Fetal Loss in Pregnant Rabbits Infected with Genotype 3 Hepatitis E Virus Is Associated with Altered Inflammatory Responses, Enhanced Virus Replication, and Extrahepatic Virus Dissemination with Positive Correlations with Increased Estradiol Level.

Hepatitis E virus (HEV) causes adverse clinical outcomes in pregnant women, but the underlying mechanisms remain poorly understood. To delineate the mechanisms of pregnancy-associated adverse effects during HEV infection, we utilized a genotype 3 HEV from rabbit (HEV-3ra) and its cognate host (rabbits) to systematically investigate the clinical consequences, viral replication dynamics, and host immune and hormonal responses of HEV infection during pregnancy. We found a significant fetal loss of 23% in HEV-infected pregnant rabbits, indicating an early-stage miscarriage. HEV infection in pregnant rabbits was characterized by higher viral loads in feces, intestinal contents, liver, and spleen tissues, as well as a longer and earlier onset of viremia than in infected nonpregnant rabbits. HEV infection altered the pattern of cytokine gene expressions in the liver of pregnant rabbits and caused a transient increase of serum interferon gamma (IFN-γ) shortly after a notable increase in viral replication, which may contribute to early fetal loss. Histological lesions in the spleen were more pronounced in infected pregnant rabbits, although moderate liver lesions were seen in both infected pregnant and nonpregnant rabbits. Total bilirubin was elevated in infected pregnant rabbits. The serum levels of estradiol (E2) in HEV-infected pregnant rabbits were significantly higher than those in mock-infected pregnant rabbits at 14 days postinoculation (dpi) and correlated positively with higher viral loads in feces, liver, and spleen tissues at 28 dpi, suggesting that it may play a role in extrahepatic virus dissemination. The results have important implications for understanding the severe diseases associated with HEV infection during pregnancy. IMPORTANCE HEV causes adverse pregnancy outcomes, with a mortality rate of >30% in pregnant women, but the underlying mechanisms are poorly understood. In this study, we utilized HEV-3ra and its cognate host (pregnant rabbit) to delineate the potential underlying mechanisms of pregnancy-associated adverse outcomes during HEV infection. We found that infected pregnant rabbits had a fetal loss of 23%, which coincided with enhanced viral replication and an elevated systemic IFN-γ response, followed by longer viremia duration and extrahepatic viral dissemination. Estradiol levels were increased in infected pregnant rabbits and correlated positively with higher fecal viral shedding and higher viral loads in liver and spleen tissues. Infected pregnant rabbits had more pronounced splenic lesions, higher serum total bilirubin, and an altered cytokine gene expression profile in the liver. The results will contribute to our understanding of the mechanisms of HEV-associated adverse pregnancy outcomes.

Ribavirin Treatment Failure-Associated Mutation, Y1320H, in the RNA-Dependent RNA Polymerase of Genotype 3 Hepatitis E Virus (HEV) Enhances Virus Replication in a Rabbit HEV Infection Model.

Chronic hepatitis E virus (HEV) infection has become a significant clinical problem that requires treatment in immunocompromised individuals. In the absence of an HEV-specific antiviral, ribavirin (RBV) has been used off-label, but treatment failure may occur due to mutations in the viral RNA-dependent RNA polymerase (RdRp), including Y1320H, K1383N, and G1634R. Chronic hepatitis E is mostly caused by zoonotic genotype 3 HEV (HEV-3), and HEV variants from rabbits (HEV-3ra) are closely related to human HEV-3. Here, we explored whether HEV-3ra, along with its cognate host, can serve as a model to study RBV treatment failure-associated mutations observed in human HEV-3-infected patients. By utilizing the HEV-3ra infectious clone and indicator replicon, we generated multiple single mutants (Y1320H, K1383N, K1634G, and K1634R) and a double mutant (Y1320H/K1383N) and assessed the role of mutations on replication and antiviral activity of HEV-3ra in cell culture. Furthermore, we also compared the replication of the Y1320H mutant with the wild-type HEV-3ra in experimentally infected rabbits. Our in vitro analyses revealed that the effects of these mutations on rabbit HEV-3ra are altogether highly consistent with those on human HEV-3. Importantly, we found that the Y1320H enhances virus replication during the acute stage of HEV-3ra infection in rabbits, which corroborated our in vitro results showing an enhanced viral replication of Y1320H. Taken together, our data suggest that HEV-3ra and its cognate host is a useful and relevant naturally occurring homologous animal model to study the clinical relevance of antiviral-resistant mutations observed in human HEV-3 chronically-infected patients. IMPORTANCE HEV-3 causes chronic hepatitis E that requires antiviral therapy in immunosuppressed individuals. RBV is the main therapeutic option for chronic hepatitis E as an off-label use. Several amino acid changes, including Y1320H, K1383N, and G1634R, in the RdRp of human HEV-3 have reportedly been associated with RBV treatment failure in chronic hepatitis E patients. In this study, we utilized an HEV-3ra from rabbit and its cognate host to investigate the effect of these RBV treatment failure-associated HEV-3 RdRp mutations on viral replication efficiency and antiviral susceptibility. The in vitro data using rabbit HEV-3ra was highly comparable to those from human HEV-3. We demonstrated that the Y1320H mutation significantly enhanced HEV-3ra replication in cell culture and enhanced virus replication during the acute stage of HEV-3ra infection in rabbits. The rabbit HEV-3ra infection model should be useful in delineating the role of human HEV-3 RBV treatment failure-associated mutations in antiviral resistance.

