The development of a recombinant hepatitis E vaccine HEV 239

Abstract

Hepatitis E virus (HEV) infection is one of the main causes of acute hepatitis worldwide. A recombinant hepatitis E vaccine, HEV 239, has been licensed in China for immunizing adults of 16 y old and above. The vaccine antigen contains pORF2 aa 368 - 606 of the HEV genotype 1 expressed in E. coli. The quality of the vaccine is controlled through a combination of biophysical, biochemical and immunochemical methods. The vaccine is well tolerated in adults. The efficacy of the HEV 239 vaccine against symptomatic and asymptomatic infection had been proven to be high during a Phase III clinical trial and long-term follow up. The safety and efficacy of HEV 239 vaccine in certain high-risk populations remains to be further investigated.

Keywords:

• efficacy
• Hecolin
• Hepatitis E virus
• HEV 239
• vaccine

Introduction

Hepatitis E (HE) is caused by infection with the hepatitis E virus (HEV). The awareness of this disease has increased a lot in recent years.¹ HEV infection remains a serious threat to life and productivity in the developing world. In the developed countries, an increasing number of autochthonous HE cases have been reported, suggesting that this threat is not limited to developing regions. Patients with underlying liver disease and patients who are immunosuppressed or immunocompromised are at particular risk.²

HEV is a non-enveloped, single-stranded, positive-sense RNA virus. It is the only member of the genus Hepevirus in the family Hepeviridae.³ Four genotypes of HEV are associated with human diseases: genotypes 1 and 2 infect only humans, while genotypes 3 and 4 infect many animal species in addition to humans.^3,4 All 4 genotypes are characterized as one serotype, making the development of a univalent hepatitis E vaccine reasonable. Two candidate hepatitis E vaccines have been evaluated in clinical trials. The first of these, rHEV,⁵ was developed by GlaxoSmithKine (GSK, Rixensart, Belgium). The other, HEV 239 (trade name Hecolin®),^6-8 was developed by Innovax (Xiamen, China) (Table 1). HEV 239 was launched in China in 2012. In this review, we focus on the development of the HEV 239 vaccine.

Table 1. Characteristics of the 2 existing hepatitis E vaccines that have been evaluated in clinical trials

	rHEV	HEV 239 (Hecolin®)
Manufacturer	GlaxoSmithKine, Belgium	Innovax, China
Origin	HEV genotype 1	HEV genotype 1
Expression system	Baculavirus	E. coli
Gene location	ORF2 aa 112–607	ORF2 aa 368–606
Antigen content	20 μg	30 μg
Adjuvant	Aluminum hydroxide	Aluminum hydroxide
Immunization schedule	0, 1, and 6 month	0, 1, and 6 month
Sample size of the efficacy trial	2 000	112 604
Participants enrolled	Mostly male (99.6%), aged from 18–62 (mean age 25.2)	Female and male, aged from 16–65 (mean age 44.1)
Efficacy against hepatitis E in the PPS cohort	96% (95% CI, 86–99)	100% (95% CI, 72–100)
Efficacy against hepatitis E in the ITT cohort	89% (95% CI, 77–94)	96% (95% CI, 66–99)
Development stage	Phase II clinical trial finished	Licensed in China
Advantages and disadvantages	Efficacy in women and in some special population remains to be determined	Efficacy in some special population remains to be determined

PPS: per-protocol set; ITT: intention to treat.

