A novel linear neutralizing epitope of hepatitis E virus.

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A novel linear neutralizing epitope of hepatitis E virus

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Zi-Min Tang a,1 , Ming Tang b,1 , Min Zhao b , Gui-Ping Wen b , Fan Yang a , Wei Cai b , Si-Ling Wang a , Zi-Zheng Zheng a,∗ , Ning-Shao Xia a,b,∗ a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, Fujian 361005, PR China b School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, PR China

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Article history: Received 17 January 2015 Received in revised form 14 May 2015 Accepted 23 May 2015 Available online xxx

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Keywords: Hepatitis E virus Linear epitope Nonimmunodominant Vaccine Neutralization

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1. Introduction

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Hepatitis E virus (HEV) is a serious public health problem that causes acute hepatitis in humans and is primarily transmitted through fecal and oral routes. The major anti-HEV antibody responses are against conformational epitopes located in a.a. 459–606 of HEV pORF2. All reported neutralization epitopes are present on the dimer domain constructed by this peptide. While looking for a neutralizing monoclonal antibody (MAb)-recognized linear epitope, we found a novel neutralizing linear epitope (L2) located in a.a. 423–437 of pORF2. Moreover, epitope L2 is proved non-immunodominant in the HEV-infection process. Using the hepatitis B virus core protein (HBc) as a carrier to display this novel linear epitope, we show herein that this epitope could induce a neutralizing antibody response against HEV in mice and could protect rhesus monkeys from HEV infection. Collectively, our results showed a novel nonimmunodominant linear neutralizing epitope of hepatitis E virus, which provided additional insight of HEV vaccine. © 2015 Published by Elsevier Ltd.

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Hepatitis E is one of the major acute viral hepatitides and is caused by hepatitis E virus (HEV). Outbreaks usually occur in developing countries [1,2], but sporadic cases have also been identified in Europe, the United States and Japan [3–7]. HEV is a naked, positivestrand RNA virus. HEV has nonenveloped, 32–34 nm, icosahedral virions, and the native virion has a T = 3 symmetry and was composed of 180 copies of the capsid protein [8,9]. The 7.2 kb RNA genome contains 3 open reading frames (ORFs), of which ORF2 (nt 5147–7127) encodes the main structural protein constructing the viral capsid, pORF2 [10]. The capsids of HEV constructed with pORF2 are the main target of host antibody responses. Both prokaryotic- and eukaryoticexpressed recombinant pORF2 antigens efficiently induce a host antibody response and immune protection against HEV [11,12]. The main antibody responses induced by recombinant pORF2 depend

∗ Corresponding authors at: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, 422nd Siming South Road, Xiamen 361005, PR China. Tel.: +86 592 2880626; fax: +86 592 2181258. E-mail addresses: [email protected] (Z.-Z. Zheng), [email protected] (N.-S. Xia). 1 These authors contributed equally to this work.

on the presence of conformational epitopes located in a.a. 459–606 of pORF2 [13,14]. Additionally, in a dimeric domain constructed by pORF2, a.a. 459–606 was the target in sera from HEV infected patients [15–17]. A study of HEV capsid structures (PDB, 2ZTN, 3YIO, 2ZZQ, and 3HAG) further showed that the dimeric domain (E2s/P2 domain) constructed by a.a. 459–606 of pORF2 was highly exposed on the surface of the HEV capsid [18–22]. A prokaryotic-expressed recombinant pORF2 peptide (a.a. 368–606), p239, can self-assemble a 23 nm virus-like particle (VLP) by first constructing dimers [12]. The HEV vaccine, which uses p239 recombinant particle as a unique antigen, was demonstrated to be safe and efficacious during a large scale clinical trial [23]. In this study, we generated 30 conformational dependent and 6 linear dependent HEV-specific mouse monoclonal antibodies (MAbs) by immunization with p239 VLP (Table 1S) and found a novel neutralizing MAb 12A10. The neutralization sites had been characterized in HEV VLPs with a large panel of monoclonal antibodies [14,16,24–29]. Different from all identified neutralizing sites were conformational and were mapped in the a.a. 459–606 of pORF2 and are composed with discontinuous peptide stretches, the epitope recognized by MAb 12A10 was a linear determinant (L2) and located in a.a. 423–437 of pORF2. To improve the immunogenicity of antigens, virus-like particles (VLPs) were employed as particulate carriers. Among these, HBc protein VLP has been well characterized and used as carrier for different foreign sequences [30–32]. HBc protein consists roughly

