Intranasal co-administration of recombinant active fragment of Zonula occludens toxin and truncated recombinant EspB triggers potent systemic, mucosal immune responses and reduces span of E. coli O157:H7 fecal shedding in BALB/c mice.

Escherichia coli O157:H7 with its traits such as intestinal colonization and fecal-oral route of transmission demands mucosal vaccine development. E. coli secreted protein B (EspB) is one of the key type III secretory system (TTSS) targets for mucosal candidate vaccine due to its indispensable role in the pathogenesis of E. coli O157:H7. However, mucosally administered recombinant proteins have low immunogenicity which could be overcome by the use of mucosal adjuvants. The quest for safe, potent mucosal adjuvant has recognized ΔG fragment of Zonula occludens toxin of Vibrio cholerae with such properties. ΔG enhances mucosal permeability via the paracellular route by altering epithelial tight junction structure in a reversible, ephemeral and non-toxic manner. Therefore, we tested whether recombinant ΔG intranasally co-administered with truncated EspB (EspB + ΔG) could serve as an effective mucosal adjuvant. Results showed that EspB + ΔG group induced higher systemic IgG and mucosal IgA than EspB alone. Moreover, EspB alone developed Th2 type response with IgG1/IgG2a ratio (1.64) and IL-4, IL-10 cytokines whereas that of EspB + ΔG group generated mixed Th1/Th2 type immune response evident from IgG1/IgG2a ratio (1.17) as well as IL-4, IL-10 and IFN-γ cytokine levels compared to control. Sera of EspB + ΔG group inhibited TTSS mediated haemolysis of murine RBCs more effectively compared to EspB, control group and sera of both EspB + ΔG, EspB group resulted in similar levels of efficacious reduction in E. coli O157:H7 adherence to Caco-2 cells compared to control. Moreover, vaccination with EspB + ΔG resulted in significant reduction in E. coli O157:H7 fecal shedding compared to EspB and control group in experimentally challenged streptomycin-treated mice. These results demonstrate mucosal adjuvanticity of ΔG co-administered with EspB in enhancing overall immunogenicity to reduce E. coli O157:H7 shedding.

Intranasal immunization of cocktail/fusion protein containing Tir along with ΔG active fragment of Zot as mucosal adjuvant confers enhanced immunogenicity and reduces E. coli O157:H7 shedding in mice.

Ruminants are the major reservoirs of Escherichia coli O157:H7 and its fecal shedding mainly act as a source of entry of this pathogen into the human food chain. In humans, E. coli O157:H7 infection causes diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. Intimate adherence of E. coli O157:H7 is mediated by Translocated intimin receptor (Tir) to which intimin binds in the host cell. Since E. coli O157:H7 colonizes intestinal epithelium, the mucosal vaccine has a potential to prevent its colonization. Zonula occludens toxin (Zot) of Vibrio cholerae transiently, reversibly alters epithelial tight junction structure to increase mucosal permeability of macromolecules via paracellular route. The C-terminal region of Zot (ΔG) responsible for this function could be used for mucosal antigen delivery. Therefore, we employed individual (Tir), cocktail (ΔG + Tir), fusion protein (ΔG-Tir) and assessed the efficacy of its intranasal immunization on immunogenicity and fecal shedding of E. coli O157:H7 in streptomycin treated mouse model. Compared to control, ΔG + Tir, ΔG-Tir immunized mice elicited significant antigen specific antibody titers in serum (IgG, IgA) and feces (IgA), whereas Tir immunized mice induced only serum IgG titer. Cytokine analysis revealed mixed Th1/Th2 type immune response in case of ΔG + Tir, ΔG-Tir group while that of Tir group was solely Th2 type. Tir, ΔG + Tir and ΔG-Tir immunized mice showed reduction in shedding of E. coli O157:H7 compared to control group. However, ΔG-Tir immunized group performed better than ΔG + Tir, Tir group in reducing fecal shedding. Overall, our results demonstrate that intranasal immunization of ΔG-Tir induces effective systemic, mucosal, cellular immune responses and represents a promising mucosal subunit vaccine to prevent E. coli O157:H7 colonization.

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.

Application of monoclonal antibodies generated against Panton-Valentine Leukocidin (PVL-S) toxin for specific identification of community acquired methicillin resistance Staphylococcus aureus.

Panton-Valentine Leukocidin (PVL) produced by community acquired methicillin Staphylococcus aureus (CA-MRSA) involved in skin and soft-tissue infections and necrotizing pneumonia comprised of two fractions, namely PVL S and PVL F. In the present study, three monoclonal antibodies designated as MAb1, MAb9 and MAb10 were generated against recombinant PVL-S (35kDa) protein of S. aureus. All the three MAbs specifically reacted to confirm PVL-S positive strains of S. aureus recovered from clinical samples in Western blot analysis. Similarly all the three MAbs did not show any binding to other tested 14 different pathogenic bacteria belonging to other genera and species in Western blot analysis. Furthermore, a simple dot-ELISA method was standardized for the identification of PVL-S toxin containing S. aureus strains. Initially in dot-ELISA, Protein A (Spa) of S. aureus posed background noise problems due to the non-specific binding of antibodies resulting in false positive reactions. With the addition of 10mM diethylpyrocarbonate (DEPC) along with 5% milk in PBS in the blocking step prevented this non-specific binding of Spa to mouse anti-PVL monoclonal antibodies in dot-ELISA. Once standardized, this simple dot-ELISA was evaluated with nine PVL positive strains recovered from food, environmental and clinical samples and the results were compared with PCR assay for the presence of PVL toxin genes and also with Western blot analysis. A 100% correlation was found between dot-ELISA, PCR assay and Western blot analysis. Collectively our results suggest the newly developed simple dot-ELISA system can be of immense help in providing, rapid detection of the PVL toxin containing S. aureus strains at a relatively low cost and will be a valuable tool for the reliable identification of CA-MRSA.

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 SE's in different wells of same PCR plate using common DNA substrate.

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.