Structural basis for the HEV 239 vaccine

The HEV genome consists of 3 open reading frames (ORFs). ORF2 encodes a single structural protein (pORF2) for the HEV capsid. Structurally, the HEV capsid is arranged as a T = 3 icosahedral lattice, which assembles with the coordination of the HEV RNA genome.⁹ The approximately 110 amino acid residues (aa) of the N-terminal region are responsible for RNA binding, given the positively charged arginine-clustered region here. With truncations in the 111 N-terminal aa and the 53 C-terminal aa, the protein can self-assemble into a T = 1 icosahedral VLP via a baculovirus / insect cell expression system.¹⁰ The VLP form of genotypes 3 and 4 can be crystallized to allow the structural determination at a resolution of 3.5 Å.^11,12 The analysis of these structures has revealed 3 functional domains of pORF2, including the S domain (aa 118–313), which contributes to the 5-fold interaction and the formation of the basal shell; the P1 domain (aa 314–453), which is important for the 3-fold interaction; and the P2 domain (aa 454–606), which is involved in the 2-fold interaction and is responsible for host interactions.^11,12 The P2 domain, also called the E2s domain, was further analyzed, and its high-resolution crystal structure was determined. The crystal structure showed that two E2s form a tight homodimer through the strong hydrophobic interaction maintained by side chains of residues Val503, Trp548, Thr552, Ala555, Tyr557, Tyr561, Val598 and Val600 of both subunits of the dimer.¹³ There is a unique groove region located on the most protrusive moiety along the 2-fold axis. This groove region was verified to be involved in virus-host interactions using site-directed mutagenesis and the binding of neutralizing mAbs.¹³ The structure of the E2s domain in a complex with neutralizing mAb 8C11 further pinpointed the neutralization sites at Asp496-Thr499, Val510-Leu514, and Asn573-Arg578. Mutation and cell-model assays identified Arg512 as the most crucial residue for 8C11 interaction with and neutralization of HEV.¹⁴ It should be noted that the epitope recognized by 8C11 represents a major immune dominant epitope. As such, it is capable of blocking naturally acquired anti-HEV from binding with the E2 antigen.¹⁵

HEV 239 was designed as ORF2 aa368–606 fragment from a further N-terminal truncation of T = 1 VLP and was adapted to be expressed with a high efficiency in E.coli.¹⁶ During the development of the HEV 239 vaccine, some historic constructs based on antigenicity and immunogenicity were produced, including E2s (aa459–606), E2(aa394-606), HEV239(aa368-606) and T=1 VLP(112-607). E2s forms a dimer with 8C11 binding activity (Fig. 1). E2 (aa394–606) forms a hexamer with considerable antigenicity and marked immunogenicity in rhesus monkeys. The N-terminal 26 aa extension of E2, called HEV 239, self-assembles into shrunken version of the virus-like particles of the ∼20 nm diameter. These particulate particles verified with plausible antigenicity and immunogenicity.^{16, 17} Although HEV 239 (aa 368–606) protein is N-terminally shorter than T = 1 VLP (aa 112–606) in aa sequence, but they share the structural feature of outmost moiety of viral capsid (Fig. 1), which, E2s, or called P2 domain, is responsible for virus-host interaction and subsequently accounts for antigenicity and immunogenicity in vaccine design. Compared with the complex expression in insect cell, production in E.coli is easy to handle and scale-up. HEV 239 can self-assemble into particles in vitro. However, T = 1 VLP was only found forming particle in insect cell (rHEV vaccine,⁵ GSK). In clinical trials, HEV 239 and T = 1 VLP conferred comparable hepatitis E protection in vaccine recipients.^5,7 In terms of nanoscale molecules used for antigen presenting cell processes, VLPs were verified to be the key characteristics for immune recognition.^17,18 Based on this, successful recombinant vaccines for hepatitis B and human papillomavirus were brought to market.^17,18 Thus, HEV 239 was considered for further preclinical study as a HEV vaccine.

Figure 1. Structural interpretation for HEV 239 vaccine. The structure of HEV ORF2 aa112–606 is rendering in surface mode (P2 domain) and cartoon mode (P1 and S domain), colored by key regions for several HEV historic constructs (left panel), i.e., aa112–367 in cyan, aa368–393 in green, aa394–458 in yellow and aa459–606 in orange. To elucidate the spatial configuration, 3 crystal structures were in superimposition to demonstrate major neutralization sites ( Neutralizing mAb 8C11 binding sites colored in red, by PDB (protein database) no. 3RKD), outmost protrusion domain (E2s domain in orange surface, by PDB no. 3GGQ) and P1&S domains (contributing for virus capsid assembly, by PDB no. 3HAG). The figure was prepared by the program PyMol.

