| Hand Foot And Mouth Disease(HFMD) is a common epidemic-prone disease in the Asia-Pacific region in young children caused by viral infection. In 2008, HFMD was classified as one of category C infectious diseases. HFMD is usually mild and self-limiting, it may lead to severe neurological complications including poliomyelitis-like paralysis, encephalitis and aseptic meningitis or even death. Mortality rate in severe cases is increasing. HFMD has become a serious problem to affect public health of young children in China. Human enterovirus 71(EV71) and coxsackievirus A16(CVA16)have been identified as the two major etiological agents of HFMD. EV71 is reported as the major cause for severe cases and death cases. However, no appropriate techniques are yet available for the prevention or treatment against EV71 infection. In the present study, our research interest is focused on drug development against HFMD, which is divided into the following two parts.For the first one, the specific chimeric antibody against a peptide in VP4 protein highly conserved in both EV71 and CVA16 was isolated by phage display technique and expressed in 293 cells. In vitro neutralization assay showed that the chimeric antibody is able to cross-neutralize both EV71 and CVA16 in vitro, suggesting a broad antiviral activity of monoclonal chimeric antibody.For the second one, human anti-EV71 monoclonal antibody was isolated by using single-cell based gene amplification from EV71 infected patients. 137 monoclonal antibodies were successfully isolated and 4 of them had variable anti-EV71 neutralization activity. Among them, antibody hmAb136 has the highest anti-EV71 activity. It is able to neutralize both EV71-A and-C4 subtype viruses and the IC50 were 0.040 μg/m L and 0.071 μg/m L, respectively. Otherwise, this antibody fully protected mice from EV71 lethal challenge at dose of 300 ng/g and the IC50 is 0.053 μg/m L in vivo. However, this antibody is not able to neutralize CVA16. Western blot analysis indicated that this antibody do not interacted with linear epitope but neutralized EV71 virus through recognizing a conformational epitope. No similar datas were reported yet worldwide at present.Coxsackievirus B3(CVB3) is non-enveloped RNA virus of the Picornaviridae family, which can produce pathological syndromes in central nervous system similar with severe cases of HFMD, such as aseptic meningitis and encephalitis. CVB3 is also etiological agent of viral myocarditis and viral pancreatitis-associated type I diabetes. Viral myocarditis(VMC) can develop into dilated cardiomyopathy(DCM) which has a higher mortality rate caused by persistent infection of CVB3 virus. However, there is no vaccine available for CVB3 control. Therefore, for the third part in the present study, recombinant vaccine was constructed to control CVB3 infection.For the third one, to construct virus-like particle(VLP) of CVB3, VLPs were formed by co-expression of P1 protein and 3CD protein or co-expression of VP1, VP2, VP3 and VP4 in yeast cells. VLP is composed of intact virus capsid proteins without viral genome with good immunogenicity and biosafety. In the present study, CVB3 VLPs were expressed in saccharomyces cerevisiae yeast cells. The successful construction of CVB3 VLPs in two methods provided a series of reliable data and a novel strategy for CVB3 control. |
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