- Email: [email protected]
Lessons from hepatitis E vaccine design
Available online at www.sciencedirect.com
ScienceDirect Lessons from hepatitis E vaccine design Shaowei Li1,2, Jun Zhang1,2 and Ningshao Xia1,2 Acute hepatitis E is still a major public health issue, especially in developing countries, and hepatitis E virus (HEV) infection will likely only be preventable through prophylactic vaccines. In this review, we describe the lessons learnt from developing the first commercial hepatitis E vaccine (Hecolin), launched to market in China in 2012. The antigenicity and immunogenicity of VLP immunogens concomitant with the scalable Escherichia coli system and our large-scale clinical verification resulted in the success of our vaccine. The structures of the HEV capsid protein in complex with different antibodies provide important molecular insights into capsid assembly and antibody neutralization of the virus, providing a paradigm for B-cell epitope-based vaccine design. Addresses 1 State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen 361005, China 2 National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen 361005, China Corresponding author: Xia, Ningshao ([email protected])
Current Opinion in Virology 2015, 11:130–136 This review comes from a themed issue on Preventive and therapeutic vaccines Edited by Mansun Law
http://dx.doi.org/10.1016/j.coviro.2015.04.003 1879-6257/# 2015 Elsevier B.V. All rights reserved.
Introduction Hepatitis E virus (HEV) causes both the epidemic and sporadic forms of acute hepatitis [1]. HEV infection is prevalent in approximately one-third of the world’s population and causes a wide spectrum of clinical manifestations, from self-limited acute hepatitis to lethal liver failure [2,3]. Mortality rates of HEV infection are about 0.5% but can be as high as 25% in pregnant women [4]. The lack of a robust cell culture method to propagate HEV makes it impracticable to develop inactive or attenuating vaccines [5]. Instead, the B-cell epitopes of HEV have been mapped biochemically using recombinant capsid proteins or overlapping peptides. Using these analyses, a recombinant hepatitis E vaccine — the first human vaccine — was designed and created using an E. coli expression system [6,7]. The odyssey of the Current Opinion in Virology 2015, 11:130–136
development of this vaccine [8] provides a paradigm for B-cell epitope-based vaccine design and, plausibly, offers future strategies for the development of other non-enveloped virus vaccines.
Hepatitis E virus biology Hepatitis E virus (HEV) resembles the calicivirus in virion morphology, and similarly comprises the S, P1 and P2 domains in the T = 3 icosahedral capsid structure [9,10]. Because of its unique gene organization, however, it was classified as its own Hepevirus genus in the Hepeviridae family [11]. HEV infects a wide range of mammalian species, including humans, and has, over time, evolved into two major groups comprising four genotypes. Genotypes 1 and 2 infect humans and non-human primates only, and often lead to epidemic outbreaks through the fecal–oral route. Genotypes 3 and 4 can infect a wide range of animals including humans in a sporadic manner [12]. Serologically, the naturally acquired anti-HEV is indistinguishable among the four genotypes and ascribes to a single serotype [13]. HEV is a small, non-enveloped RNA virus, the genome for which is a positive-sense, single-stranded, 7.2-kb RNA. The HEV genome contains three open reading frames (ORFs): ORF1 encodes non-structural proteins; ORF2 encodes the viral structural protein of 660 amino acids (aa), which assembles into a T = 3 icosahedral capsid of 35 nm in diameter; and ORF3 encodes a small protein associated with virion morphogenesis and release [14]. Considering all of the structures of the HEV capsid protein, including the E2s domain [15] and VLPs with T = 1 [16,17] and T = 3 [10], the capsid protein is thus considered to consist of three domains: an S domain (aa 112–313), a P1 or M domain (aa 314–454) and a P2 or P domain (aa 455–606) [16,17]. The P2 domain, also referred to as the E2s domain, forms an intimate homodimer in solution [15] that protrudes from the basal shell of the virus capsid to form a spike (Figure 1). This spike is responsible for host interactions, and harbors the major neutralization B-cell epitopes that present immunogenicity. The crystal structures of the E2s in complex with the Fab fragments of the genotype-preferred 8C11 [18] and cross-genotype 8G12 [19] neutralizing monoclonal antibodies (mAbs) have highlighted the immunodominant epitope site on the capsid spike. Yet, how to maintain these conformation-dependent B-cell epitopes and their immunogenicity during vaccination remain critical concerns in hepatitis E vaccine design.
B-cell epitopes of hepatitis E virus Despite belonging to a single serotype, HEV associated with human diseases can be classified into four main www.sciencedirect.com
Lessons from hepatitis E vaccine design Li, Zhang and Xia 131
Figure 1
(a)
(b)
mAb1323 4 mAbs 8H3/Fab224 8C11 8G12 mAb272 12A10
90°
90° (c)
(d)
mAb 8C11 8G12 8H3 mAb1323 mAb272 4 mAbs Fab224 12A10
B-cell epitope sites E479,Y485,D496,R512 K534,H577,R578 E549,K554,G591 E479, Y485,I529,K534 S487,S488,T489,P491, N562,T564 D496,T497,G591,P592 T489,P491,Y561,N562 T585,T586 E479,Y485,I529,K534 D430,L433 Current Opinion in Virology
The key amino acid residues involved in the interaction of functional antibodies and the HEV capsid protein. The dimeric pORF2 in the crystal structure of hepatitis E virus (HEV) virus-like particles (VLP) for genotype 3 (PDB no. 2ZTN) was rendered in surface mode and is depicted in front view (a), side view (b) and top view (c). The residues are colored according to the monoclonal antibody (mAb) binding sites, although some overlap. All of the epitope residues were listed in panel (d). The figures related to the structure were prepared using PyMol.
genotypes: genotypes 1 and 2 infect humans, whereas genotypes 3 and 4 are zoonotic [20]. Previous evidence suggests that these four genotypes share a common epitope(s), which elicits major, neutralizing antibodies that manifest as a single serotype in the humans [19,21]. Yet, the capsid proteins of zoonotic and non-zoonotic HEVs may contain distinct host receptor binding sites, and this may, in turn, lead to the production of genotypespecific immune responses [18]. The epitopes recognized by the cross-genotype and genotype-specific antibodies can be grouped into conformational and linear epitopes according to their structural features [18,19]. The B-cell epitope of the HEV structural protein, pORF2, was identified retrospectively using recombinant www.sciencedirect.com
proteins, overlapping peptides and their corresponding immune sera or mAbs; some of these mAbs were characterized by virus neutralization using HEV-infection cell model and/or through the use of a virus-challenging animal model [22,23]. The immune epitopes of HEV and the B-cell and T-cell types are summarized in Table 1. Moreover, to provide a clearer understanding of their spatial location on the HEV capsid protein, the key amino acid residues involved in the interaction of pORF2 or its partial segments with mAbs are structurally rendered in Figure 1. The crystal structure of the immune complex (PDB no. 3RKD) highlights several of the amino acids (chiefly, Current Opinion in Virology 2015, 11:130–136
132 Preventive and therapeutic vaccines
Table 1 The immune epitopes of hepatitis E virus (HEV) identified by monoclonal antibodies (mAb), truncated capsid protein and overlapping peptides Immune epitope or region E479,Y485,D496,R512,K534,H577,R578 E549,K554,G591 E479,Y485,I529,K534 S487,S488,T489,P491,N562,T564 D496,T497,G591,P592 T489,P491,Y561,N562,T564,T585,T586 E479,Y485,I529,K534 D430,L433 Linear epitope 578–607 aa 394–457 Within aa 394–604 aa 477–613 Within aa 452–617 aa 394–470; aa 546–580; aa 490–579 aa 477–492; aa 389–410; aa 583–600 aa 73–156; aa 289–372; aa 361–444; aa 505–588 P34 (aa 532–546); P35 (aa 537–551)
Method used to identify
Reference
8C11
[18]
8G12
[19]
8H3 mAb1323 mAb272 4 mAbs Fab224 12A10 HEV#4 and HEV#31 mAb 1C7, 3B2, 1E6, 1E7, 4B5 mAb 13D8, 16D7 mAb 1G10 N/A N/A N/A N/A N/A
[15] [17] [17] [17] [24] [19,25] [22,26] [27] [23,28] [29] [30] [31] [32] [33] [34]
Overlapping peptide, T-cell epitope
N/A
[35]
R512) involved in the virus–host interaction, and reveals the specificity of mAb 8C11 for genotypes 1 and 2. This interaction is particularly dependent on residue 497, a serine residue (S497) for genotypes 1/2, and a threonine (T497) for genotypes 3 and 4 [18]. Others have also shown that L477 and L613 are critical in the neutralization epitope of genotype 4 [29]. Another complex crystal structure of the 8G12 cross-genotype neutralizing antibody with the HEV E2s domain of genotypes 1 and 4 (PDB no. 4PLK and 4PLJ) pinpoint several other conserved residues (E549, K554 and G591) that are essential for common HEV neutralization. Moreover, mAb 8G12 is the predominant antibody identified in naturally acquired anti-HEV antibodies in human and monkey sera [19]. For vaccine design, these major neutralization epitopes on the capsid protein should be taken into consideration and exploited in the intended immunogens. Several of the designed constructs for pORF2 (Figure 2) were used to identify the B-cell epitopes on the HEV capsid; this circumvented the lack of an efficient cell culture system for HEV. The recombinant subunit ORF2.1 antigen (aa 394–660) induced antibodies against the immunodominant, conformational epitopes [36]. Indeed, Zhang et al. (2001) described a dimeric antigen E2 that harbored a dimer-dependent conformational epitope [28] and conferred immune protection against HEV in monkeys [37]. Li et al. (2005) further narrowed down this dimer-dependent epitope to the E2s domain (aa 454–606) [7] and solved the high-resolution structure of E2s using crystallization; this knowledge revealed that the dimerization of the E2s domain was essential for the virus–host interaction [15]. The antigens with VLP form, p495 (aa Current Opinion in Virology 2015, 11:130–136
Corresponding mAb
Crystal structure combined with mutation assay Crystal structure combined with mutation assay Mutation assay Density fractionation assay Density fractionation assay Binding to host cell model assay Mutation assay Mutation assay Truncation binding assay Truncated proteins Homodimer protein Truncated proteins Overlapping proteins, peptides Overlapping peptides Overlapping peptides Overlapping synthetic peptides T-cell epitope mapping
112–606) and p239 (aa 368–606), also contain some known functional epitopes (Figures 1 and 2) [6,7,38–40].
