Study On The Antigenicity Of HEV ORF2 And ORF3 Polypeptides And The Immunogenicity Of ORF3 Polypeptide

HEV is a small,non-enveloped virus with a single-stranded,positive-sense RNA genome of approximately 7.2 kb and contains three partially overlapping open reading frames(ORFs):ORF1,ORF2 and ORF3.Four genotypes of HEV that can infect mammals are recognized currently but there is only one serotype.Due to the lack of an efficient cell-culture system for HEV,little is known about the mechanisms of HEV pathogenesis and replication.Several methods have been available for the diagnosis of hepatitis E.These include immune electron microscopy (IEM),PCR and EIA.Amongst these methods,EIA is the most convenient for the detection of HEV IgM and/or IgG antibody and its advantage is inexpensive and suitable for clinical diagnosis and epidemiological studies of serology.However,this method also has some limitations,such as the relatively low sensitivity and so on.In addition,the test results from different reagents may be inconsistent due to different antigens are used by different manufacturers.In view of this,one important part of the following study is on the antigenicity of HEV ORF2 and ORF3 polypeptides and the immunogenicity of ORF3 polypeptide.Cloning and sequence analysisFour HEV strains were isolated from patient's stool suspensions and their full-length cDNA clones were constructed and sequenced.Sequence analysis results indicated that the strain named W2 from a Xinjiang sporadic patient was genotype 1, while all of the other 3 strains W3,W5 and MO from sporadic cases in Beijing were genotype 4 but belong to 3 different subgenotypes 4f,4d and 4a respectively.The genome sequence of W5 which was from swine was very similar with a human strain T1 from Beijing region reported previously and they have a high nucleotide identity as 92.1%.This result strongly supported the zoonosis hypothesis of hepatitis E.Analysis of the antigenicity of genotype 1 and 4 HEV ORF2 and ORF3 polypeptidesW2 and W3 isolates represented the genotypes 1 and 4 HEV respectively were selected in this study.Six and three overlapping polypeptides from open reading frames(ORFs) 2 and 3,respectively,of them were expressed as fusion proteins in Escherichia coli.And all of them were named by turns from N-terminus to C-terminus as pS1-1 to pS1-6/ pS4-1 to pS4-6 for ORF2 from genotype 1/4 HEV and pS1-7 to pS1-9/ pS4-7 to pS4-9 for ORF3 from genotype 1/4 HEV.All the polypeptides were purified successfully using ion-exchange chromatography and were then freeze-dried.Following purification,each product showed one clear band of the expected molecular mass on SDS-PAGE.The reactivities of each recombinant polypeptide with both IgG and IgM of anti-HEV antibody were determined by immunoblotting.The results showed that the reaction abilities were different for different peptides.Some polypeptides,such as pS1-1/ pS4-1(1-110/ 1-124aa) from N-terminus of ORF2,pS1-6/ pS4-6(371-660/ 385-674aa) from C-terminus of ORF2, pS1-8/ pS4-8(56—123/ 47-114aa) from C-terminus of ORF3 and the full length ORF3 showed strong reactivity with anti-HEV antibody.Moreover,the peptides from the same location got the same reaction abilities between genotypes 1 and 4. Furthermore,the reactivities of the polypeptides with anti-HEV antibody were evaluated using enzyme mmunoassays(EIAs).1.Reactivity to HEV IgM antibodyThe reactivities of the six polypeptides,pS1-1,pS1-6,pS1-8,pS4-1,pS4-6 and pS4-8 with anti-HEV IgM were evaluated using EIA to detect serial sera from ten rhesus monkeys,infected experimentally with different genotypes of HEV(JE11, JE12,JE47 and JE48 were infected with genotype 1 HEV from humans,JE13 and JE14 were infected with genotype 4 from humans,JE15,JE16,JE51 and JE52 were infected with genotype 4 from swine) and three serial sera from HE patients.The serial sera from seven of ten monkeys were positive for ORF3 IgM antibody. And those from nine of ten monkeys were positive for IgM antibody against both termini of ORF2.Anti-ORF2 IgM antibody was present earlier than the elevated ALT level while the peak value of anti-ORF3 IgM antibody was present almost at the same time with the peak value of the elevated ALT.Thus,the N- and C-terminal polypeptides of ORF2 may provide epitopes which are highly reactive with sera from the early acute phase of HEV infection,while the C-terminal polypeptides of ORF3 may contain epitopes which are reactive with early convalescent-phase sera,or from the later period of the acute phase.The polypeptides used in most anti-HEV EIAs are derived from the C-terminus of ORF2(and ORF3).However,in this study, polypeptide pS1-1 from the N-terminus of genotype 1 ORF2 showed a more ideal pattern of immunoreactivity than that of pS1-6 from the C-terminus of genotype 1 ORF2.The reactive antibodies against polypeptide pS1-1 were detectable earlier and shorter-lasting in sera from JE12,JE52 and JE15.Polypeptide pS4-1 from genotype 4 ORF2 showed a better ability to detect anti-HEV IgM in sera from JE11,JE12 and JE15 than pS4-6 from genotype 4 ORF2.In some cases,such as with sera from JE11, JE13 and JE14,the polypeptide pS4-6 from the C-terminus of genotype 4 ORF2 had greater reactivity than pS4-1 from the N-terminus.Thus,using only a single polypeptide from either the N-or C-terminus in EIAs for the