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Recombinant vaccine against hepatitis E: Duration of protective immunity in rhesus macaques
Abstract
Hepatitis E virus (HEV) is an enterically transmitted virus that causes acute hepatitis. A candidate vaccine containing recombinant HEV capsid protein (56kDa) expressed in insect cells was shown previously to be highly immunogenic when administered in two doses to rhesus monkeys and to protect them from hepatitis E when challenged with a large intravenous dose of homologous or heterologous HEV. In the present study, the effect of a third dose of the same vaccine lot was evaluated and more importantly, the duration of protection following two doses was determined. Rhesus monkeys vaccinated twice with the 56kDa capsid protein were challenged with homologous virus 6 or 12 months after the last vaccination: 3 of 4 monkeys challenged 6 or 12 months later, respectively were protected against viral hepatitis. Similarly, all four of the rhesus monkeys given a third dose of vaccine 1 month prior to challenge were protected against hepatitis. In contrast, all four monkeys given placebo developed hepatitis following challenge. In summary, two doses of HEV vaccine partially protected rhesus monkeys from hepatitis E following intravenous challenge 6 or 12 months after vaccination.
... The efficacy of the generated vaccines was assessed by immunization in different animal models. Some surveys only tested the generation of anti-HEV titers without challenging with wild-type strains [49,50,[52][53][54][55][56][57][58][59][60][61][62], whereas some preclinical studies also analyzed the protective effects of the vaccine after rechallenge in primates (Tables 1 & 2) [41,[44][45][46][47][48][63][64][65][66]. Among the different developed HEV vaccines, one recombinant vaccine, which was derived from the Sf9 insect cell expression system using baculovirus overexpression of ORF2, showed a superior protection efficiency compared with other vaccines [65]. ...
... Among the different developed HEV vaccines, one recombinant vaccine, which was derived from the Sf9 insect cell expression system using baculovirus overexpression of ORF2, showed a superior protection efficiency compared with other vaccines [65]. This 56-kDa HEV vaccine was designed based on the HEV ORF2 of the SAR-55 Pakistani isolate (amino acid 112-607), and showed proper immunization outcome in a preclinical trial in Rhesus monkeys after challenging with different wild-type strains [64][65][66]. After two doses of immunization, Rhesus monkeys were protected from HEV infection by Pakistani (homologous) and Mexican (heterologous) isolates [65]. ...
... After two doses of immunization, Rhesus monkeys were protected from HEV infection by Pakistani (homologous) and Mexican (heterologous) isolates [65]. The administration of three doses of vaccination resulted in a longer period of protection compared with two doses of the vaccine [66]. In the last part of the preclinical development, immunized monkeys were challenged by three different viruses (strains Sar-55, Mex-14 and USA-2), and anti-HEV titers were then quantified against a WHO standard. ...
HEV, a positive ssRNA and nonenveloped virus, is endemic In many developing countries and one of the most frequent causes of acute hepatitis after fecal-oral transmission. Pregnant women are at particular risk for a fatal course of disease, including maternal and fetal mortality. Rocent reports Indicate that HEV genotype 3, possibly related to zoonotic transmission, may cause chronic hepatitis In some immunosuppressed organ transplant patients. Various approaches have been conducted to develop HEV vaccines, but only one candidate, a recombinant HEV (rHEV) vaccine generated from Spodoptera frugiperda-9 cells by baculoviruses expressing the HEV capsid antigen, has reached clinical Phase I and II trials so for. These trials suggest that the rHEV vaccine Is safe and can prevent clinically overt acute hepatitis E In high-risk populations. We herein review the different approaches in HEV-vaccine development and critically discuss the current status and future directions of the rHEV vaccine used in clinical trials.
... The truncated ORF2 proteins derived from swine, avian and rat HEV were each expressed as insoluble inclusion bodies with an approximate predicted size of 58 kDa [41]. Following nickel affinity chromatography purification, the his-tag fused to each protein was properly identified on each of the recombinant fusion proteins using Western blot with IRDy800 (LI-COR Biosciences) conjugated antibodies to poly-histidine tags (Fig. 1A). ...
... Vaccine trials of HEV ORF2 antigens have been performed in chickens [30], non-human primates [36,41], and humans [27,28]. A recombinant truncated capsid protein expressed in baculovirus induced complete protection in rhesus monkeys against both hepatitis and infection after challenge with genotype 1 or 3 HEV. ...
... In conclusion, vaccination of pigs with recombinant capsid antigens derived from three animal strains of HEV induced strong IgG anti-HEV antibody response in vaccinated pigs, which is consistent with other studies that showed a robust seroconversion to IgG anti-HEV after vaccination with HEV ORF2 antigens [29,30,41]. However, the recombinant antigens from the 3 animal strains of HEV appear to confer only partial cross-protection against a genotype 3 mammalian HEV. ...
Hepatitis E virus (HEV), the causative agent of hepatitis E, is primarily transmitted via the fecal-oral route through contaminated water supplies, although many sporadic cases of hepatitis E are transmitted zoonotically via direct contact with infected animals or consumption of contaminated animal meats. Genotypes 3 and 4 HEV are zoonotic and infect humans and other animal species, whereas genotypes 1 and 2 HEV are restricted to humans. There exists a single serotype of HEV, although the cross-protective ability among the animal HEV strains is unknown. Thus, in this study we expressed and characterized N-terminal truncated ORF2 capsid antigens derived from swine, rat, and avian HEV strains and evaluated their cross-protective ability in a pig challenge model. Thirty, specific-pathogen-free, pigs were divided into 5 groups of 6 pigs each, and each group of pigs were vaccinated with 200μg of swine HEV, rat HEV, or avian HEV ORF2 antigen or PBS buffer (2 groups) as positive and negative control groups. After a booster dose immunization at 2 weeks post-vaccination, the vaccinated animals all seroconverted to IgG anti-HEV. At 4 weeks post-vaccination, the animals were intravenously challenged with a genotype 3 mammalian HEV, and necropsied at 4 weeks post-challenge. Viremia, fecal virus shedding, and liver histological lesions were compared to assess the protective and cross-protective abilities of these antigens against HEV challenge in pigs. The results indicated that pigs vaccinated with truncated recombinant capsid antigens derived from three animal strains of HEV induced a strong IgG anti-HEV response in vaccinated pigs, but these antigens confer only partial cross-protection against a genotype 3 mammalian HEV. The results have important implications for the efficacy of current vaccines and for future vaccine development, especially against the novel zoonotic animal strains of HEV.
... There have been many attempts to evaluate the efficacy of a vaccine by expressing HEV VLPs using the Escherichia coli expression system and the baculovirus insect cell expression system [12][13][14][15][16]. Using the E. coli expression system, a large amount of protein can be obtained quickly at a low production cost. ...
... In addition, the HEV 239 VLP has been confirmed to be effective in humans through several clinical trials and was released as a preventive measure in China in 2012 [13,16,19]. The immunogenicity and protective efficacy of the HEV VLP vaccine produced using the baculovirus insect cell expression system, based on 496 amino acids of ORF2, were established in experiments using primates [14,20]. Clinical trials in humans have indicated that the baculovirus-expressed 56-kDa HEV vaccine was effective in preventing the occurrence of hepatitis E [15]. ...
Here, rabbits were immunized with a virus-like particle (VLP) vaccine prepared by expressing 239 amino acids of the swine hepatitis E virus (HEV)-3 capsid protein using a baculovirus system. Thirty specific-pathogen-free rabbits were divided into five groups (negative and positive control and 10, 50, and 100 μg VLP-vaccinated). Positive control group rabbits showed viremia and fecal viral shedding, whereas rabbits vaccinated with 10 μg VLP showed transient fecal viral shedding, and rabbits vaccinated with 50 and 100 μg VLP did not show viremia or fecal viral shedding. Serum anti-HEV antibody titers increased in a dose-dependent manner. Anti-HEV antibody titers were significantly higher (p < 0.05) in 100 μg VLP-vaccinated rabbits than in the negative control rabbits at week 4. Anti-HEV antibody titers were significantly higher in 50 and 10 μg VLP-vaccinated rabbits than in the negative control rabbits at weeks 8 and 11, respectively. Serum IFN-γ and IL-12 levels were significantly higher (p < 0.01) in rabbits vaccinated with 50 and 100 μg VLP than in the negative control rabbits at weeks 4 and 6. Liver tissues of 50 and 100 μg VLP-vaccinated rabbits displayed significantly less (p < 0.05) fibrosis than those of the positive control rabbits. The prepared VLP vaccine demonstrated dose-dependent immunogenicity sufficient for inducing anti-HEV antibody production, thus protecting rabbits against swine HEV-3.
