Development of new hepatitis E vaccines

ABSTRACT

Hepatitis E virus (HEV) infection is an emerging zoonotic disease posing a severe threat to public health in the world, especially to pregnant women. Currently, no specific treatments are available for HEV infection. Therefore, it is crucial to develop vaccine to prevent this infection. Although several potential candidate vaccines against HEV have been studied for their immunogenicity and efficacy, only Hecolin® which is developed by Xiamen Innovax Biotech Co., Ltd. and approved by China Food and Drug Administration (CFDA) in 2012, is the licensed HEV vaccine in the world so far. Extensive studies on safety, immunogenicity and efficacy in phase III clinical trials have shown that Hecolin® is a promising vaccine for HEV prevention and control. In this article, the advances on HEV vaccine development and research are briefly reviewed.

KEYWORDS:

• Hepatitis E
• HEV
• Vaccine
• Advance

Introduction

Hepatitis E virus (HEV) belongs to the genus of orthohepevirus within the family of Hepeviridae. As an emerging causative agent for Hepatitis E, this virus infection poses severe threats to the public health worldwide, especially in developing countries.^1-3 HEV is the main cause of enterically transmitted hepatitis worldwide, and responsible for more than half of the cases with acute hepatitis in endemic countries such as India and China.^3-7 In Europe, 5%-15% of the acute hepatitis of unknown origin are caused by HEV.^8,9 In recent years, outbreaks of HEV on a large scale have been revealed among displaced people in South Sudan, Chad, Uganda and Nepal.^10-12 Hepatitis E infection has a relatively high seropositive rate for HEV IgG ranging from 10.0% to 33.5% and a low fatality rate ranging from 0.5% to 4%, while infections in pregnant women induces acute liver failure, leading to approximately 10%-25% mortality.^13-15 Due to the limitations of the detection methods, actual incidence of HEV exceeded the clinical positive carrier among general population. According to the estimates by World Health Organization, one third of the world population has been exposed to HEV, and 35 million people are infected by HEV annually, resulting in 70,000 deaths.^16-19 The sporadic infection in industrialized countries and outbreaks in developing countries are becoming more and more serious. As the disease affects mainly young people and results in higher mortality among pregnant women, especially in the developing countries, much attention should be paid to people at high risk of severe disease and priority be given to the prevention and control of HEV infection in this crowd during outbreaks.^20,21

HEV classification and global distribution

HEV is currently classified to four major genotypes I-IV and twenty-four subtypes, while all genotypes belong to one serotype.²² HEV genotypes I and II are restricted to humans, whereas genotypes III and IV are zoonotic and mainly responsible for sporadic cases. In addition, genotype IV is restricted to Asia and genotype III is spread worldwide.^23,24 Genotype I isolated from the Delhi epidemic from 1955 to 1956 is responsible for the epidemic and sporadic hepatitis E in the developing countries such as India and China (Xinjiang).^25-27 Genotype II was initially identified from Mexico, and subsequently from Chad and Nigeria in Africa.²⁸ Genotype III was firstly isolated from the swine population in mid-western United States in 1997, and now is prevalent in the swine herd globally. The genotype III isolates were subsequently identified from human in the developed world including Italy, France, the United States and Japan.^29-33 Genotype IV was first isolated from patients in Taiwan and subsequently found in China, Japan and India.^34-37 The presence of acute viral hepatitis caused by HEV in developing countries has been documented for decades. Since the late 1990s, endemic hepatitis E caused by genotype III and genotype IV has been increasingly reported in developed countries.^38,39

HEV genome organization

HEV is a non-enveloped virus with a diameter of 27-34 nm, which has a single-stranded positive-sense RNA genome. The genome is approximately 7.2 kb in length containing a relatively conserved 5′non-coding region (NCR), a variable 3′NCR, and three open reading frames (ORFs).^40-42 The organization of the ORFs differs slightly among four HEV genotypes. However, the proteins they encoded have the same functions.

