Browsing by Author "Coffin, Carla"
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- ItemOpen AccessDeveloping SINrep5 as a Viral Vector Platform to Test SARS-CoV-2 Vaccine Candidates(2023-06) Chen, Michelle; Marle, Guido van; Coffin, Carla; Devinney, RebekahThe COVID-19 pandemic, caused by SARS-CoV-2, serves as a stark reminder of the importance of vaccines and immunization in the arms race against novel viral agents. SARS-CoV-2 vaccines target the mutation-prone Spike Protein, leading to the rise of viral variants, and warranting additional research into the potential of diversifying vaccine target candidates, as well as vaccine options. One particular vaccine of interest is the adenovirus vector-based vaccine, which has raised apprehension since its use against SARS-CoV-2. Public hesitation and unclear human-use risks surrounding adenovirus vector vaccines have warranted exploration into other vectors—not just as ready-for-use vaccines, but also as expression platforms for vaccine study. SINrep5, derived from the Sindbis alphavirus, is one expression platform that has been studied for its unique protein expression abilities. A workflow was developed to use SINrep5 as an expression platform for SARS-CoV-2 proteins of interest—Nucleocapsid-N, Envelope-E, and Membrane-M. The N gene was subcloned into a mammalian SINrep5 plasmid (pmSINrep5) to test the vector’s protein expression in vitro. Vector transfections were optimized for protein expression and Virus-Like Particles (VLP) production. Techniques to purify, concentrate, and quantify VLP titres were also optimized. C57Bl/6 Mice were received VLP vaccines and humoral immunity was assessed. An efficient cloning system was developed for the SINrep5 mammalian expression plasmid (pmSINrep5) construct. Applying the system to N, E, and M resulted in successful pmSINrep5-N, pmSINrep5-E, and pmSINrep5-M creation. Transfections optimized using pmSINrep5-eGFP achieved high protein expression in nearly all transfected cells. Applied to the other pmSINrep5 constructs, low E and M Protein expression was detected, likely due to altered subgenomic promoter activity and cytotoxicity. Conversely, high N Protein expression was detected from Western Blot analysis. Transfections were able to produce mSINrep5(SV)-eGFP and SV-N VLPs. Purification was achieved by adjusting the optimized transfection protocol to eliminate contaminating proteins, and SV VLP quantification was subsequently optimized in a two-pronged approach involving VLP infection titration and RT-qPCR. The development of a working pipeline to efficiently construct and test the SINrep5 viral vector expression vector highlights its utility in studying viral proteins and antigens, diversifying the tools already proffered by alphaviruses in immunotherapy research.
- ItemOpen AccessHepatitis B Virus (HBV) Infection in Peripheral Blood Mononuclear Cells of HBV Mono-infected and HBV/Human Immunodeficiency Virus Type-1 Co-infected Patients(2014-07-17) Lee, Zengina; Coffin, Carla; Czub, MarkusIt is unknown whether HIV-1 co-infection impacts HBV lymphotropism. We hypothesize that concomitant HIV-1 infection will affect HBV detection in PBMC subsets. We compared HBV genome detection in whole PBMC and CD4+ T, CD8+ T, CD14+ monocyte, CD19+ B and CD56+ NK cell subsets isolated from 14 HBV mono-infected (4/14 with a second sample collected after starting antivirals) and 6 HBV/HIV-1 co-infected patients on antivirals using nested PCR/nucleic hybridization and/or quantitative PCR assays. HBV DNA was detected in most target PBMC subsets regardless of HIV-1 co-infection and antiviral treatment; with the exception of the CD4+ T cell subset from HBV/HIV-1 + patients. All whole PBMC analyzed (13/13 HBV treatment naïve mono-infected, 4/4 follow-up cases on antivirals, and 3/3 HBV/HIV-1 co-infected) were HBV genome positive. The data suggests that HBV infection in CD4+ T cells is affected by concomitant HIV-1 infection.
