本文選題：HIV-1 + 中和抗體　； 參考：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文

【摘要】：中和抗體的產(chǎn)生是機(jī)體針對(duì)病毒感染的主要免疫應(yīng)答,同時(shí)也是設(shè)計(jì)研發(fā)有效疫苗的基礎(chǔ)。就HIV-1而言,中和抗體在機(jī)體對(duì)抗病毒感染中起著尤其重要的作用,誘導(dǎo)機(jī)體產(chǎn)生有效的中和抗體也是疫苗免疫反應(yīng)的一個(gè)重要組成部分。在遏制HIV-1感染的疫苗設(shè)計(jì)中,一個(gè)關(guān)鍵目標(biāo)是通過疫苗免疫能夠誘導(dǎo)機(jī)體產(chǎn)生中和不同HIV-1毒株的廣譜中和抗體。因此,分離和鑒定具有廣譜中和活性的抗體,在抗HIV-1感染的研究中正得到越來越多的關(guān)注。 然而,HIV-1在長(zhǎng)期的進(jìn)化中產(chǎn)生出許多拮抗機(jī)體免疫應(yīng)答的方法,從而逃避機(jī)體免疫系統(tǒng)的抗病毒反應(yīng)。這就使得機(jī)體在感染HIV-1時(shí),產(chǎn)生的中和抗體往往中和病毒的活性弱,而且這些抗體只能中和某種特定亞型的病毒。 迄今為止,只有為數(shù)不多的中和抗體具有廣譜交叉中和活性,包括2F5.4E10、2G12、b12、VRC01、PG9、PG16、X5、17b等。最近,一個(gè)新的針對(duì)HIV-1gp41膜鄰接區(qū)域(membrane-proximal external region, MPER)靶位的強(qiáng)有效的廣譜中和抗體10E8被發(fā)現(xiàn),它是繼2F5、4E10后另外一個(gè)針對(duì)此區(qū)域的中和抗體。相比較于2F5、4E10局限的中和反應(yīng)譜,10E8可以中和接近98%的病毒。這也使它成為目前擁有最為廣譜中和活性的抗體。 HIV-1廣泛的基因變異、快速的增殖、頻繁的重組使其成為自然界進(jìn)化最快的、擁有最為復(fù)雜的進(jìn)化分枝及亞型的微生物之一,而且這一特點(diǎn)在中國艾滋病流行中表現(xiàn)的尤為突出。HIV-1在中國的流行病株主要包括三個(gè)亞型：CRF01_AE、B'和C/CRF07_BC/CRF08_BC/B'C (C/07/08/BC)。目前已知的廣譜中和抗體對(duì)中國流行毒株的中和活性并不理想,其中包括最近才發(fā)現(xiàn)的對(duì)90%HIV-1具有中和活性的VRC01,這無疑給針對(duì)中國流行株的疫苗研發(fā)帶來了更大的挑戰(zhàn)。此外,美國、南非、歐洲、肯尼亞以及印度HIV-1感染者血清中和抗體的研究為全世界艾滋病疫苗的研制提供了重要的參考信息。然而,針對(duì)中國HIV-1感染者血清中和抗體反應(yīng)的研究非常薄弱。最近,中國疾病預(yù)防控制中心性病艾滋病預(yù)防控制中心報(bào)道了國內(nèi)103例HIV-1B'亞型感染者血清針對(duì)25株不同亞型HIV-1假病毒的中和反應(yīng),其中包括主要的中國流行株CRF07_BC、CRF01_AE等亞型。這些被列入研究的感染者沒有接受過抗病毒藥物治療并且長(zhǎng)期攜帶病毒超過10年未發(fā)病,即艾滋病精英控制者(elite controllers)。研究發(fā)現(xiàn),29%(30例)的血清能夠中和80%的檢測(cè)病毒,其中血清樣本F524能夠中和全部25株病毒,這提示具有中和活性的感染者血清內(nèi)可能存在廣譜的中和抗體,也為設(shè)計(jì)研發(fā)針對(duì)中國流行株的艾滋病疫苗帶來了新的希望。因此,本實(shí)驗(yàn)室試圖從F524感染者中分離具有廣譜中和活性的抗體,力圖通過對(duì)新抗體進(jìn)行深入研究,為開發(fā)有效的HIV疫苗奠定理論基礎(chǔ)。我們前期的工作利用F524感染者的外周血單個(gè)核淋巴細(xì)胞(PBMC)標(biāo)本構(gòu)建了噬菌體展示抗體庫,并通過交叉利用來源于A和C亞型HIV-1的三聚體形式包膜蛋白富集篩選的方法,成功分離得到一個(gè)新的具有廣譜反應(yīng)活性的單克隆Fab抗體A16。本課題聚焦于研究A16的廣譜中和活性、中和機(jī)制和抗原識(shí)別表位。通過基于假病毒中和實(shí)驗(yàn)的方法對(duì)A16抗體的中和活性做了鑒定,利用受體結(jié)合阻斷實(shí)驗(yàn)和抗體競(jìng)爭(zhēng)實(shí)驗(yàn)對(duì)A16的中和機(jī)制進(jìn)行了初步分析,同時(shí)利用噬菌體隨機(jī)肽庫結(jié)合生物信息學(xué)方法預(yù)測(cè)并確定了抗體所識(shí)別的表位,對(duì)抗體的中和機(jī)制進(jìn)行了解釋。研究發(fā)現(xiàn),A16能夠中和包括A, B, C, B', CRF_AG, CRF07_BC和CRF01_AE等亞型在內(nèi)的多株HIV毒株。有意義的是,A16針對(duì)中國流行株有著更好的中和活性,能夠中和HIV-1中國流行株BC和B’亞群中對(duì)另兩個(gè)針對(duì)CD4結(jié)合區(qū)的廣譜中和抗體b12和VRC01抵抗的多株病毒。通過對(duì)A16抗原表位的研究發(fā)現(xiàn),A16識(shí)別位于gp120的CD4結(jié)合區(qū)的保守位點(diǎn)：主要由loop D區(qū),p20-21發(fā)夾結(jié)構(gòu)的N425,及W96,I109,R480位點(diǎn)組成。其表位有別于另外兩個(gè)同樣針對(duì)該區(qū)域的廣譜中和抗體VRC01及b12。推測(cè)A16主要通過與病毒競(jìng)爭(zhēng)受體CD4的結(jié)合,進(jìn)而發(fā)揮其廣譜的中和活性。A16另一突出的特點(diǎn)是,其重鏈和輕鏈可變區(qū)(VH和VL)都具有相對(duì)較長(zhǎng)的CDR3序列和較低的體細(xì)胞突變率。 綜上所述,A16抗體能夠廣譜并且有效地中和不同HIV-1亞型的代表毒株,尤其是在中國流行的BC和B’亞型,其中一些毒株難于被其他一些廣譜中和抗體中和,提示A16可能可以與這些抗體聯(lián)合使用,用于治療HIV-1感染。另外,我們的研究結(jié)果表明,A16識(shí)別的位于CD4結(jié)合區(qū)loop D為主的表位能夠被機(jī)體免疫系統(tǒng)識(shí)別并且.產(chǎn)生具有廣譜活性的抗體。本研究可能為開發(fā)有效的誘導(dǎo)機(jī)體產(chǎn)生廣譜中和抗體的疫苗提供線索。
