A phase 1 randomized open-label clinical study to evaluate the safety and tolerability of a novel recombinant hepatitis E vaccine.

BACKGROUND: This study aimed to evaluate the safety and tolerability for variable dosages of a novel hepatitis E vaccine p179. METHODS: The randomized open-label parallel control phase 1 clinical trial enrolled 120 eligible participants aged 16-65years in Jiangsu Province, China. The experimental groups were randomized to receive different dosages of 20µg, 30µg, and 40µg Hepatitis E Virus (HEV) p179 vaccines, with the 30µgHEV vaccine p239 Hecolin as control, and vaccinated at 0, 1 and 6month intervals. Participants were observed for solicited local and systemic adverse reactions (ARs) occurring within 7days after each vaccination, and any serious adverse events (SAEs) occurring within 6months post-vaccination. Blood samples were collected from participants 3days before and after each injection, to determine the blood routine and serum biochemical indexes. RESULTS: The solicited local ARs incidence in experimental groups were significantly lower than that of the control group (P=0.027). The difference between solicited total and systemic ARs incidence of experimental groups and the control group were not significant (P>0.05). Similar patterns were observed when the analyses were performed on the group having ARs of varying grades and symptoms. All changes in blood biochemical indexes and routine blood tests before and after different vaccinations were mild (grade 1) or moderate (grade 2), and the difference in experimental groups and the control group were not statistically significant. No vaccine related SAEs occurred in any of the subjects during the study. CONCLUSION: Three different dosages of HEV p179 vaccine were deemed safe and well tolerated. No vaccine-associated SAEs were identified, and the 30µg dosage formulation was selected for further investigation for efficacy. Clinical trials registration number: 2012L01657.

A single amino acid substitution changes antigenicity of ORF2-encoded proteins of hepatitis E virus.

Extensive genomic diversity has been observed among hepatitis E virus (HEV) strains. However, the implication of the genetic heterogeneity on HEV antigenic properties is uncertain. In this study, monoclonal antibodies (Mabs) against truncated ORF2-encoded proteins (aa452-617, designated p166 proteins) derived from HEV strains of Burma (genotype 1a, p166Bur), Pakistan (1b, p166Pak) and Morocco (1c, p166Mor) were raised and used for identification of HEV antigenic diversity. Six Mabs reacted to these 3 p166 proteins as well as p166 proteins constructed from strains derived from Mexico (genotype 2), US (genotype 3) and China (genotype 4), indicating the existence of pan-genotypic epitopes. Two Mabs, 1B5 and 6C7, reacted with p166Bur and p166Mor, but not p166Pak or p166s derived from genotypes 2, 3, and 4, indicating that these 2 Mabs recognized strain-specific HEV epitopes. Both the common and specific epitopes could not be mapped by 23 synthetic peptides spanning the p166Bur sequence, suggesting that they are confirmation-dependent. Comparative sequence analysis showed that p166Bur and p166Mor shared an identical aa sequence along their entire lengths, whereas for p166Pak the aas occupying positions 606 and 614 are different from aas at corresponding positions of p166Bur and p166Mor. Reactivity between 1B5 and p166Bur was abrogated with mutation of p166Bur/A606V, whereas p166Pak acquired the reactivity to 1B5 with mutation of p166Pak/V606A. However, mutations of p166Bur/L614M and P166Pak/M614L did not affect the immunoreactivity. Therefore, the aa occupying position 606 plays a critical role in maintaining the antigenicity of the HEV p166 proteins.

[Design and application of a set of universal PCR primers for genotyping of hepatitis E virus].

