Browsing by Author "Coffin, Carla S."

Now showing 1 - 5 of 5
  • Thumbnail Image
    ItemOpen Access
    Characterization of Hepatitis B Virus (HBV) and Host Cytokine Patterns in a Multiethnic Cohort of Patients with Non-alcoholic Fatty Liver Disease (NAFLD) and Chronic Hepatitis B (CHB)
    (2020-01-06) Lucko, Aaron Michael; Coffin, Carla S.; Chadee, Kris C.; Raman, Maitreyi
    Studies have reported conflicting data on the relationship between non-alcoholic fatty liver disease (NAFLD) and chronic hepatitis B (CHB). We aimed to identify how metabolic factors associated with NAFLD (diabetes, hypertension, central obesity and dyslipidemia) affects the hepatitis B virus (HBV) in patients with CHB. Patients with CHB and NAFLD were prospectively enrolled from 3 Canadian liver clinics. Patients underwent standardized liver tests (liver stiffness measurement [LSM] by transient elastography, controlled attenuation parameter [CAP]) and HBV clinical tests (quantitative [q] HBV surface antigen [HBsAg], HBeAg). Plasma levels of HBV DNA and RNA were measured by quantitative (q)PCR. Viral genotype was identified by population and next generation sequencing of the precore (C)/C and presurface (S)/S genes and analyzed using MEGA 7. Peripheral blood mononuclear cells (PBMCs) were stimulated ex vivo for 72h by HBV core antigen (HBcAg) or HBsAg peptides. PBMC supernatant and serum were analyzed for cytokine/chemokine markers using a 13-plex immunoassay. Kruskal-Wallis, multiple linear regression, Chi-square, and Fischer’s exact tests were performed using R commander. Of 48 subjects enrolled (median age 44.5 [IQR 16.8]), most were male (n=31), of Asian descent (n=29), and HBeAg negative (n=45). In HBeAg negative patients, the mean CAP was 30652 dB/m, ALT was 4026 IU/mL, and LSM was 5.82.0 kPa, indicating high steatosis without fibrosis. In all patients, the HBV genotypes were 13% A, 16% B, 46% C, 17% D, 6% E. Mutations associated with severe liver disease, anti-viral drug resistance, immune escape, and HBeAg negativity were identified in all subjects. Obese patients had increased qHBsAg levels, while diabetic patients had increased S gene diversity. Hepatic steatosis severity did not relate to viral factors analyzed. Ex vivo PBMC responses to HBcAg or HBsAg stimulation were not different to unstimulated controls. In this study, a multi-ethnic cohort of CHB and NAFLD patients were prospectively evaluated with novel virologic and host immunological markers. We found that metabolic factors associated with NAFLD correlated to inflammatory cytokine levels, viral genetic characteristics, and HBV replication markers. These viral and host factors can influence the risk of liver disease progression in patients with both NAFLD and CHB, warranting further study.
  • Thumbnail Image
    ItemOpen Access
    Epitope Mapping of Bovine Viral Diarrhea Virus Antigens E1, E2, Erns, and NS3 using Phage Display and Peptide Scanning
    (2020-12-21) Bremner, William T. R.; van der Meer, Frank; Schryvers, Anthony Bernard; Coffin, Carla S.; van Marle, Guido
    Bovine viral diarrhea virus is a cattle pathogen with global distribution, and substantial economic impact. Control of viral transmission is challenging by the lifelong viral shedding by persistently infected (PI) animals, and the significant diversity of the virus. While surveillance and removal of PI animals is the primary focus of control programs, insight into the antigenicity of the virus could be valuable for developing broadly protective vaccines to reduce the spread of the virus within farms and in this way the incidence of persistently infected animals (PI’s). This study aims to characterise the viral proteins of BVDV which elicit specific antibody responses. These proteins are the surface glycoproteins E1, E2 and Erns, as well as the non-structural protein NS3. This work employs the techniques of phage display, and peptide scanning to identify epitope structures on these four antigens and puts them into the context of existing structural models. Linear epitopes of potential interest for future vaccine development were identified on each of the four antigens. While attempts at characterizing conformational epitopes highlighted shortcomings in the computational workflow for such efforts, valuable insight into the limitations and future directions are drawn.
