本文選題：HIV-1 + 病毒適應性　； 參考：《吉林大學》2014年博士論文

【摘要】：人類免疫缺陷病毒（human immunodeficiency virus, HIV）是導致獲得性免疫缺陷綜合癥（acquired immunodeficiency syndrome, AIDS）的主要病原體。HIV-1感染宿主后，能以高復制速率、短復制周期和極高的突變率迅速的增殖和進化，這些特點對于病毒能夠迅速適應并克服宿主的免疫反應具有重要意義。而HIV-1在不同的免疫攻擊環(huán)境中能夠完成繁殖、進化和傳播過程的能力，被稱為HIV-1適應性。病毒適應性決定了病毒在宿主體內(nèi)的復制能力。作為HIV-1最重要的固有性質之一，病毒適應性微小的改變就能顯著地影響HIV-1的進化趨勢、致病機制、流行傳播以及病程進展等。 在HIV-1感染的初期，宿主免疫系統(tǒng)能迅速產(chǎn)生大規(guī)模的細胞毒性T細胞（cytotoxic T lymphocyte; CTL）免疫反應遏制HIV-1。這些有效的CTL免疫反應產(chǎn)生的持續(xù)的選擇壓力，能壓迫病毒產(chǎn)生特異的突變從而使病毒逃避CTL免疫反應，而代價是這些免疫逃逸突變將對病毒適應性造成損害。在以往報道中研究最廣泛的CTL免疫逃逸突變之一，是位于Gag蛋白的TW10（TSTLQEQIGW, Gag240~249）表位內(nèi)的T242N突變，其產(chǎn)生與宿主表達的B*57/5801型人類白細胞抗原（human leukocyte antigen; HLA）介導的CTL免疫反應密切相關。普遍認為，CTL免疫逃逸突變T242N能顯著損害HIV-1適應性，因而在臨床上可能使感染者獲得較好的病情控制效果；此外，T242N突變體病毒的傳播能力，也可能由于病毒適應性的損失而降低；同時，即使T242N突變體病毒傳染了新的宿主，其病程進展可能比較緩慢。因此，TW10表位被認為是抗HIV/AIDS疫苗的理想靶點。然而最近的研究表明，CTL逃逸突變T242N產(chǎn)生常常伴隨著TW10表位內(nèi)部或外部的相關病毒適應性補償突變的產(chǎn)生，從而能部分程度或完全修復病毒適應性的損失；另外，如果新宿主體內(nèi)沒有特異性的CTL免疫反應，T242N突變體也可能在感染后發(fā)生回復突變，但常常需要幾個月甚至幾年時間。 但是由于實驗條件和技術方法的限制，以往的研究并沒有在自然選擇出T242N突變的奠基（transmitted/founder, T/F）病毒基因組中研究其對病毒適應性的影響，而是以實驗室適應毒株作為替代，甚至還加入與HIV-1病毒基因不相關的報告基因。因此，這些研究無法避免病毒基因組中預存的已知或未知的補償性氨基酸對T242N突變的修復作用，從而導致不準確甚至不正確的實驗結果。在本研究中，我們通過單基因組擴增法（single genome amplification, SGA）分析了4位HIV-1急性感染者的長期臨床監(jiān)測樣品，獲得了HIV-1在感染者體內(nèi)的詳細進化過程，并成功分離得到了4位感染者體內(nèi)的T/F病毒。在4株T/F病毒中，CH58T/F在感染HLA-B*57/5801陽性的宿主后產(chǎn)生T242N突變；CH131T/F本身攜帶T242N突變，并在感染HLA-B*57/5801陰性的宿主后回復為N242T；而CH470T/F和CH40T/F感染HLA-B*57/5801陰性的宿主后沒有產(chǎn)生T242N突變。以這4株T/F病毒為樣本，我們?nèi)娴匮芯苛薚242N突變在不同的T/F病毒基因組中對于病毒適應性的影響。 此外，以往報道的HIV-1適應性檢測方法也有很多局限性。它們或者由于采用了平行感染培養(yǎng)法而導致檢測靈敏度有限，或是由于采用傳統(tǒng)聚合酶鏈反應（polymerase chain reaction; PCR）而導致結果的穩(wěn)定性和精確性有限，同時還費時費力。為了突破這些局限，我們建立了基于HIV-1的T/F病毒和平行等位基因定點測序法（the parallel allele-specific sequencing, PASS）的新型HIV-1病毒適應性檢測方法，即PFA法（PASS fitness assay, PFA）。應用PFA法，我們靈敏而精確地檢測出T242N突變在不同的T/F病毒基因組中對病毒適應性造成的不同程度的損害；同時，還檢測出HIV-1基因組中已存的相關補償性氨基酸I247或Q219能大幅修復由于T242N導致的病毒適應性損失；此外，我們還發(fā)現(xiàn)在預存補償性氨基酸的作用下，攜帶T242N突變的CH131T/F與其回復突變體N242T的病毒適應性并無顯著差異。這些結果揭示了攜帶T242N突變的HIV-1仍然能相對有效地傳播的原因，，以及T242N突變體感染HLA-B*57/5801陰性的宿主數(shù)月乃至幾年以后才出現(xiàn)，甚至不出現(xiàn)回復突變N242T的原因。更重要的是，這些關于CTL逃逸突變T242N對病毒適應性影響的系統(tǒng)性研究結果，對設計以細胞免疫為基礎的HIV/AIDS疫苗具有重要的指導意義。 最后，HIV-1在宿主體內(nèi)進化的兩個最重要的外界壓力是宿主的CTL免疫反應和中和抗體。在本研究中，我們發(fā)現(xiàn)了位于CH131T/F的Gag蛋白中的選擇突變K43R，并對其產(chǎn)生的原因和病毒適應性影響進行了研究。用CH131感染者的外周血單核細胞（peripheral blood mononuclear cells, PBMC）樣品進行體外酶聯(lián)免疫斑點（Enzyme-Linked Immuno-Spot, ELISpot）分析，我們對特異性識別含有K43R突變的T細胞表位的CTL免疫反應進行了檢測，結果表明K43R突變的產(chǎn)生與宿主的CTL免疫選擇壓力沒有關系。但隨后的病毒適應性研究結果顯示，K43R突變能嚴重損害病毒適應性，提示該突變的產(chǎn)生能嚴重地影響病毒的復制能力。這些結果表明，在宿主對抗HIV-1感染的過程中，很可能存在我們?nèi)匀晃粗哪軌驅Σ《靖腥井a(chǎn)生強烈抑制效果的宿主因素。 本研究對CTL逃逸突變T242N對HIV-1適應性的影響，以及病毒基因組內(nèi)已存的補償性氨基酸的修復作用進行了深入、精確及系統(tǒng)的研究。這些發(fā)現(xiàn)對于我們進一步了解HIV-1適應性這一重要的病毒學特征，及其與病程進展之間的關系具有重要意義。同時，對于設計以誘導CTL免疫反應為靶向的HIV/AIDS疫苗具有指導意義。
