The Enhancement Of DNA Mucous Vaccine By HMGB1for Viral Myocarditis And Its Mechanism

Viral myocarditis (VMC), an infectious myocardial disease, is a common clinicalcardiovascular system disease caused by infections of coxsackievirus, ECHO, polio,adenovirus40,41, the influenza virus and so on. Among enterovirus infection,coxsackievirus especially group B type3(CVB3), is the most common infection. Atpresent there is no effective treatment for VMC and it is necessary to develop anefficient vaccine for VMC. Because CVB3mainly enter the body via gastrointestinalmucosa, induction of effective mucosa immune response for control of viral infectionand subsequent myocarditis has the important meaning. Considering mucosal vaccinesare the most effective way to induce mucosa immune response, the CVB3specificmucosal vaccine become an important strategy for the prevention and treatment of viralmyocarditis.Our laboratory previously generated the eukaryotic expression plasmid encodingCVB3advantage antigen VP1. Through the intranasal immunization, it induced acertain degree of immune response and protected more than30%of CVB3infectedmice from dying. However, the immune effect still needs to be further strengthen andthe improvement of protective effect of this vaccine will be the focus in this study.Here, we described HMGB1molecule could be a new immune mucosa adjuvantwith VP1encoding plasmid packaged with chitosan for intranasal immunization. Theserums of immunizated mice were collected timely and test for the immune responseduring immunization. Results showed that the co-immunization group (VP1/HMGB1)mice had systemic high level of serum IgG than the VP1or HMGB1individual groupand the control vector group. At the same time, in order to verify the immune responsewas Th1or Th2response, we tested the subtypes of IgG: IgG2a and IgG1, and foundthat expression of Ig2a in serum was highter than IgG1indicating that the body immuneresponse tended to be Th1response as well as the VP1immunization group. However,HMGB1as a mucosal adjuvant can enhance Th1immune response as increased both of IgG2a and IgG1levels comparing to VP1immunization group. Therefore, it seemsmore beneficial for the body against virus. Moreover, HMGB1induced high level ofintestinal secretion IgA antibody which is important to clean the virus at intestinal tract.Cytotoxic T cell (CTL) respone is very important to mediate antiviral function, sowe took and measured the spleen and mesenteric lymph node CTL cells in immunizedmice. Results showed that HMGB1co-immunzaition group (VP1/HMGB1) CTLfuction was higher than that of individual immuization group or vector group in themesenteric lymph nodes or spleen, suggesting that HMGB1can effectively enhance Tcell CTL function.The IFN-γ secreted by lymphatic cells also plays an important role for cleaningvrius. In order to verify HMGB1as a new mucosal adjuvant could affect T cells forIFN-γ secretion, we tested IFN-γ secretion in the spleen cells and mesenteric lymphnode cells immunized mice. ELISPOT results showed HMGB1co-immunization groupwas304SFC/106is obviously higher than other group mice(VP1group isSFC188/106;HMGB1group is46SFC/106；vector group is9SFC/106). IFN-γ+T cellsfrequency plays a important role in cleaning the virus in the body. We also investigatedthe ablility of spleen and mesenteric lymph node T cells proliferation by Brdu assay.Itwas found that HMGB1co-immunization group(OD370nm spectrophotometry was1.24)of mice spleen lymphocyte spectrophotometry was higher than any other group of micespleen lymphocyte (VP1OD370nm of mice in spectrophotometry was0.75; theHMGB1mice OD370nm place spectrophotometry was0.36; the mice OD370nm vectorgroup in absorbency of0.25) proliferation rate. At the same time the mesenteric lymphnode lymphocyte of co-immunization group(OD370nm spectrophotometry in0.72)proliferation rate is also significantly greater than the other group mice (VP1groupOD370nm of mice in spectrophotometry was0.36; HMGB1group OD370nm placespectrophotometry was0.16; the vecor OD370nm in absorbency was0.10) mesentericlymph cell proliferation. Togenther, HMGB1can enhane humoral Th1response, CTLfunction, T cell proliferation and IFN-γ serration upon co-immunized with VP1DNAvaccine in musoca.In order to evaluate the protective effects of HMGB1co-immunization, we monited28days survival rate of fours groups mice immunized with HMGB1/VP1, VP1,HMGB1and control vector followed by leatheal dose CVB3infection two weeks afterimmunization. Suvrial rate of HMGB1co-immuzation group was80%, greater thanthose of VP1immunized mice60%, HMGB1group50%. To be note, all of the miceinfected with CVB3died10days post infection. Our results indicated that HBGB1asmucosa adjuvant impoves the protection effects of CVB3DNA vaccine upon viralinfection. Heart tissue of pathological slices demonstrated that less inflammation cellassault and better myocardial structure were observed in HMGB1co-immunizationgroup indicating the pathological changes between these groups.In order to further understand the way with which HMGB1works as mucosaladjuvants, we examined the dendritic cell (DC) maturation in spleen and mesentericlymph node by CD80and CD11C double staining. Flow cytometric detection showedthat co-immuization with HMGB1could promote DC maturation which might lead tothe potent immune reponse against viral infection.In this study, we detected enhanced humoral immune response as well as cellularimmune response with HMGB1as immune adjuvant in CVB3DNA vaccine. We alsoachived better survival rate in HMGB1co-immunized mice after viral infection. Furtherstudy indicated that HBGB1was able to promote spleen and mesenteric lymph node DCmaturation. All of these results indicate HMGB1has its unique features and could be anovel adjuvant for DNA musoca vaccine development.