Live recombinant Lactococcuslactis expressing avian hepatitis virus ORF2 protein: Immunoprotection against homologous virus challenge in chickens

Avian hepatitis E virus (aHEV) is a pathogen associated with hepatitis-splenomegaly syndrome in chickens. To date, no commercial vaccine is available for preventing aHEV infection. In this study, three recombinant LactococcuslactisNZ9000experimental live vaccines expressing cytoplasmic, secreted, and cell wall-anchored forms of aHEV truncated ORF2 protein spanning amino acids 249–606 (ΔORF2) were constructed using pTX8048 vector and characterized. Each chicken was immunized three times at two-week intervals with one of the three live aHEV ORF2 vaccines (experimental group) or with live vaccine containing empty vector only (control group). Both groups were then challenged with aHEV and evaluated to compare immune responses and immunogenic effects. Serum IgG levels, secretory IgA (sIgA) levels in bile and jejunal lavage fluid, and mRNA expression levels ofIL-2 and IFN-γ in liver and spleen were significantly higher in experimental chickens than in controls. Meanwhile, post-challenge serum and fecal virus loads were significantly lower in experimental chickens versus controls. Moreover, on day 7 post infection (PI), serum lactose dehydrogenase (LDH) levels were significantly higher in controls than experimental chickens. Furthermore, at day 28 PI, obvious gross pathological lesions and histopathological changes typical for aHEV infection were observed in control livers and spleens, with only moderate pathological changes observed in the experimental group. The results of this study collectively demonstrate that an oral vaccineusing L.lactisNZ9000 as a delivery vector for aHEV immunogenic antigen could effectively control aHEV infection of chickens.     

Randomized-controlled phase II clinical trial of a bacterially expressed recombinant hepatitis E vaccine

The candidate recombinant hepatitis E vaccine, HEV 239, protect monkeys against infection by hepatitis E virus (HEV). The safety and immunogenicity of the vaccine for humans was assessed in a randomized controlled phase II clinical trial. The study was conducted in an endemic area of southern China and consisted of a dose scheduling, involving 457 adults and a dose escalation component involving 155 high school students. The results showed that the vaccine is safe and immunogenic for humans and suggest that it could prevent new HEV infection.     

Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection

Avian hepatitis E virus (HEV) is the etiologic agent of big liver and spleen disease in chickens. In 2010, the Chinese avian HEV (CaHEV) strain was isolated from chickens and demonstrated to cause the decreased egg production in layer hens. No avian HEV commercial vaccine has yet been developed to prevent virus infection in China. In this study, recombinant CaHEV truncated ORF2 and complete ORF3 proteins were evaluated separately for immunoprotection of chickens against CaHEV infection. First, truncated ORF2 and complete ORF3 proteins were expressed in Escherichia coli. Next, 48 specific-pathogen-free chickens were randomly divided into three groups. One group was immunized with truncated ORF2 protein, the second group was immunized with recombinant ORF3 protein, while the third group (control) was mock-immunized with PBS. After booster immunization, chickens in all three groups were challenged intravenously with CaHEV infectious stock and assessed for viremia, fecal virus shedding, seroconversion, and gross hepatic lesions. In the ORF2 protein-immunized group, no chickens showed evidence of avian HEV infection. In the ORF3 protein-immunized group, nine chickens exhibited viremia and seven had fecal virus shedding. In the control group, all 16 chickens showed viremia and fecal virus shedding. However, the durations in chickens from the ORF3 protein group (2–4 weeks) were shorter than the ones from the control group (4–8 weeks). Moreover, no gross liver lesions emerged in the ORF2 protein group, while lesions observed in the ORF3 protein group were milder than in controls. Therefore, the ORF2 protein can confer complete immunoprotection against chicken CaHEV infection, while the ORF3 protein only confers partial immunoprotection.     

