HIV-1血浆RNA与前病毒DNA准种变异的比较和吡啶酮化合物抗HIV-1活性的研究

发布时间：2018-07-05 11:29

本文选题：艾滋病病毒 + 前病毒DNA　；参考：《中国疾病预防控制中心》2016年博士论文

【摘要】：研究目的和意义：HIV-1感染由于持续存在的病毒储藏库而难以治愈。在实现HIV-1治愈之前,最有效的治疗方法仍然是高效抗逆转录病毒治疗(Highly Active Antiretroviral Therapy, HAART)。2003年,我国开始在全国范围内实行免费的抗病毒治疗,至2015年底累计47万余艾滋病患者接受了抗病毒治疗。但是HIV-1的耐药性是抗病毒治疗过程中面临的严重问题。HIV-1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,处于动态的变化过程。在HIV-1感染发展过程中,血浆中病毒变异会持续地进入整合的前病毒中。目前,对于全血前病毒DNA与血浆病毒RNA耐药准种变异差异的研究较为有限。因此,本研究采用深度测序技术结合血浆病毒RNA和全血前病毒DNA分析两者之间耐药准种的变异及其对长期抗病毒治疗结果的影响,并对吡啶酮化合物抗HIV-1活性进行了研究。该研究将为HIV-1耐药监测,优化抗病毒治疗策略及药物的研发提供重要的科学依据。研究方法：1、本研究回顾性的纳入我国河南、安徽地区长期接受抗病毒治疗的既往献血HIV-1感染者为研究对象。初始治疗方案为以3TC为主的后期一线治疗药物。共纳入43例HIV-1感染者,根据一线治疗方案的治疗结果将其划分为病毒抑制成功组(23人)和病毒抑制失败组(20人)。2、以HIV-1 pol基因逆转录酶区为研究指标,利用深度测序方法获得每例病人全血前病毒DNA和血浆病毒RNA的耐药准种序列并进行比较。建立Taqman荧光定量PCR方法,对全血前病毒DNA进行定量检测。分析治疗前耐药准种与长期抗病毒治疗结果的相关性。3、利用CorMut软件和Bayesian网络分析全血前病毒DNA和血浆病毒RNA准种中的治疗失败相关特征性位点,并以此特征性位点和治疗结果为变量分析与治疗失败直接相关的突变位点,并进一步分析其在长期抗病毒治疗过程中的动态变化。4、利用基于TZM-bl细胞系的HIV-1病毒表型耐药性检测方法,对不同的吡啶酮化合物抗HIV-1的活性,特别是抗HIV-1耐药株的活性进行研究。研究结果：1、基于深度测序技术的前病毒DNA和血浆病毒RNA耐药准种变异的比较本研究通过深度测序技术获得43例HIV-1感染者感染前后不同时间点共66份样本的血浆病毒RNA和全血前病毒DNA的pol基因逆转录酶区序列。37份样本中检测到耐药位点,其中67.57%(25/37)的样本中检测到劣势耐药位点。不论在血浆RNA还是前病毒DNA,耐药位点K103N和Y181C均具有较高的发生频率。血浆RNA和前病毒DNA中的耐药位点和频率存在差异。优势耐药位点出现的M41L、M184I、 E138A、G190E和M230I仅在全血DNA中检测到。在劣势耐药位点中出现的M184V/I、 T215I、K219R、E138K、H22IY和M2301仅在全血DNA中检测到。在进行药物敏感性分析时,分析优势耐药位点时,有32.43%(12/37)样本的血浆RNA和前病毒DNA呈现出对一线治疗药物不同程度的耐受,其中33.33%(4/12)样本的前病毒DNA对一线治疗药物的耐受高于血浆病毒RNA。分析劣势耐药位点时,发现有62.16%(23/37)样本的血浆RNA和前病毒DNA呈现出对一线治疗药物不同程度的耐受,其中69.57%(16/23)样本的前病毒DNA对一线治疗药物的耐受程度高于血浆病毒RNA。2、治疗前耐药准种与长期抗病毒治疗结果相关性分析治疗前HIV-1感染者全血前病毒DNA载量与血浆病毒载量(viral load, VL)呈显著的正相关关系(p=0.003)。治疗失败的15例HIV-1感染者在治疗过程中HIV-1DNA载量的动态变化与血浆RNA VL呈现相同的变化趋势。将治疗前血浆病毒RNA和全血前病毒DNA中优势耐药位点和劣势耐药位点统称为治疗前耐药(RNADNA)。利用logistic回归分析与长期抗病毒治疗结果相关的基线临床特征。纳入变量包括CD4+T细胞数,血浆VL,前病毒DNA载量,治疗前有无耐药(RNADNA),治疗前RNA中有无耐药,治疗前DNA有无耐药。结果显示在多因素logistic回归中,治疗前有无耐药(RNADNA)是与抗病毒治疗失败相关的变量[OR=5.9(95%CI 1.4-24.7),p=0.01]。进一步分析发现,当血浆病毒RNA耐药位点的频率增加1%时,病毒抑制失败率增加了1.3%。3、HIV-1 pol基因逆转录酶区治疗失败直接相关位点分析治疗前,血浆病毒RNA中治疗失败直接相关位点包括非核苷类耐药位点K101E、K103N、E138K和V179D,以及多态性位点K64R、K102Q、V111I、D123N、I135T、 S162Y、E169D、Q174K、I202V、R211G、R211T、R211M、S251C。全血前病毒DNA中治疗失败直接相关位点包括非核苷类耐药位点G190A和V179D,以及多态性位点E42K、K64R、K102Q、D123N、R125K、Q174K、V189I、R211T、R211M、R211Q、 V245E、D250N。在治疗过程中,血浆病毒RNA中新出现的治疗失败直接相关位点包括非核苷类耐药位点K103S、H221Y、Y181C,核苷类耐药位点M184V,以及多态性位点I31L、K32R、V35I、T39A、K173R、I178M、Q197E和E203D。全血前病毒DNA中新出现的治疗失败相关位点包括非核苷类耐药位点K103S、V179I、G190E,核苷类耐药位点D67N、M184V和M184I,以及多态性位点K32R、V35I、T39A和G51R。经分析发现,在长期治疗过程中,治疗失败直接相关位点包括耐药位点K103N、Y181C,和多态性位点K102Q、E169D、R211G、I202V、R211T、V245E以较高频率存在。4、吡啶酮化合物抗HIV-1活性的研究由于在血浆RNA和前病毒DNA中,耐药位点K103N和Y181C均具有较高的发生频率,为此,我们和北京大学药学院合作设计合成一类NNRTIs,即吡啶酮化合物3-碘-4-(2-环己基甲基氧)-6-苯乙基-2(2H)-吡啶酮的5种构象化合物7-cis、7-mix、 7-trans、7-trans-1、7-trans-2,对其抗HIV-1耐药株的活性进行了检测。结果显示,该5种化合物对TZM-bl细胞均具有较低的毒性,对HIV-1 SF33双嗜性实验室标准株和HIV-1 A17非核苷类逆转录酶耐药的标准株具有较好的抑制活性,EC50分别为0.003-0.503州和0.117-19.657μM,治疗指数分别为在323-100000和8.2-2564之间。其中化合物7-mix、7-trans、7-trans-1、7-trans-2对我国临床分离HIV-I流行株CRF07_BC(XJDC6371, XJDC6291和CBJB256)、CRF01_AE(BJM543)、B'(020100968和020100096)3种亚型毒株均有不同程度抑制活性(EC50:0.4-90nM)。其中7-trans-2化合物能够显著的抑制实验室适应株和携带K103N和Y181C耐药突变的耐药株HIV-1 A17, EC50分别为3 nM和117 nM(SI2500),对所有的临床毒株的EC50值为0.4-57 nM,SI为5263-750000。结论：本研究从血浆RNA和全血前病毒DNA方面研究了HIV-1准种变异对长期抗病毒治疗结果的影响。深度测序揭示了全血前病毒DNA和血浆病毒RNA具有不同的耐药准种。治疗前血浆病毒RNA和前病毒DNA耐药准种的存在将会导致抗病毒治疗的失败,血浆病毒RNA耐药位点的频率升高会增加抗病毒治疗失败的风险。基于深度测序技术的血浆RNA和前病毒DNA的耐药检测应该考虑应用于HIV-1耐药监测。吡啶酮化合物对携带K103N和Y181C的HIV-I耐药株具有一定的抑制活性,同时对我国HIV-I主要流行株也具有广泛的抑制作用。本研究对于耐药监测的指导和药物的研发具有重要的意义。
