Hepatitis E vaccine development

Abstract

The first prophylactic vaccine, Hecolin®, against hepatitis E virus (HEV) infection and the HEV associated disease was approved by China's State Food and Drug Administration (SFDA) in December 2011. Key milestones during the 14-year HEV vaccine development are summarized in this commentary.  After years of innovative research the recombinant virus-like particle (VLP) based antigen with virion-like epitopes was successfully produced in E. coli production platform on a commercial scale. Safety and efficacy of this vaccine was demonstrated in a large scale phase III clinical trial.

Ever since the publication of the 2010 Lancet paper⁷ with 112,604 volunteers during a large Phase III clinical trial, a lot of attention and enthusiasm were put on the highly expected first human vaccine against Hepatitis E. Here we recount the main milestones during the development of this vaccine for Hepatitis E disease. Vaccine development since 1998 has been a long journey in our laboratories side by side with the development of diagnostic kits. Here we focus on key progress, particularly the milestones (Fig. 1) of bioprocessing, clinical trials and regulatory interface with Chinese State of Food and Drug Administration (SFDA) during the 14-y development of this HE vaccine.

Figure 1. Process (A), (pre-)clinical (B) and regulatory (C) milestones during Hecolin® development from 1998 to 2012. * Final developmental process defined. **Final manufacturing process defined and implemented.

Optimization of Antigen For Prophylactic Vaccination

The HEV genome contains three open reading frames (ORFs), ORF1, ORF2 and ORF3, among which ORF2 encodes the only capsid protein, i.e., pORF2. Therefore, pORF2 is the virion capsid protein, with recombinant pORF2 alone being capable of particle assembly into virus-like or subviral particles. This protein was also shown to be crucial for binding to host cells and eliciting of neutralizing and protective antibodies during preclinical studies.^8,9 Along with human serum samples from Hepatitis E patients during acute phase or post recovery, a large panel of hundreds of mouse monoclonal antibodies were developed for epitope characterization of HEV capsid. These reagents and clinical samples were used to understand and define the key epitopes on HEV virions and on different lengths of wild-type and mutated recombinant pORF2. In-depth characterization of the immune-reactivity of mono- and polyclonal antibodies (Abs) against HEV showed that the immunodominant epitopes reside on pORF2. Further immunochemical and structural analyses revealed that the immunodominant epitopes are at the unique groove regions near the dimeric interface in the E2 homodimers – the building blocks of recombinant virus-like-particles (VLPs) or native viral capsids.^10,11

The dimer form of pORF2 showed high affinity to polyclonal Abs in patient sera of HEV-infected individuals.¹² To further enhance immunogenicity, a particulate form or VLPs of the pORF2 (referred to as “239” in reflection of the number of aa residues) is used as the immunogen. Much higher (~240-fold) immunogenicity and efficacy of the particulate form of this HEV 239 vaccine were demonstrated in non-human primates, as compared with a shortened (by 26 amino acids) dimer form or E2.⁸ Rhesus monkeys twice vaccinated with a 10μg or a 20μg formulation of this vaccine showed essentially the same antibody response, whereas the response to a 5μg formulation was delayed but reached similar antibody levels. Proof of concept of this vaccine was demonstrated in that all three vaccine formulations afford complete protection against infection with 10⁴ genome-equivalent doses of the homologous genotype 1 virus and heterogeneous genotype 4 virus. At higher virus dose of 10⁷, the same vaccine formulation partially protected against infection and completely protected against hepatitis.

Clinical Milestones

All clinical studies were conducted in China during 2005–2009. A Phase I clinical trial was initiated in early 2005 in Guangxi province.¹³ We recruited 44 volunteers of ages 21–55. A two-dose vaccination regimen was given at 0–1 mo with 20 μg HE vaccine per injection. After safety of the vaccine was preliminarily demonstrated in Phase I, Phase II trials were performed with the purpose of testing dose schedule and dose escalation. A total of 457 subjects were enrolled in a Phase IIa trial. Two dose schedules of 0,-6 and 0–1-6 mo were tested in this trial. Data showed that in patients of the ‘3-dose group’, the anti-HEV IgG seroconversion rate is higher than those in the ‘2-dose group’ (100% vs. 98%) but the difference was not statistically significant. In addition, the geometric mean concentration (GMC) of anti-HEV IgG was significantly higher in the ‘3-dose group’ than ‘2-dose group’ (15.9 vs. 8.6 WHO units/ml, p < 0.05).¹³