A dual vaccine candidate against norovirus and hepatitis E virus

Norovirus (NoV) and hepatitis E virus (HEV) are both enterically-transmitted viruses causing gastroenteritis and hepatitis, respectively, in humans. While a vaccine against HEVs recently became available in China, there is no prophylactic or therapeutic approach against NoVs. Both NoV and HEV have surface protrusions formed by dimers of the protruding (P) domains of the viral capsids, which is responsible for virus–host interactions and eliciting viral neutralizing antibody. We developed in this study a bivalent vaccine against the two viruses through a recently developed polyvalent complex platform. The dimeric P domains of NoV and HEV were fused together, designated as NoV P⁻-HEV P, which was then linked with the dimeric glutathione-S-transferase (GST). After expression and purification in E. coli, the GST-NoV P⁻-HEV P fusion protein assembled into polyvalent complexes with a mean size of 1.8 μm, while the NoV P⁻-HEV P formed oligomers ranging from 100 to 420 kDa. Mouse immunization study demonstrated that both GST-NoV P⁻-HEV P and NoV P⁻-HEV P complexes induced significantly higher antibody titers to NoV P⁻ and HEV P, respectively, than those induced by a mixture of the NoV P⁻ and HEV P dimers. Furthermore, the complex-induced antisera exhibited significantly higher neutralizing activity against HEV infection in HepG2/3A cells and higher blocking activity on NoV P particles binding to HBGA receptors than those of the dimer-induced antisera. Thus, GST-NoV P⁻-HEV P and NoV P⁻-HEV P complexes are promising dual vaccine candidates against both NoV and HEV.     

Immunization of human hepatitis E viruses conferred protection against challenge by a camel hepatitis E virus

Dromedary camel hepatitis E virus is a novel HEV that belongs to the family Hepeviridae, and is classified as genotype 7 HEV (HEV-7). Since HEV-7 is transmitted from camels to humans and causes acute hepatitis E, this virus is a non-negligible pathogen for zoonosis, and a vaccine against HEV-7 infection is urgently needed. Here, we first intravenously inoculated HEV-7 to rhesus monkeys to explore the susceptibility, and we established an animal model. We then used virus-like particles (VLPs) of HEV-1 (HEV-1 VLPs) and HEV-3 (HEV-3 VLPs), a candidate hepatitis E vaccine, to intramuscularly inoculate rhesus monkeys. The monkeys elicited IgG antibody titers as high as >1:102,400 against heterologous HEV-7 without any adjuvants. The HEV-1 VLPs and HEV-3 VLPs-immunized monkeys were challenged intravenously with HEV-7, and they were protected completely from the infection, demonstrating that these VLPs could be a usable vaccine against HEV-7 infection. We also observed that HEV-7-infected rhesus monkeys did not show any liver damage during these experiments. Further efforts are necessary to establish an animal model for investigation of the pathogenesis of hepatitis E caused by HEV-7 infection.     

Alphavirus-vectored hemagglutinin subunit vaccine provides partial protection against heterologous challenge in pigs

Influenza A virus in swine (IAV-S) is an important pathogen in pigs in the United States, in addition to posing a potential risk to humans through zoonotic events. Intervention strategies continue to be explored to better control virus circulation. Improved surveillance efforts has led to significantly increased sequence data available on circulating strains, vastly improving our understanding of the genetic and antigenic diversity of IAV-S. IAV-S in North America is characterized by repeated spillover events of human viruses into pigs followed by genetic and antigenic diversification. An important gap that needs to be addressed is our understanding of the role that various vaccine platforms have on efficacy against antigenically heterologous challenge. Currently licensed vaccines often update their components to adapt to a dynamic antigenic landscape and newly developed technologies continue to be approved. Hence, it remains critical to test the performance of vaccines against challenge with antigenically distinct viruses. We tested the level of protection conferred by an alphavirus-vectored hemagglutinin (HA) subunit vaccine, delivered as a monovalent or bivalent formulation, against challenge with IAV-S. Monovalent alphavirus-vectored HA vaccines provided efficient protection against challenge with viruses with matched and mismatched HA, although in one mismatched HA challenge group there was a trend for reduced protection. A bivalent vaccine, in which two HA’s were simultaneously delivered, was effective in producing antibody response against both antigens and provided protection against challenge. The alphavirus platform is a promising new technology available to swine producers to help reduce the burden of disease caused by IAV-S.     

戊型肝炎疫苗研究进展

戊型肝炎(戊肝)病毒(HEV)经粪-口途径传播,有嗜肝性,发病时表现为急性肝炎.在亚洲及非洲发展中国家均有传播,中国亦有分布.在发达国家,HEV主要影响猪和禽的养殖,密切接触生猪及鲜猪肉的人群中,抗-HEV抗体阳性率较高.虽然在发达国家仅有零星病例,但是普通人群中抗体阳性率比预期要高[1].在中亚和东南亚,HEV是急性临床型肝炎病例最主要的病原体,而在中非、东非和北非,HEV是造成肝病的第二位原因(仅次于HBV)[1].HEV感染的病死率为1%～4%[1],孕妇感染后病死率可高达20%[1,2].HEV分5个基因型,其中1、2型感染人类,3、4型可感染人和猪,5型感染鸟类[1].     

