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.     

CD8+ T细胞对HIV-1合成表位的免疫主导应答研究

目的研究CD8^＋ T细胞对人免疫缺陷病毒1型（HIV-1）表位的免疫主导应答。方法分别采用酶联免疫斑点技术（ELtSPOT）和羧乙基锗倍半氧化物（CFSE）标记流式分析技术，以覆盖HIV-1 Env、Pol、Gag、Vif、Nef、Tat区的701个重叠肽段组成的34个肽段库及其部分单肽段作为刺激表位，对一例感染HIV-1的长期不进展者（LTNP）的外周血单个核细胞（PBMC）中CD8^＋ T细胞的了γ-干扰素（IFN-γ）分泌细胞频率和细胞增殖率进行了测定研究。结果HIV-1 Gag区域肽段诱导产生的CD8^＋ T细胞的IFN-γ分泌细胞频率最高，Nef、Tat、Vif区域依次顺减，Env和Pol区域不能诱导产生显著性应答；在IFN-γ ELISPOT实验中，肽段和相应肽段库刺激产生的结果一致；CD8^＋ T细胞在单肽段刺激下，用ELISPOT技术测定的IFN-γ分泌细胞频率和CFSE标记流式分析技术测定的细胞增殖率显示出较好的相关性。结论CD8^＋ T细胞能特异性识别某些HIV-1抗原表位，诱导出免疫主导应答；当进行HIV-1特异性CD8^＋ T细胞反应增殖测定和免疫主导应答研究时，ELISPOT是值得称道的标准实验，同时，推荐一种新颖的CFSE标记流式分析技术。     

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/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.     

中国HIV-1 C/B'重组毒株和B'毒株感染者Nef特异性T细胞免疫应答的研究

目的 研究中国主要流行的HIV-1 C/B'重组毒株和B'亚型毒株感染者Nef特异性T细胞反应特征,确定两种亚型感染者共同识别的免疫优势区.方法 本研究以59名HIV-1 C/B'重组毒株、27名B'亚型毒株感染者为研究对象,用ELISPOT检测针对HIV-1型C/B'Nef重叠多肽产生IFN-γ的特异性T细胞反应.结果 44例(74.58%)HIV-1 C/B'重组毒株感染者产生Nef特异性T细胞反应,主要识别EVA7081.1、5、6、7、43、44、45、47、48、49这10条多肽,氨基酸序列为Nef63～115和117～139的区域.20例(74.07%)的HIV-1 B'毒株感染者产生Nef特异性T细胞反应,主要识别EVA7081.1、2、43、49这4条多肽,氨基酸序列为Nef 63～77和87～119的区域.两种亚型感染者特异性T细胞反应的强度和广度与病毒载量和CIM细胞数不相关.结论 中国HIV-1 C/B'重组毒株和B'亚型毒株感染者共同识别氨基酸序列为Nef63～77和87～115的免疫优势区,提示此区域可用于疫苗的设计.     

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/AIDS患者特异性细胞毒性T细胞功能的研究

目的 了解中国HIV／AIDS患者HIV特异性细胞毒性T细胞(CTL)功能。方法 将覆盖HIV-1 P15、P17和P24 Gag全长的94个重叠多肽作为抗原，用IFN-γ ELISPOT方法检测HIV／AIDS患者HIV-1特异性CTL功能。结果 HIV-1抗原多肽P17-15、P17-16、P24-7、P17-8，P24-28最易被HIV／AIDS患者特异性CTL识别。HIV感染者识别HIV-1多肽的数量和强度均高于AIDS患者。结论 我国HTV／AIDS患者体内存在识别不同HIV-1 Gag多肽的特异性CTL，且HIV特异性CTL功能与疾病进展相关。     

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.     

HIV-1高暴露持续血清阴性者对Nef、Gag肽段库的特异性CTL应答的研究

目的 研究中国HIV-1高暴露持续血清阴性（highly exposed persistently seronegative,HEPS）者的Nef、Gag特异性细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL）应答特点,探讨HIV-1特异性CTL应答在这类特殊人群中抵抗感染的作用机制.方法 选取10例HIV-1高暴露持续血清阴性者,11例经性接触感染且从未接受抗病毒治疗的HIV/AIDS患者及4例未经暴露的健康志愿者.以覆盖HIV-1 gag全长和部分nef的14个肽段库为刺激原,应用IFN-γ ELISPOT法测定3组人群的特异性CTL应答,并对3组的应答强度、宽度以及对肽段库识别比例进行比较.结果 50%（5/10）的HEPS,100%（11/11）的HIV/AIDS患者均存在Nef及Gag特异性CTL应答,而4例健康对照均为阴性.存在应答的HEPS者对14个肽段库的平均应答强度和宽度分别是HIV/AIDS患者的4.3%和37.7%.在HEPS者中主要识别的肽段库均为HIV/AIDS患者中识别比例相对较低的肽段库.结论 与HIV/AIDS患者相比,HEPS者中的HIV-1特异性CTL应答存在着不同的特点和规律,可能在保护机体免于HIV-1感染中发挥着重要作用.     

