HIV-1膜抗原改造及免疫原性评价

发布时间：2019-06-24 20:59

【摘要】： 人类免疫缺陷病毒(HIV)的膜蛋白(Env)位于病毒的表面，是中和抗体作用的主要靶位，但野生型Env在机体免疫压力下易发生免疫逃逸，这可能与其复杂的构象和表面寡糖链有关。本课题试图通过删除寡糖链、复杂氨基酸侧链及高变区，来暴露目前已知的及CD4i(CD4 induced)区潜在的中和抗体表位，以期诱导广谱有效的中和抗体，同时将体液免疫与细胞免疫结合起来，力图寻找一种能够诱导均衡、有效的体液和细胞免疫反应的免疫原。本课题根据HIV-1 Env三维结构中四种广谱中和单抗的结合表位及前人经验，设计四组突变，通过四组之间不同搭配设计五种突变组合：M2、M5-1、M5-2、M4和M7。在原始株及各突变组合的基础上做V1V2区的删除，设计另六种突变组合：dWt、dM2、dM5-1、dM5-2、dM4和dM7。通过环形诱变，DpnI筛选的方式得到这11种突变体，酶切初步鉴定，测序确定突变结果。提取原始及突变质粒并转染293T细胞，经WesternBlot检测体外表达情况。原始及突变质粒免疫家兔，用五个不同厂家ELISA抗体检测试剂盒检测血清抗体水平，用假病毒单周期感染中和试验检测其中和抗体水平，将ELISA抗体检测结果与中和试验检测结果做相关性分析。原始及突变质粒免疫小鼠，取血分离血清，检测中和抗体水平；取脾分离脾白细胞，以SHIVchn19肽库和单肽Env34作为刺激物，用IFN—γELISPOT方法评价细胞免疫反应。将原始及改造后env基因克隆入pcDNA~(TM)3.1 D／V5-His-TOPO(?)载体，WB检测体外表达情况，与pSG3~(△Env)共转染293T细胞，收集上清感染TZM-bl细胞，检测改造对功能性假病毒形成的影响。 WB结果显示D-GPEi的原始株及各突变克隆，以及克隆入pcDNA~(TM)3.1 D／V5-His-TOPO(?)载体的原始及突变env在体外都能正常表达，且表达量没有明显区别，突变株的Env条带较原始株泳动速率提高，进一步佐证了改造结果。家兔血清ELISA检测结果表明，电穿孔介导DNA免疫家兔，与单纯DNA免疫相比，其所用质粒量少，免疫周期短，抗体水平高。不同厂家抗体检测试剂盒检测家兔四次免疫后血清中抗体水平，检测的结果数值上虽然存在差异，但总体的趋势相似。Wt、N2010、G2、M2、M5-1和dM5-1组抗体水平较高，但各组与Wt组比较没有明显优势。对于同一质粒免疫的两只家兔，其抗体水平不一致。中和试验结果显示，Wt、N2010、G2、M2和dM2中和抗体水平较高，但同一组两只家兔中和抗体水平不一致。将ELISA抗体检测结果与假病毒中和试验检测结果，做相关性分析，两者之间没有统计学意义上的相关性，但对于同一组两只家兔，其中和抗体水平高的总抗体水平也高。小鼠ELISPOT结果显示，与原始组相比，，改造组细胞免疫反应大部分是降低的，包括M5-1、dM5-1、M5-2、dM5-2、M7、dM7、dWt、M4和dM4，其中与Wt组相比降低有统计学意义(p＜0.05)的包括dM5-1、M5-2、M7、dM7和dM4，其余各组与Wt组相比虽有降低但无统计学意义。改造组M2、dM2、G2与Wt组相比细胞免疫反应增高，其中M2组的增高有统计学意义(p＜0.05)。针对四个肽库的细胞免疫反应由强到弱：肽库1＞肽库3＞肽库2＞肽库4，肽库1在四肽库总反应中所占比例最高，＞75％。各肽库反应与总反应的相关性由高到低肽库1＞肽库3＞肽库2＞肽库4，针对Env34的细胞免疫反应与总反应有较高的相关性，相关系数为0.942(p＜0.01)。在Wt和74-2两假病毒中和试验中，dWt、M2、M5-2、M5-1、dM7、N201Q组与Wt组相比，其中和抗体水平有增高趋势，只有dWt组和M5-2组的增高在两假病毒试验中都有有统计学意义(p＜0.05)，此外，在Wt假病毒中和试验中，M5-1组的增高有统计学意义，在74-2假病毒中和试验中，M2和N2010组的增高有统计学意义。从两假病毒中和试验结果可以看出，dM2、dM5-2、M4、dM4和M7组中和抗体水平与Wt组相比有降低趋势，其中只有在Wt假病毒中和试验中，M4、dM4组的降低有统计学意义。单周期感染实验结果显示，除G2改造外，其它改造都使功能性假病毒形成能力大为下降。从动物实验结果可以得出：不同改造对Env免疫原性的影响不同，M2组改造使Env诱导细胞与体液免疫的能力都有一定的提高；dWt组改造，使诱导体液免疫的能力有一定幅度提高，同时诱导细胞免疫的能力没有明显变化；N2010、M5-1、M5-2和dM7虽然诱导细胞免疫能力有所下降，但诱导体液免疫能力有一定程度提高；dM2和G2诱导体液免疫能力降低，但诱导细胞免疫能力有小幅度提高或基本不变；dM5-2、M4、dM4和M7组改造使其诱导细胞及体液免疫的能力都降低。从单周期感染实验结果可以得出：G2(N620Q，N632Q)改造不影响功能性假病毒的形成，而F174A和N2010两个单点的改造使Env丧失了形成功能性假病毒的能力。
[Abstract]:The membrane protein (Env) of the human immunodeficiency virus (HIV) is located on the surface of the virus, is the main target for neutralizing the neutralizing antibody, but the wild-type Env is susceptible to immune escape under the body's immune pressure, which may be associated with its complex conformation and the surface oligose chain. The present subject attempts to expose potential neutralizing antibody epitopes in the currently known and CD4i (CD4-induced) region by removing the oligosaccharide chain, the complex amino acid side chain and the high-transformation region, with a view to inducing a broad-spectrum and effective neutralizing antibody, while the humoral immunity is combined with cellular immunity, The invention seeks to find an immunogen capable of inducing a balanced, effective humoral and cellular immune response. According to the binding epitopes of four broad-spectrum