ORF1 encodes a polyprotein of approximately 1693 amino acids, which is post-translationally processed into multiple non-structural proteins including methyltransferase, putative papain-like cysteine protease, RNA helicase and RNA-dependent RNA polymerase that are required for virus replication.⁴³

HEV ORF2 encodes the capsid protein, a structural protein consisting of 660 amino acids with a predicted molecular mass of 72 kDa.⁴⁴ The N-terminal 112 residues of structural protein encoded by ORF2 are responsible for the packaging of the viral RNA genome.⁴⁵ The virus-like particles (aa 112-608, VLPs) have been made up of three definitive domains: S domain (aa 129-319), M domain (aa320-455), and P domain (aa 456-606). S domain forms the viral shell. M domain is involved in the formation of the 2, 3, and 5-fold icosahedral symmetries of the HEV capsid. P domain forms the protrusions that extend outward from the viral shell.^46-48 The HEV capsid protein induces the antibodies that can neutralize the HEV in vitro, and protect primates against HEV infection, thus the complete and truncated capsid proteins are considered as vaccine candidate against HEV infection in humans.^49-51

ORF3 overlaps with ORF2 and encodes a phosphoprotein of 123 amino acids that interacts with the cytoskeleton protein, suggesting it plays a role in the assembly of virus particles.²⁰

Hepatitis E vaccines

Since no specific treatment is available for hepatitis E at present, development of an effective vaccine against HEV infections is the only pragmatic approach to prevent and control this disease.^52,53 The inability to reproducibly produce HEV in culture system makes it impossible to develop traditional live or inactivated vaccines. However, significant progress has been made in developing recombinant vaccines based on the ORF2 viral capsid protein VLPs (Table 1). Protein-based VLPs are self-assembling bionanoparticles that expose multiple epitopes on their surface and faithfully mimic the native virions.⁵⁴ Particle size of the nanometer scale particulate antigen and presence of key epitopes on the particle surface are two prerequisites for an effective VLP-based vaccine.⁵⁵ Progress and detailed information was summarized as follows.

Proteins expressed in prokaryotic system

Escherichia coli is the most economical and commonly used expression system for its high production of heterologous protein. Purdy et al. expressed a fusion protein TrpE-C2 containing aa 221- 660 of ORF2 protein from Burmese strain (genotype I) and tryptophan synthetase. The TrpE-C2 fusion protein can protect the cynomolgus macaques from wild-type homologous (genotype I) or heterologous (genotype II) HEV in preclinical challenge experiments.⁵⁶ Meng et al. mapped the neutralization epitopes for HEV at the minimal size fragment of 166 aa (p166) located in ORF2.⁵⁷ The HEV p179 (aa 439-617) vaccine with 13 aa extension at the N-terminus of p166, was demonstrated to self-assembly as VLPs and manufactured by Changchun Institute of Biological Products Co. Ltd.^58,59 The immunogenicity and protective assessment showed that the HEV p179 vaccine could protect rhesus macaques from challenging with genotypes I and IV HEV. A randomized open-label controlled and a dose escalation scheduling of 20μg, 30μg and 40μg phase I clinical trial indicated that the HEV p179 vaccine was safe and well-tolerated for the 16-65 year adult population.¹⁹ Another HEV p239 vaccine Hecolin® developed by Xiamen Innovax Biotech and expressed in Escherichia coli with truncated ORF2 (aa 368–606) was consisted of 23 nm VLPs and launched on market in China in 2012.^60-62 The vaccine HEV p239 was tested in a randomized, controlled trial involving 112,604 participants in China. The vaccine showed an efficacy >99% in preventing clinical hepatitis E among population who were inoculated 3-doses of HEV p239 (n = 48,693) compared with a placebo (a commercial hepatitis B vaccine, n = 48,663).⁶³ Immunization with Hecolin® induced antibodies against HEV and provided protection against hepatitis E for up to 4.5 years.⁶⁴