HIV-1感染过程中CD8~+T细胞PD-1水平增高与其活化状态正相关

目的分析人类免疫缺陷病毒1型(HIV-1)感染者CD8+T细胞程序性死亡蛋白1(PD-1)、CD38、人白细胞抗原DR(HLA-DR)及KI-67抗原(ki67)的表达水平,探讨HIV-1感染过程中CD8~+T细胞PD-1表达水平与免疫活化和免疫耗竭的关系及意义。方法收集HIV-1感染者87例,健康志愿者22例,密度梯度法分离外周血单个核细胞;采用流式细胞术分析其CD8+T细胞PD-1、CD38、HLA-DR、ki67的表达水平;观察用抗PD-L1 m Ab阻断PD-1/PD-L1通路后CD8~+T细胞分泌肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)的水平变化。结果 HIV-1感染者CD8~+T细胞PD-1表达水平显著高于健康对照组;CD8~+T细胞PD-1水平与病毒载量呈正相关,与CD4~+T细胞数呈负相关;CD8~+T细胞PD-1与CD38、HLA-DR表达呈正相关,与ki67表达无相关性;体外阻断PD-1/PD-L1通路后TNF-α、IFN-γ的表达量增加。结论 HIV-1感染者外周血CD8~+T细胞PD-1表达增加;PD-1过表达与HIV感染过程中CD8~+T细胞功能受抑、免疫耗竭及疾病进程相关;体外阻断PD-1/PD-L1通路可恢复CD8~+T细胞功能。     

Characterization of HIV-1 Gag-specific T cell responses in chronically infected Indian population

India is at the epicentre of the global HIV/AIDS epidemic in South-east Asia, predominated by subtype C infections. It is important to characterize HIV-1-specific T cell responses in this particular population with the aim of identifying protective correlates of immunity to control HIV-1 infection. In this study, we performed a comprehensive analysis of the breadth and magnitude of T cell responses directed at HIV-1 subtype C Gag, one of the most conserved HIV-1 proteins. The study population consisted of antiretroviral naive, chronic HIV-1 subtype C-infected individuals at various stages of infection. We used recent advanced techniques such as enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining to quantify the total CD4(+) and CD8(+) T cell response to HIV-1 gag at single peptide level, regardless of HLA haplotype of the infected individual. The p24-Gag was identified as the most frequently recognized subunit protein with the greatest magnitude of CD4(+) and CD8(+) T cell responses. Stronger and broader CD8 T cell responses were recognized, contrasting with the weaker and narrower CD4 T cell responses with regard to Gag protein subunits. The magnitude of the HIV-specific interferon (IFN)-gamma responses was observed to be higher than the corresponding interleukin (IL)-2 response, indicating the persistence of antigenic load in chronically infected Indian population due to the probable dysfunction of HIV-specific, IFN-gamma-secreting CD8 T cells in absence of IL-2 help.     

Interactive association of proviral load and IFN-gamma-secreting T cell responses in HIV-1C infection

We investigated the interactive relationship between proviral DNA load and virus-specific IFN-gamma-secreting T cell responses in HIV-1C infection. The presence or absence of correlation, and inverse or direct type of correlation, if any, were dependent on targeted viral gene product. Responses to Gag p24 or to Pol were associated with lower proviral DNA load. Associations between proviral DNA load and T cell responses did not necessarily mirror relationships between plasma RNA load and T cell responses. An interaction analysis showed a synergy in that lower proviral DNA and lower plasma RNA load were associated with high Gag p24-specific IFN-gamma-secreting T cell response (interaction test P = 0.0003). Our findings support the idea that HIV proteins have differential value for vaccine design, and suggest that, for HIV-1C, Gag p24 may be one of the most attractive regions to include in vaccine designs to control both plasma RNA load and cell-associated proviral DNA load.     

Polyfunctional T cell responses are a hallmark of HIV-2 infection

HIV-2 is distinguished clinically and immunologically from HIV-1 infection by delayed disease progression and maintenance of HIV-specific CD4(+) T cell help in most infected subjects. Thus, HIV-2 provides a unique natural human model in which to investigate correlates of immune protection against HIV disease progression. Here, we report a detailed assessment of the HIV-2-specific CD4(+) and CD8(+) T cell response compared to HIV-1, using polychromatic flow cytometry to assess the quality of the HIV-specific T cell response by measuring IFN-gamma, IL-2, TNF-alpha, MIP-1beta, and CD107a mobilization (degranulation) simultaneously following Gag peptide stimulation. We find that HIV-2-specific CD4(+) and CD8(+) T cells are more polyfunctional that those specific for HIV-1 and that polyfunctional HIV-2-specific T cells produce more IFN-gamma and TNF-alpha on a per-cell basis than monofunctional T cells. Polyfunctional HIV-2-specific CD4(+) T cells were generally more differentiated and expressed CD57, while there was no association between function and phenotype in the CD8(+) T cell fraction. Polyfunctional HIV-specific T cell responses are a hallmark of non-progressive HIV-2 infection and may be related to good clinical outcome in this setting.     

