本文選題：HIV-1 + 膜蛋白�。� 參考：《中國(guó)疾病預(yù)防控制中心》2016年博士論文

【摘要】：自發(fā)現(xiàn)艾滋病和其病原體人類免疫缺陷病毒(HIV)30多年來(lái),HIV已經(jīng)感染和殺死超過(guò)7000萬(wàn)人,是威脅人類健康最嚴(yán)重的傳染病。盡管因抗HIV藥物取得重大突破,艾滋病己從死亡率最高的“超級(jí)癌癥”演化為可控制的慢性病,但因無(wú)法治愈而需長(zhǎng)期服藥,感染者終身攜帶病毒并可能感染他人。每年全球仍有數(shù)百萬(wàn)人被HIV感染,我國(guó)每年發(fā)現(xiàn)的HIV感染和艾滋病人也從十年前的幾萬(wàn)人增加到現(xiàn)在的十余萬(wàn)人。國(guó)際共識(shí)是人類需要有效的疫苗才能最終控制艾滋病。HIV-1膜蛋白是HIV疫苗研究的重要免疫原,因?yàn)樗且詮V譜中和抗體為主的保護(hù)性抗體的的唯一靶位。由于HIV高度變異,其在全球流行過(guò)程中產(chǎn)生出眾多的亞型、簇和流行株。如何誘導(dǎo)廣譜中和抗體反應(yīng)一直是HIV疫苗研究的一個(gè)重點(diǎn)也是最大的難點(diǎn)。許多研究顯示,在自然感染中僅有少數(shù)人可產(chǎn)生抗HIV廣譜中和抗體。在疫苗研究中,天然HIV-1膜蛋白和常規(guī)的免疫策略也不能產(chǎn)生廣譜中和抗體和其他具有免疫保護(hù)性的抗體反應(yīng)。因此,本論文研究針對(duì)HIV疫苗研究的這一難點(diǎn),沿著免疫原改造和優(yōu)化免疫策略的兩條緊密聯(lián)系的技術(shù)路線,展開(kāi)課題的研究,力求獲得能提高HIV膜蛋白保護(hù)性抗體反應(yīng)和優(yōu)化的免疫策略。本研究以中國(guó)主要流行的B'/C重組亞型的CN54株作為疫苗研究的出發(fā)毒株,在第一條技術(shù)路線中對(duì)兩個(gè)基于馬傳染性貧血病毒弱毒疫苗的突變體gp140免疫原2M和5M與CN54 gp140免疫原通過(guò)DNA初免-蛋白加強(qiáng)誘導(dǎo)的抗體免疫反應(yīng)進(jìn)行了系統(tǒng)的比較,然后純化不同結(jié)構(gòu)的CN54gp140蛋白,比較不同結(jié)構(gòu)的gp140蛋白免疫豚鼠誘導(dǎo)的抗體免疫反應(yīng),隨后選擇三種亞型的HIV-1膜蛋白和HIV-2的膜蛋白研究交叉免疫誘導(dǎo)的抗體免疫反應(yīng)。為了比較兩個(gè)突變體gp140 2M和CN54 gp140免疫原誘導(dǎo)產(chǎn)生的抗體免疫反應(yīng)情況,三種gp140蛋白在真核表達(dá)系統(tǒng)得到表達(dá)并純化,通過(guò)DNA初免-蛋白加強(qiáng)的方式免疫小鼠和豚鼠,最后一次免疫后三周采集血清檢測(cè)誘導(dǎo)產(chǎn)生的抗體免疫反應(yīng)。檢測(cè)結(jié)果顯示相比于野生型gp140免疫組,gp140 2M免疫組誘導(dǎo)更高水平的膜蛋白特異性IgG、IgG1和IgG3結(jié)合抗體反應(yīng),gp140 5M免疫組誘導(dǎo)更高水平的膜蛋白特異性IgG2a、IgG3和IgA結(jié)合抗體反應(yīng)。進(jìn)一步分析顯示gp140 2M免疫組誘導(dǎo)Th1型免疫反應(yīng),而gp140和gp140 5M免疫組誘導(dǎo)Th0型免疫反應(yīng)。在誘導(dǎo)膜蛋白特異性IgG2b結(jié)合抗體和IgM結(jié)合抗體上,三組沒(méi)有顯著差異。與未突變gp140以及gp140 5M相比,gp140 2M能誘導(dǎo)產(chǎn)生更好的IgG3,IgA等保護(hù)性結(jié)合抗體,以及更高水平的針對(duì)tier 1假病毒的中和抗體反應(yīng)。提示gp140 2M可能是更好的候選疫苗。為了研究不同結(jié)構(gòu)的gp140蛋白的免疫原性,我們表達(dá)純化了多聚體gp140、三聚體gp140和單體gp140蛋白,并免疫豚鼠檢測(cè)其抗體免疫反應(yīng)情況。免疫結(jié)果顯示,三聚體gp140蛋白可以誘導(dǎo)產(chǎn)生比多聚體和單體gp140蛋白更高水平的結(jié)合抗體和中和抗體反應(yīng)。提示三聚體gp140蛋白可能是更好的免疫原。在第二條技術(shù)路線中,為了提高免疫原誘導(dǎo)產(chǎn)生的具有交叉免疫反應(yīng)的中和抗體反應(yīng),選擇了三種不同HIV-1亞型的膜蛋白和HIV-2膜蛋白用于評(píng)價(jià)交叉免疫產(chǎn)生的抗體免疫反應(yīng)。結(jié)果顯示相比于BC亞型單獨(dú)免疫組,不同亞型混合免疫組可以提高中和抗體的廣譜性和中和強(qiáng)度,對(duì)于B亞型和AE亞型的膜蛋白特異性結(jié)合抗體反應(yīng)和V1V2特異性結(jié)合抗體反應(yīng)也顯著提高。HIV-1和HIV-2免疫原的交叉免疫也能提高中和抗體的廣譜性和中和強(qiáng)度,對(duì)于B亞型和AE亞型的膜蛋白特異性結(jié)合抗體反應(yīng)和AE亞型的V1V2特異性結(jié)合抗體反應(yīng)也顯著提高。交叉免疫可以提高保護(hù)性結(jié)合抗體和中和抗體反應(yīng),不同的免疫順序會(huì)影響誘導(dǎo)產(chǎn)生的抗體免疫反應(yīng)。為了提高保護(hù)性抗體免疫反應(yīng),我們從本實(shí)驗(yàn)室已經(jīng)分離到廣譜中和抗體DRVIA7的HIV-1 B亞型病毒感染者DRVI01不同時(shí)間點(diǎn)擴(kuò)增相應(yīng)的膜蛋白抗原,克隆表達(dá)后免疫豚鼠研究其免疫原性,然后采取不同的免疫程序以求提高免疫原誘導(dǎo)的保護(hù)性抗體反應(yīng)。結(jié)果顯示不同免疫程序可以提高針對(duì)易中和的tier 1的HIV-1假病毒的中和抗體反應(yīng),但對(duì)于難中和的tier 2的HIV-1假病毒的中和抗體反應(yīng)影響不大。這些結(jié)果顯示廣譜中和患者體內(nèi)可以分離出具有一定中和活性的膜蛋白抗原,不同的免疫順序會(huì)影響誘導(dǎo)產(chǎn)生的抗體免疫反應(yīng)。
