血浆RNA和PBMCs DNA HIV-1耐药准种多态性和分子进化研究
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摘要
人免疫缺陷病毒(Human Immunodeficiency Virus,HIV)是高度变异的人类病原体,在抗逆转录病毒药物压力下,快速突变产生耐药性,导致抗逆转录病毒治疗(antiretroviral therapy,ART)的失败。耐药HIV毒株还可以传播,使得从未接受过抗病毒治疗的艾滋病(acquired immunodeficiency syndrome,AIDS,获得性免疫缺陷综合征)病人具有对药物的耐受性。HIV的耐药性已成为严重的公共卫生问题,是艾滋病防治的主要障碍。了解HIV耐药的分子进化规律并理解新耐药突变的作用,是全面认识HIV耐药分子机制的基础,将为有效遏制耐药毒株、做好针对耐药毒株的艾滋病防治提供科学依据。
为了揭示血浆和外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)中HIV耐药的分子进化规律、阐明新型耐药突变对耐药的贡献,本研究以河南某乡接受抗病毒治疗艾滋病患者队列为基础,开展了系列研究。首先对河南某乡接受抗病毒治疗的艾滋病患者更换二线药物前后的病毒耐药发生和演变情况进行了调查,对部分患者进行了病毒分离培养和基因型耐药鉴定;然后,基于长期随访获得的部分患者系列血浆和PBMCs标本,采用大量克隆测序及基因型耐药分析的方法,揭示血浆和PBMCs中耐药准种进化的规律;针对患者中大量出现的尚未有明确表型解释的H221Y突变,通过构建系列突变病毒并进行表型耐药测定的方式,阐明H221Y及其它突变联合对耐药的作用。
第一部分河南某乡接受抗病毒治疗的艾滋病患者更换二线治疗方案前后的耐药性研究
河南某乡是我国最早开展艾滋病免费抗病毒治疗的地区之一,采用的是使用统一的治疗方案、同时开始治疗,由村医管理患者并监督服药的治疗模式。自初始治疗(2003年9月)起,我们实验室对这个地区艾滋病患者的HIV耐药情况进行了连续7年的监测。根据患者的临床表现、病毒载量、CD4+T淋巴细胞计数检测的结果及病毒耐药监测的结果,2010年2月起,部分患者由一线治疗方案更换为二线治疗方案。本部分研究的目的是了解更换二线治疗方案后的HIV耐药发生发展的特点和规律,同时,观察更换二线治疗方案后的病毒学和免疫学应答,从而为抗病毒治疗方案的改进提供依据。
对抗病毒治疗患者队列中的77名患者进行了两次随访,时间为2009年10月和2010年6月,共调查和采血154人次。其中43例患者在首次随访时(2010年2～3月)更换了二线治疗方案,34例患者仍然使用一线治疗方案。对所有患者使用统一的调查表进行流行病学调查,了解服药依从性、临床表现等信息,采集血液标本进行病毒载量和CD4+T淋巴细胞计数检测,采用耐药基因型方法进行耐药性检测和分析。主要结果和结论有:
1.换药组患者在换药3个月后病毒载量显著下降(P值=0.005)。病毒抑制率由治疗前的46.34%(19/41)上升至换药后的65.80%(27/41);未换药组患者的病毒载量也显著下降(P值<0.05),病毒抑制率由首次随访的21.86%(7/32)上升至第二次随访的40.63%(13/32)。
2.换药组患者在换药前后CD4+T淋巴细胞计数没有显著变化(P=0.279),换药前CD4+T淋巴细胞计数>350cells/μl的患者有39.53%(17/43),换药后有44.19%(19/43),比换药前略有增加;未换药组在两次随访中CD4+T淋巴细胞计数也无显著变化(P值=0.608),首次随访时CD4+T淋巴细胞计数>350cells/μl的患者有82.35%(28/34),第二次随访时CD4+T淋巴细胞计数>350cells/μl的患者的比例是70.59%(24/34)。
3.换药组和未换药组患者在第二次随访中耐药的比例均有所下降:换药患者的耐药率由换药前的7/9降至换药后的6/11;未换药患者的耐药率人群由首次随访的12/20降至第二次随访的7/14。换药的患者在换药前后耐药毒株的演变情况较未换药患者更为复杂。
总体来看,换药显著提高了患者的病毒抑制率,长久也将观察到免疫功能的改善,对耐药的影响还需要进一步观察。
第二部分血浆RNA和PBMCs DNA HIV-1耐药准种多态性和分子进化
HIV在体内以复杂的准种形式存在,在不同种类和强度的药物选择压力下,HIV-1的耐药性随时间序贯发展、逐渐增强,病毒准种的组成和分布始终处于动态变化的过程中。耐药不是全或无的现象,优势与劣势耐药准种共存,某些劣势种群可以持续进化,最终发展成为优势种群。在HIV感染发展的过程中,病毒基因组的各种变化被持续地记录在潜伏整合的前病毒中,耐药突变持续不断地进入潜伏的病毒库。耐药毒株的进化在不同组织是独立的,有研究报道血浆与外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)、淋巴及神经组织中耐药准种的分布显著不同,通常认为血浆可即时反应体内病毒的耐药进化,是世界卫生组织(World Health Organization,WHO)指定的HIV-1耐药检测和监测常规使用的样本。尽管目前对血浆中耐药毒株的分子进化已经有了比较深入的认识,但对长期治疗患者血浆中耐药病毒准种的进化特征、PBMCs中耐药病毒准种的进化特点、血浆与PBMCs中耐药病毒准种的动态进化关系还没有系统的研究。
本研究的目的是,通过对河南省某农村长期接受抗病毒治疗艾滋病患者的系列血浆和PBMCs标本的大量克隆测序、基因型耐药和分子进化分析,阐明我国农村特殊抗病毒治疗模式下血浆和PBMCs中病毒耐药准种的分子进化规律,揭示血浆和PBMCs中病毒耐药准种的进化关系。从河南省某乡HIV耐药监测队列中选出6名长期接受抗病毒治疗的艾滋病患者,这些患者最少的随访8次、最多的随访10次,累计随访54人次、时间跨度5年。每次随访均对研究对象进行访谈,了解临床表现、抗病毒治疗方案、服药依从性及其他社会人口学特征。对血浆和PBMCs分别采用RT-PCR和直接巢式PCR方法扩增HIV-1蛋白酶和逆转录酶区2162bp基因片段,然后克隆测序。每份样品对20～30个克隆测序,共获得序列1588条,其中来自血浆样本的序列967条,来自PBMCs前病毒的序列621条,对序列进行耐药突变和进化分析,主要结果和结论如下:
1.血浆与PBMCs中耐药准种的分布:血浆中蛋白酶抑制剂(protease Inhibitors,PIs)耐药突变、核苷类逆转录酶抑制剂(nucleoside reverse transcriptase inhibitors,NRTIs)、非核苷类逆转录酶抑制剂(non-nucleoside reverse transcriptase inhibitors,NNRTIs)的耐药突变种类比PBMCs中的突变种类多。多数患者血浆和PBMCs样本中共同出现的耐药突变也是两种样本中发生频率较高的突变,即血浆和PBMCs两种组织中占优势地位的耐药准种形式差别不大;
2.血浆与PBMCs中耐药准种的进化:多数患者PBMCs中PIs耐药突变、NRTIs、NNRTIs耐药突变的发生晚于血浆。少数患者血浆和PBMCs两种组织呈现完全不同的耐药进化路径。6例患者血浆和(或)PBMCs中NRTIs耐药谱和NNRTIs耐药谱的进化路径均不同程度地显示出,随着接受治疗时间的延长,主要突变谱携带的突变种类增加,即突变谱的进化都有突变位点累积的趋势,并且随之产生的耐药程度也逐渐增加;
3.血浆与PBMCs中NRTIs耐药谱的特点:6例患者几乎所有血浆和(或)PBMCs NRTIs耐药谱中都有T215Y,而且该突变在随访早期即单独或同其它突变一起出现,早期联合出现的以M41L居多,这两种突变通常稳定存在于随访的整个过程, L210W常在随访中晚期累加进NRTI突变谱,累加的其它突变还有D67N、K70R、E44D(A)等,个别患者T215Y突变模式到晚期有被T215F取代的趋势。NRTIs耐药谱的耐药程度常与其携带的突变种类有关,往往携带的突变种类越多,耐药程度也越高;
4.血浆与PBMCs中NNRTIs耐药谱的特点:总的来讲,NNRTIs耐药谱不如NRTIs耐药谱复杂,携带的突变种类不如NRTIs耐药谱多。6例患者中,除2例患者的血浆样本之外,其他患者的样本都出现了Y181C,该位点一旦出现,就会稳定存在至随访末期;K103N是另外一个重要的NNRTIs突变,在4例患者中都有出现,值得注意的是,该位点在1例患者的血浆样本和1例患者的PBMCs样本中只是在随访早期一过性出现,在其他样本中早期出现后可持续存在至随访末期;除1例患者的血浆和PBMCs样本以及1例患者的PBMCs样本之外,其它样本中都出现了H221Y突变,不同样本中该突变出现的时间不同,有的在随访早期或中期出现,有的在随访晚期才累加入NNRTIs耐药谱,该位点也是一旦出现,就会持续存在于突变谱中。NNRTIs耐药出现得较早,耐药程度往往很高,单位点突变也可导致对NVP的高度耐药;
