A hepatitis B virus core antigen-based virus-like particle vaccine expressing SARS-CoV-2 B and T cell epitopes induces epitope-specific humoral and cell-mediated immune responses but confers limited protection against SARS-CoV-2 infection.

The hepatitis B virus core antigen (HBcAg) tolerates insertion of foreign epitopes and maintains its ability to self-assemble into virus-like particles (VLPs). We constructed a ∆HBcAg-based VLP vaccine expressing three predicted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B and T cell epitopes and determined its immunogenicity and protective efficacy. The recombinant ∆HBcAg-SARS-CoV-2 protein was expressed in Escherichia coli, purified, and shown to form VLPs. K18-hACE2 transgenic C57BL/6 mice were immunized intramuscularly with ∆HBcAg VLP control (n = 15) or ∆HBcAg-SARS-CoV-2 VLP vaccine (n = 15). One week after the 2nd booster and before virus challenge, five ∆HBcAg-SARS-CoV-2 vaccinated mice were euthanized to evaluate epitope-specific immune responses. There is a statistically significant increase in epitope-specific Immunoglobulin G (IgG) response, and statistically higher interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) expression levels in ∆HBcAg-SARS-CoV-2 VLP-vaccinated mice compared to ∆HBcAg VLP controls. While not statistically significant, the ∆HBcAg-SARS-CoV-2 VLP mice had numerically more memory CD8+ T-cells, and 3/5 mice also had numerically higher levels of interferon gamma (IFN-γ) and tumor necrosis factor (TNF). After challenge with SARS-CoV-2, ∆HBcAg-SARS-CoV-2 immunized mice had numerically lower viral RNA loads in the lung, and slightly higher survival, but the differences are not statistically significant. These results indicate that the ∆HBcAg-SARS-CoV-2 VLP vaccine elicits epitope-specific humoral and cell-mediated immune responses but they were insufficient against SARS-CoV-2 infection.

Hepatitis E Virus in Pigs from Slaughterhouses, United States, 2017-2019.

Hepatitis E virus (HEV) RNA was detected in 6.3% and HEV IgG in 40% of 5,033 serum samples from market-weight pigs at 25 slaughterhouses in 10 US states. The prevalent HEV genotype was zoonotic genotype 3, group 2. Blood of HEV-viremic pigs from slaughterhouses may contaminate pork supply chains.

The U-Rich Untranslated Region of the Hepatitis E Virus Induces Differential Type I and Type III Interferon Responses in a Host Cell-Dependent Manner.

Hepatitis E virus (HEV), a single-strand positive-sense RNA virus, is an understudied but important human pathogen. The virus can establish infection at a number of host tissues, including the small intestine and liver, causing acute and chronic hepatitis E as well as certain neurological disorders. The retinoic acid-inducible gene I (RIG-I) pathway is essential to induce the interferon (IFN) response during HEV infection. However, the pathogen-associated motif patterns (PAMPs) in the HEV genome that are recognized by RIG-I remain unknown. In this study, we first identified that HEV RNA PAMPs derived from the 3' untranslated region (UTR) of the HEV genome induced higher levels of IFN mRNA, interferon regulatory factor-3 (IRF3) phosphorylation, and nuclear translocation than the 5' UTR of HEV. We revealed that the U-rich region in the 3' UTR of the HEV genome acts as a potent RIG-I PAMP, while the presence of poly(A) tail in the 3' UTR further increases the potency. We further demonstrated that HEV UTR PAMPs induce differential type I and type III IFN responses in a cell type-dependent fashion. Predominant type III IFN response was observed in the liver tissues of pigs experimentally infected with HEV as well as in HEV RNA PAMP-induced human hepatocytes in vitro In contrast, HEV RNA PAMPs induced a predominant type I IFN response in swine enterocytes. Taken together, the results from this study indicated that the IFN response during HEV infection depends both on viral RNA motifs and host target cell types. The results have important implications in understanding the mechanism of HEV pathogenesis.IMPORTANCE Hepatitis E virus (HEV) is an important human pathogen causing both acute and chronic viral hepatitis E infection. Currently, the mechanisms of HEV replication and pathogenesis remain poorly understood. The innate immune response acts as the first line of defense during viral infection. The retinoic acid-inducible gene I (RIG-I)-mediated interferon (IFN) response has been implicated in establishing antiviral response during HEV infection, although the HEV RNA motifs that are recognized by RIG-I are unknown. This study identified that the U-rich region in the 3' untranslated region (UTR) of the HEV genome acts as a potent RIG-I agonist compared to the HEV 5' UTR. We further revealed that the HEV RNA pathogen-associated motif patterns (PAMPs) induced a differential IFN response in a cell type-dependent manner: a predominantly type III IFN response in hepatocytes, and a predominantly type I IFN response in enterocytes. These data demonstrate the complexity by which both host and viral factors influence the IFN response during HEV infection.

