| Hepatitis E(HE)is a liver disease caused by the hepatitis E virus(HEV).Unusually,the clinical manifestations are limited to an acute hepatitis.However,chronic liver disease patients with HEV infection may result in liver failure and other serious consequences,and the mortality rate of HEV in pregnant women is as high as 20%.The World Health Organization(WHO)data show that about 20.1 million people worldwide are infected with HEV every year,with 3.3 million acute patients and about 70,000 deaths.About 44,000 people worldwide died from HEV infection in 2015.HEV infection is prevalent in China,and sporadic cases are on the rise.Hepatitis E has become a serious public health problem globally and in China.HEV is a positive-stranded RNA virus,with icosahedron particles of ?35 nm in diameter.It is a non-enveloped virus with a positive-stranded RNA genome of 7.2 kb in length.The viral genome contains three open reading frames(ORF): ORF1,ORF2 and ORF3.The ORF2 encodes a single structural protein(pORF2)with a total length of 660 amino acids(aa).Given the difficulties to propagate HEV in cell culture,the efforts on investigating its structural characteristics were focused on the recombinant ORF2 proteins expressed in different expression systems.Furthermore,by using genetic engineering to express truncated proteins of different lengths in different host cells,it has been shown that the ORF2 proteins could assemble into two different virus-like particles: T=1 and T=3.However,up to now,the mechanism of the full-length ORF2(aa1-660)self-assembly into virus particles is still not clear,especially the role of C-terminal region(aa606-660)in the assembly process.The HEV strains were divided into four genotypes,namely genotype 1,genotype 2,genotype 3 and genotype 4.The genotypes 1 and 2 only infect humans,while genotypes 3 and 4 infect both humans and animals,hence the name zoonotic genotypes.Pig is the primary host of HEV,and a zoonotic transmission of genotypes 3 and 4 HEV infections exists between the pig and human.Since pigs are the main source of human HEV infection,this human infection could be prevented and controlled by eliminating the source of infection.However,the HEV infection in pigs is a self-limiting infection with very mild or no symptoms,therefore it is very likely that pig breeders would not approve vaccination against HEV,because it will rise the cost-benefice ratio.Therefore,the development of a combined vaccine for HEV and another serious disease in pigs can effectively solve this problem.Foot and mouth Disease(FMD)is a highly infectious disease of cloven-hoofed animals caused by Foot and mouth Disease virus(FMDV).FMD is a worldwide burden,and the rapid spread of the infection through susceptible animal populations leads to large-scale animal death,which can cause huge economic losses and political impact.The world animal health organization(OIE)lists FMD as the first class A animal disease.The FMDV belonging to genus Aphthovirus of the family Picornaviridae is responsible for this disease.Seven FMDV serotypes(A,O,C,Asia 1,SAT 1,SAT 2 and SAT 3)have been identified so far.Serotype O is the most prevalent serotype in swine in China.In 2011,the invasion of O/Mya/98 strain led to the large-scale outbreak of FMD in pigs in Eastern Asian countries including China,Japan,Mongolia,Russia and South Korea.The porcinophilic strain(Cathay topotype)of O type FMDV still exists in several provinces and autonomous regions of China and its neighboring countries.Currently,the strains of inactivated vaccines are mainly O/MYA/98/BY/2010 and O/GX/09-7 +O/XJ/10-11.In the present study we first investigated the role of C-terminal of HEV ORF2 in the formation of VLPs.Then,we screened out the best HEV vaccine candidate.Finally,we developed and assessed the first combined vaccine candidate against HEV and FMDV.Such a combined vaccine would prevent the pig FMDV and HEV infections at the same time,and this would allow the control of the zoonotic HEV infection in its main source1.Construction and expression of different HEV ORF2 truncated proteins in a prokaryotic expression system The Laboratory of Genetically Engineered Vaccines mainly focuses on the study of HEV C-terminally truncated ORF2 proteins.Previously,four ORF2 truncated proteins(p179,p216,p222 and p231)have been produced and studied in the laboratory.We have found that the oligomerization and the morphology of the formed of p216,p222 and p231 VLPs were different from those of p179.To further investigate these differences,we expressed five more other ORF2 truncated proteins with different N-terminal and/or C-terminal truncations,namely: p188,p196,p189,p199 and