【作者】 耿庆茂；

【导师】 李敬云；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2010， 硕士

【摘要】 HIV-1毒株的耐药性是艾滋病抗病毒治疗的主要障碍,阐明耐药毒株的流行状况及分子进化规律,对于认识HIV耐药的机制、指导抗病毒治疗具有重要的意义。本研究首先对国内使用的in-house HIV基因型耐药性检测方法进行了评价,然后利用经过评价的in-house耐药检测方法对2009年河南省某地的艾滋病患者进行了耐药毒株流行状况的横断面研究,分析了耐药发生情况及对治疗的影响,为改进治疗方案提供依据。引入并改进了HIV准种的单基因组扩增法(Single-Genome Amplification,SGA),并用这种方法分析了一名接受蛋白酶抑制剂治疗的艾滋病患者体内印地那韦耐药准种的发生、发展过程,确定了新型印地那韦耐药突变模式。最后,根据在河南HIV耐药分子流行病学研究的结果,对筛选出来的与抗病毒治疗失败相关的新型突变位点进行了鉴定,通过构建突变病毒及表型耐药性研究,分析这些突变位点对耐药的作用。第一部分In-house HIV-1基因型耐药性检测方法的评价目的:评价国内建立并广泛使用的in-house HIV-1基因型耐药检测方法的准确性与敏感性。方法:以美国FDA批准的HIV-1基因型耐药性检测系统(ViroSeq? v2.0,雅培)为参比,两种方法平行检测130份艾滋病患者的血浆样本,比较二者在耐药突变位点检测以及耐药性解释方面的一致性。结果:两种方法均得到耐药结果的样本有110例。在已知的14850个HIV-1耐药突变位点中,两种方法可同时检出99.34%(14752/14850)的耐药突变位点(kappa值为0.9488,P值<0.00001)。在对不同类型的突变位点检测中,两种方法对蛋白酶抑制剂耐药突变位点、逆转录酶抑制剂耐药突变位点检测的一致率分别为99.70%和99.01%(kappa值分别为0.9099和0.9521,P值均<0.00001)。两种方法出具耐药检测报告结果的一致率为94.55%(kappa值为0.6374,P值<0.0001),表明两种方法在药物敏感性评价方面具有高度一致性。两种方法共检测到34个ViroSeq?数据库(ViroSeq?软件v2.7)未收录的位点,这些突变位点在SHDB数据库中对耐药性有一定影响。其中2个突变位点(逆转录酶区的V179F和K238T)对耐药的影响较大,提示in-house方法的数据库(SHDB)与ViroSeq?相比具有一定优势。结论:In-house基因型耐药性检测方法与ViroSeq?基因型耐药性检测系统在耐药突变位点检测以及药物敏感性评价上具有高度一致性,是一种快速准确、性价比高的HIV-1基因型耐药检测方法。第二部分河南省某地HIV-1耐药毒株流行状况的横断面研究目的:了解我国河南省某县艾滋病患者的抗逆转录病毒治疗的效果、免疫重建的情况和耐药性发生情况,为改进抗病毒治疗方案、提高疗效提供依据。方法:对某县120名2003年或2004年开始接受抗病毒治疗的艾滋病患者进行横断面研究。对每一名患者进行了面对面访谈,了解患者的临床表现、服药依从性等情况。经患者知情同意,采集10ml抗凝血。通过测定患者的病毒载量和CD4+T计数评价抗病毒治疗效果和免疫恢复情况,采用in house方法对治疗失败的患者进行了基因型耐药性检测。结果:120名艾滋病患者使用了三种抗病毒治疗方案,分别为AZT/DDI/NVP组(n=102);AZT/3TC/NVP组(n=12);D4T/3TC/NVP组(n=6)。患者的CD4+T细胞计数平均值为377±218 cells/ml,中位数为367 cells/ml,其中CD4+T细胞计数大于350 cells/ml的患者有64例(53.3%)。33例患者的HIV-1病毒载量小于检测限,其余患者病毒载量平均值为4.09±1.10 lg拷贝/毫升(中位数为3.87 lg拷贝/毫升)。在114例具有病毒载量数据的患者中,67例(58.77%)患者治疗失败(病毒载量大于1000拷贝/毫升)。经Fisher精确检验,三种治疗方案治疗效果之间没有显著性差异(P=0.53)。对治疗失败的患者(病毒载量大于1000拷贝/毫升)进行基因型耐药性检测,共67份标本,得到58份(86.6%)基因型耐药结果。其中40份产生了逆转录酶抑制剂耐药性,治疗失败患者中的耐药发生率为69.0%(40/58);蛋白酶抑制剂耐药发生率为0.0%(0/58)。治疗失败患者中核苷类逆转录酶抑制剂的耐药发生率为53.4%(31/58);非核苷类逆转录酶抑制剂耐药发生率为67.2%(39/58)。核苷类逆转录酶抑制剂中,AZT的耐药发生率最高(39.66%,23/58),非核苷类逆转录酶抑制剂中,NVP的耐药发生率最高(67.24%,39/58)。分析耐药患者的耐药水平,发现患者产生的耐药性基本上为中高度耐药,并且具有交叉耐药性。M41L、T215Y是耐药发生率最高的NRTIs类突变; K103N、Y181C是耐药发生率最高的NNRTIs类突变。结论:河南某县目前艾滋病患者的抗病毒治疗效果和免疫恢复均不理想,耐药毒株流行严重,目前的治疗方案已不适用,建议改变治疗方案,换用蛋白酶抑制剂。第三部分新型HIV-1耐药相关突变位点的鉴定由于HIV-1的流行毒株亚型不同、抗病毒治疗模式不同及长期的病毒/宿主/药物相互作用,新型HIV耐药相关突变位点不断被发现。逆转录酶是抗病毒药物最重要的靶点,已经鉴定了很多对药物敏感性影响非常大的突变位点,但这些突变位点主要位于具有催化活性的聚合酶区,连接区的耐药突变位点近几年才被发现。目的:鉴定前期研究筛选出来的新型逆转录酶基因突变位点对于耐药的作用。方法:利用定点突变和质粒转染技术,构建含有相关突变的HIV-1 pNL4.3病毒株。通过测定3种药物(AZT、EFV和NVP)对突变型和野生型病毒株对的50%抑制浓度(50% inhibitory concentration,IC50),确定突变位点对药物敏感性的影响。结果:成功构建7个突变病毒株,分别含有HIV-1逆转录酶基因的D123E、V292I、K366R、T369A、T369V、A371V和I375V突变。(1)A371V和T369V可使病毒对AZT的IC50分别增加3.60和2.83倍,其它5个突变却在一定程度上提高病毒对AZT的敏感性,特别是T369A,使病毒对AZT的IC50值降低35.67倍。(2)T369V和A371V可以导致EFV的低度耐药,IC50分别增加4.55和2.87倍数,其它5个突变可提高病毒对EFV的敏感性,特别是I375V,单独出现时可使病毒的IC50由8.11nM降低到1.23nM,降低了6.7倍。(3)突变T369V可导致病毒对NVP的耐药性,IC50增加11.55倍。其它突变对NVP耐药性的影响很小。结论:A371V可以导致低度AZT耐药(FC = 3.60),T369V分别可以导致EFV、NVP的低度和中度耐药(FC = 4.55,11.55)。首次证实HIV-1连接区的T369位点对AZT、EFV和NVP耐药性的影响均较大,T369A突变可提高病毒对AZT和EFV的敏感性,而T369V突变却导致对AZT、EFV和NVP的耐药性。第四部分单基因组扩增法分析HIV-1印地那韦耐药准种的分子进化HIV-1在感染者体内以准种形式存在,耐药毒株与野生毒株共存,在不同的药物压力下,呈动态的进化和发展过程。认识HIV-1耐药准种在体内的进化路径是阐明耐药机制的基础。目的:利用单基因组扩增法(Single-Genome Amplification,SGA)分析一名长期服用逆转录酶抑制剂、刚刚将奈韦拉平(Nevirapine,NVP)改为印地那韦(Indinavir,IDV)的艾滋病患者(XLF)体内IDV耐药准种的产生及发展情况。方法:对患者进行连续随访,采集6份系列血浆标本,包括服用IDV之前的1次样本(XLF1)和服用IDV之后的5次(XLF2至XLF6)样本。利用SGA法得到病毒聚合酶区(包括逆转录酶和蛋白酶全长)的准种序列,将得到的序列提交斯坦福大学HIV-1耐药数据库,得到耐药信息。结果:从6份系列样本中共得到了149条蛋白酶全长和171条逆转录酶全长序列,所有序列均为B亚型。患者服用IDV之前,病毒的蛋白酶区只含一些多态性突变位点。服用IDV之后,含蛋白酶区多态性位点的病毒比例下降,而含有次级突变位点G73S的病毒开始上升,并成为优势毒株。随着治疗时间延长,G73S与M46I/L90M组成一个连锁的耐药突变模式——M46I/G73S/L90M,并逐渐取代G73S成为优势毒株。在第6次随访的样本中,97.9%的病毒含有M46I/G73S/L90M突变模式。由于一直服用逆转录酶抑制剂,患者体内存在高比例的对逆转录酶抑制剂高度耐药的病毒株,并没有随着IDV耐药株的出现和发展产生大的变化。结论:引入并改进了SGA的方法,利用SGA方法确定了一种相对稀有的IDV耐药突变模式,即在突变G73S的基础上加入突变M46I和L90M,形成M46I/G73S/L90M突变模式。更多还原