[Abstract]:Since the discovery of AIDS and its pathogen, human immunodeficiency virus (HIV) for more than 30 years, HIV has infected and killed more than 70 million people and is the most serious infectious disease that threatens human health. Despite a major breakthrough in anti HIV drugs, AIDS has evolved from the highest "supercancer" to a controlled chronic disease, but it is not cured. The infected people carry the virus for a long time and can infect others for a long time. Millions of people worldwide are still infected with HIV every year. The HIV infection and aids people found in our country have increased from tens of thousands of people ten years ago to more than 10, 000 people. The international consensus is that human need effective vaccine to control AIDS.HIV-1 membrane protein finally. It is the important immunogen of HIV vaccine research, because it is the only target of protective antibody with broad spectrum and neutralization antibody. Because of the high variation of HIV, it produces numerous subtypes, clusters and epidemic strains in the global epidemic process. How to induce the broad-spectrum neutralization antibody reaction has been a key and the most difficult point in the study of the HIV vaccine. Many studies have shown that only a few people can produce anti HIV broad-spectrum neutralizing antibodies in natural infections. In vaccine studies, natural HIV-1 membrane proteins and conventional immunization strategies do not produce broad-spectrum neutralizing antibodies and other immune protective antibodies. Therefore, this paper aims at the difficulty of the study of HIV vaccines. The two closely related technical routes of immunization and optimization of immunization were carried out in order to obtain the immunization strategy which could improve the protective antibody response and optimization of HIV membrane protein. This study took the CN54 strain of B'/C recombinant in China as the starting strain of the vaccine research, and two in the first technical route. A systematic comparison of the immune response of the mutant gp140 immunogen 2M and 5M based on the vaccine of the equine infectious anemia virus (5M) and CN54 gp140 immunogen was systematically compared with the Immunogenicity Induced by the DNA primer protein immunogen, and then the CN54gp140 protein of different structures was purified to compare the immune response induced by the different structural gp140 protein immunized guinea pigs. Then select three subtypes of HIV-1 membrane protein and HIV-2 membrane protein to study the antibody immunoreaction induced by cross immunization. In order to compare the antibody immunoreaction induced by the two mutant gp140 2M and CN54 gp140 immunogen, three gp140 proteins were expressed and purified in eukaryotic expression system, and through the strengthening of DNA first immune protein. Immune mice and guinea pigs were immunized three weeks after the last immunization. The results showed that compared to the wild type gp140 immune group, the gp140 2M immune group induced a higher level of membrane protein specific IgG, IgG1 and IgG3 combined with antibody reaction, and the gp140 5M immune group induced a higher level of membrane protein specific I. GG2a, IgG3 and IgA combined with antibody reaction. Further analysis showed that gp140 2M immune group induced Th1 type