Construction And Comparative Evaluation Of Chlamydia Abortus Subunit Candidate Vaccine RVCG-Pmp18N In A Mouse Model And Immune Mechanism Of Dc Pulsed With Pmp18N

Chlamydia abortus (C. abortus) is an obligate intracellular gram-negative bacterium, and a major cause of abortion in sheep, goats, pigs, horses and cattle leading to considerable economic losses in animal husbandry worldwide. On the other hand, C. abortus also poses a zoonotic risk in pregnant women. To date, the commercial vaccine, based on an attenuated temperature-sensitive strain IB, was used to prevent and control the abortion caused by C. abortus in sheep and goats. But the protection was not complete and there was potential risk for the strain to be virulent, underlining the need for the development of safe effective vaccines.The Polymorphic outer membrane protein (Pmps) of C. abortus, which have been associated with virulence, have been identified in several pathogenic Chlamydiae and resemble autotransporters of the type V secretion system. Pmps are highly immunogenic and there has been much interest in their exploitation as vaccine and diagnostic candidates. The previous work in our lab has proved the diagnostic value of Pm1l8N, an indirect ELISA with high specificity and sensitivity was established to check clinical serum. In this study, we try to evaluate the immunogenicity of Pmp18N with three different kinds of formulae. Furthermore, the machanism of DC pulsed with Pmp18N in vitro was analysed, which was valuable for the relevant vaccine development and design. The work was described in detail as following.1. Constraction of rVCG-Pmp18NThe Pmp18N gene was cloned into pSTV66to generate pST-18N and expressed in Vibrio cholera V912. The protein expression was induced by5.0mM IPTG, and the lysis was induced by5.0mM3-MB. After washing with different reagents, the rVCG-Pmp18N was lyophilized. Totally, we got518.90mg lyophilized powder made in two batches. The Western Blot results showed that the Pmpl8N could be highly expressed in Vibrio cholera and delivered by rVCG2. Evaluation of Pmp18D based vaccines with different formulaeThree different kinds of vaccines, including rPmpl8N+CpG+FL, rVCG-Pmp18N, and rVCG-Pmp18N+FL, were evaluated for their abilities to stimulate host immune responses. The results showed that rVCG-Pmp18N could stimulate humoral, cellular, and mucosal immunity against C. abortus infection. There is no neccesory to add any adjuvant because of the adjuvant property of VCG itself, the efficiency of VCG as an adjuvant is higher than CpG+FL. If FL was added as a co-delivery adjuvant with VCG, a better immune response could be induced to protect the mice from C. abortus infection. Furthermore, these self-adjuvanting properties, ease and low cost of production and absence of a cold chain requirement are invaluable for the rapid development and production of a cost-effective C. abortus vaccine for veterinary use.3. The immune mechanism in vitro of DC pulsed with Pmp18NRecombinant Pmp18N is able to promote the maturation of DC when DC pulsed with Pmp18N in vitro. The expression of co-stimulate moleculars CD40, CD80, CD86, and MHC II, are highly up-regulated, and the concentration of cytokines IL-1β、IL-6, IL-12p70, and chemokine MCP-1in the cell supernatant also increase, underlining that Pmp18N promoted T cell proliferated to Thl type immune response, which had been proved to be neccesery for Chlamydia immunity. This result was incident with the high concentration of IFN-y produced by T cells in vivo. Furthermore, the mechanism of phenotypic change of DC pulsed with Pmp18N was investigated.In conclusion, Pmp18N could increase the innate immunity via TLR4/MyD88/NF-KB signaling pathway and NLRP3inflammasome activation, and regulate the adaptive immunity through the T cells activation, so that it could be used as a vaccine candidate against C. abortus infection.