Proteins expressed in insect cells

The HEV capsid antigen expressed in Baculovirus-infected insect cell system has been proposed as a candidate subunit vaccine for the prevention of hepatitis E. Most of the baculovirus-mediated ORF2 proteins have been expressed in Sf21, Sf9, Tn5 and Spodoptera Litura Larvae insect cell lines.⁶⁵ Expression of the HEV ORF2 capsid gene produced multiple immunoreactive proteins with molecular weight size ranging from 30 to 100 kDa, and the most abundant HEV proteins had molecular weights of 72, 63, 56, and 53 kDa.⁶⁶ The 56 kDa candidate vaccine showed highly immunogenic when administered to rhesus monkeys with two doses, and protected them from hepatitis E when challenged with a large intravenous dose of homologous or heterologous HEV. In this study, the immune procedure with two doses of HEV vaccine partially protected rhesus monkeys from hepatitis E following intravenous challenge at 6 or 12 months post-vaccination.⁶⁷ A randomized, double-blind, placebo-controlled phase II clinical trial was launched to evaluate the safety and efficacy of this candidate vaccine. A total of 1794 subjects received three vaccine doses, with 898 of them as vaccine group and 896 as placebo group. After three doses of immunization, 69 subjects developed in hepatitis E, of whom 66 were from the placebo group, with vaccine efficacy 95.5%.⁶⁸ Qi et al. constructed and expressed HEV ORF2 capsid protein using Baculovirus-infected insect cell system. In the study, two conformations of the HEV capsid proteins VLPs and non-VLPs were expressed in insect cells, and purified separately. The humoral immune response research showed that the immunogenicity of VLPs was better than HEV non-VLPs in mice.⁶⁹

Proteins expressed in yeast

The N-glycosylation plays an important role in the stability, immunogenicity and biological activity of protein. HEV ORF2 capsid protein contains several potential N-glycosylation sites, with N137 and N310 located in the S domain, and one site N562 in the P domain. The P domain forms a protruding spike from the shell with N562 in the apical center of the spike, which is also a cell-attachment region and neutralizing antigenic site.⁷⁰ Consideration of the influence of glycosylation, many research fellows attempted to express truncated HEV ORF2 protein in yeast.⁷¹ Suet al. expressed truncated genotype IV HEV ORF2 (aa112–607) in a system of methylotrophic yeast Hansenula polymorpha. The 56 kDa truncated protein showed high immunoreactivity, providing foundation for the further development of recombinant vaccine against hepatitis E.⁷² Xu et al. expressed the recombinant polypeptide p179 comprising of the region (aa 439-617) within the HEV ORF2 and a set of 4 mutant proteins where N at position 562 were substituted with Q, D, P and Y respectively, in Pichia pastoris. In vitro neutralization experiments showed the mice antibodies generated against p179N562P and p179N562Y were more efficient than the antibodies generated against the other 3 proteins.⁷⁰ Tong et al. studied the immunogenicity of the recombinant HEV ORF2 protein expressed in Pichia pastoris by immunizing BALB/c mice in different routes with different adjuvants. The study demonstrated high immunogenicity of this kind of ORF2 recombinant protein.⁷³

Vectored vaccine against HEV

A novel candidate vectored vaccine against HEV through adeno-associated virus (AAV) vector expressing the truncated capsid protein of HEV ORF2 (aa 112-660) in Sf9 cells using the Baculovirus Expression System was developed. The recombinant baculovirus expressing the truncated capsid protein was constructed and the capsid protein was confirmed by Western blot in transducted HEK 293 EBNA cells with rAAV.⁷⁴ Vaccinia virus was also used as vector to express recombinant HEV truncated△_(aa1-111)-ORF2 protein in BHK-21 cells, facilitating the study of its glycosylation patterns and subcellular localization. The expressed proteins have good antigenicity for diagnosis and good immunogenicity to induce long-lasting specific antibodies for anti-HEV in mice.⁷⁵ In another study, a synthetic codon-optimized gene cassette encoding tPAsp-PADRE-truncated ORF2 (aa 112-660) protein was sub-cloned into the pVAX1 expression vector and expressed in eukaryotic cells with truncated ORF2 constructs as control. The expression of truncated ORF2 protein in eukaryotic cells was proven by Immunofluorescence assay (IFA) and RT-PCR, which can be used as a novel DNA candidate vaccine in future investigations.⁷⁶