http://dx.doi.org/10.1016/j.vaccine.2015.05.065 0264-410X/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Tang Z-M, et al. A novel linear neutralizing epitope of hepatitis E virus. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.065

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of an assembly domain (residues 1–149) and an arginine-rich protamine-like domain (residues 150–183) [33]. The C-terminal deletion form which is truncated at 149 still can self-assemble into practically indistinguishable particles from full-length HBc particles [34,35]. And the immunogenicity of inserted foreign epitope can be considerably enhanced owing to repetitive arrays and multiple potent T helper epitopes of HBc VLPs [36]. As the 15-mer peptide lacks of immunogenicity, we used the HBc protein as a carrier to display L2 epitope as the procedure described in other studies [37,38]. Further investigation in this study indicated that the L2 epitope is a novel linear neutralizing non-immunodominant determinant on the HEV capsid.

replaced by a linker (G4 SG4 T-GS-G4 SG4 ), in which GS-encoding sequence was a BamH I site [40]. To express the HBcL1/L2/L3 fusion protein, the L1/L2/L3 gene was inserted into the pC149/mut plasmid vector [40] by anneal oligos to generate a plasmid HBcL1/L2/L3 with the primers (Table 2S), which inserted 15 amino acids between a.a. 78 and 83 of pC149/mut. Assemble the annealing reaction by mixing 1 ␮L of each oligo with 48 ␮L annealing buffer (100 mM NaCl and 50 mM HEPES pH 7.4), Incubate the mixture at 90 ◦ C for 4 min, and then at 70 ◦ C for 10 min. Slowly cool the annealed oligos to 10 ◦ C. The annealed oligo inserts can be used immediately in a ligation reaction. Recombinant HBcL1/L2/L3 and pC149/mut were prepared as described previously [40].

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2. Materials and methods

2.6. Murine polyclonal antibody preparation

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2.1. Cell lines, antibodies and hepatitis E virus

Six-week-old BALB/c female mice were immunized subcutaneously with 100 ␮g of the peptide fusion protein mixed with Freund’s complete adjuvant (Sigma, St. Louis, USA). This was followed by two booster immunizations with 100 ␮g of protein in an incomplete adjuvant (Sigma, St. Louis, USA) at 1 week intervals. Blood samples were taken from immunized mice before every booster immunization.

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HepG2 cells (HB-8065, obtained from the ATCC, MD, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (both from GIBCO, California, USA) and antibiotics (100 unit/ml ampicillin and 100 unit/ml streptomycin) (Lukang, Shandong, China) at 37 ◦ C with 5% CO2 . HEV capsid protein-specific MAbs (15B2, 12A10, 1B7, and 8G12) were produced in our laboratory. Mouse MAb anti-␤-tubulin was from Santa Cruz Biotechnology. Goat anti-mouse polyclone antibody (PAb) conjugated with Alexa Fluor 680 was from Invitrogen. The virus source was stool samples from rhesus monkeys infected with genotype 1 virus (strain Xinjiang), and genotype 4 virus (strain Ch-S-1), respectively.

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2.2. In vitro neutralization test of HEV by MAb/PAb in HepG2 cells

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MAb/PAb (100 ␮g) were mixed with 105 copies of HEV virus and incubated at 37 ◦ C for 30 min. Then, the mixture was added to HepG2 cells (106 cells per well) and incubated at 37 ◦ C for 30 min. After washing three times with PBS, the presence of HEV RNA in the cells was detected by RT-PCR [39].

