The Biological Analysis And Immunogenicity Evaluation Of A New Varicella Attenuated Live Vaccine VZV-7D

Varicella-zoster virus(VZV)is a pathogen that can couses chickenpox(varicella)and shingles(herpes zoster,HZ).Chickenpox is caused by primary infection with VZV,then the virus can establish long-term latency in the host ganglia,in the case of trauma,fatigue,senility,immunosuppression and other conditions will trigger reactivation and couses herpes zoster.Varicella is a global epidemic with highly contagious and frequently found in children.Herpes zoster mostly occurs in the middle-aged and elderly people and usually accompanies with a strong neuralgia.Currently,there is still no specific medicine for cure VZV-induced diseases.Vaccination is among the most cost-effective measures for preventing chickenpox and shingles.The Oka strain of live attenuated VZV(vOka)is now the only varicella-zostervaccine on the market.Although studies have shown that vOka is impaired for replication in human skin,vOka can infecthuman neurons and establishlatency like the wild-type virus,and thus poses security risks.Therefore,there is a strong need for a safer and more effective new-type varicella vaccine.In the previous study,VZV orf7 had been identified that it is a decisive influence on the VZV infection in human skin and ganglion tissue.Subsequently,VZV-7D attenuated live vaccine has been made as a new type of VZV vaccine.In this study,I carried out the biological analysis and immunogenic ity research of the new VZV-7D vaccine to provide important basis and support for the follow-up clinical study and clinical trial of the new VZV-7D vaccine.In the first part of this study,variety of methods were applied for biological analysis of VZV-7D that centre on mutation characteristics of VZV ORF7,particle assembly and structure of VZV virion and the expression of main antigen protein.Firstly,this study applies MGB real-time fluorescent PCR technology,double antibody sandwich enzyme-linked immunosorbent method,western blotting and immunofluorescence technique to analysis and determine the mutation of ORF7 expression deletion in VZV-7D on gene level and protein level.After that,the TME was used to observe VZV-7D infected ARPE-19 cells and purified VZV-7D complete virus particles to analysis the procedure and location of the virus in the infected cells and the structure of the virus particles,find that there is no significant difference between VZV-7D and vOka.Finally,the differences of protein expression between VZV-7D and vOka were analysised by western blotting,showed that the expression of main viral antigen proteins including glycoprotein,main capsid protein and typical cortical proteins of VZV-7D are the same as vOka.This indicates that VZV-7D can normally express the main antigen protein of VZV.In the second part,the immuno genicity of VZV-7D was evaluated in this study.Using the same dose of VZV-7D and vOka to immunize the Balb/c mice,SD rats,guinea pigs and rabbits to evaluate the level of humoral immunity and cellular immune response.In the humoral immune response,the gpELIS A and plaque reduction test were used to detect anti-VZV antibody of animal serum after immunization.The results show that the titer of VZV antibody in animal serum was significantly increased after immunization,and the level of humoral immune response that VZV-7D activated was comparable with vOka.Then the analysis result of IgG subtypes shows that the immune response that activated by VZV-7D and vOka both are TH2 cell-mediated humoral immune response.In cellular immune response,different antigens were used to stimulate the spleen cells of mice and rats,and then the secretion of cytokines was detected.Test results display that various inflammatory correlation factors and antiviral factors of splenocyte from mice and rat that immuned by VZV-7D and vOka were significantly higher than those of the negative control group,such as IL-6?TNF??FN ?,and the elevated level are the same between with VZV-7D and vOka.It is shows that the immunogenicity of VZV-7D and vOka in activate cellular immunity are the same.In summary,in this study,the biological analysis of VZV-7D was carried out,and the method system applied to VZV-7D biological analysis was preliminarily established,which provided theoretical basis and technical support for the quality control analysis of VZV-7D.In addition,this study also analysis and determine that VZV-7D and vOka have the same immuno genicity That provides some research foundation for the preclinical study and assessment of immune efficacy of VZV7D.