诱导HIV-1中和抗体的膜蛋白抗原的优化和改造

发布时间：2018-04-26 01:14

本文选题：HIV + Env　；参考：《中国药品生物制品检定所》2011年硕士论文

【摘要】：自1981年确认首例艾滋病以来,艾滋病已成为对人类健康和社会稳定威胁最大的感染性疾病之一。研制有效的艾滋病疫苗是控制艾滋病流行甚至根除AIDS的理想途径。选择合适的抗原作为免疫原以诱导强效、广谱的中和抗体一直是疫苗研究的重要的基础。HIV-1膜抗原位于HIV的表面,是中和抗体主要作用靶位。但天然HIV-1膜蛋白免疫原性低,很难诱导出强效广谱的中和抗体,因而需要对其进行优化改造以提高中和表位的免疫原性,增强其诱导中和抗体的能力。本研究以一株CRF07_BC亚型的假病毒株S939为原始株,对其膜区进行改造以暴露某些保守中和表位,通过单抗、阳性血清的中和试验来验证改造效果,最终获得一株对中和性单克隆抗体和HIV-1感染阳性血清均敏感、中和特性强的假病毒,为候选疫苗的抗原选择和优化提供依据。本课题根据已知的单抗识别表位、已报道的能增强病毒对单抗敏感性的位点和N-连接糖基化位点为依据,对S939 Env设计突变,通过环形诱变,DpnI酶筛选的方式得到突变体,酶切初步鉴定,测序确定突变结果。将含有突变env的真核表达质粒与HIV-1骨架质粒pSG3△env共转染293FT细胞,收集上清获得假病毒,通过假病毒单轮感染TZM-bl细胞检测假病毒滴度和突变位点对假病毒感染能力的影响。用HIV-1中和性单抗、HIV-1感染的阳性血清来检测改造位点对假病毒中和特性的影响。单抗2F5、4E10识别gp41近膜区(MPER)上的线性表位,在S939上对该表位进行联合改造,改造后的假病毒FE相比原始株S939感染力无显著变化。FE对单抗2F5敏感性较S939提高11倍以上,对单抗4E10敏感性无明显变化。在FE上进行单抗2G12表位的改造(I295N、N334S和D386N)获得假病毒株FE-2G12,其感染力较FE略微增强。2G12表位的改造没有使假病毒FE-2G12对单抗2G12敏感,反而降低了对单抗b12和部分阳性血清的敏感性。在FE上进行了8个已报道的增强病毒中和活性位点的改造,其中6个位点的突变I309L、L669S、G458A、T569A、I675V和D180N显著降低了病毒的感染力,因而不能进行其对病毒中和特性影响的评价。其余两个突变中,S365A增强了病毒的感染力,F22L对病毒感染力无影响,这两个突变均没有增强病毒的中和活性。以FE为模板,对25个N-连接的糖基化位点进行单独删除,共获得25个含有单个糖基化删除的Env表达质粒,并与pSG3△env质粒共转染293FT细胞收获假病毒。对这25个假病毒感染力的检测发现,其中5个分布在V1/V2,C1/C2区糖基化位点的删除严重影响了病毒的感染力,含有这5个删除位点的假病毒检测不到其感染能力。分布在免疫反应静默面(immunologically-silent face domains)V4、C4和V5区域中的糖基化位点的删除(除了N392 (V4))对假病毒的感染性有一定的增强。其他糖基化位点的删除均不同程度的减弱了假病毒的感染能力。在假病毒中和特性方面,糖基化位点N197 (C2),N301 (V3),N442 (C4)和N625(gp41)的删除使假病毒对部分HIV-1阳性血清、抗CD4结合位点抗体b12和抗gp41抗体2F5和4E10更加敏感。N142 (V1)的删除使假病毒对单抗b12,2F5和4E10敏感性增强,但对HIV-1阳性血清敏感性没有影响。N355 (C3)和N463 (V5)的删除使假病毒对2F5和4E10敏感性增强。根据前几部分的改造结果,选择其中能对病毒中和活性显著增强的位点,结合V5(N463、N466)和C3(N339)区的糖基化位点以及与N442临近的N448的删除,在FE上按不同组合进行了联合改造,共获得了7株联合突变假病毒,其中除197M-4外,均显著降低了病毒的感染力。对其中6株假病毒的中和特性分析发现,相比于原始株S939、改造株FE以及单个糖基化位点的改造,联合突变株对单抗2F5、4E10和b12,以及大部分阳性血清,均显示出了很强的敏感性。本实验通过对55个氨基酸位点的突变改造,发现了一些能够显著影响病毒感染能力的位点,发现了一些能显著增强病毒中和特性的位点。通过联合改造,最终获得了几株对HIV-1中和单抗和阳性血清表现出较强中和特性的假病毒株,为HIV-1膜蛋白的优化改造以及候选膜抗原的选择提供了依据。
[Abstract]:Since the first AIDS was identified in 1981, AIDS has become one of the most infectious diseases that threaten human health and social stability. The development of an effective AIDS vaccine is an ideal way to control the epidemic and even eradicate AIDS. The important basic.HIV-1 membrane antigen is located on the surface of HIV, which is the main target of neutralizing antibody. But the natural HIV-1 membrane protein is low immunogenicity, it is difficult to induce the strong and broad-spectrum neutralization antibody. Therefore, it needs to be optimized to improve the immunogenicity of neutralizing epitopes and enhance the ability to induce neutralization antibody. The strain CRF07_BC subtype S939 was the original strain, and its membrane region was reformed to expose some conservative neutralization epitopes. The effect was verified by neutralization test of mAb and positive sera. Finally, a strain of neutralizing monoclonal antibody and HIV-1 infection positive sera, a pseudo virus with strong neutralization characteristics, was obtained as a candidate vaccine. The original selection and optimization provide the basis.
According to the known epitopes of the known monoclonal antibody, it has been reported that the virus can enhance the sensitivity of the virus to the loci of the monoclonal antibody and the glycosylation site of the N- connection. The mutation is designed for the S939 Env, the mutation is obtained through the ring mutation and the DpnI enzyme screening. The mutation results are determined by the enzyme digestion and the mutation results are sequenced. The eukaryotic expression plasmid containing the mutant env is expressed. The HIV-1 cytoskeleton plasmid pSG3 delta env was co transfected with 293FT cells, and the pseudo virus was collected from the supernatant. The pseudo virus titer and the mutation site were detected by the pseudo virus single wheel infection. The effect of the HIV-1 neutralization monoclonal antibody and the positive serum of HIV-1 infection on the neutralization characteristics of the pseudo virus was detected by the HIV-1 neutralization monoclonal antibody and the positive serum of HIV-1 infection.
