Intraperitoneal administration of a novel chimeric immunogen (rOP) elicits IFN-γ and IL-12p70 protective immune response in BALB/c mice against virulent Brucella

Abstract

Recombinant engineering of immunologically active chimeric protein consisting of Omp19 and P39 domains of B. abortus (rOP), was purified under denaturing conditions upon expression in E. coli BL21 (DE3) and refolded to dynamic form. The immuno-protective efficacy of rOP was evaluated by challenging the BALB/c mice intraperitoneally (I.P) with the infective species of Brucella in the absence or presence of adjuvants, such as Aluminum hydroxide gel (Al), or Freund's Complete Adjuvant (FCA)/Incomplete Freund's Adjuvant (IFA). Surprisingly, after second boosting, mice received rOP per se were found to be immunogenic in terms of IgG response with the dominant expression of IgG2a and significant IFN-γ by splenic T cells, suggesting that rOP is a strong inducer of anti-Brucella immunity. The resulted anti-rOP antibodies recognized native Omp19 and P39 among species of Brucella with distinct double bands and single band against chimera in immunoblotting. An enhanced and comparable antibody response with varied IgG isotype combinations were noticed in the mice primed and boosted with rOP in adjuvants. However, rOP+FCA/IFA formulation was found to be the most effective in lymphocyte recall assays at inducing significant (P<0.001) proliferation index (P.I.) as well as increased Th1-coupled cytokines (IFN-γ, IL-2 and IL-12p70) than rOP+Al in response to rOP re-stimulation. Furthermore, in vitro defensive assay revealed that compared to anti-rOP antisera, the polyclonal anti-sera from rOP+adjuvants exhibited enhanced protection of RAW264.7 cells against virulent challenge by B. melitensis 16M and B. abortus 544. In addition, compared to sham group, enumeration of Brucella CFU after challenge with the above species showed a significant (P<0.01) reduction of bacteria (log CFU) in the macrophage cell lines and organs of vaccinated mice. On the whole, a relatively higher and faster reduction was noticed in the mice vaccinated with similar amount of purified antigen in Freund's adjuvant. Ability of inducing Th1 directed immune protection in the absence of adjuvant support, postulated rOP as a plausible entrant for developing a chimeric based subunit vaccine against Brucella.

Keywords: Adjuvant; Chimeric immunogen; Infective Brucella; rOP.