Heterologous expression of Intimin and IpaB fusion protein in Lactococcus lactis and its mucosal delivery elicit protection against pathogenicity of Escherichia coli O157 and Shigella flexneri in a murine model.

Escherichia coli O157:H7 and Shigella flexneri are the predominant diarrhoeal pathogens and those strains producing Shiga toxins cause life-threatening sequelae including hemolytic uremic syndrome (HUS) upon their entry into the host. Intimate adherence of E. coli O157 and invasion of S. flexneri in the host intestinal epithelial cells is mainly mediated by Intimin and IpaB proteins, respectively. In this study, we have synthesized chimera of immunodominant regions of Intimin (eae) and IpaB (ipaB) designated as EI and expressed it in Lactococcus lactis (LL-EI) to develop a combinatorial oral vaccine candidate. Immune parameters and protective efficacy of orally administered LL-EI were assessed in the murine model. Significant EI-specific serum IgG, IgA, and fecal IgA antibody titer were observed in the LL-EI group. Considerable increase in EI-specific splenocyte proliferation and a concurrent upregulation of both Th1 and Th2 cytokines was observed in LL-EI immunized mice. Flow cytometry analysis also revealed a significant increase in CD4 and CD8 cell counts in LL-EI immunized group compared to PBS, LL control group.In vitro studies using LL-EI immunized mice sera showed substantial protection against bacterial adhesion and invasion caused by E. coli O157 and Shigella flexneri¸ respectively. LL-EI immunized group challenged with E. coli O157 ceased fecal shedding within 6 days, and mice challenged with S. flexneri showed 93% survival with minimal bacterial load in the lungs. Our results indicate that LL-EI immunization elicits systemic, mucosal and cell-mediated immune responses, and can be a promising candidate for oral vaccine development against these pathogens.

Genome-wide unique insertion sequences among five Brucella species and demonstration of differential identification of Brucella by multiplex PCR assay.

Brucellosis is a neglected zoonotic disease caused by alpha proteobacterial genus Brucella comprising of facultative intracellular pathogenic species that can infect both animals and humans. In this study, we aimed to identify genome-wide unique insertion sequence (IS) elements among Brucella abortus, B. melitensis, B. ovis, B. suis and B. canis for use in species differentiation by conducting an intensive in silico-based comparative genomic analysis. As a result, 25, 27, 37, 86 and 3 unique ISs were identified respectively and they had a striking pattern of distribution among them. To explain, a particular IS would be present in four species with 100% identity whereas completely absent in the fifth species. However, flanking regions of that IS element would be highly identical and conserved in all five species. Species-specific primers designed on these flanking conserved regions resulted in two different amplicons grouping the species into two: one that possesses IS and the other that lacks it. Seeking for species-specific amplicon size for particular species was sufficient to identify it irrespective of biovar. A multiplex PCR developed using these primers resulted in successful differentiation of the five species irrespective of biovars with significant specificity and sensitivity when examined on clinical samples.

Protective and therapeutic efficacy study of divalent fusion protein rL7/L12-Omp25 against B. abortus 544 in presence of IFNγ.

Brucella as intracellular pathogen requires a coordinate interaction between Th1 subset of gamma interferon-secreting CD4 T cells and CD8 T cells for optimal protective immunity. It was previously recognized that L7/L12 as T cell-reactive antigen from the pathogen. On other hand, Omp25 was found as another antigen to provide protection against the Brucella infection by eliciting both Th1 and Th2 type of immune responses in mice. Here, we analyzed the prophylactic and therapeutic efficacy of a divalent fusion protein (rL7/L12-Omp25) comprising these two promising immunogens of Brucella in the presence of murine IFN-gamma in mice against B. abortus 544 challenge. rIFN-gamma with rL7/L12-Omp25 resulted in superior immune response when compared to the animal vaccine strain B. abortus S19. The vaccine candidate caused dominance of IgG1 over IgG2a and upregulated cytokine secretion (IFN-gamma, TNF-α, and IL-10) among immunized mice. Moreover, the antigen in combination with murine IFN-gamma elicited stronger cell-mediated immune response among the immunized animals when compared to standard vaccine (S19). The registered log protection unit among challenged mice with B. abortus 544 pathogen was 2.16, p = 0.0001 when rL7/L12-Omp25 was administered alone and 2.4, p = 0.0001 when it was administered along with rIFN-gamma. However, the molecule upon administration with murine IFN-gamma imparted very minimal or no therapeutic effect against brucellosis. To conclude, our study demonstrates the potential of rL7/L12-Omp25 as an immunogen of prospective and efficient prophylaxis as it is capable of eliciting both cell-mediated and humoral immune responses against brucellosis.

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

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.