... Formal proof of its activity against genotype 2 and 3 HEV is, however, still lacking. Another recombinant HEV vaccine, consisting of a 56 kDa protein derived from capsid antigen ofthe gt 1 Pakistani Sar55 strain was expressed in insect cells from a baculovirus vector [62,67,68]. Several consecutive studies using this vaccine in cynomolgus and rhesus macaques showed the immunogenicity and efficacy in preventing HEV infection upon challenge with genotypes 1-3 HEV [67,68]. ...
... Another recombinant HEV vaccine, consisting of a 56 kDa protein derived from capsid antigen ofthe gt 1 Pakistani Sar55 strain was expressed in insect cells from a baculovirus vector [62,67,68]. Several consecutive studies using this vaccine in cynomolgus and rhesus macaques showed the immunogenicity and efficacy in preventing HEV infection upon challenge with genotypes 1-3 HEV [67,68]. Subsequently, the vaccine was tested in human trials and showed 95% protection against genotype 1 HEV in military troops in Nepal [69]. ...
Hepatitis E virus (HEV) is an underdiagnosed pathogen with approximately 20 million infections each year and currently the most common cause of acute viral hepatitis. HEV was long considered to be confined to developing countries but there is increasing evidence that it is also a medical problem in the Western world. HEV that infects humans belongs to the Orthohepevirus A species of the Hepeviridae family. Novel HEV-like viruses have been observed in a variety of animals and some have been shown to be able to cross the species barrier, causing infection in humans. Several cell culture models for HEV have been established in the past years, but their efficiency is usually relatively low. With the circulation of this virus and related viruses in a variety of species, several different animal models have been developed. In this review, we give an overview of these animal models, indicate their main characteristics, and highlight how they may contribute to our understanding of the basic aspects of the viral life cycle and cross-species infection, the study of pathogenesis, and the evaluation of novel preventative and therapeutic strategies.
... Zhang a spol. publikovali v roce 2002 [11] výsledky testování vakcíny založené na rekombinantním proteinu z ORF2 exprimovaném v bakulovirovém systému. Takto připravený protein se samovolně uspořádal do VLP částic. ...
... Rekombinantní vakcíny jsou vysoce imunogenní -očkované opice byly odolné vůči HEV i po šesti měsících, v některých případech i po roce [11]. R. H. Purcell konstruoval vakcínu proti HEV obsahující zkrácenou formu rekombinantního kapsidového proteinu o velikosti 56 kDa [7]. ...
... Nevertheless, recently, an ex-vivo model was set up to study the replication of HEV genotypes 1 and 3 at the maternal-foetal interface using the decidua basalis and fetal placenta [343]. Rhesus macaques and cynomolgus monkeys show protective immunity against HEV therefore they can be used to identify vaccine candidates [307,315,344]. Since NHP are not a natural host for HEV, this presents a drawback in this model. ...
Hepatitis E virus (HEV) is the major cause of acute hepatitis worldwide. Currently,no effective anti-viral treatment against HEV is available and an HEV vaccine is restrictedto China. HEV is a positive-sense RNA virus expressing 3 open readingframes (ORFs). ORF1 encodes the ORF1 non-structural polyprotein, the viral replicasewhich transcribes the full—length genome and a subgenomic RNA that encodesthe structural ORF2 and ORF3 proteins. Our aim is to better characterize ORF1 :to determine whether it is processed during the HEV lifecycle and to identify thecompartment of replication. As no commercial antibody recognizes ORF1 in HEVreplicatingcells, we aimed at inserting epitope tags within the ORF1 sequence of thecell—culture selected genotype 3 p6 strain. First, a HEV replicon where the Gaussialuciferase reporter gene lies under the control of ORF1 was used to assess the replicationefficacy of the tagged genome by luminometry. The insertion of the 2 V5 tagsin the hypervariable région did not significantly impact the replication of genomic andproduction of subgenomic RNA, whereas the insertion of an HA tag at the C-terminusof the RNA-dependent RNA polymerase reduced the production of subgenomic RNA.Tags were next inserted both into the infectious full—length p6 HEV genome and intoORF1 heterologously expressed. Western-blot and immunoprecipitation analyses oftagged ORF1 expressed in the 3 systems showed a high molecular weight protein(>180kDa) that likely corresponds to the unprocessed form of ORF1 and that canbe detected up to 25 days after electroporation of the p6 infectious RNA in PLC3cells. Additionally, lower molecular weight proteins (90-180kDa) were detected inlower abundance, suggesting that ORF1 might be processed. Mass spectrometryanalyses were performed to identify the sequence of the potential cleavage productsobserved by western blot and colloidal blue staining. It appears that the N-terminusof ail V5-tagged proteins of lower molecular weight is stable and corresponds to theN-terminus of the full length ORF1 protein. However, the C-terminus was not yetidentified. Interestingly, western blot analysis of subcellular fractions as well as confocalmicroscopy revealed cytoplasmic and nuclear localization of the tagged ORF1,indicating that the ORF1 might pass through the nucleus during infection.Finally, we used the RNAscope technique to visualize the intracellular HEV RNA.Probes were designed against positive and negative-stranded HEV RNA. Genomicand subgenomic RNA were found in close proximity to each other and to viral proteinsin perinuclear substructures that may correspond to replication complexes.
... 184 Additionally, animal studies suggested anti-HEV ORF2 is protective. [185][186][187][188][189][190][191] ...
Viral hepatitis is caused by a heterogenous group of viral agents representing a wide range of phylogenetic groups. Many viruses can involve the liver and cause liver injury but only a subset are delineated as "hepatitis viruses" based upon their primary site of replication and tropism for hepatocytes which make up the bulk of the liver cell population. Since their discovery, beginning with the agent that caused serum hepatitis in the 1960s, the alphabetic designations have been utilized. To date, we have five hepatitis viruses, A through E, though it is postulated that others may exist. This chapter will focus on those viruses. Note that hepatitis D is included as a subset of hepatitis B, as it cannot exist without concurrent hepatitis B infection. Pregnancy has the potential to affect all aspects of these viral agents due to the unique immunologic and physiologic changes that occur during and after the gestational period. In this review, we will discuss the most common viral hepatitis and their effects during pregnancy.
... Ces modèles animaux sont également utilisés pour des études vaccinales contre le HEV en utilisant des formes tronquées de la protéine ORF2 (Tsarev et al. 1994;Zhang, M. et al. 2002). Malheureusement, les modèles d'étude utilisant les primates non-humains ne sont pas représentatifs de ce qui se passe chez la femme enceinte. ...
L’infection par le virus de l’hépatite E (HEV) est un problème majeur de santé publiquequi touche plus de 20 millions de personnes et tue environ 70 000 chaque année à travers lemonde. Le HEV est la cause majeure d’hépatite virale aiguë dans le monde. En France, laséroprévalence du HEV est de 22,4% dans la population générale. Ce virus se transmet parvoie féco-orale ou par consommation de viande contaminée mal cuite. Très récemment, nousavons décrit un système de culture cellulaire permettant d’amplifier efficacement le HEV. Cesystème représente un outil unique pour caractériser les différentes étapes du cycle infectieuxdu HEV qui sont très mal connues à ce jour. Dans le cadre de ma thèse, je me suis intéresséplus particulièrement à la protéine de capside ORF2 qui est l’unité structurale des particulesvirales et est donc un acteur central du cycle infectieux du HEV. La protéine ORF2 est uneprotéine de 660 acides aminés (aa) qui possède un peptide signal (PS) et trois sites potentielsde N-glycosylation (N1, N2 et N3). J’ai donc étudié le rôle de la N-glycosylation de cetteprotéine ORF2 dans le cycle infectieux du HEV. Pour atteindre cet objectif, la soucheinfectieuse HEV-p6 génotype 3 a été utilisée pour infecter les cellules de la lignéehépatocytaire PLC3, sous clone de la lignée PLC/PRF/5. Les résultats obtenus ont montré quela protéine ORF2 est N-glycosylée uniquement au niveau des sites N1 et N3 ; le site N2 estdéfavorable à la N-glycosylation. Nos résultats ont montré que la N-glycosylation de laprotéine ORF2 n’est pas importante pour sa stabilité, son oligomérisation, sa reconnaissancepar des anticorps, l’assemblage et l’infectiosité des particules virales. Nous avons aussimontré que la protéine ORF2 est importée dans le noyau des cellules hôtes au cours du cycleinfectieux du HEV de manière indépendante de la N-glycosylation. Nous avons identifié pourla première fois un signal de localisation nucléaire (NLS) fonctionnel en position N-terminalede la protéine ORF2 lui permettant d'interagir probablement avec l’importine alpha1. Demanière surprenante, ce NLS régule non seulement l’import nucléaire de cette protéine virale,mais aussi sa translocation réticulaire. Nous avons également démontré que la protéine ORF2est exportée du noyau des cellules hôtes en interagissant avec l’exportine1 et possède 3signaux d’export nucléaire (NES) en position C-terminale. Ces résultats améliorent laconnaissance du trafic intracellulaire de la protéine ORF2 et pourraient orienter les stratégiesantivirales dirigées contre le HEV.