Modulation of SOCS3 Levels via STAT3 and Estrogen-ERαp66 Signaling during Hepatitis E Virus Replication in Hepatocellular Carcinoma Cells.

Hepatitis E virus (HEV) infection usually results in a self-limiting acute disease; however, in infected pregnant women, it is associated with increased mortality and fulminant hepatic failure. Estrogen is known to be elevated during pregnancy, and estrogen signaling via classical estrogen receptor-ERα is known to regulate hepatocyte function and host innate immune response, including the STAT3 pathway. In this study, we investigated whether the estrogen classical signaling pathway via ERαp66 has any effect on STAT3 activation during HEV replication and HEV-induced IFN response. We first demonstrated that Huh7-S10-3 liver cells expressed the nonfunctional estrogen receptor ERαp36 isoform and lack the functional ERαp66 isoform. We further showed persistent phosphorylated-STAT3 levels in genotype 3 human HEV (Kernow P6 strain) RNA-transfected cells at later time points. In Huh7-S10-3 cells, estrogen at first-to-third trimester concentration (7.3 to 73 nM) did not significantly affect HEV replication; however, blocking of STAT3 activation led to a decrease in the HEV ORF2 protein level. Our mechanistic study revealed that STAT3 differentially regulates SOCS3 and type-III interferon (IFN) levels during HEV replication and the presence of estrogen-ERαp66 signaling stabilizes SOCS3 levels in vitro. We also demonstrate that HEV infection in pregnant and nonpregnant rabbits led to a significant increase in IFN response as measured by increased levels of IFN-stimulated-gene-15 (ISG15) mRNA levels irrespective of pregnancy status. Collectively, the results indicate that estrogen signaling and STAT3 regulate SOCS3 and IFN responses in vitro during HEV replication. The results have important implications for understanding HEV replication and HEV-induced innate immune response in pregnant women. IMPORTANCE Hepatitis E is usually a self-resolving acute disease; however, in pregnant women, HEV infection is associated with high mortality and fulminant hepatic failure. During pregnancy, estrogen levels are elevated, and in the liver, the estrogen receptor ERα is predominant and estrogen signaling is known to regulate hepatocyte metabolism and leptin-induced STAT3 levels. Viruses can module host innate immune response via STAT3. Therefore, in this study, we investigated whether STAT3 and estrogen-classical signaling via the ERαp66 pathway modulate HEV replication and HEV-induced innate immune response. We demonstrated that estrogen signaling did not affect HEV replication in human liver cells, but blocking of STAT3 activation reduced HEV capsid protein levels in human liver cells. We also showed that inhibition of STAT3 activation reduced SOCS3 levels, while the presence of the estrogen-ERαp66 signaling pathway stabilized SOCS3 levels. The results from this study will aid our understanding of the mechanism of HEV pathogenesis and immune response during pregnancy.

Two mutations in the ORF1 of genotype 1 hepatitis E virus enhance virus replication and may associate with fulminant hepatic failure.