To improve the reliability and credibility of genotyping hepatitis E virus (HEV) and to explore the possibility of unifying standards of HEV genotyping by designing HEV universal primers for amplification of a long genomic fragment of different HEV genotypes. A set of universal primers (HEVuPrimer) was designed based on conserved regions determined by alignment analysis of 82 HEV strains with complete genome in GenBank. HEVuPrimer was compared with a set of previously used primers (MXJ primers) for their sequence-matching to different HEV strains and applied to amplify HEV genomic fragments from HEV reference strains with known different genotypes and clinical serum samples with anti-HEV-IgM by RT-nPCR. HEV genotyping based on the fragments amplified with HEVuPrimer was compared and validated with that based on HEV full genome and fragments obtained with MXJ primers. HEV genotyping by the phylogenetic analysis supplemented with the percent of nucleotide identity of the HEVuPrimer-determined fragments showed good correspondence with that based on HEV full-length genome. In addition, HEVuPrimer was much better than MXJ primers in matching sequences of HEV strains available from GenBank, and was able to amplify all the reference HEV strains with different genotypes. Among 124 samples with anti-HEV-IgM, 60 were positive for HEV RNA determined by a 644bp amplicon of RT-nPCR with the HEVuPrimenr. All the positive isolates belonged to HEV genotype 4 with nucleotide homology of 80.0%-99.9%, and could be further divided into 4 subgenotypes. Moreover, a novel subtype was identified with 6 HEV strains isolated very recently. The RT-nPCR using the HEVuPrimer and phylogenetic analysis of the amplified region provided strong evidences for its feasibility in HEV genetic classification. Our data have new implication for the consensus of genotype classification of HEV.

[Identification of a novel antigenic epitope on GST fusion-expressed and ORF2-encoded proteins of hepatitis E virus].

AIM: To investigate the effect of a GST tag on the antigenic structure of GST fusion-expressed and ORF2-encoded recombinant proteins of hepatitis E virus (HEV). METHODS: The monoclonal antibodies (mAb) were prepared with a GST fusion protein, p166Chn-GST, which was derived from a Chinese HEV strain. Then they were tested by indirect ELISA, competition ELISA and Western blot with different GST fusion, His fusion or non-fusion recombinant proteins derived from HEV reference strains of all 4 genotypes and other non-HEV recombinant proteins. RESULTS: Three mAb named 1A8, 9B4 and 8H10 were obtained. All of them reacted to p166Chn-GST but did not react to GST. mAb 1A8 and 9B4 reacted to 4 p166-GST proteins of different HEV genotypes and 2 N- or C-terminal truncated p166Chn-GST proteins named p146Chn-GST and p137Chn-GST, but they did not react to 4 p166-His proteins of different HEV genotypes and a non-fusion p179Chn protein. No detectable signals were found when 1A8 and 9B4 were subjected to HEV antigen competition ELISA or Western blot after SDS-PAGE. No cross reaction was observed between the two mAb and HEV-irrelevant GST fusion proteins, either. CONCLUSION: A novel antigenic epitope recognized by mAb 1A8 and 9B4 appears on the GST fusion-expressed and ORF2-encoded HEV recombinant proteins and it is dependent on the conformational folding of both GST and HEV sequences.

[Leukemia cell surface expression of E-cadherin and its correlation with membrane localization of beta-catenin].

OBJECTIVE: To investigate the expression of cell surface E-cadherin in leukemia cell and the correlation of cell membrane localization of beta-catenin with E-cadherin expression. METHODS: Bone marrow samples from 46 patients with acute leukemia and 17 normal donors were analyzed. Cell surface expression of E-cadherin and membrane localization of beta-catenin were labeled by immunofluorescence and analyzed with a laser scanning confocal fluorescence microscope in 14 specimens. RESULTS: Cell surface E-cadherin expression level was significantly lower in leukemia cells (with the median fluorescent intensity of 16.78) than in normal hematopoietic progenitors (26.03). Correlation analysis showed that cell membrane localization of beta-catenin was correlated with E-cadherin expression (r = 0.74, P = 0.002). After E-cadherin was induced to express in leukemic cell by 5-Aza-CdR, membranous expression of beta-catenin was elevated while the nuclear expression reduced, indicating that E-cadherin-mediated adhesions could recruit beta-catenin to cell membrane. CONCLUSION: The loss of E-cadherin in leukemia cells may result in beta-catenin translocating to the nuclear and transcriptional activation of its target genes.

[Expression of pig7 in acute leukemia and its clinical significance].

