HIV-1的免疫逃逸与包膜蛋白的糖基化修饰的相关性研究

The N-glycans on HIV-1 gp120 surface could perform protections in part to human immune system. We are trying to research on the N-glycosylated positions in gp120 which could avoid triggering the strong neutralization reaction. For this purpose, we isolated the N-linked deglycosylation mutants from cultivations in vitro. The cultivation in vitro is lack of human inevitable immune suppression on N-glycans of HIV-1 envelopes in vivo. The cultivations in vitro were used to push the diversity and emergence of viral resistance in the presence of CCR5-antagonists. The selected deglycosylation mutants in vitro could be regards as the essential modified positions without human immune stress. Therefore, these glyco-positions are supposed to be taken the restrictions under the immune response in vivo, rather than confer contributions to gp120 functions. Our previous results supported this speculation. One of these mutants had been detected the viral replication and CCR5-antagonist resistance. The relative N-glycan removal could not confer the resistance to CCR5-antagonist and have preferential enhancement on viral replication capacity. Consequently, the effect of this N-glycan on viral immune escape might be one of its major works in this case. Following this speculation, we are planning to test the mutant sensitivity to neutralization of human positive serum and monoclone antibodies against gp120. Furthermore, the viral susceptibility to CCR5-antagonists, replication capacity, CCR5 binding affinity will be involved into considerations on the functions of the mutated N-glycans. Altogether, the key epitopes in gp120 covered by the N-glycans could be addressed in detail. This work is focusing on the N-glycan functions on viral immune escape. A novel strategy of the eliciting neutralization antibodies has been reported. The stronger neutralization reaction was induced by N443-removed gp120 in small animal model. This implied on the usage of our findings in vaccine design. And delineation on the epitopes covered by N-glycans is also helpful for understanding the immune response and viral evolution. Besides, the resistance relied on relative N-glycan modifications to CCR5-antagonists could be determined in our work. This is an ideal thought to research on the N-glycans in viral background contributing to the resistance against CCR5-antagonists.

HIV-1包膜蛋白可以糖基化修饰掩盖抗原位点，规避宿主免疫系统的中和反应。我们采用体外培养体系，加入CCR5拮抗剂类药物，筛选到6个糖基化缺失突变株。通过分析数据库中的序列，发现对应位点的糖基化在HIV-1的原始分离株中是比较保守的。因此推测，体外、无免疫压力的培养条件下，获得的糖基化缺失突变位点可能是受到机体免疫系统监视的位点，还可能是体外培养的适应性位点。我们拟通过测定突变体的免疫敏感性，综合考虑它们的药物抗性、CCR5募集能力、病毒复制力等数据，分析这6个N-糖基化突变对抗原表位覆盖的影响。分析该6个位点覆盖抗原表位的情况，将加深我们对HIV-1的免疫逃逸机制的理解，并且为抗体疫苗的设计、开发提供候选方案。此外，分辨HIV-1的包膜蛋白糖基化对CCR5拮抗剂类药物抗性的贡献，是国际上首例从N-糖基化角度考虑复杂的包膜蛋白背景对药物抗性的影响。

为了更准确的判断并且使用适宜的抗病毒药物，HIV-1病毒的抗性突变的基础研究需要大量的数据积累。本研究中，讨论了CCR5拮抗剂马拉若唯的抗性突变的产生，并且集中探讨了马拉若唯抗性相关的糖基化突变。在体外培养体系中，为了更容易的得到马拉若唯的抗性突变，我们构建了V3的突变体病毒库，序列测序后确定了多个特定的抗性突变位点，其中包括合理的包膜蛋白gp120的V3区的突变，特别的筛查得到的马拉若唯抗性突变更倾向于糖基化修饰位点发生突变。造成这样的结果可能有体外情况下没有免疫系统压制的原因，也有可能就是糖基化突变与改变的受体的结合方式有关。通过构建病毒表达载体，将各个突变位点嫁接入HIV-1 R5的JR-FL骨架中，测定病毒生长率，HIV-1各个包膜蛋白突变体均未发生明显的生长缺陷；相反的，在MVC处理的细胞中，突变体病毒的生长量有5-10倍的升高。继而，我们分析了MVC结合态的CCR5构象的变化，通过CCR5单克隆抗体识别CCR5在细胞表面暴露的抗原肽段的变化，推测胞外的CCR5第2和第3环区可能发生了比较显著的构象变化。并且，HIV-1包膜蛋白的C4区的糖基化突变位点的突变，与MVC-CCR5的结合态的结合能力有所升高，推测结合力的变化与HIV-1病毒的受体拮抗剂的抗性适应性突变相关，糖基化突变体更易于识别变化了构象的MVC-CCR5结合态的CCR5的第2和第3环区。国际上其他研究组的研究结果显示，V3区的突变体的CCR5拮抗剂突变体的受体结合位点存在于CCR5分子的游离的N-末端的部分肽段。但是，本研究中的结果显示CCR5的胞外域的构象变化使得第2和第3环区更加暴露，此外，结果中涉及的糖基化修饰位点的突变在CCR5拮抗剂抗性的产生的影响也尚未有相关的报道的可以参考的研究工作。