Hepatitis E virus (HEV) infection in pregnant women has a high incidence of developing fulminant hepatic failure (FHF) with significant mortality. Multiple amino acid changes in genotype 1 HEV (HEV-1) are reportedly linked to FHF clinical cases, but experimental confirmation of the roles of these changes in FHF is lacking. By utilizing the HEV-1 indicator replicon and infectious clone, we generated 11 HEV-1 single mutants, each with an individual mutation, and investigated the effect of these mutations on HEV replication and infection in human liver cells. We demonstrated that most of the mutations actually impaired HEV-1 replication efficiency compared with the wild type (WT), likely due to altered physicochemical properties and structural conformations. However, two mutations, A317T and V1120I, significantly increased HEV-1 replication. Notably, these two mutations simultaneously occurred in 100% of 21 HEV-1 variants from patients with FHF in Bangladesh. We further created an HEV-1 A317T/V1120I double mutant and found that it greatly enhanced HEV replication, which may explain the rapid viral replication and severe disease. Furthermore, we tested the effect of these FHF-associated mutations on genotype 3 HEV (HEV-3) replication and found that all the mutants had a reduced level of replication ability and infectivity, which is not unexpected due to distinct infection patterns between HEV-1 and HEV-3. Additionally, we demonstrated that these FHF-associated mutations do not appear to alter their sensitivity to ribavirin (RBV), suggesting that ribavirin remains a viable option for antiviral therapy for patients with FHF. The results have important implications for understanding the mechanism of HEV-1-associated FHF.

Hepatitis E virus infects brain microvascular endothelial cells, crosses the blood-brain barrier, and invades the central nervous system.

Hepatitis E virus (HEV) is an important but understudied zoonotic virus causing both acute and chronic viral hepatitis. A proportion of HEV-infected individuals also developed neurological diseases such as Guillain-Barré syndrome, neuralgic amyotrophy, encephalitis, and myelitis, although the mechanism remains unknown. In this study, by using an in vitro blood-brain barrier (BBB) model, we first investigated whether HEV can cross the BBB and whether the quasi-enveloped HEV virions are more permissible to the BBB than the nonenveloped virions. We found that both quasi-enveloped and nonenveloped HEVs can similarly cross the BBB and that addition of proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has no significant effect on the ability of HEV to cross the BBB in vitro. To explore the possible mechanism of HEV entry across the BBB, we tested the susceptibility of human brain microvascular endothelial cells lining the BBB to HEV infection and showed that brain microvascular endothelial cells support productive HEV infection. To further confirm the in vitro observation, we conducted an experimental HEV infection study in pigs and showed that both quasi-enveloped and nonenveloped HEVs invade the central nervous system (CNS) in pigs, as HEV RNA was detected in the brain and spinal cord of infected pigs. The HEV-infected pigs with detectable viral RNA in CNS tissues had histological lesions in brain and spinal cord and significantly higher levels of proinflammatory cytokines TNF-α and interleukin 18 than the HEV-infected pigs without detectable viral RNA in CNS tissues. The findings suggest a potential mechanism of HEV-associated neuroinvasion.

Killed whole-genome reduced-bacteria surface-expressed coronavirus fusion peptide vaccines protect against disease in a porcine model.

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible gram-negative autotransporter to express vaccine antigens on the surface of genome-reduced bacteria to enhance interaction of vaccine antigen with the immune system. As a proof-of-principle, we utilized genome-reduced Escherichia coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as killed whole-cell vaccines. The FP sequence is highly conserved across coronaviruses; the six FP core amino acid residues, along with the four adjacent residues upstream and the three residues downstream from the core, are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated interferon-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine-appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.

