Effect And Mechanism Of Mucosal Immune Adjuvant On Mucosal And Systemic Immune Responses In Chicken

Most of infectious diseases spread through the digestive and respiratory tract. Some experiments showed that orally mucosal immunization could induce mucosal and systematic immune responses. However, oral vaccination need more and repeated antigen doses to achieve the protective immune response level because the antigen degradation by gastric acid and proteases which present in the gastrointestinal tract. In recent years, many researchers have found that mucosal immune adjuvant could increases the level of antibody in mucosal and systematic immune. However, to choose the correct adjuvant is hard because of different types and effects of adjuvant. Therefore, they are urgent to identify the functional mechanism of adjuvant and develop high-effect and safe mucosal immune adjuvant. Five mucosal immune adjuvants (daidzein, CpG DNA, recombinant IL-2 (rlL-2), lactic acid bacteria(LAB) and sodium fluoride(NaF)) were selected as object and their effects and mechanism were studied systemically in the research. The better effective adjuvants of them were compounded and the development of new-type mucosal immune adjuvant and the part mechanism of it were worked out. The details were divided into five parts as follows:1. Effects of different mucosal immune adjuvants on local mucosal and systematic immune responses in chickenChicken were orally vaccinated by ND vaccine containing different mucosal immune adjuvants (daidzein, CpG DNA, rIL-2, LAB, NaF). The changes of mucosal and systematic immune response were observed by histochemical and serological methods and differences of effects and part mechanism among adjuvants were worked out. Our results showed that (1)the numbers of intestine intraepithelial lymphocyte (ilEL) of Group daidzein, CpG DNA and rlL-2 were gradually increased during the whole immune period, and significantly higher than that of Group ND at week 5(P<0.01), the number of ilELs were the most at week 7. (2)the adjuvants could activate mast cells in mucosal lamina propria. And the effects were different in the type of adjuvant and part of intestine, the better one was LAB in the duodenum, CpG DNA in the jejunum and CpG DNA and LAB in the Peyer's patch. (3)markedly increased numbers of intestine IgA secreting cells in daidzein, CpG DNA, rlL-2 and LAB were found. The effect of LAB was significantly in the initial stage of immunization. (4)significantly raised level of SIgA in mucosa in daidzein and CpG DNA, especially in the final phase of immunization. (5)the titers of ND specific antibody in serum were elevated in Group daidzein, CpG DNA, rlL-2 and NaE The NaF could keep the level of antibody during the whole immune period.2. Mechanism of different mucosal immune adjuvants on antigen presentingIn order to study the mechanism that adjuvant activate immune system and the way of presenting antigen, the change of dendritic cells (DC) and MHCâ…¡molecular in daidzein, CpG DNA and LAB were determined by rat legated and perfused intestinal segments model. Our results showed that (1)significantly increased numbers of DC in intestine were found in CpG DNA and LAB. It demonstrated that adjuvants could activate DC, promote antigen presenting and induce specific immune responses in local mucosa. (2)the expression of MHCâ…¡molecular in Group adjuvants were markedly increased within 24 hours. It demonstrated that adjuvants could induce the expression of MHCâ…¡molecular in antigen presenting cells.3. Effects of different mucosal immune adjuvants on nuclear transcription factorIn order to study the pathway that adjuvant activate immune system, the change of transcription factor NF-κB in daidzein, CpG DNA and LAB were determined by rat legated and perfused intestinal segments model. Our results showed that (1)significantly increased expression of NF-κB protein in CpG DNA, especially in 0.25 hour. It indicated that CpG DNA could activate the transcription of NF-κB, induce T and B lymphocytes reaction and enhance the nonspecific immune responses. (2)significantly increased expression of NF-κB protein in LAB with 6 hours. It indicated that LAB could also activate NF-κB pathway. (3) no significant changes were found in daidzein. It demonstrated that the transduction factor in cAMP/PKA signal transduction pathway of daidzein may not NF-κB.4. Effects of compound mucosal immune adjuvants on local mucosal and systematic immune responses in chicken According to the study on effect and mechanism of different adjuvants, we investigated the adjuvant effects of two kinds of new compound mucosal immune adjuvant (cMIA, cMIAâ… and cMIAâ…¡) with respect to mucosal and systemic immune responses. cMIAâ… was made by daidzein, LAB and NaF, cMIAâ…¡by CpG DNA and NaF. Chicken were orally vaccinated by ND vaccine containing cMIAs. The changes of immune response were observed by histochemical, molecular biological and serological methods and differences of effects and mechanism among compound adjuvants were worked out. Our results showed that (1)significantly increased number of ilELs, CD3⁺ T lymphocytes and mast cells were found in cMIAs. The expression of IFN-γmRNA were decreased, and no markedly changes were found in cMIAs. It demonstrated that cMIAs could induce the cellular immune responses in local mucosa. The effect was different between them in different intestine fragments. The better one was cMIAâ…¡in the jejunum. (2)significantly increased number of IgA secreting cells in intestine and level of SIgA in intestine content, but there were no markedly changes were found in SIgA in dejecta. It indicated that cMIAs could induce humoral immune responses in local mucosa, and the effects were kept during the whold immune period. (3)significantly elevated level of specific IgG, IgA in serum were found in cMIAs. It demonstrated that cMIAs could induce systematic immune reponses, and cMIAâ…¡was better in keeping the level of antibody.5. The investigation on the ability for resisting the challenge of ND F₄₈E₂ strong virusIn order to study the protection of cMIAs in animals, chickens were vaccinated orally with ND vaccine containing different cMIA and orally challenged by ND F₄₈E₂ strong virus. Our results showed that 2 chickens in Group cMIAs had light nervous symptoms at Day 4 after challenge (D4), the others were normal; the livability in Group cMIAs was 100%. 3 chickens in Group ND died at D4, 2 at D5, all died at D6. The chickens in Group control all died until D4. It demonstrated that chicken vaccinated with cMIAs could resist the challenge of ND strong virus at the level of antibody (≤4 log2). The cMIAs can be used as a new-type mucosal immune adjuvant in industry.