A structural analysis of the hepatitis E vaccine Recombinant VLPs usually mimic native virions in structure but without the viral genome. They are well-recognized by the immune system and are highly immunogenic because of their size and the repetitive pattern of B cell epitopes displayed on the particle surface [41]. Thus, VLPs often manifest sufficient antigenicity and immunogenicity for vaccine development. In the human vaccine field, two recombinant human vaccines against hepatitis B (HB) and human papillomavirus (HPV) have shown high efficacy in their VLP applicability [42]. During the immunogenic design for the HEV vaccine, we comprehensively interrogated the amino acids from the N-termini to C-termini to identify domains involved in dimer and particulate formation using the E. coli expression system. The minimal dimerization domain was located at aa 459–601, with the particulate domain further extending to aa 368 at the N-terminus [7]. The crystal structure and cryo-EM structure of T = 1 [17] (Figure 2a) and T = 3 [10] (Figure 2b) VLPs indicated that the E2s domain is equivalent to the P2 domain, serving as a major B-cell epitope reservoir, and its copies are arranged by the P1 (in the case of both p239 and p495) and the S (p495 only) domains to form particles and subsequently present high immunogenicity [7,15,16,17]. Viewed inward along the threefold axis of the T = 1 VLP (Figure 2a), three E2 dimers form a hexamer around the threefold axis by interaction from the partial P1 domain (aa 394–453) and the N-terminal 26-aa extension www.sciencedirect.com
Lessons from hepatitis E vaccine design Li, Zhang and Xia 133
Figure 2
(a)
(b)
(c) 660
ORF2.1
C
p495 p239
E2
E2s
606
P2
5 454
2
3 394 368
3 3 3
454
P1
394 394 368
314
2 5
S
112 112
1
HEV T=1 VLP
HEV T=3 VLP
N
HEV ORF2 Current Opinion in Virology
Structural interpretation of previously assessed truncated pORF2 constructs by the structure of hepatitis E virus (HEV) virus-like particles (VLP). (a) The molecular segmentation of domains or constructs on T = 1 HEV VLP (PDB no. 2ZTN) and (b) T = 3 HEV VLP (PDB no. 2ZTN fitted to 3IYO). The twofold, threefold and fivefold axes of the icosahedral lattice are denoted as solid black ovals, triangles and pentagons, respectively. (c) Schematic regions for the truncated pORF2 constructs. The immunogen p239 in the Hecolin vaccine is shown covering the P2 domain (in orange) and partially covering the P1 domain (in green, the rest portion in white) of the capsid. The color scheme is consistent with the structure rendering.
further to p239 (aa 368–606), which forms a shrunken version of the T = 1 VLP (Figure 2c) [6,7,42]. In addition to the N-terminal extension for particulate formation, the immunogenicity of p239 dramatically increases up to 200-times with respect to that of E2 [6,35].
Expression system and the manufacturing process HEV capsid proteins have been expressed in various expression systems, including E. coli, yeast, plant, insect and mammalian cells [28,43–46]. Two of these recombinant proteins have been the subject of clinical trials, the first of which is p495, which spans aa 112–606. Using baculovirus insect cells, the resultant proteins were found to present T = 1 VLPs in the cell lysate [38,47]. Previous strategies for purification have included PEG precipitation [48], chromatography [49] and ultra-centrifugation [39,47], and the purified VLPs were crystalized for X-ray crystallography [16,17] or vitrified for electron cryomicroscopy [10,38,50]; a scalable purification process that is suitable for manufacturing the p495 vaccine has not been reported to date. In addition, there is no evidence to show that p495 can self-assemble into VLPs in vitro. The second recombinant protein is p239 (aa 368–606), which is expressed in E. coli via a well-established manufacturing process that includes VLP assembly in vitro. In our investigations, we showed that the fermentation scale for manufacturing the p239 vaccine is up to 50 L [51], and demonstrated that the proteins formed inclusion bodies in bacteria and dissolved in 4 M urea after pan-washing with Triton-X 100. p239 exclusively www.sciencedirect.com
presented as dimer in 4 M urea, and, under these conditions, almost all of the contaminants were removed before the self-assembly process. In the presence of adequate salt (e.g., 0.5 M amide sulfate), p239 then assembled into particles. This assembly mechanism was clarified using time-lapse analytical ultracentrifugation and the assembly procedure can be modulated by adjusting the salt concentration and incubation temperature [52]. After buffer exchange, the assembled particles were further fractionated by ion-exchange chromatography instead of traditional size-exclusion. All of the sub-processes are scalable using chromatography and tangential flow techniques and the manufacturing process can be comprehensively evaluated by a combination of biophysical, biochemical and immunochemical means. Thus, this technique provides a robust, scalable manufacturing process and, consequently, product lot consistency, for the p239 vaccine. In our evaluations of vaccine quality, the particulate nature and the structural integrity of the major B-cell epitopes by mAb-based assay are emphasized and, thus, the use of E. coli to manufacture the p239 vaccine seems accessible and affordable for its wider applicability [51].
Clinical trials of hepatitis E vaccine The success of any vaccine design is determined by its efficacy in clinical trials. To date, both of the recombinant vaccines described above (p495 and p239 vaccines) have undergone clinical trials for this purpose. The p495 vaccine has been proven to be safe and immunogenic in young male adults, and conferred 95% protection against hepatitis E in Nepal, where only genotype Current Opinion in Virology 2015, 11:130–136
134 Preventive and therapeutic vaccines
1 HEV has been isolated [53]. The p239 vaccine was verified to be safe, immunogenic and efficacious against subclinical HEV infection in sero-negative subjects in a Phase 2 clinical trial in China [54], and was later tested in a randomized, double-blind, placebo-controlled Phase 3 clinical trial. This Phase 3 trial comprised 112 604 participants who were randomized 1:1 into vaccine or control groups, and were administered with three doses of p239 vaccine or a control vaccine (hepatitis B vaccine) at 0, 1 and 6 months. In the per-protocol analysis, 15 subjects in the control group developed hepatitis E versus none in the p239 vaccine group over the 12 months after the third vaccination. Vaccine efficacy in that year after the three doses was determined to be 100.0% (95%CI, 72.1–100.0) for those who received the three doses in the requested time frame (per-protocol analysis) [55]. The long-term follow-up of the participants in the Phase 3 trial has indicated that this antibody efficacy persists for at least 4.5 years with an efficacy of 93.3% (95%CI, 78.6–97.9) in the per-protocol cohort and 86.8% (95%CI, 71.0–94.0) in those who received at least one dose of the vaccine [56]. The HEV isolates from the identified hepatitis E patients were predominantly genotype 4, with a few genotype 1, indicating that the p239 vaccine of genotype 1 provides cross-protection against HEV genotype 4. Because of the limited breakthrough cases in p239 vaccines observed in the clinical trial, the protective antibody level against hepatitis E has not been determined. In preclinical efficacy studies in rhesus monkeys, the p239 vaccine induced an anti-HEV IgG titer of 1384 Wu/mL (WHO Unit per mL), which can prevent symptomatic hepatitis E after challenging with 104 or 107 genome equivalences of HEV. However, this antibody level can only partially protect (75%) the monkeys from HEV infection, even when challenging with the 107 dose. In the human study, the data showed that marginal levels of anti-HEV IgG (0.077–0.25 Wu/mL) could decrease the risk of asymptomatic HEV infection in immunocompetent persons (Relative risk = 0.26) [57]. The pre-infection antibody levels of the subjects with post-vaccination, asymptomatic infection varied widely. However, although the dose-effect is not significant, subjects with an anti-HEV level >1.0 Wu/mL are significantly better protected than those with marginal antibody levels. In the immunogenicity analysis, p239 induced a strong anti-HEV response in the vaccine recipients, with a seroconverted ratio of 99.9% and a geometric mean antibody concentration of 14.96 Wu/mL at one month after the third vaccination [55]. Collectively, these results demonstrate that the hepatitis E vaccine in its VLP form is highly efficacious in protecting against hepatitis E, and that the B-cell epitopes are sufficiently immunogenic to elicit protective antibodies with some capacity for crossgenotype neutralization. It will be important to continue to monitor the potential immune pressure of the vaccine Current Opinion in Virology 2015, 11:130–136
in these protected subjects to ascertain if there is any capacity for epitope-escape or mutation of HEV.
