Study On Enterovirus 71 Virus-like Particle Vaccine Against Hand, Foot, And Mouth Disease

Hand, foot and mouth disease (HFMD), a disease caused by Enterovirus, continues to be a common infectious disease among young children. It was reported in the 1960s that HFMD occurred, but HFMD was first described in Canadan children in 1957. Coxsackievirus 16 (CA16) was isolated by J. Alsop from the vesicle samples in 1958 and the disease was designated as HFMD in 1959. Enterovirus 71 (EV71) was first isolated from patients in California in 1969. Since then outbreaks of HFMD occurred every year. Recently the incidence of HFMD increases rapidly in the Asia-Pacific region. For example, a large outbreak of HFMD occurred in China during 2008. Approximately half million cases of HFMD occurred and 120 cases were dead, which made people panic. HFMD is categorized into one of the third infectious diseases since May 2008. The morbidity and mortality of HFMD increases rapidly, which threats the health of young children and pubic health safety. So people are more concerned with HFMD.Enterovirus is a major etiologic agent responsible for seasonal epidemics of HFMD. Classically the enteroviruses consisted of Coxsackie group A (types 16, 4, 5, 7, 9 ,10), Coxsackie group B(types 2, 5, 13), Enterovirus (68, 71) and echoviruses. EV71 and CA16 during HFMD epidemics were apparent. They became the dominant viruses. EV71 infection sometimes manifests the most severe forms of neurological disease, including aseptic meningitis, brainstem encephalitis and acute flaccid paralysis. Pulmonary edema and hemorrhage caused by EV71 infection often leads to quick deaths in children. However, CA16 infection occasionally causes myocarditis, cardiopericarditis, and so on. EV71 was divided into three genetic lineages A, B, C, of which B was further divided into sub genotype B1-B5 and C into C1-C4. Genotype C4 of EV71 was the dominant sub genotype recently in China. Immune responses against EV71 include innate and adaptive immunity, of which humoral immune responses mediated by B cells were important in the prevention and control of EV71 infection.Vaccination has been one of the key and cheap means in the control of the infectious diseases. Many of molecular mechanism on virulence and immunity are far from clear, which block developing vaccines against EV71. No effective vaccines are available yet, although many tentative vaccines are developed. Inactivated EV71 vaccines induce strong protection, however, because they will be used for children, their safety must be more considered, including the residue of nucleic acid, the overreaction of immunity, and so on. DNA vaccines and subunit vaccines based on VP1 of EV71 elicited lower degrees of protection than inactivated EV71 vaccines. Virus-like particles (VLPs) are empty particles comprised of viral capsid proteins yet devoid of viral nucleic acids. The repetitive, high density display of viral antigens and epitopes on the VLPs surface usually elicit strong immune responses with little difference to that stimulated by authentic viruses. To date, the human papillomavirus VLP has been approved for use as vaccine. EV71 vaccines based on VLPs have been reported, yet no further studies were performed. Based on the epidemiology of EV71, EV71 VLPs vaccine was developed and assayed in this study. This study paved the way for further EV71 research on developing a safe, effective new type vaccine against EV71.In this study, immunology, virology, molecular epidemiology, molecular biology and vaccinology were used to develop and assay EV71 vaccines based on VLPs, which will provide useful knowledge for EV71 vaccines.Contents:1. Analyze the molecular epidemiology of EV71 in China and screen the EV71 strains for immunization and challenge, and then do the comparative analysis of genomic difference.2. EV71 VLPs antigen was expressed by the Bac-to-Bac system and purified through a new way.3. After mixed with alum or SP01 adjuvant, EV71 VLPs and inactivated EV71 vaccines were prepared.Mice were immunized with EV71 vaccines and challenged with virulent EV71. The immunogenicity and protective efficacy were assayed.Methods:1. Viruses were isolated from the anal swabs, throat swabs, vesicle samples of HFMD cases in Vero cells. RT-PCR, plaque purification, virus titers, electron microscope observation, sequence homology of VP1 of positive viruses were performed in order.2. The candidate vaccine strains were screened based on immunogenicity, cross protection and stability of the available EV71 strains. The EV71 virulent strains for challenge were screened and adapted in the ICR suckling mice by intracerebrally inoculating lethal EV71 into the mice. Sequencing and analyzing the EV71 strains for vaccine and challenge, which provide useful viruses and knowledge for developing vaccines and studying molecular pathogenic mechanism.3. One candidate vaccine strain was selected for vaccine development in this study. The gene fragments coding for P1 and 3CD were amplified by RT-PCR from EV71 and subcloned separately into multiple cloning site (MCS) I and II of pFastBacDUAL plasmids (Invitrogen). Likewise, the P1 gene fragment was cloned into MCS I under the polyhedrin promoter and the 3CD gene fragment was inserted into MCS II under the p10 promoter. The resultant plasmid carrying two gene cassettes was designated pDual-P1-3CD. The recombinant baculoviruse Bac-P1-3CD was subsequently generated using Bac-to-Bac? system. The recombinant viruse was propagated, amplified, stored and tittered. EV71 VLPs were identified by