The Pathogenesis And Protection From Lethal Challenge In A Mouse Mode Of Vaccine Candidate With Recombinant Form Coxsachievirus A16

Hand, foot and mouth disease (HFMD) is a global infectious disease caused bymultiple enterovirus and commonly affects children who are younger than5years old.Most patients have mild symptoms and recover spontaneously within2weeks, but asmall proportion of them may suffer from severe complications such as asepticmeningitis, encephalitis, acute flaccid paralysis, pulmonary edema, myocarditis andeven death. In recent years, the prevalence of HFMD in the Asia-Pacific region hasincreased greatly and there were several epidemics in mainland China in the past fewyears. It is a serious threat to health of infants and young children. As HFMD is lifethreatening for children, the disease was listed as one of the category “C” notifiablediseases in China since2nd May2008. The epidemic may further expand, and how toprevent the disease is still very important in future. At present, there is no specifictreatment, specific vaccines and antiviral drugs. Therefore, the clinical characteristicsof HFMD and the research of biology, pathogenesis and prevention of enterovirus areextremely necessary.The enterovirus which cause hand, foot and mouth disease spread popular insoutheast Asia and the Pacific. EV71and CA16are the two main pathogens. EV71and CA16belong to Ribonucleic Acid (RNA), Picornaviridae and Enterovirus, whichcan cause a variety of human disease, including conjunctivitis, febrile, rash, HFMDand herpangina, myocarditis, cardiomyopathy and aseptic meningitis, encephalitis,diseases such as pneumonia and acute flaccid paralysis, seriously affects infants andchildren’s health. EV71and CA16infection or alternately appear at the same time,become the main pathogens causing, foot and mouth disease epidemic.The RNA genome of EV71and CA16consists of7400nucleotides and iscomposed of a5′-untranslated region (UTR) and a3′-UTR, and an open reading frame (ORF), encoding a polyprotein precurosor that is proteolytically cleaved to11endproducts. The translation of CA RNAs generally proceeds by a cap-independentmechanism, in which ribosomes bind directly to an internal ribosome entry site (IRES)in the5′-UTR of the RNA. It might play an important role in viruses binding cellularreceptor in the infectious cycle. Various cellular receptors were demonstratedincluding coxsackievirus-adenovirus receptor (CAR), the complement system (DAF),Polio virus receptors (CD155), α2β3, αvβ3and αvβ6and decay accelerating factorand so on. Meanwhile, the interactions between cellular receptor and some accessoryfactors are thought to be crucial factors in tissue tropism.In the study， we have investigated the molecular biology, the biologicalcharacteristic, adaptation to cell culture, neutralization of antibody in vitro andanimal model of CA16which was isolated from FHMD patients. The CA16strainswere identified recombinant form by genome sequencing and compared with otherknown virus gene. Moreover, circulating CA16viruses from HFMD patients aregenetically distinct from the prototype CA16(G10), especially in5′UTR, P2and P3region. The currently circulating CA16viruses are actually complex recombinantviruses involving multiple types A HEV, including CA4, CA16, and possiblyEV71/CA2/CA3/CA6/CA10/CA12. The enterovirus can form different genotypesbecause it mutates extremely easily.In neutralization test in vitro, we found the antibody of CA16can neutralizemultiple strains of heterologous CA16virus, even including G10virus, and theneutralizing antibody titer is relatively high. However, the antibody of CA16can notneutralize EV71and the antibody of EV71can not neutralize CA16, these resultswere demonstrated by lethal challenge neonatal mouse model.Circulating recombinant forms of CA16(CA16) is a major cause of HFMD inSoutheast Asia. At present, there is no vaccine against CA16viruses. Pathogenicanimal models that are sensitive to diverse circulating CA16viruses would bedesirable for vaccine development and evaluation. In this study, we isolated andcharacterized several circulating CA16viruses from recent HFMD patients. TheseCA16viruses currently circulating in humans were highly pathogenic in a newly developed neonatal mouse model; we also observed and analyzed the pathogenesis ofrepresentative circulating recombinant form (CRF) CA16viruses.In this animal model, the pathogenicity of the circulating recombinant formCA16viruses to neonatal mice was stronger than that of the prototype CA16(G10)and EV71virus. Although all the viruses were susceptible to the same organ andtissue including muscle and so on, the mice muscle was the main room where theCA16and EV71viruses replicated. These results were demonstrated throughpathological analysis, immunohistochemistry and viral load. We found in the firsttims that the recombinant form CA16viruses were very susceptible to lung inneonatal mouse model.An inactivated CA16vaccine candidate, formulated with alum adjuvant andcontaining sub-microgram quantities of viral proteins, well protected neonatal miceborn to immunized female mice from lethal-dose challenge with a series of CA16viruses. Further analysis of humoral immunity showed that antibody elicited fromboth the immunized dams and their pups can neutralize various lethal viruses bycytopathic effect (CPE) in vitro. Moreover, viral titers and loads in the tissues ofchallenged pups in the vaccine group were far lower than those in the control group,and some were even undetectable. This lethal challenge model using pathogenicCA16viruses and the vaccine candidates that mediated protection in this model couldbe useful tools for the future development and evaluation of CA16vaccine.