Oral Immunization against ETEC with Recombinant Protein-Loaded Chitosan Nano-Structure and its Immunogenicity in Comparison with Subcutaneous Vaccine

Background: Enterotoxigenic E. coli (ETEC) can be considered the main cause of traveler’s diarrhea, which is affecting children in developing countries. The bacterium has several virulence factors, including colonization factors (CFs), heat-labile (LT), and heat-stable (ST) toxins. The World Health Organization has designated the development of an ETEC vaccine one of its top goals due to the disease's rising antibiotic resistance and deteriorating access to sources of clean drinking water. Objective: The objective of this study is to investigate the oral immunogenicity of chitosan nanoparticles (CNPs) encapsulated CCL protein containing CfaB along with STa toxoid, CfaE, and LtB. Methods: The E. coli BL21DE3 harboring pET-28a-ccl vector was used for protein expression. After purification and confirmation, the protein was encapsulated in CNPs and the particle size was measured. Immunogenicity was assessed by evaluating antibody titers after BALB/c mice vaccination. Finally, the neutralization efficiency of immunized mice sera was evaluated by a rabbit ileal loop test. Results: The purified protein (~57kDa) was confirmed by Western blotting and the size of CCLCNPs was measured with an average of 112.0nm with 98.8% of encapsulation efficiency. CCLCNPs are able to stimulate the immune system by providing suitable titers of antibodies. The fluid accumulation in the rabbit’s intestine was significantly reduced. Conclusion: The CCL-CNPs can be considered a candidate for producing oral nanovaccine.