發(fā)布時間：2018-05-03 08:50

  本文選題：HIV-1 + gp160��； 參考：《中國疾病預(yù)防控制中心》2013年博士論文

【摘要】：研究意義和目的： HIV的高復(fù)制和高突變性使得患者體內(nèi)存在復(fù)雜混合的病毒準(zhǔn)種。高度復(fù)雜性是HIV病毒能夠快速適應(yīng)各種選擇壓力的基礎(chǔ),從而適應(yīng)不斷變化的宿主環(huán)境,進(jìn)行自我調(diào)節(jié),保持復(fù)制和致病的能力。HIV變異和耐藥株的出現(xiàn)造成了病毒的逃逸和適應(yīng)性變化,是抗病毒治療失敗的主要原因。HIV準(zhǔn)種序列的變異對抗病毒治療效果的反應(yīng)及適應(yīng)性的影響作用目前仍不完全清楚。因此,本研究針對目前我國基于3TC組方藥物治療方案存在的耐藥問題,以我國長期接受ATR的HIV感染者為研究對象,從病毒基因變異和適應(yīng)性方面探討病毒準(zhǔn)種變異對抗病毒治療效果的影響。該研究對于監(jiān)測我國HIV的流行、實施有效的抗病毒治療方案,繼而進(jìn)行疫苗和藥物的研發(fā)有重要的指導(dǎo)意義。 研究方法： 1、本研究利用已建立的全國HIV耐藥性調(diào)查現(xiàn)場隊列,回顧性的納入感染途徑較為一致、初始治療方案為我國后期一線治療方案,長期接受抗病毒治療的中國安徽、河南HIV-1感染者為研究對象。本研究共納入抗病毒治療平均63月HIV-1感染者46例。依據(jù)治療效果劃分,病毒抑制組20例,治療失敗組26例。 2、以HIV-1env為研究指標(biāo),通過從血漿中提取總RNA,運用單基因組擴(kuò)增測序法獲得每例患者的準(zhǔn)種序列,分別從橫斷面水平及縱向隊列水平分析HIV-1env準(zhǔn)種,獲得準(zhǔn)種變異與治療效果的關(guān)系以及其在治療過程中的變化特點。 3、利用生物信息技術(shù),進(jìn)行治療失敗相關(guān)的陽性選擇位點的篩選,關(guān)聯(lián)上述陽性選擇位點與治療效果的關(guān)系,獲得治療效果相關(guān)的潛在適應(yīng)性位點。 4、通過構(gòu)建攜帶潛在適應(yīng)性突變位點的重組病毒,通過感染能力測定,競爭性復(fù)制生長試驗確定以體外復(fù)制適應(yīng)性為主要指標(biāo)的病毒致病性對治療效果可能的影響。 研究結(jié)果： 一、治療前基線HIV-1env準(zhǔn)種變異對抗病毒治療效果影響 本研究通過單基因組擴(kuò)增測序法獲得286條和352條治療失敗組和病毒抑制組治療前基線的gp160準(zhǔn)種序列。從多樣性、選擇壓力、氨基酸長度、糖基化位點數(shù)、輔助受體利用和特征性氨基酸位點突變等多個方面對不同治療效果的兩組人群的HIV-1env準(zhǔn)種特征進(jìn)行比較。首先,治療失敗組的gp160, gp120及gp41的多樣性及同義替換率和非同義替換率顯著高于病毒抑制組,說明治療失敗患者在開始治療前體內(nèi)已存在高復(fù)雜度的準(zhǔn)種；其次治療失敗組的C3區(qū)更長并存在更多的糖基化位點,而兩組共受體選擇并無差異。長度多態(tài)性的形成與糖基化位點的增加可能與維持治療失敗患者體內(nèi)準(zhǔn)種的高復(fù)雜度有關(guān)；最后,通過比較兩組氨基酸序列差異,得到了9個氨基酸特征性位點,主要分布在V1/V2區(qū)和gp41區(qū),表明治療失敗組與治療成功組在這兩個區(qū)域的優(yōu)勢氨基酸不同。 根據(jù)以上不同效果的準(zhǔn)種差異,利用logistic回歸和ROC曲線來進(jìn)行效果預(yù)測的分析與評價。納入變量包括HIV-1CD4+T細(xì)胞數(shù),VL, gp160復(fù)雜度,治療前有無耐藥,及是否存在CXCR4共受體。結(jié)果顯示gp160復(fù)雜度是最終引入模型的唯一有意義變量,高gp160復(fù)雜度將顯著增加治療失敗的風(fēng)險。本研究中,gp160的復(fù)雜度是預(yù)測治療失敗的唯一最佳變量,可正確預(yù)測75.6%的本隊列個體治療效果,其在ROC曲線中的曲線下面積(AUC)為0.794(CI95%,0.657-0.931),選擇0.50的cut-off值,其對應(yīng)敏感度和陽性預(yù)測值為75.0%,特異度和陰性預(yù)測值為80.0%。 