基于包膜蛋白gp41的HIV免疫原设计

Human Immunodeficiency Virus (HIV) is the etiological agent of Acquired Immune Deficiency Syndrome (AIDS). Once infected by HIV, the individuals can't clean up the virus and most of the individuals will lose the control to the virus eventually even with extensive theraputic intervention. An effort to develop a sterile vaccine against AIDS has been initiated from the beginning of HIV identification. While with 30 years passed, most of trials for vaccine development have failed due to the high mutation rate and effective immune evasion of the virus. The vaccine against EIAV, which is developed more than 30 years ago by Chinese scientists, have been used as the reference of HIV vaccine study based on the similar life cycles between two viruses. The glycoprotein gp45 in EIAV plays similar roles as gp41 in HIV by forming a hetero-dimer with glycoprotein gp90 (ortholog of HIV gp120) on surface of viral particles, mediating the membrane fusion during viral invasion. The high conservation on sequence and low extent of glycosylation make gp45/gp41 a good target for vaccine development to induce broad spectrum neutralizing antibodies. Actually, several identified antibodies against gp41 in HIV, such as 2F5, 4E10 and 10E8, have been proven to neutralize HIV with wide spectrum. By comparasion of gp45 of wild type and vaccine strains of EIAV, a critical point mutation is identified, which is highy relevant to the evolution status of EIAV during viral attenuation. From the crystal structure of EIAV gp45 we determined recently, this point mutation is found to be localized in a/d position in heptad repeat, where natural mutation rarely occures. Our biochemical analysis reveals this type of mutations alter the stability of gp45 and influence the membrane fusion induced by viral infection. In this project, by multiple techniques such as crystallography, circular dichroism, and dectection of viral infection, we wish to uncover the underlying mechanism of EIAV vaccine derived from gp45, especially the importance of point mutations in a/d position in heptad repeat. The corresponding finding will be employed in HIV gp41 and the consequence on HIV replication and attenuation will be quantitated. By refering to EIAV, we hope our study could shed new light on the current stagnation on HIV vaccine development.

由于艾滋病病毒（HIV）的高突变率和免疫逃逸等特征，疫苗是对付艾滋病的最有效手段。目前仍然没有成功的艾滋病疫苗，根本原因是目前所用的免疫原在体内不能诱导出高效广谱的中和抗体，这需要我们有更有效的免疫原设计。马传贫病毒（EIAV）与HIV同属慢病毒属，有相似的基因组结构和生活周期。我们已有的数据显示在EIAV疫苗株中，gp45（相当于HIV gp41）异于野生株的关键位点定向突变可影响包膜蛋白的稳定性和病毒的侵染能力，但具体的EIAV gp45疫苗化机制还需要进一步阐明。本研究希望借助于EIAV疫苗研制成功的经验，针对HIV的包膜蛋白gp41,借助于结构、生化和病毒侵染等技术，仔细地比较EIAV野生株和疫苗株gp45的异同点，寻找其疫苗化基础，并将发现移植到HIV上进行验证，力求找到基于HIV gp41的免疫原设计方法，为目前停滞不前的艾滋病疫苗研制提供新的思路。

目前针对艾滋病的主要问题就是我们没有很好的HIV疫苗，这与我们对HIV的疫苗化机制理解不够有关。我们通过对已经获得疫苗的EIAV进行深入比较研究，设计多个关联突变体，研究慢病毒属多种病毒的gp41/gp45蛋白的结构、稳定性和它们对病毒侵染能力的影响，了解EIAV的疫苗化机制和应用于HIV疫苗研制的手段，从而能够设计有效诱导产生高效广谱抗体的免疫原，并加深我们对这些病毒致病机制的理解。目前已经解析HIV gp41，EIAV gp45，HIV gp120/CD4 complex等多个蛋白结构，并通过结构的分析，为免疫原的改造提出了方案。对EIAV gp45野生株和疫苗株的晶体结构比较发现，一个在HR1 heptad repeat区d位置从疏水到极性氨基酸的突变与EIAV的疫苗化紧密相关。我们证明这种突变提高了病毒对温度的敏感性。我们将类似的突变在HIV上进行了验证，据此提出了在HR1区进行突变来尝试HIV疫苗株研发的设计思路。对HIV CRF07 gp120进行了大量的构建、表达、以及去糖化处理，得到了HIV CRF07 gp120/CD4复合物的晶体，据此发现了一个新的受体结合表面。获得高稳定性gp140三体免疫原，与原有的gp140三体相比，均一性更好，抗原反应性更高，且针对目前中国流行株，非常适合作为免疫原用于艾滋病亚单位疫苗或多价联合疫苗的研发。这些研究成果对我们目前抗艾滋病疫苗的研发具有重要意义。