HIV-1准种变异对抗病毒治疗效果的影响及其适应性研究

发布时间：2018-05-03 08:50

本文选题：HIV-1 + gp160　；参考：《中国疾病预防控制中心》2013年博士论文

【摘要】：研究意义和目的： HIV的高复制和高突变性使得患者体内存在复杂混合的病毒准种。高度复杂性是HIV病毒能够快速适应各种选择压力的基础,从而适应不断变化的宿主环境,进行自我调节,保持复制和致病的能力。HIV变异和耐药株的出现造成了病毒的逃逸和适应性变化,是抗病毒治疗失败的主要原因。HIV准种序列的变异对抗病毒治疗效果的反应及适应性的影响作用目前仍不完全清楚。因此,本研究针对目前我国基于3TC组方药物治疗方案存在的耐药问题,以我国长期接受ATR的HIV感染者为研究对象,从病毒基因变异和适应性方面探讨病毒准种变异对抗病毒治疗效果的影响。该研究对于监测我国HIV的流行、实施有效的抗病毒治疗方案,继而进行疫苗和药物的研发有重要的指导意义。研究方法： 1、本研究利用已建立的全国HIV耐药性调查现场队列,回顾性的纳入感染途径较为一致、初始治疗方案为我国后期一线治疗方案,长期接受抗病毒治疗的中国安徽、河南HIV-1感染者为研究对象。本研究共纳入抗病毒治疗平均63月HIV-1感染者46例。依据治疗效果划分,病毒抑制组20例,治疗失败组26例。 2、以HIV-1env为研究指标,通过从血浆中提取总RNA,运用单基因组扩增测序法获得每例患者的准种序列,分别从横断面水平及纵向队列水平分析HIV-1env准种,获得准种变异与治疗效果的关系以及其在治疗过程中的变化特点。 3、利用生物信息技术,进行治疗失败相关的阳性选择位点的筛选,关联上述阳性选择位点与治疗效果的关系,获得治疗效果相关的潜在适应性位点。 4、通过构建携带潜在适应性突变位点的重组病毒,通过感染能力测定,竞争性复制生长试验确定以体外复制适应性为主要指标的病毒致病性对治疗效果可能的影响。研究结果：一、治疗前基线HIV-1env准种变异对抗病毒治疗效果影响本研究通过单基因组扩增测序法获得286条和352条治疗失败组和病毒抑制组治疗前基线的gp160准种序列。从多样性、选择压力、氨基酸长度、糖基化位点数、辅助受体利用和特征性氨基酸位点突变等多个方面对不同治疗效果的两组人群的HIV-1env准种特征进行比较。首先,治疗失败组的gp160, gp120及gp41的多样性及同义替换率和非同义替换率显著高于病毒抑制组,说明治疗失败患者在开始治疗前体内已存在高复杂度的准种；其次治疗失败组的C3区更长并存在更多的糖基化位点,而两组共受体选择并无差异。长度多态性的形成与糖基化位点的增加可能与维持治疗失败患者体内准种的高复杂度有关；最后,通过比较两组氨基酸序列差异,得到了9个氨基酸特征性位点,主要分布在V1/V2区和gp41区,表明治疗失败组与治疗成功组在这两个区域的优势氨基酸不同。根据以上不同效果的准种差异,利用logistic回归和ROC曲线来进行效果预测的分析与评价。纳入变量包括HIV-1CD4+T细胞数,VL, gp160复杂度,治疗前有无耐药,及是否存在CXCR4共受体。结果显示gp160复杂度是最终引入模型的唯一有意义变量,高gp160复杂度将显著增加治疗失败的风险。本研究中,gp160的复杂度是预测治疗失败的唯一最佳变量,可正确预测75.6%的本队列个体治疗效果,其在ROC曲线中的曲线下面积(AUC)为0.794(CI95%,0.657-0.931),选择0.50的cut-off值,其对应敏感度和阳性预测值为75.0%,特异度和阴性预测值为80.0%。二、抗病毒治疗过程中治疗失败患者的HIV-1env准种特征动态变化治疗失败患者在治疗前即存在高复杂度的HIV-1env准种。为研究这些HIV-1env准种在抗病毒治疗过程中的的动态变化,12例平均治疗59月的治疗失败患者被纳入本部分研究。分别从多样性、选择压力、氨基酸长度、糖基化位点数、辅助受体利用和特征性氨基酸位点突变等方面对治疗过程中的HIV-1env准种特征进行分析。通过SGA/S法,共获得抗病毒治疗过程中每人2个治疗时间点的gp160序列552条。首先,抗病毒治疗开始后,治疗失败患者的HIV-1env基因多样性,同义替换率和非同义替换率随治疗时间较治疗前未发生改变。说明治疗前已存在的高复杂度准种并未因药物压力和宿主免疫反应而逐渐降低。其次,治疗失败患者的HIV-1env准种随着治疗时间的延长,gp120区的长度逐渐增长PNGS数逐渐增多。治疗失败患者的HIV-1准种可能会通过改变长度多态性和N-糖基化位点的增加来适应增添了药物压力的宿主环境。抗病毒治疗的开展也没有改变治疗失败患者的HIV-1准种辅助受体选择。最后,治疗失败患者在治疗后出现了22个差异性氨基酸位点,依旧主要分布在V1/V2区和gp41区,且各出现一个插入性突变。治疗后这两个区的优势氨基酸随治疗的进展在发生改变。三、与治疗效果相关的HIV-1env种变异对病毒适应性的影响根据已获得的研究结果,选用基于选择压力法筛选治疗失败相关适应性位点,以探讨HIV-1env基因的变异是否对病毒适应性产生一定影响。将治疗基线env区的gp120与gp41分别进行治疗失败相关的阳性选择位点的筛选,共获得36个gp120区,10个gp41区的治疗失败相关的潜在适应性位点。然后通过贝叶斯网络建模确定4个(G145N, E150S,1371V, N392S)和5个(A96N, E151K, N166K, E223G和V321L)分别位于gp120和gp41的治疗效果相关的适应性位点。随后选择G145N, I371V, A607N, E662K, N677K等5个治疗后频率较高的位点用于突变病毒的构建,并用已建立的生长竞争试验体系来评估突变对病毒适应性的影响。结果显示当野生病毒突变为A607N与N677K后,虽然病毒滴度较野生型低,但相对适应性却较野生型显著增高。结论：本研究首次从病毒基因变异和适应性方面证实了HIV-1env的准种复杂度对抗病毒治疗的影响。治疗前基线HIV-1env准种的高复杂度将增加治疗失败的风险,表现为在治疗过程中HIV-1env准种依旧保持高复杂度并存在不同程度的进化,可能与其在治疗前就携带一些相对适应性较高的突变位点有关,这将有助于病毒适应宿主环境的改变,进而高复杂度为药物压力下耐药位点的出现提供了必要条件。因此HIV-1准种复杂度也应被考虑作为影响治疗效果的重要因素。该研究对合理设计临床用药,提高艾滋病治疗效果具有重要的指导意义。
[Abstract]:Research significance and purpose:
The high replication and high mutagenicity of HIV leads to the presence of complex mixed virus quasispecies in the patient. The high complexity is that the HIV virus can adapt to a variety of selective pressure bases, adapt to the changing host environment, adjust itself, maintain the ability of replication and pathogenicity, and cause the escape of the virus and the emergence of the drug resistant strain. And adaption changes are the main causes of the failure of antiviral therapy. The effect of.HIV quasi species sequence variation against the effect of viral therapy and its adaptability is still not completely clear. Therefore, this study aims at the problem of drug resistance based on the 3TC group based drug therapy in our country, with the long-term acceptance of ATR HIV infection in our country. The study is to explore the effect of virus quasi species variation against the effect of virus therapy from the variation and adaptability of virus gene. This study has important guiding significance for monitoring the epidemic of HIV in China, implementing effective antiviral treatment schemes, and developing vaccines and drugs.
Research methods:
