Abstract
BackgroundIn our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant.Key ResultsIn the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPO® vector and expressed using induction with isopropyl thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines.ConclusionThese results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associated with the marked protective efficacy of immunized mice after intranasal shigellae infection.
Highlights
Shigellosis, an important etiological agent of bacillary dysentery in humans is caused by Shigella, a Gram-negative bacterium which belongs to the family Enterobacteriaceae [1]
These results suggest that the Outer Membrane Protein A (OmpA) of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses
To confirm that the 34 kDa outer membrane protein (OMP) of S. flexneri 2a is OmpA, the pore-forming activity of 34 kDa OMP from S. flexneri 2a was determined by reconstitution into proteoliposomes and by the osmotic swelling of these vesicles detected by following the optical density of the suspension
Summary
Shigellosis, an important etiological agent of bacillary dysentery in humans is caused by Shigella, a Gram-negative bacterium which belongs to the family Enterobacteriaceae [1]. The World Health Organization has given high priority to the development of a safe and effective vaccine against shigellosis [1]. Many approaches for the development of Shigella vaccines have been attempted [4,5,6,7,8,9,10]. The current vaccine candidates are either not sufficiently attenuated or immunogenic enough [5,11], demonstrating the identification of further attenuation or novel protective antigen is indispensable. Immunoproteome analysis of S. flexneri has shown that more protective antigens, which can be screened from immunogenic outer membrane protein, may be selected as vaccine candidates [12,13,14]. In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant
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