基于马赛克HA序列的H9亚型禽流感灭活疫苗的免疫效力分析

H9亚型禽流感在我国家禽中广泛流行，给养禽业造成巨大经济损失的同时，也严重威胁着公共卫生安全。H9亚型禽流感病毒（avian influenza virus，AIV）具有高度遗传变异性，导致流行株和疫苗株之间抗原匹配性差，从而影响疫苗的临床保护效果，急需研发一种高效、具有交叉保护性的通用型H9亚型禽流感疫苗。马赛克疫苗是针对遗传多样性病原体设计，通过整合所有抗原序列获得一条抗原表位覆盖最广泛的嵌合蛋白，并制备疫苗。本研究参考mosaic疫苗设计原则，设计、优化并合成了一条H9亚型禽流感病毒的mosaic血凝素（hemagglutinin，HA）基因序列，采用反向遗传操作技术，以H1N1亚型流感病毒PR8株为骨架，以mosaic H9HA序列替换PR8株的HA片段，获得重组病毒rPR8-HAm/H9。将其制备为灭活疫苗并免疫SPF雏鸡，监测抗体水平、攻毒保护效果，评价其交叉保护效果。结果表明，重组病毒rPR8-HAm/H9灭活疫苗免疫SPF雏鸡，可诱导机体产生较高水平的HI抗体和中和抗体，可显著抑制攻毒后病毒的脱落，对H9N2 AIV JM0305株的攻毒保护率为80%。rPR8-HAm/H9灭活疫苗可以对异源H9N2 AIV JM0305株产生较好的交叉攻毒保护，为研发基于马赛克技术的禽流感通用疫苗提供了前期基础。

Evaluation of Immune Efficacy of H9 Subtype Avian Influenza Virus Inactivated Vaccine Based on Mosaic HA Sequence

H9 subtype avian influenza is widely prevalent in poultry in China, which has caused huge economic losses to the poultry industry and also seriously threatened the public health safety. The high genetic variability of H9 subtype avian influenza virus (AIV) leads to poor antigenic matching between the circulating and vaccine strains, thus affecting the clinical protective effect of the vaccine. Therefore, it is urgent to develop a highly effective cross-protective vaccine against H9 subtype AIV. Mosaic vaccine is designed for the genetic diversity of pathogens by integrating all antigenic sequences to obtain a mosaic protein with the most extensive epitopes coverage, and to generate vaccine. In order to develop an efficient and universal H9 subtype avian influenza vaccine, a mosaic HA sequence of H9 AIV HAm/H9 was designed, optimized and synthesized according to the principles of mosaic vaccine. Utilizing the reverse genetic technology, a recombinant virus rPR8-HAm/H9 was constructed and rescued by replacing the HA segment of influenza virus PR8 strain (H1N1) with the HAm/H9 gene. The SPF chickens were immunized with the inactivated rPR8-HAm/H9 virus, and the effect of cross-protection was evaluated by detecting the antibody titer, the protection rate and viral shedding. The recombinant virus rPR8-HAm/H9 could induce high level of HI antibody and neutralizing antibody, as well as reduce virus shedding, and the protection rate against H9N2 AIV JM0305 strain was 80%. Our results showed that rPR8-HAm/H9 inactivated vaccine could provide efficient cross-protection against H9N2 AIV strain JM0305, and provided a preliminary basis for the research and development of universal vaccine for avian influenza based on mosaic technology.