| Hepatitis E virus (HEV) is non-enveloped, positive-strand RNA virus that is transmitted mainly through oral-route and causes human acute hepatitis E (HE). Hepatitis E is generally a mild disease but may be severe, especially in pregnant women, among whom the mortality rate reaches 20%. Recently, chronic HEV infection has been reported in the organ transplanted and immunosuppressed patients. HEV causes epidemic and sporadic cases of acute hepatitis E in developing countries of Asia, Africa and Latin America, where sanitary conditions are suboptimal. Since the HEV infects human and animals, and can be transmitted from animals to human beings, in recent years, the incidence of hepatitis e infection in the developed countries also increased significantly, and this indicates that HEV poses a public health threat across the globe.HEV isolates, which could infect human, derived from different area all over the world could be classified into four major genotypes. Diverse behaviors have been found among different genotypes, such as isolates from genotype 1 and 2 only infect human beings and responsible for severe HE outbreaks. Whereas, genotype 3 and genotype 4 do not only infect human but also cause sub-clinical infection in animals. The data indicate that divergence exists between HEV isolates from different genotype and from different hosts. To date, no in vitro culture system for HEV replication is available. Therefore, it is difficult to characterize the antigenic epitopes of HEV without viral particles. The major HEV structural protein is encoded by HEV open reading frame (ORF) 2 of the viral genome. Predominant immunogenic regions have been localized at C-terminal 2/3 region of pORF2, including epitopes that represent protective neutralizing activity,which is the main target of HE vaccine development. In China, two recombinant vaccines, based on human-HEV sequences, have undergone clinical trials. One of these vaccines, HEV 239, is already commercialized in China. It was derived from HEV genotype 1 and produced in bacterial cells. The second vaccine, HEV p179, was derived from HEV genotype 4 and expressed in E. coli; and was developed by the Changchun institute of biological products and our laboratory, phase 1 clinical trial has been completed.Our previous study revealed that recombinant protein p166,consisting of amino acid(aa) residues 452 to 617 of HEV ORF2 at the C-terminus, could model HEV conformational neutralization epitope. When performing neutralization assay, antibodies induced by p166 could protect rhesus monkeys from experimental infection of HEV in vivo. Mapping of p166truncated proteins revealed that neutralizing epitopes are located between aa 460-605. So the p166 protein can be used as a packet of antigen to detect anti-HEV antibodies in the sera of infected or vaccine immunized subjects. Sensitivity difference has been noticed when proteins from different HEV genotypes and subtypes were used in ELIS A to detect anti-HEV antibodies in sera from HE patients.This data indicated that genotype associated specific epitopes may exist in the p166 recombinant protein. Sensitivity difference has been noticed when p166 proteins, generated from different HEV genotypes, were used for detection of anti-HEV antibodies in sera from patients infected with different HEV genotypes. But it was unknown whether the genetic differences of IgG induced by immunization with p239 or p179, that is to say, the immunogenicity differences between the two vaccines, exist. The main research work is listed as follows:1. The immunogenic differences of p239 and p179 vaccines derived from different genotypes.To study the immunogenic differences of p239 and p179 vaccines, anti-HEV IgG in serum samples after immunization with p239 or p179 were detected by ELIS A using p166 proteins derived from different genotypes as antigens. Serum samples were obtained from eight rhesus monkeys inoculated intravenously with105 HEV genome equivalents of genotype 1 strain (W01) or HEV genotype 4 strain (NJ703). Sixty human serum samples were collected in a clinical trial of HEV vaccine at Danyang, Jiangsu province after three vaccination shots with p239 or p179. Mice serum samples were obtained from twelve mice experimentally immunized with p239 or p179. All sera were analyzed for the presence of anti-HEV IgG using indirect ELISA, based on HEV genotypes 1-4 truncated ORF2 antigens (pl66W01, p166Mex, p166US and p166Chn). Two sets of indirect sandwich ELIS As were developed, the first assay used a mixture (p166mix) of human HEV antigens originating from genotype 1,2,3 and 4 in equal concentration and the second assay based on p166w01, p166Mex, p166US and p166Chn respectively. The results are as shown below:A. Serial dilutions of serum samples from 12 mice immunized with p239 (n=6) or p179 (n=6) were tested for the presence of anti-HEV IgG using p166w01, p166Mex, p166US and p166Chn.Interestingly, The results showed that the antigens generated from HEV genotypes 1 and 2 (p166w01 and p166Mex) reacted strongly and similarly with the anti-HEV IgG in sera from mice and human vaccinated with genotype 1-generated vaccine (p239) and showed weak immunoreactivity against anti-HEV antibodies raised against genotype 4-generated vaccine (p179). By contrast, the antigens generated from HEV genotypes 3 and 4 (p166US and p166Chn) showed an almost identical higher ability for detection of anti-HEV IgG in sera from mice and human vaccinated with p179.B. The detection of anti-HEV IgGs in human sera collected from vaccinees with either p239 (n=30) or p179 (n=30), were tested for the presence of anti-HEV IgG using p166w01, p166Mex, p166US, p166Chn. In both sera groups (from human vaccinated by p239 or p179) p166w01 and p166Mex presented an almost identical ability to detect anti-HEV IgG; and so did p166US and p166Chn.16 out of the 30 sera from p239-vaccinated subjects have shown OD values at least 2 times higher with genotypes 1 and 2 antigens than with genotypes 3 