The preclinical development of HEV 239 vaccine

HEV 239 was over expressed under the regulatory of T7 promoter in E. coli, the gene was cloned into a non-fusion plasmid vector pTO-T7 and subsequent expression was induced by IPTG. HEV 239 forms inclusion bodies in bacteria. These inclusion bodies were harvested by liquid-solid separation and pan-washing with Triton-X100 before chaotropic agent denaturation. After harvesting, the resulting ∼70% pure HEV 239 inclusion bodies were dissolved in 4 M urea. The HEV 239 protein was further purified to a final purity of 95–98% by 2 polishing chromatography steps and then subjected to particle reassembly with a scalable tangential flow technique, using a membrane cassette with a cutoff size of 30kD.¹⁶ The reassembly condition was optimized for a robust range of pH, ion-strength and temperature. Essentially, HEV 239 can self-assemble in 4 M urea in the presence of salt, which facilitates the assembly control during this process.¹⁹ Self-assembled HEV 239 was further fractionalized using capture chromatography, and the component with an average radius of ∼10 nm was collected and absorbed onto an aluminum adjuvant.¹⁶

The pilot process scale was developed in 10 L fermentation batches, which generated several thousands of vials of vaccine for investigational new drug (IND) purposes. Briefly, the conditions for the serial Essentially, HEV 239 processing were gauged using a variety of biochemical and biophysical approaches, including SDS-PAGE, Western blotting, DLS (Dynamic Light Scattering), SEC HPLC (Size-Exclusion Chromatography), AFM (Atom Force Microscopy), AUC (Analytical Ultracentrifugation), DSC (Differential Scanning Calorimetry) and TEM (Transmission Electronic Microscopy).²⁰ Antigenicity was monitored using a sandwich ELISA with mAbs 8C11 and 8H3. This mAb couple identified the E2s moiety of HEV 239 on the groove region of the E2s crystal structure.¹³ As a rare event in virology, the binding of 8C11 with an antigen will significantly enhance the binding affinity of another mAb 8H3 by 2 log in SPR (Surface Plasmon Resonance) assay. The synergistic action of this mAb couple also was discernible in authentic HEV.¹⁵ All residual contaminants were evaluated and quantified under the regulatory limits issued by the China Food and Drug Administration (CFDA). The immunogenicity of the formulated HEV 239 was measured in vivo with the ED₅₀ (50% effective dose) test in mice and with the anti-HEV levels in rhesus monkeys. The ED₅₀ value for HEV 239 in mice was approximately 0.05 μg, demonstrating that HEV 239 has a high level of immunogenicity. Indeed, the immunogenicity of HEV 239 was 200 times greater than that of the E2 protein.^16,21 The protection efficacy of HEV 239 was primarily evaluated in the rhesus monkey model. Two doses of 5 μg, 10 μg or 20 μg HEV 239 were administered 4 weeks apart. The induced antibody response peaked at week 7 in all 3 dosage groups with comparable antibody titers of around 1384 IU. The anti-HEV levels were measured by ELISA assay and calibrated against a World Health Organization (WHO) reference standard for anti-HEV (National Institute for Biological Standards and Control, NIBSC catalog no. 95/584), which was obtained from an recovered hepatitis E patients with an arbitrary assigned value of 100 IU.¹⁶ The monkeys were then challenged with 10⁴ or 10⁷ genome equivalences of homologous HEV (genotype 1) or heterologous HEV (genotype 4). All of the HEV 239 vaccinated monkeys (n = 24 ) were completely protected from symptomatic hepatitis E and were also completely protected from subclinical infection by 10⁴ HEV (n = 12 ). When challenged with 10⁷ HEV, 9 of the 12 monkeys (75%) were protected from subclinical infection. Among the 3 monkeys with breakthrough HEV infection, 2 monkeys (with anti-HEV level of 368 IU and 196 IU) shed virus during the first week post infection and the third monkey (anti-HEV level 961 IU) had detectable HEV RNA in feces during week 3. The vaccine protection efficacy against homologous HEV (genotype 1) or heterologous HEV (genotype 4) infection was similar.¹⁶ After the further safety evaluation of HEV 239 in animals, which including intramuscular irritation in rabbits, acute toxicity testing in KM (Kunming) mice, deformity testing in gravid white KM mice and repetitive immune toxicity testing in SD (Sprague dawley) rats, the HEV 239 vaccine was approved by the CFDA for further clinical trials.^8,22

Assessment the HEV 239 vaccine critical quality attributes (CQAs)

The product consistency of the HEV 239^8,20,23 vaccine was demonstrated using a combination of biophysical, biochemical and immunochemical methods. Results from these methods demonstrated that different vaccine batches have comparable antigen characteristics or CQAs.