p209.Then,we used all these proteins together to analyze the key amino acids involved in the oligomerization and VLP formation processes.The results were as follows:1.1 Five ORF2 truncated proteins(p188,p196,p189,p199 and p209)were successfully over-expressed in E.coli.The soluble fractions of these proteins have been successfully purified using His-tag Ni-NTA purification technology.1.2 The results of non-reducing SDS-PAGE and Western blot showed that p179,p188 and p196 proteins,sharing the same C-terminal truncation at aa617,formed mainly dimers with very few tetramers;while the other proteins p189,p199,p209,p216,p222 and p231,sharing the same N-terminal truncation but different C-terminal truncations,all formed dimers,tetramers and higher oligomerization states.All the proteins and their oligomers reacted against the HEV neutralizing 5G5 monoclonal.These results indicated that aa618-627 region was crucial for the formation of higher oligomers other than dimers and tetramers.1.3 Transmission electron microscopy(TEM)revealed that the formation of higher oligomers was directly related to the formation of VLPs.Proteins with a C-terminal extending to aa627 or farther(p189,p199,p209,p216,p222 and p231)formed abundant particles with a clear spherical structure,while the other three proteins(p179,p188 and p196,C-terminal up to aa617)have fewer particles with a different shapes.2.Structural analysis of ORF2 truncated protein self-assembly into VLPsIn order to further study key amino acids involved in the oligomers and VLPs formation,we analyzed a second set of proteins spanning the aa618-627 domain.We expressed the second series of recombinant proteins(pC618,pC619,pC620,pC621,pC622,pC623,pC624,pC625 and pC626),and then analyzed them simultaneously with p179 and p189.In addition,the mutation of Cys627 in P189 protein to Ala(p189A),the mutation of Cys627 and Cys630 in P199 protein to Ala(p199A),and the mutation of Cys627,Cys630 and Cys638 in P222 protein to Ala(p222A),in depth it's further verified the important role of Cys in protein formation polymers and VLPs.The results were as follows:2.1 The nine proteins(pC618,pC619,pC620,pC621,pC622,pC623,pC624,pC625 and pC626)were successfully expressed in E.coli.The results of non-reducing SDS-PAGE and Western blotting showed that p179,pC618,pC619,pC620,pC621,pC622,pC623,pC624,pC625,pC626 formed only dimers,while p189,containing the Cys627,formed higher oligomers,hence suggesting that Cys627 was a key amino acid that played a role in the formation of higher oligomerization states.2.2 The morphology of p189 A,p199A and p222 A showed only dimer and a small amount of tetramer,while the three unmutated proteins showed polymer,with significant difference before and after mutation;After the proteins were treated by DTT,the p179,p188 and p196 were still found as dimers,while all the oligomers of p189,p199,p209,p216,p222 and p231 were reduced into dimers and a small amount of tetramers,as observed by non-reducing SDS-PAGE and Western blotting analysis.This suggested that unlike the dimers,the higher oligomers required the formation of disulfide bonds.2.3 The visualization of the proteins under transmission electron microscopy without DTT treatment revealed that p179,p188 and p196 VLPs were fewer with greater shape heterogeneity,while the six proteins containing the Cys627,p189,p199,p209,p216,p222 and p231 were abundant and homogenous,with shapes and size similar to the native virions.After DTT treatment,the morphology of the particles was destroyed and the number drastically decreased in all the proteins samples.These results suggested that the oligomers enhanced the assembly into virus-like particles through the formation disulfide bonds that maintained stabilized the morphology of the particles.2.4 Further,the particle size and particle size distribution of different VLPs were evaluated by dynamic laser scattering experiment before and after treatment with a reducing agent TCEP.The size of p189 and p222 particles decreased significantly after the treatment,while the negative control p179 VLPs had a negligible effect.At the same time,the scattering light intensity results showed that the strength of p189 and p222 VLPs decreased after adding the reducing agent,but the effect was not noticed in p179 samples.2.5 Native HEV virions were isolated from feces of infected animals by using 5G5 monoclonal antibody and immunomagnetic beads.The samples were treated by DTT and observed under transmission electron microscopy.No particles were visible compared to the non-treated samples indicating that the capsid of the native virions was also reduced by DTT and suggesting therefore that