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.

Recombinant outer membrane protein 25c from Brucella abortus induces Th1 and Th2 mediated protection against Brucella abortus infection in mouse model.

Development of a safe and efficacious vaccine for brucellosis is a long standing challenge for scientists. Recognizing potential antigens towards developing vaccine candidate is crucial. Omp25c, a porin protein of Brucella, is a paralog of two previously identified promising vaccine candidates namely, Omp25 and Omp31, with notable sequence identity. Also, Omp25c is conserved in all major Brucella species. This highlights the possibility of employing this protein in multivalent subunit vaccine based approach of Brucella management. In this study, we were interested in examining the immunogenicity and protective efficacy of Omp25c against Brucella infections. Recombinant unlipidated form of this antigen (rOmp25c) produced, upon intraperitoneal immunization in BALB/c mice along with Freund's adjuvant, was confirmed to be highly immunogenic; leading to high IgG antibody titers during the study duration. The IgG2a/IgG2b ratio of anti-rOmp25c antibodies revealed elicitation of Th2 based humoral immunity. Lymphocyte proliferation study divulged induction of specific memory response and secretion of both Th1-type (IFN-γ, GM-CSF and TNF-α) and Th2-type cytokine (IL-5) from restimulated splenocytes of rOmp25c immunized mice. CD4 T-cell subpopulation was comparatively increased than total B cell subpopulation in case of immunized mice, indicating the induction of strong cell-mediated (Th1 biased) immunity than humoral (Th2) immunity. The collective Th1 plus Th2 immune response specific to rOmp25c could be the reason for protection against Brucella challenge observed in mice groups that was comparable with S19 vaccine strain. Thus, the study encourages rOmp25c as a potent candidate vaccine against brucellosis.

Evaluation of fimC and bdha based duplex PCR for specific identification and differentiation of Burkholderia pseudomallei from near-neighbor Burkholderia species.

Assays for the rapid detection and accurate differentiation of Burkholderia pseudomallei from near-neighbor species are urgently needed in melioidosis endemic regions due to the high associated mortality and biowarfare importance of the pathogen. PCR-based methods have revolutionized this field due to the accuracy, sensitivity, and specificity that are achievable in a rapid way. In this study, a compound molecular detection system, consisting of a duplex PCR assay, was developed for the specific identification of Burkholderia pseudomallei and differentiation from other Burkholderia species. For accurate identification of B. pseudomallei, we deciphered and adopted a novel gene termed putative fimbrial chaperone (fimC). d-beta hydroxybutyrate dehydrogenase (bdha), reported previously by our group for sequence-based differentiation of B. pseudomallei from other Burkholderia species, was employed as a genus-specific target. Enforcement of an internal amplification control in the PCR format ruled out possible false negative results in the assay. Thus, the developed PCR assay was highly specific (100%) in its detection features, and a clear detection sensitivity of 10 pg/µl for purified gDNA and 3 × 103 CFU/ml for B. pseudomallei spiked urine was recorded. Successful identification of B. pseudomallei from an experimental mouse model at both the genus and species level revealed the accurate diagnostic efficiency of the duplex PCR method.

Prevalence and Identification of Burkholderia pseudomallei and Near-Neighbor Species in the Malabar Coastal Region of India.

Accurate identification of pathogens with biowarfare importance requires detection tools that specifically differentiate them from near-neighbor species. Burkholderia pseudomallei, the causative agent of a fatal disease melioidosis, is one such biothreat agent whose differentiation from its near-neighbor species is always a challenge. This is because of its phenotypic similarity with other Burkholderia species which have a wide spread geographical distribution with shared environmental niches. Melioidosis is a major public health concern in endemic regions including Southeast Asia and northern Australia. In India, the disease is still considered to be emerging. Prevalence surveys of this saprophytic bacterium in environment are under-reported in the country. A major challenge in this case is the specific identification and differentiation of B. pseudomallei from the growing list of species of Burkholderia genus. The objectives of this study included examining the prevalence of B. pseudomallei and near-neighbor species in coastal region of South India and development of a novel detection tool for specific identification and differentiation of Burkholderia species. Briefly, we analyzed soil and water samples collected from Malabar coastal region of Kerala, South India for prevalence of B. pseudomallei. The presumptive Burkholderia isolates were identified using recA PCR assay. The recA PCR assay identified 22 of the total 40 presumptive isolates as Burkholderia strains (22.72% and 77.27% B. pseudomallei and non-pseudomallei Burkholderia respectively). In order to identify each isolate screened, we performed recA and 16S rDNA sequencing. This two genes sequencing revealed that the presumptive isolates included B. pseudomallei, non-pseudomallei Burkholderia as well as non-Burkholderia strains. Furthermore, a gene termed D-beta hydroxybutyrate dehydrogenase (bdha) was studied both in silico and in vitro for accurate detection of Burkholderia genus. The optimized bdha based PCR assay when evaluated on the Burkholderia isolates of this study, it was found to be highly specific (100%) in its detection feature and a clear detection sensitivity of 10 pg/µl of purified gDNA was recorded. Nucleotide sequence variations of bdha among interspecies, as per in silico analysis, ranged from 8 to 29% within the target stretch of 730 bp highlighting the potential utility of bdha sequencing method in specific detection of Burkholderia species. Further, sequencing of the 730 bp bdha PCR amplicon of each Burkholderia strain isolated could differentiate the species and the data was comparable with recA sequence data of the strains. All sequencing results obtained were submitted to NCBI database. Bayesian phylogenetic analysis of bdha in comparison with recA and 16S rDNA showed that the bdha gene provided comparable identification of Burkholderia species.

Functional characterization and evaluation of in vitro protective efficacy of murine monoclonal antibodies BURK24 and BURK37 against Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of melioidosis has been recognized by CDC as a category B select agent. Although substantial efforts have been made for development of vaccine molecules against the pathogen, significant hurdles still remain. With no licensed vaccines available and high relapse rate of the disease, there is a pressing need for development of alternate protection strategies. Antibody-mediated passive protection is promising in this regard and our primary interest was to unravel this frontier of specific mAbs against Burkholderia pseudomallei infections, as functional characterization of antibodies is a pre-requisite to demonstrate them as protective molecules. To achieve this, we designed our study on in vitro-based approach and assessed two mAbs, namely BURK24 and BURK37, reactive with outer membrane proteins and lipopolysaccharide of the pathogen respectively, for their ability to manifest inhibitory effects on the pathogenesis mechanisms of B. pseudomallei including biofilm formation, invasion and induction of apoptosis. The experiments were performed using B. pseudomallei standard strain NCTC 10274 and a clinical isolate, B. pseudomallei 621 recovered from a septicemia patient with diabetic ailment. The growth kinetic studies of the pathogen in presence of various concentrations of each individual mAb revealed their anti-bacterial properties. Minimal inhibitory concentration and minimal bactericidal concentration of both the mAbs were determined by using standards of Clinical and Laboratory Standards Institute (CLSI) and experiments were performed using individual mAbs at their respective bacteriostatic concentration. As an outcome, both mAbs exhibited significant anti-Burkholderia pseudomallei properties. They limited the formation of biofilm by the bacterium and completely crippled its invasion into human alveolar adenocarcinoma epithelial cells. Also, the mAbs were appreciably successful in preventing the bacterium to induce apoptosis in A549 cells. The present study design revealed the protection attributes possessed by BURK24 and BURK37 that has to be further substantiated by additional in vivo studies.