Risk factors and sources of foodborne hepatitis E virus infection in the United States.

Hepatitis E virus (HEV) is an important human pathogen with pigs and other species serving as natural animal reservoirs. Ample evidence documents sporadic cases of hepatitis E acquired via consumption of undercooked meat. Chronic hepatitis E cases in immunosuppressed individuals are mostly caused by zoonotic HEV of swine origin. We report here the identification of genotype 3 HEV from non-liver commercial pork from local grocery stores in southwest Virginia, and association of HEV seropositivity to the consumption of undercooked meat in healthy young adults at a university in the United States. These results raise concerns about foodborne HEV transmission in the United States. J. Med. Virol. 88:1641-1645, 2016. © 2016 Wiley Periodicals, Inc.

Replacement of the hepatitis E virus ORF3 protein PxxP motif with heterologous late domain motifs affects virus release via interaction with TSG101.

The ORF3 protein of hepatitis E virus (HEV) contains a "PSAP" amino acid late domain motif, which allows for interaction with the endosomal sorting complexes required for transport (ESCRT) pathway aiding virion release. Late domain motifs are interchangeable with other viral late domain motifs in several enveloped viruses, however, it remains unknown whether HEV shares this functional interchangeability and what implications this might have on viral replication. In this study, by substituting heterologous late domain motifs (PPPY, YPDL, and PSAA) for the HEV ORF3 late domain (PSAP), we demonstrated that deviation from the PSAP motif reduces virus release as measured by viral RNA in culture media. Virus release could not be restored by insertion of a heterologous late domain motif or by supplying wild-type ORF3 in trans, suggesting that the HEV PSAP motif is required for viral exit which cannot be bypassed by the use of alternative heterologous late domains.

Membrane-bound IL-12 and IL-23 serve as potent mucosal adjuvants when co-presented on whole inactivated influenza vaccines.

BACKGROUND: Potent and safe adjuvants are needed to improve the efficacy of parenteral and mucosal vaccines. Cytokines, chemokines and growth factors have all proven to be effective immunomodulatory adjuvants when administered with a variety of antigens. We have previously evaluated the efficacy of membrane-anchored interleukins (IL) such as IL-2 and IL-4 co-presented as Cytokine-bearing Influenza Vaccines (CYT-IVACs) using a mouse model of influenza challenge. FINDINGS: Here, we describe studies evaluating the parenteral and mucosal adjuvanticity of membrane-bound IL-12 and IL-23 CYT-IVACs in young adult mice. Mucosal immunization using IL-12 and IL-23 bearing whole influenza virus vaccine (WIV) was more effective at eliciting virus-specific nasal IgA and reducing viral lung burden following challenge compared to control WIV vaccinated animals. Both IL-12 and IL-23 bearing WIV elicited the highest anti-viral IgA levels in serum and nasal washes. CONCLUSIONS: This study highlights for the first time the mucosal adjuvant potential of IL-12 and IL-23 CYT-IVAC formulations in eliciting mucosal immune responses and reducing viral lung burden. The co-presentation of immunomodulators in direct context with viral antigen in whole inactivated viral vaccines may provide a means to significantly lower the dose of vaccine required for protection.

Tailored vaccines targeting the elderly using whole inactivated influenza vaccines bearing cytokine immunomodulators.

Influenza and its complications disproportionately affect the elderly, leading to high morbidity and mortality in this ever-increasing population. Despite widespread vaccination efforts, the current influenza vaccines are less effective in the elderly; hence newer vaccine strategies are needed to improve their efficacy in this age group. We have previously shown that co-presentation of cytokines on the surface of inactivated influenza virus particles affords better protection from lethal homotypic viral challenge in young adult mice than conventional non-adjuvanted whole inactivated vaccine. Here, we determined the efficacy of these vaccine formulations in Balb/c mice "aged" to 17 months ("aged mice") along with the addition of a membrane-bound interleukin-12 (IL-12) vaccine formulation. Our investigations found that a single low-dose intramuscular vaccination with inactivated whole influenza vaccine co-presenting IL-12 was sufficient to provide enhanced protection from subsequent influenza challenge as compared with non-adjuvanted whole inactivated vaccine. Our results indicate that incorporation of cytokines such as IL-12 in a membrane-bound formulation in whole inactivated vaccine may provide a means to lower the vaccine dose while eliciting enhanced protective responses in the elderly, an age group that responds poorly to current vaccination regimens.