【Abstract】 Drug resistance of HIV-1 is the main obstacle for antiretroviral therapy (ART). The information about the prevalence and molecular evolution of HIV-1 drug resistance is significant for understanding drug resistance mechanism and the supervision of ART. At the beginning, in-house genotyping method was evaluated. Then this method was used in the cross-sectional monitoring of drug resistance in Henan province. The prevalence of drug resistance and its impact on ART were analyzed and some advices about ART were provided. Combined with the information about the HIV-1 drug resistance epidemiology in Henan province, some new mutations associated with ART failure were screened and viruses with these mutations were constructed by site-directed mutagenesis and transfection strategies. And the impacts of these mutated viruses on antiretroviral drugs were measured by phenotypic resistance test. At last, a new method for the analysis of quasispecies drug resistance was successfully constructed. Single-Genome Amplification (SGA) was used to analyze the molecular evolution of Indinavir (IDV) drug resistance in one patient, and the new IDV resistance mutation pattern was identified.PartⅠEvaluation of an in-house method for HIV-1 genotyping resistance test used in ChinaObjective: Evaluating the sensitivity and accuracy characteristics of an in-house genotypic resistance system widely used in China. Methods: This in-house method was evaluated by comparing with an US FDA-approved genotyping system, ViroSeq? v2.0 (Abbott, Switzerland). The characteristics of the detection and interpretation of drug resistance mutations were validated through drawing a parallel between these two tests in 130 plasma samples. Results: Out of the 14850 mutations detected, 14752 (99.34%) mutations was concordant between these two methods (kappa value was 0.9488; P value was less than 0.00001). Through the comparison between these two methods, the rates of concordance in protease inhibitors-, reverse transcriptase inhibitors-resistance mutations were 99.70% and 99.01% respectively, and kappa values were 0.9099 and 0.9521 respectively (P values were all less than 0.00001). The comparison of drug resistance reports showed similar results (Kappa value was 0.6374, P value was less than 0.0001), suggesting there was high concordance between these two tests in drug resistance mutation interpretation. There were 34 mutations excluded from ViroSeq? drug resistance mutation database (ViroSeq? software v2.7) which were detected by one or both tests. Two mutations, V179F and K238T in RT region, had significance in drug resistance, indicating the database used by in-house assay was superior to ViroSeq?. Conclusions: The in-house genotyping system is an accurate, cost-effective method and had high concordance with commercial ViroSeq? genotyping system in the detection and interpretation of drug resistance mutations.PartⅡCross-sectional monitoring of the prevalence of drug resistance in Henan provinceObjective: The impact of current treatment regimens were investigated through this cross-sectional monitoring of drug resistance in Henan province, such as the treatment efficacy, immune system rebuilding and the prevalence of drug resistance variants, which could provide evidence to improve treatment regimen. Methods: This cross-sectional study included 120 HIV-1 infected patients who began to ART from 2003 or 2004. The information of patients’clinical manifestation and drug adherence was obtained through face to face review. All patients’CD4+ T cells counts and HIV-1 viral loads were measured to evalutate the ART efficacy and immune rebuilding, and in-house drug resistance test was performed in treatment-failure patients. Results: There were three ART regimens used in these patients, AZT/DDI/NVP group (n=102), AZT/3TC/NVP group (n=12), D4T/3TC/NVP group (n=6). The average of CD4+ T cell counts was 377±218 cells/ml and the median was 367 cells/ml, and 64 (53.3%) patients’CD4+ T cell counts were higher than 350 cells/ml. Out of 114 patients with viral load data, 33 patients’viral loads were less than 50 copies/ml, and the remaining patients’viral loads had an average of 4.09±1.10 lg copies/ml (the median was 3.87 lg copies/ml). Effective viral inhibition (viral load is less than 1000 copies/ml) was found in 47 patients (41.23%). No statistics significance was found among these three ART regimens’efficacies (P=0.53) through Fisher Exact test. In treatment failure patients, the ratio of reverse transcriptase inhibitors (RTIs) resistance was 69.0% (40/58), and there was no protease inhibitors (PIs) resistance variants. The ratio of nucleoside reverse transcriptase inhibitors (NRTIs) resistance and non-nucleoside reverse transcriptase inhibitors (NNRTIs) resistance were 53.4% (31/58) and 67.2% (39/58), respectively. NVP had the highest drug resistance ratio (67.24%, 39/58) in these NNRTIs used in these patients, and AZT was the highest in NRTIs (53.4%, 31/58). In the patients who had drug resistant variants, variants which showed high- and intermediate-level resistance were much more than those with low-level resistance, indicating variants existed in treatment failure patients were mostly high- and intermediate-level and had cross-drug resistance. M41L and T215Y in RT region were the most prevalence NRTIs mutations; K103N and Y181C were the most prevalence NNRTIs mutations. Conclusions: The treatment regimens currently used in these patients were not suitable, because these regimens did not gain realistic antiretroviral efficacy. Drug resistance variants existed in most treatment failure patients, suggesting protease inhibitors should be involved in new regimens. PartⅢIdentification of new drug resistance associated HIV-1 mutationsBecause of the prevalence of different HIV-1 subtypes, ART and the interaction among virus, host and drugs, more and more drug resistance mutations were identified. Reverse transcriptase (RT) is the most important target for ART, and many mutations which had significance in drug resistance had been reported and applied for drug resistance detection and monitoring. But almost all of mutations are located in the polymerase domain of RT; the mutations in ligation domain are rare. Objective: Some new selected RT mutations’impact on drug resistance was analyzed. Methods: The mutated pNL4.3 viruses were constructed by site-directed mutagenesis and transfection strategies. The 