NanH and PknG putative virulence factors as a recombinant subunit immunogen against Corynebacterium pseudotuberculosis infection in mice.

Despite the economic and zoonotic relevance of caseous lymphadenitis, a competent immunoprophylaxis tool is still necessary. Here, we evaluated two putative virulence factors of Corynebacterium pseudotuberculosis, rNanH, and rPknG, as recombinant subunit vaccines in a murine model against the infection by C. pseudotuberculosis. Three groups of ten Balb/c mice each were inoculated with a sterile 0.9% saline solution (G1), rNanH (G2), or rPknG (G3) in formulations containing saponin as an adjuvant. The mice received two vaccine doses intercalated by a 21-day interval and were challenged with 2 × 10 CFU/mL of the C. pseudotuberculosis MIC-6 strain 21 days after the last immunization. The total IgG, IgG1, and IgG2a production levels increased significantly in the experimental groups (G2 and G3) on day 42. The highest levels of IgG2a antibodies in G2 and G3 were observed compared to IgG1 levels. G3 showed a significant (p < 0.05) humoral response through higher production of total IgG at day 42 when compared to G2. A significant increase of mRNA expression levels of interleukin (IL)-17, tumor necrosis factor, and interferon-γ was observed only in G2, while IL-4 was significantly produced only by G3. The levels of IL-10 and IL-12 obtained were not significant in any group. The survival rates after the challenge were 20% for G3 and 60% for G2 (p < 0.05). Our findings suggest that the formulation containing rNanH and saponin (G2) resulted in the best protection against the challenge and was able to elicit a Th1 immune response in mice, and can be considered as a promising antigen in the development of an effective vaccine against caseous lymphadenitis.