... For recombinant vaccine development, efforts made till date focused mostly on ORF2 capsid protein. This includes mostly truncated forms of ORF2 protein such as trpE-C2 (aa 221-660) (Purdy et al. 1993), pE2 (aa 394-607) (Im et al. 2001), HEV 239 (aa 368-606) (Zhang et al. 2013), 53 kDa (aa 112-578) , 56 kDa (aa 112-607) (Zhang et al. 2002), 62 kDa (aa 112-660), rHEV VLP (aa 112-608) (Li et al. 2004) and T1-ORF2 (aa 126-621) (Huang et al. 2009) vaccines. Among these, only two vaccine candidates (HEV 239 and 56 kDa) have been tested for human clinical trials . ...
This study presents nanoparticle-based vaccine development for Hepatitis E virus (HEV). Gold nanoparticles (GNP) of average size 12 nm were synthesized by citrate reduction method followed by functionalization with cysteamine hydrochloride for nano-conjugation. Immune response of nano-conjugates of GNP with 26 kDa protein (368-606 amino acids) and 54 kDa protein (112-606 amino acids) were evaluated. In vitro release kinetics of GNP-conjugated 54 kDa (GNP54) and 26 kDa (GNP26) proteins showed slower rate of release of 54 kDa protein as compared to 26 kDa protein. Humoral immune response of mice immunized intramuscularly with GNP54, GNP26 and GNP alone, exhibited HEV-specific IgG titer of 7.9 ± 2.9, 5.686 ± 4.098 and 0.698 ± 0.089, respectively, after 14 days of booster immunization. In addition to this, HEV-specific cell-mediated immune response was demonstrated by splenocyte proliferation assay. Analysis of results using one-way ANOVA, showed statistically significant (p value < 0.05) increase in splenocyte proliferation for GNP54- and GNP26-immunized mice in comparison to GNP alone immunized mice. Stimulation index of HEV ORF2 proteins in GNP54/GNP26-immunized mice were comparable to Concanavalin A-treated positive control. These results indicate GNP-based vaccine as a promising candidate for efficiently mediating both humoral and cell-mediated immune response against HEV.
... [12] Full-length and truncated open reading frame 2 of HEV capsid proteins were expressed in various expression systems including E. coli, yeast, plant, insect and mammalian cells. [13][14][15][16][17][18][19][20][21] It was demonstrated that the full-length ORF2 protein was not the ideal target to develop diagnostic tests, such as ELISA, since the important epitopes constituting the capsid protein, located in the C-terminal region and specifically between amino acids 394-457, were hidden. [22] Thus, more interest was brought to truncated forms of ORF2 (tORF2). ...
Hepatitis E virus (HEV) is a nonenveloped virus causing an emerging zoonotic disease posing a severe threat to the public health in the world, especially to pregnant women. In this study, a truncated form (aa 368-606) of the open reading frame 2 of the capsid protein (tORF2-HEV), a major structural protein of HEV, was expressed in Escherichia coli. This work characterizes for the first time, the fused Glutathione-S-Transferase-tagged tORF2 (GST-tORF2) and tORF2-HEV forms in E. coli. The fusion protein was purified by affinity chromatography with a purity higher than 90% and to yield about 27% after thrombin digestion. The purified GST-tORF2 protein was then characterized by western blot, using anti-GST antibodies, and CD spectroscopy. The GST-tORF2 and tORF2-HEV proteins were shown to be efficient to develop an ELISA test to detect anti-HEV IgG in mice sera immunized with a recombinant full length ORF2 protein. Sera showed a significant increase of the absorbance signal at 450 nm, in plate wells coated with a quantity of 0.5, 1 and 2 mg of proteins. ELISA plates coated with the purified GST-tORF2 and tORF2-HEV showed similar response when compared to the HEV ELISA where total insect cell lysate, infected with the recom-binant baculovirus expressing full ORF2, was used as positive control.
... In this study, the immune procedure with two doses of HEV vaccine partially protected rhesus monkeys from hepatitis E following intravenous challenge at 6 or 12 months post-vaccination. 67 A randomized, double-blind, placebo-controlled phase II clinical trial was launched to evaluate the safety and efficacy of this candidate vaccine. A total of 1794 subjects received three vaccine doses, with 898 of them as vaccine group and 896 as placebo group. ...
Hepatitis E virus (HEV) infection is an emerging zoonotic disease posing a severe threat to public health in the world, especially to pregnant women. Currently, no specific treatments are available for HEV infection. Therefore, it is crucial to develop vaccine to prevent this infection. Although several potential candidate vaccines against HEV have been studied for their immunogenicity and efficacy, only Hecolin® which is developed by Xiamen Innovax Biotech Co., Ltd. and approved by China Food and Drug Administration (CFDA) in 2012, is the licensed HEV vaccine in the world so far. Extensive studies on safety, immunogenicity and efficacy in phase III clinical trials have shown that Hecolin® is a promising vaccine for HEV prevention and control. In this article, the advances on HEV vaccine development and research are briefly reviewed.
... Interestingly, postexposure immunization provided no protection from hepatitis, a clear distinction from inactivated HAV vaccine. Additional studies showed that three of four rhesus macaques were protected against hepatitis when challenged intravenously with homologous virus 6 or 12 months after immunization (Zhang et al. 2002). In further preclinical trials, immunized rhesus macaques were challenged with three different HEV strains, including the homologous gt1 Pakistani SAR55 virus, the gt2 Mex-14 isolate, and gt3 US-2 virus. ...
Although phylogenetically unrelated, human hepatitis viruses share an exclusive or near exclusive tropism for replication in differentiated hepatocytes. This narrow tissue tropism may contribute to the restriction of the host ranges of these viruses to relatively few host species, mostly nonhuman primates. Nonhuman primate models thus figure prominently in our current understanding of the replication and pathogenesis of these viruses, including the enterically transmitted hepatitis A virus (HAV) and hepatitis E virus (HEV), and have also played major roles in vaccine development. This review draws comparisons of HAV and HEV infection from studies conducted in nonhuman primates, and describes how such studies have contributed to our current understanding of the biology of these viruses.
... Thus, non-human primates have been useful models for studying hepatitis E clinical progression and for immunological studies 220,[226][227][228] . Both cynomolgus and rhesus monkeys have been used to test the efficacy of pORF2-derived anti-HEV vaccines [229][230][231] The species permissive to hepatitis E virus (HEV) infection include (but are not limited to) humans, rabbits, swine, deer, wild boar and camel. To better study HEV infection in physiologically relevant in vitro models, it will be desirable to generate co-cultures that recapitulate the complexity of the liver including endothelial, stellate, cholangiocytic, Kupffer and hepatic cells in the appropriate ratios. ...
At least 20 million hepatitis E virus (HEV) infections occur annually, with >3 million symptomatic cases and ∼60,000 fatalities. Hepatitis E is generally self-limiting, with a case fatality rate of 0.5–3% in young adults. However, it can cause up to 30% mortality in pregnant women in the third trimester and can become chronic in immunocompromised individuals, such as those receiving organ transplants or chemotherapy and individuals with HIV infection. HEV is transmitted primarily via the faecal–oral route and was previously thought to be a public health concern only in developing countries. It is now also being frequently reported in industrialized countries, where it is transmitted zoonotically or through organ transplantation or blood transfusions. Although a vaccine for HEV has been developed, it is only licensed in China. Additionally, no effective, non-teratogenic and specific treatments against HEV infections are currently available. Although progress has been made in characterizing HEV biology, the scarcity of adequate experimental platforms has hampered further research. In this Review, we focus on providing an update on the HEV life cycle. We will further discuss existing cell culture and animal models and highlight platforms that have proven to be useful and/or are emerging for studying other hepatotropic (viral) pathogens.
... The HEV neutralizing antibody recognition site was mapped to the E2s (I) domain (26). Both HEV495 and HEV239 form virus-like particles (VLPs), contain immunodominant epitopes, and are highly immunogenic when administered with alum as the adjuvant (33,40,(42)(43)(44)(45). In fact, an addition of 26 amino acids at the N terminus of E2 protein was shown to improve immunogenicity when administered as a single dose of VLP preparation (27). ...