The presence of clinically relevant epitopes can be used as a surrogate marker for vaccine potentcy.^17,24,25 A large panel of monoclonal antibodies (mAbs) against the HEV capsid protein were developed to improve the understanding of HEV virology and to analyze the CQAs of the vaccine during bioprocessing and in the lot release and stability testing.^{14,15,20,26,27} Key epitopes of the particulate antigen were analyzed using a panel of murine anti-HEV mAbs against different epitopes, analogous to the approach used for other vaccines.^24,25,28-31 Epitope mapping using cross-inhibitory ELISA, surface plasmon resonance biosensor chip based binding assay, and sandwich ELISA using HEV 239 specific mAbs were used to assure vaccine potency.^20,26,27 Overall, our findings showed that the antigen production process is robust and scalable in Hecolin® manufacturing.

A successful and robust scale-up (i.e., 50-fold) in the production process (1.0 L, 10 L and 50 L) was demonstrated.²⁰ Multiple HEV 239 batches were produced at a commercial scale (50 L) and were characterized using several different methods (Table 2).

Table 2. Analytical toolbox for the comprehensive characterization of a recombinant HEV 239 antigen to demonstrate the process reproducibility and product consistency during Hecolin® production.^20,26,27

Method
Biochemical methods	Characteristics	Parameters of HEV 239 or Notes (from multiple lots, n = 6 )
SDS-PAGE	Purity, integrity, molecular weight (kDa)	29.32 ± 0 .08
MALDI-TOF MS	Molecular weight (Da)	25,561.64 ± 1 .37
LC-MS	Peptide mapping (coverage, %)	100 (3 lots)
icIEF	pI, whole column detection capillary isoelectric focusing	6.55 ± 0 .03
Biophysical methods
HPSEC	Size (as reflected by retention time, min)^b	14.02 ± 0 .08
TEM	Size, morphology	20–30 nm particles with certain degree of irregularity
DLS	Hydrodynamic diameter (nm)	28.92 ± 0 .20
AUC	Sedimentation coefficient (S)	21.42 ± 0 .13
DSC	Thermal stability (℃)	75.58 ± 0 .10
Cloud point	Aggregation propensity (℃)	71.83 ± 0 .16
CD	Secondary structure	The secondary structure, including α-helices and β-sheets, is
UV	Trp/Tyr absorption	consistent with the UV spectrum of a globular protein with a maximum absorption wavelength at 280 nm
Immunochemical methods
SPR With mAbs	affinity to mAbs	The binding activity to 5 mAbs for different batches of aqueous products with the RU(Ag)/ RU(Ab)
Sandwich ELISA^d	Antigenicity	8C11 (capture) and 8H3 (detection) to analyze the consistency of the antigenicity among different batches of HEV 239 aqueous products
Immunological assessment
Mouse potency	Immunogenicity ED50 (μg)	0.025–0.060

Note: 1. For product lot release of prefilled syringe, mouse potency assay is used for lot release (spec < 1.0 μg). Lot release of HEV 239 purity (spec > 95.0%) and molecular weight (spec 27.5 ± 2.6 kDa) by SDS-PAGE was performed on aqueous product batches.