5.血浆与PBMCs中多重耐药谱的特点:血浆样本中的多重耐药(multidrug resistance,MDR)谱的构成形式比PBMCs中的复杂,多重耐药谱的种类也比PBMCs样本多;NRTI+NNRTI双重耐药谱是MDR的主要构成形式,在所有样本中都有出现;其次是PI+NRTI+NNRTI三重耐药谱,6例患者的血浆样本中都有出现;MDR的耐药程度往往比较高;
6.血浆与PBMCs中准种序列的离散率:2例患者血浆准种的离散率大于PBMCs样本中的离散率,1例患者血浆准种的离散率小于PBMCs样本中的离散率,1例患者历次血浆和PBMCs的平均离散率没有有统计学意义差异,但是这些患者血浆中耐药谱的复杂程度都高于PBMCs样本,提示基因离散率不能代表样本中耐药准种的复杂程度。
第三部分体外无药物选择压力条件下HIV-1耐药突变的演变
HIV-1耐药准种在体内药物选择压力下呈现复杂的动态变化和进化,在体外感染细胞的过程中,携带各种耐药突变的病毒准种在靶细胞中生长,在没有药物压力的情况下,病毒产生复杂的变化,有可能发生回复突变,也可能发生各种准种的竞争性生长,最终具有最佳复制能力和复制适应性的病毒能够有效生长,成为在体外稳定传代生长的病毒,这种变化在一定程度上反映了各种突变对病毒造成的复制适应性损失的程度。
本部分研究通过对接受抗病毒治疗的艾滋病患者HIV-1毒株的体外传代培养,观察体外无药物压力条件下HIV-1耐药毒株的体外生长以及主要耐药突变的演化趋势,从而揭示各种耐药性基因突变对病毒复制能力和复制适应性的影响。我们采集了15例服用拉米夫定+司他夫定+萘韦拉平(3TC+D4T+NVP)的艾滋病患者的外周血单核细胞,用体外共培养的方法分离HIV-1毒株,观察这些毒株在体外无药物压力下的生长情况及耐药基因突变的演变。主要结果如下:
1.15例患者中病毒载量>1000拷贝/毫升的有8例,均成功分离出稳定传代的原代毒株,对这些毒株的体外复制动力学进行分析,有3株病毒呈快/高型生长方式,有5株呈慢/高型生长方式。
2.8株病毒中有2株为耐药毒株,所携带的主要耐药突变分别是K103N/K238T和M184V/K103N/Y181C/H221Y,分别对NVP和3TC/NVP高度耐药。无药物压力情况下,携带K103N突变的毒株具有较好的复制适应性,可稳定存在;携带M184V和K103N/Y181C/H221Y的毒株也能够稳定复制;K238T耐药突变稳定性差,易发生回复突变。
第四部分H221Y突变及与其它突变联合对NVP耐药作用的研究
H221Y突变是近年来发现的与NVP治疗相关的的NNRTI类耐药相关突变,在我国接受抗病毒治疗的艾滋病患者中广泛流行。本论文第二部分研究中6例患者均出现了该突变,并且该突变在准种中出现的频率多大于30%,到随访后期(初始治疗31个月后)出现频率多大于90%。值得注意的是,该部分研究中H221Y突变常伴随Y181C突变一起出现。此外,本论文第三部分分离出的原代耐药毒株HNNY15携带的NNRTI耐药突变组合为K103N/Y181C/H221Y,并且该突变组合在体外无药物压力下可稳定传代。目前,H221Y突变对耐药作用尚没有确定,对H221Y突变与其它突变的相互作用研究的也很少。
本部分研究的目的是阐明H221Y突变单独及在各种NNRTI耐药突变组合中对耐药的作用。将来自患者的携带耐药突变的pol区646bp的基因片段连接至pNL4-3骨架质粒,在此基础上进行H221Y和(/或)Y181C的定点回复诱变,包装出携带各种NNRIs耐药突变组合的毒株,在TZM-bl细胞上进行NVP表型耐药检测和分析,结果和结论如下:
1.构建和包装出20株携带不同突变组合的HIV毒株,分别是:以K101Q为基础的4株病毒,携带的突变分别是K101Q/Y181C/H221Y(1-1)、K101Q/Y181C(1-2)、K101Q/H221Y(1-3)、K101Q(1-4);以K101E为基础的4株病毒,携带的突变分别是K101E/Y181C/H221Y(2-1)、K101E/Y181C(2-2)、K101E/H221Y(2-3)、K101E(2-4);以V179E为基础的4株病毒,携带的突变分别是V179E/Y181C/H221Y(3-1)、V179E/Y181C(3-2)、V179E/H221Y(3-3)、V179E(3-4);以V179D为基础的4株病毒,携带的突变分别是V179D/Y181C/H221Y(4-1)、V179D/Y181C(4-2)、V179D/H221Y(4-3)、V179D(4-4);以K103N为基础的4株病毒,携带的突变分别是K103N/Y181C/H221Y(5-1)、K103N/Y181C(5-2)、K103N /H221Y(5-3)、K103N(5-4)。
2.测定了12株病毒的TCID50和对NVP的IC50。毒株1-1、1-2、1-3、1-4、4-1、4-2、4-3、4-4、5-1、5-2、5-3、5-4对NVP的IC50分别是108.150±1.909μM、47.807±18.131μM、0.141±0.052μM、0.037±0.010μM、162.633±47.497μM、44.980±12.763μM、0.415±0.014μM、0.142±0.022μM、191.100±91.358μM、89.180±22.656μM、4.408±0.915μM、2.013±0.360μM、0.031±0.005μM。
3.Y181C突变可大幅度提高毒株对NVP耐药的程度,在6种突变或突变组合(K101Q/H221Y、K101Q、V179D/H221Y、V179D、K103N/H221Y、K103N)的基础上,Y181C可使毒株对NVP的IC50分别提高759.144±41.903倍、1296.979±289.108倍、390.039±101.646倍、312.469±45.255倍、41.879±8.403倍和47.878±4.197倍。
4.H221Y可在一定程度上增加各种NNRTIs耐药突变组合对NVP的耐药程度。在6种突变或突变组合(K101Q/Y181C、K101Q、V179D/Y181C、V179D、K103N/Y181C、K103N)的基础上,H221Y可使毒株对NVP的IC50分别提高2.237±0.910倍、3.238±0.317倍、3.644±0.469倍、2.969±0.435倍、2.080±0.496倍和2.183±0.091倍。
5.Y181C/H221Y突变组合使携带K101Q、V179D和K103N突变毒株对NVP的IC50分别提高3444.646±834.542倍、1132.624±180.368倍和100.621±32.478倍。
Human immunodeficiency virus (HIV) is highly variable human pathogen. Under the pressure of the antiviral drugs, HIV can rapidly make drug resistance mutations, resulting in the failure of antiviral therapy (ART). Resistant HIV strains can be disseminated so that they can present in antiretroviral treatment–Na?¨ve populations and compromise initial antiretroviral therapy. Drug resistance has become a very important public health problem, and has become the mostly obstacle of AIDS (acquired immunodeficiency syndrome) prevention and cure. Finding out the molecular evolution rule of HIV drug resistance and comprehending the effect of new drug resistant mutations are bases of comprehensive study of the resistance molecular mechanism, and this can provide a scientific basis for effective prevention and restraint of HIV drug-resistant strains.
