Design And Development Of Chimeric Vaccine Candidates Against Hepatitis E And Foot-and-mouth Disease

Hepatitis E is a liver disease caused by the hepatitis E virus(HEV).An estimated 20 million HEV infections and 3.3 million acute cases occur annually worldwide with an estimated 56600 deaths.Four major genotypes of HEV have been identified to infect humans,with a single serotype: genotypes 1 and 2 are human viruses that circulate only in humans causing epidemics in developing areas,whereas genotypes 3 and 4 are zoonotic causing sporadic cases in developed countries.Hepatitis E is a common infection in China,caused mainly(in recent years)by the HEV genotype 4.The prevention remains the most effective approach against HEV infection.In China,two recombinant vaccines based on human-HEV sequences have undergone clinical trials and even one of them is already in commercialized(Hecolin).However,since hepatitis E is usually self-limiting,there are no recommendations yet for the introduction of the HEV vaccine for routine use in national programs in populations where epidemic and sporadic hepatitis E disease is common.Therefore,herein we present the control of HEV in the nonhuman reservoirs as an alternative and effective approach for the prevention and control of HEV infection: swine is considered the primary host of HEV genotype 4 in China,and therefore,a swine vaccine against HEV would strongly reduce the source of HEV genotype 4 and thus reduce,probably eradicate,HEV genotype 4 infection in human populations.In order to achieve the above-mentioned objective,the HEV vaccine should be effective in terms of benefit/cost ratio and attractive to the swine breeding industry.Therefore,the development of a combined vaccine with bivalent protection against HEV and foot and mouth disease virus(FMDV)will increase the benefit/cost ratio and allow a double protection.Foot and mouth disease virus has been chosen because it is the causative agent of foot-and-mouth disease(FMD),one of the highly infectious diseases of cloven-hoofed animals that cause huge economic consequences during the outbreaks.Moreover,vaccination is still the most common method of controlling FMD in China and it is mandatory in government disease control programs in China.In order to develop this HEV-FMDV bivalent vaccine,we proceeded as follows: Bioinformatics analysis and selection of the best HEV-FMDV chimeric proteinsMethods: to develop the HEV-FMDV combined protein,we first selected 4 fragments of HEV ORF2 protein(genotype 4)that we have previously investigated and evaluated their antigenicity and immunogenicity,namely: p166(aa 452-617),p179(aa 439-617),p216(aa 420-637)and p222(aa 439-660).Then,we designed the FMDV Seq8 antigen by combining three entire immunodominant VP1 G–H loop domains from different topotypes of O-type FMDV: the O/Mya/98(Southeast Asia topotype)strain;the O/HN/CHA/93 vaccine strain(of Cathay topotype)and the O/IRN/2010(PanAsia 2 topotype)strain.Next,we designed a set of combined proteins by combining the Seq8 antigen to the N-or C-terminal of the four HEV proteins.These combined proteins were then subjected to different computational analysis to evaluate and compare their antigenicity: prediction and refinement of the 3D structural models,prediction of the exposition of both HEV and FMDV key epitope sites in the different models,comparing the epitopes exposition and selection of the best antigenic combined proteins and finally,flexibility analysis of the selected HEV-FMDV combined vaccine candidates.Results and discussion: The 3D structures of the individual proteins,as well as the HEV-FMDV chimeric antigens,have been predicted refined and the best model for each protein has been selected for further analysis.The analysis of the structural features revealed that attaching the Seq8 antigen to the N-terminal of the HEV proteins had no or minimal effect on the exposition of both HEV and FMDV key epitope sites.More specifically,Seq8-P216 and Seq8-PP222 showed the best conservation of the antigenicity and a better exposition of the key epitopes.Therefore,these two proteins have been selected to undergo the flexibility analysis.The molecular dynamics simulation revealed that Seq8-P216 was more flexible than Seq8-P222 in aqueous solution,which affected the exposure of the epitopes sites in most conformations of Seq8-P216.Meanwhile,the fluctuations of Seq8-P222 had minimal effect on the key epitope residues and their protrusions were even increased in most conformational states.Conclusions: According to the computational analyses,attaching the FMDV Seq8 antigen to the N-terminal of HEV P216 and P222 fragment would permit the conservation of the antigenic composition of both HEV and FMDV fragments.Therefore,we have chosen to express Seq8-P216 and Seq8-P222 combined proteins and experimentally evaluate their antigenicity and immunogenicity.Expression,purification,and characterization of the HEV-FMDV chimeric proteinsMethods: In this chapter,we evaluated: the expressivity of the selected HEVFMDV chimeric proteins in Escherichia coli(E.coli),their solubility and thermal stability as well as their assembly into virus-like particles.The FMDV Seq8 gene was synthesized according to the most commonly occurring codons in E.coli,while HEV target genes were amplified from HEV ORF2 coding DNA(HEV genotype 4 strain NJ-703,GenBank No: AY789228).Then,the Seq8 gene has been attached to the HEV p166,p216 and p222 coding sequences by linear DNA ligation.Next,the combined genes were amplified,purified and inserted into the E.coli expression vector pET28a(+).These expression constructs were verified by sequence