In a Phase IIb clinical trial, 155 subjects were recruited and randomly assigned to four groups, with each group receiving 3 doses of one of four dosage levels - 10, 20, 30, or 40 μg HE vaccine per injection. All four groups showed 100% seroconversion. Detailed analysis showed that the high-dosage groups (20, 30, 40 μg) showed higher anti-HEV IgG GMV levels than the 10-μg dosage group (p < 0.05). The adverse event rates were similar among the four dosage groups, indicating comparable reactogenicity or tolerability of the vaccine at all levels.¹³

From August 2007 to May 2009, a large-scale randomized double-blind placebo-controlled Phase III trial was conducted in Dongtai, Jiangsu Province.⁷ We recruited 112,604 volunteers with ages ranging 16–65 y. These volunteers were randomly assigned into two groups, receiving 3 doses of 30 μg HE vaccine or 5 μg recombinant Hepatitis B virus (HBV) vaccine (as “placebo”) at 0–1-6 mo. HEV vaccine, similar to placebo, was well tolerated and no vaccine-related serious adverse events (SAE) were reported.⁷ As part of this large-scale Phase III trial, there were cases of pregnancy occurred post-screening stage with the volunteers having received at least one dose of HE vaccine. Safety of the vaccine for pregnant women was demonstrated by preliminary analysis in 37 pregnant women who inadvertently received 1, 2 or 3 doses of HE vaccine during pregnancy. The vaccine was well tolerated, and the babies born in the vaccine group were as healthy as those born in the control group.¹⁴

An active acute hepatitis surveillance system comprising 205 sentinels was established for the trial. An independent data and safety monitoring board reviewed the clinical and laboratory results of hepatitis-like patients and confirmed the diagnoses of hepatitis E before unblinding. The Hepatitis E case definition included three conditions that should all be fulfilled: (1) clinical symptoms: acute hepatitis-like illness lasting for at least three days; (2) diagnosis of acute hepatitis: alanine transaminase (ALT) level exceeding 2.5-times the upper limit of normal range (ULN); (3) two positive HEV markers: anti-HEV IgM, HEV RNA, ≥ 4-fold increase in anti-HEV IgG. During the 12 mo after 30 d from receipt of the third dose, 15 per-protocol participants in the placebo group developed hepatitis E compared with none in the vaccine group. Vaccine efficacy after three doses was 100% (95% CI, 72.1–100).⁷ Due to lack of international gold-standard for hepatitis E case-definition where a specific ALT criterion is used, efficacy calculations based on different ALT criterion were described in Table 2. Since most viral liver disease in China is due to chronic hepatitis and not acute hepatitis, the conventional standard of care is to look for lower levels of ALT that are consistent with chronic liver injury. Acute hepatitis E has a different pathophysiology. In India, clinicians suggest that hepatitis E patients should be diagnosed only if the ALT level is ≥ 10-times ULN.¹⁵ Lower levels of ALT were considered to offer an excessive risk of falsely confirming acute liver injury. The abnormal ALT criterion in our study is 2.5 ULN, which is similar to that for the Phase II clinical trial in Nepal of another HE vaccine candidate by GlaxoSmithKline,¹⁶ based on the consideration that all clinically consequential cases associated with substantial liver injury during the acute phase were counted, while mild syndromes having little or no clinical consequences were excluded.

Table 2. Different scoring of efficacy of Hecolin^® vaccine against hepatitis E based on four different criteria for the disease or its protection against overall HEV infection in participants who received all three doses

