The Preparation And Immunogenicity Of EV71Virus-Like Particles

Enterovirus71(EV71), a member of the Enterovirus genus in the Picornaviridae family, is generally recognized as the main pathogen responsible for hand-foot-and-mouth disease(HFMD) in human. However71infections can sometimes induce a variety of severe neurological complications including brainstem encephalitis and myelitis associated with cardiopulmonary, which can lead to high morbility rates in children.Since its association with HFMD was verified in1974, several epidemic outbreaks of HFMD involving several large population cohorts have occurred around the globe, particularly in Asian countries. EV71infections have increasingly become a major public health issue, which can cause more severe neurological complications than other enterovirus. Until now there are no virtual antiviral drugs against EV71infection. The development of effective vaccines is a top priority in terms of control strategies.Recently, several new formats of EV71candidate vaccines have been developed. Although the inactivated virus vaccine can induce stronger immune responses than such subunit and DNA vaccine, whole virus vaccines raise higher safety concerns. Virus-like particles (VLPs) are empty particles consisting of all major structural proteins, mimicking the organization and conformations of the native particle, but devoid of viral nucleic acids, hence they are non-infectious and have captured increasing attention as potential vaccine candidates. A potent vaccine has been achieved by VLPs mixed with complete or incomplete Freund’s adjuvant. This VLP can induce potent humoral immune responses as evidenced by the neutralization titer and the VLPs immunization of mother mice conferred protection to neonatal mice against the lethal viral challenge. But so far there is noresearch shows that how the immune responses induced by EV71VLPs confer specific protective effect in vivo. So, in this study we tested a potential EV71vaccine candidate based on VLPs prepared from an EV71strain belonging to C4sub-genotype. In addition to assessing the vaccine’s efficacy in mice, ourdata further elucidate the significance and value of assessing the immunogenicity and immunoprotection of vaccine candidates in ICR mice by relying on a range of analyses, including pathological, etiological and lethal challenge analyses. At last, our study showed that EV71VLPs immune treatment resulted in effective inhibition of viral replication in some susceptible organs(cardiac muscle, skeletal muscle, lung, intestine) of the animals and the immunoprotective efficacy reached nearly100%. Hence, the EV71VLPs have great potential for vaccine development.Part1The Preparation of EV71VLPs expressed in BaculovirusIn China, EV71activity was first detected during a HFMD epidemic in Shanghai in1981. Since1998, the C4subgroup was isolated from sporadic infections in mainland China, and become the solo causation of EV71epidemics in recent years. In this study we used an EV71strain belonging to C4for EV71VLPs construction.To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1and3CD genes of enterovirus71(EV71), the P1and3CD genes were cloned into the same baculovirus shuttle vector(Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of Sf9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected sf9cells. Electron microscopy showed that the structural protein capsid P1was cut by virus-encoded protease3CD into3individual proteins (VPO, VP1and VP3) and assembled into EV71virus like particles(VLPs) about27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used Sf9cells with serum-free medium in CellSTACK-10Culture Chambers to produce EV71VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1(39KD), VP0(34KD) and VP3(26KD) as well as the intact structure, which can be used for further immunogenicity evaluation study.Part2The immunogenicity and immnoprotection elicited by EV71VLPs vaccine.Three groups of4-to5-old female ICR mice were immunized via the intramuscular injection (i.n.) route with5Ｌg/mouse of one of the following samples adjuvanted with alum:purified VLPs, inactivated whole-virus and PBS. The immunization schedule consisted of three inoculations given2weeks apart. We detected the EV71-specific antibodies, neutralization titers, ELISPOT, and T cell response to find their immune responses to EV71VLPs. We also tested the immune protection provided by the vaccine by using a lethal challenge assay in neonatal mice models. Besides the neonatal mice for mortality study, three neonatal mice out of each group are used for histopathology analysis. Tissue sections were stained with hematoxylin and eosin, and immunohistochemical analysis were performed.These mice developed both specific humoraland cellular immune responses to EV71VLPs vaccine. Despite lower neutralizing antibodies to EV71were found insera of VLP-immunized mice than mice vaccinated with inactivated EV71, VLP-based vaccine generated a good memory cell immune response to EV71. Splenocytes of mice immunized with VLPs produced a high level of EV71-specific IFN-y-producing T cells number and did not demonstrate a significant difference with the inactivated EV71group. But in response to EV71antigens, the IL-4-producing T cells number in VLPs immunized mice were apparently higher than formalin-inactivated EV71 immunized mice. The positive result of the ELISPOT assay against IFN-y and IL-4in both groups suggested that both formalin-inactivated EV71and VLPs could both induce memory Thl/Th2cellular response.The IgA-memory B cells number in both VLPs group and formalin-inactivated EV71group were increased after immunization as compared with PBS group, which also suggested a similar cellular immune response between these groups. Passive protection experiments indicated that the vaccine-immunized mice showed no characteristic clinical manifestations compared with control groups and no marked lesions or obvious signs of inflammation in other organs expected slight myatrophy in the limb musculature as well as the results of virus load in these major organs. The vaccine-immunized mice displayed slight myofiber regeneration with nuclear rowing, suggesting signs of recovery from the virus infection. This study indicates that the EV71candidate vaccine is capable of generating protective levels of EV71neutralizing antibodies in neonatal mice and primes the immune system effectively against a subsequent exposure to EV71by inhibiting the proliferation of the virus in the tissues and organs of mice. This VLPs vaccine can effectively protect the body from the pathological damage caused by viral infection. These approaches may be applied for the development of vaccines against EV71infection in humans. Hence, the EV71VLPs vaccine is a strong candidate for EV71human vaccine.