OBJECTIVE: To investigate pig7 expression level in acute leukemia (AL) and its clinical significance and explore the possible mechanisms for pig7 silence in terms of methylation control. METHODS: Expression levels of pig7 mRNA in bone marrow samples from 138 patients with de novo AL and 21 normal controls and in 6 leukemic cell lines were detected by quantitative real-time reverse transcription PCR (RT-PCR). Differentiation induction effect by all-trans retinoic acid (ATRA) and concomitant change in pig7 expression were also monitored in NB4 cells. Endonuclease analysis was employed to determined the identity of pig7 transcript present in AL samples. Methylation specific PCR (MSP) was used to elucidate if hypermethylation was responsible for pig7 silence in AL. RESULTS: Compared with that in normal control, pig7 expression was markedly decreased (0.62 vs 18.30, median, P < 0.01) in AL patients on progression (at diagnosis, relapse or refractory). No significant difference was observed between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). AL at diagnosis had a higher pig7 level than those with relapsed or refractory disease (1.43 vs 0.16, median, P < 0.05). The complete remission (CR) rate after chemotherapy was found to be significantly correlated with pig7 expression levels (P < 0.05). Differentiated NB4 cells showed an increased level of pig7 expression (from 1.61 +/- 0.72 to 44.75 +/- 3.93, P < 0.01). Only one form of pig7 transcripts i.e., Small integral membrane protein of late endosome (SIMPLE), was detected in AL patients. Hypermethylation of pig7 promoter was identified in K562 and HL-60 cells, in contrast to non-methylation predominant in U937 cells. CONCLUSION: Aberrant down-regulation of pig7 provides novel insights into leukemogenesis and therapy response prediction in AL.

[Characterization of antigenic epitopes of ORF2 encoded proteins of hepatitis E virus genotype 4 identified in China].

To characterize antigenic epitopes of hepatitis E virus (HEV) genotype 4 that was first identified in China a few years ago, a recombinant protein, p166Chn, encoded by HEV genotype 4 ORF2 was used to prepare anti-p166Chn McAbs. Simultaneously, twenty N- or C-terminal truncated p166Chn proteins were generated. Immunoreactivity between the McAbs and the truncated proteins as well as seven p166 recombinant proteins derived from different HEV genotypes and subgenotypes was detected by indirect ELISA, Western blot and competition inhibition ELISA. Two reactive profiles were observed with different McAbs and different truncated proteins. The McAbs, represented by 1G10, reacted with those N-terminal truncated proteins beginning at upstream of aa477 and those C-terminal truncated proteins ending at down-stream of aa613, suggesting that the epitope recognized by 1G10 relied on the region of aa477aa613 and was conformation-dependent. While McAb 2F11 was reactive to those truncated p166Chn proteins beginning at upstream of aa474 or ending at downstream of aa617, indicating that the epitope recognized by 2F11 was also conformation-dependent and relied on a longer peptide of aa474aa617. However, the two groups of McAbs didn't inhibit each other when tested by a competition inhibition ELISA, which confirmed the different spatial positions of the two epitopes. Furthermore, when p166 proteins derived from different HEV genotypes and subtypes were applied, all of the McAbs prepared against pChn166 of genotype 4 identified in China could react with the proteins of genotype 1, 2 and 3 distributed worldwide. The data suggested that the two identified epitopes were HEV genotype-common and played significant effects on cross immunoreactivity between different HEV genotypes.

[Immunogenicity of experimentally combined hepatitis A and hepatitis E vaccine at different dosage ratio].