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2.3. Indirect enzyme-linked immunosorbent assay (ELISA)

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Purified recombinant proteins were used as coating antigens and were added (100 ng per well) to carbonate buffer (pH 9.6) at 37 ◦ C for 4 h. After washing once with 0.025% (v/v) Tween 20 in PBS (PBST), the proteins were blocked with 2% BSA in PBS at 37 ◦ C for 2 h. MAbs (100 ␮l) were added to the wells and incubated at 37 ◦ C for 30 min. The plates were washed five times and incubated with HRP-conjugated goat anti-mouse antibodies (Wantai, Beijing, China) at 37 ◦ C for 30 min. After washing five times, the color was developed. The absorbance was measured at 450 nm and 620 nm.

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2.4. Competitive ELISA assay of MAb and serum

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Microtiter plate wells were coated with 100 ng per well of purified recombinant protein as indirect ELISA. Patient serum was diluted 1:100 (v/v) in PBS prior to use. The serum (100 ␮l per well) was added to the well and incubated at 37 ◦ C for 1 h. After washing five times with PBST, the plates were incubated with 100 ␮l/well HRP-conjugated competition antibody (8G12HRP and 12A10-HRP) at 37 ◦ C for 40 min. The method of detection is described in the Indirect ELISA section.

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2.5. Plasmid construction and production of proteins

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N-terminus of HBc antigen (a.a.1-149) protein was used for present the linear epitope. In pC149/mut plasmid vector, amino acid 79–81-encoding sequence of HBc antigen (a.a. 1–149) was

2.7. In vitro neutralization test of genotype 1 p239 (p239(1)) and genotype 4 p239 (p239(4)) by MAb and PAb in HepG2 cells Antibodies (100 ␮g) were mixed with 15 ␮g of p239(1) or p239(4) and incubated at 37 ◦ C for 30 min. The mixture was added to the HepG2 cells and incubated at 4 ◦ C for 30 min. The cells were washed three times with PBS, and the proteins in the cells were tested by Western blotting. 2.8. Western blotting The protein concentration was assessed using the Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of proteins were separated by SDS-PAGE and subsequently blotted onto a nitrocellulose membrane (Whatman, Dassel, Germany). After transfer, the membrane was immersed for 30 min in a blocking solution (5% non-fat milk in PBS) and washed with PBST. The membrane was incubated with the primary antibody and then washed three times with PBST. Alex Fluor 680 conjugated polyclonal antibodies (PAbs) were used as the secondary antibody. The reaction signals were quantitatively examined with an Odyssey imaging system (Li-COR, Lincoln, Nebraska). 2.9. Protection test in rhesus monkeys by using HBcL2 recombinant chimeric particles Six rhesus monkeys were used in this study. The animals were screened using an HEV Ab ELISA kit (Wantai, Beijing, China), and none showed detectable serum HEV antibodies at 1:10 serum dilution. The 6 rhesus monkeys were divided into 3 groups and immunized with 20 ␮g of p239 particles, HBc-L2 particles or HBc particles separately, in 3 doses by a deltoid muscle injection on the left forelimb (0, 30 and 120 d). Proteins were mixed with Freund’s complete adjuvant in the first dose and with an incomplete adjuvant for the two remaining doses. All of the monkeys were infected with 107 copies of genotype 1 HEV by intravenous injection. Stool samples were collected every day and tested for HEV viral genomes by RT-PCR as described. Serum samples were collected twice a week to assess levels of alanine aminotransferase (ALT) and anti-HEV IgM and IgG by ELISA (Wantai, Beijing, China). The animal experiment was designed based on the principles expressed in the “Guide for the Care and Use of Laboratory Animals” by the National Research Council of the National Academies and “Guidance for Experimental


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Fig. 1. Characterization of a typical antibody. (A) The typical MAbs used in this study and the location of the epitopes recognized by these MAbs. 15B2 is the MAb recognizing the linear epitope L1 located in a.a. 403–417, 12A10 is the MAb recognizing the linear epitope L2 located in a.a. 423–437, 1B7 is the MAb recognizing the linear epitope L3 located in a.a. 443–457, and 8G12 is the MAb recognizing the conformational epitope located in a.a. 459–606. (B) The capacity to block HEV infection of HepG2 cells, as described in the text. HEV infection was shown by detection of a 190 bp product from infected cells by RT-PCR. The G3PDH gene was co-amplified to serve as an internal control.