[Abstract]:The purpose and significance of the study: HIV-1 infection is difficult to cure because of the persistent virus storage. Before the HIV-1 cure, the most effective treatment is the Highly Active Antiretroviral Therapy (HAART).2003 year, and our country begins to carry out free antiviral treatment throughout the country to 2015 More than 47 million AIDS patients received antiviral therapy at the end of the year. But HIV-1 resistance is a serious problem in the process of antiviral treatment,.HIV-1 is in a complex quasi species, under the pressure of different types and intensity of drug selection, in the dynamic process of change. In the development of HIV-1 infection, the virus variation in plasma will be in the process of development of HIV-1 infection Continuing to enter the integrated pre virus. Currently, there is limited study on the difference between pre - blood virus DNA and plasma virus RNA - resistant quasi species variation. Therefore, this study uses deep sequencing technology combined with plasma virus RNA and pre - blood virus DNA analysis of the variation of drug resistance quasi species and their effects on long-term antiviral treatment results, The anti HIV-1 activity of pyridone compound is studied. This study will provide an important scientific basis for HIV-1 resistance monitoring, optimization of antiviral therapy strategies and drug development. 1. This study was reviewed in Henan, China, and in the Anhui region, who had been treated for a long time in the blood donation of HIV-1. 43 cases of HIV-1 infection were included in the initial treatment program, which was mainly 3TC in the late first line treatment. According to the treatment results of the first line treatment, they were divided into virus inhibition group (23 people) and virus inhibition failure group (20 people).2, and HIV-1 pol gene reverse transcriptase region was used as the study index, and each case was obtained by deep sequencing method. The sequence and comparison of the drug resistance quasispecies of human pre blood virus DNA and plasma virus RNA were compared. The Taqman fluorescence quantitative PCR method was established for quantitative detection of the pre blood virus DNA. The correlation between the pre treatment and the long-term antiviral treatment results was analyzed, and the CorMut software and Bayesian network were used to analyze the pre whole blood virus DNA and the plasma virus RNA. The treatment failure related characteristic loci in the quasi species, and the characteristic loci and the therapeutic results as variables, analyze the mutation sites that are directly related to the treatment failure, and further analyze the dynamic changes in the long-term antiviral therapy,.4, using the TZM-bl cell line based HIV-1 virus phenotype resistance detection method, to different pyridoidin. The anti HIV-1 activity of acetone compounds, especially the activity of anti HIV-1 resistant strains. 