本文選題：HIV-1 + 病毒適應(yīng)性　； 參考：《吉林大學(xué)》2014年博士論文

【摘要】：人類免疫缺陷病毒（human immunodeficiency virus, HIV）是導(dǎo)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(fù)制速率、短復(fù)制周期和極高的突變率迅速的增殖和進(jìn)化，這些特點(diǎn)對(duì)于病毒能夠迅速適應(yīng)并克服宿主的免疫反應(yīng)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(jìn)化和傳播過(guò)程的能力，被稱為HIV-1適應(yīng)性。病毒適應(yīng)性決定了病毒在宿主體內(nèi)的復(fù)制能力。作為HIV-1最重要的固有性質(zhì)之一，病毒適應(yīng)性微小的改變就能顯著地影響HIV-1的進(jìn)化趨勢(shì)、致病機(jī)制、流行傳播以及病程進(jìn)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(xì)胞毒性T細(xì)胞（cytotoxic T lymphocyte; CTL）免疫反應(yīng)遏制HIV-1。這些有效的CTL免疫反應(yīng)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(yīng)，而代價(jià)是這些免疫逃逸突變將對(duì)病毒適應(yīng)性造成損害。在以往報(bào)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(dá)的B*57/5801型人類白細(xì)胞抗原（human leukocyte antigen; HLA）介導(dǎo)的CTL免疫反應(yīng)密切相關(guān)。普遍認(rèn)為，CTL免疫逃逸突變T242N能顯著損害HIV-1適應(yīng)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(yīng)性的損失而降低；同時(shí)，即使T242N突變體病毒傳染了新的宿主，其病程進(jìn)展可能比較緩慢。因此，TW10表位被認(rèn)為是抗HIV/AIDS疫苗的理想靶點(diǎn)。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(guān)病毒適應(yīng)性補(bǔ)償突變的產(chǎn)生，從而能部分程度或完全修復(fù)病毒適應(yīng)性的損失；另外，如果新宿主體內(nèi)沒(méi)有特異性的CTL免疫反應(yīng)，T242N突變體也可能在感染后發(fā)生回復(fù)突變，但常常需要幾個(gè)月甚至幾年時(shí)間。 但是由于實(shí)驗(yàn)條件和技術(shù)方法的限制，以往的研究并沒(méi)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(duì)病毒適應(yīng)性的影響，而是以實(shí)驗(yàn)室適應(yīng)毒株作為替代，甚至還加入與HIV-1病毒基因不相關(guān)的報(bào)告基因。因此，這些研究無(wú)法避免病毒基因組中預(yù)存的已知或未知的補(bǔ)償性氨基酸對(duì)T242N突變的修復(fù)作用，從而導(dǎo)致不準(zhǔn)確甚至不正確的實(shí)驗(yàn)結(jié)果。在本研究中，我們通過(guò)單基因組擴(kuò)增法（single genome amplification, SGA）分析了4位HIV-1急性感染者的長(zhǎng)期臨床監(jiān)測(cè)樣品，獲得了HIV-1在感染者體內(nèi)的詳細(xì)進(jìn)化過(guò)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(yáng)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(fù)為N242T；而CH470T/F和CH40T/F感染HLA-B*57/5801陰性的宿主后沒(méi)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?cè)诓煌腡/F病毒基因組中對(duì)于病毒適應(yīng)性的影響。 此外，以往報(bào)道的HIV-1適應(yīng)性檢測(cè)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(dǎo)致檢測(cè)靈敏度有限，或是由于采用傳統(tǒng)聚合酶鏈反應(yīng)（polymerase chain reaction; PCR）而導(dǎo)致結(jié)果的穩(wěn)定性和精確性有限，同時(shí)還費(fèi)時(shí)費(fèi)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(diǎn)測(cè)序法（the parallel allele-specific sequencing, PASS）的新型HIV-1病毒適應(yīng)性檢測(cè)方法，即PFA法（PASS fitness assay, PFA）。應(yīng)用PFA法，我們靈敏而精確地檢測(cè)出T242N突變?cè)诓煌腡/F病毒基因組中對(duì)病毒適應(yīng)性造成的不同程度的損害；同時(shí)，還檢測(cè)出HIV-1基因組中已存的相關(guān)補(bǔ)償性氨基酸I247或Q219能大幅修復(fù)由于T242N導(dǎo)致的病毒適應(yīng)性損失；此外，我們還發(fā)現(xiàn)在預(yù)存補(bǔ)償性氨基酸的作用下，攜帶T242N突變的CH131T/F與其回復(fù)突變體N242T的病毒適應(yīng)性并無(wú)顯著差異。這些結(jié)果揭示了攜帶T242N突變的HIV-1仍然能相對(duì)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(fù)突變N242T的原因。更重要的是，這些關(guān)于CTL逃逸突變T242N對(duì)病毒適應(yīng)性影響的系統(tǒng)性研究結(jié)果，對(duì)設(shè)計(jì)以細(xì)胞免疫為基礎(chǔ)的HIV/AIDS疫苗具有重要的指導(dǎo)意義。 最后，HIV-1在宿主體內(nèi)進(jìn)化的兩個(gè)最重要的外界壓力是宿主的CTL免疫反應(yīng)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(duì)其產(chǎn)生的原因和病毒適應(yīng)性影響進(jìn)行了研究。用CH131感染者的外周血單核細(xì)胞（peripheral blood mononuclear cells, PBMC）樣品進(jìn)行體外酶聯(lián)免疫斑點(diǎn)（Enzyme-Linked Immuno-Spot, ELISpot）分析，我們對(duì)特異性識(shí)別含有K43R突變的T細(xì)胞表位的CTL免疫反應(yīng)進(jìn)行了檢測(cè)，結(jié)果表明K43R突變的產(chǎn)生與宿主的CTL免疫選擇壓力沒(méi)有關(guān)系。但隨后的病毒適應(yīng)性研究結(jié)果顯示，K43R突變能嚴(yán)重?fù)p害病毒適應(yīng)性，提示該突變的產(chǎn)生能嚴(yán)重地影響病毒的復(fù)制能力。這些結(jié)果表明，在宿主對(duì)抗HIV-1感染的過(guò)程中，很可能存在我們?nèi)匀晃粗哪軌驅(qū)Σ《靖腥井a(chǎn)生強(qiáng)烈抑制效果的宿主因素。 本研究對(duì)CTL逃逸突變T242N對(duì)HIV-1適應(yīng)性的影響，以及病毒基因組內(nèi)已存的補(bǔ)償性氨基酸的修復(fù)作用進(jìn)行了深入、精確及系統(tǒng)的研究。這些發(fā)現(xiàn)對(duì)于我們進(jìn)一步了解HIV-1適應(yīng)性這一重要的病毒學(xué)特征，及其與病程進(jìn)展之間的關(guān)系具有重要意義。同時(shí)，對(duì)于設(shè)計(jì)以誘導(dǎo)CTL免疫反應(yīng)為靶向的HIV/AIDS疫苗具有指導(dǎo)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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.91

