Murali Harishchandra Sripathy

Evaluation of IgY capture ELISA for sensitive detection of Alpha hemolysin of Staphylococcus aureus without staphylococcal protein A interference

Staphylococcal protein A (Spa) secreted by all Staphylococcus aureus strains is the major hindrance in development of specific immunoassays for detecting S. aureus antigens, because of its characteristic feature of binding to Fc region of most mammalian immunoglobulins and also to Fab region of certain classes of mammalian immunoglobulins. Immunoglobulin Y (IgY) is the avian equivalent of mammalian IgG which does not have any affinity to Spa. In the present study we report that using chicken egg yolk IgY over mammalian IgG as capture antibody prevents both soluble and surface bound protein A from causing false positives quantified by chicken anti-protein A antibodies. This was demonstrated by development of sandwich ELISA for detection of alpha hemolysin toxin from culture supernatants of S. aureus strains with anti alpha hemolysin IgY as capture and rabbit anti alpha hemolysin IgG as revealing antibody. This indirect sandwich ELISA was evaluated onto a large number of S. aureus isolates recovered from clinical sources for alpha hemolysin secretion. Results of sandwich ELISA were compared with PCR and Western blot analysis. The immunoassay is highly specific and has high sensitivity of detecting less than 1 ng/ml. This procedure is highly effective in eliminating Spa interference and can be extended to detection of other important superantigen toxins of S. aureus.

Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference

In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SEs in different wells of same PCR plate using common DNA substrate.

Development and evaluation of a novel combinatorial selective enrichment and multiplex PCR technique for molecular detection of major virulence‐associated genes of enterotoxigenic Staphylococcus aureus in food samples

To develop a multiplex PCR assay coupled with selective enrichment step to detect major virulence‐associated genes of enterotoxigenic Stap‐hylococcus aureus and evaluate the same directly on contaminated food samples.

Immunization with recombinant bivalent chimera r-Cpae confers protection against alpha toxin and enterotoxin of Clostridium perfringens type A in murine model

Clostridium perfringens type A, an anaerobic pathogen is the most potent cause of soft tissue infections like gas gangrene and enteric diseases like food poisoning and enteritis. The disease manifestations are mediated via two important exotoxins, viz. myonecrotic alpha toxin (αC) and enterotoxin (CPE). In the present study, we synthesized a bivalent chimeric protein r-Cpae comprising C-terminal binding regions of αC and CPE using structural vaccinology rationale and assessed its protective efficacy against both alpha toxin (αC) and enterotoxin (CPE) respectively, in murine model. Active immunization of mice with r-Cpae generated high circulating serum IgG (systemic), significantly increased intestinal mucosal s-IgA antibody titres and resulted in substantial protection to the immunized animals (100% and 75% survival) with reduced tissue morbidity when administered with 5×LD(100) doses of αC (intramuscular) and CPE (intra-gastric gavage) respectively. Mouse RBCs and Caco-2 cells incubated with a mixture of anti-r-Cpae antibodies and αC and CPE respectively, illustrated significantly higher protection against the respective toxins. Passive immunization of mice with a similar mixture resulted in 91-100% survival at the end of the 15 days observation period while mice immunized with a concoction of sham sera and respective toxins died within 2-3 days. This work demonstrates the efficacy of the rationally designed r-Cpae chimeric protein as a potential sub unit vaccine candidate against αC and CPE of C. perfringens type A toxemia.

Studies on recombinant glucokinase (r-glk) protein of Brucella abortus as a candidate vaccine molecule for brucellosis.

Brucellosis is one of the most prevalent zoonotic diseases of worldwide distribution caused by the infection of genus Brucella. Live attenuated vaccines such as B. abortus S19, B. abortus RB51 and B. melitensis Rev1 are found most effective against brucellosis infection in animals, contriving a number of serious side effects and having chances to revert back into their active pathogenic form. In order to engineer a safe and effective vaccine candidate to be used in both animals and human, a recombinant subunit vaccine molecule comprising the truncated region of glucokinase (r-glk) gene from B. abortus S19 was cloned and expressed in Escherichia coli BL21DE3 host. Female BALB/c mice immunized with purified recombinant protein developed specific antibody titer of 1:64,000. The predominant IgG2a and IgG2b isotypes signified development of Th1 directed immune responses. In vitro cell cytotoxicity assay using anti-r-glk antibodies incubated with HeLa cells showed 81.20% and 78.5% cell viability against lethal challenge of B. abortus 544 and B. melitensis 16M, respectively. The lymphocyte proliferative assay indicated a higher splenic lymphocyte responses at 25μg/ml concentration of protein which implies the elevated development of memory immune responses. In contrast to control, the immunized group of mice intra-peritoneal (I.P.) challenged with B. abortus 544 were significantly protected with no signs of necrosis and vacuolization in their liver and spleen tissue. The elevated B-cell response associated with Th1 adopted immunity, significant in vitro cell viability as well as protection afforded in experimental animals after challenge, supplemented with histopathological analysis are suggestive of r-glk protein as a prospective candidate vaccine molecule against brucellosis.