immune response, while gp140 and gp140 5M immunization group induced Th0 type immune response. There was no significant difference between the three groups in inducing membrane specific IgG2b binding antibody and IgM binding antibody. IgG3, IgA and other protective binding antibodies, as well as a higher level of neutralizing antibody response to the tier 1 pseudo virus, suggest that gp140 2M may be a better candidate vaccine. In order to study the immunogenicity of gp140 proteins of different structures, we purified the gp140, three polymer gp140 and mono gp140 protein, and immunized guinea pigs. The antibody immune response was measured. The immune results showed that the trimer gp140 protein could induce a higher binding antibody and neutralizing antibody reaction than the polymer and the monomer gp140 protein. It suggests that the trimer gp140 protein may be a better immunogen. In the second technical route, the interaction between the immunogen and the immunogen is improved. Three different HIV-1 subtypes of membrane proteins and HIV-2 membrane proteins were selected to evaluate the antibody immunoreaction produced by cross immunization. The results showed that the different subtypes of mixed immune groups could improve the broad-spectrum and neutralization intensity of neutralizing antibodies compared to the BC subtype individual immune group, and for the B subtype and the AE subtype. The membrane protein specific binding antibody reaction and V1V2 specific binding antibody reaction also significantly increase the cross immunization of.HIV-1 and HIV-2 immunogens and improve the broad-spectrum and neutralization strength of neutralizing antibodies. The specific binding antibody reaction of the B subtype and AE subtype and the V1V2 specific binding antibody reaction of the AE subtype also significantly increased. Cross immunization improves the protective binding antibody and neutralizing antibody response, and the different immune sequence affects the induced antibody immunoreaction. In order to improve the protective antibody immune response, we have isolated the HIV-1 B subtype infection of the broad-spectrum neutralizing antibody DRVIA7 from our laboratory and amplified the corresponding DRVI01 at different time points. Membrane protein antigen, after cloning and expression, immunized guinea pigs to study its immunogenicity, and then adopted different immunization programs to improve immunogen induced protective antibody response. The results showed that different immunization programs could improve the neutralizing antibody response to the HIV-1 pseudo virus of the easily neutralized tier 1, but for the difficult neutralizing HIV-1 pseudo virus of the tier 2 The neutralization antibody reaction is not affected. These results show that the membrane protein antigen with certain neutralization activity can be isolated in the broad spectrum and in the patient's body, and the different immune order may affect the induced antibody immune response.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R392