In the developing countries, Hepatitis E virus (HEV) is a predominant cause of sporadic acute hepatitis in adults and waterborne epidemics leading to high mortality in pregnant women. Vaccine development mainly focuses on the structural capsid protein open-reading-frame-2 (ORF-2) of the virus. We successfully evaluated liposome-adjuvanted recombinant neutralizing epitope protein (rNEp), a part of ORF-2, 458-607aa, in mice and rhesus macaques. We compared immune response to adjuvants alone, rNEp alone, or adjuvanted with liposome (lipo-rNEp)/alum (al-rNEp) in mice following intramuscular administration of two doses of 5 μg each. IgG anti-HEV titers (enzyme-linked immunosorbent assay), immunophenotyping (flow cytometry, CD3(+)CD4(+), CD3(+)CD8(+), CD11c(+), CD11b(+), CD19(+) cells; costimulatory markers CD80, CD86, MHC-I, MHC-II, and early activation marker CD69), and levels of Th1/Th2 cytokines (IL-2/IFN-γ/IL-4/IL-5 and additionally IL-1β/IL-6/IL-10/TNF for early time points) were determined at early (4/12/24-h postdose-1) and later time points (2 weeks post-both doses). IgG anti-HEV titers were higher in the lipo-rNEp group than al-rNEp post-both doses (p < 0.05). At early time points, cell type proportions were comparable at the site of injection; IL-Iβ levels increased in lipo-rNEp, 24 h, while IL-6 levels rose in lipo-rNEp/al-rNEp/alum-alone groups, 4 h, compared to controls. In the draining lymph nodes (DLNs), CD11c(+)CD86(+) cells increased at 24 h in liposome-alone/lipo-rNEp groups. A rise in the CD11c(+)CD69(+) cells was noted in the lipo-rNEp group compared to other groups (p < 0.05). Cytokine levels in the spleen/sera remained unchanged in all the groups (p > 0.05). At 2 weeks postdose-2, CD11c(+)MHC-II(+)/CD11b(+)MHC-II(+) cells increased in the spleen in the lipo-rNEp and al-rNEp groups, respectively. In the DLNs, CD19(+)MHC-II(+) cells increased in rNEp/al-rNEp/lipo-rNEp groups post-both doses and CD11c(+)CD86(+) cells in the lipo-rNEp group. A balanced Th1/Th2 response was evident in the lipo-rNEp, while a Th2 bias was noted in al-rNEp. Different immune response gene clustering patterns were noted in uncultured spleens from immunized mice and cultured-stimulated splenocytes. In conclusion, lipo-rNEp is a better immunogen, works through dendritic cells, and elicits a balanced Th1/Th2 response, while alum functions through macrophages and induces a Th2 response.
... Currently, most research on HEV vaccines is focused on ORF-2-derived proteins or peptides that contain neutralizing epitopes common to all different genotypes ( Emerson and Purcell 2001 ;Meng et al. , 2001 ;Wang and Zhuang, 2004 ;Deshmukh et al. , 2007 ). A recombinant HEV baculovirus vaccine candidate that protects against intravenous administration of heterologous HEV strains has entered clinical trials and appears very promising ( Stevenson 2000 ;Zhang et al. , 2002 ;Purcell et al. , 2003 ). ...
Hepatitis viruses comprise a group of very diverse pathogens that primarily infect the liver, but belong to very different virus families with very different replication strategies (hepatitis A virus (HAV) Picornaviridae, hepatitis B virus (HBV) Hepadnaviridae, hepatitis C virus (HCV) Flaviviridae, hepatitis delta virus (HDV) genus Deltavirus, not assigned to a family yet, and hepatitis E virus (HEV) Hepeviridae). All of them have in common a high genome plasticity, and have received special attention because of their worldwide distribution in human population, infecting hundreds of million people, causing either acute and/or chronic infections that in many cases lead to liver cirrhosis and hepatocellular carcinoma, which is one of the leading causes of death worldwide. The huge number of infected people all over the world is the best proof of how different replication and transmission strategies, with the common factor of variability, may succeed in terms of viral persistence.
... Additionally, the utilization of immunoglobulins for prophylaxis after the exposure to infection have constrained worth. [ 42,43] Indeed, for the treatment of Vol.5; Issue: 4; April 2015 ...
Background: In Egypt Hepatitis E virus (HEV) is considered as an endemic infection. The aim of the present study was to determine the prevalence of HEV infection among HD patients and to study the risk factors associated with such infection. Methods: The study included ninety six cohort HD patients in Mansoura University Hospital, Egypt in addition to one hundred sixty seven healthy blood donors. Full virological markers assay for hepatitis B, C and E viruses were performed. Positive samples for serological markers for hepatitis E were subjected to nested PCR for HEV. Results: HCV IgG was the predominant serological markers among HD (42.7%) followed by HEV IgG (22.9%). Serological virological markers for HCV and HEV were significantly higher in HD patients compared to healthy blood donors (18.6%-5.9%, P=0. 0001, P=0. 02 respectively). HEV viremia was detected in statistically significantly higher percent of HD patients (36.4%) compared to blood donors (20%), P=0. 07. The duration of dialysis and the number of blood transfusion units had no statistically significant association with HEV viremia. HCV antibodies were prevalent among 50% of patients with HEV viremia. Conclusion: From this study we can reason that HEV is common infection among hemodialysis patients. Further studies are obliged to clear up the wellspring of HEV disease in dialysis units in Egypt.
... Therefore, pregnant women from endemic countries are considered the ideal category for hepatitis E immunization. So far, 10 vaccine candidates including ours were shown to be efficacious in the preclinical trial in rhesus monkeys [2][3][4][5][6][7][8][9][10][11] and two have undergone clinical trials [12,13]. However, except for incidental immunization of pregnant women during a clinical trial [14], none of these were evaluated during pregnancy. ...
Pregnant women from developing countries are at high-risk of hepatitis E-associated high mortality and constitute priority population for vaccination. So far, candidate vaccines have not been evaluated during pregnancy. We evaluated our vaccine candidate, recombinant Neutralizing Epitope protein (rNEp) encapsulated in liposomes, in pregnant mice.
A single dose (10 μg) of the formulation was administered intramuscularly on day 7 of pregnancy. Titres of serum IgG antibodies to hepatitis E virus (IgG-anti-HEV), levels of cytokines and biochemical parameters were determined. Spleens were harvested from pregnant and non-pregnant mice for immunophenotyping (flow cytometry), cytokines (cytometric bead array, CBA) and gene expression of immune response genes (Taqman low density array, TLDA). Histopathology studies of spleen, liver, kidneys, brain and muscle was carried out.
The vaccine was well-tolerated during pregnancy as evidenced by histopathology and serum biochemical parameters. Anti-HEV titres were significantly higher in the pregnant balb/c and C57BL/6 mice (3592 ± 802 and1016 ± 138 respectively, than in non-pregnant groups (634 ± 191 and 320 ± 55 respectively, p < 0.001 for both)suggesting that the higher antibody response in pregnant mice was independent of the genetic makeup of the host but immunogen-driven. Pups receiving vertically transferred antibodies developed lower anti-HEV antibodies (p < 0.05) when immunized with the formulation after seronegativity than in the age-matched mice without such antibodies. In non-pregnant mice, a Th1 response and discordance between splenic and serum cytokines was evident while in pregnancy, a Th2 bias was observed irrespective of immunization. Increased CD19 levels correlated with higher anti-HEV titres in pregnant mice.
The single dose of the vaccine was safe and highly immunogenic in pregnant mice. Degree and type of immune response to vaccination during pregnancy is immunogen-driven. In-depth studies are needed to understand the underlying immunologic mechanism(s).These encouraging results for a vaccine intended for use in pregnant women should be confirmed in higher animals.
... Additionally, the utilization of immunoglobulins for prophylaxis after the exposure to infection have constrained worth. [ 42,43] Indeed, for the treatment of Vol.5; Issue: 4; April 2015 ...
... Additionally, the utilization of immunoglobulins for prophylaxis after the exposure to infection have constrained worth. [ 42,43] Indeed, for the treatment of Vol.5; Issue: 4; April 2015 ...
Background: In Egypt Hepatitis E virus (HEV) is considered as an endemic infection. The aim of the present study was to determine the prevalence of HEV infection among HD patients and to study the risk factors associated with such infection. Methods: The study included ninety six cohort HD patients in Mansoura University Hospital, Egypt in addition to one hundred sixty seven healthy blood donors. Full virological markers assay for hepatitis B, C and E viruses were performed. Positive samples for serological markers for hepatitis E were subjected to nested PCR for HEV. Results: HCV IgG was the predominant serological markers among HD (42.7%) followed by HEV IgG (22.9%). Serological virological markers for HCV and HEV were significantly higher in HD patients compared to healthy blood donors (18.6%-5.9%, P=0. 0001, P=0. 02 respectively). HEV viremia was detected in statistically significantly higher percent of HD patients (36.4%) compared to blood donors (20%), P=0. 07. The duration of dialysis and the number of blood transfusion units had no statistically significant association with HEV viremia. HCV antibodies were prevalent among 50% of patients with HEV viremia. Conclusion: From this study we can reason that HEV is common infection among hemodialysis patients. Further studies are obliged to clear up the wellspring of HEV disease in dialysis units in Egypt. INTRODUCTION Hepatitis E virus (HEV) is a non enveloped, positive sense single-stranded RNA virus that is approximately 27 to 34 nm in diameter. It has been classified as the single member of the genus Hepevirus and has a similar structure to the viruses of the Caliciviridae and Tombusviridae families.