detection of anti-HEV IgM may be insufficient,and may result in failure to diagnose acute hepatitis E.Furthermore,we developed EIAs based on a combination of C- and N-terminal polypeptides.These EIAs were evaluated using serial sera from monkeys infected experimentally with different HEV genotypes.Anti-HEV IgM was detected using dual-antigen EIAs at the same time as detection with EIAs based on either terminus of ORF2,and before elevation of ALT levels or anti-HEV IgM became detectable by EIAs based on ORF3 polypeptides.Moreover,the detection rate based on combination antigen was higher than that just based on either N-terminal or C-terminal of ORF2.These EIAs also were evaluated using serial serum samples from patients with HEV infection.As expected,the results indicated that anti-HEV IgM against N- and C-terminal polypeptides from ORF2 were detected and decreased gradually,as seen in the data from monkeys.However,the titer of anti-HEV IgM against ORF3 polypeptides gradually peaked in two patients,and lasted longer than antibodies against N- and C-terminal polypeptides from ORF2.Regarding genotype,there were no significant differences between genotypes using EIAs based on the ORF2 protein.But all monkeys infected with genotype 1 HEV were negative for anti-HEV IgM when tested with pS4-8,while two out of four animals were positive when tested with pS1-8.This suggests that the ORF3 protein is genotype-specific.2.Reactivity to HEV IgG antibodyAnti-HEV IgM usually is thought to develop earlier during infection than anti-HEV IgG,and is considered to be an important serological marker for the diagnosis of acute hepatitis E.An ideal anti-HEV IgM test should be able to detect HEV IgM antibody in the early HEV infection phase to increase the rate of early diagnosis,and should not be long-lasting in the convalescent phase to improve its diagnostic accuracy of acute infection.The results of this study indicate that anti-HEV IgM against polypeptides from the N-terminus and C-terminus of ORF2 may be the best markers for the diagnosis of the early stages of hepatitis E infection.Anti-HEV IgG is an significant marker of the epidemiological investigations and clinical diagnosis.In this study,the dynamic changes of the anti- both termini of HEV ORF2 antibodies in the serial sera from monkeys infected with HEV were analyzed based on EIA.The result showed that anti-ORF2 N-terminal antibody were detectable earlier and peaked soon followed by a gradual decline in antibody levels.Furthermore, the peak value of antibody level was present almost at the same time with the peak value of the elevated ALT.On the other hand,compared with the anti-ORF2 N terminal antibody,the anti-ORF2 C terminal antibody produced at similar time,but the antibody level was weaker at the earlier period,increased gradually,and in the observation of 14 weeks,no clear downward trend.In generally,anti-HEV IgG may develop early,levels may be very high during the acute phase and it may remain detectable for many years.Therefore,although it is a valuable diagnostic marker,IgG is not suitable for distinguishing between recent and past HEV infections.In this study, the results suggest that anti-ORF2 N-terminal antibody was present primarily in acute phase and short duration.Thus,it was significant for clinical diagnosis of acute phase. On the other hand,anti-ORF2 C-terminal antibody was long duration after present and related to past infection.So it was applied to be used in epidemiological investigation. In summary,we should optimize the antigen fragment according to the different purposes of study.It is known that the ORF2 polypeptides were equally efficient for detecting HEV IgM antibody by enzyme immtmoassay.Similarly,there was no significant difference between genotype 1 and 4 HEV correspond to the location of HEV ORF2 antigen to detect HEV IgG antibody.However,as different genotypes ORF3 antigen had different IgM antibodies binding capacity,the ORF3 antigen showed also different IgG antibody binding capacity between genotype 1 and 4 HEV.The reactivities of both genotype 1 and 4 HEV ORF3 polypeptides with anti-HEV IgG were evaluated using EIA to detect serial sera from eleven rhesus monkeys,nine of them infected experimentally with different genotypes of HEV(JE12,JE67 and JE68 were infected with genotype 1 HEV from humans,JE13,JE14,JE53 and JE54 were infected with genotype 4 from humans,JE51 and JE52 were infected with genotype 4 from swine) and two of them vaccinated with ORF3 peptide from genotype 4 HEV.In the seria sera such as JE12,JE67 and JE68,the positive rate was 68.8%when evaluated with the genotyep 1 ORF3 capture antigen versus 37.5%when evaluated with the genotype 4 ORF3 capture antigen.Polypeptides from ORF3 of genotype 4 gave negative results with serial sera from monkeys JE12 which reacted positively to the polypeptides from ORF3 of genotype 1 beginning 4 weeks after infection,with peak S/CO value of 7.8. The HEV IgG antibody against the genotype 1 ORF3 polypeptides appeared 3 and 5.7 weeks earlier than those against genotype 4 ORF3 polypeptides in JE67 and JE68, respectively.And the former got 3 and 5.7 times the peak S / CO values of the latter's, respectively.Detection anti-HEV IgG antibody in serial sera from monkeys infected with genotype 4 HEV,both from human and swine,showed