H5亚型禽流感病毒同义HA的设计及其免疫特性研究

Current H5N1 avian influenza virus (AIV) vaccines in China provide good protection against disease in poultry, and the vaccine seed has to be updated timely due to antigenic drift of the virus. While, the challenge, including vaccine seeds development behind the emergence of dominant strains, neglecting the non-dominant strains and the dilemma between the cost of the vaccine and the trivalent vaccine against the coexist of two more antigenic drift virus in poultry in the same district, influenced the vaccination efficacy against the H5 AIV in China. These limitations clearly highlight the importance of developing a universal avian influenza vaccine. Consensus HA of AIV could induce neutralization antibody against AIV and be a potential antigen for development of universal vaccine. In this study, we will analysis the HA sequences of H5 influenza viruses available in the GenBank and our lab between 1996 and 2016, and design and identify a series of consensus HA for different clade virus to select the consensus HA that could induce broad neutralization antibody based on HI test and neutralization test. We further construct ressortant AIV vaccine seed and confirm the immune properties of consensus HA through animal expriments. Finally, we will clarify the key section or epitope of consensus HA for induction of the broad neutralization antibody against different antigenic drift virus by molecular cloning technique. This project will help develop the H5 universal vaccine against different antigenic drift virus in poultry and elaborate the molecular base for induction of the broad neutralization antibody against different antigenic drift virus.

尽管我国H5亚型禽流感防控疫苗随着病毒进化，不断地更换疫苗毒株，但应对新变异毒株的疫苗总是滞后于流行毒株的出现，而且也容易忽视潜在的流行毒株，同时局部区域多个抗原群同时存在，给防控疫苗的研制、生产和应用都带来挑战。通用疫苗是应对这一挑战的理想策略，以同义HA为靶抗原具有研制通用疫苗的潜在优势。本研究在系统分析1996年至2016年H5亚型不同clade病毒HA基因基础上，设计针对不同clade病毒的一系列同义HA并进行鉴定，筛选出可诱导H5亚型广谱性中和抗体的同义HA，接着构建出表达同义HA的重组疫苗种毒株，通过动物实验，验证同义HA诱导抗体的广谱性，最后通过定点突变技术，确定其诱导广谱性中和抗体的关键区域或位点。本项研究的开展有助于研制针对H5亚型多个抗原群AIV的通用疫苗，具有巨大的应用价值，同时有助于完善对HA基因诱导H5亚型广谱性中和抗体分子机制的认识，具有重要的学术意义。

针对严重危害我国养禽业的H5亚型主流分支（2.3.4.4分支和2.3.2.1分支）禽流感病毒（Avian influenza virus, AIV）设计出同义HA基因序列，并构建出同义HA重组H5亚型AIV株(rConsH5-1)，以该毒株研制的灭活疫苗具有一定广谱性，能够有效对多个分支H5亚型AIV攻击提供完全免疫保护。但随着AIV不断进化，近两年出现新的亚分支（2.3.4.4d）病毒与已设计构建的H5亚型禽流感同义HA疫苗株存在明显抗原性差异，该同义HA基因的重组禽流感疫苗不能有效抵御该分支H5亚型AIV攻击。为进一步探索设计同义HA基因疫苗的可能性，我们针对近期流行的H5N6亚型2.3.4.4d分支和H5N1亚型2.3.2.1分支AIV，设计同义HA基因，同时从家禽中监测到的H5亚型AIV中筛选毒株，进行灭活疫苗种毒株的构建；构建成功的重组H5N1亚型Re-11株和Re-12株疫苗株均具有相对广谱的免疫保护效果，Re-11株疫苗能够有效抵御近期监测到的H5亚型2.3.4.4b,c和d分支AIV攻击，Re-12株疫苗能够有效抵御H5亚型2.3.2.1d和2.3.2.1e分支AIV攻击；但该2株疫苗株与新近我国野鸟出的H5N8亚型AIV具有明显的抗原性差异。初步推测HA基因200E和205K位点可能是诱导部分广谱性抗体的关键位点。对我国1996年～2020年出现的不同分支H5亚型AIV抗原性系统分析发现，病毒抗原性持续发生变异，难以预测病毒抗原性变异的方向。综上所述，基于本研究策略和手段，难以设计和研制出对当前和将来出现的AIV均能够提供完全广谱性保护的疫苗株，为禽流感通用疫苗研究提供参考；本研究构建的H5亚型Re-11株和Re-12株疫苗株具有重要应用价值，已用于我国H5亚型禽流感灭活疫苗和诊断试剂的研制和生产；提示禽流感疫苗种毒株和国际上使用的人流感疫苗种毒株一样，需要根据病毒抗原变异情况进行及时更新，才能持续有效保障禽流感疫苗的免疫防控效果。