-TRIVN的免疫原性最强;DNA疫苗混合免疫倾向于促进特异性T细胞反应在TRIVN融合抗原上的均匀分布.

Abstract：

0bjective To determine the immunogenicities of DNA vaccines expressing tat-rev-integrase(c-half)-vif-neffusion genes(TRIVN) derived from prevalent B', B'/C and AE recombinant subtypes of HIV-1 in China. Methods Two DNA vaccines were constructed by inserting the codon optimized tat-revintegrase(c-half)-vif-nef fusion genes derived from B' and B'/C subtype of HIV-1 into mammalian expression vector pSVI. 0. DNA vaccine containing tat-rev-integrase (c-half)-vif-nef fusion gene derived from HIV-1AE2f has been constructed previously. In vitro expression efficiencies of three DNA vaccines were determined by Western blot and their immunogenicities were compared by immunizing female BALB/c mice. IFN-γ ELISPOT assay was used to read out the specific T cell immunity. Results The constructed DNA vaccines were validated by restriction enzyme digestion and DNA sequencing. Western blot assay showed three constructed DNA vaccines could be expressed at a comparable level in vitro. After vaccination, AE-TRIVN mounted T cell immune responses at (948.0 ± 330.0) SFCs/106 splenocytes, followed by the mixed DNA vaccine[ (500.0 ± 155.0) SFCs/106 splenocytes ], RL-TRIVN r[ ( 195. 1 ± 44.0) SFCs/106 splenocytes ]and CN-TRIVN [ (89.5 ± 17.0) SFCs/106 splenocytes]. Interestingly, we observed that single DNA vaccination induced specific T cell responses predominantly targeting Integrase (C-half) and Vif, whereas the mixed DNA could significantly improve T cell responses against Nef. Conclusion AE-TRIVN was the most immunogenic among the three DNA vaccines and the mixed DNA vaccination could change the immunogenic hierarchy of T cell epitopes across the fusion genes vaccine.     

Explosive HIV-1 subtype B' epidemics in Asia driven by geographic and risk group founder events

We explored the timescale, spatial spread, and risk group population structure of HIV-1 subtype B', the cause of explosive blood-borne HIV-1 epidemics among injecting drug users (IDUs) and former plasma donors (FPDs) in Asia. Sequences from FPDs in China formed a distinct monophyletic cluster within subtype B'. Further analysis revealed that subtype B' was founded by a single lineage of pandemic subtype B around 1985. Subsequently, the FPD cluster appears to have derived from a single subtype B' lineage around 1991, corroborating the hypothesis that FPD outbreaks stemmed from the preceding epidemic among IDUs in Southeast Asia, most likely from the Golden-Triangle region.     

Ancestral and consensus envelope immunogens for HIV-1 subtype C

Immunogens based on "centralized" (ancestral or consensus) HIV-1 sequences minimize the genetic distance between vaccine strains and contemporary viruses and should thus elicit immune responses that recognize a broader spectrum of viral variants. However, the biologic, antigenic and immunogenic properties of such inferred gene products have to be validated experimentally. Here, we report the construction and characterization of the first full-length ancestral (AncC) and consensus (ConC) env genes of HIV-1 (group M) subtype C. The codon-usage-optimized genes expressed high levels of envelope glycoproteins that were incorporated into HIV-1 virions, mediated infection via the CCR5 co-receptor and retained neutralizing epitopes as recognized by plasma from patients with chronic HIV-1 subtype C infection. Guinea pigs immunized with AncC and ConC env DNA developed high titer binding, but no appreciable homologous or heterologous neutralizing antibodies. When tested by immunoblot analysis, sera from AncC and ConC env immunized guinea pigs recognized a greater number of primary subtype C envelope glycoproteins than sera from guinea pigs immunized with a contemporary subtype C env control. Mice immunized with AncC and ConC env DNA developed gamma interferon T cell responses that recognized overlapping peptides from the cognate ConC and a heterologous subtype C Env control. Thus, both AncC and ConC env genes expressed functional envelope glycoproteins that were immunogenic in laboratory animals and elicited humoral and cellular immune responses of comparable breadth and magnitude. These results establish the utility of centralized HIV-1 subtype C Env immunogens and warrant their continued evaluation as potential components of future AIDS vaccines.     

我国西南西北地区吸毒人群重组人类免疫缺陷病毒1？…

目的 寻找人类免疫缺陷病毒１型在中国可能的重组。方法从流行２种以上ＨＩＶ－１亚型的地区收集ＨＩＶ感染者的血样。从ＰＭＣｓ中应用套式聚合酶链反应方法,对ＨＩＶ病毒的ｔａｔ和ｅｎｖ基因进行扩增,ＰＣＲ产物直接测序并进行序列分析。结果 对中国Ｂ’亚型和Ｃ亚型浒区域收集的１４个ＨＩＶ－１毒株进行序列分析,对ｅｎｖ基因进行测序后没有发现重组毒株的证据。     

Amplification of T-Cell and Antibody Responses in DNA-Based Immunization with HIV-1 Nef by Co-Injection with a GM-CSF Expression Vector

Intradermal inoculation of mice with naked plasmid DNA encoding the regulatory HIV-1 Nef protein was shown to induce Nef-specific T and B cell responses. Co-inoculation with an expression vector encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine known to facilitate the induction of primary immune responses, resulted in a markedly enhanced response to Nef. This was manifested both as an increase in Nef-specific T cell responses and antibody levels. DNA immunization with the Nef and GM-CSF vectors induced primarily a Th1 response as judged by the raised levels of both IFN-γ and IL-2 from re-stimulated T cells. The immunostimulatory activity of GM-CSF DNA was locally restricted and was observed only if both plasmid vectors were injected at the same site.