人源HIV-1广谱中和Fab抗体的鉴定

发布时间：2018-05-11 04:24

本文选题：HIV-1 + 中和抗体　；参考：《北京协和医学院》2013年博士论文

【摘要】：中和抗体的产生是机体针对病毒感染的主要免疫应答,同时也是设计研发有效疫苗的基础。就HIV-1而言,中和抗体在机体对抗病毒感染中起着尤其重要的作用,诱导机体产生有效的中和抗体也是疫苗免疫反应的一个重要组成部分。在遏制HIV-1感染的疫苗设计中,一个关键目标是通过疫苗免疫能够诱导机体产生中和不同HIV-1毒株的广谱中和抗体。因此,分离和鉴定具有广谱中和活性的抗体,在抗HIV-1感染的研究中正得到越来越多的关注。然而,HIV-1在长期的进化中产生出许多拮抗机体免疫应答的方法,从而逃避机体免疫系统的抗病毒反应。这就使得机体在感染HIV-1时,产生的中和抗体往往中和病毒的活性弱,而且这些抗体只能中和某种特定亚型的病毒。迄今为止,只有为数不多的中和抗体具有广谱交叉中和活性,包括2F5.4E10、2G12、b12、VRC01、PG9、PG16、X5、17b等。最近,一个新的针对HIV-1gp41膜邻接区域(membrane-proximal external region, MPER)靶位的强有效的广谱中和抗体10E8被发现,它是继2F5、4E10后另外一个针对此区域的中和抗体。相比较于2F5、4E10局限的中和反应谱,10E8可以中和接近98%的病毒。这也使它成为目前拥有最为广谱中和活性的抗体。 HIV-1广泛的基因变异、快速的增殖、频繁的重组使其成为自然界进化最快的、拥有最为复杂的进化分枝及亚型的微生物之一,而且这一特点在中国艾滋病流行中表现的尤为突出。HIV-1在中国的流行病株主要包括三个亚型：CRF01_AE、B'和C/CRF07_BC/CRF08_BC/B'C (C/07/08/BC)。目前已知的广谱中和抗体对中国流行毒株的中和活性并不理想,其中包括最近才发现的对90%HIV-1具有中和活性的VRC01,这无疑给针对中国流行株的疫苗研发带来了更大的挑战。此外,美国、南非、欧洲、肯尼亚以及印度HIV-1感染者血清中和抗体的研究为全世界艾滋病疫苗的研制提供了重要的参考信息。然而,针对中国HIV-1感染者血清中和抗体反应的研究非常薄弱。最近,中国疾病预防控制中心性病艾滋病预防控制中心报道了国内103例HIV-1B'亚型感染者血清针对25株不同亚型HIV-1假病毒的中和反应,其中包括主要的中国流行株CRF07_BC、CRF01_AE等亚型。这些被列入研究的感染者没有接受过抗病毒药物治疗并且长期携带病毒超过10年未发病,即艾滋病精英控制者(elite controllers)。研究发现,29%(30例)的血清能够中和80%的检测病毒,其中血清样本F524能够中和全部25株病毒,这提示具有中和活性的感染者血清内可能存在广谱的中和抗体,也为设计研发针对中国流行株的艾滋病疫苗带来了新的希望。因此,本实验室试图从F524感染者中分离具有广谱中和活性的抗体,力图通过对新抗体进行深入研究,为开发有效的HIV疫苗奠定理论基础。我们前期的工作利用F524感染者的外周血单个核淋巴细胞(PBMC)标本构建了噬菌体展示抗体库,并通过交叉利用来源于A和C亚型HIV-1的三聚体形式包膜蛋白富集筛选的方法,成功分离得到一个新的具有广谱反应活性的单克隆Fab抗体A16。本课题聚焦于研究A16的广谱中和活性、中和机制和抗原识别表位。通过基于假病毒中和实验的方法对A16抗体的中和活性做了鉴定,利用受体结合阻断实验和抗体竞争实验对A16的中和机制进行了初步分析,同时利用噬菌体随机肽库结合生物信息学方法预测并确定了抗体所识别的表位,对抗体的中和机制进行了解释。研究发现,A16能够中和包括A, B, C, B', CRF_AG, CRF07_BC和CRF01_AE等亚型在内的多株HIV毒株。有意义的是,A16针对中国流行株有着更好的中和活性,能够中和HIV-1中国流行株BC和B’亚群中对另两个针对CD4结合区的广谱中和抗体b12和VRC01抵抗的多株病毒。通过对A16抗原表位的研究发现,A16识别位于gp120的CD4结合区的保守位点：主要由loop D区,p20-21发夹结构的N425,及W96,I109,R480位点组成。其表位有别于另外两个同样针对该区域的广谱中和抗体VRC01及b12。推测A16主要通过与病毒竞争受体CD4的结合,进而发挥其广谱的中和活性。A16另一突出的特点是,其重链和轻链可变区(VH和VL)都具有相对较长的CDR3序列和较低的体细胞突变率。综上所述,A16抗体能够广谱并且有效地中和不同HIV-1亚型的代表毒株,尤其是在中国流行的BC和B’亚型,其中一些毒株难于被其他一些广谱中和抗体中和,提示A16可能可以与这些抗体联合使用,用于治疗HIV-1感染。另外,我们的研究结果表明,A16识别的位于CD4结合区loop D为主的表位能够被机体免疫系统识别并且.产生具有广谱活性的抗体。本研究可能为开发有效的诱导机体产生广谱中和抗体的疫苗提供线索。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R392;R512.91

【共引文献】