Conclusions The hepatitis E vaccine, Hecolin, is the first human vaccine using the E. coli-expressed VLP antigen. The B-cell epitope profile of the capsid protein along with mAb-based binding assays will aid in ensuring quality control of the p239 vaccine during manufacturing. In protected subjects and during future vaccine applications worldwide, it will be important to continue to screen for potential epitope-escape HEVs. These lessons learned from the HEV vaccine design could be extrapolated for the investigations of other non-enveloped, capsid viruses.
Acknowledgements We acknowledge funding support from the Chinese Government: Fujian Provincial Science Fund for Distinguished Young Scholars (Grant no. 2011J06015), Fujian Provincial Fund for Key Platform of Scientific Innovation (Grant no. 2014Y2101), Major National Project ‘‘863’’ (Grant no. 2014AA021302), Xiamen Science and Technology Project (Grant no. 3502Z201410045).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Kamar N, Bendall R, Legrand-Abravanel F, Xia NS, Ijaz S, Izopet J, Dalton HR: Hepatitis E. Lancet 2012, 379:2477-2488.
2.
Aggarwal R, Kini D, Sofat S, Naik SR, Krawczynski K: Duration of viraemia and faecal viral excretion in acute hepatitis E. Lancet 2000, 356:1081-1082.
3.
Hoofnagle JH, Nelson KE, Purcell RH: Hepatitis E. N Engl J Med 2012, 367:1237-1244.
4.
Kumar A, Beniwal M, Kar P, Sharma JB, Murthy NS: Hepatitis E in pregnancy. Int J Gynaecol Obstet 2004, 85:240-244.
5.
Okamoto H: Culture systems for hepatitis E virus. J Gastroenterol 2013, 48:147-158.
6.
Li SW, Zhang J, Li YM, Ou SH, Huang GY, He ZQ, Ge SX, Xian YL, Pang SQ, Ng MH et al.: A bacterially expressed particulate hepatitis E vaccine: antigenicity, immunogenicity and protectivity on primates. Vaccine 2005, 23:2893-2901. The E. coli-derived p239 vaccine was characterized by particulation, antigenicity and immunogenicity assay. p239 vaccine confers protection against HEV infection and hepatitis E disease in monkeys.
7.
Li SW, Zhang J, He ZQ, Gu Y, Liu RS, Lin J, Chen YX, Ng MH, Xia NS: Mutational analysis of essential interactions involved in the assembly of hepatitis E virus capsid. J Biol Chem 2005, 280:3400-3406. The elaborated mutational analysis identifies key regions for dimerization, particle assembly and neutralization of B-cell epitope formation of the HEV capsid protein, which was the basis for the successful HEV vaccine design. 8.
Wu T, Li SW, Zhang J, Ng MH, Xia NS, Zhao Q: Hepatitis E vaccine development: a 14 year odyssey. Hum Vaccin Immunother 2012, 8:823-827.
9.
Prasad BV, Hardy ME, Dokland T, Bella J, Rossmann MG, Estes MK: X-ray crystallographic structure of the Norwalk virus capsid. Science 1999, 286:287-290.
10. Xing L, Li TC, Mayazaki N, Simon MN, Wall JS, Moore M, Wang CY, Takeda N, Wakita T, Miyamura T et al.: Structure of hepatitis E virion-sized particle reveals an RNA-dependent viral assembly pathway. J Biol Chem 2010, 285:33175-33183. www.sciencedirect.com
Lessons from hepatitis E vaccine design Li, Zhang and Xia 135
11. Smith DB, Purdy MA, Simmonds P: Genetic variability and the classification of hepatitis E virus. J Virol 2013, 87:4161-4169. 12. Krain LJ, Nelson KE, Labrique AB: Host immune status and response to hepatitis E virus infection. Clin Microbiol Rev 2014, 27:139-165. 13. Worm HC, Wirnsberger G: Hepatitis E vaccines: progress and prospects. Drugs 2004, 64:1517-1531. 14. Yamada K, Takahashi M, Hoshino Y, Takahashi H, Ichiyama K, Nagashima S, Tanaka T, Okamoto H: ORF3 protein of hepatitis E virus is essential for virion release from infected cells. J Gen Virol 2009, 90:1880-1891. 15. Li S, Tang X, Seetharaman J, Yang C, Gu Y, Zhang J, Du H, Shih JW, Hew CL, Sivaraman J et al.: Dimerization of hepatitis E virus capsid protein E2s domain is essential for virus-host interaction. PLoS Pathog 2009, 5:e1000537. 16. Guu TS, Liu Z, Ye Q, Mata DA, Li K, Yin C, Zhang J, Tao YJ: Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding. Proc Natl Acad Sci U S A 2009, 106:12992-12997. The crystal structure of the HEV VLP of genotype 4 reveals three key domains within the HEV capsid, of which the protruding P2 domain serves as a receptor binding site and potentially harbors major neutralization sites. 17. Yamashita T, Mori Y, Miyazaki N, Cheng RH, Yoshimura M, Unno H, Shima R, Moriishi K, Tsukihara T, Li TC et al.: Biological and immunological characteristics of hepatitis E virus-like particles based on the crystal structure. Proc Natl Acad Sci U S A 2009, 106:12986-12991. The crystal structure of HEV VLP of genotype 3 reveals three key domains within the HEV capsid, identifying several B-cell epitopes of HEV by mutational analysis. 18. Tang X, Yang C, Gu Y, Song C, Zhang X, Wang Y, Zhang J, Hew CL, Li S, Xia N et al.: Structural basis for the neutralization and genotype specificity of hepatitis E virus. Proc Natl Acad Sci U S A 2011, 108:10266-10271. The first crystal structure of the HEV immune complex shows the footprint of a neutralizing mAb 8C11 at the atomic level and identifies key points of virus-host interactions using the HEV cell model. 19. Gu Y, Tang X, Zhang X, Song C, Zheng MH, Wang KH, Zhang J, Ng MH, Hew CL, Li SW et al.: Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody. Cell Res 2015, 25 http://dx.doi.org/10.1038/cr.2015.34. The crystal structures of HEV E2s domains of genotypes 1 and 4 in complex with cross-genotype neutralizing mAb 8G12 reveals several conserved amino acids for common HEV neutralization. This supports the phenomenon of a single serotype for the four main HEV genotypes in human sera and the ability for cross-protection with one genotype vaccine. 20. Zhang J, Li SW, Wu T, Zhao Q, Ng MH, Xia NS: Hepatitis E virus: neutralizing sites, diagnosis, and protective immunity. Rev Med Virol 2012, 22:339-349. 21. Emerson SU, Clemente-Casares P, Moiduddin N, Arankalle VA, Torian U, Purcell RH: Putative neutralization epitopes and broad cross-genotype neutralization of hepatitis E virus confirmed by a quantitative cell-culture assay. J Gen Virol 2006, 87:697-704. 22. Schofield DJ, Glamann J, Emerson SU, Purcell RH: Identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis E virus capsid protein. J Virol 2000, 74:5548-5555. 23. Zhang J, Gu Y, Ge SX, Li SW, He ZQ, Huang GY, Zhuang H, Ng MH, Xia NS: Analysis of hepatitis E virus neutralization sites using monoclonal antibodies directed against a virus capsid protein. Vaccine 2005, 23:2881-2892. 24. Xing L, Wang JC, Li TC, Yasutomi Y, Lara J, Khudyakov Y, Schofield D, Emerson SU, Purcell RH, Takeda N et al.: Spatial configuration of hepatitis E virus antigenic domain. J Virol 2011, 85:1117-1124. 25. He S, Miao J, Zheng Z, Wu T, Xie M, Tang M, Zhang J, Ng MH, Xia N: Putative receptor-binding sites of hepatitis E virus. J Gen Virol 2008, 89:245-249. www.sciencedirect.com