重组戊型肝炎疫苗应用于16~65岁人群的安全性及免疫原性研究

目的 评价长春生物制品研究所有限责任公司研制的重组戊型病毒性肝炎（戊肝）疫苗用于16~65周岁人群免疫的临床安全性与免疫原性。方法 先筛选出戊肝抗体阴性者作为试验目标人群,再采用单中心、随机、盲法、同类制品平行对照的试验设计,选择戊肝抗体阴性的16~65周岁健康受试者60人,按1：1的比例随机接种试验疫苗和阳性对照疫苗,免疫程序均为0,1,6月程序,记录每针次接种后28 d内的不良反应/事件发生情况。采集免疫接种前和全程免疫接种后1个月的血液标本,进行戊肝IgG浓度检测。结果 招募369名受试者,经筛查戊肝抗体阴性者有187人,最终入组60人。试验组和阳性对照组免疫接种后总体征集性不良反应发生率分别为43.33%和40.00%,严重程度以轻、中度为主,最常见接种部位和全身不良反应分别为疼痛和头痛,不良反应的发生在组间差异均无统计学意义（均有P>0.05）。免疫后试验组与阳性对照组戊肝抗体阳性率均达到100%,抗体几何平均浓度（geometric mean concentration,GMC）分别为72.53 U/ml和87.49 U/ml。结论 试验疫苗具有良好的安全性和免疫原性,可以开展Ⅱ期临床试验。     

Long-term protection provided by hepatitis B vaccine and need for booster dose: A meta-analysis

The duration of protection provided by hepatitis B vaccine is still unknown but can be estimated through long-term follow-up studies. Electronic databases and conference databases to December 2008 were searched. Reference lists of articles were screened and the studies authors and manufacturers were contacted for additional unpublished references. Randomized clinical trials and prospective cohort studies addressing the long-term protective effect of hepatitis B vaccine were included in this meta-analysis. We assessed 42 separate cohorts involving overall 11,090 subjects; 34 cohorts involving 9356 subjects were included in the final meta-analysis. Results indicate that the overall cumulative incidence of HBV breakthrough infection 5–20 years post-primary vaccination was 0.007 [95% CI: 0.005 to 0.010] with a variation among studies from 0 to 0.094. Available data do not allow us to exclude an increased risk for infection with time since vaccination. We conclude that the protection provided by three or four doses of monovalent HB vaccine persists for at least two decades in the great majority of immunocompetent individuals. Additional studies are needed for assessing vaccine efficacy for longer periods of time and the need of booster doses in different subgroups of population.     

Yeast expressed classical swine fever E2 subunit vaccine candidate provides complete protection against lethal challenge infection and prevents horizontal virus transmission

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) is a highly contagious swine disease resulting in large economical losses worldwide. The viral envelope glycoprotein E(rns) and E2 are major targets for eliciting antibodies against CSFV in infected animals. A Pichia pastoris yeast expressed E2 protein (yE2) has been shown to induce a protective immune response against CSFV challenge. The purpose of this study is to determine the optimal dose of yE2 and its efficacy on the prevention of virus horizontal transmission. A yeast-expressed E(rns) (yE(rns)) protein was also included to evaluate its immunogenicity. The yE(rns) vaccinated pigs seroconverted to CSFV-E(rns)-specific antibody but no neutralizing antibody was detected and none survived after challenge infection, suggesting yE(rns) and yE2 retain correct immunogenicity but only the yE2 is able to induce a protective immune response. All three doses of yE2 (200, 300, and 400μg) could elicit high titers of neutralizing antibodies and protective responses after challenge. The yE2/200 group demonstrated a mild fever response but recovered soon, and none of the yE2/300 and yE2/400 pigs became febrile. The optimal dose of yE2 was recommended to be 300μg of the total amount of secreted proteins. In addition, the yE2 vaccine could cross-protect from all three genotypes of viruses. Further, the yE2 vaccine efficacy in preventing virus horizontal transmission was evaluated by cohabitation of unimmunized sentinels 3 days after challenge infection. All the sentinel pigs were alive and had no clinical symptoms confirming yE2 vaccine could confer a protective immune response and prevent horizontal transmission of CSFV.     

