Preliminary Study On The Role Of Dengue Virus Non-structure Protein 1 And Its Antibody In The Pathogenesis Of Severe Dengue Virus Infection

Nearly 3.9 billion people around the worldwide at risk of infection with Dengue viruses(DENV).In recent years,the incidence of infection has increased in China.Only in Guangdong Province,about 45,000 cases were reported in 2014.There are four serotypes in DENV.The main clinical manifestation of DENV infection is self-limiting Dengue Fever(DF),while dengue hemorrhagic fever/dengue shock syndrome(DHF/DSS)may occur in a few cases,accounting for about 5%to 10%of DF patients.The incidence is low but the mortality rate is high.However,the pathogenesis of severe dengue infection has not been fully elucidated,although there are some possible causes such as antibody-dependent enhancement(ADE),cytokine storm,complement-dependent cytotoxicity,and so on.Resently,much attention has been paid to the role of dengue virus nonstructural protein 1(DENV-NS1)and its antibodies in the pathogenesis of DHF/DSS.Since a higher concentration of NS1 protein and the corresponding level of IgG antibodies often have been detectecd in the serum of patients with severe dengue virus infection,we focus on whether the immune complexes formed by NS1 and its IgG antibodies could cause shock.In addition,the occurrence of DHF may be associated with anti-NS1 antibodies that can be cross-reactive to platelets and other blood system cells in vivo,resulting in thrombocytopenia and cause bleeding.Based on the work focused on the diagnosis of dengue serology in our laboratory,particularly,148 strains of monoclonal antibodies against 4 serotypes DENV-NS1 were prepared and preserved,a preliminary exploration of the mechanism of shock and bleeding related to severe dengue virus infection of DSS and DHF were performed in this work.The study is divided into two chapters as follows.Part I IgG Antibodies against Dengue Virus Non-structure Protein 1 Mediate Passive Systemic Anaphylaxis in MiceDENV1 NS1 protein was purified by affinity chromatography from concentrated virus supernatant,the immune complexes(ICs)were prepared with purified NS1 protein and the monoclonal antibodies(mAbs)or mAb cocktails from 20 IgG mAbs of DENV1 NS1.The passive systemic anaphylaxis(PSA)and passive cutaneous anaphylaxis(PCA)modes were established by the ICs prepared with two kinds of antibody cocktails,respectively.Meanwhile,the effects of GdCl3 and platelet activating factor receptor(PAFR)antagonist CV-3988 on PSA were observed.The monoclonal antibody cocktails(3B1+5D25)and(3C65+5D25)with purified NS1 were proved to be capable of provoking PCA and PSA in mice.The murine PSA could be significantly inhibited by in vivo treatment with GdCl3 or PAFR antagonist CV-3988.For PSA trigged by IgG pathway,PAF was proved as major effector mediator and monocyte/macrophages are proved as the major effector cells.This could be considered as one of the possible mechanisms of DSS.Part ?.Study on anti-DENV-NS1 antibody binding with human platelets.We identified 27 strains of anti-DENV-NS1 mAbs capable of binding to human platelets from 113 strains of anti-DENV-NS1 mAbs by indirect ELISA,flow cytometry and Westenr Blot.Among them,14 strains were able to bind with protein disulfide isomerase(PDI),and 12 strains were able to recognize the conformational epitopes of platelet membrane protein crude extracts.This result indicated that only a part of mAb againt NS1 can bind with human platelet,and recognize different epitope,which would provide evidence to elucidation of pathogenesis for DHF/DSS,and also some consult for vaccine design.