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本研究使用EV-A71灭活疫苗或CV-A16灭活疫苗免疫BALB/c小鼠，每种疫苗通过肌肉注射和皮内注射的方式分别两针免疫小鼠后14d，检测动物体内的中和抗体和细胞免疫应答水平。随后我们筛选CD19+EV-A71+B细胞或CD19+CV-A16+B细胞，对分选获得的特异性B细胞的BCR抗体重链基因进行巢式PCR扩增。随后对获取的抗体基因打靶，获得重链IGHV、IGHD、IGHJ基因的胚系来源，并对抗体IGH基因分类，对可变区CDR3区域氨基酸长度进行分析。结果表明，EV-A71实验性疫苗和CV-A16实验性疫苗两次免疫后，均能诱导较好的中和抗体水平，但是皮内免疫效果优于肌肉注射效果。EV-A71实验性疫苗或CV-A16实验性疫苗皮内注射小鼠与肌肉注射小鼠相比，IGHV基因谱系更加丰富。其中EV-A71疫苗通过两种接种方式产生的共有基因谱系为IGHV1-63*02,CV-A16疫苗通过两种接种方式产生的共有基因谱系为IGHV3-8*02。皮内注射比肌肉注射诱导的抗体重链可变区CDR3结构域的氨基酸长度变化更为明显，特别是CV-A16皮内注射小鼠重链可变区的CDR3区域长度主要集中在13个氨基酸，最长达到17个氨基酸。综上，皮内注射和肌肉注射诱导产生的特异性中和抗体滴度不同，CVA16通过皮内免疫诱导的中和抗体滴度高于肌肉注射诱导的抗体滴度，后续的BCR重链基因进化特征也存在明显的差异，这些发现为后续的联合疫苗设计提供参考。
Abstract：This study investigates the immune responses induced in Balb/c mice immunized with either the EVA71 or CV-A16 inactivated vaccine. Each vaccine was administered via intramuscular and intradermal injections, with two doses given for each route. Fourteen days post-immunization, the levels of neutralizing antibodies and cellular immune responses were evaluated. CD19+ EV-A71+ B cells or CD19+ CV-A16+ B cells were then isolated, and the BCR heavy chain genes from the specific B cells were amplified using nested PCR. The resulting antibody genes were targeted to determine the germline origins of the IGHV, IGHD, and IGHJ regions. Lineage classification and analysis of the variable region CDR3 amino acid lengths were also conducted. The results show that both EV-A71 and CV-A16 experimental vaccines successfully induced neutralizing antibodies after two rounds of immunization; however, intradermal injection produced better immune responses compared to intramuscular injection. Mice immunized with either vaccine via intradermal injection exhibited a more diverse IGHV gene lineage compared to those injected intramuscularly. Specifically, the common gene lineage for EV-A71 immunized mice was IGHV1-6302, and for CV-A16 immunized mice, it was IGHV3-802. The variation in the amino acid length of the heavy chain variable region CDR3 domain was more pronounced in intradermal immunization compared to intramuscular injection. Notably, the CDR3 region of the heavy chain variable region in CV-A16-immunized mice predominantly concentrated around 13 amino acids, with a maximum length of 17 amino acids. In conclusion, the titers of specific neutralizing antibodies induced by intradermal and intramuscular injections differ, with intradermal immunization of CV-A16 yielding higher neutralizing antibody levels. Furthermore, there are significant differences in the evolutionary characteristics of the BCR heavy chain genes between the two immunization routes, offering valuable insights for the future design of combined vaccines.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.240331
中图分类号:R392-33
引用信息:
[1]陈可琪,廖芸,蒋国润等.EV-A71或CV-A16灭活疫苗通过两种不同接种途径免疫小鼠诱导的BCR重链可变区基因特征比较[J].病毒学报,2025,41(02):317-325.DOI:10.13242/j.cnki.bingduxuebao.240331.
基金信息:
云南省科技计划-基础研究专项（项目号：202401AS070047）,题目：EV-A71和CV-A16抗原预存免疫对CVA10和CV-A6抗原诱导的B细胞和T细胞免疫的影响; 云南省科技计划-生物医药专项（项目号：202402AA310018）,题目：EV71-CA16-CA10-CA6灭活疫苗的制备、评价及多价疫苗关键抗原表位筛选;云南省科技计划-生物医药专项（项目号：202202AA100001）,题目：EV71-CA16联合疫苗临床前研究; 云南省科技人才与平台计划（项目号：202305AC160008）,题目：中青年学术和技术带头人后备人才项目~~