the HEV capsid was maintained by disulfide bonds.2.6 The dimer structures of p189,p199,p209,p216,p222 and p231 were predicted by Phyre2 server.The 3D structures of the proteins were visualized and analyzed by the Py Mol software.The positions of Cys627,Cys630 and Cys638 in the dimer structure were located.Further,a total of 162 sequences of HEV ORF2 of different genotypes were retrieved from Gen Bank and the amino acid sequences were aligned using MEGA 5software.The results showed that the three C-terminal cysteines Cys627,Cys630 and Cys638 were highly conserved.3.Preparation and immunogenicity study of a combined vaccine candidate against HEV and FMDV Among the truncated HEV ORF2 proteins we expressed,the best candidate for HEV vaccine was selected by analyzing the antigenicity,thermal stability,VLP formation and immunogenicity of the different proteins.The different formulations of recombinant HEV vaccine and the inactivated FMDV vaccine were prepared,and the preliminarily immunogenicity study was carried out.In order to detect the FMDV-specific IgG levels in mice immunized with the combined vaccine formulations,a recombinant protein containing G-H loop of FMDV VP1 protein was prepared by molecular cloning technology.The results were as follows:3.1 The results of protein stability assays showed that p179 and p222 were the most stable proteins at high temperature(37?).Ellipro Web server was used to calculate the protrusion index of the key epitopes sites in both p179 and p222,and the area under curve(AUC)showed that p222 was more antigenic than p179.Monoclonal antibody 5G5 reacted similarly with p179 and p222 antigens.However,after immunizing the mice with an equal dose,the immunogenicity of p222 was higher than that of p179(p=0.007**).Therefore,HEV p222 recombinant antigen was selected as the best candidate to be combined with the FMDV vaccine.3.2 According to the O/GX/09-7 strain and O/Mya98/XJ/2010 strain contained in FMDV inactivated vaccine,an antigen LMYA-GX was designed as an antigen to detect the FMDV-specific IgG antibodies.The G–H loop domain of FMDV VP1 protein has been previously reported to contain the dominant neutralization epitopes.Therefore,the entire G–H loops of O/GX/09-7 strain and O/Mya/98 were linked together by two glycine(G)residues,then the target gene(LMYA-GX)was inserted into the plasmid p GEX-4T-2 and was successfully expressed and purified.3.3 Monitoring of HEV-and FMDV-specific antibodies in the mice immunized with HEV or FMDV vaccines separately:1)Total anti-FMDV IgG antibodies were monitored by indirect ELISA in the serum samples from mice immunized with three different concentrations of the inactivated FMDV vaccine(0.5,1,2?g/ml).Anti-FMDV antibodies were detected in all the immunized mice at week 2 but the levels were very low.As expected,the antibody titers increased overtime to reach the highest level at week 6 for the group F0.5 and at week 10 for the groups F1 and F2.The antibody titers in the group F0.5 were lower than in the two other groups all throughout the experiment,which was expected given that these latter groups received a higher dose of the FMDV vaccine.However,the results were not dose-dependent in the first four weeks in the F1 and F2 groups:the levels of the anti-FMDV antibodies were higher in the F1 group at weeks 2 and 4;then they reached similar levels at week 10 in both groups.The responses were negative in the control group 1-C that received only the adjuvant during the whole experiment.Overall,during the 12 weeks of the experiment and taking into account the four groups,there was a positive correlation between the dose and titers of the induced FMDV-specific antibodies.Therefore,according to these results,the concentration of 25?g/ml of the HEV immunogen was selected for the preparation of HEV-FMDV combined vaccine.2)Likewise,the sera collected from the mice immunized with different dosages of the HEV recombinant vaccine(25,50,100 ?g/ml)were analyzed for the presence of anti-HEV antibodies by indirect ELISA(Figure 3.B and Table 2.B).The anti-HEV antibodies were not detectable at week 2 in all groups.Starting from week 4,the HEV-specific IgGs started increasing in a dose-independent manner in the vaccine groups and reached the highest levels at week 10 in the E25 and E100 groups,and at week 12 in the E50 group.At week 4,the antibody titers in the E25 and E100 were4-fold higher than in the E50 groups.However,at week 10,the levels of the anti-HEV antibodies in the E25 group were 5-fold higher than in the E50 group and2-fold higher than in the E100 group.Therefore,according to these results,the concentration of 1?g/ml of the FMDV immunogen was selected