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊景偉;魏煜程;沈毅;;肺癌免疫逃逸研究進(jìn)展[J];青島大學(xué)醫(yī)學(xué)院學(xué)報(bào);2007年03期

2 潘潤(rùn)存;;從哲學(xué)角度對(duì)腫瘤發(fā)生發(fā)展與免疫逃逸的研究[J];中國(guó)免疫學(xué)雜志;2012年09期

3 胡高裕;黃贊松;;肝癌免疫逃逸與免疫治療的研究進(jìn)展[J];世界華人消化雜志;2014年15期

4 楊長(zhǎng)青;Fas/FasL與免疫逃逸[J];醫(yī)學(xué)信息;1999年07期

5 黃云勝;施志明;;肺積方對(duì)肺癌免疫逃逸干預(yù)作用的臨床研究[J];中國(guó)中西醫(yī)結(jié)合雜志;2007年06期

6 任紅;乙型肝炎病毒表面抗原免疫逃逸突變基因在人肝癌細(xì)胞的表達(dá)[J];中華醫(yī)學(xué)雜志;1995年07期

7 朱海宏;免疫逃逸現(xiàn)象與HLA相關(guān)性的研究現(xiàn)狀[J];青海醫(yī)學(xué)院學(xué)報(bào);2002年02期

8 項(xiàng)榮;梁龍;劉艷平;;病毒miRNA與免疫逃逸[J];中國(guó)細(xì)胞生物學(xué)學(xué)報(bào);2012年09期

9 荊雪寧,張玲;CD44與腫瘤的免疫逃逸[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2004年03期

10 康璇;王琦;楊潔;;吲哚胺2,3雙-加氧酶介導(dǎo)肝癌細(xì)胞免疫逃逸的實(shí)驗(yàn)研究[J];中國(guó)生物工程雜志;2008年05期

