长期抗病毒治疗患者HIV耐药毒株的进化研究

发布时间：2018-01-26 01:42

本文关键词： HIV 抗逆转录病毒治疗耐药低病毒载量非核苷类抑制剂补偿性位点病毒进化　出处：《中国疾病预防控制中心》2013年硕士论文　论文类型：学位论文

【摘要】：艾滋病抗逆转录病毒治疗(Antiretroviral Therapy, ART)自20世纪90年代起在全世界范围内已广泛开展,已由最初的齐多夫定(Zidovudine, AZT)发展至七大类33种药物。ART的开展给全世界的艾滋病患者带来了福音,帮助其延缓了病情的发展,并提高了生活质量。我国自2003年开始逐步推广免费的抗逆转录病毒治疗,艾滋病的病死率已由2003年的9.5死亡/100人年降低至了2009年的5.2死亡/人年。虽然我国的ART已初步取得了一定的成绩,但是由于抗病毒治疗早期药物资源有限,相当数量患者在接受ART治疗后不得不长期使用同一种治疗方案。而众多研究表明长期治疗下HIV耐药株的产生则是影响抗病毒治疗效果的主要因素。HIV病毒的生物学特点之一是较高的遗传突变率,它使HIV的基因序列产生较多的突变,这些突变病毒以准种形式在患者体内存在。在HIV患者接受抗病毒治疗的过程中,HIV的高突变率使病毒能够产生有效的突变位点,携带耐药位点的病毒株可以从药物作用下逃逸。中国疾病预防控制中心性病艾滋病预防控制中心与河南省疾病预防控制中心于2003年底即在河南省某确山县对抗病毒患者进行了长期的随访观察,本文利用该队列尝试探寻HIV耐药突变对长期抗病毒治疗的影响。本文应用科室自建的基因型耐药检测方法(in-house)和单模板扩增方法(SingleGenomeAmplification,SGA)获得HIV-1病毒poL区的蛋白酶和逆转录酶基因区片段(2253bp-3553bp,1.3kb).应用Sequencher (4.10.1)和Bioedit(7.0)软件对序列进行编辑并利用美国斯坦福大学HIV耐药数据库(Stanfrod University HIV Drug Resistance Database, HIVDB)进行耐药程度分析。世界卫生组织(WHO)公布的HIV耐药检测指南中,HIV耐药检测的病毒载量检测线为1000拷贝/ml,故国内外对于病毒载量在50-1000拷贝/ml时患者的耐药研究报道较少,而这一范围往往是药物抑制效果开始降低的时期。对低载量耐药的研究可能会有助于提早预警病毒抑制失败和耐药的发生,有助于提高患者的治疗效果。本研究利用上述耐药队列中曾出现过50-1000拷贝/ml的患者,研究其发生耐药后对后期治疗效果的影响及耐药的发展。结果发现在28例病毒抑制失败的患者中有18人在低病毒载量时期发生了耐药。在耐药毒株出现的初期,非核苷类抑制剂(Non-Nucleoside Reverse Transcriptase Inhibitor, NNRTI)是主要耐受药物,对奈韦拉平(Nevirapine NVP)的耐药是导致患者耐药的主要因素,且耐药水平已达到高度。在病毒载量大于150拷贝/mL低于1000拷贝/mL的患者中,随着治疗时间延长其耐药位点增加,耐药谱扩大(耐受药物种类与大于1000拷贝/mL的治疗失败患者相似)并且原本耐药程度较低的核苷类抑制齐(Nucleoside Reverse Transcriptase Inhibitor, NRTI)类药物的耐药水平有所增加。 NVP是我国早期一线抗病毒治疗方案中NNRTI类药物的重要组成部分,在早期药物缺乏的情况下部分患者在产生NVP耐药后仍在服用。许多研究已证实NVP相关的耐药位点可以叫持久的存在于患者体内,但另外一些较新的论文则发现了NVP相关位点K103N随治疗时间而产生的减少。对于这一现象,本研究针对长期治疗的患者,研究NVP相关耐药位点的变化和发展情况,并希望借此帮助患者在更换新NNRTI类药物时了解自身的优势株NNRTI类耐药位点,避免或延迟再次耐药的发生。此外,本研究希望能够借助对NNRTI类主要耐药位点变化发展的研究,帮助现阶段以PCR探针技术为基础的快速检测手段,更好更准确的针对不同治疗时间的患者使用与其优势耐药株符合的探针。本文以上述建立的耐药队列为基础,应用in-house基因型检测技术对全部研究对象进行耐药分析,并应用单模板扩增技术(Single Genome Amplification, SGA),对其中三例患者的连续血浆样本进行准种毒株的耐药进化分析。结果显示存活的患者群体中K103N1位点的频率保持相对稳定,而Y181C和G190A位点的频率则从5%左右升高至了15%左右。3例随访患者中的情形则略有不同,K103N位点在准种毒株中的频率随治疗时间显著降低,在两例患者中甚至从100%降到了0%。Y181C和G190A位点则同人群中相似,其频率有较明显的升高。耐药位点在降低毒株对药物敏感性的同时,会降低毒株的复制适应性,但是补偿性位点可以部分恢复这种被降低的复制能力。这种补偿性位点虽然自身不能产生耐药性,但有助于耐药毒株在药物压力下存活和发展,因此其会对HIV抗病毒治疗,特别是长期治疗带来不利因素和风险。本文对3例患者(HENDRC604, HENDRC622, HENDRC593)连续6年的系列血浆样本进行单模板扩增分析,并获得HIV-1准种毒株pol区序列,每个时间点至少分析20条以上序列,每个患者累计100条以上的序列,使用贝叶斯软件(Beast, v1.7.0)结合分子钟方法构建Bayesian MCMC系统进化树,按样本采样时间完成分子钟的计算,并利用核苷酸替代数学模型对病毒序列数据进行计算和模拟,结合3D结构模拟、Ka/Ks率等方面的计算描述患者体内HIV准种的进化和耐药株的发展。本研究发现了两个与主要耐药位点相关的补偿性突变位点,K43E和L228R。推测K43E是一个能够与邻近的M41L耐药位点协同产生作用的补偿性突变位点。它可能通过提高HIV-1毒株适应性从而使毒株在抗病毒药物压力下生存并进化。此外,还发现L228R位点是一个与常见非核苷类耐药位点H221Y功能相近的补偿性位点。它可以通过协同作用,协同其他非核苷类耐药位点产生耐药作用。本文针对长期抗病毒治疗下HIV耐药株变异的特点及其对抗病毒治疗影响的研究,将为进一步提高我国的HIV抗病毒治疗效果提供参考数据。总之,为实现“十二五”降低艾滋病新发感染和病死率的目标,治疗作为预防正在我国逐步推广,接受终生治疗的患者会越来越多,寻找措施如何更好的预防HIV耐药的发生和传播的工作将面临着新的挑战。