for the preparation HEV-FMDV combined vaccine formulations.3.4 Monitoring of HEV-and FMDV-specific antibodies in the mice immunized with different HEV-FMDV combined vaccine formulations:In order to determine whether combining the HEV and FMDV immunogens together could affect their respective immunogenicity we set the following experiments:1)The FMDV immunogen(1?g/ml)was combined with three different doses of the HEV p222 protein(25,50 and 100?g/ml).After injection into mice,we monitored the production of FMDV-specific antibodies.The results revealed that all three combined vaccine formulations induced the production of anti-FMDV IgGs detectable at week 2 post-immunization.Interestingly,the levels of anti-FMDV antibodies in the three combined vaccine groups were significantly higher than the control group F1 that received only the inactivated FMDV vaccine.In addition,anti-FMDV IgG titers were increasing as the combined HEV immunogen dose was increasing.The antibody levels reached a peak at week 12 in the F1+E25 and F1+E50 groups,and at week 10 in the F1+E100 group.At the end of the experiment(week 12),the antibody titers remained as high as at week 10 in the F1+E100 group.These results indicated that the addition of the HEV p222 immunogen enhanced the immunogenicity of the FMDV vaccine.1)The 25?g/ml HEV dosage was selected to be combined with three different concentration of FMDV vaccine(0.5,1 and 2?g/ml),and injected into mice.The results of the detection of the HEV-specific IgG showed that the HEV-FMDV formulations induced a stronger humoral response than the control group that received only 25?g/ml of HEV p222(Figure 3.D and Table 3.B).The anti-HEV antibodies were detectable at week 4 post-immunization and their levels were increased to reach a peak at week 10 in all the groups.During the 12 weeks of experimentation,the E25+F0.5 group registered the highest anti-HEV antibody titers followed by E25+F2 and E25+F1 groups.Although the addition of the FMDV vaccine seems to enhance the immunogenicity of the HEV immunogen,the increase in the anti-HEV antibodies was not dependent on the increase of the FMDV vaccine dose.A negative reaction for the presence of both anti-HEV and anti-FMDV antibodies was registered all throughout the experiment in the negative control groups that received only the adjuvant.Conclusions:(1)The p179,p188 and p196 formed only dimers,while p189,p199,p209,p216,p222 and p231 formed higher oligomers,indicating that aa618-627 is the key amino acid region for the formation of high oligomerization states.The number of VLPs formed by the three proteins p179,p188 and p196 was fewer and heterogeneous;the VLPs formed by the p189,p199,p209,p216,p222 and p231 proteins were more abundant with higher uniformity.(2)By further expressing a second set of proteins(pC618 to pC626),the Cys627 residue was identified to be the key amino acid involved in the formation higher oligomers and VLPs.Non-reducing SDS-PAGE,Western blot,DLS,TEM were used to verify the existence of disulfide bonds in the higher oligomers or VLPs by adding reducing agents(DTT or TECP);the results pointed towards the formation of inter-dimer disulfide bonds.The natural HEV capsid may also contain disulfide bonds that enhances the structural stability of capsid.Bioinformatics was used to predict the dimer structure and showed that Cys627 Cys630 and Cys638 were located at the edge of the dimer structure and the protein sequence alignment of 162 HEV strains showed that Cys627,Cys630 and Cys638 were highly conserved,with conservation rates of 99.4%,100% and 100%,respectively.(3)The best recombinant HEV vaccine candidate was selected according to protein stability,structural analysis by computational methods,antigenicity assay,VLPs formation,and immunogenicity assessment.Accordingly,HEV-p222 was selected as the best vaccine candidate for subsequent HEV-FMDV combined vaccine investigation.(4)A preliminary immunogenicity analysis was carried out to compare three different doses of HEV-p222 recombinant vaccine and FMDV inactivated vaccine,injected separately and the optimal immune dose was selected for each vaccine alone.Next,the immunogenicity assessment of the different formulations of the HEV-FMDV combined vaccine was carried out.The HEV-p222 recombinant vaccine and FMDV inactivated vaccine were combined into F1+E25,F1+E50,F1+E100,F2+E25 and F0.5+E25(?g/ml)formulations.The combined vaccine groups were compared to monovalent HEV and FMDV vaccine groups.The results showed that there was no negative interaction between the combined immunogens and synergistic effect on their respective immunogenicity was noticed. |
PDF Full Text Request