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.

Evaluation of recombinant SEA-TSST fusion toxoid for protection against superantigen induced toxicity in mouse model.

Treatment of Staphylococcus aureus infections has become complicated owing to growing antibiotic resistance mechanisms and due to the multitude of virulence factors secreted by this organism. Failures with traditional monovalent vaccines or toxoids have brought a shift towards the use of multivalent formulas and neutralizing antibodies to combat and prevent range of staphylococcal infections. In this study, we evaluated the efficacy of a fusion protein (r-ET) comprising truncated regions of staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST-1) in generating neutralizing antibodies against superantigen induced toxicity in murine model. Serum antibodies showed specific reactivity to both SEA and TSST-1 native toxins. Hyperimmune serum from immunized animals protected cultured splenocytes from non-specific superantigen induced proliferation completely. Passive antibody administration prevented tissue damage from acute inflammation associated with superantigen challenge from S. aureus cell free culture supernatants. Approximately 80% and 50% of actively and passively immunized mice respectively were protected from lethal dose against S. aureus toxin challenge. This study revealed that r-ET protein is non-toxic and a strong immunogen which generated neutralizing antibodies and memory immune response against superantigen induced toxic effects in mice model.

A simple and universal ligation mediated fusion of genes based on hetero-staggered PCR for generating immunodominant chimeric proteins.

We developed a simple T4 DNA ligase mediated strategy for inframe splicing of two or more cohesive genes generated by hetero-staggered PCR and directionally cloning the spliced product bearing sticky overhangs in to a correspondingly cut vector. For this, two pairs of primers are used in two different parallel PCRs, for generation of each cohesive gene product. We exemplified this strategy by splicing two major super-antigen genes of Staphylococcus aureus, namely, staphylococcal enterotoxin A (sea), and toxic shock syndrome toxin (tsst-1) followed by its directional cloning into pre-digested pRSET A vector. The fusion gene encoding chimeric recombinant SEA-TSST protein (32kDa) was expressed in E. coli BL21(DE3) host strain. The recombinant chimeric protein retained the antigenicity of both toxins as observed by the strong immunoreactivity with commercial antibodies against both SEA and TSST-1 toxin components by Western blot analysis. We observed that the present method for gene splicing with cohesive ends is simple since it does not require elaborate standardization and a single fusion product is obtained consistently during nested PCR with forward primer of first gene and reverse primer of second gene. For comparison, we fused the same genes using splicing by overlap extension PCR (SOE-PCR) and consistently obtained DNA smearing and multiple non-specific bands even after several rounds of PCRs from gel excised product. Moreover, the newly described method requires only two to six complimentary sticky ends between the genes to be spliced, in contrast to long stretch of overlapping nucleotides in case of SOE-PCR.

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.

Evaluation of Recombinant Multi-Epitope Outer Membrane Protein-Based Klebsiella pneumoniae Subunit Vaccine in Mouse Model

Safety and protective efficacy of recombinant multi-epitope subunit vaccine (r-AK36) was evaluated in a mouse model. Recombinant AK36 protein comprised of immunodominant antigens from outer membrane proteins (Omp’s) of Klebsiella pneumoniae namely OmpA and OmpK36. r-AK36 was highly immunogenic and the hyperimmune sera reacted strongly with native OmpA and OmpK36 proteins from different K. pneumoniae strains. Hyperimmune sera showed cross-reactivity with Omp’s of other Gram-negative organisms. Humoral responses showed a Th2-type polarized immune response with IgG1 being the predominant antibody isotype. Anti-r-AK36 antibodies showed antimicrobial effect during in vitro testing with MIC values in the range of 25–50 μg/ml on different K. pneumoniae strains. The recombinant antigen elicited three fold higher proliferation of splenocytes from immunized mice compared to those with sham-immunized mice. Anti-r-AK36 antibodies also exhibited in vitro biofilm inhibition property. Subunit vaccine r-AK36 immunization promoted induction of protective cytokines IL-2 and IFN-γ in immunized mice. When r-AK36-immunized mice were challenged with 3 × LD100 dose, ∼80% of mice survived beyond the observation period. Passive antibody administration to naive mice protected them (67%) against the lethal challenge. Since the targeted OMPs are conserved among all K. pneumoniae serovars and due to the strong nature of immune responses, r-AK36 subunit vaccine could be a cost effective candidate against klebsiellosis.