26. Schofield DJ, Purcell RH, Nguyen HT, Emerson SU: Monoclonal antibodies that neutralize HEV recognize an antigenic site at the carboxyterminus of an ORF2 protein vaccine. Vaccine 2003, 22:257-267. 27. Riddell MA, Li F, Anderson DA: Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies. J Virol 2000, 74:8011-8017. 28. Zhang JZ, Ng MH, Xia NS, Lau SH, Che XY, Chau TN, Lai ST, Im SW: Conformational antigenic determinants generated by interactions between a bacterially expressed recombinant peptide of the hepatitis E virus structural protein. J Med Virol 2001, 64:125-132. 29. Zhang H, Dai X, Shan X, Meng J: The Leu477 and Leu613 of ORF2-encoded protein are critical in forming neutralization antigenic epitope of hepatitis E virus genotype 4. Cell Mol Immunol 2008, 5:447-456. 30. Meng J, Dai X, Chang JC, Lopareva E, Pillot J, Fields HA, Khudyakov YE: Identification and characterization of the neutralization epitope(s) of the hepatitis E virus. Virology 2001, 288:203-211. 31. Khudyakov Yu E, Favorov MO, Jue DL, Hine TK, Fields HA: Immunodominant antigenic regions in a structural protein of the hepatitis E virus. Virology 1994, 198:390-393. 32. Khudyakov YE, Lopareva EN, Jue DL, Crews TK, Thyagarajan SP, Fields HA: Antigenic domains of the open reading frame 2encoded protein of hepatitis E virus. J Clin Microbiol 1999, 37:2863-2871. 33. Guo H, Zhou EM, Sun ZF, Meng XJ, Halbur PG: Identification of B-cell epitopes in the capsid protein of avian hepatitis E virus (avian HEV) that are common to human and swine HEVs or unique to avian HEV. J Gen Virol 2006, 87:217-223. 34. Aggarwal R, Shukla R, Jameel S, Agrawal S, Puri P, Gupta VK, Patil AP, Naik S: T-cell epitope mapping of ORF2 and ORF3 proteins of human hepatitis E virus. J Viral Hepat 2007, 14:283-292. 35. Wu T, Wu XL, Ou SH, Lin CX, Cheng T, Li SW, Ng MH, Zhang J, Xia NS: Difference of T cell and B cell activation in two homologous proteins with similar antigenicity but great distinct immunogenicity. Mol Immunol 2007, 44:3261-3266. 36. Li F, Riddell MA, Seow HF, Takeda N, Miyamura T, Anderson DA: Recombinant subunit ORF2.1 antigen and induction of antibody against immunodominant epitopes in the hepatitis E virus capsid protein. J Med Virol 2000, 60:379-386. 37. Im SW, Zhang JZ, Zhuang H, Che XY, Zhu WF, Xu GM, Li K, Xia NS, Ng MH: A bacterially expressed peptide prevents experimental infection of primates by the hepatitis E virus. Vaccine 2001, 19:3726-3732. 38. Xing L, Kato K, Li T, Takeda N, Miyamura T, Hammar L, Cheng RH: Recombinant hepatitis E capsid protein self-assembles into a dual-domain T = 1 particle presenting native virus epitopes. Virology 1999, 265:35-45. 39. Li TC, Yoshimatsu K, Yasuda SP, Arikawa J, Koma T, Kataoka M, Ami Y, Suzaki Y, Mai le TQ, Hoa NT et al.: Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses. J Gen Virol 2011, 92:2830-2837. 40. Wei M, Zhang X, Yu H, Tang ZM, Wang K, Li Z, Zheng Z, Li S, Zhang J, Xia N et al.: Bacteria expressed hepatitis E virus capsid proteins maintain virion-like epitopes. Vaccine 2014, 32:28592865. 41. Grgacic EV, Anderson DA: Virus-like particles: passport to immune recognition. Methods 2006, 40:60-65. The review describes the importance of virus-like particles as an ideal immunogen form for immune recognition. 42. Zhao Q, Li S, Yu H, Xia N, Modis Y: Virus-like particle-based human vaccines: quality assessment based on structural and functional properties. Trends Biotechnol 2013, 31:654-663. Current Opinion in Virology 2015, 11:130–136
136 Preventive and therapeutic vaccines
43. Tyagi S, Korkaya H, Zafrullah M, Jameel S, Lal SK: The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2. J Biol Chem 2002, 277: 22759-22767. 44. Ma Y, Lin SQ, Gao Y, Li M, Luo WX, Zhang J, Xia NS: Expression of ORF2 partial gene of hepatitis E virus in tomatoes and immunoactivity of expression products. World J Gastroenterol 2003, 9:2211-2215. 45. He J, Tam AW, Yarbough PO, Reyes GR, Carl M: Expression and diagnostic utility of hepatitis E virus putative structural proteins expressed in insect cells. J Clin Microbiol 1993, 31:2167-2173. 46. Jameel S, Zafrullah M, Ozdener MH, Panda SK: Expression in animal cells and characterization of the hepatitis E virus structural proteins. J Virol 1996, 70:207-216. 47. Li TC, Takeda N, Miyamura T, Matsuura Y, Wang JC, Engvall H, Hammar L, Xing L, Cheng RH: Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus. J Virol 2005, 79:12999-13006. 48. Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T: Expression and self-assembly of empty virus-like particles of hepatitis E virus. J Virol 1997, 71:7207-7213. 49. Robinson RA, Burgess WH, Emerson SU, Leibowitz RS, Sosnovtseva SA, Tsarev S, Purcell RH: Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells. Protein Expr Purif 1998, 12:75-84. 50. Mori Y, Matsuura Y: Structure of hepatitis E viral particle. Virus Res 2011, 161:59-64.
Current Opinion in Virology 2015, 11:130–136
51. Zhang X, Wei M, Pan H, Lin Z, Wang K, Weng Z, Zhu Y, Xin L, Zhang J, Li S et al.: Robust manufacturing and comprehensive characterization of recombinant hepatitis E virus-like particles in Hecolin((R)). Vaccine 2014, 32:4039-4050. 52. Yang C, Pan H, Wei M, Zhang X, Wang N, Gu Y, Du H, Zhang J, Li S, Xia N: Hepatitis E virus capsid protein assembles in 4 M urea in the presence of salts. Protein Sci 2013, 22:314-326. 53. Shrestha MP, Scott RM, Joshi DM, Mammen MP Jr, Thapa GB, Thapa N, Myint KS, Fourneau M, Kuschner RA, Shrestha SK et al.: Safety and efficacy of a recombinant hepatitis E vaccine. N Engl J Med 2007, 356:895-903. 54. Zhang J, Liu CB, Li RC, Li YM, Zheng YJ, Li YP, Luo D, Pan BB, Nong Y, Ge SX et al.: Randomized-controlled phase II clinical trial of a bacterially expressed recombinant hepatitis E vaccine. Vaccine 2009, 27:1869-1874. 55. Zhu FC, Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, Wang H, Yang CL, Jiang HM, Cai JP et al.: Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults: a largescale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet 2010, 376:895-902. Convincing evidence from this clinical trial confirms the efficacy of the HEV p239 vaccine. The clinical trial is unprecedented in its large scale of over 110 000 subjects. 56. Zhang J, Zhang XF, Huang SJ, Wu T, Hu YM, Wang ZZ, Wang H, Jiang HM, Wang YJ, Yan Q et al.: Long-term efficacy of a hepatitis E vaccine. N Engl J Med 2015, 372:914-922. Immunization with HEV p239 vaccine induced antibodies against HEV and provided protection against hepatitis E for up to 4.5 years. 57. Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, Yao X, Liang ZL, Wu T, Li JX, Yan Q et al.: Protection against hepatitis E virus infection by naturally acquired and vaccine-induced immunity. Clin Microbiol Infect 2014, 20:O397-O405.
www.sciencedirect.com
ScienceDirect Lessons from hepatitis E vaccine design Shaowei Li1,2, Jun Zhang1,2 and Ningshao Xia1,2 Acute hepatitis E is still a major public health issue, especially in developing countries, and hepatitis E virus (HEV) infection will likely only be preventable through prophylactic vaccines. In this review, we describe the lessons learnt from developing the first commercial hepatitis E vaccine (Hecolin), launched to market in China in 2012. The antigenicity and immunogenicity of VLP immunogens concomitant with the scalable Escherichia coli system and our large-scale clinical verification resulted in the success of our vaccine. The structures of the HEV capsid protein in complex with different antibodies provide important molecular insights into capsid assembly and antibody neutralization of the virus, providing a paradigm for B-cell epitope-based vaccine design. Addresses 1 State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen 361005, China 2 National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen 361005, China Corresponding author: Xia, Ningshao ([email protected])
Current Opinion in Virology 2015, 11:130–136 This review comes from a themed issue on Preventive and therapeutic vaccines Edited by Mansun Law
http://dx.doi.org/10.1016/j.coviro.2015.04.003 1879-6257/# 2015 Elsevier B.V. All rights reserved.
