Construction and characterization of Francisella tularensis vaccine strain with a single copy of iglC gene and lacking recA gene

Abstract

Live vaccine used in Russia against tularemia is based on Francisella tularensis subsp. holarctica NIIEG vaccine strain 15. This vaccine is highly effective, but fairly unstable. Therefore, the development of stable live tularemia vaccine with minimal side effects is rather pressing. F. tularensis iglC gene is required for intracellular production of the microbe, and recA gene is crucial for homologous recombination. Using methodology for allele removal, we deleted one copy of iglC gene and both copies of recA gene in F. tularensis vaccine strain. To achieve replacement of intact F. tularensis chromosome segments with modified segments, we constructed a novel pGM5 suicide vector based on pHV33 bireplicon plasmid. Modified chromosome segments contained a F. tularensis DNA fragment without 545 bp of iglC structural gene segment (in pGMΔiglC plasmid) and lacked a 1060-bp DNA fragment containing recA structural gene segment (pGMΔrecA plasmid). The constructed 15/23-lΔrecA mutant was capable to reproduce in macrophage-like J77A.l cell line with proliferation efficiency eight to ten times higher than parent vaccine strain 15. BALB/c mouse responded to immunization by 15/23-lΔrecA strain with a slight (about 2%) weight decrease compared to infection with strain 15 (about 14%). Bacteria of 15/23-lΔrecA strain were virtually incapable of maintaining germination in BALB/c murine spleen 14 days after invasion. In turn, bacteria of strain 15 were detected in murine spleen even after 21 days. Thus, F. tularensis 15/23-lΔrecA strain, which results in a lighter organismal reaction, can be successfully used as a basis for construction of stable live tularemia vaccines with minimal side effects.