1, this study uses the established national HIV drug resistance survey field cohort, and the retrospective approach is more consistent. The initial treatment scheme is the first line treatment in China, Anhui in China and the HIV-1 infected people in Henan, China, who have long received antiviral treatment as the research object. This study included the average of 63 months HIV-1 infection in the antiviral treatment 4. 6 cases. According to the therapeutic effect, there were 20 cases in the viral suppression group and 26 cases in the treatment failure group.
2, using HIV-1env as the study index, by extracting the total RNA from plasma and using the single genome amplification sequencing method to obtain the quasi species sequence of each patient, analyze the HIV-1env quasi species from the horizontal level and the vertical queue level respectively, and obtain the relationship between the quasi species variation and the therapeutic effect and the characteristics of the changes in the treatment process.
3, using bioinformatics technology to screen the positive selection sites related to treatment failure, related to the relationship between the positive selection site and the therapeutic effect, and to obtain the potential adaptable sites related to the therapeutic effect.
4, by constructing a recombinant virus carrying potential adaptable mutation sites, the effects of viral pathogenicity on the therapeutic effect of the virus in vitro replication are determined through the determination of the infectious ability and the competitive replication growth test.
The results of the study:
1. The influence of baseline HIV-1env quasispecies on the efficacy of anti viral therapy before treatment
In this study, a single genome amplification sequencing method was used to obtain the gp160 quasispecies sequence of 286 and 352 treatment failure groups and virus inhibition groups before treatment. From the diversity, selection of pressure, amino acid length, glycosylated point number, auxiliary receptor utilization and characteristic amino acid site mutation, the H of two groups of people with different therapeutic effects IV-1env quasispecies were compared. First, the diversity of gp160, gp120, and gp41 in the treatment failure group was significantly higher than that of the virus suppressing group, indicating that the patients who failed in the treatment had a high degree of complexity before the treatment, and then the C3 area of the treatment failure group was longer and had more glycosylation. There is no difference in the selection of the two groups. The formation of the length polymorphism and the increase of glycosylation sites may be related to the high complexity of maintaining the quasi species in the patients with the failure of the treatment. Finally, by comparing the differences in the amino acid sequence of the two groups, the 9 amino acid characteristic loci are obtained, mainly in the V1/V2 and gp41 regions, indicating that the treatment is lost. The dominant amino acids in the two regions were different from those in the treatment group.
Logistic regression and ROC curves were used to analyze and evaluate the effect prediction based on the differences of the above effects. The variables included the number of HIV-1CD4+T cells, the VL, the gp160 complexity, the resistance before treatment, and the existence of CXCR4 co receptor. The results showed that the gp160 complexity was the only meaningful variable of the final introduction of the model, high GP1. The 60 complexity will significantly increase the risk of treatment failure. In this study, the complexity of gp160 is the only optimal variable to predict the failure of the treatment. It can correctly predict the effect of 75.6% of this cohort on the individual treatment. The area under the curve in the ROC curve (AUC) is 0.794 (CI95%, 0.657-0.931), and the cut-off value of the selection is selected for its sensitivity and positive prediction. The value is 75%, and the specificity and negative predictive value are 80.0%.
Two, dynamic changes of HIV-1env quasispecies in patients with failed treatment during antiviral therapy.