Immunization with recombinant ORF2 p551 protein protects common marmosets (Callithrix jacchus) against homologous and heterologous hepatitis E virus challenge

BackgroundHepatitis E virus (HEV) is a major causative agent of acute hepatitis worldwide, prompting continuous HEV vaccine efforts. Vaccine development is hampered by the lack of convenient animal models susceptible to infection with different HEV genotypes. We produced recombinant open reading frame 2 protein (pORF2; p551) of HEV genotype (GT) 3 and assessed its immunogenicity and protectivity against HEV challenge in common marmosets (Callithrix jacchus, CM).Methodsp551 with consensus sequence corresponding to amino acid residues 110–660 of HEV GT3 pORF2 was expressed in E. coli and purified by affinity chromatography. CMs were immunized intramuscularly with 20 μg of p551 VLPs with alum adjuvant (n = 4) or adjuvant alone (n = 2) at weeks 0, 3, 7 and 19. At week 27, p551-immunized and control animals were challenged with HEV GT1 or GT3 and thereafter longitudinally screened for markers of liver function, anti-HEV IgG and HEV RNA in feces and sera.ResultsPurified p551 formed VLPs with particle size of 27.71 ± 2.42 nm. Two immunizations with p551 induced anti-HEV IgG mean titer of 1:1810. Immunized CMs challenged with homologous and heterologous HEV genotype did not develop HEV infection during the follow-up. Control CMs infected with both HEV GT1 and GT3 demonstrated signs of HEV infection with virus shedding and elevation of the levels of liver enzymes. High levels of anti-HEV IgG persisted in vaccinated CMs and control CMs that resolved HEV infection, for up to two years post challenge.ConclusionsCMs are shown to be a convenient laboratory animal model susceptible to infection with HEV GT1 and GT3. Immunization with HEV GT3 ORF2/p551 triggers potent anti-HEV antibody response protecting CMs from homologous and heterologous HEV challenge. This advances p551 in VLPs as a prototype vaccine against HEV.     

Design and immunogenicity analysis of the combined vaccine against zoonotic hepatitis E and foot-and-mouth disease

AimDesign and immunogenicity assessment of the combined vaccine candidate against zoonotic hepatitis E virus (HEV) and foot-and-mouth disease virus (FMDV).MethodsUsing the molecular cloning technology, we produced and purified 9 HEV ORF2-truncated proteins (HEV genotype 4). Then, we compared their thermal stability, antigenicity, and immunogenicity to select the best HEV immunogen. Next, we used the adjuvant Montanide ISA-206 to prepare different formulations of HEV vaccine alone, FMDV vaccine alone and HEV-FMDV combined vaccine. The formulations were injected into mice and the induced humoral immune responses were monitored up 12 weeks post-immunization.ResultsThe HEV p222 protein could self-assemble into VLPs (∼34 nm) and showed higher stability and better antigenicity/immunogenicity than the other HEV antigens, thus it was selected as the best HEV immunogen. Mice immunization with the FMDV vaccine alone induced high FMDV-specific antibody titers in a dose-dependent manner; the HEV p222 protein also induced high levels of anti-HEV antibodies but in a dose-independent manner. The HEV-FMDV combination induced anti-FMDV antibody titers 7–16-fold higher than the titers induced by the FMDV vaccine alone, and HEV-specific antibody titers 2.4-fold higher than those induced by the HEV p222 antigen alone.ConclusionHerein, we proposed a new approach for the control of zoonotic HEV infection through its control in its main host (pig). We also designed the first HEV-FMDV combined vaccine and the preliminary analyses revealed a synergistic effect on the immunogenicity of both HEV and FMDV antigens.     

Bat influenza vectored NS1-truncated live vaccine protects pigs against heterologous virus challenge