[Abstract]:The production of neutralizing antibodies is the main immune response to the virus infection and is also the basis for the design and development of effective vaccines. As far as HIV-1 is concerned, neutralizing antibodies play a particularly important role in the body against virus infection, and the induction of effective neutralizing antibodies is also an important part of the immune response of the vaccine. In the design of HIV-1 infection vaccine, one of the key objectives is to induce broad-spectrum neutralizing antibodies against different HIV-1 strains by immunization by vaccine. Therefore, the isolation and identification of antibodies with broad-spectrum neutralization activity are being paid more and more attention in the study of anti HIV-1 infection.
However, in a long period of evolution, HIV-1 produces many ways to antagonize the immune response of the body, thus escaping the antiviral response of the body's immune system, which makes the neutralizing antibodies produced by the body when infected with HIV-1 are often weak in the activity of the virus, and these antibodies are only associated with a specific subtype of virus.
So far, only a few neutralization antibodies have broad-spectrum cross neutralization activity, including 2F5.4E10,2G12, B12, VRC01, PG9, PG16, X5,17b and so on. Recently, a powerful and effective broad-spectrum neutralization antibody 10E8 was discovered for the HIV-1gp41 membrane adjacent area (membrane-proximal external region, MPER) target, and it is the other after another. Compared with the 2F5,4E10 localized neutralization response spectrum, the 10E8 can neutralize the virus close to 98%, which makes it the most broad-spectrum neutralization antibody at present.
HIV-1's extensive genetic variation, rapid proliferation, and frequent recombination make it the fastest evolving nature in nature, with one of the most complex evolutionary branches and subtypes of microbes, and this characteristic is particularly prominent in the epidemic of AIDS in China. The epidemic of.HIV-1 in China mainly includes three subtypes: CRF01_AE, B'and C/CRF0 7_BC/CRF08_BC/B'C (C/07/08/BC). The neutralization activity of the known broad-spectrum neutralization antibody to Chinese epidemic strains is not ideal, including the recently discovered VRC01 with neutralization activity to 90%HIV-1, which undoubtedly poses a greater challenge to the development of vaccines for Chinese epidemic strains. In addition, the United States, South Africa, Europe, Kenya, and The study of serum neutralization antibodies in India HIV-1 infected people has provided important reference information for the development of the worldwide AIDS vaccine. However, the study of serum neutralization antibody response to HIV-1 infected people in China is very weak. Recently, the center for STD and AIDS prevention and control of the CDC has reported 103 HIV-1B'subtypes in China. The neutralization response of infected persons to 25 different subtypes of HIV-1 pseudo viruses, including the major Chinese epidemic strains CRF07_BC, CRF01_AE and other subtypes, had not been treated with antiviral drugs and had long carried the virus for more than 10 years, that is, the elite controllers (elite controllers). It was found that the serum of 29% (30 cases) could neutralize 80% of the virus, in which the serum sample F524 could neutralize all 25 strains of virus, suggesting that the neutralization activity might have a broad-spectrum neutralization antibody in the sera and the design and development of the AIDS vaccine for Chinese epidemic strains. 