AIM: To evaluate the immunogenicity of experimentally combined hepatitis A and hepatitis E (cHA+E) vaccine and analyze the interactions between the two antigenic components in vaccine. METHODS: Nine different dosage ratio of the cHA+E vaccine, A500 + E200, A500 + E100, A500 + E50, A250 + E200, A250 + E100, A250 + E50, A125 + E200, A125 + E100 and A125 + E50 (U+mg)/L, was prepared from an inactivated hepatitis A (HA) vaccine and a hepatitis E (HE) vaccine. 120 mice were immunized with the cHA+E vaccine, HA vaccine and HE vaccine alone in different doses, respectively. Mice were bled before immunization and 2, 4, 6, 8, 10, 12 and 14 weeks post immunization. Specific antibodies against hepatitis A and E viruses were detected by ELISA and neutralization assay. RESULTS: After immunization with different dosages of cHA+E vaccine, all animals developed hepatitis A virus antibody (anti-HAV) and hepatitis E virus antibody (anti-HEV). Anti-HAV responses increased with the concentration of the HA vaccine component in the cHA+E vaccine. Anti-HAV neutralizing antibody titers reached 1:1 024 when mice were immunized with cHA+E vaccine prepared from higher dosage (500 U/mL) of the HA vaccine. The titers only reached 1:512 when lower dosages (250 and 125 U/mL) of HA vaccine were applied. However, different dosages (200, 100 and 50 mg/L) of the HE vaccine component in the cHA+E vaccine developed no effects on the production of anti-HAV neutralizing antibody. In addition, comparing with the single HE vaccine, the cHA+E vaccine induced higher level of anti-HEV response. The anti-HEV level increased with the concentration of HA vaccine component in the cHA+E vaccine. However, there was no obvious relationship between the anti-HEV levels and the different dosages (200, 100 and 50 mg/L) of the HE vaccine component in the cHA+E vaccine. Immune sera elicited by the cHA+E vaccine neutralized HEV infectivity when detected by an in vitro RT-nPCR-based HEV neutralization assay. CONCLUSION: In cHA+E vaccine, the HA vaccine component can increase the immunogenicity of the HE vaccine component, and the HE vaccine component plays no adverse effects on the immunogenicity of the HA vaccine component.

The first experimental study on a candidate combined vaccine against hepatitis A and hepatitis E.

To test the possibility of developing a combined vaccine against hepatitis A and E, groups of mice were immunized with different formulations containing different dosages of a commercially inactivated hepatitis A vaccine and a candidate recombinant hepatitis E vaccine. Monovalent vaccine components were used as controls. The experimental results showed that the combined vaccine could induce neutralizing antibodies against both hepatitis A virus (HAV) and hepatitis E virus (HEV) effectively in mice. Moreover, the inactivated hepatitis A vaccine could increase the immunogenicity of the recombinant HEV protein, and the recombinant HEV protein had no adverse effects on the immunogenicity of the inactivated HAV vaccine. Thus, the present study demonstrates an important first step for the further development of a combined hepatitis A and E vaccine.

[Hepatitis E virus of different genotypes contains multiple-type antigenic epitopes].

Monoclonal antibodies (McAbs) were prepared against a recombinant protein p166Us derived from US-1 strain of hepatitis E virus (HEV). The immune reactivity of the McAbs to seven p166s derived from different genotypes and subtypes of HEV, which included p166Bur (genotype I a), p166Pak (genotype I b), p166Mor (genotype I c), p166Mex (genotype II), p166Us (genotype III), p166Nz (swine HEV, genotype III) and p166Chn (genotype IV), was tested by an indirect enzyme-linked immunosorbent assay (ELISA) and a Western blotting assay. The immunological relationship between the McAbs and native HEV particles or anti-HEV positive serum samples was analyzed by an antigen-competitive or antibody-competitive ELISA. Totally, six McAb-producible hybridoma cell lines, designated by the name of 4D3, 2E3, 11E11, 12H5, 3A3 and 1F1 respectively, were cloned and obtained in this study. The McAb of 4D3 could react to all of the seven p166 recombinant proteins. This kind of reaction could be inhibited by each of 4 genotypes of native HEV particles or anti-HEV positive serum samples. The McAbs produced by 2E3, 11E11 and 12H5 reacted to p166Us, p166Nz and p166Chn, but did not react to p166Bur, p166Pak, p166Mor and p166Mex. The reaction of McAb of 2E3, as an example of the McAbs of 2E3, 11E11 and 12H5, could be only inhibited by genotype III and IV HEV or anti-HEV positive serum. The McAb of 3A3 could bind to p166Us as well as p166Nz. The McAb produced by 1F1 was reactive to the p166Us only. However, neither of I , II , IV genotype HEV particles or antisera could inhibit both of their reactions to p166Us. The data as mentioned above suggested that there are multiple-type antigenic epitopes such as genotype I, II, III and IV common, III and IV common, and III specific epitopes within HEV ORF2 encoded p166 proteins of different genotypes and subtypes of HEV. Moreover, the antigenic epitopes on recombinant protein p166s and these on native HEV particles possess identical immunological characteristics.