Swine influenza is an important disease for the swine industry. Currently used whole inactivated virus (WIV) vaccines can induce vaccine-associated enhanced respiratory disease (VAERD) in pigs when the vaccine strains mismatch with the infected viruses. Live attenuated influenza virus vaccine (LAIV) is effective to protect pigs against homologous and heterologous swine influenza virus infections without inducing VAERD but has safety concerns due to potential reassortment with circulating viruses. Herein, we used a chimeric bat influenza Bat09:mH3mN2 virus, which contains both surface HA and NA gene open reading frames of the A/swine/Texas/4199–2/1998 (H3N2) and six internal genes from the novel bat H17N10 virus, to develop modified live-attenuated viruses (MLVs) as vaccine candidates which cannot reassort with canonical influenza A viruses by co-infection. Two attenuated MLV vaccine candidates including the virus that expresses a truncated NS1 (Bat09:mH3mN2-NS1-128, MLV1) or expresses both a truncated NS1 and the swine IL-18 (Bat09:mH3mN2-NS1-128-IL-18, MLV2) were generated and evaluated in pigs against a heterologous H3N2 virus using the WIV vaccine as a control. Compared to the WIV vaccine, both MLV vaccines were able to reduce lesions and virus replication in lungs and limit nasal virus shedding without VAERD, also induced significantly higher levels of mucosal IgA response in lungs and significantly increased numbers of antigen-specific IFN-γ secreting cells against the challenge virus. However, no significant difference was observed in efficacy between the MLV1 and MLV2. These results indicate that bat influenza vectored MLV vaccines can be used as a safe live vaccine to prevent swine influenza.     

A trivalent vaccine candidate against hepatitis E virus,norovirus, and astrovirus

Hepatitis E virus (HEV), norovirus (NoV), and astrovirus (AstV) are enterically-transmitted viral pathogens causing epidemic or endemic hepatitis (HEV) and gastroenteritis (NoV and AstV) respectively in humans, leading to significant morbidity and mortality worldwide. While a recombinant subunit vaccine against HEVs is available in China, there is no commercial vaccine or antiviral against NoV or AstV. We report here our development of a trivalent vaccine against the three viral pathogens through our new polymer vaccine technology. All HEV, NoV, and AstV are non-enveloped RNA viruses covered by a protein capsid, featuring surface protruding (P) proteins that are responsible for virus–host interaction. These dimeric P proteins elicit neutralizing antibody and are good targets for subunit vaccine development. The trivalent subunit vaccine was developed by fusion of the dimeric P domains of the three viruses together that formed tetramers. This trivalent vaccine elicited significantly higher antibody responses in mice against all three P domains than those induced by a mixture of the three free P domains (mixed vaccine). Furthermore, the post-immune antisera of the trivalent vaccine showed significantly higher neutralizing titers against HEV infection in cell culture and higher blocking activity against NoV binding to HBGA ligands than those of the post-immune sera of the mixed vaccine. Thus, the trivalent vaccine is a promising vaccine candidate against HEV, NoV, and AstV.     

母牛分枝杆菌疫苗与乙肝疫苗联合免疫对慢性乙肝患者效果观察

目的：用母牛分枝杆菌疫苗联合乙肝疫苗免疫慢性乙肝患者,观察乙肝患者免疫水平的变化。方法：将慢性乙肝患者随机分成对照组和试验组,试验组按0、1、2免疫程序,在不同部位同时接种22．5μg母牛分枝杆菌疫苗和10μg酵母乙肝疫苗,对照组接种疫苗稀释液。于免疫前后分别采血,对其CD3＋CD4＋、CD3＋CD8＋和淋巴细胞进行计数。结果：试验组与对照组之间CD3＋CD4＋、CD3＋CD8＋、淋巴细胞、CD3＋CD4＋／CD3＋＋CD8＋之数值比较有显著性差异。结论：母牛分枝杆菌疫苗联合乙肝疫苗免疫慢性乙肝患者,能够提高其细胞免疫的应答能力。     

天津铁路疾病预防控制所辖区内戊型肝炎病毒感染状况的血清分析

目的了解北京铁路局天津铁路疾病预防控制所辖区内人群戊型肝炎病毒IgM(HEV-IgM)抗体的水平和分布状况及丙氨酸转氨酶(ALT)和总胆红素(TBil)2项生化指标的基本情况,为今后预防工作提供科学依据。方法采用血清流行病学调查方法,对辖区内9 297名参加预防性健康检查的观察对象,利用酶联免疫吸附试验(ELISA),对其血清中HEV-IgM抗体进行检测,同时利用生化分析仪检测其ALT和TBil水平,并进行统计分析。结果 9 297名观察者中,HEV-IgM抗体阳性率为0.25%。HEV-IgM抗体阳性者平均ALT及TBil水平无显著增高。对1例HEV-IgM抗体阳性者进行跟踪检测,其HEV-IgM抗体在8个月内未见下降,其ALT及TBil水平也无显著增高。结论该辖区内HEV呈隐性潜伏感染,健康人群内有传播HEV病毒的风险。     

