The Selection Of Candidate Strain Of Attenuated Live Coxsackievirus A16Vaccine

Coxsackievirus A16(CA16) is one of the most important pathogens that can cause hand, foot and mouth disease (HFMD). The main clinical symptoms are fever, rash or herpes on hands, feet, mouth and other body parts of infants. CA16infection also can induce aseptic meningitis, encephalitis, acute flaccid paralysis, neurogenic pulmonary edema and myocarditis, and even death. In recent years, the incidence of HFMD shows an increasing trend. So, HFMD becomes a serious threat to the health of infants and young children.Currently, there is no effective drug for HFMD treatment. Although the prognosis of the most patients is good, a few severe cases accompany with myocarditis, encephalitis and so on. Thus, vaccine is the most effective strategy of preventing such disease. The results of Enterovirus71(EV71) vaccine clinical trials to verify the protective effect were very satisfactory. The EV71vaccine is expected to be used for the prevention of HFMD that caused by EV71. In order to fully prevent the occurrence of HFMD, how to develop CA16vaccine has become an urgent problem to be solved.In this study,7CA16strains isolated from stool and throat swab of clinical specimens that were collected from HFMD patients, and another3CA16strains (FY18, KMM/08, KM208) provided by other lab were studied. The whole sudy was divided into two parts.The first part is designed to research the biological characteristics of CA16strains. In this part, we analyzed the biological characteristics of the10CA16strains, including the growth characteristics, the plaque morphology, the virulence and the pathological changes in suckling mice. The genotype of FY18strain is A type, the other9strains belong to B1subtype, and5are B1b type,4are B1a type; By analyzing the amino acid sequence, we found that there are several important amino acid diversity sites, especially the98th and the241th; These viruses proliferated rapidly in Vero cells, the majority of these strains could reach peak of proliferation in4to6days, but the infectious titer was significantly different, the lowest titer was4.751gCCID50/ml, and the highest was up to7.781g CCID50/ml; After infecting Vero cells and given the same amount of time for proliferation, these strains formed different plaques. The plaques of KM15, KM154and KM168are round, needle-like size and their edges were clear. FY18’s plaques were smaller and more regular than those three strains refered above; In contrast, KM1, KM881, KM263and KM165’s plaques were large, irregular and blurry; The virulence of these strains were strong in suckling mice after intracranial injection. These mice began to develop the disease in3to6days in succession and the mortality could reach100%, except KM168, KM154and KM15groups. Each strain could cause varying degrees of damage to brain, heart, lungs, muscles, spinal cord, liver and other tissues of suckling mice, especially the brain and muscle tissue.The second part is for the selection of candidate strains of attenuated live CA16vaccine. The10viral strains mentioned above were continuously passaged in KMB17cells at low temperature to decease the virulence. Then the satisfactory strains were selected according to the infectious titer, the antigen content, the pathogenicity, immunogenicity et al. KM165was quit because its infectious titer(3.751gCCID50/ml) was still too low when passaged to KP2; When passaged to KP4, the virulence of these CA16strains were weaken with different degrees except for FY18. But the immunogenicity of FY18, KM1, KM263was poor, so these3strain were desisted from further study; After the measurement of viral load in suckling mice tissues, we found that the CA16viruses could spread to other tissues and organs of suckling mice via the blood after viral infection, and the muscle was the major growth areas,then the viruses were excreted through the intestinal tract; To KP7, the infection titers of the remaining6strains were greater than6.OOlgCCID50/ml, and KM154, KM168were greater than7.OOlgCCID50/ml. In addition, the6strains were capable of inducing neutralizing antibodies; After the plaque purification,73clones were obtained from these6strains. According to the infectious titer, the antigen content, the immunogenicity, the residual virulence and the results of preliminary study, we selected KM168-8, KM154-6both were passaged to KP11as the candidate strains for subsequent studies. So the future work should focus on the study of the characteristics of these2chones, including the infectious titer, the antigen content, the pathogenicity, the immunogenicity, the genetic stability, the heat stability and the second plaque purification to establish the valid attenuated live vaccine candidate strain.