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Animal Welfare and Ethical Treatment” by the Ministry of Science and Technology of the People’s Republic of China. The experimental procedures and the animal use and care protocols were approved by the Committee on Ethical Use of Animals of Xiamen University.

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3. Results

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3.1. Linear determinant located at a.a. 394–458 on HEV ORF2

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MAbs recognizing the HEV capsid were obtained using a standard murine MAb preparation protocol [25] from mice immunized with the recombinant-expressed HEV capsid VLP p239 (amino acids 368–606) (Table 1S). MAbs recognizing the linear epitopes were identified by detecting the reaction of these MAbs with completely denatured p239 and 15-mer peptides (Figs. 1S and 2S). The reactions with the 15-mer peptides showed the major linear epitopes recognized by these MAbs. These linear epitopes included 3 major independent peptides, a.a. 403–417 of pORF2, a.a. 423–437 of pORF2 and a.a. 443–457 pORF2, which were named linear epitopes L1 (a.a. 403–417), L2 (a.a. 423–437) and L3 (a.a. 443–457) in this study. Three MAbs, 15B2, 12A10 and 1B7,were chosen as the typical MAbs for further detection (Fig. 1A). 15B2 recognizes linear epitope L1, 12A10 recognizes linear epitope L2, and 1B7 recognizes linear epitope L3. 8G12, a protective and neutralizing MAb which recognizes the conformational determinant located at the dimeric domain (E2s domain, a.a. 459–606), was used as a typical antibody control [41,42]. We first detected the neutralization of these MAbs in HEV genotypes 1 and 4 after in vitro HEV infection in HepG2 cells. As shown

in Fig. 1B, 12A10 and 8G12 significantly blocked the infections, but 15B2 and 1B7 did not. The results show that 12A10 neutralized HEV genotypes 1 and 4 as well as the reported neutralizing MAb 8G12, which recognizes the conformational epitope located in a.a. 459–606 (E2s domain) of pORF2.

Fig. 2. MAb 12A10 and 8G12 binding to p239 in the presence of HEV-infected or uninfected donor sera. Competitive ELISA test for MAb 12A10 binding to p239. The sera were incubated with p239 in plates prior to the addition of HRP-conjugated 12A10 or 8G12 to block their binding. Acute- and convalescent-phase patient sera have been shown to block more than 50% of MAb 8G12 binding to p239. However, the sera could not block MAb 12A10. The negative sera were used as negative controls.