[Abstract]:AIDS antiretroviral treatment (Antiretroviral, Therapy, ART) since 1990s in the world has been widely carried out, from the initial set of AZT (Zidovudine, AZT) to carry out the development of seven kinds of 33 kinds of drug.ART for World AIDS patients brought the gospel, to help the development of delayed disease. And improve the quality of life. China began to gradually promote free antiretroviral treatment of AIDS since 2003, the mortality rate from 9.5 in 2003 to reduce deaths /100 person years in 2009 5.2 deaths per person year. Although China's ART has made some preliminary results, but due to antiviral treatment of early drug resources Co., a considerable number of patients receiving ART after treatment to long-term use of the same kind of treatment. Many studies show that long-term treatment of HIV resistant strains produced is the impact of antiviral treatment One of the main factors of the biological characteristics of fruit.HIV virus is a high genetic mutation rate, which makes the sequence of HIV gene to produce more mutations, these mutant viruses exists as quasispecies in patients. The process of receiving antiretroviral treatment in patients with HIV in HIV, the high mutation rate of the virus can produce mutations effectively. Carrying the virus mutation can escape from drugs. Chinese Center for Disease Control and prevention center of STD and HIV / AIDS prevention and control in Henan Province Center for disease prevention and control at the end of 2003 in a Henan Province on antiviral patients were observed the long-term follow-up in this paper attempts to explore the influence on the resistance of HIV cohort of long-term antiviral therapy mutation.
Genotypic resistance testing method used in this paper were built (in-house) and single template amplification method (SingleGenomeAmplification, SGA) HIV-1 virus poL protease and reverse transcriptase gene fragment (2253bp-3553bp, 1.3kb). The application of Sequencher (4.10.1) and Bioedit (7) software to edit the sequence and the use of the United States HIV resistance database of Stanford University (Stanfrod University HIV Drug Resistance Database, HIVDB) of degree of drug resistance analysis.