Introduction Hepatitis E virus (HEV) causes both the epidemic and sporadic forms of acute hepatitis [1]. HEV infection is prevalent in approximately one-third of the world’s population and causes a wide spectrum of clinical manifestations, from self-limited acute hepatitis to lethal liver failure [2,3]. Mortality rates of HEV infection are about 0.5% but can be as high as 25% in pregnant women [4]. The lack of a robust cell culture method to propagate HEV makes it impracticable to develop inactive or attenuating vaccines [5]. Instead, the B-cell epitopes of HEV have been mapped biochemically using recombinant capsid proteins or overlapping peptides. Using these analyses, a recombinant hepatitis E vaccine — the first human vaccine — was designed and created using an E. coli expression system [6,7]. The odyssey of the Current Opinion in Virology 2015, 11:130–136
development of this vaccine [8] provides a paradigm for B-cell epitope-based vaccine design and, plausibly, offers future strategies for the development of other non-enveloped virus vaccines.
Hepatitis E virus biology Hepatitis E virus (HEV) resembles the calicivirus in virion morphology, and similarly comprises the S, P1 and P2 domains in the T = 3 icosahedral capsid structure [9,10]. Because of its unique gene organization, however, it was classified as its own Hepevirus genus in the Hepeviridae family [11]. HEV infects a wide range of mammalian species, including humans, and has, over time, evolved into two major groups comprising four genotypes. Genotypes 1 and 2 infect humans and non-human primates only, and often lead to epidemic outbreaks through the fecal–oral route. Genotypes 3 and 4 can infect a wide range of animals including humans in a sporadic manner [12]. Serologically, the naturally acquired anti-HEV is indistinguishable among the four genotypes and ascribes to a single serotype [13]. HEV is a small, non-enveloped RNA virus, the genome for which is a positive-sense, single-stranded, 7.2-kb RNA. The HEV genome contains three open reading frames (ORFs): ORF1 encodes non-structural proteins; ORF2 encodes the viral structural protein of 660 amino acids (aa), which assembles into a T = 3 icosahedral capsid of 35 nm in diameter; and ORF3 encodes a small protein associated with virion morphogenesis and release [14]. Considering all of the structures of the HEV capsid protein, including the E2s domain [15] and VLPs with T = 1 [16,17] and T = 3 [10], the capsid protein is thus considered to consist of three domains: an S domain (aa 112–313), a P1 or M domain (aa 314–454) and a P2 or P domain (aa 455–606) [16,17]. The P2 domain, also referred to as the E2s domain, forms an intimate homodimer in solution [15] that protrudes from the basal shell of the virus capsid to form a spike (Figure 1). This spike is responsible for host interactions, and harbors the major neutralization B-cell epitopes that present immunogenicity. The crystal structures of the E2s in complex with the Fab fragments of the genotype-preferred 8C11 [18] and cross-genotype 8G12 [19] neutralizing monoclonal antibodies (mAbs) have highlighted the immunodominant epitope site on the capsid spike. Yet, how to maintain these conformation-dependent B-cell epitopes and their immunogenicity during vaccination remain critical concerns in hepatitis E vaccine design.
B-cell epitopes of hepatitis E virus Despite belonging to a single serotype, HEV associated with human diseases can be classified into four main www.sciencedirect.com
Lessons from hepatitis E vaccine design Li, Zhang and Xia 131
Figure 1
(a)
(b)
mAb1323 4 mAbs 8H3/Fab224 8C11 8G12 mAb272 12A10
90°
90° (c)
(d)
mAb 8C11 8G12 8H3 mAb1323 mAb272 4 mAbs Fab224 12A10
B-cell epitope sites E479,Y485,D496,R512 K534,H577,R578 E549,K554,G591 E479, Y485,I529,K534 S487,S488,T489,P491, N562,T564 D496,T497,G591,P592 T489,P491,Y561,N562 T585,T586 E479,Y485,I529,K534 D430,L433 Current Opinion in Virology
The key amino acid residues involved in the interaction of functional antibodies and the HEV capsid protein. The dimeric pORF2 in the crystal structure of hepatitis E virus (HEV) virus-like particles (VLP) for genotype 3 (PDB no. 2ZTN) was rendered in surface mode and is depicted in front view (a), side view (b) and top view (c). The residues are colored according to the monoclonal antibody (mAb) binding sites, although some overlap. All of the epitope residues were listed in panel (d). The figures related to the structure were prepared using PyMol.
genotypes: genotypes 1 and 2 infect humans, whereas genotypes 3 and 4 are zoonotic [20]. Previous evidence suggests that these four genotypes share a common epitope(s), which elicits major, neutralizing antibodies that manifest as a single serotype in the humans [19,21]. Yet, the capsid proteins of zoonotic and non-zoonotic HEVs may contain distinct host receptor binding sites, and this may, in turn, lead to the production of genotypespecific immune responses [18]. The epitopes recognized by the cross-genotype and genotype-specific antibodies can be grouped into conformational and linear epitopes according to their structural features [18,19]. The B-cell epitope of the HEV structural protein, pORF2, was identified retrospectively using recombinant www.sciencedirect.com
proteins, overlapping peptides and their corresponding immune sera or mAbs; some of these mAbs were characterized by virus neutralization using HEV-infection cell model and/or through the use of a virus-challenging animal model [22,23]. The immune epitopes of HEV and the B-cell and T-cell types are summarized in Table 1. Moreover, to provide a clearer understanding of their spatial location on the HEV capsid protein, the key amino acid residues involved in the interaction of pORF2 or its partial segments with mAbs are structurally rendered in Figure 1. The crystal structure of the immune complex (PDB no. 3RKD) highlights several of the amino acids (chiefly, Current Opinion in Virology 2015, 11:130–136
132 Preventive and therapeutic vaccines
Table 1 The immune epitopes of hepatitis E virus (HEV) identified by monoclonal antibodies (mAb), truncated capsid protein and overlapping peptides Immune epitope or region E479,Y485,D496,R512,K534,H577,R578 E549,K554,G591 E479,Y485,I529,K534 S487,S488,T489,P491,N562,T564 D496,T497,G591,P592 T489,P491,Y561,N562,T564,T585,T586 E479,Y485,I529,K534 D430,L433 Linear epitope 578–607 aa 394–457 Within aa 394–604 aa 477–613 Within aa 452–617 aa 394–470; aa 546–580; aa 490–579 aa 477–492; aa 389–410; aa 583–600 aa 73–156; aa 289–372; aa 361–444; aa 505–588 P34 (aa 532–546); P35 (aa 537–551)
Method used to identify
Reference
8C11
[18]
8G12
[19]
8H3 mAb1323 mAb272 4 mAbs Fab224 12A10 HEV#4 and HEV#31 mAb 1C7, 3B2, 1E6, 1E7, 4B5 mAb 13D8, 16D7 mAb 1G10 N/A N/A N/A N/A N/A
[15] [17] [17] [17] [24] [19,25] [22,26] [27] [23,28] [29] [30] [31] [32] [33] [34]
Overlapping peptide, T-cell epitope
N/A
[35]
R512) involved in the virus–host interaction, and reveals the specificity of mAb 8C11 for genotypes 1 and 2. This interaction is particularly dependent on residue 497, a serine residue (S497) for genotypes 1/2, and a threonine (T497) for genotypes 3 and 4 [18]. Others have also shown that L477 and L613 are critical in the neutralization epitope of genotype 4 [29]. Another complex crystal structure of the 8G12 cross-genotype neutralizing antibody with the HEV E2s domain of genotypes 1 and 4 (PDB no. 4PLK and 4PLJ) pinpoint several other conserved residues (E549, K554 and G591) that are essential for common HEV neutralization. Moreover, mAb 8G12 is the predominant antibody identified in naturally acquired anti-HEV antibodies in human and monkey sera [19]. For vaccine design, these major neutralization epitopes on the capsid protein should be taken into consideration and exploited in the intended immunogens. Several of the designed constructs for pORF2 (Figure 2) were used to identify the B-cell epitopes on the HEV capsid; this circumvented the lack of an efficient cell culture system for HEV. The recombinant subunit ORF2.1 antigen (aa 394–660) induced antibodies against the immunodominant, conformational epitopes [36]. Indeed, Zhang et al. (2001) described a dimeric antigen E2 that harbored a dimer-dependent conformational epitope [28] and conferred immune protection against HEV in monkeys [37]. Li et al. (2005) further narrowed down this dimer-dependent epitope to the E2s domain (aa 454–606) [7] and solved the high-resolution structure of E2s using crystallization; this knowledge revealed that the dimerization of the E2s domain was essential for the virus–host interaction [15]. The antigens with VLP form, p495 (aa Current Opinion in Virology 2015, 11:130–136
Corresponding mAb
Crystal structure combined with mutation assay Crystal structure combined with mutation assay Mutation assay Density fractionation assay Density fractionation assay Binding to host cell model assay Mutation assay Mutation assay Truncation binding assay Truncated proteins Homodimer protein Truncated proteins Overlapping proteins, peptides Overlapping peptides Overlapping peptides Overlapping synthetic peptides T-cell epitope mapping
112–606) and p239 (aa 368–606), also contain some known functional epitopes (Figures 1 and 2) [6,7,38–40].