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3.2. Characterization of reactivity against epitope L2 and E2s domain in sera from HEV-infected patients We determined the variation in reactivity of 12A10 and 8G12 in the presence of HEV- or non-HEV-donor sera to determine whether the reactivity against epitope L2 and/or E2s domain in the sera. As shown in Fig. 2, pre-incubation with both acute phase and convalescent HEV patient sera interfered with the reaction of 8G12 with pORF2 (at 47.29% with acute phase HEV patient sera and 23.9% with convalescent HEV patient sera), but showed no effect on the reactivity of 12A10 (at 93.6% with acute phase HEV patient sera and 97.7% with convalescent HEV patient sera). This indicates that there was no competition between 12A10 and the HEV-infected patient sera when binding to HEV pORF2. Thus, in both acute phase and convalescent HEV patient sera, the major anti-pORF2 antibodies were against the immunodominant conformational determinant located in the E2s domain (a.a. 459–606), and the novel neutralization linear epitope L2 (recognized by 12A10) was non-immunodominant. The anti-L2 responses in sera from HEV infected patients and vaccinated donors were also detected. The result showed that sera from HEV infected patients and vaccinated donors had no responses with L2 peptide (Fig. 3S). 3.3. Immunogenicity of recombinant chimeric particles (vector: HBc 149) displaying the linear epitopes (L1, L2 and L3) To further understand the effect of epitopes L1, L2 and L3 on the active immunization process, the peptides were displayed on the HBc149 vector as described [43] to induce an anti-peptide specific antibody response in vivo. The chimeric proteins were expressed in E. coli (the identification process of the chimeric particles is described in supplemental material 6, Fig. 4S) and were used to immunize mice via subcutaneous injection. The p239(1) and p239(4) particles were used for immunization as an antiHEV immunogenicity positive control, and the HBc 149 particle was used as the negative control. The sera were obtained after 2 booster doses for further analysis of the reaction and neutralization. The reaction of sera with peptides (L1/L2/L3) was analyzed by ELISA (Fig. 3A). The reaction of sera induced by HBcL1/L2/L3 particle with their corresponding linear peptide yielded optical density (OD) readings between 1.389 and 1.907, while the sera induced by HBc particle with L1/L2/L3 peptides yielded OD readings under 0.1. The results showed that the PAbs specifically recognized the corresponding L1, L2 and L3 peptides. The neutralization of PAbs to p239 particle and the virus were analyzed as described previously. The p239 particles and virus on and in the cells were detected by Western blotting (Fig. 3B) and RTPCR, respectively (Fig. 3C). The p239 particles binding on cells were interfered to 21.85 ± 14.6% (p239(1)) and 22.97 ± 16.6% (p239(4)) by anti-HBcL2 PAbs compared with control group in Fig. 3B. The anti-HBcL2 PAb significantly blocked both the binding of p239 particle (Fig. 3B) and infection of the virus (Fig. 3C) on cells as well as in the sera of mice immunized with p239(1) and p239(4). The anti-HBcL1, anti-HBcL3, and anti-HBc-vector PAbs showed no significant influence on the binding of p239 particle and viral infection. The results from both genotypes 1 and 4 were similar, which suggested that the HBc 149 vector displayed epitope L2 well, and the novel potential neutralizing epitope L2 successfully induced an anti-HEV neutralizing antibody response in vivo. 3.4. HEV challenge of rhesus monkeys immunized with HBcL2 particles As mentioned above, the L2 peptide displayed on the HBc 149 particle showed good immunogenicity in mouse immunization. However, mice do not provide an HEV-infectable model.

Therefore, this study evaluated the protection of the HBcL2 particle against HEV infection in rhesus monkeys, which are sensitive to HEV infection. Six rhesus monkeys were divided into 3 groups and immunized with p239(1) particle (the only antigen in the HEV vaccine Hecolin® ), HBcL2 particle, or HBc particle in 3 doses by deltoid muscle injection in the left forelimb. The sera of the monkeys were collected weekly, and the anti-HEV IgG, anti-HEV IgM, anti-L2 Abs and anti-HBc Abs were examined to identify the effect of immunization. As shown in Fig. 4, immunization with HBcL2 particle induced specific anti-L2 (Fig. 4C, blue points and lines) and anti-HBc antibodies (Fig. 4D, blue points and lines) in monkeys as expected. The HBcL2 group sera got positive with L2 peptide at −5 log10 dilution at day 120. P239 particle also exhibited a low-level anti-L2 response (positive at 1:100 or 1:1000 dilution at day 120) (Fig. 4C, red points and lines). Anti-HEV IgG was efficiently induced both in monkeys immunized with p239 particle and those immunized with HBcL2 particle, and the sera from these monkeys got positive at least −4 log10 dilution after second boost. This result indicated that the HBc 149 particle displaying the L2 peptide showed good immunogenicity in rhesus monkey immunization. Thirty days after the second booster dose, all of the monkeys were infected with 107 copies of genotype 1 HEV virus by intravenous injection. The sera were collected twice per week for ALT detection, and viral RNA in the stools was detected every day until all of the parameters became negative. As shown in Fig. 5, HBcL2 particle did not protect as well as p239(1) particle. In the monkeys immunized with HBcL2 particle, viral RNA was still detected in the stools 3–15 d.p.i. (days post infection) and 5–23 d.p.i., but the fecal shedding period of HEV was much shorter than in the monkeys immunized with HBc particle (2–49 d.p.i. and 1–34 d.p.i.). The number of HEV virus copies present in stools from the rhesus monkeys immunized with HBcL2 particle was also lower than that of the HBc particle-immunized control group. The pre-peak ALT values for each monkey were: 1.27 (p239(1)-1), 1.18 (p239(1)-2), 3.81 (HBcL2-1), 2.71 (HBcL2-2), 4.53 (HBc-1), and 6.44 (HBc-2). The ALT elevation in the HBcL2 group was much lower than in the HBc particle group. The protection offered by HBcL2 particle was not as effective as candidate vaccine p239 particle; we did not use the virus in a further titer experiment. However, the results showed that active immunization with HBcL2 particle protected monkeys from HEV infection to some degree.