... In animal studies, the use of HEV capsid proteins and HEV DNA has resulted in the induction of virus-specific protective antibodies [154][155][156][157]. Human vaccines are not commercially available yet. ...
Awareness of the natural history of chronic hepatitis B infection in children is central to any decisions regarding treatment.
A child with chronic HBV infection should have serological markers of HBV infection (HBsAg, HBeAg, and/or HBV DNA) on two occasions at least 6 months apart to be considered for treatment.
A liver biopsy may be helpful before starting treatment.
Therapeutic goals include long-term suppression of HBV replication, cessation of liver injury, and reduction of risk for cirrhosis and hepatocellular carcinoma.
Currently, six agents in two classes (interferons and nucleot(s)ide analogs) are available for treatment of chronic HBV infection but only interferon-alfa and lamivudine are licensed in USA for children less than 12 years of age.
There is limited information regarding the use of other nucleot(s)ide analogs in children, although studies are ongoing.
Co-infections with hepatitis C or human immunodeficiency virus require special approaches to treatment of chronic HBV infection.
Given concerns about the long-term natural history, the limited therapeutic options, and the serious risk of resistance to antiviral agents, an optimal treatment regimen for children with chronic hepatitis B infection has not yet been developed, and clinicians should use these agents prudently.
... In animal studies, the use of HEV capsid proteins and HEV DNA has resulted in the induction of virus-specific protective antibodies [154][155][156][157]. Human vaccines are not commercially available yet. ...
Identification of children with hepatitis B or hepatitis C requires a heightened awareness of whom to screen, and which tests to obtain.
Recent recommendations for hepatitis B case identification include screening of any person born in an area of the world where the prevalence exceeds 2%, or children born in the USA to parents who have emigrated from endemic regions.
Chronic hepatitis B is a life-long disease that requires monitoring that is best managed as a partnership between the primary practitioner and the specialist.
Vaccination is the best method of preventing hepatitis B transmission and titer measurement post-vaccination is recommended for close contacts.
Like hepatitis B, hepatitis C in childhood frequently is undetected without knowing whom to screen.
The best method of testing for hepatitis C is determination of antibody (anti-HCV).
Children in whom HCV RNA is detected by PCR should be referred for consideration of treatment. Quantitative hepatitis C RNA and genotype testing are helpful in making treatment decisions and determining the likelihood of treatment response.
Social aspects of both hepatitis B and C are essential areas of discussion for the practitioner, who should address alcohol use, illicit drugs, and sexual transmission.
Family members of children with hepatitis B or hepatitis C should undergo appropriate testing and teaching about strategies to reduce transmission within the home.
Children with hepatitis B or hepatitis C should not be excluded from school or extracurricular activities. Universal precautions that should already be in place in these settings are sufficient.
... In animal studies, the use of HEV capsid proteins and HEV DNA has resulted in the induction of virus-specific protective antibodies [154][155][156][157]. Human vaccines are not commercially available yet. ...
Patients in countries with high HBV endemicity tend to have perinatal transmission of hepatitis B, whereas those in countries with low endemicity have horizontal infection in adolescence or early adulthood.
Universal infant immunization could effectively reduce the prevalence of HBV infection to approximately 10% of the prevalence before the vaccination program.
Important factors affecting HBsAg seroconversion included maternal HBeAg status, virus genotypes, and host effects.
... In animal studies, the use of HEV capsid proteins and HEV DNA has resulted in the induction of virus-specific protective antibodies [154][155][156][157]. Human vaccines are not commercially available yet. ...
Effective treatments for children infected with HCV are available.
Children over the age of 2 years with chronic hepatitis C should be considered for treatment.
Treatment should be encouraged in children with chronic hepatitis C, without contraindications, especially in those with genotype 2 or 3.
Treatment for chronic hepatitis C has a number of side effects, and treatment of children should be supervised by those experienced with these medications.
New medications for the treatment of chronic hepatitis C are currently in development.
Animal models are one of the most important tools in the study of human hepatitis E virus (HEV) infection. They are particularly important in light of the major limitations of the cell culture system for HEV. Besides nonhuman primates, which are extremely valuable because of their susceptibility to HEV genotypes 1-4, animals like swine, rabbit, and humanized mice are also potential models for studies of pathogenesis, cross-species infection, and the molecular biology of HEV. Identification of a useful animal model for human HEV infection studies is crucial to further investigations into this ubiquitous yet poorly understood virus and facilitate the development of antiviral therapeutics and vaccines.
The hepatitis E has been increasingly recognized as an underestimated global disease burden in recent years. Subpopulations with more serious infection associated damage or death include pregnant women, patients with basic liver diseases, and elderly persons. Vaccine would be the most effective means for prevention of HEV infection. The lack of an efficient cell culture system for HEV makes the development of classic inactive or attenuated vaccine infeasible. Hence, the recombinant vaccine approaches are explored deeply. The neutralizing sites are located almost exclusively in the capsid protein, pORF2, of the virion. Based on pORF2, many vaccine candidates showed potential of protecting primate animals, two of them were tested in human and evidenced to be well-tolerated in adults and highly efficacious in preventing hepatitis E. The world's first hepatitis E vaccine, Hecolin® (HEV 239 vaccine), was licensed in China and launched in 2012.
Hepatitis E virus (HEV) usually causes acute self-limiting hepatitis but sometimes leads to chronic infection in immunocompromised persons. HEV is not directly cytopathic. Immunologically mediated events after HEV infection are believed to play important roles in the pathogenesis and clearance of infection. The anti-HEV antibody responses have been largely clarified since the determination of major antigenic determinant of HEV, which is located in the C-terminal portion of ORF2. This major antigenic determinant also forms the conformational neutralization epitopes. Robust anti-HEV immunoglobulin M (IgM) and IgG responses usually develop 3-4 weeks after infection in experimentally infected nonhuman primates. In humans, potent specific IgM and IgG responses occur in the very early phase of the disease and are critical in eliminating the virus, in concert with the innate and adaptive T-cell immune responses. Testing anti-HEV IgM is valuable in the diagnosis of acute hepatitis E. The long-term persistence and protection of anti-HEV IgG provide the basis for estimating the prevalence of HEV infection and for the development of a hepatitis E vaccine. Although human HEV has four genotypes, all the viral strains are considered to belong to a single serotype. It is becoming increasingly clear that the innate and adaptive T-cell immune responses play critical roles in the clearance of the virus. Potent and multispecific CD4+ and CD8+ T cell responses to the ORF2 protein occur in patients with acute hepatitis E, and weaker HEV-specific CD4+ and CD8+ T cell responses appear to be associated with chronic hepatitis E in immunocompromised individuals.
Hepatitis E virus (HEV) is associated with acute hepatitis disease, which may lead to chronic disease in immunocompromised individuals. The disease is particularly severe among pregnant women (20–30% mortality). No vaccine is available to combat the HEV except Hecolin, which is available only in China. Virus-like particle (VLP) generated from the capsid protein (ORF2) of HEV is known to be a potent vaccine antigen against HEV. Hecolin consists of 368–606 amino acid (aa) region of the capsid protein of HEV, which forms a VLP. It is expressed and purified from the inclusion bodies of E. coli. Here, we describe a method to express the 112-608aa region of the capsid protein (ORF2) of genotype-1 HEV in Pichia pastoris (P. pastoris) and purify VLPs from the culture medium. 112-608aa ORF2 VLPs are secreted into the culture medium in a methanol inducible manner. The purified VLPs are glycosylated and induce robust immune response in Balb/c mice. Further, 112-608aa ORF2 VLPs are bigger than the 368–606 VLP present in Hecolin, which may help them in inducing a superior immune response. P. pastoris offers a robust and economical heterologous expression system to produce large quantities of glycosylated 112-608aa ORF2 VLP, which appears to be a promising vaccine candidate against the HEV.
Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis globally. HEV belongs to the Hepeviridae family and at least five genotypes (gt) infect humans. Several animal species are reservoirs for different HEV strains, and they are the source of infection for humans. Some HEV strains are species specific, but other strains could cross species and infect many hosts. The study of HEV infection and pathogenesis was hampered due to the lack of an in vitro and in vivo robust model system. The cell culture system has been established for certain HEV strains, especially gt3 and 4, but gt1 strains replicate poorly in vitro. To date, animal models are the best tool for studying HEV infection. Non-human primates (NHPs) and pigs are the main animal models used for studying HEV infection, but ethical and financial concerns restrict the use of NHPs in research. Therefore, new small animal models have been developed which help more progress in HEV research. In this review, we give updates on the animal models used for studying HEV infection, focusing on the applicability of each model in studying different HEV infections, cross-species infection, virus-host interaction, evaluation of anti-HEV therapies and testing potential HEV vaccines.