A structural analysis of the hepatitis E vaccine Recombinant VLPs usually mimic native virions in structure but without the viral genome. They are well-recognized by the immune system and are highly immunogenic because of their size and the repetitive pattern of B cell epitopes displayed on the particle surface [41]. Thus, VLPs often manifest sufficient antigenicity and immunogenicity for vaccine development. In the human vaccine field, two recombinant human vaccines against hepatitis B (HB) and human papillomavirus (HPV) have shown high efficacy in their VLP applicability [42]. During the immunogenic design for the HEV vaccine, we comprehensively interrogated the amino acids from the N-termini to C-termini to identify domains involved in dimer and particulate formation using the E. coli expression system. The minimal dimerization domain was located at aa 459–601, with the particulate domain further extending to aa 368 at the N-terminus [7]. The crystal structure and cryo-EM structure of T = 1 [17] (Figure 2a) and T = 3 [10] (Figure 2b) VLPs indicated that the E2s domain is equivalent to the P2 domain, serving as a major B-cell epitope reservoir, and its copies are arranged by the P1 (in the case of both p239 and p495) and the S (p495 only) domains to form particles and subsequently present high immunogenicity [7,15,16,17]. Viewed inward along the threefold axis of the T = 1 VLP (Figure 2a), three E2 dimers form a hexamer around the threefold axis by interaction from the partial P1 domain (aa 394–453) and the N-terminal 26-aa extension www.sciencedirect.com
Lessons from hepatitis E vaccine design Li, Zhang and Xia 133
Figure 2
(a)
(b)
(c) 660
ORF2.1
C
p495 p239
E2
E2s
606
P2
5 454
2
3 394 368
3 3 3
454
P1
394 394 368
314
2 5
S
112 112
1
HEV T=1 VLP
HEV T=3 VLP
N
HEV ORF2 Current Opinion in Virology
Structural interpretation of previously assessed truncated pORF2 constructs by the structure of hepatitis E virus (HEV) virus-like particles (VLP). (a) The molecular segmentation of domains or constructs on T = 1 HEV VLP (PDB no. 2ZTN) and (b) T = 3 HEV VLP (PDB no. 2ZTN fitted to 3IYO). The twofold, threefold and fivefold axes of the icosahedral lattice are denoted as solid black ovals, triangles and pentagons, respectively. (c) Schematic regions for the truncated pORF2 constructs. The immunogen p239 in the Hecolin vaccine is shown covering the P2 domain (in orange) and partially covering the P1 domain (in green, the rest portion in white) of the capsid. The color scheme is consistent with the structure rendering.
further to p239 (aa 368–606), which forms a shrunken version of the T = 1 VLP (Figure 2c) [6,7,42]. In addition to the N-terminal extension for particulate formation, the immunogenicity of p239 dramatically increases up to 200-times with respect to that of E2 [6,35].
Expression system and the manufacturing process HEV capsid proteins have been expressed in various expression systems, including E. coli, yeast, plant, insect and mammalian cells [28,43–46]. Two of these recombinant proteins have been the subject of clinical trials, the first of which is p495, which spans aa 112–606. Using baculovirus insect cells, the resultant proteins were found to present T = 1 VLPs in the cell lysate [38,47]. Previous strategies for purification have included PEG precipitation [48], chromatography [49] and ultra-centrifugation [39,47], and the purified VLPs were crystalized for X-ray crystallography [16,17] or vitrified for electron cryomicroscopy [10,38,50]; a scalable purification process that is suitable for manufacturing the p495 vaccine has not been reported to date. In addition, there is no evidence to show that p495 can self-assemble into VLPs in vitro. The second recombinant protein is p239 (aa 368–606), which is expressed in E. coli via a well-established manufacturing process that includes VLP assembly in vitro. In our investigations, we showed that the fermentation scale for manufacturing the p239 vaccine is up to 50 L [51], and demonstrated that the proteins formed inclusion bodies in bacteria and dissolved in 4 M urea after pan-washing with Triton-X 100. p239 exclusively www.sciencedirect.com
presented as dimer in 4 M urea, and, under these conditions, almost all of the contaminants were removed before the self-assembly process. In the presence of adequate salt (e.g., 0.5 M amide sulfate), p239 then assembled into particles. This assembly mechanism was clarified using time-lapse analytical ultracentrifugation and the assembly procedure can be modulated by adjusting the salt concentration and incubation temperature [52]. After buffer exchange, the assembled particles were further fractionated by ion-exchange chromatography instead of traditional size-exclusion. All of the sub-processes are scalable using chromatography and tangential flow techniques and the manufacturing process can be comprehensively evaluated by a combination of biophysical, biochemical and immunochemical means. Thus, this technique provides a robust, scalable manufacturing process and, consequently, product lot consistency, for the p239 vaccine. In our evaluations of vaccine quality, the particulate nature and the structural integrity of the major B-cell epitopes by mAb-based assay are emphasized and, thus, the use of E. coli to manufacture the p239 vaccine seems accessible and affordable for its wider applicability [51].
Clinical trials of hepatitis E vaccine The success of any vaccine design is determined by its efficacy in clinical trials. To date, both of the recombinant vaccines described above (p495 and p239 vaccines) have undergone clinical trials for this purpose. The p495 vaccine has been proven to be safe and immunogenic in young male adults, and conferred 95% protection against hepatitis E in Nepal, where only genotype Current Opinion in Virology 2015, 11:130–136
134 Preventive and therapeutic vaccines
1 HEV has been isolated [53]. The p239 vaccine was verified to be safe, immunogenic and efficacious against subclinical HEV infection in sero-negative subjects in a Phase 2 clinical trial in China [54], and was later tested in a randomized, double-blind, placebo-controlled Phase 3 clinical trial. This Phase 3 trial comprised 112 604 participants who were randomized 1:1 into vaccine or control groups, and were administered with three doses of p239 vaccine or a control vaccine (hepatitis B vaccine) at 0, 1 and 6 months. In the per-protocol analysis, 15 subjects in the control group developed hepatitis E versus none in the p239 vaccine group over the 12 months after the third vaccination. Vaccine efficacy in that year after the three doses was determined to be 100.0% (95%CI, 72.1–100.0) for those who received the three doses in the requested time frame (per-protocol analysis) [55]. The long-term follow-up of the participants in the Phase 3 trial has indicated that this antibody efficacy persists for at least 4.5 years with an efficacy of 93.3% (95%CI, 78.6–97.9) in the per-protocol cohort and 86.8% (95%CI, 71.0–94.0) in those who received at least one dose of the vaccine [56]. The HEV isolates from the identified hepatitis E patients were predominantly genotype 4, with a few genotype 1, indicating that the p239 vaccine of genotype 1 provides cross-protection against HEV genotype 4. Because of the limited breakthrough cases in p239 vaccines observed in the clinical trial, the protective antibody level against hepatitis E has not been determined. In preclinical efficacy studies in rhesus monkeys, the p239 vaccine induced an anti-HEV IgG titer of 1384 Wu/mL (WHO Unit per mL), which can prevent symptomatic hepatitis E after challenging with 104 or 107 genome equivalences of HEV. However, this antibody level can only partially protect (75%) the monkeys from HEV infection, even when challenging with the 107 dose. In the human study, the data showed that marginal levels of anti-HEV IgG (0.077–0.25 Wu/mL) could decrease the risk of asymptomatic HEV infection in immunocompetent persons (Relative risk = 0.26) [57]. The pre-infection antibody levels of the subjects with post-vaccination, asymptomatic infection varied widely. However, although the dose-effect is not significant, subjects with an anti-HEV level >1.0 Wu/mL are significantly better protected than those with marginal antibody levels. In the immunogenicity analysis, p239 induced a strong anti-HEV response in the vaccine recipients, with a seroconverted ratio of 99.9% and a geometric mean antibody concentration of 14.96 Wu/mL at one month after the third vaccination [55]. Collectively, these results demonstrate that the hepatitis E vaccine in its VLP form is highly efficacious in protecting against hepatitis E, and that the B-cell epitopes are sufficiently immunogenic to elicit protective antibodies with some capacity for crossgenotype neutralization. It will be important to continue to monitor the potential immune pressure of the vaccine Current Opinion in Virology 2015, 11:130–136
in these protected subjects to ascertain if there is any capacity for epitope-escape or mutation of HEV.