1, comparison of pre virus DNA and plasma virus RNA resistant quasi species variation based on deep sequencing technology, this study was used to obtain the plasma virus RNA of 66 samples in 43 cases of HIV-1 infected persons at different time points in different time points by deep sequencing technology. The resistance loci were detected in.37 samples from the pol gene reverse transcriptase sequence of the pre blood virus DNA, of which 67.57% (25/37) samples were detected at the disadvantaged loci. Both the plasma RNA and the pre viral DNA, the resistance loci K103N and Y181C have high frequency. The drug resistance loci and frequencies in the plasma RNA and the virus DNA are found. M41L, M184I, E138A, G190E and M230I were detected only in the whole blood DNA. M184V/I, T215I, K219R, E138K, H22IY, and M2301 were detected only in the whole blood. In the analysis of drug sensitivity, the analysis of the dominant loci was 32.43% Virus DNA showed tolerance to a different degree of first-line treatment, of which 33.33% (4/12) samples were tolerant to first-line therapy than in plasma viral RNA. analysis at inferior resistance sites, and 62.16% (23/37) samples of plasma RNA and pre viral DNA showed tolerance to different levels of first-line treatment, of which 69.57% (16/23) the tolerance of the pre viral DNA to the first-line treatment is higher than that of the plasma virus RNA.2. The correlation between the pre treatment drug resistance quasi species and the long-term antiviral treatment results is a significant positive correlation between the DNA load of the pre blood virus DNA and the plasma viral load (viral load, VL) before the treatment of HIV-1 infection (p=0.003). 15 cases of the failure of the treatment of HIV- The dynamic changes of the HIV-1DNA load and the plasma RNA VL were the same trend during the treatment process. The pretreatment plasma virus RNA and the predominant virus DNA were called pretreatment resistance (RNADNA), and the baseline associated with the long-term antiviral treatment was associated with the logistic regression analysis. The variables included the number of CD4+T cells, the plasma VL, the DNA load of the pre virus, the drug resistance (RNADNA) before treatment, the drug resistance in RNA before treatment, and the resistance to DNA before treatment. The results showed that in the multiple factor Logistic regression, no drug resistance (RNADNA) was a variable (95%CI 1.4-24.7) that was associated with the failure of antiviral therapy (95%CI 1.4-24.7) before treatment. Further analysis showed that when the frequency of the RNA resistance loci of the plasma virus increased by 1%, the failure rate of virus inhibition increased by 1.3%.3. Before the direct correlation site analysis of the HIV-1 pol gene reverse transcriptase region treatment failure, the