neutralizing monoclonal antibodies in the three-dimensional structure of HIV-1 Env and the previous experience, four groups of mutations were designed, and five mutation combinations were designed by different collocations among the four groups: M2, M5-1, M5-2. on the basis of the original plant and each mutation combination, the deletion of the V1V2 region is carried out, and the other six mutation combinations are designed: dWt, dM2, dM5-1, dM5-2 and d M4 and dM7. The 11 mutants were obtained by ring-directed mutagenesis and DpnI screening, and the enzyme was initially identified and sequenced. The mutation results were determined. The original and mutant plasmids were extracted and the 293T cells were transfected and tested by Western Blot In vitro expression, the original and the mutant plasmid were immunized with rabbit, the serum antibody level was detected by using five different manufacturers of ELISA antibody detection kit, and the ELISA antibody detection result is compared with the neutralization test detection result by using the false virus single-cycle infection and the test to detect the antibody level. Carry out the correlation analysis. The original and mutant plasmid was used to immunize the mice, and the serum, the serum and the antibody level were isolated from the blood, and the spleen and the white blood cells were isolated from the spleen, and the SHIVchn19 peptide library and the single peptide Env34 were used as the stimulus, and the method was evaluated using the method of the method of the IFN-CREELISPOT. Cellular immune response. The original and modified env gene was cloned into pcDNA ~ (TM) 3.1D/ V5-His - In vitro expression of TOPO (?) vector and WB, 293T cells were co-transfected with pSG3 ~ (+ Env), and the supernatant was collected to infect TZM-bl cells. The effects of virus formation. WB results showed that the original strain of D-GPEi and the mutant clones were cloned and cloned into pcDNA ~ (TM) 3.1D / The original and mutant env of the V5-His-TOPO (?) vector can be expressed in vitro, and the expression amount is not obviously different, and the Env band of the mutant strain is higher than that of the original plant. The results of the modified rabbit serum ELISA showed that the electroporation-mediated DNA-immunized rabbit, compared with the pure DNA immunization, used the plasmid quantity. in that invention, the antibody level in the serum after four immunization of the rabbit is detected by the antibody detection kit of different manufacturers, and the result value of the detection is There was a difference, but the overall trend was similar. The levels of antibodies in Wt, N2010, G2, M2, M5-1, and dM5-1 groups were high, but each There was no significant advantage between the group and the Wt group. For the same plasmid, it was immune to the same plasmid. In both rabbits, the antibody levels were not consistent. The neutralization test showed that the levels of neutralizing antibodies in Wt, N2010, G2, M2, and dM2 were high, but the same group The level of the neutralizing antibodies