and 4 antigens; and 19 out of the 30 sera from p179-vaccinated subjects demonstrated OD values at least 2 times higher with genotypes 3 and 4 antigens than with genotype 1 or 2 ones.C. Sera collected from rhesus monkeys inoculated intravenously with HEV genotype 1 strain (W01) or HEV genotype 4 strain (NJ703), were tested for anti-HEV IgG using p166W01, p166Mex, p166US and p166Chn, respectively. The antibodies induced by genotype 4 strain reacted strongly with genotypes 3 and 4 antigens (p166US, p166Chn) than with genotypes 1 or 2 ones (p166W01, p166Mex). Furthermore, p166US and p166Chn have shown a similarity in their immunoreactivity that was statically different from p166w01 and p166Mex immunoreactivity which was also very similar (p166w01 and p166Mex reactivity). By contrast, antibodies induced by genotype 1 strain showed a stronger immunoreactivity against p166w01and p166Mex than against p166US or p166Chn.All of the 4sera from p239-vaccinated subjects have shown OD values at least 4 times higher with genotypes 1 and 2 antigens than with genotypes 3 and 4 antigens; and all of the 4 sera from p179-vaccinated subjects demonstrated OD values at least 4 times higher with genotypes 3 and 4 antigens than with genotype 1 or 2 ones.These data suggest that the genetic differences exist between anti-HEV IgGs in sera after either an immunization with HE vaccines or infection with HEV. That is to say, p239 and p179 vaccines as well as wild HEVs of different genotypes have immunogenicity differences.2. The mechanism of the immunogenic differences of p239 and p179 vaccines derived from different genotypes.To reveal the mechanism of the immunogenic differences of p239 and p179 vaccines, McAbs against p166s derived from HEV of genotype 1 and 4 were prepared. ELISA, Western blot, in vitro neutralization assay and competitive ELISA were used to identify and characterize the McAbs.A. The genotype 1 strain p166Sar was used to immunize female Balb/c mice. Then, the mouse spleen cells were fused with SP2/0. Three hybridoma cell lines screening McAbs 3G1, 4H4 and 2B1 against p166 derived from HEV genotype 1 were obtained. The generated McAbs were tested with p166 recombinant proteins derived from HEV genotypes 1,2,3 and 4 by ELISA and Western blot. They were evaluated for their ability to neutralize human HEV genotypes 1-4 strains by an in vitro PCR-based HEV neutralization assay. The McAb 3G1 could bind to p166 recombinant proteins derived from HEV genotypes 1,2,3 and 4. However,3B1 only reacted to p166 recombinant proteins derived from HEV genotypes 1,2. To further study the neutralizing activity of mAbs against the four HEV strains w01 (genotype1), Mexico-14 (genotype2), US-1 (genotype 3) and NJ703 (genotype4), an in vitro neutralization assay was performed. This latter demonstrated that 2B1 could only neutralize HEV strains w01 and Mexico-14, but did not neutralize HEV strains US-1 and NJ703. On the other hand, the 3G1 can neutralize HEV strains of the four genotypes.B. Three McAbs 4C5,6A1,5G5 were obtained by immunizing Balb/c mice with pChn166 which is derived from genotype 4.The McAbs prepared above were used to characterize HEV epitopes by using an indirect ELISA and western-blot assay. An in vitro PCR-based neutralization assay was used to define the neutralizing activity of the epitopes. The results revealed that McAb 4C5 reacted only with p166US and p166Chn, but not with p166W01 or p166Mex. Meantime, McAb 5G5 well reacted against the four p166 proteins. These observations suggest that there might be at least three different epitopes in the p166 proteins. The first one is specific to genotypes 1 and 2 as characterized by mAb 3B1; the second is specific to genotypes 3 and 4 as recognized by McAb 4C5; and the last one is common to the four genotypes since it was recognized by two different Mabs3G1 and 5G5 which were raised against two different antigens generated from HEV genotypes 1 and 4 respectively.C. A competition assay was performed to determine whether the McAbs recognize similar or overlapping epitopes on the HEV capsid. McAbs 2B1,3G1,4C5,5G5 were produced, purified and conjugated with HRP. McAb 2B1 could react with p166W01 but not p166Chn, the McAb 4C5 could react against p166Chn but not p166W01, McAbs 3G1,5G5 could react with both p166W01 and p166Chn. Two competitive ELISA were established and the results showed that the binding of 2B1 to p166w01 could not be inhibited by 5G5 or 3G1.Similarly, the binding of 4C5 to p166Chn could not be inhibited by 5G5 or 3G1. But the binding of 5G5 to p166W01 or p166Chn could be inhibited by 3G1.In conclusion, we demonstrated that the p166 recombinant proteins contain neutralizing epitopes common to the 4 genotypes and also contain genotypes 1 and 2 specific neutralizing epitopes (for p166 proteins derived from genotypes 1 and 2) and genotypes 3 and 4 specific neutralizing epitopes (for p166 proteins derived from genotypes 3 and 4). Since both p239 and p179 vaccines contain the p166 fragment, p166 genotype-related immunoreactivity differences could perfectly explain not only p239 and p179 immunogenicity differences, but also the immunoreactivity differences of the anti-HEV antibodies in sera from rhesus monkeys infected with different HEV strains (genotypes 1 and 4 strains). The HEV genotype-specific neutralizing epitopes are not only present in the engineered recombinant proteins, but also in the natural HEV particles. The immunogenicity differences of HE vaccines and their underlying mechanisms, reported in this study are new insights into the field of HEV research. Therefore, we believe that these new findings could be of crucial importance for the evaluation of HEV vaccines efficacy and opens a new exciting door in the HEV research field for development of new diagnostic kits and more effective vaccines that would engage both common and specific epitopes for a more powerful protection such as a genotypes 1 and 4 combined vaccine for both humans and consumed animals. |
