细胞免疫逃逸突变及已存补偿性氨基酸对HIV-1适应性的影响机制

发布时间：2018-07-11 18:55

本文选题：HIV-1 + 病毒适应性　；参考：《吉林大学》2014年博士论文

【摘要】：人类免疫缺陷病毒（human immunodeficiency virus, HIV）是导致获得性免疫缺陷综合症（acquired immunodeficiency syndrome, AIDS）的主要病原体。HIV-1感染宿主后，能以高复制速率、短复制周期和极高的突变率迅速的增殖和进化，这些特点对于病毒能够迅速适应并克服宿主的免疫反应具有重要意义。而HIV-1在不同的免疫攻击环境中能够完成繁殖、进化和传播过程的能力，被称为HIV-1适应性。病毒适应性决定了病毒在宿主体内的复制能力。作为HIV-1最重要的固有性质之一，病毒适应性微小的改变就能显著地影响HIV-1的进化趋势、致病机制、流行传播以及病程进展等。在HIV-1感染的初期，宿主免疫系统能迅速产生大规模的细胞毒性T细胞（cytotoxic T lymphocyte; CTL）免疫反应遏制HIV-1。这些有效的CTL免疫反应产生的持续的选择压力，能压迫病毒产生特异的突变从而使病毒逃避CTL免疫反应，而代价是这些免疫逃逸突变将对病毒适应性造成损害。在以往报道中研究最广泛的CTL免疫逃逸突变之一，是位于Gag蛋白的TW10（TSTLQEQIGW, Gag240~249）表位内的T242N突变，其产生与宿主表达的B*57/5801型人类白细胞抗原（human leukocyte antigen; HLA）介导的CTL免疫反应密切相关。普遍认为，CTL免疫逃逸突变T242N能显著损害HIV-1适应性，因而在临床上可能使感染者获得较好的病情控制效果；此外，T242N突变体病毒的传播能力，也可能由于病毒适应性的损失而降低；同时，即使T242N突变体病毒传染了新的宿主，其病程进展可能比较缓慢。因此，TW10表位被认为是抗HIV/AIDS疫苗的理想靶点。然而最近的研究表明，CTL逃逸突变T242N产生常常伴随着TW10表位内部或外部的相关病毒适应性补偿突变的产生，从而能部分程度或完全修复病毒适应性的损失；另外，如果新宿主体内没有特异性的CTL免疫反应，T242N突变体也可能在感染后发生回复突变，但常常需要几个月甚至几年时间。但是由于实验条件和技术方法的限制，以往的研究并没有在自然选择出T242N突变的奠基（transmitted/founder, T/F）病毒基因组中研究其对病毒适应性的影响，而是以实验室适应毒株作为替代，甚至还加入与HIV-1病毒基因不相关的报告基因。因此，这些研究无法避免病毒基因组中预存的已知或未知的补偿性氨基酸对T242N突变的修复作用，从而导致不准确甚至不正确的实验结果。在本研究中，我们通过单基因组扩增法（single genome amplification, SGA）分析了4位HIV-1急性感染者的长期临床监测样品，获得了HIV-1在感染者体内的详细进化过程，并成功分离得到了4位感染者体内的T/F病毒。在4株T/F病毒中，CH58T/F在感染HLA-B*57/5801阳性的宿主后产生T242N突变；CH131T/F本身携带T242N突变，并在感染HLA-B*57/5801阴性的宿主后回复为N242T；而CH470T/F和CH40T/F感染HLA-B*57/5801阴性的宿主后没有产生T242N突变。以这4株T/F病毒为样本，我们全面地研究了T242N突变在不同的T/F病毒基因组中对于病毒适应性的影响。此外，以往报道的HIV-1适应性检测方法也有很多局限性。它们或者由于采用了平行感染培养法而导致检测灵敏度有限，或是由于采用传统聚合酶链反应（polymerase chain reaction; PCR）而导致结果的稳定性和精确性有限，同时还费时费力。为了突破这些局限，我们建立了基于HIV-1的T/F病毒和平行等位基因定点测序法（the parallel allele-specific sequencing, PASS）的新型HIV-1病毒适应性检测方法，即PFA法（PASS fitness assay, PFA）。应用PFA法，我们灵敏而精确地检测出T242N突变在不同的T/F病毒基因组中对病毒适应性造成的不同程度的损害；同时，还检测出HIV-1基因组中已存的相关补偿性氨基酸I247或Q219能大幅修复由于T242N导致的病毒适应性损失；此外，我们还发现在预存补偿性氨基酸的作用下，携带T242N突变的CH131T/F与其回复突变体N242T的病毒适应性并无显著差异。这些结果揭示了携带T242N突变的HIV-1仍然能相对有效地传播的原因，，以及T242N突变体感染HLA-B*57/5801阴性的宿主数月乃至几年以后才出现，甚至不出现回复突变N242T的原因。更重要的是，这些关于CTL逃逸突变T242N对病毒适应性影响的系统性研究结果，对设计以细胞免疫为基础的HIV/AIDS疫苗具有重要的指导意义。最后，HIV-1在宿主体内进化的两个最重要的外界压力是宿主的CTL免疫反应和中和抗体。在本研究中，我们发现了位于CH131T/F的Gag蛋白中的选择突变K43R，并对其产生的原因和病毒适应性影响进行了研究。用CH131感染者的外周血单核细胞（peripheral blood mononuclear cells, PBMC）样品进行体外酶联免疫斑点（Enzyme-Linked Immuno-Spot, ELISpot）分析，我们对特异性识别含有K43R突变的T细胞表位的CTL免疫反应进行了检测，结果表明K43R突变的产生与宿主的CTL免疫选择压力没有关系。但随后的病毒适应性研究结果显示，K43R突变能严重损害病毒适应性，提示该突变的产生能严重地影响病毒的复制能力。这些结果表明，在宿主对抗HIV-1感染的过程中，很可能存在我们仍然未知的能够对病毒感染产生强烈抑制效果的宿主因素。本研究对CTL逃逸突变T242N对HIV-1适应性的影响，以及病毒基因组内已存的补偿性氨基酸的修复作用进行了深入、精确及系统的研究。这些发现对于我们进一步了解HIV-1适应性这一重要的病毒学特征，及其与病程进展之间的关系具有重要意义。同时，对于设计以诱导CTL免疫反应为靶向的HIV/AIDS疫苗具有指导意义。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.91

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