[Abstract]:Human immunodeficiency virus (HIV), which is the host of the main pathogen.HIV-1 infection of acquired immunodeficiency syndrome, AIDS, can rapidly proliferate and evolve at high replication rate, short replicative cycle and high mutation rate, and these features can be rapid for the virus. It is of great significance to adapt and overcome the immune response of the host. And the ability of HIV-1 to reproduce, evolve and propagate in different immune attacks is called HIV-1 adaptability. Virus adaptability determines the replication ability of the virus in the host. As one of the most important intrinsic properties of HIV-1, the virus adapts little. Changes can significantly affect the evolution trend, pathogenesis, epidemic spread and progression of HIV-1.
In the early stage of HIV-1 infection, the host immune system can rapidly produce large scale cytotoxic T cells (cytotoxic T lymphocyte; CTL) immune response to contain the persistent selective pressure produced by the effective CTL immune response of HIV-1., which can oppress the virus to produce specific mutations and make the virus escape the CTL immune response at the cost of these immune responses. Escape mutation will damage the adaptability of the virus. One of the most extensive CTL immune escape mutations in the previous reports is a T242N mutation in the epitopes of the TW10 (TSTLQEQIGW, Gag240~249) of the Gag protein, which produces a CTL immune response mediated by the B*57/5801 type human leukocyte antigen (human leukocyte antigen; HLA) expressed by the host. It is closely related. It is generally believed that the CTL immune escape mutation T242N can significantly damage the adaptability of HIV-1, and thus may make the infected person get better control effect; in addition, the transmission ability of the T242N mutant virus may also be reduced by the loss of the virus adaptation; at the same time, even the T242N mutant virus infect new lodging. TW10 epitopes are considered to be an ideal target for anti HIV/AIDS vaccines. However, recent studies have shown that the CTL escape mutation T242N is often associated with the production of adaptive compensation mutations within or outside the TW10 epitopes, which can partially or completely repair the adaptive damage of the virus. In addition, if there is no specific CTL immune response in the main body of the Shinjuku, the T242N mutant may also have a recovery mutation after infection, but it often takes months or even years.