Construction and efficacy evaluation of novel swine leukocyte antigen (SLA) class I and class II allele-specific poly-T cell epitope vaccines against porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease. Here we report the development of subunit PRRSV-2 vaccines by expressing swine leucocyte antigen (SLA) class I and class II allele-specific epitope antigens in a robust adenovirus vector. SLA I-specific CD8 and SLA II-specific CD4 T cell epitopes of PRRSV-2 NADC20 were predicted in silico. Stable murine leukaemia cell lines (RMA-S), which are TAP-deficient and lacking endogenous class I epitope loading, were established to express different SLA I alleles. The binding stability of PRRSV T cell epitope peptides with SLA I alleles expressed on RMA-S cells was characterized. Two PRRSV poly-T cell epitope peptides were designed. NADC20-PP1 included 39 class I epitopes, consisting of 8 top-ranked epitopes specific to each of 5 SLA I alleles, and fused to 5 class II epitopes specific to SLA II alleles. NADC20-PP2, a subset of PP1, included two top-ranked class I epitopes specific to each of the five SLA I alleles. Two vaccine candidates, Ad-NADC20-PP1 and Ad-NADC20-PP2, were constructed by expressing the polytope peptides in a replication-incompetent human adenovirus 5 vector. A vaccination and challenge study in 30 piglets showed that animals vaccinated with the vaccines had numerically lower gross and histopathology lung lesions, and numerically lower PRRSV RNA loads in lung and serum after challenge compared to the controls, although there was no statistical significance. The results suggested that the Ad-NADC20-PP1 and Ad-NADC20-PP2 vaccines provided little or no protection, further highlighting the tremendous challenges faced in developing an effective subunit PRRSV-2 vaccine.

Cytotoxic T lymphocyte epitopes identified from a contemporary strain of porcine reproductive and respiratory syndrome virus enhance CD4+CD8+ T, CD8+ T, and γδ T cell responses.

Immuno-stimulatory class I-restricted cytotoxic T lymphocytes (CTL) epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) are important for vaccine development. In this study we first determined the expression frequency of swine leukocyte antigen (SLA) class I alleles in commercial pigs in the United States. The SLA genotyping result allowed us to predict potential CTL epitopes from a contemporary strain of PRRSV (RFLP 1-7-4) by using bioinformatic tools. The predicted epitopes were then evaluated in an ex vivo stimulation assay with peripheral blood mononuclear cells isolated from pigs experimentally-infected with PRRSV. Using flow-cytometry analysis, we identified a number of immuno-stimulatory CTL epitopes, including two peptides from GP3 and two from Nsp9 that significantly improved both degranulation marker CD107a and IFN-γ production in cytotoxic CD4+CD8+ T cells, CD8+ T cells, and γδ T cells, and two peptides that inhibited IFN-γ production. These CTL epitopes will aid future vaccine development against PRRSV.

Evaluation of the pathogenicity of mammalian orthoreovirus type 3 (MRV3) in germ-free gnotobiotic pigs and of the efficacy of an inactivated vaccine against MRV3 infection in neonatal conventional piglets.

A novel U.S. strain of mammalian orthoreovirus type 3 (MRV3) isolated from diarrheic pigs in 2015 was reportedly highly pathogenic in pigs. In this study, we first developed an inactivated MRV3 vaccine and determined its protective efficacy against MRV3 infection in conventional neonatal piglets. A pathogenicity study was also conducted in gnotobiotic pigs to further assess the pathogenicity of MRV3. To evaluate if piglets could be protected against MRV3 infection after immunization of pregnant sows with an inactivated MRV3 vaccine, pregnant sows were vaccinated with 2 or 3 doses of the vaccine or with PBS buffer. Four-day-old piglets born to vaccinated and unvaccinated sows were subsequently challenged with MRV3. The results showed that piglets born from vaccinated sows had lower levels of fecal viral RNA shedding at 1, 3, and 4 days post-challenge, suggesting that the inactivated MRV3 vaccine can reduce MRV3 replication. Surprisingly, although the conventional piglets were infected, they did not develop severe enteric disease as reported previously. Therefore, in an effort to further definitively assess the pathogenicity of MRV3, we experimentally infected gnotobiotic pigs, a more sensitive model for pathogenicity study, with the wild-type MRV3 virus. The infected gnotobiotic piglets all survived and exhibited only very mild diarrhea in some pigs. Taken together, the results indicate that the novel strain of MRV3 recently isolated in the United States infected but caused only very mild diarrhea in pigs, and that maternal immunity acquired from sows vaccinated with an inactivated vaccine can reduce MRV3 replication in neonatal pigs.