Conclusions The hepatitis E vaccine, Hecolin, is the first human vaccine using the E. coli-expressed VLP antigen. The B-cell epitope profile of the capsid protein along with mAb-based binding assays will aid in ensuring quality control of the p239 vaccine during manufacturing. In protected subjects and during future vaccine applications worldwide, it will be important to continue to screen for potential epitope-escape HEVs. These lessons learned from the HEV vaccine design could be extrapolated for the investigations of other non-enveloped, capsid viruses.
Acknowledgements We acknowledge funding support from the Chinese Government: Fujian Provincial Science Fund for Distinguished Young Scholars (Grant no. 2011J06015), Fujian Provincial Fund for Key Platform of Scientific Innovation (Grant no. 2014Y2101), Major National Project ‘‘863’’ (Grant no. 2014AA021302), Xiamen Science and Technology Project (Grant no. 3502Z201410045).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Kamar N, Bendall R, Legrand-Abravanel F, Xia NS, Ijaz S, Izopet J, Dalton HR: Hepatitis E. Lancet 2012, 379:2477-2488.
2.
Aggarwal R, Kini D, Sofat S, Naik SR, Krawczynski K: Duration of viraemia and faecal viral excretion in acute hepatitis E. Lancet 2000, 356:1081-1082.
3.
Hoofnagle JH, Nelson KE, Purcell RH: Hepatitis E. N Engl J Med 2012, 367:1237-1244.
4.
Kumar A, Beniwal M, Kar P, Sharma JB, Murthy NS: Hepatitis E in pregnancy. Int J Gynaecol Obstet 2004, 85:240-244.
5.
Okamoto H: Culture systems for hepatitis E virus. J Gastroenterol 2013, 48:147-158.
6.
Li SW, Zhang J, Li YM, Ou SH, Huang GY, He ZQ, Ge SX, Xian YL, Pang SQ, Ng MH et al.: A bacterially expressed particulate hepatitis E vaccine: antigenicity, immunogenicity and protectivity on primates. Vaccine 2005, 23:2893-2901. The E. coli-derived p239 vaccine was characterized by particulation, antigenicity and immunogenicity assay. p239 vaccine confers protection against HEV infection and hepatitis E disease in monkeys.
7.
Li SW, Zhang J, He ZQ, Gu Y, Liu RS, Lin J, Chen YX, Ng MH, Xia NS: Mutational analysis of essential interactions involved in the assembly of hepatitis E virus capsid. J Biol Chem 2005, 280:3400-3406. The elaborated mutational analysis identifies key regions for dimerization, particle assembly and neutralization of B-cell epitope formation of the HEV capsid protein, which was the basis for the successful HEV vaccine design. 8.
Wu T, Li SW, Zhang J, Ng MH, Xia NS, Zhao Q: Hepatitis E vaccine development: a 14 year odyssey. Hum Vaccin Immunother 2012, 8:823-827.
9.
Prasad BV, Hardy ME, Dokland T, Bella J, Rossmann MG, Estes MK: X-ray crystallographic structure of the Norwalk virus capsid. Science 1999, 286:287-290.
10. Xing L, Li TC, Mayazaki N, Simon MN, Wall JS, Moore M, Wang CY, Takeda N, Wakita T, Miyamura T et al.: Structure of hepatitis E virion-sized particle reveals an RNA-dependent viral assembly pathway. J Biol Chem 2010, 285:33175-33183. www.sciencedirect.com
Lessons from hepatitis E vaccine design Li, Zhang and Xia 135
11. Smith DB, Purdy MA, Simmonds P: Genetic variability and the classification of hepatitis E virus. J Virol 2013, 87:4161-4169. 12. Krain LJ, Nelson KE, Labrique AB: Host immune status and response to hepatitis E virus infection. Clin Microbiol Rev 2014, 27:139-165. 13. Worm HC, Wirnsberger G: Hepatitis E vaccines: progress and prospects. Drugs 2004, 64:1517-1531. 14. Yamada K, Takahashi M, Hoshino Y, Takahashi H, Ichiyama K, Nagashima S, Tanaka T, Okamoto H: ORF3 protein of hepatitis E virus is essential for virion release from infected cells. J Gen Virol 2009, 90:1880-1891. 15. Li S, Tang X, Seetharaman J, Yang C, Gu Y, Zhang J, Du H, Shih JW, Hew CL, Sivaraman J et al.: Dimerization of hepatitis E virus capsid protein E2s domain is essential for virus-host interaction. PLoS Pathog 2009, 5:e1000537. 16. Guu TS, Liu Z, Ye Q, Mata DA, Li K, Yin C, Zhang J, Tao YJ: Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding. Proc Natl Acad Sci U S A 2009, 106:12992-12997. The crystal structure of the HEV VLP of genotype 4 reveals three key domains within the HEV capsid, of which the protruding P2 domain serves as a receptor binding site and potentially harbors major neutralization sites. 17. Yamashita T, Mori Y, Miyazaki N, Cheng RH, Yoshimura M, Unno H, Shima R, Moriishi K, Tsukihara T, Li TC et al.: Biological and immunological characteristics of hepatitis E virus-like particles based on the crystal structure. Proc Natl Acad Sci U S A 2009, 106:12986-12991. The crystal structure of HEV VLP of genotype 3 reveals three key domains within the HEV capsid, identifying several B-cell epitopes of HEV by mutational analysis. 18. Tang X, Yang C, Gu Y, Song C, Zhang X, Wang Y, Zhang J, Hew CL, Li S, Xia N et al.: Structural basis for the neutralization and genotype specificity of hepatitis E virus. Proc Natl Acad Sci U S A 2011, 108:10266-10271. The first crystal structure of the HEV immune complex shows the footprint of a neutralizing mAb 8C11 at the atomic level and identifies key points of virus-host interactions using the HEV cell model. 19. Gu Y, Tang X, Zhang X, Song C, Zheng MH, Wang KH, Zhang J, Ng MH, Hew CL, Li SW et al.: Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody. Cell Res 2015, 25 http://dx.doi.org/10.1038/cr.2015.34. The crystal structures of HEV E2s domains of genotypes 1 and 4 in complex with cross-genotype neutralizing mAb 8G12 reveals several conserved amino acids for common HEV neutralization. This supports the phenomenon of a single serotype for the four main HEV genotypes in human sera and the ability for cross-protection with one genotype vaccine. 20. Zhang J, Li SW, Wu T, Zhao Q, Ng MH, Xia NS: Hepatitis E virus: neutralizing sites, diagnosis, and protective immunity. Rev Med Virol 2012, 22:339-349. 21. Emerson SU, Clemente-Casares P, Moiduddin N, Arankalle VA, Torian U, Purcell RH: Putative neutralization epitopes and broad cross-genotype neutralization of hepatitis E virus confirmed by a quantitative cell-culture assay. J Gen Virol 2006, 87:697-704. 22. Schofield DJ, Glamann J, Emerson SU, Purcell RH: Identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis E virus capsid protein. J Virol 2000, 74:5548-5555. 23. Zhang J, Gu Y, Ge SX, Li SW, He ZQ, Huang GY, Zhuang H, Ng MH, Xia NS: Analysis of hepatitis E virus neutralization sites using monoclonal antibodies directed against a virus capsid protein. Vaccine 2005, 23:2881-2892. 24. Xing L, Wang JC, Li TC, Yasutomi Y, Lara J, Khudyakov Y, Schofield D, Emerson SU, Purcell RH, Takeda N et al.: Spatial configuration of hepatitis E virus antigenic domain. J Virol 2011, 85:1117-1124. 25. He S, Miao J, Zheng Z, Wu T, Xie M, Tang M, Zhang J, Ng MH, Xia N: Putative receptor-binding sites of hepatitis E virus. J Gen Virol 2008, 89:245-249. www.sciencedirect.com