A high complexity HIV-1env quasi species was present before treatment. To study the dynamic changes of these HIV-1env quasispecies during the antiviral treatment, 12 patients with an average of 59 months of treatment were included in this part of the study. From the diversity, the selection of pressure, the length of the amino acid, the number of glycosylated sites, and the utilization of the auxiliary receptor. The characteristics of HIV-1env quasispecies during the treatment process were analyzed with the characteristic amino acid site mutation. 552 gp160 sequences of 2 time points for each person in the process of antiviral therapy were obtained by SGA/S method. First, after the antiviral treatment began, the HIV-1env gene diversity, the synonymous replacement rate and the non synonymous replacement rate of the patients with the treatment failed. The exchange rate was not changed with the treatment time before treatment. It showed that the high complexity of the quasi species before treatment did not gradually decrease because of the drug pressure and the host immune response. Secondly, the HIV-1env quasi species in the patients with the treatment failure gradually increased with the prolongation of the treatment time, the length of the gp120 region increased gradually, and the number of PNGS increased gradually. The HIV-1 of the treatment failed patients. The quasispecies may be adapted to the host environment that adds to the drug stress by changing the length polymorphism and the increase of the N- glycosylation site. The development of antiviral therapy does not alter the HIV-1 quasispecid receptor selection of the patients with the treatment failure. Finally, 22 differential amino acid sites have appeared after the treatment. There was an insertion mutation in the V1/V2 and gp41 regions. The dominant amino acids in these two regions changed with the progress of treatment.
Three, the effects of HIV-1env variants associated with treatment effects on viral adaptation.
Based on the results obtained, the selection pressure method was selected to screen the adaptive loci of treatment failure related to whether the mutation of the HIV-1env gene had a certain effect on the adaptability of the virus. A total of 36 gp120 regions and 10 g were obtained from the screening of the positive selection loci related to the treatment failure of the gp120 and gp41 at the baseline of the treatment. 4 (G145N, E150S, 1371V, N392S) and 5 adaptive loci (A96N, E151K, N166K, E223G and V321L) were determined by Bayesian network modeling, and 5 treatment frequencies were then selected. High sites were used for the construction of mutant viruses, and the effects of mutation on the adaptability of the virus were evaluated with the established growth competition test system. The results showed that when the wild virus was mutated to A607N and N677K, although the virus titer was lower than the wild type, the relative adaptability was significantly higher than that of the wild type.
Conclusion:
This study is the first to confirm the effect of HIV-1env's quasi species complexity against virus therapy from the variation and adaptability of the virus gene. The high complexity of the pre treatment baseline HIV-1env quasispecies will increase the risk of treatment failure, showing that the HIV-1env quasispecies remain high complex and have different degrees of evolution during the treatment process. It is associated with relatively high adaptable mutation sites before treatment, which will help the virus adapt to changes in the host environment, and the high complexity provides the necessary conditions for the emergence of drug resistant loci under drug pressure. Therefore, the HIV-1 quasispecies complexity should also be considered as an important factor in the effect of response therapy. It is very important to design clinical medication to improve the therapeutic effect of AIDS.

【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】：2013
【分类号】：R512.91

【参考文献】