WHO (WHO) HIV drug resistance testing guidelines published in the HIV resistance detection of viral load testing line for 1000 copies of /ml, both inside and outside the country for viral load in patients with 50-1000 copies /ml drug resistance study reported less, and this range is often begins to decrease the inhibitory effect of drugs on the amount of low load period. Drug resistance may contribute to the early warning of viral suppression failure and the occurrence of drug resistance, help to improve patient outcomes. This study uses the queue had resistant 50-1000 copies of /ml patients, the occurrence of drug resistance development research on the treatment effect and drug resistance later. Results the occurrence of drug resistance 18 people in the low load period failed in 28 cases of virus inhibition of virus patients. Early in resistant strains, non nucleoside inhibitors (Non-Nucleoside Reverse, Transcriptase Inhibitor, NNRTI) is The main drug resistance to nevirapine (Nevirapine, NVP) resistance is a major factor in the patients with drug resistance, and the resistance level has reached a high degree. More than 150 copies of /mL less than 1000 copies of /mL in patients with viral load, with prolonged treatment resistant sites increased drug resistance (tolerance of drugs and expand the spectrum of more than 1000 copy of the /mL treatment failure patients were similar and had low level resistance) nucleoside (Nucleoside Reverse Transcriptase Inhibitor inhibitor, NRTI) levels of resistance to drugs increased.
NVP is an important part of China's first NNRTI drugs early antiviral therapy in the absence of drugs in the early part of patients with drug-resistant NVP still in use. Many studies have demonstrated that NVP mutation related call lasting in patients, but also in some of the newer found reduce NVP related sites of K103N due to the time of treatment. For this phenomenon, this study for patients with long-term treatment, the change and development of NVP related drug resistance loci, and hopes to help patients understand their dominant strains of NNRTI resistant loci in replacement of NNRTI drugs, to avoid or delay the resistance again. In addition, this study hopes to help the research and development of NNRTI main types of resistance loci changes, help at the stage of rapid detection method of PCR probe technology as the foundation, better and more It is accurate to use a probe that conforms to its dominant resistant strain for patients with different time of treatment.
This paper is based on the resistance of the queue, to investigate the drug resistance of all subjects using in-house genotyping techniques, and the application of single template amplification technology (Single Genome Amplification, SGA), of which the continuous plasma samples of three patients were analyzed for quasi strains resistance evolution. The results indicated that the survival of patients in the group the frequency of K103N1 locus remained relatively stable, while the Y181C and G190A loci frequency is from about 5% to about 15%..3 patients in the situation is slightly different, the frequency of K103N locus in quasi strains with treatment time significantly decreased in two patients and even dropped from 100% to 0%.Y181C and similar G190A sites are the same people, its frequency is obviously increased.
The mutation in reducing the strain sensitivity to drugs at the same time, will reduce the fitness of replication of viruses, but could partially restore the ability to replicate compensatory sites this is reduced. Although this compensatory site does not produce drug resistance, but help in drug resistant strains under the pressure of survival and development, so the treatment of HIV virus, especially long term treatment of adverse factors and risks. On the 3 patients (HENDRC604, HENDRC622, HENDRC593) for 6 consecutive years, a series of plasma samples were single template amplification analysis, and obtain the HIV-1 quasispecies sequence of strain pol, each time point of at least more than 20 sequences, each with more than 100 cumulative sequences, using Bayesian the software (Beast, v1.7.0) to construct Bayesian MCMC phylogenetic tree with molecular clock method, calculated according to the sampling time to complete the molecular clock, and the use of nucleotide substitution Calculation and Simulation of algebra model of virus sequence data, combined with 3D structure simulation, calculation of Ka/Ks rate for the description of the evolution and development of resistant strains of quasi HIV patients. This study found two main resistance loci associated with compensatory mutations, K43E and L228R. suggest that K43E is a compensatory M41L mutation can cooperative effect and adjacent mutation. It can increase the adaptability of HIV-1 strains so that the strains survival and evolution in antiviral drugs under pressure. In addition, also found that the L228R locus is a common function and non nuclear glycosides resistance loci H221Y similar sites. It can be through compensatory synergy, together with other non nucleoside the mutation of drug resistance.
This paper researches the long-term antiviral treatment characteristics and antiviral treatment effect of HIV resistant strain variation, will provide the reference data for the further improvement of HIV antiviral treatment in our country. In short, reduce new HIV infections and the mortality rate for the realization of "12th Five-Year" treatment as prevention is gradually spread in China, receiving a lifetime patients will be more and more, looking for the occurrence and spread of the measures on how to better prevent the resistance of HIV work will face new challenges.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.91

【参考文献】