524 of the 524 infected people are isolated with broad-spectrum and active antibodies, trying to establish a theoretical basis for the development of an effective vaccine through in-depth study of the new antibodies. Our previous work used F524 infected peripheral blood mononuclear lymphocytes (PBMC) to construct a phage display antibody library and cross utilization from A and C. The method of enrichment and screening of subtype HIV-1's tripolymer form envelope protein, a new monoclonal Fab antibody A16. with broad-spectrum reactivity was successfully isolated. This topic focused on the broad-spectrum neutralization activity, neutralization and antigen recognition epitopes of A16, and the neutralization activity of A16 antibody in pseudo virus and experimental methods. The neutralization mechanism of A16 was preliminarily analyzed by the receptor binding blocking experiment and the antibody competition experiment. At the same time, the epitopes identified by the phage random peptide library combined with the bioinformatics method were predicted and identified, and the neutralization mechanism of the antibody was explained. The study found that A16 could neutralize the neutralization of A, B, C, B', CRF_A. Multiple HIV strains, such as G, CRF07_BC and CRF01_AE subtypes. It is significant that A16 has better neutralization activity against Chinese epidemic strains, and can neutralize two other strains of HIV-1 in BC and B 'subsets against the broad-spectrum neutralizing antibody B12 and VRC01 resistance to CD4 binding regions. 16 identify the conserved sites in the CD4 binding area of gp120, mainly composed of the loop D region, the N425 of the p20-21 hairpin structure, and the W96, I109, and R480 sites. The epitopes are different from the other two equally wide spectrum neutralizing antibodies VRC01 and b12. speculates that A16 mainly through the combination of the virus competitive receptor, and then exerts its broad-spectrum neutralization activity. Another prominent feature of sex.A16 is that its heavy chain and light chain variable regions (VH and VL) have relatively long CDR3 sequences and lower somatic mutation rates.
To sum up, A16 antibodies can be broad-spectrum and effectively neutralizing the representative strains of different HIV-1 subtypes, especially in China's prevalent BC and B 'subtypes, some of which are difficult to be neutralized by some other broad-spectrum neutralization antibodies, suggesting that A16 may be combined with these antibodies for the treatment of HIV-1 infection. In addition, our findings table The epitopes, identified by A16, located in the CD4 binding area loop D, can be identified by the immune system and produce broad-spectrum antibodies. This study may provide clues to the development of an effective vaccine to induce broad-spectrum neutralization antibodies.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R392;R512.91