To illustrate the HIV-1 resistance molecular evolution of plasma and peripheral blood lymphocytes (PBMCs) and clarify the contribution of new drug resistant mutations, we developed a series of study on the base of AIDS patients’cohort receiving free ART in countryside of Henan province. First of all, we investigated the development and evolvement of HIV drug resistance among the AIDS patients in countryside of Henan province, part of them instead of second-line antiretroviral regimen, then isolated primary HIV-1 strains from several patients and identified the genotype drug resistance. Afterwards on the basis of plasma and PBMCs samples from partial patients followed up long-term, we revealed the resistant evolutionary relationship of virus quasispecies between plasma and PBMCs through the large number of clone sequencing and genotype resistance analysis. Finally, aim at H221Y, which emerged frequently and had not yet definite phenotype explanation, we elucidated the contributions to drug resistance of H221Y and united with other mutations by constructing serial strains with different drug resistant mutations and phenotypic identification.
PartⅠDrug resistance study of second-line antiretroviral regimen from AIDS patients receiving ART in countryside of Henan province
The countryside of Henan province was one of the regions receiving free ART earliest. The patients took the same antiretroviral regimen at the same time and supervised by doctor in rural. Our lab has monitored the HIV drug resistance of this region for 7 years since September 2003. From February 2010, partial patients replaced the first-line antiretroviral regimen with second-line, according to clinical symptoms, viral loads, the numbers of CD4+T lymphocyte, and the results of HIV drug resistance survey.
77 patients among the ART cohort followed up twice in October 2009 and June 2010, and 154 person-times were investigated and collected plasma. 43 of the 77 patients replaced the antiretroviral regimen with second-line regimen in February to March in 2010; the rest 34 patients still took first-line regimen. Questionnaires related to ART including clinical manifestations, anti-viral management, medication compliance and other socio-demographic characteristics were collected every follow-up, at the same time plasma were collected to detect VL and CD4+T lymphocyte number, in addition genotypes of drug resistance were analyzed. Main results and conclusions include:
1. VL of regimen changed group declined remarkably (P=0.005) three months later. After replacement, the viral inhibit rate ascended to 65.80% (27/41) from 46.34% (19/41) before replacement. VL of regimen unchanged group declined remarkably too (P<0.005), and the viral inhibit rate ascended to 40.63% (13/32) of second follow-up from 21.86% (7/32) of first follow-up.
2. The CD4+T lymphocyte number of regimen changed group altered invisibly (P=0.279). 39.53% (17/43) patients’CD4+T lymphocyte numbers exceeded 350cells/μl before regimen transformation, and 44.19% (19/43) after regimen replacement which increased appreciably compared to before regimen instead. The CD4+T lymphocyte number of regimen unchanged group changed unclearly (P=0.608) too. In the first follow-up, 82.35% (28/34) patients’CD4+T lymphocyte number exceeded 350cells/μl before regimen instead, and 70.59% (24/34) in the second follow-up.
3. Drug resistance rate declined little in second follow-up among both regimen changed and unchanged group. After regimen replacement, drug resistance rate of changed group descended to 6/11 from 7/9 of before regimen instead. As to regimen unchanged group, during second follow-up, drug resistance rate descended to 7/14 from12/20 during first follow-up.
PartⅡThe polymorphism and molecular evolution of HIV-1 drug resistant quasispecies in plasma RNA and PBMCs DNA
HIV exists as complex quasispecies in the HIV infection individual. Under different types and intensities of drug selection pressures, HIV-1 drug resistance develops over time and gradually increases as well as the virus composition and distribution of quasispecies are always in the process of dynamic change. The drug resistance populations consist of major and minor resistant HIV strains, and the low-abundance HIV drug resistant variants can out-compete other virus in presence of drug pressure and become the major resistant population. In the process of HIV infection, various changes in the virus genome are continuously recorded in the latent integrate provirus. At the same time, the drug resistant viruses continuously become well established in long-term reservoirs that allow drug resistance to persist. The evolutions of resistant strains are independent in different organizations, and it has been reported that resistant quasispecies distributions are significantly different in plasma and peripheral blood lymphocytes (PBMCs), lymph and nerve tissues. Generally, plasma is considered to reflect instantly the status in vivo and is the appointed sample of drug resistance detection and supervision by World Health Organization. Although molecular evolution of resistant strains in the plasma has been studied more profoundly, evolutionary characteristics of resistant virus quasispecies in plasma and peripheral blood lymphocytes(PBMCs) of long-term treatment patients and their dynamic evolution relations has no systematic research.
The objective of this study was to illustrate the molecular evolution rule of HIV-1 drug resistance quasispecies in plasma and peripheral blood lymphocytes (PBMCs) and to reveal the resistance evolutionary relationship between plasma and PBMCs virus quasispecies under the special rural anti-viral treatment mode through the large number of clone sequencing, genotype resistance analysis from AIDS patients receiving long-term ART in rural parts of Henan province. We investigated 6 AIDS patients from the drug resistance supervision cohort in the rural part of Henan province who had received ART for a long time. Follow-up times of these patients were at least 8 times, while the most was 10 times and cumulative follow-up was 54 times covering 5 years. Questionnaires related to ART including clinical manifestations, anti-viral management, medication compliance and other socio-demographic characteristics were collected every follow-up. 2162bp gene fragments (2147 ~ 4308) containing HIV-1 protease and reverse transcriptase regions of plasma and PBMCs were obtained by RT-PCR or direct PCR respectively, and then cloned and sequenced. 20-30 clones selected from each sample were sequenced. 1588 sequences were obtained in which 967 sequences from plasma samples and the others from PBMCs. Drug resistant mutations and evolution were analyzed. The head results and conclusions as follow:
1. The distribution of drug resistant quasispecies in plasma and PBMCs. The varieties of PIs (protease inhibitors), NRTIs (nucleoside reverse transcriptase inhibitors) mutations and NNRTIs (non-nucleoside reverse transcriptase inhibitors) mutations in plasma are more than those in PBMCs. In most patients, drug resistant mutations coexisting in plasma and PBMCs often occur with high-frequency, which reflects that predominant drug resistant mutations of two types of tissue are similar.
