Genome stability of the vaccine strain VACΔ6

Abstract

Due to cessation of mass smallpox vaccination in 1980, the collective immunity of humans against orthopoxvirus infections has virtually been lost. Therefore, the risk of spreading zoonotic human orthopoxvirus infections caused by monkeypox and cowpox viruses has increased in the world. First-generation smallpox vaccines based on Vaccinia virus (VAC) are reactogenic and therefore not suitable for mass vaccination under current conditions. This necessitates the development of modern safe live vaccines based on VAC using genetic engineering. We created the VACΔ6 strain by transient dominant selection. In the VACΔ6 genome, f ive virulence genes were intentionally deleted, and one gene was inactivated by inserting a synthetic DNA fragment. The virus was passaged 71 times in CV-1 cells to obtain the VACΔ6 strain from the VAC LIVP clonal variant. Such a long passage history might have led to additional off-target mutations in VACΔ6 compared to the original LIVP variant. To prevent this, we performed a genome-wide sequencing of VAC LIVP, VACΔ6, and f ive intermediate viral strains to assess possible off-target mutations. A comparative analysis of complete viral genomes showed that, in addition to target mutations, only two nucleotide substitutions occurred spontaneously when obtaining VACΔ4 from the VACΔ3 strain; the mutations persisting in the VACΔ5 and VACΔ6 genomes. Both nucleotide substitutions are located in intergenic regions (positions 1431 and 189738 relative to LIVP), which indicates an extremely rare occurrence of off-target mutations when using transient dominant selection to obtain recombinant VAC variants with multiple insertions/deletions. To assess the genome stability of the resulting attenuated vaccine strain, 15 consecutive cycles of cultivation of the industrial VACΔ6 strain were performed in 4647 cells certif ied for vaccine production in accordance with the "Guidelines for Clinical Trials of Medicinal Products". PCR and sequencing analysis of six DNA fragments corresponding to the regions of disrupted genes in VACΔ6 showed that all viral DNA sequences remained unchanged after 15 passages in 4647 cells.

Keywords: genome stability; targeted gene inactivation; transient dominant selection; vaccinia virus.

Copyright © AUTHORS.

Figures

Table 1.. Oligonucleotide primers for PCR analysis of modif ied regions in the VAC genome used in the VACΔ6 vaccine development

Table 2.. Lengths of PCR products for the original LIVP strain and VAC variants with inactivated genes

Fig. 1.. General scheme for obtaining VACΔ1 virus with a targeted B8R gene deletion

а – scheme of hybrid pΔB8R plasmid design; b – recombinant insertion of the hybrid plasmid into the viral genome; c – division of the recombinant virus progeny into two variants after removal of selective pressure on the gpt gene (see explanations in the text).

Fig. 2.. Scheme of recombinant VACΔ6 production

Table 3.. Passage history of producing the VACΔ6 strain from LIVP in CV-1 cells

Fig. 3.. Electrophoretic separation of the DNA products of PCR analysis of the regions of viral genes A35R, A56R, B8R, C3L, N1L, and J2R

0 – PCR product obtained using genomic DNA of the original strain VACΔ6 as a template; 15 – PCR product obtained using genomic DNA of the VACΔ6 strain after 15 passages in 4647 cells as a template; M – DNA marker (length in bp is indicated on the left); “+” – PCR product obtained using VAC LIVP DNA; ”–” – negative control.