Clade7．2H5N1亚型禽流感重组病毒疫苗株的构建及免疫效力评价

Construction and immunity evaluation of recombinant vaccine strain of clade7.2 H5N1 avian influenza virus

致病性禽流感病毒(AIV)编码HA抗原基因的不断进化,进而出现抗原性变异是导致原有疫苗无法提供完全免疫保护的主要原因,因此需要构建并更新疫苗株。本研究在系统分析AIV病毒抗原性基础上,采用反向遗传操作系统,以A/Puerto Rico/8/34(PR8)的内部基因为骨架,以clade7.2 H5亚型A/chicken/He Bei BD/SC007/2012(CK/BD/2012)的c DNA为模板经SOE-PCR扩增,将高致病性AIV的HA基因编码裂解位点序列336PQIEGRRRKR348突变为低致病性特征的336PQRETR343序列,以A/chicken/Shanxi/2/2006(CK/SX/2/2006)作为NA基因的供体,构建clade7.2 AIV疫苗候选株r PR8-BD/2012,并对其进行生物学特性和免疫原性评估。结果显示,该疫苗候选株对鸡胚无致病性,能够在鸡胚中高滴度生长(9 log2),静脉内接种致病指数(IVPI)为0,对雏鸡无致病性。将r PR8-CK/BD/2012作为种毒,按照常规方法制备灭活疫苗,免疫4周龄SPF雏鸡,免疫3周后对亲本强毒株的HI平均抗体效价达7.2 log2;采用亲本强毒A/chicken/He Bei BD/SC007/2012和同分支异源强毒株A/chicken/He Bei/SD001/2013以105 EID50剂量攻击,免疫组均能够得到完全保护。以上结果表明r PR8-CK/BD/2012疫苗候选株具有鸡胚适应性、生物安全性和良好的免疫原性,可以作为有效防控clade7.2 H5N1 AIV的疫苗株。

Outbreaks of avian influenza （AI） pose significant health threats to animals and human beings due to the constant evolution or mutation in hemagglutinin （HA） gene of highly pathogenic AI virus （AIV） and resulting in the available vaccines were unable to provide complete immune protection. In order to construct the candidate vaccine strain, the recombinant virus of rPR8-CK/BD/2012 was generated using the reverse genetic system. The virus contained modified HA gene of clade7.2 H5N1 AIV A/chicken/HeBei BD/SC007/2012 （CK/BD/2012） which the encoding sequence for cleavage site of 336PQIEGRRRKR348 was replaced with low pathogenic motif of 336PQRETR343 and NA gene of A/chicken/Shanxi/2/2006 （CK/SX/2/2006） based on the internal genes of A/Puerto Rico/8/34 （PR8） as the backbone. The identifications showed that the recombinant virus was not lethal to chicken embryos and capable to replicate efficiently in chicken embryos with a titer around 9log2. In addition, the intravenous pathogenicity index （IVPI） of the virus was 0, which showed the virus was no pathogenicity to chickens. Furthermore, 4-week SPF chickens were vaccinated twice with inactivated virus and the average titer of HI antibody was 7.2log2 after 3 weeks post primary immunization, and the immunized chickens were completely protected when challenged with the homologous and heterologous clade7.2 H5N1 A/Vs. These results suggest that the rPR8-CK/BD/2012 is potential to be used as the efficient candidate vaccine against clade7.2 H5N1 AIVs in chicks.