Generation of Live Attenuated Novel Influenza Virus A/California/7/09 (H1N1) Vaccines with High Yield in Embryonated Chicken Eggs
Generation of Live Attenuated Novel Influenza Virus A/California/7/09 (H1N1) Vaccines with High Yield in Embryonated Chicken Eggs
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DOI：
10.1128/jvi.02106-09
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发表时间：
2010-01-01
影响因子：
5.4
通讯作者：
Jin, Hong
中科院分区：
文献类型：
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作者：
Chen, Zhongying;Wang, Weijia;Jin, Hong
采用反向遗传方法，获得了多个甲型流感病毒/加州/7/09 (H1N1) （CA09）减毒活疫苗变体，这些变体具有来自CA09病毒的血凝素（HA）和神经氨酸酶（NA）基因片段，以及来自甲型流感病毒/安娜堡/6/60 （H2N2）病毒的6个内部蛋白基因片段。重组病毒在有胚胎的鸡蛋中复制相对较差。为了提高病毒在鸡蛋中的生长，将表达CA09的HA和NA的重组体在MDCK细胞中传代，并分离出具有大斑块形态的变体。这些变异在鸡胚蛋中的复制率比亲本菌株的复制率高约10倍。序列分析表明，第119、153、154和186位的单氨基酸变化是MDCK细胞和卵子生长性能改善的原因。此外，在残基155处引入一种突变，这种突变先前被证明可以增强1976年猪流感病毒的复制，也显著提高了CA09病毒在鸡蛋中的复制。每个变异进一步评估受体结合偏好，抗原性，衰减表型和免疫原性。残基153、154和155的突变极大地降低了病毒的抗原性，这使得这些突变体不适合作为候选疫苗。然而，残基119和186的变化并不影响病毒的抗原性或免疫原性，因此有理由将它们纳入候选减毒活疫苗中，以预防目前流行的2009年猪源H1N1病毒。
Several live attenuated influenza virus A/California/7/09 (H1N1) (CA09) candidate vaccine variants that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the CA09 virus and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (H2N2) virus were generated by reverse genetics. The reassortant viruses replicated relatively poorly in embryonated chicken eggs. To improve virus growth in eggs, reassortants expressing the HA and NA of CA09 were passaged in MDCK cells and variants exhibiting large-plaque morphology were isolated. These variants replicated at levels approximately 10-fold higher than the rate of replication of the parental strains in embryonated chicken eggs. Sequence analysis indicated that single amino acid changes at positions 119, 153, 154, and 186 were responsible for the improved growth properties in MDCK cells and eggs. In addition, the introduction of a mutation at residue 155 that was previously shown to enhance the replication of a 1976 swine influenza virus also significantly improved the replication of the CA09 virus in eggs. Each variant was further evaluated for receptor binding preference, antigenicity, attenuation phenotype, and immunogenicity. Mutations at residues 153, 154, and 155 drastically reduced viral antigenicity, which made these mutants unsuitable as vaccine candidates. However, changes at residues 119 and 186 did not affect virus antigenicity or immunogenicity, justifying their inclusion in live attenuated vaccine candidates to protect against the currently circulating 2009 swine origin H1N1 viruses.