Immunological and protective evaluation of purE/purK gene-deletion mutant of Brucella melitensis M5 strain.

Brucellosis is listed by the World Health Organization as one of the seven most neglected global zoonotic diseases. A live-attenuated vaccine is still the main strategy used to prevent the spread of brucellosis. In this study, we constructed a two-gene (purE and purK)-deletion vaccine in Brucella melitensis vaccine strain M5 with homologous recombination. We evaluated its safety and immunogenicity in vitro and vivo, and its immunoprotective effect against B. melitensis wild-type strain 16M infection. In vitro, strain M5-ΔpurEK induced 10.50% lactose dehydrogenase (LDH) release, only half that induced by the vaccine strain M5 (29.92% LDH). The ability of M5-ΔpurEK to invade macrophages also decreased gradually, with a reduction of 1.02-log. In vivo, the hepatosplenic load and hepatosplenomegaly decreased significantly over time in mice inoculated with M5-ΔpurEK (P < 0.05). It elicited anti-Brucella-specific IgG responses and induced the secretion of γ-interferon (IFN-γ) and interleukin-6 (IL-6). Serum samples inoculated with M5-ΔpurEK were only 20% positive on the Bengal plate test. Hematoxylin-eosin staining showed only slight infiltration of neutrophils in the spleens of M5-ΔpurEK-infected mice on days 14 and 42 after infection. The hepatocytes in the hepatic parenchyma were sparse and lightly stained cytoplasm, with few lymphocytes around the confluent area, which gradually returned to normal within 42 days. Like parental strain M5, strain M5-ΔpurEK conferred protection against infection with B. melitensis wild-type strain 16M. In conclusion, we have shown that the deletion of purE and purK diminishes the cytotoxic effect of vaccine strain M5 on cells and organs, while leaving the degree of protection against infection with virulent wild-type Brucella strain 16M unchanged.