IntroductionViral hepatitisHighly pathogenic RNA viruses causing hemorrhagic feversHighly pathogenic RNA viruses causing encephalitisSmallpox and monkeypoxRabiesRespiratory tract infectionsHerpesviral infectionsViral gastroenteritisHuman T-lymphoma/leukemia virus 1MeaslesHuman polyomavirusesHuman enterovirusesReferences
Background:
Liposome encapsulated neutralizing epitope protein of Hepatitis E virus (HEV), rNEp, our Hepatitis E vaccine candidate, was shown to be immunogenic and safe in pregnant and non-pregnant mice and yielded sterilizing immunity in rhesus monkeys.
Methods:
The current study in Balb/c mice assessed the levels and persistence of anti-HEV IgG antibodies by ELISA, frequencies of B, memory B, T and memory T cells by flow cytometry and HEV-specific IgG secreting memory B cells by ELISPOT till 420days post immunization (PI) with 5?g rNEp encapsulated in liposome based adjuvant (2 doses, 4weeks apart). Mice immunized with a lower dose (1?g) were assessed only for anamnestic response post booster dose.
Results:
Vaccine candidate immunized mice (5?g dose) elicited strong anti-HEV IgG response that was estimated to persist for lifetime. At day 120 PI, frequency of memory B cells was higher in immunized mice than those receiving adjuvant alone. Anti-HEV IgG titers were lower in mice immunized with 1?g dose. A booster dose yielded a heightened antibody response in mice with both high (>800GMT, 5?g) and low (?100GMT, 1?g) anti-HEV IgG titers. At day 6th post booster dose, HEV-specific antibody secreting plasma cells (ASCs) were detected in 100% and 50% of mice with high and low anti-HEV IgG titers, respectively, whereas the frequencies of CD4+ central and effector memory T cells were high in mice with high anti-HEV IgG titers only.
Conclusions:
Taken together, the vaccine candidate effectively generates persistent and anamnestic antibody response, elicits participation of CD4+ memory T cells and triggers memory B cells to differentiate into ASCs upon boosting. This approach of assessing the immunogenicity of vaccine candidate could be useful to explore the longevity of HEV-specific memory response in future HEV vaccine trials in human.
Hepatitis E has been increasingly recognized as an underestimated global disease burden in recent years. Subpopulations with more serious infection-associated damage or death include pregnant women, patients with basic liver diseases, and elderly persons. Vaccine would be the most effective means for prevention of HEV infection. The lack of an efficient cell culture system for HEV makes the development of classic inactive or attenuated vaccine infeasible. Hence, the recombinant vaccine approaches are explored deeply. The neutralizing sites are located almost exclusively in the capsid protein, pORF2, of the virion. Based on pORF2, many vaccine candidates showed potential of protecting primate animals; two of them were tested in human and evidenced to be well tolerated in adults and highly efficacious in preventing hepatitis E. The world’s first hepatitis E vaccine, Hecolin® (HEV 239 vaccine), was licensed in China and launched in 2012.
Animal models are one of the most important tools in the study of human hepatitis E virus (HEV) infection. They are particularly important in light of the major limitations of the cell culture system for HEV. Besides nonhuman primates, which are extremely valuable because of their susceptibility to HEV genotypes 1–4, animals like swine, rabbit, and chicken are also potential models for studies of pathogenesis, cross-species infection, and the molecular biology of HEV. Identification of the most useful animal model for human HEV infection studies is crucial to further investigations into this ubiquitous yet poorly understood virus.
Hepatitis E virus (HEV) causes acute self-limiting hepatitis in most cases and chronic infection in rare circumstances. It is believed to be noncytopathic, so immunologically mediated events should play important roles in its pathogenesis and infection outcomes. The anti-HEV antibody response was clarified when the major antigenic determinants on the ORF2 polypeptide were determined, which are located in its C-terminal portion. This subregion also forms the conformational neutralization epitopes. Robust anti-HEV immunoglobulin M (IgM) and IgG responses usually develop 3–4 weeks after infection in experimentally infected nonhuman primates. In humans, potent specific IgM and IgG responses occur in the very early phase of the disease and are critical in eliminating the virus, in concert with the innate and adaptive T-cell immune responses. They are also very valuable in the diagnosis of acute hepatitis E, when patients are tested for both anti-HEV IgM and IgG. The long-term persistence and protection of anti-HEV IgG provide the basis for estimating the prevalence of HEV infection and for the development of a hepatitis E vaccine. Although HEV has four genotypes, all the viral strains are considered to belong to a single serotype. It is becoming increasingly clear that the innate and adaptive T-cell immune responses play critical roles in the clearance of the virus. Potent and multispecific CD4+ and CD8+ T-cell responses to the ORF2 protein occur in patients with acute hepatitis E, and weaker HEV-specific CD4+ and CD8+ T-cell responses appear to be associated with chronic hepatitis E in immunocompromised individuals.
Hepatitis E virus infections have been continuously reported in Indian subcontinent, Africa, southeast and central Asia, posing great health threats to the public, especially to pregnant women. Hecolin® is the only licensed HEV vaccine developed by Xiamen Innovax Biotech Co.,Ltd. Extensive characterizations on antigenicity, physicochemical properties, efficacy in clinical trials, and manufacturing capability have made Hecolin® a promising vaccine for HEV control. However, there are many obstacles in large scale application of Hecolin®. Efforts are needed to further evaluate safety and efficacy in HEV risk populations, and to complement HEV standards for quality control. Passing World Health Organization prequalification and licensing outside China are priorities as these are also hindering Hecolin® promotion. Multilateral cooperation among Chinese vaccine manufacturers, Chinese National Regulatory Authorization (NRA) and WHO will expedite the entrance of Hecolin® into international market, so that Hecolin® could play its due role in global hepatitis E control.
Hepatitis E virus (HEV) is the cause of human hepatitis E. Hepatitis E is endemic in many developing countries, including China, and represents a major public health problem. In this article, we will review the current knowledge on humoral and cellular immune responses and mechanisms of immunologic injury in HEV infection as well as the development of HEV vaccines.
Simian Virology is the first text to comprehensively cover all currently known simian viruses. Chapters provide an overview of nonhuman primate models of medically important viral diseases as well as natural infections of nonhuman primates with human and animal viruses. The text covers a variety of topics including primate models of medically important viral diseases such as AIDS, hypotheses on the origins of epidemic forms of HIV, and viral diseases caused by non-simian viruses in both wild and captive primates.
Hepatitis E accounts for the major part of enterally transmitted non-A, non-B hepatitis worldwide. Its agent, the hepatitis E virus (HEV), is a small, single-stranded RNA virus. Only one serotype of HEV is recognised. Infection results in protective immunity with long-lived neutralising antibodies. In developing countries with poor sanitary conditions and high population density, hepatitis E causes water-borne epidemics with substantial mortality rates in pregnant women. In addition, more than 50% of cases of acute hepatic failure and sporadic acute hepatitis are due to hepatitis E. The overall prevalence rates of antibodies to the HEV in populations native to these areas rarely exceed 25%. Hence, many individuals remain susceptible to hepatitis E infection, making hepatitis E an important public health concern.
In this context, the development of an HEV vaccine is warranted. Because HEV does not grow adequately in cell cultures the development of a vaccine based on inactivated or attenuated whole-virus particles is not feasible. HEV vaccines currently under study are based on recombinant proteins derived from immunogenic parts of the HEV capsid gene. Other approaches such as DNA-based vaccines or transgenic tomatoes have also been developed. Several recombinant protein-based vaccines elicited neutralising antibodies and protective immunity in vaccinated non-human primates. One such vaccine has passed phase I trial and is currently under further evaluation in field trials. Even so, several questions remain to be answered before vaccination programmes could be implemented.
Enterically transmitted hepatitis represents the most common manifestation of acute hepatitis worldwide. Enteric hepatitis include two types: hepatitis A and hepatitis E. Hepatitis A infection may develop asymptomatically. This type of subclinical infection is most common among young children, while in older children and in the adulthood the infection usually proceeds with symptoms. The clinical course of hepatitis A is indistinguishable from that of other types of acute viral hepatitis. The clinical case definition for hepatitis A is an acute illness with moderate onset of symptoms (fever, malaise, anorexia, nausea, abdominal discomfort, and dark urine) and jaundice, and elevated serum bilirubin and aminotransferases levels later on. Hepatitis E is an infection with clinical and epidemiological features of acute hepatitis. The clinical presentation of hepatitis E is basically similar to that of hepatitis A, but cholestatic jaundice is more common. For detection and quantification of hepatitis A virus (HAV) immunological and, particularly, molecular techniques should be used. The availability of an increased number of Hepatitis E virus (HEV) sequences from different sources and geographical regions has enabled the design of specific oligonucleotide primers that match conserved regions of the HEV genome and allows the detection of HEV in acute phase sera, stools, and contaminated water and sewage. Inactivated HAV vaccines are available that provide long-lasting immunity against hepatitis A infection. Cross-reactive vaccines are also available that provides immunity against HEV.