SDS-PAGE, WB, indirect immunofluoresence and electron microscope observation.4. EV71 VLPs were produced by infecting Sf-9 cells with Bac-P1-3CD and purified by ultrafiltration, sucrose density gradient centrifugation, DEAE anion-exchange chromatography, 4FF gel chromatography one by one. Purified EV71 VLPs were identified by SDS-PAGE, WesternBlot, and TEM..5. EV71 VLPs and inactivated EV71 antigen were mixed with adjuvants. The 6-8 week-old mice were immunized by these candidate vaccines. ELISA, NA, MTT, FACS assays were used to analyze the immunogenicity of these vaccines. NA assays were used to evaluate the cross protection against the dominant EV71 strains. The mother mice were immunized with these vaccines and the suckling mice were challenged by the virulent EV71 strain. The protective efficacy was evaluated by observation of the survival rate.Results:1. Thirty five EV71 strains (virus titer more than 105CCID₅₀/ml) were isolated from 441 samples from HFMD cases. In a phylogenetic tree, based on the complete VP1 gene sequence, all strains grouped into the C4 genotype. Two candidate EV71 strains for vaccine development were obtained, designated as BJ01 and LCH02, respectively. The titers of two candidate EV71 strains were beyond 10^7.5CCID₅₀/ml. Titers of total IgG and neutralizing antibody were more than 2¹¹,2⁸, respectively. Within 15 passages, the sequence homologies of VP1 gene, amino acid were 99.99%, 100%, respectively. So BJ01 was selected as the candidate vaccine strain and LCH02 as backup in this study. One virulent EV71 strain that can cause 1 day old suckling mice dead were obtained and designated as LCH01. One-day-old ICR mice were inoculated with LCH01 by intracerebral routes. All of the mice inoculated with LCH01 had the typical signs and symptoms of EV71 infection by day 5 postinoculation, such as paralysis of limbs. By day 6 post-inoculation, some of the mice died. These mice died by 14 days post-inoculation. The LCH01 strain virus titer was 10⁸ CCID₅₀/ml in Vero cells and 10⁸LD₅₀/ml in 1-day-old suckling ICR mice.2. The titer of Bac-P1-3CD was 10⁸PFU/ml. The expression of EV71 VLPs was identified by SDS-PAGE, WB and indirect immunofluoresence. Virus-like particles were observed in the cytoplasm. These particles measured 25-30 nm in diameter and appeared icosahedral, thus resembling the authentic enterovirus in size and appearance. EV71 VLPs with more than 95% purification were obtained by ultrafiltration, sucrose density gradient centrifugation, DEAE anion-exchange chromatography, 4FF gel chromatography one by one.3. Six candidate vaccines were prepared. Purified inactivated whole virus vaccine and EV71 VLPs vaccine were mixed with 1 mg/ml aluminum hydroxide and SP01 adjuvants at the volume ratio of 1:1, respectively.Titers of neutralizing antibodies elicited by EV71 VLPs vaccine were more than ones that elicited by purified inactivated whole virus vaccine with or without adjvants. Meanwhile, titers of neutralizing antibodies elicited by adjuvanted vaccines were more than ones that elicited by vaccines without adjvants. Titers of neutralizing antibodies elicited by vaccines with SP01 were more than ones that elicited by vaccines with aluminum hydroxide. Titers of total IgG and neutralizing antibody that elicited by EV71 VLPs vaccine with SP01 were more than 2¹⁵, 2¹¹, respectively. Antisera generated by immunization with these vaccines can neutralize current clinical dominate isolates from different times and regions. The splenocytes collected from the EV71 VLPs vaccine-immunized mice exhibited more significant cell proliferation, more CD4+T cells and produced higher levels of IFN-γand IL-4 than ones that from the purified inactivated EV71 vaccine-immunized mice after stimulation, indicating stronger induction of Th1 and Th2 immune responses.4. At a dose of 50 LD₅₀, the survival rate of all vaccines was 100%, while no mice in the control group receiving only PBS survived. However, increasing the EV71 dose to 500 LD₅₀ dramatically decreased the survival rates to 90%,77%,72%,60%,55% and 50% for the groups that received EV71 VLPs vaccine with SP01, EV71 VLPs vaccine with aluminum hydroxide, inactivated whole virus vaccine with SP01, inactivated whole virus vaccine with aluminum hydroxide, EV71 VLPs vaccine and inactivated whole virus vaccine, respectively.Conclusions:1. Two candidate EV71 strains for vaccine development were obtained, designated as BJ01 and LCH02, respectively. They have the biology characteristics of strong immunogenicity, broad spectrum cross protection and high stability. BJ01 was selected as the candidate vaccine strain and LCH02 as backup in this study. One virulent EV71 strain that can cause 1 day old suckling mice dead were obtained and designated as LCH01. It has the characteristics of high virus titer in Vero cells and strong virulent to one day old suckling ICR mice, which can be used as evaluate the protective efficacy of candidate vaccines.2. EV71 VLPs were expressed in Sf-9 cells and purified with more than 95% purification by ultrafiltration, sucrose density gradient centrifugation, DEAE anion-exchange chromatography, 4FF gel chromatography one by one.3. Six candidate vaccines were prepared. EV71 VLPs vaccine can elicit stronger humoral and cellular immune responses than inactivated whole virus vaccine. Aluminum hydroxide and SP01 adjuvants can enhance immune responses. Compared with aluminum hydroxide, SP01 adjuvant can elicit stronger immune responses. Among these vaccines, EV71 VLPs vaccine with SP01 can elicit the strongest protective efficacy.