相關(guān)會(huì)議論文 前8條

1 馬強(qiáng);王業(yè)富;;乙型肝炎病毒表面抗原免疫逃逸分子研究分析[A];2012年湖北生物產(chǎn)業(yè)發(fā)展高端論壇暨湖北省生物工程學(xué)會(huì)2012年度學(xué)術(shù)交流會(huì)論文匯編[C];2012年

2 徐凱;郭劍明;張沂南;劉宇軍;徐志兵;朱延軍;王國(guó)民;;冬凌草甲素對(duì)雄激素非依賴性前列腺癌裸鼠移植瘤免疫逃逸相關(guān)蛋白的影響[A];第十五屆全國(guó)泌尿外科學(xué)術(shù)會(huì)議論文集[C];2008年

3 李道睿;林洪生;吳皓;于明薇;祁鑫;裴迎霞;;扶正培元方對(duì)Lewis肺癌小鼠免疫逃逸調(diào)控作用的初步機(jī)理研究[A];第六次全國(guó)中西醫(yī)結(jié)合中青年學(xué)術(shù)研討會(huì)論文集[C];2008年

4 左錢飛;曾浩;;MRSA感染的免疫逃逸及免疫防治研究進(jìn)展[A];第五次全國(guó)免疫診斷暨疫苗學(xué)術(shù)研討會(huì)論文匯編[C];2011年

5 韓巖梅;張明剛;郭秋莉;曹雪濤;;IL-6在新型髓樣抑制性細(xì)胞亞群誘導(dǎo)腫瘤轉(zhuǎn)移和免疫逃逸中的作用及相關(guān)機(jī)制研究[A];第六屆全國(guó)免疫學(xué)學(xué)術(shù)大會(huì)論文集[C];2008年

6 闕祖俊;劉建文;;靈芝酸Me干預(yù)IDO誘導(dǎo)肺癌微環(huán)境免疫逃逸的分子機(jī)理研究[A];第三屆中國(guó)藥理學(xué)會(huì)補(bǔ)益藥藥理專業(yè)委員會(huì)學(xué)術(shù)研討會(huì)論文集[C];2013年

7 趙軍;鄭世營(yíng);葛錦峰;邵峰;紀(jì)勇;蔣東;;FasL表達(dá)與食管癌免疫逃逸關(guān)系的研究[A];中華醫(yī)學(xué)會(huì)第六次全國(guó)胸心血管外科學(xué)術(shù)會(huì)議論文集（胸外科分冊(cè)）[C];2006年

8 趙軍;鄭世營(yíng);葛錦峰;邵峰;紀(jì)勇;蔣東;;FasL表達(dá)與食管癌免疫逃逸關(guān)系的研究[A];第八屆華東六省一市胸心血管外科學(xué)術(shù)會(huì)議論文匯編[C];2005年

相關(guān)博士學(xué)位論文 前4條

1 黃德東;中國(guó)人群HIV-1 Gag區(qū)特異性CTL應(yīng)答和免疫逃逸[D];第四軍醫(yī)大學(xué);2010年

2 劉曉艷;HPV相關(guān)miRNA參與HPV免疫逃逸的分子機(jī)制及基于miRNA骨架的RNA干擾載體的效應(yīng)研究[D];浙江大學(xué);2013年

3 劉東來(lái);細(xì)胞免疫逃逸突變及已存補(bǔ)償性氨基酸對(duì)HIV-1適應(yīng)性的影響機(jī)制[D];吉林大學(xué);2014年

4 竇愛(ài)霞;cAMP在小劑量環(huán)磷酰胺逆轉(zhuǎn)NHL動(dòng)物模型免疫逃逸過(guò)程中的作用研究[D];山東大學(xué);2012年

相關(guān)碩士學(xué)位論文 前4條

1 高曉寧;放療對(duì)膠質(zhì)瘤B7-H1及細(xì)胞免疫逃逸影響的研究[D];寧波大學(xué);2012年

2 張超雄;HMGB1在結(jié)腸癌免疫逃逸中的作用及其機(jī)制研究[D];第二軍醫(yī)大學(xué);2009年

3 遠(yuǎn)麗芳;肺癌患者外周血NKG2A和NKG2D表達(dá)及其臨床意義的初步研究[D];山西醫(yī)科大學(xué);2012年

4 紀(jì)傳彪;從T細(xì)胞免疫應(yīng)答的初始階段研究TLR-4和B7-H1在膀胱癌免疫逃逸中的作用及臨床意義[D];青島大學(xué);2013年

本文編號(hào)：2116289