  • Thumbnail Image
    ItemEmbargo
    Kupffer Cells In Action: Understanding The Mechanisms Of Hepadnaviral Persistence
    (2023-06) Al-Yasiri, Layla; Coffin, Carla S.; Jenne, Craig N.; van der Meer, Frank
    Hepatitis B virus (HBV) is the prototypical member of Hepadnaviridae family, and a causative agent of liver disease. Despite the availability of a vaccine, an estimated 1.5 million new individuals become infected each year. HBV can establish persistent infection in hepatocytes, which can disrupt hepatic homeostasis. Kupffer cells (KCs) are liver-resident macrophages that act as the first of line of defense against infectious agents. Woodchuck hepatitis virus (WHV) is a member of Hepadnaviridae. North American woodchucks (Marmota monax) infected with WHV present a natural immunocompetent model and demonstrate comparable infection outcomes and progression to hepatocellular carcinoma. We hypothesized that KC absence in acute viral hepatitis leads to chronicity by impacting virus processing and immune responses. To investigate this, we sought to uncover the early host-virus interactions in acute WHV infection. We designed and optimized in-house WHV and woodchuck-specific assays to assess intrahepatic and systemic virus presence following WHV challenge (<24 hours) and infection (6 weeks). An intravital imaging protocol was developed to capture real-time events in a living woodchuck using purified and fluorescently labelled WHV. Clodronate liposome-mediated depletion of KCs in woodchucks was performed to evaluate pre-acute challenge and acute infection in either the presence or absence of KCs. Intravital imaging of woodchucks challenged with WHV showed first appearance of virus in the liver within 30 seconds and capture by KCs within 5-10 minutes. In addition, depletion of KCs did not impact the localization of WHV during the initial hour or 24 hours of challenge. Assessment of WHV markers, serology and liver histology revealed differences in viral and biochemical markers between the KC-depleted and non-depleted animals over the initial 6 weeks. KC depletion, prior to acute WHV infection, resulted in faster onset of acute hepatitis (elevated liver enzymes) and greater suppression of WHV DNA (in circulation and intrahepatically). However, there was no difference in WHV DNA suppression between KC-depleted and non-depleted cohorts after 6 weeks p.i, as all assayed woodchucks achieved suppression of plasma WHV DNA. This may suggest that KCs normally operate under tolerogenic conditions to prevent hepatocyte damage and hepatitis, allowing for greater viral replication and immune evasion.
  • Thumbnail Image
    ItemOpen Access
    Novel Approaches to Fight Prion Diseases
    (2020-04-29) Thapa, Simrika; Schaetzl, Hermann M.; Gilch, Sabine; Trang, Tuan; van Marle, Guido; Coffin, Carla S.; Telling, Glenn C.