Combined vaccine and recombinant chimeric vaccine

Hepatitis A virus (HAV) and HEV are causative agents of acute viral hepatitis transmitted via the fecal-oral route. To relieve burden on the public health in developing countries, approach of combined vaccine was adopted extensively. Dong et al. developed a combined vaccine against hepatitis A and E. Different groups of mice immunized with different formulations containing a commercial inactivated hepatitis A vaccine and a candidate recombinant hepatitis E vaccine. The experimental results showed that the combined vaccine can induce neutralizing antibodies against both HAV and HEV effectively in a mouse model.⁷⁷ The other chimeric recombinant vaccine HAV-HEp148 was created as a vector to express an HEV neutralization epitope HEp148 (aa 459-606 of HEV capsid protein). Immunization with the HAV-HEp148 virus induced a strong HAV- and HEV-specific immune response in mice.⁷⁸ Another novel combined protein vaccine by conjugating tuftsin to HEV-ORF2 (aa368-607) and HAV-VP1 (aa 1-198) used synthetic CpG oligodeoxynucleotides (ODNs) as the adjuvant. Mice in group intranasally immunized with tuftsin showed higher levels of IFN-γ-secreting splenocytes (Th1 response) and CD4⁺/CD8⁺ T cells than mice in group without tuftsin, demonstrating the tuftsin group generated stronger humoral and cellular immune responses than the no-tuftsin group. Moreover, intranasal immunization generated some enhanced immune responses compared with intramuscular injection to the combined protein vaccine.⁷⁹

Li et al. constructed the fusion gene fragment of HEV capsid protein (aa 551-607) and gene of HBsAg via synthetic glycine linker. The resulting fusion gene was then integrated into the genome of Pichia pastoris through transformation and expressed intracellularly. The chimeric VLPs showed high immunoreactive activity in ELISA assay, indicating that the chimeric VLPs may be a potential HBV/HEV bivalent vaccine candidate.⁸⁰

Norovirus (NoV) and HEV are both enteric-transmitted viruses causing gastroenteritis and hepatitis, respectively. Both NoV and HEV have surface protrusions formed by dimers of the protruding (P) domains of the viral capsids, which are responsible for virus-host interactions and inducing neutralizing antibody. A bivalent vaccine against the two viruses fusing the dimeric P domains of NoV and HEV together linked with the dimeric glutathione-S-transferase (GST). Both of the GST-NoV P(-)-HEV P and NoV P(-)-HEV P complexes vaccines can induce significantly higher antibody titers, indicating they are promising chimeric vaccine candidates against both NoV and HEV.⁸¹

HEV, rotavirus (RV) and astrovirus (AstV) are important pathogens that transmit via common fecal-oral route, causing hepatitis (HEV) and gastroenteritis (RV and AstV) in humans respectively. A recombinant fusion protein comprising the HEV truncated ORF2 gene (aa 112-607) and Rotavirus NSP4 was expressed in Baculovirus System as a potential candidate vaccine.⁸² In another study, two formats of subunit vaccine candidates consisted of spike protein antigens from HEV, rotavirus (RV) and astrovirus (AstV) were designed. One was fused vaccine and the other was mixed vaccine. The fused vaccine is a fusion protein of the three antigens into one molecule and the mixed vaccine is a mixture of the three free antigens together. The fused vaccine induced significantly higher antibody responses against the three viral antigens than those induced by the mixed vaccine in mice immunization experiments. The similar results were observed in neutralizing titers against HEV and blocking titers (BT50) against RV VP8-HBGA receptor interactions.⁸³