Infection Dynamics of Hepatitis E Virus in Wild-Type and Immunoglobulin Heavy Chain Knockout JH-/- Gnotobiotic Piglets.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain JH-/- knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain JH-/- knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain JH-/- knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the JH-/- knockout pigs, suggesting that the Ig heavy chain JH-/- knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and JH-/- knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected JH-/- knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems.IMPORTANCE According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain JH-/- knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel JH-/- knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and JH-/- knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected JH-/- knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

Recombinant Porcine Reproductive and Respiratory Syndrome Virus Expressing Membrane-Bound Interleukin-15 as an Immunomodulatory Adjuvant Enhances NK and γδ T Cell Responses and Confers Heterologous Protection.

Cytokines are often used as adjuvants to improve vaccine immunogenicity, since they are important in initiating and shaping the immune response. The available commercial modified live-attenuated vaccines (MLVs) against porcine reproductive and respiratory syndrome virus (PRRSV) are unable to mount sufficient heterologous protection, as they typically induce weak innate and inadequate T cell responses. In this study, we investigated the immunogenicity and vaccine efficacy of recombinant PRRSV MLVs incorporated with the porcine cytokine interleukin-15 (IL-15) or IL-18 gene fused to a glycosylphosphatidylinositol (GPI) modification signal that can anchor the cytokines to the cell membrane. We demonstrated that both cytokines were successfully expressed on the cell membrane of porcine alveolar macrophages after infection with recombinant MLVs. Pigs vaccinated with recombinant MLVs or the parental Suvaxyn MLV had significantly reduced lung lesions and viral RNA loads in the lungs after heterologous challenge with the PRRSV NADC20 strain. The recombinant MLVs SUV-IL-15 and SUV-IL-18 recovered the inhibition of the NK cell response seen with Suvaxyn MLV. The recombinant MLV SUV-IL-15 significantly increased the numbers of gamma interferon (IFN-γ)-producing cells in circulation at 49 days postvaccination (dpv), especially for IFN-γ-producing CD4- CD8+ T cells and γδ T cells, compared to the Suvaxyn MLV and SUV-IL-18. Additionally, MLV SUV-IL-15-vaccinated pigs also had elevated levels of γδ T cell responses observed at 7 dpv, 49 dpv, and 7 days postchallenge. These data demonstrate that the recombinant MLV expressing membrane-bound IL-15 enhances NK and T cell immune responses after vaccination and confers improved heterologous protection, although this was not statistically significant compared to the parental MLV.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) has arguably been the most economically important global swine disease, causing immense economic losses worldwide. The available commercial modified live-attenuated vaccines (MLVs) against PRRS virus (PRRSV) are generally effective against only homologous or closely related virus strains but are ineffective against heterologous strains, partially due to the insufficient immune response induced by the vaccine virus. To improve the immunogenicity of MLVs, in this study, we present a novel approach of using porcine IL-15 or IL-18 as an adjuvant by directly incorporating its encoding gene into a PRRSV MLV and expressing it as an adjuvant. Importantly, we directed the expression of the incorporated cytokines to the cell membrane surface by fusing the genes with a membrane-targeting signal from CD59. The recombinant MLV virus expressing the membrane-bound IL-15 cytokine greatly enhanced NK cell and γδ T cell responses and also conferred improved protection against heterologous challenge with the PRRSV NADC20 strain.

Isolation of Peripheral Blood CD8 T Cells Specific to Porcine Reproductive and Respiratory Syndrome Virus Utilizing Porcine CD137 Activation Marker.

CD137 is a costimulatory molecule transiently expressed on activated T cells after mitogen or antigen stimulation that can be exploited for isolating antigen-specific T cells as reported in mouse models. By utilizing an antiporcine CD137 monoclonal antibody (mAb, clone 3B9) developed in our laboratory, we isolated virus-specific CD8ß T cells from peripheral blood of pigs experimentally infected with different porcine reproductive and respiratory syndrome virus (PRRSV) strains. Similar to mouse, porcine CD8ß T cells also express CD137 transiently upon Concavalin A stimulation while the unstimulated cells did not. Most frequently, virus-specific CD8ß T cells were isolated at low levels from peripheral blood of pigs experimentally infected with PRRSV strains VR2385, NADC20, and MN184B at 49 and 63 days postinfection. The results suggest that porcine CD137-specific mAb is a useful tool for isolating virus-specific CD8 T cells from peripheral blood and tissues of pigs after in vitro stimulation with viral antigen.