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李丹亞;;人乳中發(fā)現(xiàn)保護(hù)性抗體[J];國(guó)外醫(yī)學(xué)情報(bào);1984年12期

2 ;丙型肝炎病毒抗體是否是保護(hù)性抗體[J];中華內(nèi)科雜志;1995年04期

3 蘇彥萍;王寶蘭;唐一清;;兒童乙型肝炎疫苗免疫和加強(qiáng)免疫后保護(hù)性抗體觀察[J];中國(guó)計(jì)劃免疫;2006年04期

4 楊長(zhǎng)貴;蔡琳;歐秀蓮;;深圳市福田區(qū)青工麻疹保護(hù)性抗體水平監(jiān)測(cè)及分析[J];社區(qū)醫(yī)學(xué)雜志;2009年01期

5 許新強(qiáng),虞福亮;乙型肝炎保護(hù)性抗體的新認(rèn)識(shí)[J];國(guó)外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊(cè);1986年03期

6 楊建農(nóng);霍亂毒素B亞單位與流感疫苗鼻內(nèi)合種可增強(qiáng)保護(hù)性抗體應(yīng)答[J];國(guó)外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊(cè);1990年04期

7 楊玉仙;余慶福;張耀喜;馬運(yùn)葵;李廷學(xué);吳麗清;張麗芳;楊曉娟;;玉溪市兒童乙型肝炎疫苗免后保護(hù)性抗體監(jiān)測(cè)分析[J];職業(yè)與健康;2010年01期

8 郭上忰;乙型肝炎疫苗接種后抗體無(wú)、弱應(yīng)答與遺傳關(guān)系的研究[J];中華醫(yī)學(xué)信息導(dǎo)報(bào);1997年16期

9 鄭琦;一次接種滅活甲型肝炎脂質(zhì)體疫苗兩周后可誘導(dǎo)產(chǎn)生保護(hù)性抗體[J];國(guó)外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊(cè);1993年03期

10 王永忠,楊大為;棗莊市市中區(qū)小學(xué)生乙肝保護(hù)性抗體檢測(cè)[J];預(yù)防醫(yī)學(xué)文獻(xiàn)信息;2003年05期

相關(guān)會(huì)議論文 前3條

1 王華菁;戴建新;;SEB中和保護(hù)性抗體的作用及其結(jié)構(gòu)基礎(chǔ)[A];第十一屆中國(guó)生物毒素研究及醫(yī)藥應(yīng)用年會(huì)論文摘要集[C];2013年

2 范黎;陳霞;王俊;楊衛(wèi)平;王保寧;;乙型肝炎病毒表面抗體(乙肝疫苗保護(hù)性抗體)快速測(cè)定方法的建立和應(yīng)用評(píng)價(jià)[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

3 魏承斌;林紅;楊康林;范黎;王俊;王保寧;;乙型肝炎病毒表面抗體(乙肝疫苗保護(hù)性抗體)快速測(cè)定方法應(yīng)用評(píng)價(jià)[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

相關(guān)重要報(bào)紙文章 前1條

1 吳志軍邋通訊員 郝成濤;SARS病人保護(hù)性抗體會(huì)消失[N];健康報(bào);2007年

相關(guān)博士學(xué)位論文 前2條

1 劉建東;誘導(dǎo)HIV-1膜蛋白保護(hù)性抗體免疫策略的研究[D];中國(guó)疾病預(yù)防控制中心;2016年

2 王華菁;高效抗SEB中和保護(hù)性抗體的作用及其機(jī)制研究[D];第二軍醫(yī)大學(xué);2013年

，

本文編號(hào)：1817203