Oral immunization HEV vaccine

HEV is an important causative agent of enterically-transmitted hepatitis, thus oral immunization may be an efficient approach for HEV vaccine. An oral administration experiment in mice was designed via detecting the response of serum IgM, IgG, and IgA and fecal IgA to evaluate the potential of recombinant HEVVLPs with its capsids protein N-terminal 111 amino acids truncated as an oral immunogen. The study indicated that serum IgG and fecal IgA were reactive to the native HEV antigen, as detected by Western blot and antigen-capture ELISA.⁸⁴ Serum IgM, IgG and IgA antibodies were also detected in cynomolgus monkeys orally inoculated with 10mg of this purified HEVVLPs. The monkeys were protected against infection or developing hepatitis after they were challenged with native HEV by intravenous injection, suggesting this recombinant HEV VLPs is a candidate for the oral hepatitis E vaccine.⁸⁵ In another study, a recombinant (inaQ-ORF2 gene fusion) Lactococcus lactis (L. lactis) strain NZ3900 expressing the HEV ORF2 antigen utilizing an ice nucleation protein-based anchor system was constructed. The BALB/c mice vaccinated orally displayed significantly higher levels of ORF2-specific mucosal IgA, serum IgG and cellular immunity, suggesting that L. lactis-based HEV antigen vaccines can be used for protection against HEV infection.⁸⁶ Tomato is used as an ideal plant carrier for transgenic oral vaccine for being consumed raw and easily transformed. The fragment of HEV ORF2 (aa 394-604) was cloned into plant binary expression vector p1301E2 containing a constitutive CaMV35s promoter and nos terminator, then directly introduced into agrobacterium tumefaciens EHA105. The production of truncated recombinant HEV ORF2 protein was 61.22 ng/g fresh weight in fruits and 6.37-47.9 ng/g fresh weight in leaves of the transformants. This study laid the foundation for further research on the development of a new type of plant-derived hepatitis E oral vaccine.⁸⁷

HEV vaccine neutralization assay

For assessing vaccine potency as a surrogate for clinical efficacy, a viral neutralization assay is needed. The host humoral immune responses for vaccination, especially the level of neutralization antibody, reflect the potency for an effective candidate vaccine. Rhesus macaque has been used as a classical animal model to evaluate the protective effect against the challenging HEV. Study revealed that 5µg of HEV p179 vaccine could induce protective IgG, while 20 µg of p179 vaccine could prevent 75% of rhesus macaques from shedding virus in feces.¹⁹ Meng et al. reported a new PCR-based seroneutralization assay for the serological diagnosis of HEV infection based on neutralization of the virus in cell culture.⁸⁸ Takahashi et al. generated one mAb (H6225) against HEV capsid protein efficiently neutralizing HEV in vitro using a hepatocarcinoma cell culture system.⁸⁹ Yang et al. developed a high-throughput neutralization titer evaluation model for HEV. In the study, the quantitative assay for neutralizing capacity of different antibodies and antiserum samples from HEV vaccine recipients was established.⁹⁰ In another study, a broad neutralizing mAb 8G12 was used to develop a competitive ELSIA assay and quantified 8G12 competitive antibody (8G12-like antibody) in serum samples. The 8G12-like antibody was approved to be a promising surrogate for vaccine induced HEV neutralizing antibody.⁹¹

Summary

HEV is one of the common cause of acute viral hepatitis globally. The HEV prevalence rates are more than 25% in many regions including Central America, large parts of Africa, and all of Asia, especially in population living in refugee camps or overcrowded temporary housing after natural disasters.⁹² Hepatitis E infection results in significant burden in some parts of the world especially in developing countries. China has been listed as one of the countries suffering the severe economic burden with HEV infection.^93,94 It is crucial to use vaccine to prevent HEV infection, thus knowledge for the structure of the HEV genome and the function of ORFs are essential for vaccine development.^95-98 Although four genotypes of HEV have been reported globally, they belong to one serotype. The antibodies induced by different genotype HEV can neutralize homologous or heterologous HEV. The finding that serum antibodies to HEV were developed and detected in human naturally infected by HEV and in the primate animal models experimentally infected with HEV ratified the capacity of the humoral responses elicited by HEV.⁹⁹ Epidemiological survey during a hepatitis E outbreak suggests that people previously infected with HEV can be protected during epidemics of the disease, further validating the vaccine as effective tools in fighting HEV infection.¹⁰⁰