Two. The reported values were calculated from the results on 6 manufacturing lots. These values are representative on process performance since similar values were reported for sizing using DLS and antigenicity test using the results from over 20 lots.²⁰

Biochemical methods

To verify the consistency of the primary amino acid sequence, analyses were carried out to determine the antigen purity, antigen integrity, molecular weight, pI and protein sequence.^20,27 The most prominent band on the SDS-PAGE gel showed an apparent molecular weight of 29.4 kDa. All three lots exhibited similar MWs by MALDI-TOF-MS, with a mean MW of 25,561.5 ± 1.5 Da (Table 1), which is consistent with the theoretical MW of HEV 239. In addition, LC-MS-based protein mapping with tryptic digestion was used to determine the peptide sequence. The overall amino acid sequence was confirmed with 100% coverage for all 3 batches.²⁰

Biophysical methods

Four different sizing methods (TEM, HPSEC, AUC and DLS) were used to monitor the particle size consistency (Table 1). The HEV 239-based VLPs were found to be 20–30 nm in diameter by TEM, with a certain degree of irregularity and heterogeneity.²⁰ HPSEC analysis resulted in nearly identical retention time for all 6 lots of HEV 239 aqueous products.²⁰ The AUC profiles demonstrated highly similar sedimentation properties for the 6 different lots, all of which peaked at approximately 21.5 s.²⁰

Both circular dichroism and UV spectroscopy methods verified the consistency in the secondary and tertiary protein structure among the different HEV 239 lots.²⁰

The thermal unfolding of the VLP antigen was analyzed using DSC by monitoring the T_m values of all HEV 239 lots, all of which ranged from ∼75–76°C. These results showed good cross lot product consistency as reflected by the thermal stability of the recombinant protein antigen during heat-induced unfolding.^20,27 In addition, the propensity for the protein to aggregate under thermal stress was assessed using cloud point analysis. The heat-induced unfolding or aggregation profiles of the VLPs were highly comparable among all 6 lots, as determined by both DSC and cloud point (Table 2).

Immunochemical methods

The in vitro antigenicity of an antigen, as probed with different mAbs, correlates in general to the in vivo immunogenicity of a vaccine.^30-33 Therefore, a one-site binding and label-free assay (i.e., surface plasmon resonance or SPR)^34-36 and a 2-site binding assay (i.e., a sandwich ELISA)^30,31,33 were carried out to evaluate the antibody binding activity of the HEV 239 antigen.

The label-free and real time sensor chip-based SPR method was used with 5 mAbs recognizing different epitopes (mouse IgGs-8C11, 8H3, 13D8, 12A10 and 16D7) to assess the immunoreactivity of the different batches.²⁰ A sandwich ELISA was implemented in a manufacturing setting with the desired robustness and reproducibility to show product consistency.²⁰ A well-characterized neutralizing and protective mAb, 8C11, was selected as the capture antibody,²⁶ and 8H3 labeled with HRP was used as the detection antibody.

Immunological assessment

To measure the in vivo potency of the vaccine immunogenicity, 6 batches of HEV 239 were evaluated by measuring the ED₅₀ in a mouse model after absorption onto an amorphous aluminum-based adjuvant. All six batches had comparable ED₅₀ values in the range of 0.025–0.060 μg (Table 2). Two of the assays used here, the SDS-PAGE and the mouse potency assay (ED₅₀ determination), are used for routine lot release testing on the final vaccine formulation and filled product. Based on assay results on recovered antigen, full antigen recovery was observed post adjuvant dissolution with no alterations on antigen characteristics.²⁷

Clinical development of HEV 239

All pre-approval clinical trials were carried out in China in accordance with guidelines and regulations issued by the CFDA and the provisions of the Declaration of Helsinki.

Efficacy of HEV 239

A randomized, double-blind, placebo-controlled, single-center Phase III clinical trial was conducted to assess the efficacy of the HEV 239 vaccine against symptomatic hepatitis E. Participants aged 16–65 (n = 112 604) were randomly assigned to receive 30 μg HEV 239 (absorbed to 0.8 mg of aluminum hydroxide suspended in 0.5 mL of buffered saline) or 5 μg of the hepatitis B vaccine control intramuscularly at 0, 1, and 6 months. The patients were followed for 19 months,⁷ and symptomatic hepatitis E cases were identified though a sensitive surveillance system containing 205 local sentinels at the site. The efficacy was demonstrated to be 100% (95% CI, 72–100) in participants receiving all 3 doses of the HEV 239 vaccine or the control vaccine (PPS, per-protocol set) during the following 12 months, starting at 30 d after the receipt of the final dose (7 m–18 m). In the PPS cohorts, no HEV 239 vaccine recipients contracted hepatitis E, whereas 15 hepatitis E cases developed in the control group during the same year. For those who received at least one dose (ITT, intention to treat cohort), the efficacy was 96% (95% CI, 66–99) during the first 18 months.