[Expression, purification and immunogenicity of a novel hepatitis E virus-like particle].

AIM: To express and characterize a novel hepatitis E virus (HEV) recombinant protein which contains HEV neutralization epitope(s). METHODS: The gene fragment encoding for amino acid 452-617 of HEV open reading frame 2 protein (pORF2) was inserted into the plasmid pET28a(+). The recombinant plasmid was used to transform the E. coli BL21(DE3) strain. The recombinant protein was expressed by IPTG induction, purified by Ni-NTA chromatography and analyzed by SDS-PAGE, Western blot and electronmicroscopy. Immune responses to the recombinant protein were determined by the immunization of mice. RESULTS: The expressed HEV recombinant protein was in a naturally-soluble form with a molecular weight of 22,000. The protein assembled into a virus like particle (VLP) with a diameter of approximate 20 nanometers. Moreover, this novel protein was reactive to serum samples obtained from the patients with HEV infection. After the mice were immunized with the protein, they developed anti-HEV antibodies which could neutralize HEV infection in cell culture. CONCLUSION: An E. coli-expressed recombinant protein containing 166 amino acid of HEV pORF2 can form VLP with good immunogenicity and antigenicity. This novel HEV VLP is valuable for the development of HEV vaccine and new diagnostic kit.

[Effects of different vectors and gene fragments on antigen expression of hepatitis E virus DNA immunization].

AIM: To investigate the effects of different vectors and gene fragments on antigen expression of hepatitis E virus (HEV) DNA immunization. METHODS: Gene fragments encoding p166 and p179, which contain the neutralization antigenic epitopes of a Chinese strain of HEV genotype IV, were cloned into two different eukaryotic expression vectors (pTR421 and pCDNA3.1), respectively. The in vitro expression level of p166 and p179 in HepG2 cells transfected by each of the recombinant plasmids with lipofectamine2000 was examined by means of immunofluorescence and Western blot. Meanwhile, the in vivo expression level in muscles of mice was examined with immunohistochemistry staining. RESULTS: Four recombinant plasmids, pTR421-166, pTR421-179, pCDNA3.1-166 and pCDNA3.1-179, were constructed successfully and confirmed correct with restriction endonuclease analysis and nucleotide sequencing. The antigen expression was only detected in HepG2 cells transfected by pTR421-179 and in myocytes of the mice injected with pTR421-179. Neither in vitro nor in vivo antigen expression was detected with pTR421-166 although p166 was only 13 amino acids shorter than p179 at N terminus. Neither pCDNA3.1-166 nor pCDNA3.1-179 was expressed in vitro and in vivo. CONCLUSION: Selection of the vectors and gene fragments is critical to HEV gene expression and HEV DNA vaccine.

[A new PCR-based in vitro neutralization assay of hepatitis B virus].

OBJECTIVE: To establish a PCR-based neutralization assay of hepatitis B virus (HBV), which may be applied for detecting neutralizing antibodies against HBV and used as an in vitro model to screen new HBV vaccines. METHODS: Immune serum was mixed with HBV stock. The mixture was incubated and then inoculated onto Hep G2 cell monolayers. After adsorption, washing and incubation, HBV DNA was extracted from the cells and detected by PCR. The neutralization effect was determined based on the PCR results. RESULTS: Two HBV stocks suitable for the neutralization assay were selected from 18 serum samples collected from patients with hepatitis B. The neutralization assay was optimized in the conditions of using 10 infectious doses of the HBV stock and incubating the cell culture for 24 hours prior to PCR detection. Four immune sera obtained from mice immunized with commercial HBV vaccine and 2 serum specimens from mice immunized with 2 new HBV vaccine candidates definitely blocked the in vitro HBV adsorption. However, 4 sera obtained from normal mice and 2 sera from mice immunized with 2 hepatitis E virus vaccine candidates did not show any neutralizing activity. CONCLUSION: The established new PCR-based in vitro HBV neutralization assay is a simple, rapid and economic assay. It may be used as a model for primary evaluation for HBV vaccine candidates prior to primate assay.