analysis to ensure that all of the genes were inserted correctly,and then,were used to transform BL21(DE3)competent E.coli cells.For each protein,several clones have been chosen and cultured.Then,protein expression was induced by the addition of IPTG and analyzed by SDS-PAGE.Both soluble and insoluble fractions of the lyzed recombinant E.coli cells were analyzed to verify the solubility of the expressed proteins.In the next step,the target proteins were purified by Ni–NTA affinity chromatography under native conditions.The purified proteins were pooled and stored at different temperatures: 37?,4?,-20? and-80?.Then,the degradation rate was analyzed by SDS-PAGE at different time points to determine their thermal stability.Finally,the target proteins were examined with a transmission electron microscope to determine whether they could assemble into virus-like particles(VLPs).Results and discussion: All of the amplified DNA fragments coding for the target proteins(individual and combined)were correctly inserted into the pET28a(+)vector and the construct successfully transformed into competent E.coli cells.All the target proteins were highly over-expressed and identified at the expected molecular weight positions on the SDS-PAGE gel: 9.5,27.5,33.4 and 34 kDa for Seq8,Seq8-P166,Seq8-P216 and Seq8-P222,respectively.The Seq8-P222 combined protein showed the highest expression level followed by Seq8 and Seq8-P216 while Seq8-P166 was less expressed than the other proteins.Moreover,the expressed proteins were found to be soluble and thus,successfully purified under native conditions.At low temperatures(-20 and-80?),all the proteins were very stable.However,at 37 and 4? Seq8-P222 was found to be much more stable than the other proteins followed by Seq8-P216;while Seq8 and Seq8-P166 were fully degraded in less than two weeks.The visualization of the combined proteins using the transmission electron microscopy,revealed that both Seq8-P216 and Seq8-P222 could selfassemble into virus likes particles of different diameters(up to ~40nm),while Seq8 formed small aggregates of less than 10 nm in diameter.Conclusions: We successfully expressed two HEV-FMDV chimeric proteins in E.coli,namely: Seq8-P216 and Seq8-P222.The proteins were highly over-expressed and soluble and were purified with high efficacy.Both proteins were very stable when stored at low temperatures(-20 and-80?)and Seq8-P222 remained stable even at 37?.More interestingly,both proteins could assemble into VLPs.Therefore,Seq8-P216 and Seq8-P222 are considered promising HEV-FMDV chimeric vaccine candidates.Evaluation of the antigenicity and immunogenicity of the HEVFMDV chimeric proteinsMethods: In order to investigate the antigenicity of the recombinant proteins Seq8,Seq8-P216,Seq8-P222,indirect ELISA was used to detect the FMDV-specific antibodies present in the sera of pigs infected with different FMDV serotype O strains,using the target proteins as coating antigens(detectors);and the same technique was used to evaluate their reactivity against the anti-HEV monoclonal antibody 4C5.The antigenicity of the target proteins was further confirmed by Western blot analysis.Concerning the immunogenicity,BALB/c mice were divided into experimental groups,each group containing 6 mice,and were immunized with: 50?g of Seq8,Seq8-P216 or Seq8-P222,10?g of P222,100?l of commercialized FMD inactivated vaccine or 100?l of sterile saline solution.The animals received a second dose of the immunogens two weeks after the first one.Then,blood samples were collected on weeks 1,2,4,8 and 12 after the first immunization and the elicited HEV-and FMDV specific antibodies were detected by indirect ELISA.Results and discussion: The Western blot results revealed that combined proteins(Seq8-P216 and Seq8-P222)could react against the 4C5 monoclonal antibody and against the anti-FMDV polyclonal antibodies,while Seq8 reacted only against the anti-FMDV antibodies.These results were further confirmed in the indirect ELISA experiments: Seq8 and the two combined proteins reacted against the antibodies induced by different FMDV strains(Mya98,Cathy,JSM and Asia I strains).The reactivity was more important with the sera of pigs infected with Mya98 and Cathy strains because the two G-H loops in the Seq8 subunit were designed according to sequences of these two strains.However,the proteins also showed cross-reactivity with the antibodies directed against the JSM and Asia I strains;Seq8-P222 was more reactive than the Seq8 alone which was consistent with the computational analysis results.This indicated that the HEV and FMDV antigenic entities were well conserved in the combined proteins.All the vaccinated animals produced detectable HEV-and FMDV-specific IgGs antibodies just one week after the first immunization.Overall,the kinetics of antiHEV antibody production was similar between SEq8-P216 and Seq8-P222 and elicited high antibody titers remained almost unchanged until the end of the experiment.The levels of anti-FMDV antibodies were similar in the different groups at weeks 1,2 and 4 with the highest values registered in the Seq8-P222 group.However,the FMDV-specific IgGs levels decreased significantly at week 8 and continued to decrease at week 12 in Seq8 and Seq8P216 groups but remained high in the Seq8-P222 group.Conclusions: the HEV-FMDV chimeric proteins have shown optimal antigenicity and immunogenicity properties.They reacted against both HEV-and FMDV-specific antibodies and elicited the production of such antibodies when inoculated to mice.The Seq8-P222 has been found to be more immunogenic than Seq8-P216 and therefore represent a better vaccine candidate.