2. The evolvement of drug resistant quasispecies in plasma and PBMCs. In most patients, PI mutations, NRTI mutations and NNRTI mutations in plasma arises earlier than those in PBMCs. In few patients, HIV-1 drug resistant evolution in plasma and PBMCs are obviously differential. Evolution of HIV-1 NRTIs and NNRTIs drug resistance-associated mutations patterns of the 6 patients shows gradually accumulative process along with long-term treatment, which leads to further drug resistance.
3. The characteristics of NRTIs drug resistant patterns in plasma and PBMCs. T215Y appears solely or with other mutations at the beginning of follow-up in all samples. The combination of T215Y and M41L occurs early and stays the whole course of supervision. L210W comes into the NRTI mutation patterns at medium or terminal follow-up. Other piled mutations include D67N, K70R and E44D (A). In very few patients, T215Y tends to be replaced by T215F. The drug resistant degree of NRTI mutation patterns relates with mutation category of the patterns, that is, patterns with more mutations always show higher drug resistance.
4. The characteristics of NNRTIs drug resistant patterns in plasma and PBMCs. By and large, NNRTIs mutation patterns are simpler than NRTIs mutation patterns, and frequently include fewer mutations. Y181C exists in all samples except plasma of patient 2 & 4, moreover, the mutation can stay steadily the entire course once emergence. K103N, as the other important NNRTI mutation, occurs in patient 2, 4, 5 and 6, remarkably it appears transitorily in plasma of patient 4 and PBMCs of patient 5, but within other samples, the mutation can stay stably till the end of follow-up in case appearance. H221Y emerges in almost all samples except PBMCs of patient 5, however, the occurrence time of the mutation varies from sample to sample, and same to Y181C, the mutation can stay steadily the course once emergence. NNRTI mutations occur earlier and always show higher drug resistance to NVP, especially, single mutation can lead to high drug resistance to NVP.
5. The characteristics of MDR (multidrug resistance) drug resistant patterns in plasma and PBMCs. Multi-drug resistant patterns of plasma are more complicated than those of PBMCs, the kinds of each multi-drug resistant patterns of plasma are more complex than those of PBMCs too. NRTI+NNRTI double drug resistant patterns are the most primary form, which happen in all samples. In the next place, PI+NRTI+NNRTI trinal drug resistant patterns appear in all plasma of the six patients. Multi-drug resistant patterns often show higher drug resistant degree.
6. The mean distance of MDR (multidrug resistance) drug resistant patterns in plasma and PBMCs. Mean distance of plasma quasispecies is higher than that of PBMCs in patient 1 and 5, and it is lower in patient 3, and it is no statistical difference in patient 4. However, in these patients, drug resistant patterns of plasmas are more intricate than that of PBMCs, which indicates that mean distance can not represent the complexity degree of the drug resistance.
PartⅢEvolvement of HIV-1 Drug Resistant mutations in vitro without drug pressure
HIV-1 drug resistant quasispecies show complex dynamic transformation and evolvement in vivo under the pressure of drugs. In the process of infecting cells in vitro, virus quasispecies with various of drug resistant mutations grow in target cells, and the virus change complicatedly without the pressure of drugs, at some times they can revert to wild type or compete each other among the quasispecies, ultimately the virus with best replication capacity and fitness can vegetate richly and become exist stably, thus movement reflects the trauma degree of fitness because of different mutations.
To reveal the impact of drug resistant mutations on virus replication capacity and fitness, we observed replication dynamics of the drug resistant HIV-1 isolates and evolvement tendency of the drug resistant mutations in vitro without drug pressure through culturing strains in vitro, which isolated from AIDS patients receiving ART. PBMCs from 15 AIDS patients receiving ART (3TC+D4T+NVP) were collected, and the primary HIV-1 stains were separated utilizing co-cultivated with PBMCs from normal people. Virus growth and the evolution of the drug resistant mutations in vitro without drug pressure were observed. Primary results include:
1. Eight strong positive strains were isolated successfully from 8/15 AIDS patients with viral loads higher than 1000 copies/ml, and the replication dynamics were analyzed. Three strains grew rapidly/highly and the other five strains grew slowly/highly.
2. Two of them were drug resistant strains. Drug resistant mutations of the two strains were respectively K103N/K238T and M184V/K103N/Y181C/H221Y which show high-level resistance to NVP and 3TC/NVP, respectively. Strains with K103N shows superior fitness and can exist steadily without drug pressure. Strains with M184V and K103N/Y181C/H221Y can also replicate stably in vitro without drug pressure. NNRTIs mutation K238T reproduces astatically and reverts gradually to K238.
PartⅣThe impact of H221Y mutations and combined with other mutations on NVP drug resistance
H221Y was found to be a NNRTIs mutation in recent years which conferring resistance to NVP, and had wild prevalence in our country. All the 6 patients occurred H221Y in partⅡof this paper, and the occurrence frequency within quasispecies always exceeded 30%, especially, at follow-up anaphase, the frequency could exceed 90%. Remarkably, H221Y often appeared together with Y181C in this part. In addition, one primary HIV-1 drug resistant stain in partⅢcarried K103N/Y181C/H221Y mutation combination, and the strain can replicate stably in vitro in absence of drugs. Now, the impact of H221Y on drug resistance is still faint, and the interaction of H221Y with other mutations is yet indistinct.
Objective of this part is to clarify the impact of H221Y by itself and combination with other mutations on drug resistance. 646bp HIV-1 pol gene fragments were embedded pNL4-3 bone plasmid, and H221Y and (/or) Y181C were reverted to wild type by site-directed mutagenesis, then strains with various mutations were packed, in the end phenotype analysis was analyzed on TZM-bl cells. The results as follows:
1. 20 strains with different drug resistant mutation combinations were constructed, these mutation combinations include K101Q/Y181C/H221Y (1-1), K101Q/Y181C (1-2), K101Q/H221Y (1-3), K101Q (1-4), K101E/Y181C/H221Y (2-1), K101E/Y181C (2-2), K101E/H221Y (2-3), K101E (2-4), V179E/Y181C/H221Y (3-1), V179E/Y181C (3-2), V179E/H221Y (3-3), V179E (3-4), V179D/Y181C/H221Y (4-1), V179D/Y181C (4-2), V179D/H221Y (4-3), V179D (4-4), K103N/Y181C/H221Y (5-1), K103N/Y181C (5-2), K103N /H221Y (5-3), K103N (5-4).
2. TCID50 and IC50 to NVP of 12 strains were detected. The IC50 of strain 1-1, 1-2, 1-3, 1-4, 4-1, 4-2, 4-3, 4-4, 5-1, 5-2, 5-3 and 5-4 to NVP were separately 108.150±1.909μM, 47.807±18.131μM, 0.141±0.052μM, 0.037±0.010μM, 162.633±47.497μM, 44.980±12.763μM, 0.415±0.014μM, 0.142±0.022μM, 191.100±91.358μM, 89.180±22.656μM, 4.408±0.915μM, 2.013±0.360μM and 0.031±0.005μM.
3. Y181C conferred extraordinary resistance to NVP. As to mutation combinations such as K101Q/H221Y, K101Q, V179D/H221Y, V179D, K103N/H221Y, K103N, the IC50 to NVP improved by Y181C were respectively 759.144±41.903, 1296.979±289.108, 390.039±101.646, 312.469±45.255, 41.879±8.403 and 47.878±4.197 times.