Recombinant HEV VLPs, consisting of different lengths of the ORF2 capsid protein, were prepared to mimic the protective and neutralizing epitopes on the virion surface. These candidate vaccines were designed to elicit protective immunity when inoculated non-human primates or human volunteers in preclinical tests and clinical trials.¹⁰¹ In addition, linked recognition is considered as important mechanism for antibody production of TD antigens, so cellular immune responses may play an important role in providing protection against viruses. Thus, viral proteins containing T-cell epitopes may prove useful in stimulating T-cell immunity and providing help for antibody production. Furthermore, activation of T-cell immune responses may inhibit viral replication through a cytokine-mediated noncytolytic pathway. Aggarwal et al. present their results on T cell epitope mapping of HEV ORF2 and ORF3 proteins using overlapping peptides encompassing the entire lengths of these proteins. The immunodominant T-cell epitopes of HEV ORF2 protein were located in the regions encompassed by aa 73-156, aa 289-444 and aa 505-588.¹⁰² These findings provided structure basis for T-cell immune response of HEV candidate vaccines consisting of ORF2 proteins.

Since ORF2 protein contains T-cell and B-cell epitopes and induces neutralizing antibodies, effector cell and cytokines offering help for antibody production against the HEV infection, it becomes the focus for developing the safe and effective vaccine.^103-106 Various forms of HEV vaccines based on the ORF2 protein expressed using different heterologous systems are reported. The other new forms of vaccines such as vectored HEV vaccine, combined vaccine and recombinant chimeric vaccine were also studied. Traditional recombinant vaccines such as Hecolin®, the only licensed HEV vaccine in the world so far, can induce protective humoral immune responses well, and can be produced in economical E.coli expression system using simple technological process. Construction of DNA candidate vaccines using AAV and pVAX1 expression vectors are significant attempts to explore the novel HEV vaccines. Whereas, the unknown scruple for safety and intricate technological process are the bottleneck for these novel vaccines. Combined vaccine and recombinant chimeric vaccine have become important development direction for their good performance on safety, efficacy and economic cost. FDA approved combined hepatitis A and B vaccine for its safety and highly immunogenicity, which has facilitated the interest for scientists to develop combined vaccine and recombinant chimeric vaccine associated with hepatitis.¹⁰⁷ The development of combined vaccines to prevent multiple diseases was considered desirable, especially in developing countries where these diseases are highly endemic.

Although there are several HEV candidate vaccines synthesized, only three of them have access to clinical trial registration to date. The first one developed by GSK had completed phase II clinical trial, indicating that the vaccine was effective in the prevention of hepatitis E.^108,109 The other one is the previously mentioned Hecolin® which have completed phase III clinical trial and been licensed in China. The long-term efficacy of Hecolin® had been evaluated, which is 86.8% after 4.5 years in the modified intention-to-treat analysis.^64,110 Clinical trial data indicated that Hecolin® had provided good short-term and long-term protection against HEV infection in 16-65 years old healthy Chinese population.³ Another HEV candidate vaccines developed by Changchun Institute of Biological Products Co., Ltd (China) have finished phase I clinical trial, showing good safety and tolerance.¹⁹