Vaccination of sows with a dendritic cell-targeted porcine epidemic diarrhea virus S1 protein-based candidate vaccine reduced viral shedding but exacerbated gross pathological lesions in suckling neonatal piglets.

Porcine epidemic diarrhea virus (PEDV) poses a serious threat to swine worldwide as evidenced by its recent introduction into the USA and the devastating economic impact it caused to the USA swine industry. Commercial vaccines against PEDV are available but their efficacies are inadequate. Therefore, vaccines with improved efficacy are needed to effectively control PEDV infections. We previously determined the immunogenicity of a novel dendritic cell (DC)-targeted PEDV S1 protein-based subunit vaccine in weaned piglets in which the PEDV antigen was targeted to DCs through a porcine Langerin-specific antibody. In this study, we evaluated the protective efficacy of this DC-targeting vaccine by immunizing sows at 5 and 2 weeks prior to farrowing and by challenging the 5-day-old piglets with PEDV. The results showed that immunization of sow with DC-targeted PEDV vaccine did not eliminate faecal virus shedding in piglets but significantly reduced faecal viral RNA levels in the early days after virus challenge. The vaccine also reduced the amount of PEDV antigen in intestinal tissues presented with intestinal villi regrowth. However, the DC-targeted vaccine neither mitigated PEDV clinical signs nor affected viral RNA loads in intestinal tissues of piglets. In the vaccinated sow, DC-targeted PEDV vaccine enhanced T helper 1-like cluster of differentiation (CD)4 T cell responses and induced IgG but not IgA-specific immune responses. The suckling piglets in the DC-targeted vaccine group showed increased gross pathological lesions in the small intestine. Results in this study provide insights into the effects of sow cellular immune responses to PEDV infection in suckling piglets.

Pig model mimicking chronic hepatitis E virus infection in immunocompromised patients to assess immune correlates during chronicity.

Chronic hepatitis E virus (HEV) infection is a significant clinical problem in immunocompromised individuals such as organ transplant recipients, although the mechanism remains unknown because of the lack of an animal model. We successfully developed a pig model of chronic HEV infection and examined immune correlates leading to chronicity. The conditions of immunocompromised patients were mimicked by treating pigs with an immunosuppressive regimen including cyclosporine, azathioprine, and prednisolone. Immunocompromised pigs infected with HEV progressed to chronicity, because 8/10 drug-treated HEV-infected pigs continued fecal virus shedding beyond the acute phase of infection, whereas the majority (7/10) of mock-treated HEV-infected pigs cleared fecal viral shedding at 8 wk postinfection. During chronic infection, serum levels of the liver enzyme γ-glutamyl transferase and fecal virus shedding were significantly higher in immunocompromised HEV-infected pigs. To identify potential immune correlates of chronic infection, we determined serum levels of cytokines and cell-mediated immune responses in pigs. Results showed that HEV infection of immunocompromised pigs reduced the serum levels of Th1 cytokines IL-2 and IL-12, and Th2 cytokines IL-4 and IL-10, particularly during the acute phase of infection. Furthermore IFN-γ-specific CD4+ T-cell responses were reduced in immunocompromised pigs during the acute phase of infection, but TNF-α-specific CD8+ T-cell responses increased during the chronic phase of infection. Thus, active suppression of cell-mediated immune responses under immunocompromised conditions may facilitate the establishment of chronic HEV infection. This pig model will aid in delineating the mechanisms of chronic HEV infection and in developing effective therapeutics against chronic hepatitis E.

Efficient priming of CD4 T cells by Langerin-expressing dendritic cells targeted with porcine epidemic diarrhea virus spike protein domains in pigs.