二、抗病毒治療過程中治療失敗患者的HIV-1env準(zhǔn)種特征動態(tài)變化 治療失敗患者在治療前即存在高復(fù)雜度的HIV-1env準(zhǔn)種。為研究這些HIV-1env準(zhǔn)種在抗病毒治療過程中的的動態(tài)變化,12例平均治療59月的治療失敗患者被納入本部分研究。分別從多樣性、選擇壓力、氨基酸長度、糖基化位點數(shù)、輔助受體利用和特征性氨基酸位點突變等方面對治療過程中的HIV-1env準(zhǔn)種特征進(jìn)行分析。通過SGA/S法,共獲得抗病毒治療過程中每人2個治療時間點的gp160序列552條。首先,抗病毒治療開始后,治療失敗患者的HIV-1env基因多樣性,同義替換率和非同義替換率隨治療時間較治療前未發(fā)生改變。說明治療前已存在的高復(fù)雜度準(zhǔn)種并未因藥物壓力和宿主免疫反應(yīng)而逐漸降低。其次,治療失敗患者的HIV-1env準(zhǔn)種隨著治療時間的延長,gp120區(qū)的長度逐漸增長PNGS數(shù)逐漸增多。治療失敗患者的HIV-1準(zhǔn)種可能會通過改變長度多態(tài)性和N-糖基化位點的增加來適應(yīng)增添了藥物壓力的宿主環(huán)境�？共《局委煹拈_展也沒有改變治療失敗患者的HIV-1準(zhǔn)種輔助受體選擇。最后,治療失敗患者在治療后出現(xiàn)了22個差異性氨基酸位點,依舊主要分布在V1/V2區(qū)和gp41區(qū),且各出現(xiàn)一個插入性突變。治療后這兩個區(qū)的優(yōu)勢氨基酸隨治療的進(jìn)展在發(fā)生改變。 三、與治療效果相關(guān)的HIV-1env種變異對病毒適應(yīng)性的影響 根據(jù)已獲得的研究結(jié)果,選用基于選擇壓力法篩選治療失敗相關(guān)適應(yīng)性位點,以探討HIV-1env基因的變異是否對病毒適應(yīng)性產(chǎn)生一定影響。將治療基線env區(qū)的gp120與gp41分別進(jìn)行治療失敗相關(guān)的陽性選擇位點的篩選,共獲得36個gp120區(qū),10個gp41區(qū)的治療失敗相關(guān)的潛在適應(yīng)性位點。然后通過貝葉斯網(wǎng)絡(luò)建模確定4個(G145N, E150S,1371V, N392S)和5個(A96N, E151K, N166K, E223G和V321L)分別位于gp120和gp41的治療效果相關(guān)的適應(yīng)性位點。隨后選擇G145N, I371V, A607N, E662K, N677K等5個治療后頻率較高的位點用于突變病毒的構(gòu)建,并用已建立的生長競爭試驗體系來評估突變對病毒適應(yīng)性的影響。結(jié)果顯示當(dāng)野生病毒突變?yōu)锳607N與N677K后,雖然病毒滴度較野生型低,但相對適應(yīng)性卻較野生型顯著增高。 結(jié)論： 本研究首次從病毒基因變異和適應(yīng)性方面證實了HIV-1env的準(zhǔn)種復(fù)雜度對抗病毒治療的影響。治療前基線HIV-1env準(zhǔn)種的高復(fù)雜度將增加治療失敗的風(fēng)險,表現(xiàn)為在治療過程中HIV-1env準(zhǔn)種依舊保持高復(fù)雜度并存在不同程度的進(jìn)化,可能與其在治療前就攜帶一些相對適應(yīng)性較高的突變位點有關(guān),這將有助于病毒適應(yīng)宿主環(huán)境的改變,進(jìn)而高復(fù)雜度為藥物壓力下耐藥位點的出現(xiàn)提供了必要條件。因此HIV-1準(zhǔn)種復(fù)雜度也應(yīng)被考慮作為影響治療效果的重要因素。該研究對合理設(shè)計臨床用藥,提高艾滋病治療效果具有重要的指導(dǎo)意義。
[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.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R512.91

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