26. Schofield DJ, Purcell RH, Nguyen HT, Emerson SU: Monoclonal antibodies that neutralize HEV recognize an antigenic site at the carboxyterminus of an ORF2 protein vaccine. Vaccine 2003, 22:257-267. 27. Riddell MA, Li F, Anderson DA: Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies. J Virol 2000, 74:8011-8017. 28. Zhang JZ, Ng MH, Xia NS, Lau SH, Che XY, Chau TN, Lai ST, Im SW: Conformational antigenic determinants generated by interactions between a bacterially expressed recombinant peptide of the hepatitis E virus structural protein. J Med Virol 2001, 64:125-132. 29. Zhang H, Dai X, Shan X, Meng J: The Leu477 and Leu613 of ORF2-encoded protein are critical in forming neutralization antigenic epitope of hepatitis E virus genotype 4. Cell Mol Immunol 2008, 5:447-456. 30. Meng J, Dai X, Chang JC, Lopareva E, Pillot J, Fields HA, Khudyakov YE: Identification and characterization of the neutralization epitope(s) of the hepatitis E virus. Virology 2001, 288:203-211. 31. Khudyakov Yu E, Favorov MO, Jue DL, Hine TK, Fields HA: Immunodominant antigenic regions in a structural protein of the hepatitis E virus. Virology 1994, 198:390-393. 32. Khudyakov YE, Lopareva EN, Jue DL, Crews TK, Thyagarajan SP, Fields HA: Antigenic domains of the open reading frame 2encoded protein of hepatitis E virus. J Clin Microbiol 1999, 37:2863-2871. 33. Guo H, Zhou EM, Sun ZF, Meng XJ, Halbur PG: Identification of B-cell epitopes in the capsid protein of avian hepatitis E virus (avian HEV) that are common to human and swine HEVs or unique to avian HEV. J Gen Virol 2006, 87:217-223. 34. Aggarwal R, Shukla R, Jameel S, Agrawal S, Puri P, Gupta VK, Patil AP, Naik S: T-cell epitope mapping of ORF2 and ORF3 proteins of human hepatitis E virus. J Viral Hepat 2007, 14:283-292. 35. Wu T, Wu XL, Ou SH, Lin CX, Cheng T, Li SW, Ng MH, Zhang J, Xia NS: Difference of T cell and B cell activation in two homologous proteins with similar antigenicity but great distinct immunogenicity. Mol Immunol 2007, 44:3261-3266. 36. Li F, Riddell MA, Seow HF, Takeda N, Miyamura T, Anderson DA: Recombinant subunit ORF2.1 antigen and induction of antibody against immunodominant epitopes in the hepatitis E virus capsid protein. J Med Virol 2000, 60:379-386. 37. Im SW, Zhang JZ, Zhuang H, Che XY, Zhu WF, Xu GM, Li K, Xia NS, Ng MH: A bacterially expressed peptide prevents experimental infection of primates by the hepatitis E virus. Vaccine 2001, 19:3726-3732. 38. Xing L, Kato K, Li T, Takeda N, Miyamura T, Hammar L, Cheng RH: Recombinant hepatitis E capsid protein self-assembles into a dual-domain T = 1 particle presenting native virus epitopes. Virology 1999, 265:35-45. 39. Li TC, Yoshimatsu K, Yasuda SP, Arikawa J, Koma T, Kataoka M, Ami Y, Suzaki Y, Mai le TQ, Hoa NT et al.: Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses. J Gen Virol 2011, 92:2830-2837. 40. Wei M, Zhang X, Yu H, Tang ZM, Wang K, Li Z, Zheng Z, Li S, Zhang J, Xia N et al.: Bacteria expressed hepatitis E virus capsid proteins maintain virion-like epitopes. Vaccine 2014, 32:28592865. 41. Grgacic EV, Anderson DA: Virus-like particles: passport to immune recognition. Methods 2006, 40:60-65. The review describes the importance of virus-like particles as an ideal immunogen form for immune recognition. 42. Zhao Q, Li S, Yu H, Xia N, Modis Y: Virus-like particle-based human vaccines: quality assessment based on structural and functional properties. Trends Biotechnol 2013, 31:654-663. Current Opinion in Virology 2015, 11:130–136
136 Preventive and therapeutic vaccines
43. Tyagi S, Korkaya H, Zafrullah M, Jameel S, Lal SK: The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2. J Biol Chem 2002, 277: 22759-22767. 44. Ma Y, Lin SQ, Gao Y, Li M, Luo WX, Zhang J, Xia NS: Expression of ORF2 partial gene of hepatitis E virus in tomatoes and immunoactivity of expression products. World J Gastroenterol 2003, 9:2211-2215. 45. He J, Tam AW, Yarbough PO, Reyes GR, Carl M: Expression and diagnostic utility of hepatitis E virus putative structural proteins expressed in insect cells. J Clin Microbiol 1993, 31:2167-2173. 46. Jameel S, Zafrullah M, Ozdener MH, Panda SK: Expression in animal cells and characterization of the hepatitis E virus structural proteins. J Virol 1996, 70:207-216. 47. Li TC, Takeda N, Miyamura T, Matsuura Y, Wang JC, Engvall H, Hammar L, Xing L, Cheng RH: Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus. J Virol 2005, 79:12999-13006. 48. Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T: Expression and self-assembly of empty virus-like particles of hepatitis E virus. J Virol 1997, 71:7207-7213. 49. Robinson RA, Burgess WH, Emerson SU, Leibowitz RS, Sosnovtseva SA, Tsarev S, Purcell RH: Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells. Protein Expr Purif 1998, 12:75-84. 50. Mori Y, Matsuura Y: Structure of hepatitis E viral particle. Virus Res 2011, 161:59-64.
Current Opinion in Virology 2015, 11:130–136
51. Zhang X, Wei M, Pan H, Lin Z, Wang K, Weng Z, Zhu Y, Xin L, Zhang J, Li S et al.: Robust manufacturing and comprehensive characterization of recombinant hepatitis E virus-like particles in Hecolin((R)). Vaccine 2014, 32:4039-4050. 52. Yang C, Pan H, Wei M, Zhang X, Wang N, Gu Y, Du H, Zhang J, Li S, Xia N: Hepatitis E virus capsid protein assembles in 4 M urea in the presence of salts. Protein Sci 2013, 22:314-326. 53. Shrestha MP, Scott RM, Joshi DM, Mammen MP Jr, Thapa GB, Thapa N, Myint KS, Fourneau M, Kuschner RA, Shrestha SK et al.: Safety and efficacy of a recombinant hepatitis E vaccine. N Engl J Med 2007, 356:895-903. 54. Zhang J, Liu CB, Li RC, Li YM, Zheng YJ, Li YP, Luo D, Pan BB, Nong Y, Ge SX et al.: Randomized-controlled phase II clinical trial of a bacterially expressed recombinant hepatitis E vaccine. Vaccine 2009, 27:1869-1874. 55. Zhu FC, Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, Wang H, Yang CL, Jiang HM, Cai JP et al.: Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults: a largescale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet 2010, 376:895-902. Convincing evidence from this clinical trial confirms the efficacy of the HEV p239 vaccine. The clinical trial is unprecedented in its large scale of over 110 000 subjects. 56. Zhang J, Zhang XF, Huang SJ, Wu T, Hu YM, Wang ZZ, Wang H, Jiang HM, Wang YJ, Yan Q et al.: Long-term efficacy of a hepatitis E vaccine. N Engl J Med 2015, 372:914-922. Immunization with HEV p239 vaccine induced antibodies against HEV and provided protection against hepatitis E for up to 4.5 years. 57. Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, Yao X, Liang ZL, Wu T, Li JX, Yan Q et al.: Protection against hepatitis E virus infection by naturally acquired and vaccine-induced immunity. Clin Microbiol Infect 2014, 20:O397-O405.
www.sciencedirect.com