The monoclonal antibody 2F5,4E10 identified the linear epitopes on the gp41 near membrane region (MPER), and reformed the epitopes on the S939. The transformed pseudo virus FE had no significant changes in the S939 infection force compared with the original strain.FE, and the sensitivity of.FE to the monopagion 2F5 was 11 times higher than that of S939, and the sensitivity of the monoclonal antibody 4E10 was not changed. 334S and D386N) obtained the pseudo virus strain FE-2G12, and its infection force was slightly enhanced by a slight enhancement of the.2G12 epitopes, which did not make the pseudo virus FE-2G12 sensitive to the monoclonal antibody 2G12, but reduced the sensitivity to the monoclonal antibody B12 and the partial positive serum.
8 reported enhanced viral and active sites were reported on FE, of which mutations I309L, L669S, G458A, T569A, I675V and D180N significantly reduced the virus's infectivity and therefore failed to evaluate the effect of the virus neutralization. In the other two mutations, S365A enhanced the virus infection and F22L to the virus. The two mutations did not enhance the neutralizing activity of the virus.
The glycosylation sites of 25 N- connections were deleted by FE as a template, and 25 Env expressing plasmids containing single glycosylation were obtained, and pSG3 delta env plasmids were co transfected with 293FT cells to harvest the pseudo virus. The detection of the 25 pseudo virus infection forces was found, of which 5 were distributed in V1/V2, C1/C2 region glycosylation sites were deleted seriously. The infectivity of the virus, which contains the 5 deletion sites, is not detected. The deletion of the glycosylation sites in the immune response (immunologically-silent face domains) V4, C4 and V5 regions (except for N392 (V4)) has a certain enhancement in the susceptibility to the pseudo virus. The deletion of the glycosylation site N197 (C2), N301 (V3), N442 (C4) and N625 (gp41) in the same degree of neutralization of the pseudo virus makes the false virus to some HIV-1 positive serum, the anti CD4 binding site antibody B12 and the gp41 antibody and more sensitive. 10 sensitivity increased, but had no effect on the sensitivity of HIV-1 positive serum. The deletion of.N355 (C3) and N463 (V5) increased the sensitivity of the virus to 2F5 and 4E10.
According to the transformation results of the previous parts, we selected 7 sites with significant enhancement of virus neutralization activity, combined with the glycosylation sites of V5 (N463, N466) and C3 (N339) region and the deletion of N448 near N442. The combined transformation was carried out on FE according to different combinations, and a total of 7 joint mutant pseudo viruses were obtained, all of which were significantly reduced except 197M-4. The neutralization characteristics of 6 strains of the virus were found to be more sensitive than the original strain S939, the transformation strain FE and the modification of the single glycosylation site. The combined mutant strain showed strong sensitivity to the mAb 2F5,4E10 and B12, as well as most of the positive serum.
In this experiment, we found some sites that could significantly affect the ability of virus infection by changing the 55 amino acid sites, and found some sites that could significantly enhance the neutralization characteristics of the virus. Through the combined transformation, several strains of the HIV-1 neutralization and positive sera were obtained, which were HIV-1. It provides a basis for optimization of membrane proteins and selection of candidate membrane antigens.

【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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