Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses

Virus growth during influenza vaccine manufacture can lead to mutations that alter antigenic properties of the virus, and thus may affect protective potency of the vaccine. Different reassortants of pandemic "swine" H1N1 influenza A vaccine (121XP, X-179A and X-181) viruses as well as wild type A/California/07/2009(H1N1) and A/PR/8/34 strains were propagated in embryonated eggs and used for DNA/RNA Illumina HiSeq and MiSeq sequencing. The RNA sequences of these viruses published in NCBI were used as references for alignment of the sequencing reads generated in this study. Consensus sequences of these viruses differed from the NCBI-deposited sequences at several nucleotides. 121XP stock derived by reverse genetics was more heterogeneous than X-179A and X-181 stocks prepared by conventional reassortant technology. Passaged 121XP virus contained four non-synonymous mutations in the HA gene. One of these mutations (Lys226Glu) was located in the Ca antigenic site of HA (present in 18% of the population). Two non-synonymous mutations were present in HA of viruses derived from X-179A: Pro314Gln (18%) and Asn146Asp (78%). The latter mutation located in the Sa antigenic site was also detected at a low level (11%) in the wild-type A/California/07/2009(H1N1) virus, and was present as a complete substitution in X-181 viruses derived from X-179A virus. In the passaged X-181 viruses, two mutations emerged in HA: a silent mutation A1398G (31%) in one batch and G756T (Glu252Asp, 47%) in another batch. The latter mutation was located in the conservative region of the antigenic site Ca. The protocol for RNA sequencing was found to be robust, reproducible, and suitable for monitoring genetic consistency of influenza vaccine seed stocks.

流感疫苗生产过程中的病毒增殖可引发病毒突变，进而改变病毒的抗原特性，最终可能影响疫苗的保护效力。本研究针对大流行"猪源"甲型H1N1流感疫苗毒株的3种重配株（121XP、X-179A与X-181），以及野生型A/California/07/2009(H1N1)和A/PR/8/34毒株，通过鸡胚进行病毒增殖，并采用Illumina HiSeq与MiSeq平台完成DNA/RNA测序。本研究将美国国家生物技术信息中心（National Center for Biotechnology Information，NCBI）公开的上述病毒RNA序列作为参考序列，用于比对本研究生成的测序读段。上述病毒的共识序列与NCBI存档的参考序列存在多处核苷酸差异。通过反向遗传学（reverse genetics）技术制备的121XP病毒种子批，其异质性高于采用传统重配技术获得的X-179A与X-181病毒种子批。传代后的121XP病毒的血凝素（Hemagglutinin，HA）基因中存在4个非同义突变，其中一处突变（Lys226Glu）位于HA的Ca抗原表位，突变频率为18%。源自X-179A的病毒HA基因中存在2个非同义突变：Pro314Gln（突变频率18%）与Asn146Asp（突变频率78%）。位于Sa抗原表位的Asn146Asp突变，在野生型A/California/07/2009(H1N1)病毒中也以低频率被检出（11%），而在源自X-179A的X-181病毒中则以完全替换的形式存在。在传代的X-181病毒的HA基因中，出现了2处突变：一批次中存在沉默突变A1398G（突变频率31%），另一批次中存在G756T（对应氨基酸突变Glu252Asp，突变频率47%）。后一处突变位于Ca抗原表位的保守区域内。本研究采用的RNA测序流程稳定性强、可重复性佳，适用于流感疫苗病毒种子批的遗传一致性监测。