Efficacy of an accelerated vaccination schedule against hepatitis E virus infection in pregnant rabbits.

An important goal of the Hepatitis E virus (HEV) vaccine is to prevent adverse pregnancy outcomes caused by different HEV genotypes during pregnancy, but studies directly evaluating maternal vaccination for HEV are lacking. Here we report maternal vaccination using HEV 239 vaccine in a pregnant rabbit model. Two dose of accelerated vaccination schedule (0, 7 days) induced high titers of anti-HEV protective antibodies in a short period of time in pregnant rabbits, which could protect the pregnant rabbits from HEV infection and adverse pregnancy outcomes. In addition, the immunized rabbits transfer maternal antibodies to pups through the placenta and breast milk, which protect neonates against HEV infection. Our results suggest that, besides vaccinating nonpregnant individuals, HEV 239 vaccine may also be discreetly considered for maternal vaccination.

Immunocompromised rabbit model of chronic HEV reveals liver fibrosis and distinct efficacy of different vaccination strategies.

BACKGROUND AND AIMS: HEV infection can lead to chronicity and rapid progression to liver fibrosis and cirrhosis in immunocompromised organ transplant recipients. Robust animal models are urgently needed to study the pathogenesis and test the efficacy of vaccines and antiviral drugs in immunosuppressed settings. APPROACH AND RESULTS: Cyclosporin A was used to induce immunosuppression. Rabbits were challenged with genotype 3 or 4 HEV (i.e., the rabbit-derived HEV3 and human-derived HEV3 or HEV4). We assessed HEV markers within 13 weeks post inoculation (wpi) and pathological changes by hematoxylin and eosin and Masson staining at 4, 8, or 13 wpi. Chronic HEV infection was successfully established in immunocompromised rabbits. HEV RNA and/or antigens were detected in the liver, kidney, intestine, urine, and cerebrospinal fluid samples. Chronically infected animals exhibited typical characteristics of liver fibrosis development. Intrahepatic transcriptomic analysis indicated activation of both innate and adaptive immunity. Establishment of HEV chronicity likely contributed to the inhibited T-cell immune response. Ribavirin is effective in clearing HEV infection in immunocompromised rabbits. Most interestingly, vaccination completed before immunosuppression conferred full protection against both HEV3 and HEV4 infections, but vaccination during immunosuppression was only partially protective, and the efficacy did not improve with increased or additional vaccine doses. CONCLUSIONS: The immunocompromised rabbit model of both chronic HEV3 and HEV4 infection that was established captured the key features of chronic HEV infection in transplant patients, including liver fibrogenesis, and revealed the distinct effectiveness of vaccination administered before or under immunosuppression. This rabbit model is valuable for understanding the pathogenesis of chronic hepatitis E, as well as for evaluating antiviral agents and vaccines.

Hepatitis E virus infection activates NOD-like receptor family pyrin domain-containing 3 inflammasome antagonizing interferon response but therapeutically targetable.

BACKGROUND AND AIMS: HEV infection is the most common cause of liver inflammation, but the pathogenic mechanisms remain largely unclear. We aim to explore whether HEV infection activates inflammasomes, crosstalk with antiviral interferon response, and the potential of therapeutic targeting. APPROACH AND RESULTS: We measured IL-1ß secretion, the hallmark of inflammasome activation, in serum of HEV-infected patients and rabbits, and in cultured macrophage cell lines and primary monocyte-derived macrophages. We found that genotypes 3 and 4 HEV infection in rabbits elevated IL-1ß production. A profound increase of IL-1ß secretion was further observed in HEV-infected patients (1,733 ± 1,234 pg/mL; n = 70) compared to healthy persons (731 ± 701 pg/mL; n = 70). Given that macrophages are the drivers of inflammatory response, we found that inoculation with infectious HEV particles robustly triggered NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in primary macrophages and macrophage cell lines. We further revealed that the ORF2 capsid protein and the formed integral viral particles are responsible for activating inflammasome response. We also identified NF-κB signaling activation as a key upstream event of HEV-induced NLRP3 inflammasome response. Interestingly, inflammasome activation antagonizes interferon response to facilitate viral replication in macrophages. Pharmacological inhibitors and clinically used steroids can effectively target inflammasome activation. Combining steroids with ribavirin simultaneously inhibits HEV and inflammasome response without cross-interference. CONCLUSIONS: HEV infection strongly activates NLRP3 inflammasome activation in macrophages, which regulates host innate defense and pathogenesis. Therapeutic targeting of NLRP3, in particular when combined with antiviral agents, represents a viable option for treating severe HEV infection.

Characterization of hepatitis E virus natural infection in farmed rabbits.

Rabbit hepatitis E virus (HEV3-ra) is widely distributed in rabbits worldwide and several recent reports found that HEV3-ra can infect humans. Therefore, people exposed to rabbits are at high risk of HEV infection. This study was conducted to investigate the characteristics and outcomes of HEV3-ra natural infection in rabbits. Seventy farmed rabbits (3-month-old) were surveyed in a farm in Beijing, China. Rabbits tested positive for HEV RNA were followed weekly for testing of HEV RNA, antigen, antibody and alanine aminotransferase (ALT) level. Liver and kidney tissue was collected for histopathology. Complete genome sequencing of the isolated HEV3-ra strain was performed (CHN-BJ-r4, GenBank: MT364355). The infectivity of CHN-BJ-r4 was tested in ten naïve rabbits by intravenous injection or gavage. Anti-HEV antibody and HEV RNA were tested positive in 7.14% (5/70) and 11.4% (8/70) of rabbits, respectively. Eight naturally infected rabbits were followed, and 37.5% (3/8) of the observed rabbits were found to have fecal shedding of HEV ranging from 3-22 weeks with high viral load (105 -107 copies/g). Two out of eight rabbits showed temporary viremia. Naturally infected rabbits presented elevated ALT level, seroconversion, and liver histopathology. Complete genome of HEV3-ra isolated in this study shared 84.61%-94.36% nucleotide identity with known HEV3-ra complete genomes. The isolated HEV3-ra strain was infectious and could infect other rabbits through intravenous and fecal-oral route. Naturally infected rabbits showed up to 22-week fecal virus shedding with high viral load. These features increased the risk of rabbit-to-rabbit and rabbit-to-human transmission.