However, due to the limitations of experimental conditions and technical methods, previous studies have not studied the impact of natural selection of the T242N mutation (transmitted/founder, T/F) virus genome on virus adaptation, instead of using laboratory strains as a substitute, or even adding a reporter gene unrelated to the HIV-1 virus gene. These studies do not prevent the repair of a known or unknown compensatory amino acid stored in the virus genome for the T242N mutation, resulting in inaccurate or incorrect experimental results. In this study, we analyzed the long-term results of 4 HIV-1 acute infections by single genome amplification (SGA). The clinical monitoring samples obtained the detailed evolution process of HIV-1 in the infected person and successfully isolated the T/F virus in the 4 infected persons. In the 4 T/F viruses, CH58T/F produced a T242N mutation after the infection of the HLA-B*57/5801 positive host; CH131T/F itself carried the T242N mutation and returned to the host infected with the HLA-B*57/5801 negative host. For N242T, CH470T/F and CH40T/F did not produce T242N mutations after the infection of the HLA-B*57/5801 negative host. With these 4 T/F viruses, we studied the effect of T242N mutation on the adaptability of the virus in the different T/F virus genome.
In addition, the previously reported HIV-1 adaptive detection methods also have many limitations. They either have limited detection sensitivity due to the use of parallel infection culture method, or because of the use of traditional polymerase chain reaction (polymerase chain reaction; PCR), which lead to the limited stability and accuracy of the results, and are also time-consuming and laborious. In order to break through these limitations, we set up a new adaptive detection method for HIV-1 virus based on HIV-1 T/F virus and the parallel allele-specific sequencing (PASS), i.e. PFA (PASS fitness assay). At the same time, the related compensatory amino acid I247 or Q219 existing in the HIV-1 genome can be detected by the T242N induced virus adaptation loss. Furthermore, we also found that the CH131T/F with T242N mutation and its return under the action of preexisting compensatory amino acids are also found. There was no significant difference in the adaptability of the complex mutant N242T. These results revealed that the HIV-1 with the T242N mutation still spread relatively effectively, and that the T242N mutant infected HLA-B*57/5801 negative host only a few months or even years later, or even the reason for the response to the mutation of N242T. More importantly, these are about C. The results of systematic studies on the effects of TL escape T242N on the adaptability of viruses are important for designing HIV/AIDS vaccines based on cell-mediated immunity.
Finally, two of the most important external pressures that HIV-1 evolved in the host were the host's CTL immunoreaction and neutralizing antibody. In this study, we found the selective mutation K43R in the Gag protein in CH131T/F, and studied the causes of its production and the effect of the virus adaptation. The peripheral blood mononuclear cells (peri) infected with CH131 (peri). Pheral blood mononuclear cells, PBMC) samples carried out in vitro enzyme linked immunosorbent assay (Enzyme-Linked Immuno-Spot, ELISpot) analysis. We detected the CTL immune response to the T cell epitopes containing K43R mutations. The results showed that the occurrence of the K43R mutation was not related to the immune selection pressure of the host. The results of the toxicity study showed that the K43R mutation could seriously damage the virus adaptation, suggesting that the mutation could seriously affect the replication ability of the virus. These results suggest that in the process of host against HIV-1 infection, it is likely that there is a host factor that we are still unknown to have a strong inhibitory effect on the virus infection.
This study has conducted in-depth, accurate and systematic studies on the effect of CTL escape mutation T242N on the adaptation of HIV-1 to the adaptation of the existing compensatory amino acids in the virus genome. These findings are important for our further understanding of the important virological characteristics of the adaptation of HIV-1, as well as the relationship with the progress of the disease process. Meanwhile, it is instructive for designing HIV/AIDS vaccine targeting CTL immunoreaction.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R512.91

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