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利用TALEN技术灭活内源性逆转录病毒建立用于疫苗生产的CRFK细胞。

Establishment of CRFK cells for vaccine production by inactivating endogenous retrovirus with TALEN technology.

作者信息

Shimode Sayumi, Sakuma Tetsushi, Yamamoto Takashi, Miyazawa Takayuki

机构信息

Genome Editing Innovation Center, Hiroshima University, Higashi-Hiroshima, Japan.

Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Japan.

出版信息

Sci Rep. 2022 Apr 27;12(1):6641. doi: 10.1038/s41598-022-10497-1.

DOI:10.1038/s41598-022-10497-1
PMID:35477976
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9046391/
Abstract

Endogenous retroviruses (ERVs) are retroviral sequences present in the host genomes. Although most ERVs are inactivated, some are produced as replication-competent viruses from host cells. We previously reported that several live-attenuated vaccines for companion animals prepared using the Crandell-Rees feline kidney (CRFK) cell line were contaminated with a replication-competent feline ERV termed RD-114 virus. We also found that the infectious RD-114 virus can be generated by recombination between multiple RD-114 virus-related proviruses (RDRSs) in CRFK cells. In this study, we knocked out RDRS env genes using the genome-editing tool TAL Effector Nuclease (TALEN) to reduce the risk of contamination by infectious ERVs in vaccine products. As a result, we succeeded in establishing RDRS knockout CRFK cells (RDKO_CRFK cells) that do not produce infectious RD-114 virus. The growth kinetics of feline herpesvirus type 1, calicivirus, and panleukopenia virus in RDKO_CRFK cells differed from those in parental cells, but all of them showed high titers exceeding 10 TCID/mL. Infectious RD-114 virus was undetectable in the viral stocks propagated in RDKO_CRFK cells. This study suggested that RDRS env gene-knockout CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with no risk of contamination with infectious ERV.

摘要

内源性逆转录病毒（ERVs）是存在于宿主基因组中的逆转录病毒序列。尽管大多数ERVs已失活，但有些会作为具有复制能力的病毒从宿主细胞中产生。我们之前报道过，几种使用克兰德尔-里斯猫肾（CRFK）细胞系制备的伴侣动物减毒活疫苗被一种具有复制能力的猫ERV（称为RD-114病毒）污染。我们还发现，感染性RD-114病毒可由CRFK细胞中多个与RD-114病毒相关的前病毒（RDRSs）之间的重组产生。在本研究中，我们使用基因组编辑工具转录激活样效应因子核酸酶（TALEN）敲除RDRS env基因，以降低疫苗产品被感染性ERV污染的风险。结果，我们成功建立了不产生感染性RD-114病毒的RDRS敲除CRFK细胞（RDKO_CRFK细胞）。1型猫疱疹病毒、杯状病毒和猫泛白细胞减少症病毒在RDKO_CRFK细胞中的生长动力学与在亲代细胞中的不同，但它们的滴度均很高，超过10 TCID/mL。在RDKO_CRFK细胞中繁殖的病毒原液中未检测到感染性RD-114病毒。本研究表明，RDRS env基因敲除的CRFK细胞将作为一种细胞系用于生产无感染性ERV污染风险的减毒活疫苗或生物制品。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3795/9046391/033b26977d75/41598_2022_10497_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3795/9046391/71fd3be2d047/41598_2022_10497_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3795/9046391/4721a858e90e/41598_2022_10497_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3795/9046391/033b26977d75/41598_2022_10497_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3795/9046391/71fd3be2d047/41598_2022_10497_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3795/9046391/4721a858e90e/41598_2022_10497_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3795/9046391/033b26977d75/41598_2022_10497_Fig4_HTML.jpg

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