H9N2 AIV HA蛋白S145N变异毒株的抗原性和免疫原性

Antigenicity and immunogenicity of H9N2 avian influenza virus strains with single-amino-acid substitution of asparagine for serine at position 145(S145N) in the HA protein

在研究1998-2008年中国H9N2亚型禽流感病毒(AIV)分离株HA基因的进化时,发现在25个毒株中有2个致病性最强的毒株因HA基因第145位氨基酸的突变导致产生1个潜在的糖基化位点,从而使其不与单抗H6、F6等反应。为进一步探究这类变异毒株HA基因变异对H9亚型AIV的抗原性和免疫原性的影响,本试验对12株HA蛋白S145N变异的H9N2AIV进行了交叉中和试验和交叉攻毒试验。结果显示,不同H9N2S145N变异株与疫苗株间在抗原性上变化不大,或无显著差异(0.5≤R≤0.67)。但参照现有的H9灭活疫苗效力检验方法对HP疫苗免疫鸡进行攻毒,用HP株攻毒对照组0/5保护,免疫组保护≥9/10,达到了H9灭活疫苗质量标准要求;但用S145N变异株N3攻毒,仅保护2/10～6/10,且随免疫量剂量的增加,抗体水平的提高,攻毒保护也依次升高。对H9变异株疫苗(N1、N2、N3、N8)免疫鸡用N3攻毒,仅保护2/5～4/5,N3同源抗体也不能有效地阻止其攻毒后的排毒。用N3、N6 2个变异株交叉攻毒,采用与疫苗株攻毒相同的剂量作攻毒试验也得到类似结果。表明高于6log2的抗体能抵抗疫苗株和大多数流行毒株攻毒后的排毒,但不能抵抗S145N变异株攻毒后的排毒。这类毒株免疫原性上的变化与病毒HA基因的变异密切相关。因HA基因145～147aa位增加了1个NGT,导致三维空间构象的变化,并影响其邻近的受体结合位点,从而使这类毒株致病性提高,免原性发生改变。虽然这一类变异株或免疫逃逸毒株仅占当前流行毒株总数的5%～7%,但在强大的免疫压力和自然选择下有可能逐步成为优势毒株,造成更大的危害,这为该病的防控提出了新的挑战。

HA gene evolution of H9N2 subtype avian influenza virus（AIV） from 1998 to 2008 in China was evaluated previously and the more virulent 2（of 25 strains） with the generation of a potential glycosylation sites due to single-amino-acid substitution of asparagine for serine at position 145（S145N） in the HA protein do not reacted to the corresponding monoclonal antibody H6 and F6 against H9N2 AIV.To further explore the influence of HA gene mutation of this kind of variation strains on H9 subtype AIV antigenicity and immunogenicity,12 strains with HA S145N variants of H9N2 AIV were studied by crossing-hemagglutinin inhibition test（HI）,cells neutralization test and challenging test.The results indicated antigenicity between different H9N2 S145N variation and vaccine strains displayed no significant difference with antigen correlation（R） from 0.5 to 0.67.In the light of the authorized H9 inactivated vaccine efficacy test method,chicken vaccinated with HP vaccine were challenged with HP strain and the protected rate in the vaccine group was greater than or equal to 9/10,while 0/5 in the control.However,only 2/10-6/10 birds were protected when challenged with the N3,S145N mutation.At the same time,the protected rate increased in turn with increasing immune dose,and raised levels of antibodies.When chicken immuned with H9 variant strain vaccine（N1,N2,N3,N8） was attacked with N3,only 2/5-4/5 birds could be protected,and N3 homologous antibodies could not effectively prevent virus shedding.The similar results could be obtained in the cross challenging test with N3,N6 two variant strains.It was suggested that more than 6 log2 antibodies induced by the vaccine strain could be against virus shedding in birds challenged with HP vaccine strain and the most popular strains,which did not prevent virus shedding against HA S145N variants of H9N2 AIV.Immunogenicity change of this kind of strains was closely related to the variation of HA gene of the viruses.A potential glycosylation sites,NGT,due to single-amino-acid substitution of asparagine for serine at position 145（S145N） in the HA protein resulted in a three-dimensional space conformational change in the HA protein of the virus,and it had an effect on the near receptor binding site so that this kind of viruses displayed more pathogenic（see another newspaper） and alteration of immunogenicity.Although this kind of variation or immune escape strains accounted for only 5%-7% of the current epidemic strains,but under the stress of both strong immune and natural selection these viruses may gradually become predominant strains,causing even more damage in poultry.This has brought new challenge for prevention and control of AI.