Porcine epidemic diarrhea virus (PEDV) first emerged in the United States in 2013 causing high mortality and morbidity in neonatal piglets with immense economic losses to the swine industry. PEDV is an alpha-coronavirus replicating primarily in porcine intestinal cells. PEDV vaccines are available in Asia and Europe, and conditionally-licensed vaccines recently became available in the United States but the efficacies of these vaccines in eliminating PEDV from swine populations are questionable. In this study, the immunogenicity of a subunit vaccine based on the spike protein of PEDV, which was directly targeted to porcine dendritic cells (DCs) expressing Langerin, was assessed. The PEDV S antigen was delivered to the dendritic cells through a single-chain antibody specific to Langerin and the targeted cells were stimulated with cholera toxin adjuvant. This approach, known as "dendritic cell targeting," greatly improved PEDV S antigen-specific T cell interferon-γ responses in the CD4posCD8pos T cell compartment in pigs as early as 7days upon transdermal administration. When the vaccine protein was targeted to Langerinpos DCs systemically through intramuscular vaccination, it induced higher serum IgG and IgA responses in pigs, though these responses require a booster dose, and the magnitude of T cell responses were lower as compared to transdermal vaccination. We conclude that PEDV spike protein domains targeting Langerin-expressing dendritic cells significantly increased CD4 T cell immune responses in pigs. The results indicate that the immunogenicity of protein subunit vaccines can be greatly enhanced by direct targeting of the vaccine antigens to desirable dendritic cell subsets in pigs.

Modulation of Proinflammatory Cytokines in Monocyte-Derived Dendritic Cells by Porcine Reproductive and Respiratory Syndrome Virus Through Interaction with the Porcine Intercellular-Adhesion-Molecule-3-Grabbing Nonintegrin.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important global swine pathogen. PRRSV infects porcine dendritic cells (DCs), but the effects of the interactions with DCs are largely unknown. Current research focuses on the production and regulation of interferons and selected inflammatory cytokines in DCs, which may play key roles in immune modulation. In addition, PRRSV also downregulates swine leukocyte antigen class I (SLA-I), SLA-II, and CD80/86 costimulatory molecules in DCs. In this study, we aim to evaluate the PRRSV immunomodulatory effects on monocyte-derived DCs (MoDCs) through interactions with porcine DC-SIGN (pDC-SIGN) receptor. We demonstrated that blocking the PRRSV and pDC-SIGN interactions in MoDCs with recombinant hICAM-3 did not affect the regulatory effects of PRRSV on SLA-I, SLA-II, or CD80/86 molecules. The hICAM-3 did not affect the morphological changes on MoDCs associated with their activation and maturation after PRRSV infection, and did not impair the virus infectivity in these cells either. The mRNA levels of tumor necrosis factor alpha (TNF-α), IL-12p35, IL-1ß, and IL-6 were upregulated after hICAM-3 treatment or PRRSV infection, but in the presence of the blockage of pDC-SIGN in MoDCs with hICAM-3, PRRSV did not modulate the expression of these genes. However, in the presence of an anti-pDC-SIGN monoclonal antibody (mAb), we showed that PRRSV infection significantly reduced the mRNA expression levels of TNF-α and IL-1α, but enhanced the expression of IL-12p35 in MoDCs. Both hICAM-3-Fc and pDC-SIGN mAb treatments did not modulate proinflammatory cytokine protein levels in the culture supernatants of PRRSV-infected MoDCs. The results indicate that blocking the PRRSV-pDC-SIGN interactions by recombinant hICAM-3-Fc did not significantly affect virus infectivity, DC maturation, and proinflammatory cytokine gene expression in infected MoDCs. However, blocking the PRRSV-pDC-SIGN interactions on MoDCs with an anti-pDC-SIGN mAb revealed differential regulatory effects on specific proinflammatory gene expressions in those cells.

Interleukin-12 Immunomodulation Delays the Onset of Lethal Peritoneal Disease of Ovarian Cancer.

The omental fat band (OFB) is the predominant site for metastatic seeding of ovarian cancer. Previously, we highlighted the influx and accumulation of neutrophils and macrophages in the OFB following syngeneic ovarian cancer cell seeding as an important factor in the development of a protumorigenic cascade. Here we investigated localized immunomodulation as a means of promoting a successful protective response. As an important TH1-type immunomodulator, interleukin (IL)-12 has previously been investigated clinically as an anticancer therapeutic. However, systemic IL-12 administration was associated with serious side effects, galvanizing the development of immune or accessory cells engineered to express secreted or membrane-bound IL-12 (mbIL-12). Using an mbIL-12-expressing cell variant, we demonstrate that localized IL-12 in the tumor microenvironment significantly delays disease development. The mbIL-12-mediated decrease in tumor burden was associated with a significant reduction in neutrophil and macrophage infiltration in the OFB, and correlated with a reduced expression of neutrophil and macrophage chemoattractants (CXCL1, -2, -3 and CCL2, -7). Vaccination with mitotically impaired tumor cells did not confer protection against subsequent tumor challenge, indicating that IL-12 did not impact the immunogenicity of the cancer cells. Our findings are in agreement with previous reports suggesting that IL-12 may hold promise when delivered in a targeted and sustained manner to the omental microenvironment. Furthermore, resident cells within the omental microenvironment may provide a reservoir that can be activated and mobilized to prevent metastatic seeding within the peritoneum and, therefore, may be targets for chemotherapeutics.