[Genotyping of hepatitis E virus by PCR combining with single restriction endonuclease analysis].

OBJECTIVE: To develop a simple method for genotyping of hepatitis E virus (HEV) and to investigate HEV genotype distribution in Nanjing area. METHODS: Twenty-seven full HEV sequences currently-available in GenBank were analyzed with MegAlign and MapDraw programs of DNA STAR software. Degenerate primers were designed and applied to amplify a fragment in HEV ORF1 region. HEV genotypes were determined by the size of the PCR products and by single restriction endonuclease analysis. RESULTS: The PCR products of HEV genotype 1 and 2 were 275 bp and 269 bp in size. Distinctively, the PCR products of genotype 3 and 4 were 317 bp and 314 bp in size. Moreover, the PCR products of genotype 1 could be digested by Nae 1, but the products of genotype 2 could not. Distinctively, the PCR products of HEV genotype 3 could be digested by Not 1, but the products of genotype 4 could not. Six HEV reference strains standing for different HEV genotypes were clustered into their own types as predicted. Within 43 HEV IgM-positive clinical specimens collected in Nanjing, 19 were HEV PCR-positive and identified as genotype 4. CONCLUSION: A simple method of PCR combined with single restriction endonuclease analysis is developed for HEV genotyping. This assay allows rapid identification of a large number of HEV isolates directly from clinical specimens. Among patients with hepatitis E in Nanjing, most were infected with HEV genotype 4.

Identification of 5' capped structure and 3' terminal sequence of hepatitis E virus isolated from Morocco.

AIM: To examine 5' and 3' terminal sequences of hepatitis E virus (HEV) isolated from Morocco, to confirm 5' methylated cap structure of the genome, and to investigate whether the 3' UTR can be used to distinguish HEV genotypes instead of HEV complete genome sequence. METHODS: RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) was employed to obtain the 5' and 3' terminal sequences of HEV Morocco strain. The 3' UTR sequence of the Morocco strain was compared with that of the other 29 HEV strains using the DNAStar software. RESULTS: The 5' PCR product was obtained only from the RLM-RACE based on the capped RNA template. The 5' UTR of the Morocco strain had 26 nucleotides, and the 3' UTR had 65 nucleotides upstream to the polyA. The 5' UTR between HEV strains had only point mutations of nucleotides. The phylogenetic tree based on the sequences of 3' UTR was not the same as that based on the complete sequences. CONCLUSION: The genome of HEV Morocco strain was methylated cap structure. The 3' terminal sequence can not be used for distinguishing HEV genotype for all HEV strains in place of the whole HEV genome sequence.

[Construction and screening of hepatitis E virus-specific phage antibody combinatorial library].

AIM: To construct HEV-specific phage combinatorial anti-body library and screen anti-HEV antibodies with neutralizing activity from the library. METHODS: The total RNA was extracted from B-lymphocytes of 6 HE patients. Kappa chain and Fd segment of IgG gene were amplified respectively by RT-PCR using a set of Fab-specific primers. The amplified gene were inserted successively into vector pComb3 and electrotransformed E. coli XLI-Blue cells. Furthermore, the recombinant phage was rescued by being concultured with helper phage VCSM13 to construct HEV-specific phage anti-body library. RESULTS: Fab displayed on the surface a as fusion protein with the N terminal of coat protein III, and 1. 8 x 10(7) clone library was established. Specific antibodies to HEV ORF2 recombinant antigen were acquired after five rounds of panning with HEV ORF2 recombinant antigen including neutralizing epitope. CONCLUSION: Four clones exhibited specific binding to HEV ORF2 recombinant antigen including neutralizing epitope is identified by ELISA. The results show that we have got the recombinant phage antibodies.