4. Discussion In summary, we found a novel neutralizing linear epitope L2 (a.a. 423–437) of HEV. The novel neutralizing site present at a.a. 423–437 of pORF2 was different from the reported neutralizing epitopes on the HEV capsid located in the E2s domain (a.a. 459–606). MAb 12A10, which recognized the L2 epitope, significantly neutralized the virus in an in vitro study to the same level as the neutralizing MAbs that recognized the E2s domain. Using HBc as a carrier, L2-epitope-immunized mouse serum blocked the entry of HEV into HepG2 cells, indicating that HBcL2 particle could produce antibodies similar to 12A10. Further investigation showed that the HBcL2 particle efficiently stimulated immunogenicity and protection against HEV infection in the rhesus monkey to some degree. Fecal shedding period of HEV was 16 days and the pre-peak ALT value was 3.21 in average in HBcL2 group. In the control group, fecal shedding period of HEV was 41 days and the pre-peak ALT value was 5.4 in average, which was more severe than the HBcL2 group. HBcL2 particle immunization shortened the period of HEV presentation by 60% in rhesus monkeys. Compared with p239 particle, which is the effective protein in the HEV vaccine, HBcL2 particle did not provide complete protection to immunized monkeys. However, considering that HBcL2 particle included only a single epitope and p239


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Fig. 3. Characterization of PAbs. (A) The reaction of PAbs with peptides (L1/L2/L3) as detected by ELISA. (B) The PAbs anti-p239(1), anti-p239(4), blank sera, anti-HBcL1, anti-HBcL2 and anti-HBcL3 were pre-mixed with genotype 1 HEV VLP p239(1) (left figure) and genotype 4 HEV VLP p239(4) (right figure), and then the mixture was added to HepG2 cells. Residual VLP binding was detected by Western blotting, and the data were calculated as a percentage of the binding without sera. ␤-Tubulin was detected to serve as an internal control. (C) The PAbs were pre-mixed with genotypes 1 and 4 HEV. Residual HEV binding was detected by RT-PCR. The G3PDH gene was co-amplified to serve as an internal control. Compared with the control group (without sera and with blank sera), the PAbs anti-p239(1), anti-p239(4) and anti-HBcL2 significantly blocked the binding of particles (B) and viral infection (C) of the cells.

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particle includes multiple epitopes and that multi-epitope synergies protect more completely, the effect of the L2 epitope may be better than the results indicate. Synthetic peptides derived from other viruses, such as HAV [44], HBV [45,46], HCV [47], and EV71 [38], have been shown to elicit neutralizing antibodies, but this phenomenon has not been shown

for HEV. In a previous study by Meng’s group, 30-mer peptides spanning the a.a. 221–660 region of HEV pORF2 were tested in an in vitro neutralization assay; however, none of the pooled sera antipeptides demonstrated any neutralizing activity [16]. This result may be lack of immunogenicity with synthetic peptides, which is inefficient for stimulating high-titer antibody responses. In a


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Fig. 4. Antibody response of rhesus monkeys to HBcL2. Six rhesus monkeys were divided into 3 groups and immunized with 20 ␮g of three proteins: p239(1) (red), HBcL2

