| Hand, foot and mouth disease (HFMD) is an acute infectious disease worldwide and its emerging epidemics are serious in the Asia-Pacific regions in recent years. HFMD has became an important public health problem endangering health of infants and children in China now.Coxsackievirus A16 (CA16) and Enterovirus 71 (EV71) are the major pathogens responsible for human cases of HFMD. Researchers paid more attention to the EV71, but as demonstrated in recent studies, CA16 also leads to severe diseases and death. However, there is no effective treatment and prevention method for HFMD caoused by CA16 until now. Vaccine is hoped to be the most important tool to effectively control the HFMD. Our study identified the characteristics of CA16 candidate attenuated strains and the production process.Two CA16 cold-adapted candidates with high immunogenicity were cultured by continuous passage and their characteristics of all generations were assessed. The result as follows:(1)Two cold-adapted candidates were obtained with stable growth characteristics, the infectious titers of KM154 fluctuate within 0.5LgCCID50/ml. (2) The cold-adapted strains have good immunogenicity, Antibody positive sero conversion rate reach 100%; Maximum GMT reach 1:30; and the immunizing dose was 0.5ml. (3) High-passaged strains weakened pathogenicity of neonatal rats. Mortality and morbidity of KM 154 was significantly reduced after 12 generation. CA16 can be detected in most of the tissues in both newborn mice and 4-week mice, and the pathological examination showed inflammatory cells infiltration, whereas no tissue damage. (4) Full genetic sequence detection result showed that compared with KP1, cold-adapted strains generally keep genetic stability by continuous passage cultured. (5) in cross-protection text:we found the rabbit anti-CA16 serum can neutralize multiple strains of CA16, and protection rate reached 100%. However, the anti-CA16 serum can not neutralize EV71. (6) newborn ICR mice (i.p.) and four-week ICR mice (i.e.) infected by two cold-adapted trains can found the CA16 virus in brain, spleen, kidney, lung, liver, heart muscle and spinal cord. The result indicated that CA16 virus can be replication and spread in mouse.The second part is a basic study of manufacturing technique, study on influencing factor of strain stability revealed that, freeze-thawing within 10 times had no influence on infective titer of CA16 strains. The infective titer are still 6.58LgCCID5o/ml when strains kept in-70℃and liquid nitrogen. When CA16 strains put with MgCl2and No gelatin freeze, the infective titer declined in an obviously slow speed, Join 0.5 M MgCl2 only dropped 1.4 LgCCID50/ml to the seventh day. Study on different purification processes showed that cold-adapted trains extracted by chloroform had a better recovery on infective titer and antigen content, and it can stimulated a higher antibody level in mouse body. The result reveals that Chloroform extract is a more effective virus purification method. The experiment also found that both the protein content and degree of attenuated infective titer have influence on immunogenicity of strain. Protein content and infective titer with high degrees of strains showed better immunogenicity.This study had obtained one cold-adapted candidates, which with stable growth characteristics, good immunogenicity and genetic stability by continuous passage cultured in KMB17 cells; the anti-CA16 serum had a broad spectrum protection to CA16. We have been studied preliminarily on the thermal stability of strain, purification methods. All of this results had laid a solid foundation for the development of CA16 live attenuated vaccine research and production. |
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