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。     

表达HIV-1 gag基因的重组AAV2/1和Ad5疫苗免疫原性比较

目的 在小鼠体内比较表达HIT-1 gag基因的重组AAV2/1和Ad5疫苗的免疫原性.方法 分别用rAAV2/1-gag或rAd5-gag单独免疫BALB/c小鼠一次或两次,于免疫后不同时间点用CFSE染色法和胞内细胞因子染色法检测Gag特异性细胞免疫应答,用ELISA方法检测Gag特异性抗体反应.结果 单次免疫结果显示,在所有检测点rAd5-gag所诱导的Gag特异性CTL应答都比rAAV2/1-gag强,抗体反应在免疫后4周无明显差异.两次免疫后4周的检测结果表明,rAd5-gag所诱导的Gag特异性CTL应答比rAAV2/1-gag强,但抗体反应比rAAV2/1-gag组弱.结论 rAd5-gag诱导了很好的HIV-1特异性细胞和抗体反应,而rAAV2/1-gag诱导的HIV-1特异性抗体反应很强,但细胞免疫应答较弱.     

HIV-1 mRNA electroporation of PBMC: a simple and efficient method to monitor T-cell responses against autologous HIV-1 in HIV-1-infected patients

Efficient monitoring of HIV-1-specific T-cells is crucial for the development of HIV-1 vaccines and immunotherapies. Currently, mainly peptides and vaccinia vectors are used for detection of HIV-1-specific cytotoxic T-lymphocytes (CTL), however, as HIV-1 is a variable virus, it is unknown to what extent the T-cell response against the autologous virus is under- or overestimated by using antigens from heterologous viral strains. Therefore, we established a new method for immunomonitoring of CTL using electroporation of peripheral blood mononuclear cells (PBMC) with mRNA derived from autologous viral strains. From six HIV-1-infected patients virus derived mRNA was produced after PCR-based cloning of autologous gag (n=5) and/or nef genes (n=3) from plasma and electroporated into PBMC from patients and healthy donors. Electroporation of PBMC with mRNA resulted in efficient protein expression with good induction of γ-interferon (γ-IFN) release by specific T-cells comparable to peptide pools and better than recombinant vaccinia viruses. Three mRNA encoded autologous Gag proteins and one autologous mRNA encoded Nef protein were better recognized by autologous PBMC in comparison to heterologous mRNA encoded Gag or Nef proteins (SF2 or HXB2). However, in one case each, mRNA encoded autologous Gag or Nef, respectively, was recognized less efficiently due to the presence of CTL escape mutations. In summary, electroporation of PBMC with mRNA is a very efficient, easy and rapid method for immunomonitoring of HIV-1-specific T-cell responses against autologous viral strains. Our data demonstrate that patients' CTL responses against autologous viral strains may be under- or overestimated by using antigens from heterologous viral strains.     

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.     

Human leucocyte antigen‐Bw4 and Gag‐specific T cell responses are associated with slow disease progression in HIV‐1B‐infected anti‐retroviral therapy‐naive Chinese

In China, the majority of human immunodeficiency virus (HIV) infections are predominately subtype B. It is important to characterize the HIV‐1 subtype B‐specific and its T cell response within the Chinese population, with the aim of identifying protective correlates of immunity to control HIV‐1 infections. In this study, we performed a comprehensive analysis looking into the magnitude/strength of T cell responses directed at the Gag protein of the HIV‐1 subtype B, one of the most conserved HIV‐1 proteins. The study group consisted of anti‐retroviral native and chronic HIV‐1 subtype B‐infected individuals. We used enzyme‐linked immunospot (ELISPOT) assay to quantify the total T cell responses to HIV‐1 Gag at the single peptide level. Twenty‐eight (38%) peptides were recognized in 24 (82·8%) individuals. The p24 was identified as the most frequently recognized subunit protein with the greatest T cell response in the test, which correlated positively with CD4⁺ T cell count and inversely with viral load (VL). At the level of the human leucocyte antigen (HLA) supertypes, we detected the highest levels and a significant correlation with both the CD4⁺ T cell count and the VL with Gag T cell responses in Bw4/Bw4. These findings demonstrate that (i) the HIV‐1B Gag p24‐specific immune responses play an important role in controlling viral replication and slowing clinical progression; and (ii) HLA‐Bw4/Bw4 allele has stronger T cell responses, which is associated with slow clinical progression in Chinese HIV patients.