4. H221Y could improve the resistance degree of all mutation combinations to NVP to a certain extent. As to mutation combinations such as K101Q/Y181C, K101Q, V179D/Y181C, V179D, K103N/Y181C, K103N, the IC50 to NVP improved by H221Y were respectively 2.237±0.910, 3.238±0.317, 3.644±0.469, 2.969±0.435, 2.080±0.496 and 2.183±0.091 times.
5. According to K101Q, V179D and K103N, times of IC50 to NVP improved by Y181C/H221Y were respectively 3444.646±834.542, 1132.624±180.368 and 100.621±32.478.
为了揭示血浆和外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)中HIV耐药的分子进化规律、阐明新型耐药突变对耐药的贡献,本研究以河南某乡接受抗病毒治疗艾滋病患者队列为基础,开展了系列研究。首先对河南某乡接受抗病毒治疗的艾滋病患者更换二线药物前后的病毒耐药发生和演变情况进行了调查,对部分患者进行了病毒分离培养和基因型耐药鉴定;然后,基于长期随访获得的部分患者系列血浆和PBMCs标本,采用大量克隆测序及基因型耐药分析的方法,揭示血浆和PBMCs中耐药准种进化的规律;针对患者中大量出现的尚未有明确表型解释的H221Y突变,通过构建系列突变病毒并进行表型耐药测定的方式,阐明H221Y及其它突变联合对耐药的作用。
第一部分河南某乡接受抗病毒治疗的艾滋病患者更换二线治疗方案前后的耐药性研究
河南某乡是我国最早开展艾滋病免费抗病毒治疗的地区之一,采用的是使用统一的治疗方案、同时开始治疗,由村医管理患者并监督服药的治疗模式。自初始治疗(2003年9月)起,我们实验室对这个地区艾滋病患者的HIV耐药情况进行了连续7年的监测。根据患者的临床表现、病毒载量、CD4+T淋巴细胞计数检测的结果及病毒耐药监测的结果,2010年2月起,部分患者由一线治疗方案更换为二线治疗方案。本部分研究的目的是了解更换二线治疗方案后的HIV耐药发生发展的特点和规律,同时,观察更换二线治疗方案后的病毒学和免疫学应答,从而为抗病毒治疗方案的改进提供依据。
对抗病毒治疗患者队列中的77名患者进行了两次随访,时间为2009年10月和2010年6月,共调查和采血154人次。其中43例患者在首次随访时(2010年2～3月)更换了二线治疗方案,34例患者仍然使用一线治疗方案。对所有患者使用统一的调查表进行流行病学调查,了解服药依从性、临床表现等信息,采集血液标本进行病毒载量和CD4+T淋巴细胞计数检测,采用耐药基因型方法进行耐药性检测和分析。主要结果和结论有:
1.换药组患者在换药3个月后病毒载量显著下降(P值=0.005)。病毒抑制率由治疗前的46.34%(19/41)上升至换药后的65.80%(27/41);未换药组患者的病毒载量也显著下降(P值<0.05),病毒抑制率由首次随访的21.86%(7/32)上升至第二次随访的40.63%(13/32)。
2.换药组患者在换药前后CD4+T淋巴细胞计数没有显著变化(P=0.279),换药前CD4+T淋巴细胞计数>350cells/μl的患者有39.53%(17/43),换药后有44.19%(19/43),比换药前略有增加;未换药组在两次随访中CD4+T淋巴细胞计数也无显著变化(P值=0.608),首次随访时CD4+T淋巴细胞计数>350cells/μl的患者有82.35%(28/34),第二次随访时CD4+T淋巴细胞计数>350cells/μl的患者的比例是70.59%(24/34)。
3.换药组和未换药组患者在第二次随访中耐药的比例均有所下降:换药患者的耐药率由换药前的7/9降至换药后的6/11;未换药患者的耐药率人群由首次随访的12/20降至第二次随访的7/14。换药的患者在换药前后耐药毒株的演变情况较未换药患者更为复杂。
总体来看,换药显著提高了患者的病毒抑制率,长久也将观察到免疫功能的改善,对耐药的影响还需要进一步观察。
第二部分血浆RNA和PBMCs DNA HIV-1耐药准种多态性和分子进化
HIV在体内以复杂的准种形式存在,在不同种类和强度的药物选择压力下,HIV-1的耐药性随时间序贯发展、逐渐增强,病毒准种的组成和分布始终处于动态变化的过程中。耐药不是全或无的现象,优势与劣势耐药准种共存,某些劣势种群可以持续进化,最终发展成为优势种群。在HIV感染发展的过程中,病毒基因组的各种变化被持续地记录在潜伏整合的前病毒中,耐药突变持续不断地进入潜伏的病毒库。耐药毒株的进化在不同组织是独立的,有研究报道血浆与外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)、淋巴及神经组织中耐药准种的分布显著不同,通常认为血浆可即时反应体内病毒的耐药进化,是世界卫生组织(World Health Organization,WHO)指定的HIV-1耐药检测和监测常规使用的样本。尽管目前对血浆中耐药毒株的分子进化已经有了比较深入的认识,但对长期治疗患者血浆中耐药病毒准种的进化特征、PBMCs中耐药病毒准种的进化特点、血浆与PBMCs中耐药病毒准种的动态进化关系还没有系统的研究。
本研究的目的是,通过对河南省某农村长期接受抗病毒治疗艾滋病患者的系列血浆和PBMCs标本的大量克隆测序、基因型耐药和分子进化分析,阐明我国农村特殊抗病毒治疗模式下血浆和PBMCs中病毒耐药准种的分子进化规律,揭示血浆和PBMCs中病毒耐药准种的进化关系。从河南省某乡HIV耐药监测队列中选出6名长期接受抗病毒治疗的艾滋病患者,这些患者最少的随访8次、最多的随访10次,累计随访54人次、时间跨度5年。每次随访均对研究对象进行访谈,了解临床表现、抗病毒治疗方案、服药依从性及其他社会人口学特征。对血浆和PBMCs分别采用RT-PCR和直接巢式PCR方法扩增HIV-1蛋白酶和逆转录酶区2162bp基因片段,然后克隆测序。每份样品对20～30个克隆测序,共获得序列1588条,其中来自血浆样本的序列967条,来自PBMCs前病毒的序列621条,对序列进行耐药突变和进化分析,主要结果和结论如下:
1.血浆与PBMCs中耐药准种的分布:血浆中蛋白酶抑制剂(protease Inhibitors,PIs)耐药突变、核苷类逆转录酶抑制剂(nucleoside reverse transcriptase inhibitors,NRTIs)、非核苷类逆转录酶抑制剂(non-nucleoside reverse transcriptase inhibitors,NNRTIs)的耐药突变种类比PBMCs中的突变种类多。多数患者血浆和PBMCs样本中共同出现的耐药突变也是两种样本中发生频率较高的突变,即血浆和PBMCs两种组织中占优势地位的耐药准种形式差别不大;
2.血浆与PBMCs中耐药准种的进化:多数患者PBMCs中PIs耐药突变、NRTIs、NNRTIs耐药突变的发生晚于血浆。少数患者血浆和PBMCs两种组织呈现完全不同的耐药进化路径。6例患者血浆和(或)PBMCs中NRTIs耐药谱和NNRTIs耐药谱的进化路径均不同程度地显示出,随着接受治疗时间的延长,主要突变谱携带的突变种类增加,即突变谱的进化都有突变位点累积的趋势,并且随之产生的耐药程度也逐渐增加;
3.血浆与PBMCs中NRTIs耐药谱的特点:6例患者几乎所有血浆和(或)PBMCs NRTIs耐药谱中都有T215Y,而且该突变在随访早期即单独或同其它突变一起出现,早期联合出现的以M41L居多,这两种突变通常稳定存在于随访的整个过程, L210W常在随访中晚期累加进NRTI突变谱,累加的其它突变还有D67N、K70R、E44D(A)等,个别患者T215Y突变模式到晚期有被T215F取代的趋势。NRTIs耐药谱的耐药程度常与其携带的突变种类有关,往往携带的突变种类越多,耐药程度也越高;
4.血浆与PBMCs中NNRTIs耐药谱的特点:总的来讲,NNRTIs耐药谱不如NRTIs耐药谱复杂,携带的突变种类不如NRTIs耐药谱多。6例患者中,除2例患者的血浆样本之外,其他患者的样本都出现了Y181C,该位点一旦出现,就会稳定存在至随访末期;K103N是另外一个重要的NNRTIs突变,在4例患者中都有出现,值得注意的是,该位点在1例患者的血浆样本和1例患者的PBMCs样本中只是在随访早期一过性出现,在其他样本中早期出现后可持续存在至随访末期;除1例患者的血浆和PBMCs样本以及1例患者的PBMCs样本之外,其它样本中都出现了H221Y突变,不同样本中该突变出现的时间不同,有的在随访早期或中期出现,有的在随访晚期才累加入NNRTIs耐药谱,该位点也是一旦出现,就会持续存在于突变谱中。NNRTIs耐药出现得较早,耐药程度往往很高,单位点突变也可导致对NVP的高度耐药;
