The stability and immunogenicity of formalin-inactivated Enterovirus A71 whole virion vaccine after ten years of low temperature storage.

BACKGROUND: Vaccine stability is an important issue for vaccine development, which affects whether the vaccine product is effective within a certain period of time in each progress. Hand, foot, and mouth diseases (HFMD) is an epidemic disease in young children usually caused by Enterovirus A group viruses, and the Enterovirus A71 (EV-A71) had caused several pandemics and public health issues around the world. After two decades of research and development, formalin-inactivated EV-A71 (FI-EV-A71) vaccines are the first to complete the phase III clinical trials for protection against EV-A71 infection. Currently, the shelf life of FI-EV-A71 vaccine product is set to be within 18 months, but the stability and the effectiveness of the FI-EV-A71 whole virion when stored long-term at low temperature remains undetermined. METHODS: Assessing the long-term storage properties of viral particles facilitates flexibility in manufacturing of vaccine products. In this study, the stability profiles of FI-EV-A71 vaccine lots and bulks after long-term of low temperature storage were analyzed by protein tests, particle measurement and animal immunization study. RESULTS: After over ten years of storage, the reduction of protein concentration in the FI-EV-A71 bulk samples is less than 30 % and the antigenic content remained in a suspended, particulate state. Both the packed FI-EV-A71 final vaccine products and the FI-EV-A71 antigens adjuvant premix bulk could elicit strong neutralizing responses in mice. CONCLUSION: After ten years of low temperature storage, the FI-EV-A71 vaccine still presents decent stability and good immunogenicity.

Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system.

Coxsackievirus A10 (CVA10) is one of enteroviral pathogens that cause the hand, foot, and mouth disease (HFMD). Since CVA10 was reported to be not easily propagated in the Vero cell culture, a feasible manufacture process for producing formalin-inactivated CVA10 vaccine is urgently needed. Several cell lines that commonly used for viral vaccine production was tested for CVA10 (M2014 strain) culture in this study, and our result showed that CVA10 could be easily propagated in the HEK293A cells. A serum-free HEK293A cell culture system was developed for CVA10 production and the yields have reached over 108 TCID50/mL. The biochemical and immunogenic properties of CVA10 particles obtained from this serum-free HEK293A culture were identical to our previous study. Two major particles of CVA10 were separated by ultracentrifugation, and only the infectious mature particles were capable of inducing CVA10 neutralizing antibody responses in the mouse immunogenicity studies. Additionally, we found that coxsackievirus A6 and enterovirus A71 could also be easily propagated using this serum-free HEK293A cell culture system. Our results provide a solution to overcome the obstacle in the propagation of CVA10 and facilitate the development of multivalent vaccines for prevention of HFMD.

Intranasal Immunization with Zika Virus Envelope Domain III-Flagellin Fusion Protein Elicits Systemic and Mucosal Immune Responses and Protection against Subcutaneous and Intravaginal Virus Challenges.

Zika virus (ZIKV) infections in humans are mainly transmitted by the mosquito vectors, but human-to-human sexual transmission is also another important route. Developing a ZIKV mucosal vaccine that can elicit both systemic and mucosal immune responses is of particular interest. In this study, we constructed a recombinant ZIKV envelope DIII (ZDIII) protein genetically fused with Salmonella typhimurium flagellin (FliC-ZDIII) as a novel mucosal antigen for intranasal immunization. The results indicated that the FliC-ZDIII fusion proteins formulated with E. coli heat-labile enterotoxin B subunit (LTIIb-B5) adjuvant greatly increased the ZDIII-specific IgG, IgA, and neutralizing titers in sera, and the ZDIII-specific IgA titers in bronchoalveolar lavage and vaginal fluids. Protective immunity was further assessed by subcutaneous and intravaginal ZIKV challenges. The second-generation FliCΔD3-2ZDIII was shown to result in a reduced titer of anti-FliC IgG antibodies in sera and still retained the same levels of serum IgG, IgA, and neutralizing antibodies and mucosal IgA antibodies without compromising the vaccine antigenicity. Therefore, intranasal immunization with FliCΔD3-2ZDIII fusion proteins formulated with LTIIb-B5 adjuvant elicited the greatest protective immunity against subcutaneous and intravaginal ZIKV challenges. Our findings indicated that the combination of FliCΔD3-2ZDIII fusion proteins and LTIIb-B5 adjuvant for intranasal immunization can be used for developing ZIKV mucosal vaccines.