China is the first country where the HEV vaccine was licensed. This vaccine is demonstrated as an effective HEV vaccine. The cases of hepatitis E infection since 2012 were reported to decline by 7.58% according to the data released by Chinese Center for Disease Control and Prevention (CDC).¹¹¹ However, the application of Hecolin® worldwide was hindered by various complicated factors. First of all, the only clinical data available are based on clinical trial conducted in China, where HEV is prevalent with low morbidity about 0.002%.¹¹¹ The safety and efficacy data regarding Hecolin® need to be further verified worldwide especially in regions such as Africa, Southeast Asia and South Asia, where HEV genotype I, II are the dominated endemic types.,^112,113 Moreover, safety and efficacy data from children, elderly patients, patients with chronic liver disease, such as hepatitis B and hepatitis C, HIV and those with immune disorders, especially pregnant women are further needed for the global launch and promotion of Hecolin®.¹¹⁴ Additionally, Hecolin® has received neither WHO prequalification (PQ) nor licenses in other countries, thus it is not available to HEV endemic countries especially to the refugees at HEV high-epidemic regions.¹¹⁵ The first and only licensed vaccine was developed based on data from China where almost exclusively genotype 4 is the circulating strain. Thus more clinical data are needed to verify whether this vaccine or any vaccines being developed work effectively against other genotypes. How to meet the WHO prequalification (PQ) for Hecolin® and gather more clinical data are becoming more and more imperative.

Although steady progress has been made on research and application for HEV vaccine, the immunity mechanism has remained unarticulated. Compared with humoral immune responses, only limited research data are available on cellular immune responses for HEV vaccines. More information on cellular immune are required for illuminating the mechanism of pathogenetic injury and protective immunity response in HEV natural infection or in hepatitis E vaccination. In addition, establishment of steady and ingenious assay for neutralization antibody quantitation is indispensable for vaccines efficacy evaluation. Moreover, consideration of the huge global burden of epidemic and sporadic hepatitis E, and the public health threat caused by HEV infection in different population worldwide, available and effective approaches should be taken. With the accumulation of safety and efficacy data on pregnant women, chronic disease patients, and children under the age of 16, the application and promotion globally for HEV vaccines will be accelerated.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors would like to thank the volunteers who participated in this clinical trial.

Table 1. HEV candidate vaccines and commercialized vaccines.

Name	ORF2 range (aa)	Stage	Reference	Remark
Proteins expressed in prokaryotic system
p239	368–606	licenced	60, 61, 62	Hecolin®, genotype I strain, VLPs
P179	439-617	phase Ib clinical trial	57, 58, 59	genotype IV strain, VLPs
TrpE-C2	221–660	preclinical study	56	genotype I strain, fusion protein
Proteins expressed in baculovirus-infected system
GSK candidate vaccine	112-607	phase II clinical trial	65, 66, 67, 68	genotype I strain, 56kDa, VLPs
Bacmid-HEV ORF2	126-621	preclinical study	69	genotype IV strain, VLPs and non-VLPs
Proteins expressed in yeast
HP/HEV2.3	112-607	preclinical study	72	genotype IV, 56 kDa, VLPs
HEV ORF2	69-600	preclinical study	73	59kDa
Vectored vaccine against HEV
HEV truncated△(aa1-111)-ORF2	112-660	preclinical study	74, 75	AAV expression vector
tPAsp-PADRE-truncated ORF2	112-660	preclinical study	76	pVAX1 expression vector
Combined vaccine and recombinant chimeric vaccine
hepatitis A and E	439-617	preclinical study	77	Combined vaccine
HAV-HEp148	459-606	preclinical study	78	chimeric recombinant vaccine
HE-ORF2, HA-VP1,	368-607	preclinical study	79	chimeric recombinant vaccine
HEV-HBsAg	551-607	preclinical study	80	chimeric VLPs
GST-NoV P(-)-HEV P	452-617,	preclinical study	81	GST fusion proteins
HEV-RV-AstV	112-607	preclinical study	82	recombinant fusion protein
Oral immunization HEV vaccine
rHEV VLPs	112-660	preclinical study	84, 85	Recombinant VLPs, oral administration
plant-derived vaccine	394-604	preclinical study	87	In transgenic tomato

Additional information

Funding

This study was supported by the National Key Research and Development Program of China (2017YFD0500104, 2016YFD0500904), and the National Natural Science Foundation of China (31572531).