direct related loci of the treatment failure in the plasma virus RNA include the non nucleoside resistance loci K101E, K103N, E138K and V179D, and more. K64R, K102Q, V111I, D123N, I135T, S162Y, E169D, Q174K, I202V, R211G, R211T, R211M, including non nucleoside resistant loci and polymorphic loci K103S, H221Y, Y181C, nucleoside resistance loci, M184V, and polymorphic loci I31L, K32R, V35I, T39A, K173R, and I178M, including non nucleoside resistance loci, including non nucleoside resistance loci, are the direct related sites of the new treatment failure in the plasma virus RNA 190E, nucleoside resistant sites D67N, M184V and M184I, and polymorphic loci K32R, V35I, T39A and G51R. have been found that the direct related sites of treatment failure include K103N, Y181C, and polymorphic loci in the long-term treatment process. Because of the high frequency of drug resistance loci K103N and Y181C in plasma RNA and DNA, we collaborated with the Peking University Institute of pharmacy to design and synthesize a class of NNRTIs, namely, the 5 conformation compounds of the pyridone compound 3- iodide -4- (2- cyclohexyl methyl oxygen) -6- phenyl ethyl -2 (2H) pyridone. -1,7-trans-2, the activity of its anti HIV-1 resistant strain was detected. The results showed that the 5 compounds had low toxicity to TZM-bl cells. The standard strain of HIV-1 SF33 double tropic laboratory standard strain and HIV-1 A17 non nucleoside reverse transcriptase resistant standard strain had better inhibitory activity, EC50 was 0.003-0.503 state and 0.117-19.657 mu M, respectively. The index of treatment was between 323-100000 and 8.2-2564. Compounds 7-mix, 7-trans, and 7-trans-1,7-trans-2 had different degrees of inhibitory activity to 3 subtypes of HIV-I epidemic strains, CRF07_BC (XJDC6371, XJDC6291 and CBJB256), CRF01_AE (BJM543), and B'(020100968 and 020100096). The substance can significantly inhibit the laboratory adaptation strain and the drug resistant strain HIV-1 A17 carrying K103N and Y181C resistance mutations, EC50 is 3 nM and 117 nM (SI2500), and the EC50 value of all clinical isolates is 0.4-57 nM. The effect of treatment results. Deep sequencing revealed that the pre - blood virus DNA and the plasma virus RNA have different drug resistance para. The presence of pre - treatment plasma viral RNA and the pre - viral DNA resistance quasispecies will lead to the failure of antiviral therapy. The increase in the frequency of the drug resistance site of the plasma virus RNA will increase the risk of the failure of the antiviral therapy. The resistance detection of plasma RNA and previral DNA by sequencing technology should be considered for HIV-1 resistance monitoring. Pyridone compounds have a certain inhibitory activity for the HIV-I resistant strains carrying K103N and Y181C, and also have extensive inhibitory effects on the major HIV-I strains in China. The guidance of drug resistance monitoring and drug development in this study It is of great significance.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R512.91

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