Establish A Mouse Model Of Vaccine-enhanced Disease(VED) Of Respiratory Syncytial Virus(RSV) And Define Immune Characteristics Of Plasmid DNA Encoding G Glycoprotein Of RSV

Objective Respiratory syncytial virus(RSV) is one of the most important causes of viral lower respiratory tract illness in infants and children worldwide.It can cause bronchiolitis and pneumonia,and it is related to the development of childhood asthma. Vaccination is the most effective measure to prevent and control infection disease.But so far no secure and effective vaccine is available for prophylaxis.Infants and children who were immunized with Formalin-inactivated RSV developed vaccine enhanced disease(VED) when they infected by RSV.Some research indicated that the G protein of RSV purified by biochemistry method and recombinated G protein both can lead to VED effect.Another research reported that the immune response mediated by T lymphocyte of G protein and FI-RSV were different,although they induced similar immunopathology.The relation between G protein and VED should be more studied. In this study,we established a vaccine-enhanced disease(VED) model in mouse immunized with formalin-inactivated human respiratory syncytial virus vaccine (FI-RSV) and challenged by RSV infection.We constructed a plasmid DNA encoding G glycoprotein of respiratory syncytial virus(RSV) and investigated the protective immune response against RSV infection to approach the relation between G protein and VED effect.Methods FI-RSV vaccine was prepared from RSV Al long strain and used to intramuscularly immunize BALB/c mice;samples of lung and sera were collected before and after RSV challenge;virus titre in lung was detected by fluorescence based real time RT-PCR;sections of paraffin embedding lung tissues were stained by haematoxylin and eosin(H&E) for histological analyses;sera anti-RSV IgG levels were examined by ELBA.Recombinant plasmid DNA of pcDNA3.1~G was constructed by standard RT-PCR based cloning procedure.The immunogenicity of recombinant G protein transiently expressed in HEK293 cells was detected by Western Blotting. BABL/c mice were intramuscularly immunized with pcDNA3.1~G.Samples of lung,sera, bronchoalveolar lavage fluid(BAL) were collected before and after RSV challenge; virus titer in lung was detected by real time RT-PCR;sections of paraffin embedding lung tissues were stained by haematoxylin and eosin(HE) for histological analyses;sera anti-RSV IgG levels were examined by ELBA;Th1/Th2 cytokine were detected by ELBA kit,the T lymphocyte subsets of BAL was determined by immunefluorescence staining followed by flow cytometry.Results After challenge,FI-RSV immunized mice presented elevated levels of peribronchiolitis,bronchitis,alveolitis and interstitial pneumonitis as well as pulmonary emphysema in spite of reduced lung viral titres;those mice developed RSV specific IgG in sera,but dramatically dropped down after virus challenge.Plasmid DNA of pcDNA3.1~G was successfully constructed.The expressed target protein possesses immunogenicity.After challenge,pcDNA3.1~G immunized mice presented relieved pathological changes in lung as well as reduced lung viral titers.The RSV specific IgG was detected in sera of immunized mice.There was significantly increased number of CD25~+CD4~+ T cells in mice BAL.Conclusion We have established a mouse model of FI-RSV vaccination enhanced RSV infection which is characterized by increasing in peribronchiolitis,bronchiolitis together with alveolitis and interstitial pneumonitis.We constructed a pcDNA3.1~G plasmid DNA vaccination which can induce evident protective cellular immunity against RSV infection in mice with the increased number of CD25~+CD4~+ T cell subpopulation,and it have not induce VED effect.