Immunological Function Analysis Of Bifidobacterium Based Vaccine Expression ETEC-LTB

Enterotoxigenic Escherichia coli (ETEC) infections are a significant cause of diarrheal disease worldwide. It remains major causes of diarrheal morbidity and infant mortality in developing countries, and is also pathogenic bacteria for travelers diarrhea, and perennially associated with disease in soldiers deployed to developing countries. So a safe and effective vaccine against ETEC would be important to public health.ETEC are noninvasive and colonize the small intestines by attachment to mucosa via colonization factors (CFA). Then induce fluid and electrolyte secretion by the small intestinal epithelium in response to the production of heat-labile (LT) or heat-stable (ST) enterotoxins. CFAs are fimbrial adhesins that promote attachment to intestinal epithelium and known to provide protection against infection with ETEC expressing homologous CFs. The most common CFAs are CFA/I; In many areas of endemicity CFA/I is one of the most common CFA expressed by ETEC and so represents an important component of any vaccine. ST is encoded by the estA gene and is a monomeric polypeptide of 19 amino acids and has poorly immunogenic. ETEC strains that express CFA/I almost always express ST. LT is encoded by the eltAB,consisting of a toxic A subunit (LTA) and a pentamer of receptor-binding B subunits (LTB) similar to cholera toxin (CT). CT and LT are reported powerful immunogens and adjuvants and same to the CTB and LTB.ETEC vaccine requires the targeting of virulence factors or antigens common to large numbers of ETEC strains. There is general agreement that a logical vaccine strategy for ETEC would target the three major CFAs of ETEC (i.e.CFA/I, CFA/II, and CFA/IV) as well as the immunogenic LT. Today, the strategies for ETEC vaccine are focused on the three pathways: (a) to enhance traditional vaccine immunity by adding new adjuvants; (b) to develop mucosal immune vaccine and mucosal immune pathway; (c) to develop attenuated or atoxigenic vector expressing ETEC antigens. Bifidobacteria are natural inhabitants of the human intestinal tract and can adhere to the gut. We try to develop it as a gastrointestinal tract administers live vaccine system carrying CFA and LTB of ETEC.Objective: We aimed to construct a bifidobacteria based vaccine expression ETEC CFA/I and LTB, then to analyze its immunity by test the specific antibodies of CFA/I and LTB in SD rats post the vaccine immunize and by measure the survival rate post challenge with ETEC H10407.Methods: (1)Ltb gene was cloned to a shuttle expression vector pBES which originated from plasmid pBV220. This vector was named pBES-LTB and transformed into B. infantis. The product was tested by SDS-PAGE and its safety test by ansa intestinalis test .(2)Immunizing SD rat by recombined B. infantis–LTB (or CFA/I) vaccine. The rats were randomly placed in four groups of 12: group 1:PBS, group 2: pBES-LTB , group 3. pBES- CFA/I ,group 4:B. pBES-LTB + pBES- CFA/I. The above groups of rats were immunized intragastricly three times at 0d, 10d and 17d. Blood and fecal pellets were collected from rats at 0d, 7d, 10d, 14d, 17d, 22d and 27d. Specific antibodies were detected in sera and fecal pellets of rats by ELISA. (3) At 27d, half of SD rats in each group were challenged with a lethal dose of ETEC strain H10407 through abdominal cavity injection, and counting the mortality; Others were challenged intranasally with ETEC H10407(2×1010CFU).Results: (1) The ltb gene could stable expressed in B. infantis and the product (LTB) has a gentle toxicity of inducing intestinal juice secretory in rabbit by ansa intestinalis test. (2) The LTB,the CFA/I and the two antigens co-immunization all induced strong serum IgG and fecal IgA . The serum IgG and fecal IgA antibody from group 4 (oral inoculation with bifidobacteria–LTB + bifidobacteria–CFA/I) were significantly greater (P<0.05) than the antibody from other groups; But there were no significantly greater (P>0.05) between the group 2 (LTB) and the group 3 (CFA/I). (3) In the experiment of immunization protection assays , the SD rats got a significant protection, which immunized with bifidobacteria -LTB + bifidobacteria -CFA/I. But the LTB group and PBS group had no protection.Conclusion: (1)Bifidobacteria can be developed as oral vaccine expression system. (2) The bifidobacteria-LTB can induce specific antibodies via oral immune. (3) When bifidobacteria-LTB co- a bifidobacteria-LTB vaccine enhances bifidobacteria-CFA/I immune responses significantly as well as improves the survival rate of SD rats challenged by ETEC. This recombined bifidobacteria based vaccine pave a smooth way for ETEC vaccine development.