Influenza is a seasonal acute respiratory infection, causing millions of illnesses worldwide on a yearly basis. A common subtype, the influenza A virus (IAV), is a single stranded RNA virus, that similarly to other subtypes, targets epithelial cells. The best way to protect against the virus is through vaccination. Vaccine induced protection is mediated through the generation of adaptive CD4 and CD8 T cells, as well as antibody producing B Cells. Although generally thought of as helper cells, previous research has highlighted additional roles of memory CD4 T cells in mediating protection against IAV beyond their helper function. More specifically they have been shown to enhance innate inflammatory responses and facilitate the recruitment of innate cell populations; including a recently discovered population of Innate Lymphoid Cells (ILC). Previous research has shown ILCs to have a key role in tissue repair and limiting tissue damage following infection. Whether memory cell response, during protective recall, modulates ILC repair function is currently not well understood. To better understand the possible regulation of ILCs by memory cells, we utilized a molecular technique called reverse transcription polymerase chain reaction (RT-qPCR), to assess select innate lymphoid cell associated protein expression following IAV challenge and rechallenge. We hypothesize, that memory cells drive differential expression of ILC associated repair proteins to assist in a faster and more efficient mobilization of repair processes following pathogenic invasion. Our goal is to highlight and better understand the regulatory and inflammatory responses memory cells provide following viral infection, as these may lead to key steps in the development of long lasting and efficacious vaccines.

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The COVID-19 pandemic has been an ongoing disaster that has devasted millions of lives. With the development of COVID-19 vaccines in late 2020, there was a potential for populations to gain artificial active immunity in order to prevent future outbreaks. However, despite successful clinical trials, millions of citizens have been hesitant to receive the COVID-19 vaccines (Khubchandani et al., 2021). Demographics of the most prominent US vaccine-hesitant populations consist of ethnic/racial minorities and Republicans groups (Khubchandani et al., 2021). Little information is known about COVID-19 vaccine hesitancy in colleges and universities. Colleges provide an elevated risk for infection through their communal residencies, the reemergence of campus activities, and continuous travel to home (Sharma et al., 2021). This study explored COVID-19 vaccine hesitancy in UCF college students and explored potential pathways to achieve higher vaccination rates. Potentially believed COVID-19 misinformation was also studied. A COVID-19 opinion survey was designed and distributed to the UCF college population. Two hypotheses were made for this study: (1) There is a significant effect on vaccination status among people of different political parties, field of study, living conditions, masking frequency, and scores on the knowledge-based questions portion. (2) There will be a significant effect on knowledge-based scores with political party and field of study. The results were analyzed using Chi-square, one-way ANOVA, or two-way ANOVA on SPSS. The results showed a significant effect on vaccination status in political parties, masking frequency in class, and scores on the knowledge-based survey questions. There was no significance with race/ethnicity and field of study. There was a significant effect on the knowledge-based survey questions with political party and field of study. Potential side effects and the vaccines being seen as ineffective were the top two reasons that students choose not to vaccinate.

The emergence of viruses with zoonotic potential, i.e. with the potential ability to cross species barriers to infect unnatural hosts, poses a huge threat to humans. It is therefore essential to develop new methodologies to rapidly and efficiently generate attenuated virus vaccine candidates to attempt to control the threat. Viruses need to be able to at least partially inhibit the host’s innate defence mechanism, known as the interferon (IFN) system, to replicate efficiently in vivo and establish a productive infection. It has been previously reported that viruses that have lost their ability to circumvent the host’s IFN response, or IFN-sensitive viruses, are promising candidates for live attenuated virus vaccines. Here we report on the development of a cell-based method to attempt to rapidly select IFN-sensitive viruses that can not block IFN signalling, from wild-type virus populations. Lentivirus vectors containing selection markers (HSV-tk – Herpes Simplex virus thymidine kinase gene and pac – puromycin resistance gene) under the control of a tight IFN-inducible promoter (the murine Mx1 promoter) were generated and used to specifically engineer HEp2 cell lines, termed Mx GIPSE and Mx TIPSE, for this purpose. The developed methodology relies on the engineered cell lines and a selection procedure using exogenous IFN-α and puromycin: if a cell is infected with IFN-resistant virus, it will die in the presence of IFN-α and puromycin because IFN signalling will be blocked, thereby blocking the activation of the Mx1 promoter and consequent expression of pac; if a cell is infected with an IFN-sensitive virus, it will survive in the presence of IFN-α and puromycin because the Mx1 promoter will become activated through the IFN signalling pathway, leading to the expression of pac. IFN-sensitive viruses can then be rescued from the surviving cells, and amplified using IFN-permissive cell lines expressing viral IFN antagonist proteins (proteins that block the host’s IFN response). When tested on PIV5 strains CPI- (an IFN-sensitive virus) and CPI+ (an IFN-resistant virus), the developed method allowed the survival and amplification of cells infected with CPI-, whilst cell death was observed for cells infected with CPI+. Whilst the developed methodology seems promising, further developments of the system are required. The possibilities of using the developed methodology in combination with other techniques, such as FACS sorting and immune selection, to rapidly select IFN-sensitive mutant viruses from wild-type and mutagenised virus populations are discussed. The potential to use Mx TIPSE cells to select IFN-resistant revertant viruses from IFN-sensitive virus populations is also discussed. In addition, a high throughput screening assay has been developed using the engineered Mx GIPSE and Mx TIPSE cell lines to search for compounds that block IFN signalling or that block the action of viral IFN antagonist proteins. Compounds that block IFN signalling would potentially be useful as anti-inflammatory drugs whilst compounds that block the action of viral IFN antagonist proteins would be valuable as antiviral drugs.