In an extended efficacy study,³⁷ all participants in the Phase III trial were followed for another 3 y using the same local hepatitis surveillance system used during the Phase III trial. The long-term efficacy of HEV 239 for up to 4.5 y was demonstrated to be 93% (95% CI, 79 - 98) in the PPS cohort and 87% (95% CI, 71 - 94) in the ITT cohort. Due to the large cohorts of the Phase III trial, only participants in the immunogenicity subset were tested for anti-HEV IgG level at month 7, and as there had been no breakthrough hepatitis E cases in this immunogenicity subset with known anti-HEV level at month 7 until now, the protective level of anti-HEV can not be calculated.

As expected, 90% of the 29 HEV isolates from the identified hepatitis E patients were of genotype 4, which is the predominant HEV genotype circulating in China. The high efficacy of HEV 239, which is derived from HEV genotype 1, against HEV genotype 4 infection supported the concept that a hepatitis E vaccine containing one genotype might protect against infection from different HEV genotypes, which are capable of infecting mammals and share the same serotype.

The HEV 239 vaccine has also been shown to be effective in the prevention of subclinical HEV infection.^6,38 Asymptomatic HEV infection was identified by comparing paired serum samples from the same participant collected at 12 month intervals, starting 30 d after administration of the final dose when the antibody levels were supposed to have peaked. If the anti-HEV IgG level rose from negative to 0.154 Wu/mL (twice of the cut-off level) or increased by 4-fold or greater, patients were considered to have asymptomatic infection. The relative risk of HEV infection in the vaccine recipients decreased with increases in the anti-HEV IgG titer at month 7. Even marginal concentration of antibodies significantly lowered the infection risk. This might explain why the efficacy against subclinical HEV infection is similar in the PPS (79%) and ITT cohorts (77%).³⁸ Similar efficacy rates had been demonstrated in the Phase II trial, regardless of whether 3 doses (given at 0, 1 and 6 m) or 2 doses (given at 0, 1 and 6 m) of vaccine were given. In both groups, the tested vaccine contains only 20 μg of the HEV 239 antigen.

The administration of 2 doses of HEV 239 given at months 0 and 1 yielded efficacy rates of 100% (95% CI, 9–100) against hepatitis E during months 2 to 6, before the third shot.⁷ These data justify the possibility of emergency vaccination during epidemics. Similarly, 2 doses of HEV 239 vaccine given at one month intervals provided satisfying protection against subclinical HEV infection for at least the following 5 months.⁶

Immunogenicity of HEV 239

HEV 239 administration induced a vigorous anti-HEV IgG response in vaccine recipients, regardless of their initial antibody status. Of the baseline seronegative participants, 99.9% had seroconverted one month after the full vaccination course (7 m), and 87% remained seropositive at month 55. The geometric mean concentration (GMC) of antibody dropped quickly from a peak of 14.96 Wu/ml at month 7, which is higher than the level induced by natural HEV infection (0.6 Wu/ml) but lower than that induced after symptomatic hepatitis E disease (80.9 Wu/ml), to 1.47 Wu/ml for the first year.^37,38 The concentration then slowly decreased to 0.27 Wu/ml by month 55. Although the antibody decayed to a relative low level at this time point, the long-term efficacy until month 54 is still as high as 93% (95% CI, 79–98) in the PPS cohort, the need and timing for a booster dose of HEV 239 remains to be further investigated. For participants with pre-existing antibodies, the antibody response is more robust and sustained, with little difference between those who received 1, 2 or 3 doses of HEV 239 vaccine. This finding suggests that people with pre-existing HEV antibodies would benefit from even a single dose of the HEV 239 vaccine.^7,37 In a HBsAg positive cohort, the immunogenicity of HEV 239 was found to be similar to that of the general population.³⁹