- ItemOpen AccessHepatitis B Virus (HBV) Variants in Untreated and Tenofovir Treated Chronic Hepatitis B (CHB) Patients during Pregnancy and Post-Partum Follow-Up(PLoS One, 2015-10-16) Virine, Boris; Osiowy, Carla; Gao, Shan; Wang, Tong; Castillo, Eliana; Martin, Steven R.; Lee, Samuel S.; Simmonds, Kimberley; van Marle, Guido; Coffin, CarlaBACKGROUND: Chronic hepatitis B (CHB) is a dynamic disease that may be affected by immune changes in pregnancy. Guidelines suggest consideration of nucleos/tide analogs (NA), i.e., tenofovir, (TDF) in highly viremic mothers to reduce vertical transmission risk. HBV variability affects CHB outcome, but little is known about HBV genetic changes in pregnancy due to immune or NA selection. OBJECTIVES: To evaluate HBV diversity in NA treated or untreated pregnant vs. post-partum CHB carriers. STUDY DESIGN: In plasma collected from 21 mothers (7 matching pre/post-partum), HBV serological tests, genotype and viral load were assayed. The HBV pre-surface (S) /S overlapping polymerase (P) (N = 20), pre-core (C) /C (N = 11) and/or full genome PCR amplicons (N = 3) underwent clonal sequence analysis. RESULTS: The median age was 31 y, 71% Asian, 68% genotype B or C, 33% HBV eAg+, 5 received TDF (median HBV DNA 8.5 log IU/ml). In untreated mothers, median antepartum vs. post-partum ALT was 21 vs. 24 U/L and HBV DNA was 2.7 vs. 2.4 log(10) IU/ml. ALT and/or HBV DNA flares occurred during pregnant and/or post-partum period in 47% (10/21). Clonal sequencing antepartum showed the presence of minor "a determinant" and/or vaccine escape mutants (VEM) but drug resistant variants were infrequent. Analysis of pregnant vs. post-partum samples showed different HBV variants and viral diversity. CONCLUSIONS: Differences in immune and/or by NA selective pressures during pregnancy may affect HBV evolution during pregnancy. The presence of minor VEM warrant infant follow-up.
- ItemOpen AccessMolecular, Pathogenesis and Immunological Studies of the Canadian Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) Variant(2022-09-16) Hassan, Mohamed Saleh Hussein; Careem, Faizal; Cork, Susan; Coffin, Carla; Goldsmith, Dayna; van der Meer, Franciscus; Buret, Andre; Gallardo, RodrigoInfectious bronchitis virus (IBV) is a gammacoronavirus that infects chickens leading to economic losses globally. Although the disease caused by IBV is known as infectious bronchitis (IB), the virus also replicates and induces lesions in the renal, reproductive, and gastrointestinal systems of chickens. The control of IB relies mainly on using live attenuated and inactivated vaccines. The IBV Delmarva (DMV)/1639 variant has impacted the layer flocks in Eastern Canada over the past few years. The affected flocks showed a high incidence of false layer syndrome (FLS). The studies conducted in this thesis focused on three major areas: 1) molecular characterization of IBV DMV/1639 isolates obtained from clinical cases in Eastern Canada, 2) investigating the pathogenesis of the Canadian IBV DMV/1639 variant and the host responses to this virus in chickens, and 3) evaluating the protective efficacy of the existing IB vaccines against the infection with this virus in chickens. Following whole genome sequencing (WGS), phylogenetic and recombination analyses showed that the Canadian IBV DMV/1639 strain is a chimeric virus derived from a Connecticut (Conn) vaccine-like strain, a 4/91 vaccine-like strain, and one strain that is yet-unidentified. An experimental challenge of 1-day-old specific-pathogen-free (SPF) chicks showed that this virus has a wide tissue tropism and induces marked cystic lesions in the oviduct of growing pullets. A significant drop in egg production, accompanied by characteristic gross and microscopic lesions in the reproductive organs, followed the experimental challenge of SPF chickens at peak of egg production. Infected chickens showed significant recruitments of KUL01+ macrophages and CD4+ and CD8+ T cell subsets in the oviduct tissues. In addition, anti-IBV antibodies were detected systemically and locally in the oviduct washes. Heterologous IB vaccines that are commercially available in Canada protected laying SPF chickens against a drop in egg production induced by the Canadian IBV DMV/1639 strain. Vaccinated chickens had lower viral replication and mild reproductive tract lesions. Overall, the knowledge generated in this thesis contributed to the understanding of IBV-host interactions including virus evolution, pathogenesis, host responses, and vaccination-based control.
- ItemOpen AccessSpontaneous Regression of Hepatocellular Carcinoma and Review of Reports in the Published English Literature(2019-03-31) Chohan, Moaz B. Y.; Taylor, Nick; Coffin, Carla; Burak, Kelly W.; Bathe, Oliver F.Background. Spontaneous regression of hepatocellular carcinoma (HCC) is a rare event, although it has been described by numerous groups. The long-term fate of individuals experiencing an SR is not well described, and the underlying mechanism(s) of SR are unknown. Case Presentation: A 79-year-old Asian female with metastatic HCC taking only valsartan for hypertension had a marked reduction in tumor dimension in the primary tumor and the pulmonary metastases. Serum alpha-fetoprotein (AFP) decreased from 17,833 μg/L to 26 μg/L. Her disease progressed after 71 months, and she died shortly after. In a review of 66 patients with SR reported in the English literature, median survival was 83 months. Median survival in 37 cases that underwent resection after SR was 108 months. Conclusions. The case and a review of the literature illustrate that SR is often durable and associated with an excellent prognosis. Understanding the underlying mechanism of SR may point to novel therapeutic strategies.