Hepatitis A virus (HAV) is a nonenveloped small RNA virus in the hepatovirus genus of the picornavirus family (1) and is the most common defined cause of viral hepatitis worldwide. In the United States, the number of notified cases annually
is around 23,000, but estimates of the real number of cases of clinical disease range up to 75,000 per year. The infection
is usually transmitted via a fecal-oral route and is associated only with acute forms of viral hepatitis. Much higher virus
titers are found in bile and in stool than in blood. Whereas infection in children and the very young is most often unrecognized,
most infections in adults are symptomatic and associated with acute icteric hepatitis. Risk factors for a fulminant clinical
course include an age greater than 40 yr and some forms of preexisting liver disease. As the incidence of HAV infection among
children and adolescents has declined in many countries owing to improved socioeconomic status, these individuals are at increased
risk of disease later in life because of the lower prevalence of immunity (2). Diagnosis is made on the medical history, clinical features, and a positive anti-HAV-IgM antibody.
Along with the improvement of diagnostic techniques, hepatitis E has attracted increasing awareness in recent years. Hepatitis E virus infection leads to high mortality in pregnant women and patients with underlying liver disease. Several hepatitis E vaccine candidates have been designed and have proved their efficacy in animal models; two candidates have successfully undergone clinical trials. Having proved safe and effective in a large phase III trials, an Escherichia coli expressed particulate protein, HEV 239, has been registered in China and is now available for use in China.
Hepatitis D virus is a defective virus whose replication depends on an obligatory simultaneous infection with hepatitis B virus.
Hepatitis D virus has a worldwide but non-uniform distribution.
The clinical presentation of hepatitis D varies from asymptomatic infection to, more typically, severe icteric hepatitis.
Chronic hepatitis D is associated with rapid progression to cirrhosis and increased risk of hepatocellular carcinoma.
There is no effective therapy for HDV infection. Universal vaccination for hepatitis B is key to prevention of chronic hepatitis D.
Hepatitis E is transmitted predominantly by the fecal-oral route
The clinical presentation of acute hepatitis E is similar to that of other forms of viral hepatitis; hence the diagnosis needs to be suspected in the appropriate clinical context.
Hepatitis E therapy is supportive; HEV vaccines are being developed.
A strain of hepatitis E virus (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, was characterized extensively. Six cynomolgus monkeys (Macaca fascicularis) were infected with a strain of hepatitis E virus from Pakistan. Reverse transcription-polymerase chain reaction was used to determine the pattern of virus shedding in feces, bile, and serum relative to hepatitis and induction of specific antibodies. Virtually the entire genome of SAR-55 (7195 nucleotides) was sequenced. Comparison of the sequence of SAR-55 with that of a Burmese strain revealed a high level of homology except for one region encoding 100 amino acids of a putative nonstructural polyprotein. Identification of this region as hypervariable was obtained by partial sequencing of a third isolate of hepatitis E virus from Kirgizia.
We have recently described the cloning of a portion of the hepatitis E virus (HEV) and confirmed its etiologic association with enterically transmitted (waterborne, epidemic) non-A, non-B hepatitis. The virus consists of a single-stranded, positive-sense RNA genome of approximately 7.5 kb, with a polyadenylated 3' end. We now report on the cloning and nucleotide sequencing of an overlapping, contiguous set of cDNA clones representing the entire genome of the HEV Burma strain [HEV(B)]. The largest open reading frame extends approximately 5 kb from the 5' end and contains the RNA-directed RNA polymerase and nucleoside triphosphate binding motifs. The second major open reading frame (ORF2) begins 37 bp downstream of the first and extends approximately 2 kb to the termination codon present 65 bp from the 3' terminal stretch of poly(A) residues. ORF2 contains a consensus signal peptide sequence at its amino terminus and a capsid-like region with a high content of basic amino acids similar to that seen with other virus capsid proteins. A third open reading frame partially overlaps the first and second and encompasses only 369 bp. In addition to the 7.5-kb full-length genomic transcript, two subgenomic polyadenylated messages of approximately 3.7 and 2.0 kb were detected in infected liver using a probe from the 3' third of the genome. The genomic organization of the virus is consistent with the 5' end encoding nonstructural and the 3' end encoding the viral structural gene(s). The expression strategy of the virus involves the use of three different open reading frames and at least three different transcripts. HEV was previously determined to be a nonenveloped particle with a diameter of 27-34 nm. These findings on the genetic organization and expression strategy of HEV suggest that it is the prototype human pathogen for a new class of RNA virus or perhaps a separate genus within the Caliciviridae family.
Virtually full protection against hepatitis E and partial or complete protection against infection with hepatitis E virus (HEV) were achieved in passively or actively immunized cynomolgus monkeys. Hepatitis, viremia, and shedding of the virus in feces were detected in all nonimmunized animals that were challenged with HEV. HEV titers detected by reverse transcriptase PCR were higher in feces than in serum of nonimmunized animals. Anti-HEV antibody titers at the time of challenge ranged between 1:40 and 1:200 in animals passively immunized with convalescent plasma from a cynomolgus monkey previously infected with HEV and between 1:100 and 1:10,000 in animals actively immunized with a recombinant 55-kDa open reading frame 2 protein. The estimated 50% protective titer of passively acquired anti-HEV antibodies was 1:40. Although only one of four passively immunized animals showed histopathologic evidence of hepatitis, all four were infected after challenge; however, the titers of HEV in serum and feces were lower in the passively immunized animals than in the nonimmunized group. The actively immunized animals developed neither hepatitis nor viremia when challenged with HEV and virus was either not detected or was present in low titer in feces. The protective response was a function of the ELISA anti-HEV antibody titer at the time of challenge and the immunization schedule.
Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle.
Two monoclonal antibodies (MAbs) against the ORF2 protein of the SAR-55 strain of hepatitis E virus (HEV) were isolated by
phage display from a cDNA library of chimpanzee (Pan troglodytes) γ1/κ antibody genes. Both MAbs, HEV#4 and HEV#31, bound to reduced, denatured open reading frame 2 (ORF2) protein in a Western
blot, suggesting that they recognize linear epitopes. The affinities (equilibrium dissociation constants, Kd
) for the SAR-55 ORF2 protein were 1.7 nM for HEV#4 and 5.4 nM for HEV#31. The two MAbs also reacted in an enzyme-linked immunosorbent
assay with recombinant ORF2 protein from a heterologous HEV, the Meng strain. Each MAb blocked the subsequent binding of the
other MAb to homologous ORF2 protein in indirect competition assays, suggesting that they recognize the same or overlapping
epitopes. Radioimmunoprecipitation assays suggested that at least part of the linear epitope(s) recognized by the two MAbs
is located between amino acids 578 and 607. MAbs were mixed with homologous HEV in vitro and then inoculated into rhesus monkeys
(Macaca mulatta) to determine their neutralizing ability. Whereas all control animals developed hepatitis (elevated liver enzyme levels in
serum) and seroconverted to HEV, those receiving an inoculum incubated with either HEV#4 or HEV#31 were not infected. Therefore,
each MAb neutralized the SAR-55 strain of HEV in vitro.
A prospective field study was carried out during an epidemic of non-A non-B hepatitis for determining the incidence and severity of hepatitis in pregnant women, nonpregnant women of child bearing age and men (15 to 45 years old). In 36 (17.3 percent) of 208 pregnant women viral hepatitis developed, as compared to 71 (2.1 percent) of 3,350 nonpregnant women and 107 (2.8 percent) of 3,822 men. The incidence of disease in pregnant women was higher than in the two control groups. The incidence of viral hepatitis in the first, second and third trimesters was 8.8 percent, 19.4 percent, and 18.6 percent, respectively. The incidence in all three trimesters was higher, when compared to that in nonpregnant women. In eight pregnant women (22.2 percent) with viral hepatitis, fulminant hepatic failure developed, as compared to its occurrence in three men (2.8 percent) and in no nonpregnant women. This significantly increased incidence of fulminant hepatitis in pregnancy was indicative of a greater severity of hepatitis during pregnancy. Increased susceptibility to fulminant hepatitis was observed exclusively in the last trimester. Nonfulminant viral hepatitis did not influence the course of pregnancy or fetal well-being. Fetal loss in fatal fulminant hepatitis was a consequence of maternal death and could not be ascribed to direct effect on the fetus or pregnancy.
