Construction Of Edwardsiella Tarda Efficient Vaccines And Preliminary Study Of The Immune Mechanisms

Bacterial pathogens are one of the major causes of aquaculture diseases.Edwardsiella tarda is a Gram-negative bacterial pathogen and the etiological agent ofa systematic fsh disease called edwardsiellosis, which affects a wide range of marineand freshwater fsh. The disease caused is often fatal and can lead to heavy economiclosses.Based on this situation, we in this study have designed and constructed vaccinesagainst E. tarda and examined the protective effects of the vaccines in a Japaneseflounder (Paralichthys olivaceus) model.We have so far constructed several different types of vaccines, which includeinactivated whole cell vaccines, attenuated live vaccines, DNA vaccines, cross-genusvaccines and recombinant subunit vaccines delivered by a live bacterial host. Thesevaccines proved to produce high levels (relative percentage survival greater than70%)of protection against E. tarda. We have identfied several E. tarda antigens, Esa1,Eta2and Eta1.Esa1is a D15-like surface antigen, which, when used as a recombinant subunitvaccine and DNA vaccine, are able to induce protective immunity in Japanesefounder (Paralichthys olivaceus) against E. tarda challenge. Taking advantage of thesecretion capacity of Esa1and the natural gut-colonization ability of a fsh commensalstrain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Vaccinationanalyses showed that FP3/pJsa1as an intraperitoneal injection vaccine and an oralvaccine embedded in alginate microspheres produced relative percent survival rates of 79%and52%. We examined further the immunoprotective potential of Esa1as aDNA vaccine. The DNA vaccine plasmid pCEsa1was constructed. Hence, comparedto PBS-vaccinated fsh, pCEsa1-vaccinated fsh were signifcantly protected, with aRPS of75%and71%respectively, at one-and two-month post-vaccination (p.v.).Together these results indicate that Esa1is a protective immunogen and an effectiveoral vaccine when delivered by FP3/pJsa1as a surface-anchored antigen.Eta2, a new E. tarda vaccine candidate, we have analyzed in a comparativemanner the immune response induced by Eta2in two different formfes:d purirecombinant subunit vaccine and DNA vaccine. Recombinant Eta2(rEta2) purfiedfrom Escherichia coli was highly protective against E. tarda challenge in a Japanesefounder model and produced relative percent of survival rates of83%and78%,respectively, at4-and8-week post-vaccination (p.v.). Eta2as a DNA vaccine in theform of plasmid pCEta2also induced strong protective immunity at4-and8-week p.v.Immunological analysis indicated that (i) rEta2and pCEta2enhanced head kidneymacrophageactivation;(ii) rEta2and pCEta2induced similar patterns of serumantibody production;(iii) both rEta2and pCEta2upregulated the expression ofspecifc and nonspecifc immune factors which include. Taken together, these resultsindicate that both rEta2and pCEta2induce sfpce ciand nonspecifc immunities,however, pCEta2induces both B cell and T cell responses, whereas rEta2inducesmainly humoral response.Eta1, we identifed, via in vivo-induced antigen technology. eta1expression wasgrowth phase dependent and reached maximum at mid-logarithmic phase. Duringinfection offounder lymphocytes, eta1expression was drastically increased at theearly stage of infection. Compared to the wild type, the eta1-defective mutant, TXeta1,was unaffected in growth but exhibited attenuated overall virulence, reduced tissuedissemination and colonization capacity, and impaired ability to infvoaduenderlymphocytes and to block the immune response of host cells. Furthermore, when usedas a subunit vaccine, rEta1produced relative percent of survival rates of83.3%infounder against lethal E. tarda challenge. Taken together, these results indicate thatEta1is an in vivo-induced antigen that mediates pathogen-host nteraction and, as a result, is required for optimal bacterial infection.In this study, we also have selected TX5RM, which is an attenuated E. tardastrain with good vaccine potential and that a combination of oral and immersionvaccinations may be a good choice for the administration of live attenuated vaccines.The results showed that TX5RM administered via all four approaches producedsignifcant protection, with the highest protection levels observed with TX5RMadministered via oral feeding plus immersion, which were, in terms of relative percentof survival (RPS),80.6%and69.4%at5-and8-week post-vaccination, respectively.We have constructed a cross-genus vaccine by takeing advantage of theresidual infectivity of TX5RM and using it as a carrier host for the natural delivery ofa S. iniae DNA vaccine. The recombinant TX5RM, TX5RMS10, was created, whichharbours and retains stably the DNA vaccine plasmid pCS10that expresses Sia10.Following E. tarda and S. iniae challenge at one and2months p.v., the vaccinatedfsh exhibited relative percent survival rates of69–83%. Immunological analysisindicated that TX5RMS10-vaccinated fsh produced specifc serum antibodies andexhibited enhanced expression of a wide range of immune genes.In addition, we also examined the immune response of the vaccines and foundthat DNA vaccines stimulate stronger systematic immunity than subunit vaccines.These findings promote the research on the marine fish vaccines and provide insightsto the protection mechanism of vaccines.