Over 80% of the newly emerging infectious diseases are caused by RNA viruses. Major global problems associated with the development of vaccines against the RNA virus are their high genetic and antigenic diversity. Hence, effective control of epidemics with newly emerging RNA viruses require improved vaccines which are either specific to the new strain or broadly effective even when new viral strains emerge. The main focus of this dissertation is to develop epidemic vaccines using these two approaches. Using a newly emerged swine enteric virus called porcine epidemic diarrhea virus (PEDV) as a model, our first goal was to develop a quick and easy method for rapid response vaccines with potential applicability to a range of RNA viruses. We hypothesized that the methods which can disrupt genomic RNA without impacting the structural integrity of the virus would result in attenuated vaccine with minimum replication in the host while inducing immune responses. As hypothesized, developed rapid response PEDV vaccine induced complete protection against the virulent challenge virus, while vaccine viral shedding was not detected in vaccinated pigs. To address the second problem of rapid viral evolution leading to vaccines becoming obsolete, we used swine influenza virus (SIV) as a model to develop and test a universal vaccine composed of peptides encoding conserved antigenic epitopes which are present in most influenza A viruses. Importantly, a novel amphiphilic invertible polymer (AIP) was used to address the well-recognized problem of poor antigenicity of peptides. We hypothesized that peptides encoding conserved epitopes when conjugated with an AIP will induce strong immune responses and protect against challenge virus. While the conserved epitopes were previously tested by others in mice, we were the first to test a combination of these epitopes in pigs. Pigs vaccinated with the peptide polymer vaccine mounted strong antibody responses against the epitopes indicating that the delivery system was effective. However, protection against replication of the challenge virus was delayed. In summary, the methods developed and tested in this body of work significantly contribute to the area of emergency response management in infectious disease outbreaks. / United States Department of Agriculture, National Institute of Food and Agriculture (USDA-NIFA) / North Dakota State Agricultural Products Utilization Committee (ND APUC) / North Dakota State Board of Agricultural Research (ND SABRE)

Recombinant oncolytic virus (OV) vaccines that encode tumour-associated antigens are potent boosting agents for adoptive transfer of tumor-specific T cells (adoptive T cell therapy or ACT). Current strategies to exploit boosting vaccines for ACT rely on a priori knowledge of targetable tumour epitopes and isolation of matched epitope-specific T cells. Therefore, booster vaccines must be developed on a patient-by-patient basis, which severely limits clinical feasibility. To overcome the requirement for individualized pairing of vaccines and T cells, we propose a “universal” strategy for boosting tumor-specific T cells where the boost is provided through a synthetic receptor that can be engineered into any T cell and a matched vaccine. To this end, we are employing chimeric antigen receptors (CARs), which confer MHC-independent antigen specificity to engineered T cells, and a paired OV vaccine that encodes the CAR target. As proof-of-concept, we have developed and evaluated a model where murine TCR transgenic T cells are engineered with boosting CARs against a surrogate antigen for studies in immunocompetent hosts. In chapter 3, I optimized a murine CAR-T cell manufacturing protocol that allows for generation of highly-transduced T cells that maintain a predominantly central memory (Tcm) phenotype. This protocol leads to generation of highly functional CAR-T cell products that can be cryopreserved at the end of ex vivo culture for future use in adoptive transfer and vaccination studies. In chapter 4, I evaluated the in vivo boosting potential of our dual-specific CAR-T cells with paired OV vaccines. Adoptive transfer of these CAR-engineered tumor-specific T cells followed by vaccination with paired oncolytic vesicular stomatitis virus (VSV) vaccine leads to robust, but variable and transient, CAR-mediated expansion of tumour-specific CD8+ T-cells, resulting in delayed tumour progression in aggressive syngeneic tumour models. In chapter 5, I investigated the role of OV-induced type I interferon (IFN-I) responses on CAR-T cell boosting. I found that CAR-T cell expansion and anti-tumour function following OV vaccination is limited by the IFN-I response and can be further enhanced by blocking interferon alpha and beta receptor subunit 1 (IFNAR1). This IFN-I-mediated T cell suppression was found to be T cell-extrinsic and related to premature termination of OV infection and antigen expression in vivo. In chapter 6, I investigated the role of CD4+ T cell help in vaccine-mediated T cell boosting and evaluated different genetic engineering strategies to integrate pro-survival STAT5 signaling into the CAR-T cell product in an effort to improve persistence and long-term anti-tumour efficacy. The work presented herein describes a novel and clinically feasible approach to enhancing adoptive T cell therapies and contributes to the basic understanding of T cell biology in the context of CAR-engineering and cancer vaccination. / Thesis / Doctor of Philosophy (PhD) / Despite recent advances in cancer prevention, detection, and treatment, 2 in 5 Canadians are expected to be diagnosed with cancer in their lifetime and approximately 1 in 4 will succumb to their disease. New, more specific therapies are needed to improve responses to treatment and reduce therapy-related side effects. Cell therapy is a new way to treat cancer that uses the patient’s own immune cells as a living drug. The immune cells are taken from a patient’s blood or tumour, trained to attack cancer in the laboratory, and infused back into the patient where they will find and kill cancer cells. A major challenge with this strategy is that the trained immune cells do not always survive in the patient for long enough to get rid of the tumour. To “boost” the immune cells, we are developing a new strategy where the immune cells are genetically modified and combined with a vaccine to enhance their anti-tumor activity. Just like a vaccine against a bacteria or virus, this vaccine will tell the modified immune cells to turn on, make more of themselves, and to find and kill the cancer cells. By delivering this “go” signal through a vaccine, we think that the immune cells will be better able to survive and generate a stronger, longer-lasting immune response against the cancer. This thesis tests this approach in relevant mouse models of cancer and aims to understand how we can best design the immune cells and vaccine to work together in their tumour-killing activities.