High dose sofosbuvir and sofosbuvir-plus-ribavirin therapy inhibit Hepatitis E Virus (HEV) replication in a rabbit model for acute HEV infection.

Hepatitis E virus (HEV) is an important cause of viral hepatitis worldwide and there is currently no FDA-approved anti-HEV drug. The commonly used drug ribavirin (RBV) could not achieve viral clearance in all patients and can induce drug resistance. Recent studies showed sofosbuvir (SOF) can inhibit HEV replication in vitro and has add-on effect when combined with RBV, but the clinical effect of SOF against HEV infection remains controversial and the dosage of SOF warrants further exploration. In this study, a rabbit model for acute HEV infection was used to evaluate the effect of SOF at different doses against HEV genotype 3 and 4, and to compare the antiviral effect of SOF-plus-RBV therapy with RBV monotherapy. Virological parameters on fecal, serological and intrahepatic level were tested by real-time PCR and ELISA. Liver function tests and histopathological assays were performed. Both 200 mg/d and 300 mg/d SOF treatment inhibits HEV replication with relieved liver inflammation and declined levels of fecal HEV RNA and antigenemia. 300 mg/d SOF eliminated HEV replication while a short viral rebound was observed after 200 mg/d SOF treatment. The SOF-plus-RBV therapy also showed stronger anti-HEV effect than RBV monotherapy. Our study suggests that high dose of SOF showed anti-HEV effect in the rabbit model. Moreover, the de novo SOF-plus-RBV therapy which eliminated acute HEV infection more efficiently than RBV monotherapy may serve as an alternative treatment strategy but warrants further preclinical and clinical study.

Infectivity and pathogenicity of different hepatitis E virus genotypes/subtypes in rabbit model.

The pathogenicity of each hepatitis E virus (HEV) genotypes/subtypes may be different. This study aimed to investigate the infectivity and pathogenicity of different HEV genotypes/subtypes from different mammalian sources especially human in rabbits, and to assess whether rabbits are an appropriate animal model to study different HEV genotypes/subtypes. Thirty-seven rabbits were randomly divided into nine groups and inoculated with eight different HEV strains, including human-derived HEV3b (hHEV-3b), hHEV-4a, hHEV-4d and hHEV-4h, swine-derived HEV4d (sHEV-4d) and sHEV-4h, rabbit-derived HEV3 (HEV-3ra) and camel-derived HEV8. HEV RNA, antigen, anti-HEV and alanine aminotransferase (ALT) in serum or/and feces were monitored weekly. One rabbit from each group was euthanized at seven weeks post inoculation and the liver specimens were taken for histopathological analysis and immunofluorescence staining of HEV ORF2 proteins. hHEV-4d, sHEV-4d and HEV-3ra infections were successfully established in rabbits and typical acute hepatitis symptoms were observed, including viraemia/antigenemia, fecal virus/antigen shedding, elevated ALT level and liver histopathological changes. One rabbit infected with HEV-3ra showed chronic infection. hHEV-4d and sHEV-4d are less infectious and pathogenic than HEV-3ra in rabbits. hHEV-3b and HEV8 only caused inapparent infection in rabbits as 60% (3/5) and 20% (1/5) of the rabbits seroconverted to anti-HEV, respectively. No obvious signs of HEV infection in rabbits inoculated with hHEV-4a, hHEV-4h and sHEV-4h. The infectivity and pathogenicity of different HEV genotypes/subtypes in rabbits is different, which may be related to the species specificity of HEV. Rabbit can be used as an animal model for the study of HEV-3ra and more importantly human HEV-4d.

Hepatitis E-related adverse pregnancy outcomes and their prevention by hepatitis E vaccine in a rabbit model.

Hepatitis E virus (HEV) can lead to high mortality during pregnancy. This study was to investigate the adverse pregnancy outcomes caused by different HEV genotypes and their prevention by HEV 239 vaccine in rabbits. Forty-two female rabbits were randomly and equally divided into 7 groups (A-G). HEV 239 vaccine and a placebo were administered to groups E (10 µg×2), F (5 µg×2) and G (1 mL of PBS×2) before copulation. After pregnancy, 1 mL of 1.5×106 copies/mL rabbit HEV3 was inoculated to groups A, E, F and G, swine HEV4/human HEV3 to groups B/C, and group D was a negative control. Anti-HEV antibody, HEV RNA, and alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels were monitored. Pregnant rabbits infected by HEV manifested HEV infection symptoms including fecal virus shedding, ALT/AST elevation, and histopathological changes, and adverse pregnancy outcomes. Immunized pregnant rabbits in groups E and F showed no HEV infection symptoms and adverse outcomes. The newborn rabbits delivered by pregnant rabbits with/without immunization showed without/with HEV infection symptoms. This study demonstrated that multiple genotypes of HEV infection can cause adverse outcomes and HEV 239 vaccine can prevent HEV-related adverse outcomes in pregnant rabbits.