5.血浆与PBMCs中多重耐药谱的特点:血浆样本中的多重耐药(multidrug resistance,MDR)谱的构成形式比PBMCs中的复杂,多重耐药谱的种类也比PBMCs样本多;NRTI+NNRTI双重耐药谱是MDR的主要构成形式,在所有样本中都有出现;其次是PI+NRTI+NNRTI三重耐药谱,6例患者的血浆样本中都有出现;MDR的耐药程度往往比较高;
6.血浆与PBMCs中准种序列的离散率:2例患者血浆准种的离散率大于PBMCs样本中的离散率,1例患者血浆准种的离散率小于PBMCs样本中的离散率,1例患者历次血浆和PBMCs的平均离散率没有有统计学意义差异,但是这些患者血浆中耐药谱的复杂程度都高于PBMCs样本,提示基因离散率不能代表样本中耐药准种的复杂程度。
第三部分体外无药物选择压力条件下HIV-1耐药突变的演变
HIV-1耐药准种在体内药物选择压力下呈现复杂的动态变化和进化,在体外感染细胞的过程中,携带各种耐药突变的病毒准种在靶细胞中生长,在没有药物压力的情况下,病毒产生复杂的变化,有可能发生回复突变,也可能发生各种准种的竞争性生长,最终具有最佳复制能力和复制适应性的病毒能够有效生长,成为在体外稳定传代生长的病毒,这种变化在一定程度上反映了各种突变对病毒造成的复制适应性损失的程度。
本部分研究通过对接受抗病毒治疗的艾滋病患者HIV-1毒株的体外传代培养,观察体外无药物压力条件下HIV-1耐药毒株的体外生长以及主要耐药突变的演化趋势,从而揭示各种耐药性基因突变对病毒复制能力和复制适应性的影响。我们采集了15例服用拉米夫定+司他夫定+萘韦拉平(3TC+D4T+NVP)的艾滋病患者的外周血单核细胞,用体外共培养的方法分离HIV-1毒株,观察这些毒株在体外无药物压力下的生长情况及耐药基因突变的演变。主要结果如下:
1.15例患者中病毒载量>1000拷贝/毫升的有8例,均成功分离出稳定传代的原代毒株,对这些毒株的体外复制动力学进行分析,有3株病毒呈快/高型生长方式,有5株呈慢/高型生长方式。
2.8株病毒中有2株为耐药毒株,所携带的主要耐药突变分别是K103N/K238T和M184V/K103N/Y181C/H221Y,分别对NVP和3TC/NVP高度耐药。无药物压力情况下,携带K103N突变的毒株具有较好的复制适应性,可稳定存在;携带M184V和K103N/Y181C/H221Y的毒株也能够稳定复制;K238T耐药突变稳定性差,易发生回复突变。
第四部分H221Y突变及与其它突变联合对NVP耐药作用的研究
H221Y突变是近年来发现的与NVP治疗相关的的NNRTI类耐药相关突变,在我国接受抗病毒治疗的艾滋病患者中广泛流行。本论文第二部分研究中6例患者均出现了该突变,并且该突变在准种中出现的频率多大于30%,到随访后期(初始治疗31个月后)出现频率多大于90%。值得注意的是,该部分研究中H221Y突变常伴随Y181C突变一起出现。此外,本论文第三部分分离出的原代耐药毒株HNNY15携带的NNRTI耐药突变组合为K103N/Y181C/H221Y,并且该突变组合在体外无药物压力下可稳定传代。目前,H221Y突变对耐药作用尚没有确定,对H221Y突变与其它突变的相互作用研究的也很少。
本部分研究的目的是阐明H221Y突变单独及在各种NNRTI耐药突变组合中对耐药的作用。将来自患者的携带耐药突变的pol区646bp的基因片段连接至pNL4-3骨架质粒,在此基础上进行H221Y和(/或)Y181C的定点回复诱变,包装出携带各种NNRIs耐药突变组合的毒株,在TZM-bl细胞上进行NVP表型耐药检测和分析,结果和结论如下:
1.构建和包装出20株携带不同突变组合的HIV毒株,分别是:以K101Q为基础的4株病毒,携带的突变分别是K101Q/Y181C/H221Y(1-1)、K101Q/Y181C(1-2)、K101Q/H221Y(1-3)、K101Q(1-4);以K101E为基础的4株病毒,携带的突变分别是K101E/Y181C/H221Y(2-1)、K101E/Y181C(2-2)、K101E/H221Y(2-3)、K101E(2-4);以V179E为基础的4株病毒,携带的突变分别是V179E/Y181C/H221Y(3-1)、V179E/Y181C(3-2)、V179E/H221Y(3-3)、V179E(3-4);以V179D为基础的4株病毒,携带的突变分别是V179D/Y181C/H221Y(4-1)、V179D/Y181C(4-2)、V179D/H221Y(4-3)、V179D(4-4);以K103N为基础的4株病毒,携带的突变分别是K103N/Y181C/H221Y(5-1)、K103N/Y181C(5-2)、K103N /H221Y(5-3)、K103N(5-4)。
2.测定了12株病毒的TCID50和对NVP的IC50。毒株1-1、1-2、1-3、1-4、4-1、4-2、4-3、4-4、5-1、5-2、5-3、5-4对NVP的IC50分别是108.150±1.909μM、47.807±18.131μM、0.141±0.052μM、0.037±0.010μM、162.633±47.497μM、44.980±12.763μM、0.415±0.014μM、0.142±0.022μM、191.100±91.358μM、89.180±22.656μM、4.408±0.915μM、2.013±0.360μM、0.031±0.005μM。
3.Y181C突变可大幅度提高毒株对NVP耐药的程度,在6种突变或突变组合(K101Q/H221Y、K101Q、V179D/H221Y、V179D、K103N/H221Y、K103N)的基础上,Y181C可使毒株对NVP的IC50分别提高759.144±41.903倍、1296.979±289.108倍、390.039±101.646倍、312.469±45.255倍、41.879±8.403倍和47.878±4.197倍。
4.H221Y可在一定程度上增加各种NNRTIs耐药突变组合对NVP的耐药程度。在6种突变或突变组合(K101Q/Y181C、K101Q、V179D/Y181C、V179D、K103N/Y181C、K103N)的基础上,H221Y可使毒株对NVP的IC50分别提高2.237±0.910倍、3.238±0.317倍、3.644±0.469倍、2.969±0.435倍、2.080±0.496倍和2.183±0.091倍。
5.Y181C/H221Y突变组合使携带K101Q、V179D和K103N突变毒株对NVP的IC50分别提高3444.646±834.542倍、1132.624±180.368倍和100.621±32.478倍。
Human immunodeficiency virus (HIV) is highly variable human pathogen. Under the pressure of the antiviral drugs, HIV can rapidly make drug resistance mutations, resulting in the failure of antiviral therapy (ART). Resistant HIV strains can be disseminated so that they can present in antiretroviral treatment–Na?¨ve populations and compromise initial antiretroviral therapy. Drug resistance has become a very important public health problem, and has become the mostly obstacle of AIDS (acquired immunodeficiency syndrome) prevention and cure. Finding out the molecular evolution rule of HIV drug resistance and comprehending the effect of new drug resistant mutations are bases of comprehensive study of the resistance molecular mechanism, and this can provide a scientific basis for effective prevention and restraint of HIV drug-resistant strains.