Endpoint	Definition	No. episodes / No. subjects		Efficacy (%) (95%CI)
		Hecolin^® group	Control group
Hepatitis E (Criterion A)	(1) hepatitis-like symptoms lasting for at least 3 d; (2) ALT ≥ 2.5 × ULN*; (3) Positive for two or more acute HEV infection markers: HEV RNA, IgM anti-HEV by GL assay^& or E2 assay, ≥ 4-fold increase of IgG anti-HEV level (4) Confirmed by Date and Safety Monitoring Board	0 / 48,693	15 / 48,663	100 (72.1 - 100)
Hepatitis E (Criterion B)	(1) hepatitis-like symptoms lasting for at least 3 d; (2) ALT ≥ 2.5 × ULN; (3) Positive for two or more of acute HEV infection markers: HEV RNA, IgM anti-HEV by GL assay, ≥ 4-fold increase of IgG anti-HEV level	0 / 48,693	17 / 48,663	100 (77.3 - 100)
Hepatitis E (Criterion C)	(1) hepatitis-like symptoms lasting for at least 3 d; (2) ALT ≥ 10.0 × ULN; (3) Positive for two or more of acute HEV infection markers: HEV RNA, IgM anti-HEV by GL assay, ≥ 4-fold increase of IgG anti-HEV level	0 / 48,693	8 / 48,663	100 (41.5 - 100)
Hepatitis E (Criterion D)	(1) hepatitis-like symptoms lasting for at least 3 d; (2) ALT ≥ 1.0 × ULN; (3) Positive for two or more of acute HEV infection markers: HEV RNA, IgM anti-HEV by GL assay, ≥ 4-fold increase of IgG anti-HEV level	9 / 48,693	26 / 48,663	65.4 (26.2 - 83.8)
Overall HEV infection	(1) positive seroconversion or ≥ 4-times rising of antibody level in paired serum samples collected with 12-mo interval in two successive years after vaccination	22 / 3,567	101 / 3,548	78.3 (65.6 – 86.3)

Notes: *ULN, the upper limit of normal range. ^&GL assay produced by MP Diagnostics is based on peptides that do not overlap with the vaccine antigen, while E2 assay is based on proteins with similar conformational epitopes as the vaccine antigen.

By comparing the antibody levels of paired samples with a 12-mo interval, HEV infection episodes after vaccination were diagnosed based on positive seroconversion or ≥ 4 fold increase of antibody level. During the two successive years after vaccination, 101 episodes of HEV infection occurred among 3,548 participants in the placebo group and 22 episodes occurred among 3,567 participants in the vaccine group. All were asymptomatic, except for 3 symptomatic hepatitis E cases in placebo group. The overall vaccine efficacy against HEV infection for the two-year period after vaccination is 78.3% (95% CI, 65.6 - 86.3) (Table 1 and Table 2).

Table 1. Clinical trials of the recombinant HE vaccine during late-stage development of Hecolin®

Trial	Study Purpose	No. of patients	Key conclusion	Ref.
Phase Ia (2005.1–2005.3)	Safety	44	✓ Well tolerated*
Phase IIa (2005.4–2005.11)	Safety, Dose schedule	457	✓ Well tolerated* ✓ 3 doses with a regimen of 0–1-6 min was selected
Phase IIb (2005.4–2005.11)	Safety, Dosage escalation	155	✓ Well tolerated* ✓ Seroconversion rate was 100% in all the subjects ✓ 30-μg dose was selected for a Phase III clinical trial
Phase III (2007.8–2009.5)	Safety, Efficacy, Immunogenicity	112 604	✓ Well tolerated in the general population* and showed preliminary evidence for safety in pregnant women. *^& ✓ Efficacy after three doses was 100% (95% CI 72·1–100). ✓ 98.7% of subjects had anti-HEV IgG response after receiving HE vaccine

Notes: * No vaccine-related SAEs were reported. ^&68 pregnant women were inadvertently vaccinated with 1 or more doses of HE vaccine or placebo vaccine during Phase III clinical trial; the AEs occurred in this population were analyzed.

Regulatory Milestones

A clinical trial application for innovation drug (IND) was submitted to SFDA in June 2003. Phase I and II trials were approved in December 2004. After submitting results of the Phase I and II trials in July 2006, it took another year for SFDA to approve the initiation of the Phase III trial. Data from the Phase III trial were submitted at the end of 2009. Upon the completion of Phase III data analysis, the vaccine was named Hecolin^® (Hepatitis E vaccine made in E. coli). In December 2011, China approved the HEV Hecolin^® for use in subjects > 16 y old. The GMP inspection by SFDA for commercial vaccine production is ongoing, and product launch is expected in the second half of 2012.

Future prospects

Initial introduction of Hecolin^® will focus on high-risk populations, such as child-bearing-age women, food industry workers, aged persons, blood donors, organ transplantation receptors and travelers. The manufacturer is expected to work with non-government organizations to make the vaccine available to people in need worldwide. For high-risk populations such as pregnant women,³ chronic liver disease patients,⁴ and children o < 2 y old,⁵ more clinical trials are needed to test the benefit and safety of this HEV vaccine.