应用酵母表面展示技术筛选可诱导HIV广谱高效中和抗体的膜蛋白抗原的研究

Since the discovery of HIV-1 in the early 1980s, considerable effort has been exerted to develop a prophylactic vaccine. Recent researches suggest that the key of the development of an effective HIV vaccine is to elicit broadly neutralizing antibodies. Several broadly neutralizing antibodies have been discovered and their corresponding epitopes also have been studied. However, due to the poor exposure of these epitopes on the envelope proteins, all the effort for developing a prophylactic vaccine is failed. Thus, to enhance the exposure of HIV-1 neutralization epitopes by recombination or mutation and screening through the analysis of their affinity with broadly neutralizing antibodies, would be a reasonable method for obtaining a HIV-1 vaccine candidate. In this study, the envelope proteins gp160, gp120 and gp41 will be displayed on yeast cell surface and a mutation library will be constructed by using Error-prone PCR. The high-throughout screening of the library using FACS will be performed by analyzing the affinities with broadly neutralizing antibodies. The mutations displayed on yeast cells which were separated will be purified and studied by immunization and spectroscopic studies. Finally immunogen candidates for designing a reasonable HIV-1 vaccine will be gained by this study.

HIV疫苗是当今人类面临最难攻克的疫苗之一。现有的疫苗抗原无法诱导出广谱高效的中和抗体，成为目前有效疫苗设计的一个主要障碍。HIV完整膜蛋白、重组蛋白以及病毒样颗粒，存在抗原表位暴露程度低、非特异表位竞争、表位暴露存在时效性等缺点，不能成为理想的疫苗候选抗原。而通过重组、突变等手段对HIV包膜糖蛋白进行序列和结构修饰，并以其对广谱中和抗体的亲和程度强弱进行筛选，获得保守序列部位抗原表位高度暴露的抗原蛋白，是获得理想疫苗候选抗原的有效手段。我们将首次应用酵母表面展示技术，构建HIV病毒的gp160、gp120和gp41蛋白的序列和构象突变文库，利用流式细胞仪高通量筛选对现有广谱中和抗体具有高亲和力的抗原，通过优化免疫策略并结合HIV假病毒中和试验评价突变抗原诱导中和抗体的能力，同时应用色谱学方法对筛选出的抗原蛋白进行空间构象分析，从而希望为HIV疫苗临床研究中广谱高效的中和抗原设计提供依据。

HIV疫苗是当今人类面临最难攻克的疫苗之一。现有的疫苗抗原无法诱导出广谱高效的中和抗体，成为目前有效疫苗设计的一个主要障碍。HIV完整膜蛋白、重组蛋白以及病毒样颗粒，存在抗原表位暴露程度低、非特异表位竞争、表位暴露存在时效性等缺点，不能成为理想的疫苗候选抗原。而通过重组、突变等手段对HIV包膜糖蛋白进行序列和结构修饰，并以其对广谱中和抗体的亲和程度强弱进行筛选，获得保守序列部位抗原表位高度暴露的抗原蛋白，是获得理想疫苗候选抗原的有效手段。.在本课题中，我们应用酵母表面展示技术，构建了HIV病毒的gp120和gp41蛋白的表面展示酵母，并通过易错PCR建立了gp41的突变文库并成功展示再酵母表面。我们利用流式细胞仪高通量筛选对现有广谱中和抗体4E10和2F5具有高亲和力的展示了突变型gp41的酵母菌株，并通过动物免疫，确认了蛋白展示酵母的免疫原性，通过血清学实验，验证了经过三次免疫的豚鼠的血清当中有较高滴度的针对4E10和2F5的抗原表位的抗体，而血清中抗体对假病毒的中和能力需要进一步获得验证。在接下来的实验中我们将通过序列分析和结构分析，对我们的抗原进行进一步的分析与评价。