Hepatitis E virus (HEV) causes an enteric non-A, non-B hepatitis. The disease occurs in epidemic settings and sporadically, and viral transmission is thought to be faecal-oral. We present here a single volunteer study of HEV transmission followed by disease. Clinical and biochemical features of the infection correlated with HEV detection in the stools and sera by reverse transcription/polymerase chain amplification. IgG antibody has persisted for 2 years. The presence of HEV in serum before clinical signs appeared suggests that in endemic areas sporadic transmission of HEV may also occur parenterally.
A recombinant baculovirus containing the complete open-reading frame (ORF)-2 region of the hepatitis E virus (HEV) genome
was constructed. The major protein synthesized in insect cells infected with recombinant virus was about the size expected
for the complete ORF-2 product. This protein reacted in a Western blot assay with plasma from an HEV-infected chimpanzee.
Lysates of the recombinant virus-infected insect cells were used in ELISA to monitor seroconversion of eight primate species
(chimpanzees, four species of Old World monkeys, and three species ofNew World monkeys) inoculated with HEV. Homologous detector
anti-immunoglobulin was more sensitive than heterologous anti-immunoglobulin for detecting anti-HEV by ELISA. All primate
species except tamarins seroconverted after inoculation with HEV, although anti-HEV titers of Old World monkey species were
generally higher than those of New World monkey species. The ELISA with complete ORF-2 antigen appeared to be a sensitive
and practical method for detecting anti-HEV.
Thirty-two rhesus monkeys were used to evaluate the dose response of a recombinant HEV vaccine, and the efficacy of the vaccine based on the ORF2 protein of the Pakistani strain for pre- and post-exposure vaccination against intravenous challenge with homologous or heterologous virus was examined. Post-exposure vaccination did not protect animals against hepatitis. Although primates vaccinated twice with 50-microgram, 10-microgram, 2-microgram, or 0.4-microgram doses of the recombinant 55 kDa ORF-2 protein were infected, they were protected from hepatitis when they were challenged with very high doses of the homologous strain of HEV. Primates vaccinated twice with a 50 micrograms dose of the recombinant protein were protected from hepatitis after heterologous challenge with the Mexican strain, the strain of HEV most genetically distant from the Pakistani strain.
The hepatitis E virus (HEV) capsid antigen has been proposed as a candidate subunit vaccine for the prevention of hepatitis E. The full-length HEV ORF2 protein product is predicted to contain 660 amino acids and to weigh 72,000 daltons. Expression of the HEV ORF2 capsid gene from recombinant baculoviruses in insect cells produced multiple immunoreactive proteins ranging in size from 30 to 100 kDa. The most abundant HEV proteins had molecular weights of 72, 63, 56, and 53 kDa. Temporal expression kinetics of these viral polypeptides indicated that the 72- and 63-kDa polypeptides were produced abundantly within the initial 36 h. postinfection but were replaced by 56- and 53-kDa polypeptides in the cell and medium, respectively, by 48 h postinfection. The 53-kDa protein was secreted as early as 24 h. postinfection, and accumulation in the medium peaked by 72 h postinfection. Purification of the 53-, 56-, and 63-kDa viral polypeptides was accomplished by anion-exchange and subsequent gel filtration chromatography. Sequence analysis of the 53-, 56-, and 63-kDa HEV polypeptides indicated that the amino terminus was amino acid residue 112 of the predicted full-length protein product. The results of carboxy terminal amino acid sequencing indicated that the carboxy terminus of the 53-, 56-, and 63-kDa HEV proteins was located at amino acid residues 578, 607, and 660, respectively. The molecular masses of the 53- and 56-kDa HEV polypeptides were 53,872 and 56,144 as determined by mass spectroscopy.
The aims of this study were to examine the decline of IgG anti-HEV antibodies over a period of 7 years in rhesus monkeys experimentally infected with hepatitis E virus, and to assess the protectivity of these antibodies by challenging the monkeys with a heterologous isolate of hepatitis E virus, 5 years after the primary inoculation.
Nine rhesus monkeys (six non-pregnant and three pregnant at the time of hepatitis E virus inoculation) were followed serologically and biochemically for 7 years post-inoculation. Based on regression analysis, estimated time for IgG anti-HEV titers to reach 1:100 or 1:50 was calculated. Three of the monkeys inoculated initially with AKL-90 isolate and challenged 2 years later with PUN-85 isolate of hepatitis E virus were rechallenged with KOL-91 isolate of the virus, 5 years post-primary inoculation. Evidence of viral replication was assessed by measuring serum alanine aminotransferase levels, excretion of the virus in feces or bile (reverse-transcription polymerase chain reaction) and rise in IgG anti-HEV titers (ELISA).
None of the challenged monkeys showed evidence of disease. In contrast to extensive replication of the virus in anti-HEV-negative control monkeys, limited replication was noted in one of the challenged monkeys. The estimated time for the titers to reach 1:100 or 1:50 varied from 3.15 to 44.9 years (19.4+/-11.6 years) and 6.9 to 84.3 years (35.4+/-21.3 years), respectively. Decline in titers was independent of the pregnancy status at the time of infection or reexposure of the monkeys to HEV CONCLUSION: The results show persistence of IgG anti-HEV antibodies for a long time and protectivity of low titered antibodies against reinfection, leading to disease even after intravenous exposure to a heterologous isolate of hepatitis E virus.
Hepatitis E virus (HEV) is a non-enveloped RNA (7.5 kb) virus that is responsible for large epidemics of acute hepatitis and a proportion of sporadic hepatitis cases in southeast and central Asia, the Middle East, parts of Africa and Mexico. Hepatitis E virus infection spreads by the faecal-oral route (usually through contaminated water) and presents after an incubation period of 8-10 weeks with a clinical illness resembling other forms of acute viral hepatitis. Clinical attack rates are the highest among young adults. Asymptomatic and anicteric infections are known to occur. Chronic HEV infection is not observed. Although the mortality rate is usually low (0.07-0.6%), the illness may be particularly severe among pregnant women, with mortality rates reaching as high as 25%. Recent isolation of a swine virus resembling human HEV has opened the possibility of zoonotic HEV infection. Studies of pathogenetic events in humans and experimental animals reveal that viral excretion begins approximately 1 week prior to the onset of illness and persists for nearly 2 weks; viraemia can be detected during the late phase of the incubation period. Immunoglobulin M antibody to HEV (anti-HEV) appears early during clinical illness but disappears rapidly over a few months. Immunoglobulin G anti-HEV appears a few days later and persists for at least a few years. There is no specific treatment available for hepatitis E virus infection. Ensuring a clean drinking water supply remains the best preventive strategy. Recombinant vaccines are being developed that may be particularly useful for travellers to disease-endemic areas and for pregnant women.
It is important to monitor the long-term persistence of antibodies induced by vaccination. Four cohorts were followed for their long-term immunity after vaccination with a combined hepatitis A and B vaccine (Twinrix; SmithKline Beecham Biologicals, Rixsenart, Belgium). Two cohorts of adults (ages 17-60 years), one of 1-6-year-olds, and one of 6-15-year-olds were vaccinated following a 0, 1, and 6-month schedule. Follow-up data until month 72 (adults) and month 60 (children) are available. At month 72, antibody to hepatitis A virus (anti-HAV) seropositivity (S+) was 100% for both adult cohorts (n = 40 and n = 47) and 95% and 89% of the vaccinees were seroprotected against hepatitis B virus (HBV), respectively. The geometric mean titres (GMTs; mIU/ml) for anti-HAV were 977 and 542 and the GMTs for the antibody to hepatitis B surface antigen (anti-HBs) were 322 and 90. For 1-6-year-olds at month 60 (n = 39), anti-HAV S+ was 100% with a GMT of 479 and 97% were protected against HBV with a GMT of 195. At month 60 for the 6-15-year-olds (n = 42), anti-HAV S+ was 100% with a GMT of 990 and 95% were protected against HBV with a GMT of 263. There have been no safety issues during the follow-up. In the past 5 years, a postmarketing surveillance system was available. Using this system, all spontaneous adverse events are collected and archived. Although infrequent, the most commonly reported adverse events after more than 13 million doses were allergic-type reactions followed by fever and injection site reactions. The combined hepatitis A and B vaccine is safe and is well tolerated. Immunity provided by the vaccine remains high in adults and children with comparable results to those obtained with monovalent vaccines.
In Magnitude of the Problem of Hepatitis and Directions for Research, Prevention and Control
- S Pattanyak
Pattanyak S. In Magnitude of the Problem of Hepatitis and Directions
for Research, Prevention and Control. New Delhi (India): WHO,
1986. p. 1-13.
Incidence and severity of viral hepatitis in pregnancy
- M S Khuroo
- M R Teli
- S Skidmore
- M A Sofi
- M I Khuroo
Khuroo MS, Teli MR, Skidmore S, Sofi MA, Khuroo MI. Incidence
and severity of viral hepatitis in pregnancy. Am J Med 1981;70:252-5.