    Prion diseases are fatal neurodegenerative disorders caused by PrPSc, the misfolded and infectious isoform of the cellular prion protein (PrPC). Currently, no preventive or therapeutic measures are available. In this work, we focused on therapeutic and prophylactic strategies against prion infections. In the therapeutic approach, we targeted cellular pathways and investigated the role of the quality control (QC) proteins, ERp57 and VIP36, on prion propagation. We found that the overexpression of ERp57 or VIP36 significantly reduced PrPSc levels in persistently prion-infected cells and decreased the susceptibility of uninfected cells to de novo prion infection. Moreover, lentiviral-mediated overexpression of ERp57 prolonged the survival of prion-infected mice. Mechanistically, we found that ERp57 overexpression reduced endoplasmic reticulum (ER) stress. To translate this proof-of-concept into potential drug therapy, we investigated the anti-prion effect of Sephin1, shown to prolong the phosphorylation of eIF2α and lower ER stress in the cells. In persistently prion-infected neuronal cells, we found that treatment with Sephin1 markedly reduced PrPSc levels. Moreover, Sephin1 reduced ER stress-induced PrP aggregates in cells and significantly extended the survival of prion-infected mice. These data provide the basis for targeting these cellular pathways as novel anti-prion therapy. In our prophylactic approach, we hypothesized that active vaccination is useful to contain chronic wasting disease (CWD), a contagious and expanding prion disease of cervids. Here, we vaccinated transgenic mice expressing elk prion protein with adjuvant CpG alone, or one of four recombinant PrP (rPrP) immunogens: deer dimer (Ddi), deer monomer (Dmo), mouse dimer (Mdi), and mouse monomer (Mmo). After challenging the animals with CWD prions intraperitoneally, we found that all vaccinated groups had longer survival times than the CpG control group. Interestingly, the Mmo-immunized group revealed that survival was extended by 60%. We also observed 28.4% and 24.1% prolongation in Dmo and Ddi groups, respectively. Our preliminary study in reindeer showed substantial humoral immune response induced by Mdi and Ddi, and the sera from the Ddi-vaccinated reindeer significantly reduced CWD prions in a cell culture model. Taken together, this study describes potential vaccine candidates against CWD. However, their protective effect in the natural cervid host needs further investigation.
  • Thumbnail Image
    ItemOpen Access
    Persistent Hepatitis B Virus in Hepatic and Extrahepatic Reservoirs in Hepatitis B Virus Related Oncogenesis
    (2020-05-04) Lau, Keith Chi Kei; Coffin, Carla S.; Burak, Kelly Warren; Mahoney, Douglas J.; van Marle, Guido
    Chronic infection by Hepatitis B virus (HBV) may result in hepatocellular carcinoma (HCC). Numerous viral factors are associated with oncogenesis and development of HCC, including integration, genetic variations in the X/basal core promoter/precore (X/BCP/PC) region, and occult infection. Although primarily hepatotropic, HBV is frequently found in circulating peripheral blood mononuclear cells (PBMCs) of the lymphoid system. Few studies have characterized this extrahepatic reservoir particularly in HBV-related HCC individuals. In this thesis, we hypothesized that pro-oncogenic HBV is present in both liver and extrahepatic reservoirs in chronic HBV (CHB) carriers with and without malignant disease. A highly sensitive molecular tool for accurate detection of low-level HBV and determination of genotype is described in this thesis. Subsequently, this technique was applied to plasma and PBMCs from post-liver transplant CHB carriers in combination with next generation sequencing to identify HCC-associated viral genotypes and genetic variants. The lymphoid reservoir in 32 CHB carriers with HCC and a representative patient with an extrahepatic lymphoid malignancy (i.e., dendritic cell sarcoma [DCS]), was evaluated for pro-oncogenic HBV. Interestingly, integrated virus was identified in genes implicated in cancer development in liver, PBMCs and DCS tumor. HCC-associated X/BCP/PC variants and genotypes were also present within lymphoid cells. Functional characterization of the most commonly detected variants (Guanine-1896-Adenine and Adenine-1762-Thymine/Guanine-1764-Adenine) were evaluated in vitro. Compared to wild-type, these mutants showed reduced viral HBeAg, modulated cytokine/chemokine expression, decreased APOBEC3G protein expression. The A1762T/G1764A mutant had more robust HBV replication than the G1896A variant. Overall, the findings in this thesis contributes to the literature on the epidemiological association of occult HBV, specific HBV SNPs, and integrated virus in hepatic and extrahepatic reservoirs. Persistent carcinogenic HBV poses continual risks of HCC recurrence and viral reactivation thus advocating for long-term HBV prophylaxis use post-transplant. Pro-oncogenic HBV within lymphoid cells may contribute towards oncogenesis of extrahepatic malignancies such as DCS. HCC-associated variants show functional differences in viral replicative fitness and host immune responses. Taken together, this thesis is significant for advancing the currently limited understanding of HBV molecular virology and pathogenesis of HBV-related carcinogenesis within the hepatic and extrahepatic reservoirs.