【共引文獻(xiàn)】

相關(guān)期刊論文 前6條

1 張應(yīng)玖,金寧一,金善榮,王宏偉,沈家驄;用細(xì)菌/桿狀病毒系統(tǒng)在昆蟲細(xì)胞中表達(dá)HIV-2外膜糖蛋白gp105和跨膜糖蛋白gp36[J];免疫學(xué)雜志;2001年01期

2 張應(yīng)玖,金寧一,王宏偉,金洪濤,沈家驄;HIV-2外膜蛋白在甲醇型酵母Pichia pastoris中的表達(dá)與鑒定[J];生物化學(xué)與生物物理學(xué)報(bào);2001年04期

3 黃煜;李大金;;人早孕滋養(yǎng)細(xì)胞分泌趨化因子CXCL16促進(jìn)自身增殖和侵襲[J];分子細(xì)胞生物學(xué)報(bào);2006年01期

4 張應(yīng)玖,金寧一,殷震,沈家驄;HIV的致病機(jī)制研究進(jìn)展[J];微生物學(xué)雜志;2000年01期

5 張應(yīng)玖,金寧一,王宏偉,張東威,邵一鳴,殷霞,沈家驄;HIV-1-gp120在重組桿狀病毒系統(tǒng)中的表達(dá)[J];微生物學(xué)雜志;2000年03期

6 吳霞,李大金,袁敏敏,王明雁,程海東;人早孕期絨毛組織和滋養(yǎng)細(xì)胞趨化因子受體轉(zhuǎn)錄水平[J];中國免疫學(xué)雜志;2004年01期

相關(guān)博士學(xué)位論文 前3條

1 王聞雅;新型趨化因子MIP-2γ對(duì)造血干/祖細(xì)胞的存活、趨化和動(dòng)員作用及機(jī)制研究[D];第二軍醫(yī)大學(xué);2003年

2 吳霞;趨化因子和趨化因子受體在母—胎界面的表達(dá)及其生物學(xué)作用[D];復(fù)旦大學(xué);2004年

3 喻靜;單核巨噬細(xì)胞CC類趨化因子受體異常調(diào)控導(dǎo)致子宮內(nèi)膜異位癥發(fā)病的分子機(jī)制[D];復(fù)旦大學(xué);2008年

相關(guān)碩士學(xué)位論文 前4條

1 于潔;趨化因子CXCL16對(duì)人早孕蛻膜免疫細(xì)胞的募集及其分泌細(xì)胞因子的調(diào)節(jié)[D];青島大學(xué);2009年

2 安永輝;根際促生菌（PGPR）對(duì)紫花苜蓿幼苗生長(zhǎng)及生理的影響[D];東北師范大學(xué);2009年

3 陳柯良;去甲斑蝥素衍生物的合成及抗HIV構(gòu)效關(guān)系研究[D];中國協(xié)和醫(yī)科大學(xué);2009年

4 衛(wèi)艷萍;建立作用于CCR5啟動(dòng)子的藥物篩選方法及初步應(yīng)用[D];鄭州大學(xué);2008年

，

本文編號(hào)：1872409