Q3 (blue) or HBc (black) in 3 doses by deltoid muscle injection in the left forelimb (0, 30 and 120 d). The serum levels of anti-HEV IgG (A), anti-HEV IgM (B), anti-L2 Abs (C) and anti-HBc Abs (D) were detected weekly after primary immunization. The arrows mean the day when the booster injection was performed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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previous study, HBcL2 particle could induce anti-p239 antibodies with efficient immunization, but the peptides could not (Fig. 5S). And for analyze the conformational stability of peptides and HBcL2, a comparison of the binding activity of p239, HBcL2 and L2 peptide with MAb 12A10 was conducted in a blocking assay (Fig. 6S). It indicated that the HBcL2 displayed similar epitope L2 as p239. In this study, the HBcL2 should be a better antigen to stabilize the conformation of L2 epitope and enhance immunogenicity for stimulating 12A10-like antibodies responses. Epitope L2 is non-immunodominant in the natural HEVinfection process (Fig. 2), making it difficult to find this neutralizing epitope. However, the non-immunodominance also indicates that this epitope induces low immune responses in the natural infection process, thereby preventing mutations on the epitope sequence

more than immunodominant epitopes. Such phenomenon has been found in the influenza virus [48]. The L2 epitope is linear epitope, and the linear epitopes could be displayed and constructed according to methods which had been described and been extensively applied in numbers of investigations [31,34,36–38,40,43,49–54]. Although the immune response against immunodominant epitopes is effective in the prevention of HEV [25], epitope L2 is an alternative target for HEV vaccine development that may prevent future viral mutations with the extensive use of vaccines and the increasing anti-HEV immune pressure in the population. Moreover, this study exemplified a non-immunodominant linear neutralizing epitope against a virus. In the prevention of viruses with a single serotype, such as HEV, non-immunodominant neutralizing linear epitopes show no advantage compared with immunodominant

Fig. 5. Protection by HBcL2 assessed in a rhesus monkey infection model. The course of six infected rhesus monkeys immunized with three particles (p239(1) (red), HBcL2 (blue) or HBc (black)) was monitored for 7.5 weeks by serum ALT levels, duration of virus excretion in stool and HEV antibody responses. (A) shows that no viral shedding was found in Group p239(1). However, the viral shedding in Group HBcL2 was detected from 3 to 15 and 5 to 23 days after inoculation, and viral shedding in Group HBc was detected from 2 to 49 and 1 to 34 days after inoculation. (B) shows the levels of liver enzymes (ALT) in the serum. The baseline liver enzymes were 15–27 U of ALT per liter for rhesus monkey. The pre-peak values of ALT for each monkey were: 1.27 (p239(1)-1), 1.18 (p239(1)-2), 3.81 (HBcL2-1), 2.71 (HBcL2-2), 4.53 (HBc-1), and 6.44 (HBc-2). The serum levels of anti-HEV IgG (C) and anti-HEV IgM (D) were detected after challenge. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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neutralizing epitopes. However, in viral vaccine research with a variety of mutations in the immunodominant neutralizing epitopes, such as flu, HIV, and HCV, the search for viral nonimmunodominant linear neutralizing epitopes is a potentially effective direction. 5. Conclusion In summary, a novel non-immunodominant neutralizing linear epitope L2 present in a.a. 423–437 of HEV pORF2 was identified using a neutralizing MAb 12A10. To stabilize terminates of the peptide and to enhance immunogenicity, we used HBc as a carrier. The HBcL2 recombinant chimeric particles could elicit mouse antibodies to block the entry of HEV into HepG2 cells. And the HBcL2 chimeric particle efficiently stimulated immunogenicity and protection against HEV infection in the rhesus monkey to some degree. This study suggested that the epitope L2 was nonimmunodominant in the HEV infection, it provided an example of means to identify non-immunodominant neutralizing linear epitopes. This identification process could be a potential efficient way for vaccine research of the virus with variable immunodominant neutralizing epitopes. Conflict of interest The authors declare no conflicts of interests. Acknowledgements

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This work was supported by Chinese National High-tech R&D Program (863 program; 2011AA02A101), Xiamen Science and Technology Platform Project (3502Z20131001) and Xiamen Science and Technology Plan Project (3502Z201410045).

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Appendix A. Supplementary data

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