Influenza is a severe disease in humans and animals, causing upwards of 40,000 deaths every year in America alone. Influenza A virus (IAV) also causes periodic pandemics every 10 to 50 years, killing millions of people. Despite this, very few effective therapies are available. All strains of IAV are prone to developing resistance to antibodies due to the high mutation rate in the viral genome. Because of this mutation rate, a yearly vaccine must be generated before every flu season, and efficacy varies year to year. IAV has also mutated to escape several of the clinically-approved small molecule inhibitors. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of IAV. IAV attachment is mediated by many individually weak hemagglutinin–sialic acid interactions that all together make a strong attachment to a host cell. Polymerized sialic acid analogs can recreate these interactions and block infection. However, they are not ideal therapeutics due to solubility issues and in vivo toxicity. We used liposomes as a novel means for delivery of the sialic acid-containing glycan, sialylneolacto-N-tetraose c (LSTc). LSTcbearing decoy liposomes form multivalent, polymer-like interactions with IAV. Decoy liposomes competitively bind IAV in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. LSTc decoy liposomes co-localize with IAV, while control liposomes do not. Inhibition is specific, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind IAV or inhibit infectivity. LSTc decoy liposomes prevent the spread of IAV during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high-avidity interactions with IAV hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging strains.

While memory CD4 T cells are critical for effective immunity to pathogens, the mechanisms underlying their generation are poorly defined. Although extensive work has been done to examine the role of antigen (Ag) in shaping memory formation, most studies focus on the requirements during the first few days of the response known as the priming phase. Little is known about whether or not Ag re-encounter by effector T cells (late Ag) alters CD4 memory T cell formation. Since influenza infection produces a large, heterogeneous, protective CD4 memory T cell population, I used this model to examine the role of late Ag in promoting CD4 memory T cell formation. In the experiments presented in this thesis, I demonstrate that late Ag is required to rescue responding CD4 T cells from default apoptosis and to program the transition to long-lived memory. Responding cells that failed to re-encounter Ag had decreased memory marker expression and failed to produce multiple cytokines upon re-stimulation. Ag recognition is required at a defined stage, as short-term Ag presentation provided 6 days after infection is able to restore canonical memory formation even in the absence of viral infection. Finally, I find that memory CD4 T cell formation following cold-adapted influenza vaccination is boosted when Ag is administered at this stage. These findings imply that persistence of viral Ag presentation into the effector phase is the key factor that determines the efficiency of memory generation. They also suggest that administering Ag during the effector stage may improve vaccine efficacy.

Due to the inevitable reality that most patients diagnosed with cancer will eventually relapse, modern oncology research has been forced to tackle this outcome primitively using combination therapies. Adoptive T-cell transfer with Oncolytic Virus Vaccination represents a new class of combination therapies that can facilitate the crosstalk of multiple aspects of the immune system such that they work in concert to prevent this outcome for many types of cancer. Despite this, immunosuppressive systems like those characterized in the B16F10-gp33 melanoma model pose a new problem for this approach. Typically, this model has total regression but is subsequently followed by relapse. Previous work from the Wan lab has suggested that this may be an outcome of total target gene deletion. Here we present two approaches to tackle this through the targeting of DNA repair pathways of the host cell. Our data can show that both VSV and Vaccinia infection/ propagation does lead to the generation of DNA damage but in the case of VSV this leads to incomplete cell lysis, and ultimately target gene loss via double-stranded DNA repair mechanisms. We were able to tackle the phenomenon following VSV administration by adding DNA repair inhibitors to the mix and showed that the proportion of cells that escaped after the loss of the target antigen was decreased by half when compared to the standard procedures. Additionally, this work also gave a preliminary understanding of how Vaccinia may achieve a similar outcome to this via its unique cytoplasmic replication mechanisms. / Thesis / Master of Science (MSc)

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 4 uppsatser.