in both rabbits was not consistent. The results of the detection of the ELISA antibody and the test results of the test and the correlation analysis were not statistically significant, but for the same group of two rabbits, and The high level of the total antibody was also high in the antibody level. The mouse ELISPOT results showed that the majority of the cell immune response in the modified group was reduced compared to the original group, including M5-1, dM5-1, M5-2, dM5-2, M7, dM7, dWt, M4, and dM4, with a statistically significant decrease compared to the Wt group (p <0.05), including dM5-1, M5. -2, M7, d7 and dM4, and the remaining groups and the Wt group In the modified group M2, dM2, and G2, the immune response of M2, dM2, and G2 was higher than that of the Wt group. Statistical significance (p <0.05). The cellular immune response to the four peptide libraries is from strong to weak: peptide library 1> peptide library 3> peptide library 2> peptide library 4, peptide library 1 in total anti-peptide library The correlation between the reaction of each peptide library and the total reaction is from high to low peptide library 1> peptide library 3> peptide library 2> peptide library 4, and the cellular immune response to Env34 has a high correlation with the total reaction, and the correlation coefficient is The levels of dWt, M2, M5-2, M5-1, d7 and N201Q were higher than that of the Wt group. Significance (p <0.05), and in addition, in the Wt pseudovirus and in the test, the increase in the M5-1 group was statistically significant, in the 74-2 pseudovirus and in the test, M2 and N2 The elevation of the 010 group was statistically significant. It can be seen from both the two pseudoviruses and the test results that the levels of dM2, dM5-2, M4, dM4, and M7 have a tendency to decrease as compared to the Wt group, where only in the Wt pseudovirus and in the test, M4, The decrease of dM4 group is of statistical significance. The results of single-cycle infection show that, in addition to the transformation of G2, other modifications make work The results of animal experiments show that the effects of different modifications on the immunogenicity of Env are different, and the transformation of M2 group has a certain increase in the ability of Env to induce the immunity of Env, and the dWt group The ability of inducing humoral immunity is improved, and the ability of inducing cell immunity is not changed. N2010, M5-1, M5-2 and d7 have a certain effect on the immune competence of the cells. Increased degree of immunity; dM2 and G2 induce a decrease in humoral immunity, but the induction of cellular immunity has a small increase or substantially the same; dM5-2, M4, dM4, and M7 The ability of group modification to induce cell and humoral immunity decreased. The results of single-cycle infection showed that the transformation of G2 (N620Q, N632Q) did not affect the formation of functional pseudovirus, while F174A and N2010 were both single-point.
【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】：2007
【分类号】：R392

【共引文献】