Evaluation of the use of non-pathogenic porcine circovirus type 1 as a vaccine delivery virus vector to express antigenic epitopes of porcine reproductive and respiratory syndrome virus.

We previously demonstrated that the C-terminus of the capsid gene of porcine circovirus type 2 (PCV2) is an immune reactive epitope displayed on the surface of virions. Insertion of foreign epitope tags in the C-terminus produced infectious virions that elicited humoral immune responses against both PCV2 capsid and the inserted epitope tags, whereas mutation in the N terminus impaired viral replication. Since the non-pathogenic porcine circovirus type 1 (PCV1) shares similar genomic organization and significant sequence identity with pathogenic PCV2, in this study we evaluated whether PCV1 can serve as a vaccine delivery virus vector. Four different antigenic determinants of porcine reproductive and respiratory syndrome virus (PRRSV) were inserted in the C-terminus of the PCV1 capsid gene, the infectivity and immunogenicity of the resulting viruses are determined. We showed that an insertion of 12 (PRRSV-GP2 epitope II, PRRSV-GP3 epitope I, and PRRSV-GP5 epitope I), and 14 (PRRSV-GP5 epitope IV) amino acid residues did not affect PCV1 replication. We successfully rescued and characterized four chimeric PCV1 viruses expressing PRRSV linear antigenic determinants (GP2 epitope II: aa 40-51, ASPSHVGWWSFA; GP3 epitope I: aa 61-72, QAAAEAYEPGRS; GP5 epitope I: aa 35-46, SSSNLQLIYNLT; and GP5 epitope IV: aa 187-200, TPVTRVSAEQWGRP). We demonstrated that all chimeric viruses were stable and infectious in vitro and three chimeric viruses were infectious in vivo. An immunogenicity study in pigs revealed that PCV1-VR2385EPI chimeric viruses elicited neutralizing antibodies against PRRSV-VR2385. The results have important implications for further evaluating PCV1 as a potential vaccine delivery vector.

Expression of antigenic epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) in a modified live-attenuated porcine circovirus type 2 (PCV2) vaccine virus (PCV1-2a) as a potential bivalent vaccine against both PCV2 and PRRSV.

Co-infection of pigs in the field with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) is common and poses a major concern in effective control of PCV2 and PRRSV. We previously demonstrated that insertion of foreign epitope tags in the C-terminus of PCV2 ORF2 produced infectious virions that elicited humoral immune responses against both PCV2 capsid and inserted epitope tags. In this study, we aimed to determine whether the non-pathogenic chimeric virus PCV1-2a, which is the basis for the licensed PCV2 vaccine Fostera PCV, can express PRRSV antigenic epitopes, thus generating dual immunity as a potential bivalent vaccine against both PCV2 and PPRSV. Four different linear B-cell antigenic epitopes of PRRSV were inserted into the C-terminus of the capsid gene of the PCV1-2a vaccine virus. We showed that insertion of 12 (PRRSV-GP2 epitope II, PRRSV-GP3 epitope I, and PRRSV-GP5 epitope I), and 14 (PRRSV-GP5 epitope IV) amino acid residues did not impair the replication of the resulting PCV1-2a-PRRSVEPI chimeric viruses in vitro. The four chimeric PCV1-2a viruses expressing PRRSV B-cell linear epitopes were successfully rescued and characterized. An immunogenicity study in pigs revealed that two of the four chimeric viruses, PCV1-2a-PRRSVEPIGP3IG and PCV1-2a-PRRSVEPIEPIGP5IV, elicited neutralizing antibodies against PRRSV VR2385 as well as PCV2 (strains PCV2a, PCV2b, and mPCV2b). The results have important implications for exploring the potential use of PCV1-2a vaccine virus as a live virus vector to develop bivalent MLVs against both PCV2 and PRRSV.