To illustrate the HIV-1 resistance molecular evolution of plasma and peripheral blood lymphocytes (PBMCs) and clarify the contribution of new drug resistant mutations, we developed a series of study on the base of AIDS patients’cohort receiving free ART in countryside of Henan province. First of all, we investigated the development and evolvement of HIV drug resistance among the AIDS patients in countryside of Henan province, part of them instead of second-line antiretroviral regimen, then isolated primary HIV-1 strains from several patients and identified the genotype drug resistance. Afterwards on the basis of plasma and PBMCs samples from partial patients followed up long-term, we revealed the resistant evolutionary relationship of virus quasispecies between plasma and PBMCs through the large number of clone sequencing and genotype resistance analysis. Finally, aim at H221Y, which emerged frequently and had not yet definite phenotype explanation, we elucidated the contributions to drug resistance of H221Y and united with other mutations by constructing serial strains with different drug resistant mutations and phenotypic identification.
PartⅠDrug resistance study of second-line antiretroviral regimen from AIDS patients receiving ART in countryside of Henan province
The countryside of Henan province was one of the regions receiving free ART earliest. The patients took the same antiretroviral regimen at the same time and supervised by doctor in rural. Our lab has monitored the HIV drug resistance of this region for 7 years since September 2003. From February 2010, partial patients replaced the first-line antiretroviral regimen with second-line, according to clinical symptoms, viral loads, the numbers of CD4+T lymphocyte, and the results of HIV drug resistance survey.
77 patients among the ART cohort followed up twice in October 2009 and June 2010, and 154 person-times were investigated and collected plasma. 43 of the 77 patients replaced the antiretroviral regimen with second-line regimen in February to March in 2010; the rest 34 patients still took first-line regimen. Questionnaires related to ART including clinical manifestations, anti-viral management, medication compliance and other socio-demographic characteristics were collected every follow-up, at the same time plasma were collected to detect VL and CD4+T lymphocyte number, in addition genotypes of drug resistance were analyzed. Main results and conclusions include:
1. VL of regimen changed group declined remarkably (P=0.005) three months later. After replacement, the viral inhibit rate ascended to 65.80% (27/41) from 46.34% (19/41) before replacement. VL of regimen unchanged group declined remarkably too (P<0.005), and the viral inhibit rate ascended to 40.63% (13/32) of second follow-up from 21.86% (7/32) of first follow-up.
2. The CD4+T lymphocyte number of regimen changed group altered invisibly (P=0.279). 39.53% (17/43) patients’CD4+T lymphocyte numbers exceeded 350cells/μl before regimen transformation, and 44.19% (19/43) after regimen replacement which increased appreciably compared to before regimen instead. The CD4+T lymphocyte number of regimen unchanged group changed unclearly (P=0.608) too. In the first follow-up, 82.35% (28/34) patients’CD4+T lymphocyte number exceeded 350cells/μl before regimen instead, and 70.59% (24/34) in the second follow-up.
3. Drug resistance rate declined little in second follow-up among both regimen changed and unchanged group. After regimen replacement, drug resistance rate of changed group descended to 6/11 from 7/9 of before regimen instead. As to regimen unchanged group, during second follow-up, drug resistance rate descended to 7/14 from12/20 during first follow-up.
PartⅡThe polymorphism and molecular evolution of HIV-1 drug resistant quasispecies in plasma RNA and PBMCs DNA
HIV exists as complex quasispecies in the HIV infection individual. Under different types and intensities of drug selection pressures, HIV-1 drug resistance develops over time and gradually increases as well as the virus composition and distribution of quasispecies are always in the process of dynamic change. The drug resistance populations consist of major and minor resistant HIV strains, and the low-abundance HIV drug resistant variants can out-compete other virus in presence of drug pressure and become the major resistant population. In the process of HIV infection, various changes in the virus genome are continuously recorded in the latent integrate provirus. At the same time, the drug resistant viruses continuously become well established in long-term reservoirs that allow drug resistance to persist. The evolutions of resistant strains are independent in different organizations, and it has been reported that resistant quasispecies distributions are significantly different in plasma and peripheral blood lymphocytes (PBMCs), lymph and nerve tissues. Generally, plasma is considered to reflect instantly the status in vivo and is the appointed sample of drug resistance detection and supervision by World Health Organization. Although molecular evolution of resistant strains in the plasma has been studied more profoundly, evolutionary characteristics of resistant virus quasispecies in plasma and peripheral blood lymphocytes(PBMCs) of long-term treatment patients and their dynamic evolution relations has no systematic research.
The objective of this study was to illustrate the molecular evolution rule of HIV-1 drug resistance quasispecies in plasma and peripheral blood lymphocytes (PBMCs) and to reveal the resistance evolutionary relationship between plasma and PBMCs virus quasispecies under the special rural anti-viral treatment mode through the large number of clone sequencing, genotype resistance analysis from AIDS patients receiving long-term ART in rural parts of Henan province. We investigated 6 AIDS patients from the drug resistance supervision cohort in the rural part of Henan province who had received ART for a long time. Follow-up times of these patients were at least 8 times, while the most was 10 times and cumulative follow-up was 54 times covering 5 years. Questionnaires related to ART including clinical manifestations, anti-viral management, medication compliance and other socio-demographic characteristics were collected every follow-up. 2162bp gene fragments (2147 ~ 4308) containing HIV-1 protease and reverse transcriptase regions of plasma and PBMCs were obtained by RT-PCR or direct PCR respectively, and then cloned and sequenced. 20-30 clones selected from each sample were sequenced. 1588 sequences were obtained in which 967 sequences from plasma samples and the others from PBMCs. Drug resistant mutations and evolution were analyzed. The head results and conclusions as follow:
1. The distribution of drug resistant quasispecies in plasma and PBMCs. The varieties of PIs (protease inhibitors), NRTIs (nucleoside reverse transcriptase inhibitors) mutations and NNRTIs (non-nucleoside reverse transcriptase inhibitors) mutations in plasma are more than those in PBMCs. In most patients, drug resistant mutations coexisting in plasma and PBMCs often occur with high-frequency, which reflects that predominant drug resistant mutations of two types of tissue are similar.
2. The evolvement of drug resistant quasispecies in plasma and PBMCs. In most patients, PI mutations, NRTI mutations and NNRTI mutations in plasma arises earlier than those in PBMCs. In few patients, HIV-1 drug resistant evolution in plasma and PBMCs are obviously differential. Evolution of HIV-1 NRTIs and NNRTIs drug resistance-associated mutations patterns of the 6 patients shows gradually accumulative process along with long-term treatment, which leads to further drug resistance.