Anti-HEV IgG levels induced by different dosage and immunization schedules of the HEV 239 vaccine were also evaluated in the Phase II trial, in which only subjects who were anti-HEV seronegative at baseline were enrolled. All participants who received 3 doses of the HEV 239 vaccine seroconverted by month 7, and the level of immune response increased as the dosage increase from 10 μg to 40 μg. The difference in the geometric mean antibody concentration (GMC) was significant between the 30 μg and the other higher doses (p < 0.05). However, the difference in the GMC between 20, 30 and 40 μg was not statistically significant. The study found that the 3 doses course was more potent than the 2 doses course.⁶

Safety of HEV 239

The data from all clinical trials suggest that the HEV 239 vaccine is well tolerated.⁴⁰ In the Phase I trial, 44 healthy Chinese adults were enrolled, and all were anti-HEV IgG seronegative with normal serum biochemistry values. Then, 20 μg of the HEV 239 vaccine was administered at month 0 and 1. All participants were followed for 60 d after the first dose, and their serum biochemistry values were analyzed at day 0 and 60. No serious adverse events were reported. Furthermore, the serum biochemistry values of the participants, including fibrinogen (FBG), urea nitrogen (UREA), glucose (GLU), alanine aminotransferase (ALT), showed no abnormal changes from their baseline values by day 60.⁸

The Phase III clinical trial had 112,604 participants. In this trial, data obtained from the “reactogenicity subset” (n = 2645) within the first 72 hours following each dose showed the occurrence of more frequent local adverse events in the HEV 239 group (13.5%) compared to the control hepatitis B vaccine group (7.1%). This might be partially due to the higher antigen dose in the HEV 239 vaccine (30 μg) than the hepatitis B vaccine (5 μg). Local adverse events of level 3 or higher, solicited systemic adverse events and unsolicited adverse events occurred at similar rates between the study groups. Serious adverse event (SAE) data were collected for all 112,604 participants and categorized according to the Medical Dictionary for Regulatory Activities (MedDRA). During the Phase III trial and the extended 4.5-year follow-up period, a comparable number of participants in both the vaccine and placebo groups experienced SAEs. None of the SAEs were believed to be related to the vaccination.^7,37

There were 37 women in the HEV 239 vaccine group and 31 women in the control group who were inadvertently administered the vaccine during pregnancy.⁴¹ The rates of adverse events observed in the pregnant women were similar to those observed in matched non-pregnant women. The weights, body lengths and gestational ages of the newborns were comparable in both groups. Although this sample size is small and further study is needed, these data preliminarily suggest that the HEV 239 vaccine is well-tolerated in pregnant women. The safety of HEV 239 was also evaluated in HBsAg positive persons, and the resulting data are reassuring.³⁹

Future Challenges

To date, Hecolin® is only available in the Chinese market and been approved for use in people older than 16 years, it is indicated for vaccinating individuals at high risk of HEV infection, such as those involved in animal husbandry, students, members of the armed forces, food-handlers, women of childbearing age, and travelers to endemic regions. The Chinese CDC has an online reporting system to collect post-marketing adverse reactions/events, and this body has not identified any safety concerns with the vaccine. Although the existing 4.5 y of safety and efficacy data for Hecolin® are reassuring, data has not yet been collected in some special populations, including as pediatric subjects (< 16 y of age), the elderly (> 65 y of age), pregnant women, persons with underlying liver diseases or those who are immunosuppressed. These groups face a greater threat of HEV infection and are therefore in more urgent need of this vaccine.

On the other hand, although epidemics of hepatitis E with thousands of cases and hundreds or tens of deaths occurred now and then in low-resource countries, HEV 239 vaccine are not available to these people until it be prequalified by WHO. The deep gap between the vaccine and those who are in urgent need of this vaccine still waits to be filled.⁴²

In conclusion, the availability of Hecolin® represents an important milestone in the control and prevention of HEV. However, there is still long way to go.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

The work was supported by National Major Scientific and Technological Special Project for "Significant New Drugs Development" (2013ZX09101017), National Natural Science Foundation of China (81373061), National High-tech Research & Development Program (863 Program) (2012AA02A408).