Abstract
LC16m8 (m8), a highly attenuated vaccinia virus (VAC) strain, was developed as a smallpox vaccine, and its safety and immunogenicity have been confirmed. Here, we aimed to develop a system that recovers infectious m8 from a bacterial artificial chromosome (BAC) that retains the full-length viral genomic DNA (m8-BAC system). The infectious virus was successfully recovered from a VAC-BAC plasmid, named pLC16m8-BAC. Furthermore, the bacterial replicon-free virus was generated by intramolecular homologous recombination and was successfully recovered from a modified VAC-BAC plasmid, named pLC16m8.8S-BAC. Also, the growth of the recovered virus was indistinguishable from that of authentic m8. The full genome sequence of the plasmid, which harbors identical inverted terminal repeats (ITR) to that of authentic m8, was determined by long-read next-generation sequencing (NGS). The ITR contains x 18 to 32 of the 70 and x 30 to 45 of 54 base pair tandem repeats, and the number of tandem repeats was different between the ITR left and right. Since the virus recovered from pLC16m8.8S-BAC was expected to retain the identical viral genome to that of m8, including the ITR, a reference-based alignment following a short-read NGS was performed to validate the sequence of the recovered virus. Based on the pattern of coverage depth in the ITR, no remarkable differences were observed between the virus and m8, and the other region was confirmed to be identical as well. In summary, this new system can recover the virus, which is geno- and phenotypically indistinguishable from authentic m8.
Highlights
The virulence and immunogenicity of each vaccinia virus (VAC) strain are highly diverse
Homologous recombination was promoted by the CRISPR-Cas9 system for VAC to generate a recombinant m8, which harbors the EGFP and mini-F cassettes (m8-EGFP-bacterial artificial chromosome (BAC)) (Fig 1A). 293FT cells were infected with plasmids, pVAC-BAC11, pVAC-T7pol-Cas9.1, and m8
The m8 and modified vaccinia Ankara (MVA) are categorized into third generation smallpox vaccines that represent highly attenuated VAC strains developed as safer vaccines than those of the first and second generation smallpox vaccines [15]
Summary
The virulence and immunogenicity of each vaccinia virus (VAC) strain are highly diverse. Some strains, such as VAC, Lister, and New York City Board of Health strains (NYCBH) that were used for the smallpox eradication program caused serious adverse events, such as postvaccinial encephalitis, [1, 2]. The immunogenicity and efficacy of m8 as a vaccine relative to that of other orthopoxviral diseases, such as monkeypox in nonhuman primates, are similar to that of the other VAC strains [4,5,6,7,8,9]. After the eradication of smallpox, VAC strains were still expected as the vaccine for smallpox, which has potential use as a bioterrorism agent and as a recombinant vaccine vector. Attenuated VAC strains are naturally the first choice for these uses
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