that the EIAs based on genotype 1 and genotype 4 ORF3 antigens yielded 29.1%and 60.9%positive rate, respectively.JE51 and JE53 were negative for IgG antibodies that reacted with ORF3 antigen from genotype I HEV but positive for IgG antibodies that reacted with ORF3 antigen from genotype 4 HEV beginning 5 weeks after challenge.When the reactivity with ORF3 peptides from both genotype 1 and 4 HEV were positive,the anti-HEV IgG against ORF3 peptide from genotype 4 HEV was present earlier than that against ORF3 peptide from genotype 1 HEV or almost at the same time and the mean S/CO value of them were 3.1 and 10.6,respectively.It is thus clear that the reactivity with ORF3 antigen from genotype 1 HEV was weaker than that from genotype 4 HEV.In summary,cross-reaction would occur if the titer of antibody was as high as enough.But the reaction between the same type of antigen-antibody was stronger than that between different genotypes of the cross-reactive antibody,when the antibody titer is low,few cross-reaction could be detected.Both of HEV IgG and IgM antibody detected results suggested that although there was only one serotype of HEV,but the antigen-antibody reaction among the HEV ORF3 from different genotypes were not consistent.So there may be differences respect to serotypes based on HEV ORF3.Immunogenicity and efficacy of HEV ORF3 polypeptideORF3 encodes a small protein of unknown function but which has significant antigenicity.The purpose of this study was to investigate the immunogenicity and efficacy of the ORF3 protein as a vaccine.The entire ORF3 protein of HEV expressed as a fusion protein was vaccinated eight monkeys twice(40μg) or three times(40μg), four weeks apart.Two or three doses of placebo(saline) were similarly administered. All animals were challenged with HEV four weeks after the last vaccination.The two-dose regimen animals were consisted of JE45 and JE46,challenged with genotype 4 HEV.The three-dose regimen animals were divided into two groups.One group such as JE61,JE62 and JE63 were challenged with genotype 1 HEV.The other group including JE64,JE65 and JE66 were challenged with genotype 4 HEV.Three monkeys of the control group such as JE53,JE54 and JE69 were challenged with genotype 4 HEV and the other four monkeys such as JE47,JE48,JE67 and JE68 were challenged with genotype 1 HEV.Pre-challenge serum samples from the vaccinated monkeys were tested for HEV IgG antibody against non-fusion recombinant ORF3 polypeptide.The monkeys vaccinated with two or three 40μg doses of hepatitis E vaccine developed anti-HEV ORF3.Post-challenge sera were tested using an enzyme-linked immunosorbent assay based on the ORF2 protein.All fifteen monkeys seroconverted to anti-HEV ORF2 post-challenge,regardless of whether they were vaccinated or not,and there was no significant difference between the vaccinated groups and the control groups.The ratio of geometric mean of the peak ALT value between post challenge and pre-challenge was calculated for each group.The control monkeys had a mean ratio of 7.4 after challenge with 1.10×10~6 genomic copies of genotype 1 HEV and 13.1 after the challenge with 1.14×10~7 genomic copies of genotype 4 HEV,indicating more severe hepatitis resulting from the challenge with the higher titer virus.In contrast,the mean ALT ratio for monkeys vaccinated with two doses of vaccine was 2.9 and the values for monkeys vaccinated with three doses of vaccine were 4.6 for those challenged with genotype 1 HEV and 3.9 for those challenged with genotype 4, indicating partial but not complete protection.Furthermore,the mean duration of ALT elevation was diminished by vaccination(1.5 weeks for the two-dose regimen and 2.3 weeks for the three-dose regimens),compared to the control animals(4.9 weeks).In addition,the mean duration of ALT elevation in the vaccinated group challenged with genotype 1 was longer than for the vaccinated group challenged with genotype 4, despite the latter being at higher titer.The two-dose vaccine regimen protected one of the two animals from viremia after the challenge with genotype 4 HEV and the other monkey had viremia for 2 weeks beginning 6 days after challenge.The three-dose vaccine regimen was less effective in preventing viremia,because only two of the six monkeys were protected from viremia.Nevertheless,the mean duration of viremia was shortened by vaccination(2 weeks),compared to the control animals(5 weeks).The two-dose vaccine regimen protected one of the two animals from shedding after challenge with genotype 4 HEV and the other monkey exhibited fecal shedding for 2 weeks(6 days after challenge).The three-dose vaccine regirnens were less effective in preventing infection.Although HEV was positive in feces from all of the monkeys,the mean duration of fecal shedding was diminished by vaccination(4.5 weeks),compared to the control monkeys(6.9 weeks).Furthermore,we found that genotype 1(5 weeks) HEV was excreted in larger amounts than genotype 4 HEV(4 weeks) following the three-dose regimens.In a word,we can only draw the conclusion that the vaccine was effective to some degree,offering partial protection against infection in response to a massive viral challenge.It is expected that protection against infection and hepatitis will be more effective if pORF3 is combined with pORF2 in a vaccine.