Evaluation of the protective effects of a nanogel-based vaccine against rabbit hepatitis E virus

Infection with hepatitis E virus (HEV) has raised serious public health concerns worldwide. In this study, a nanogel-based vaccine encapsulating the capsid protein of rabbit HEV was developed and its protective efficacy was compared with a subunit vaccine. A total of 23 rabbits were divided into 5 groups: (1) negative control (n = 4), (2) positive control (n = 4), (3) nanogel control (n = 5), (4) nanogel vaccine (n = 5), and (5) subunit vaccine (n = 5). Rabbits were vaccinated two times, at weeks 0 and 1, with nanogel and subunit vaccines, respectively, and challenged with rabbit HEV at week 4. By week 11, rabbits vaccinated with the nanogel vaccine produced higher antibodies than those vaccinated with the subunit vaccine. Fecal viral shedding and viremia were identified in rabbits of the positive and nanogel control groups at weeks 6–10. However, there was no viral shedding and viremia in rabbits immunized with both the nanogel and subunit vaccines. Alanine aminotransferase and aspartate aminotransferase levels were not elevated in any rabbit. However, histopathological examination revealed much less hepatic inflammation in rabbits of the nanogel vaccine group compared to the positive and nanogel control groups. Significant increases in IL-12 and IFN–γlevels were identified from rabbits immunized with the nanogel vaccine. Collectively, these results indicate that the newly developed nanogel vaccine induced sufficient immunity leading to complete protection from HEV infection in rabbits. Application of this vaccine should be considered as a preventive measure against HEV infection in other animal species and humans.     

Development of candidate combination vaccine for hepatitis E and hepatitis B: A liposome encapsulation approach

To reduce extra injections, cost and ensure better coverage, use of combination vaccines is preferable. An attempt was made to evaluate the encapsulation of hepatitis E virus neutralizing epitope (NE) region and hepatitis B virus surface antigen (HBsAg) in liposomes as DNAs, proteins and DNA + protein. Mice groups were immunized with different liposome-encapsulated formulations and monitored for anti-HEV and anti-HBs titres, IgG subtypes, antigen-specific lymphocyte proliferation and cytokine levels. The protective levels of anti-HBs and in vitro virus-binding capacity of anti-HEV antibodies were assessed. Liposome-encapsulated DNA either singly or in combination did not elicit antibody response. Anti-HEV and anti-HBs IgG titres of individual component of protein alone (Lipo-E-P/Lipo-B-P) or DNA + protein formulations (Lipo-E-DP/Lipo-B-DP) were comparable to respective titres in combination vaccine of protein (Lipo-BE-P) and DNA + protein formulations (Lipo-BE-DP). IgG1 levels were significantly higher in Lipo-BE-P group whereas, equivalent levels of IgG1 and IgG2a were observed in Lipo-BE-DP group against both components of the vaccine. Combination vaccine group showed mixed Th1/Th2 cytokine profile. Liposome entrapped NE and HBsAg in protein and DNA + protein formats induce excellent immune response to both the components and need to be evaluated in higher animals.     

Hepatitis E vaccine immunization for rabbits to prevent animal HEV infection and zoonotic transmission

Hepatitis E virus (HEV) infection has become a significant global public health concern as increasing cases of acute and chronic hepatitis E are reported. HEV of animal origin was proved to be a possible source of human infection and a previous study showed that the recent licensed HEV 239 vaccine can serve as a candidate vaccine to manage animal sources of HEV infection. However, previous immunization strategy for rabbits was the same as that for human, which is too costly to conduct large-scale animal vaccination. In an effort to reduce the costs, three vaccination schemes were assessed in the present study. Forty specific pathogen-free (SPF) rabbits were divided randomly into five groups with eight animals for each and inoculated intramuscularly with different doses of HEV 239 and placebo, respectively. All animals were challenged intravenously with swine HEV-4 and rabbit HEV of different titers 7 weeks after the initial immunization and then fecal virus excretion was monitored for 10 weeks. The results indicated that immunizing rabbits with two 10 μg doses of the vaccine is superior to vaccination with two 20 μg doses or a single 30 μg dose, which can protect rabbits against homologous and heterologous HEV infection. These findings could enable implementation of large-scale animal vaccination to prevent rabbit HEV infection and zoonotic transmission.