Immunogenicity of a recombinant Mycobacterium smegmatis vaccine expressing the fusion protein CMX in cattle from Goiás State, Brazil

Abstract

This study aimed to evaluate the immunogenicity of a recombinant Mycobacterium smegmatis vaccine expressing the CMX fusion protein composed of immunodominant epitopes Ag85C, MPT51 and HspX of Mycobacterium tuberculosis, which are important mycobacteria virulence factors. A group of Nelore heifers that were 10 to 12 months of age and negative for the tuberculin skin test (TST) were immunized with four doses of the recombinant vaccine mc(2)-CMX (M. smegmatis-Ag85C-MPT51-HspX) during a period of one year. Before each immunization, blood was collected to obtain sera for antibody analysis. Serological analysis demonstrated that mc(2)-CMX was able to induce a humoral response with increased levels of specific IgG antibodies against CMX, despite minimum antibody levels being detected for individual Ag85C, MPT51 or HspX recombinant antigens. However, there was no significant increase in specific CD4(+) IFN-γ-positive T cells. Lymphadenomegaly was observed in superficial cervical lymph nodes adjacent to the site of vaccination among mc(2)-CMX-vaccinated bovines, and the histopathological analysis demonstrated follicular hyperplasia without inflammatory infiltrate or granuloma formation. Animals remained negative for the TST until the end of the experiments, showing no cross-reactivity with the recombinant vaccine and tuberculin proteins. We discuss the potential of mc(2)-CMX to induce an immune response in cattle.

Fig. 1.

Immune humoral response induced by the recombinant vaccine. (A) Serum levels of anti-mc²-CMX antibodies among bovine immunized with mc²-CMX, mc² and PBS. (B) Serum levels of anti-rCMX antibodies among bovine immunized with mc²CMX, mc² and PBS. Animals were immunized with 1 × 10⁷ CFU/ml of each vaccine subcutaneously. Sera were obtained at different time points following vaccination and analyzed by ELISA. Each time point represents the mean and standard error of the mean of the optical density from 10 animals per group. * Statistically significant difference between the mc²-CMX and PBS groups (P<0.05). ** Statistically significant difference between the mc² and PBS groups (P<0.05).

Fig. 2.

Induction of IFN-γ by CD4⁺ T cells. Cattle were revaccinated 200 days after the third immunization, and PBMCs were obtained. (A) Total CD4⁺ T cells from the vaccinated group were quantified by flow cytometry. The dots represent the number of CD4⁺ T cell/ml from each animal. The horizontal lines represent the average and standard deviation. (B) PBMCs from mc²-CMX-vaccinated animals were stimulated in vitro with medium, PHA or CMX, and the percentage of CD4⁺IFN-γ-positive T cells was analyzed. The bars represent the average and standard deviation per group. No significant statistical difference was observed.

Fig. 3.

Histopathological analysis of a superficial cervical lymph node from the mc²-CMX vaccinated group. The lymph node from a representative animal was sectioned and stained with hematoxylin eosin (H&E) (A, C and D) and Fite’s acid fast stain (B) 90 days after the last immunization of the group. Note the absence of inflammatory infiltrate or granuloma formation or necrosis (A). Fite’s acid fast staining revealed no presence of acid-fast bacilli (B). The structure of a normal lymph node with a germinal center, medular region and blood vessels can be seen (C and D).