50% inhibitory concentration (IC50) change folds of each mutated virus were measured through the comparison of the wild and mutated viruses. Results: All 7 mutated pNL4.3 viruses were constructed successfully, and the mutations were D123E, V292I, K366R, T369A, T369V, A371V and I375V in RT region respectively. (1) Mutations A371V and T369V in RT could confer resistance to AZT, and the IC50 change folds of variants were 3.60 and 2.83 respectively. But the other 5 mutations decreased the IC50 of AZT, especially mutation T369A which decreased the IC50 by 35.67 times. (2) Mutation A371V and T369V could increase the EFV IC50, and the change folds were 4.55 and 2.87 respectively. The other 5 mutated viruses increased the susceptibility of virus to EFV, especially the I375V mutation. When I375V mutation occurred alone, the IC50 of EFV decreased to 1.23 nM while wild virus was 8.11 nM. (3) Mutation T369V conferred resistance to NVP by increasing IC50 by 11.55 times, while other 6 mutations had no impact on NVP susceptibility. Conclusions: Mutation A371V in RT region could cause low-level resistance to AZT (IC50 change fold was 3.60). T369V could cause low- and intermidiate-level resistance to EFV and NVP respectively (IC50 change fold were 4.55 and 11.55 respectively). The impact of mutations at codon 369 in RT ligation domain on AZT, EFV and NVP susceptibility was confirmed for the first time. T369A mutation could increase the susceptibility to AZT and EFV, while T369V resulted in the resistance to AZT, EFV and NVP.PartⅣAnalysis of HIV-1 quasispecies evolution of Indinavir resistance by Single-Genome AmplificationHIV-1 exists in vivo as quasispecies, and wild variants and resistanct variants coexist. And their proportion exhibit dynamic evolution and development during the selection pressure of drugs. Understanding the molecular evolution process is fundamental to analyzing the mechanism of drug resistance. Objective: The molecular evolution of drug resistance in one patient was analyzed. This patient had received reverse transcriptase inhibitors (RTIs) for a long time and recently replaced Nevirapine (NVP) with Indinavir (IDV). Methods: The patient, XLF, was followed-up six times successively. The viral populations were amplified and sequenced by single-genome amplification (SGA). All the sequences were submitted to Stanford HIV Drug Resistance Database (SHDB) for the analysis of genotypic drug resistance. Results: 149 entire protease and 171 entire reverse transcriptase sequences were obtained from these samples, and all sequences were identified as subtype B. Before the patient received Indinavir, the viral population only had some polymorphisms in the protease sequences. After the patient began Indinavir treatment, the variants carrying polymorphisms declined while variants carrying the secondary mutation G73S gained an advantage. As therapy was prolonged, G73S was combined with M46I/L90M to form a resistance pattern M46I/G73S/L90M, which then became the dominant population. 97.9% of variants had the M46I/G73S/L90M pattern at XLF6. During the emergence of protease inhibitors resistance, reverse transcriptase inhibitors resistance always maintained at high levels. Conclusion: Indinavir-resistance evolution was observed by single-genome amplification. During the course of changing the regimen to incorporate Indinavir, the G73S mutation occurred and was combined with M46I/L90M.更多还原