3. The characteristics of NRTIs drug resistant patterns in plasma and PBMCs. T215Y appears solely or with other mutations at the beginning of follow-up in all samples. The combination of T215Y and M41L occurs early and stays the whole course of supervision. L210W comes into the NRTI mutation patterns at medium or terminal follow-up. Other piled mutations include D67N, K70R and E44D (A). In very few patients, T215Y tends to be replaced by T215F. The drug resistant degree of NRTI mutation patterns relates with mutation category of the patterns, that is, patterns with more mutations always show higher drug resistance.
4. The characteristics of NNRTIs drug resistant patterns in plasma and PBMCs. By and large, NNRTIs mutation patterns are simpler than NRTIs mutation patterns, and frequently include fewer mutations. Y181C exists in all samples except plasma of patient 2 & 4, moreover, the mutation can stay steadily the entire course once emergence. K103N, as the other important NNRTI mutation, occurs in patient 2, 4, 5 and 6, remarkably it appears transitorily in plasma of patient 4 and PBMCs of patient 5, but within other samples, the mutation can stay stably till the end of follow-up in case appearance. H221Y emerges in almost all samples except PBMCs of patient 5, however, the occurrence time of the mutation varies from sample to sample, and same to Y181C, the mutation can stay steadily the course once emergence. NNRTI mutations occur earlier and always show higher drug resistance to NVP, especially, single mutation can lead to high drug resistance to NVP.
5. The characteristics of MDR (multidrug resistance) drug resistant patterns in plasma and PBMCs. Multi-drug resistant patterns of plasma are more complicated than those of PBMCs, the kinds of each multi-drug resistant patterns of plasma are more complex than those of PBMCs too. NRTI+NNRTI double drug resistant patterns are the most primary form, which happen in all samples. In the next place, PI+NRTI+NNRTI trinal drug resistant patterns appear in all plasma of the six patients. Multi-drug resistant patterns often show higher drug resistant degree.
6. The mean distance of MDR (multidrug resistance) drug resistant patterns in plasma and PBMCs. Mean distance of plasma quasispecies is higher than that of PBMCs in patient 1 and 5, and it is lower in patient 3, and it is no statistical difference in patient 4. However, in these patients, drug resistant patterns of plasmas are more intricate than that of PBMCs, which indicates that mean distance can not represent the complexity degree of the drug resistance.
PartⅢEvolvement of HIV-1 Drug Resistant mutations in vitro without drug pressure
HIV-1 drug resistant quasispecies show complex dynamic transformation and evolvement in vivo under the pressure of drugs. In the process of infecting cells in vitro, virus quasispecies with various of drug resistant mutations grow in target cells, and the virus change complicatedly without the pressure of drugs, at some times they can revert to wild type or compete each other among the quasispecies, ultimately the virus with best replication capacity and fitness can vegetate richly and become exist stably, thus movement reflects the trauma degree of fitness because of different mutations.
To reveal the impact of drug resistant mutations on virus replication capacity and fitness, we observed replication dynamics of the drug resistant HIV-1 isolates and evolvement tendency of the drug resistant mutations in vitro without drug pressure through culturing strains in vitro, which isolated from AIDS patients receiving ART. PBMCs from 15 AIDS patients receiving ART (3TC+D4T+NVP) were collected, and the primary HIV-1 stains were separated utilizing co-cultivated with PBMCs from normal people. Virus growth and the evolution of the drug resistant mutations in vitro without drug pressure were observed. Primary results include:
1. Eight strong positive strains were isolated successfully from 8/15 AIDS patients with viral loads higher than 1000 copies/ml, and the replication dynamics were analyzed. Three strains grew rapidly/highly and the other five strains grew slowly/highly.
2. Two of them were drug resistant strains. Drug resistant mutations of the two strains were respectively K103N/K238T and M184V/K103N/Y181C/H221Y which show high-level resistance to NVP and 3TC/NVP, respectively. Strains with K103N shows superior fitness and can exist steadily without drug pressure. Strains with M184V and K103N/Y181C/H221Y can also replicate stably in vitro without drug pressure. NNRTIs mutation K238T reproduces astatically and reverts gradually to K238.
PartⅣThe impact of H221Y mutations and combined with other mutations on NVP drug resistance
H221Y was found to be a NNRTIs mutation in recent years which conferring resistance to NVP, and had wild prevalence in our country. All the 6 patients occurred H221Y in partⅡof this paper, and the occurrence frequency within quasispecies always exceeded 30%, especially, at follow-up anaphase, the frequency could exceed 90%. Remarkably, H221Y often appeared together with Y181C in this part. In addition, one primary HIV-1 drug resistant stain in partⅢcarried K103N/Y181C/H221Y mutation combination, and the strain can replicate stably in vitro in absence of drugs. Now, the impact of H221Y on drug resistance is still faint, and the interaction of H221Y with other mutations is yet indistinct.
Objective of this part is to clarify the impact of H221Y by itself and combination with other mutations on drug resistance. 646bp HIV-1 pol gene fragments were embedded pNL4-3 bone plasmid, and H221Y and (/or) Y181C were reverted to wild type by site-directed mutagenesis, then strains with various mutations were packed, in the end phenotype analysis was analyzed on TZM-bl cells. The results as follows:
1. 20 strains with different drug resistant mutation combinations were constructed, these mutation combinations include K101Q/Y181C/H221Y (1-1), K101Q/Y181C (1-2), K101Q/H221Y (1-3), K101Q (1-4), K101E/Y181C/H221Y (2-1), K101E/Y181C (2-2), K101E/H221Y (2-3), K101E (2-4), V179E/Y181C/H221Y (3-1), V179E/Y181C (3-2), V179E/H221Y (3-3), V179E (3-4), V179D/Y181C/H221Y (4-1), V179D/Y181C (4-2), V179D/H221Y (4-3), V179D (4-4), K103N/Y181C/H221Y (5-1), K103N/Y181C (5-2), K103N /H221Y (5-3), K103N (5-4).
2. TCID50 and IC50 to NVP of 12 strains were detected. The IC50 of strain 1-1, 1-2, 1-3, 1-4, 4-1, 4-2, 4-3, 4-4, 5-1, 5-2, 5-3 and 5-4 to NVP were separately 108.150±1.909μM, 47.807±18.131μM, 0.141±0.052μM, 0.037±0.010μM, 162.633±47.497μM, 44.980±12.763μM, 0.415±0.014μM, 0.142±0.022μM, 191.100±91.358μM, 89.180±22.656μM, 4.408±0.915μM, 2.013±0.360μM and 0.031±0.005μM.
3. Y181C conferred extraordinary resistance to NVP. As to mutation combinations such as K101Q/H221Y, K101Q, V179D/H221Y, V179D, K103N/H221Y, K103N, the IC50 to NVP improved by Y181C were respectively 759.144±41.903, 1296.979±289.108, 390.039±101.646, 312.469±45.255, 41.879±8.403 and 47.878±4.197 times.
4. H221Y could improve the resistance degree of all mutation combinations to NVP to a certain extent. As to mutation combinations such as K101Q/Y181C, K101Q, V179D/Y181C, V179D, K103N/Y181C, K103N, the IC50 to NVP improved by H221Y were respectively 2.237±0.910, 3.238±0.317, 3.644±0.469, 2.969±0.435, 2.080±0.496 and 2.183±0.091 times.
5. According to K101Q, V179D and K103N, times of IC50 to NVP improved by Y181C/H221Y were respectively 3444.646±834.542, 1132.624±180.368 and 100.621±32.478.
引文
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