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Browsing by Subject "Vaccine"

Now showing 1 - 19 of 19
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    Computational analysis and visualization of the evolution of influenza virus
    (2014-08) Lam, Ham Ching
    Influenza viruses can infect a large variety of birds and mammals including humans, pigs, domestic poultry, marine mammals, cats, dogs, horses, and wild carnivores \cite{Webster2002}. Surveillance for influenza viruses circulating in humans has been gradually increased and expanded to many areas around the world. These surveillance programs have produced large amount of influenza genomic data which facilitates the study of the virus by computational methods that are efficient and cost saving.The main focus of this dissertation research is the development of visualization methods to understand the evolution of influenza viruses circulating in humans and other mammals. The methods developed have been applied to different human influenza A subtypes, swine influenza viruses, and avian influenza viruses. The methods are based on unsupervised dimensional reduction techniques which can be applied to each individual genome segments or to the complete genome sequence of the virus. These methods are a departure from the traditional phylogenetic tree construction paradigm because very large number of high dimensional input sequences can be processed and results are viewed directly in a two or three dimensional Euclidean space.We reproduced the evolutionary trajectory of the seasonal human influenza A/H3N2 virus since its introduction to humans in 1968 on a 2D PCA space. The observed pathway led us to hypothesize that vaccination serves as a primary evolutionary pressure on this virus. We provided visual, simulation results, and statistical results to support this. The North American swine influenza H3N2 viruses were also studied using the developed visualization methods. The diversity of this virus is changing since the 2009 H1N1 pandemic outbreak. Five main clusters were observed from the visualization results. The mutations at two positive selected sites on the HA gene were identified as the potential driver for clusters segregation of this virus after the pandemic.A visualization method was developed to visually detect reassortant influenza virus. A reassortant influenza virus is difficult to detect because it consists of genome segments from different parental origin. As two different strains of influenza coinfect a single cell, the capability to exchange genome segments between these two strains can lead to progeny carrying different parental segments within its genome. In order to detect such progeny, a PCA projection based visualization method that is able to examine the full genome sequence of a reference and test strains simultaneously was developed in order to detect any reassorted segments within a full genome. Besides the development of visualization methods, we have also developed a compact Markov Chain model to estimate the probability of viruses with high genetic similarity found after a very large time gap. This model is a two components model where we combined a Markov Chain with a Poisson model. The Markov model uses Hamming distance as the evolution process of the virus and a computed mutation rate as the input to the Poisson model, combined together, we simulated the evolution process of the influenza virus under the neutral evolution process. The computational results from this model led us to conclude that the existence of reservoirs preserving viruses for decades cannot be completely eliminated.In short, our primary goal has been to develop visualization based approaches to understand the evolution of the influenza viruses from different hosts. The results we have so far suggested that the power of visualization paves the way to gain deeper understanding and insight of the evolution of the virus as we utilize the rapidly growing amount of the genomic data of the virus.
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    Control and characterization of influenza A viruses in swine
    (2011-07) Detmer, Susan Elisabeth
    Between 1958 and 2005, there were 37 human cases of zoonotic swine-origin influenza A virus (IAV) infection reported (Myers et al 2007; Van Reeth 2007). A majority of these infections were with classical swine H1N1 viruses and these 37 cases did not include the Fort Dix cases in 1976 that resulted in 1 death and up to 230 soldiers infected (Myers et al. 2007; Van Reeth 2007). However, a recent report of pig to human transmission was at an Ohio County Fair in 2007 (Vincent et al. 2009b). The sequence analysis of the HA gene segment of the IAVs isolated from the humans and pigs in this case revealed that it was a strain that was currently circulating in the U.S. pig population. The internal genes of this isolate were determined to be of the triple-reassortant swine influenza lineage, including a conserved avian PB2 gene sequence (Vincent et al. 2009b). On June 11, 2009 the first influenza pandemic in 41 years was declared by the World Health Organization. This virus was like no virus previously seen in the human population with gene segments from both Eurasian and North American swine viruses. The 2009 pandemic H1N1 virus was called a "quadruple-reassortant" virus because it is composed of neuraminidase (NA) and matrix (M) gene segments from Eurasian swine influenza viruses combined with triple-reassortant proteins of North American swine influenza viruses (human-origin polymerase B1 (PB1), avian-origin polymerase B2 (PB2) and polymerase A (PA), and classical swine-origin hemagglutinin (HA), nucleoprotein (NP) and non-structural (NS) (Garten et al. 2009; Smith et al. 2009). The evolutionary analysis of the 2009 pandemic H1N1 shows that the generation of this strain was not likely a recent event. In fact, in order to facilitate human-to-human spread, it probably adapted to the human host through secondary reassortments in humans (Ding et al. 2009). However, the original source of this virus has not yet been determined. The emergence of the 2009 pandemic H1N1 virus and scattered reports of human infections with swine-origin isolates underscores the importance of fully understanding the genetic, antigenic, and pathogenic characteristics of influenza A viruses so that we may limit the introduction of novel IAVs to the swine population and monitor for newly emerging and evolving viruses. In order to improve our understanding of IAVs in swine, the goal of this dissertation is to address the ability of genetic characterization to predict variations in virus phenotype, such as viral binding and antigenicity. Understanding the genetic, antigenic and pathogenic features of viruses is important to prevent introduction of human and avian viruses into swine herds and the potential spillback of those viruses to the human population, as wells as preventing the sustained transmission of IAVs within an endemically infected herd. The control of influenza viruses in pig populations continues to be dynamic and complex, and is reliant on a number of factors. Two of these factors include appropriate selection and application of (1) diagnostic tests and (2) vaccines. Routine surveillance for influenza viruses at the farm level, either syndromic or active surveillance, is often accomplished using real time RT-PCR tests on nasal swabs from live pigs and lung tissue samples from post-mortem examinations. Easily collected sample methods, such as oral fluids, could provide additional viruses for characterization of IAVs in swine. Oral fluids have been used extensively for diagnostic tests in human medicine and are now being applied in swine herds for detecting pathogens and antibodies against the pathogens (Prickett et al. 2010). As part of this dissertation, porcine oral fluids were validated as a viable sample collection method for routine RT-PCR and virus isolation tests (chapter 2). Another important control measure for influenza viruses in pigs continues to be vaccines. In order to assure continued efficacy of vaccines against currently circulating strains of virus, vaccine challenge trials are performed. In this dissertation, the efficacy of a commercial vaccine was examined against challenge with a contemporary field isolate (chapter 3). To address the genetic and phenotypic characterization of influenza A viruses from swine, two sets of viruses were selected from the influenza database at the University of Minnesota, Veterinary Diagnostic Laboratory and sequenced. The first set was isolated from a group of endemically infected farms treated by the same veterinarian from 2005 to 2009. The selected viruses were either used to produce autogenous vaccines or they were the epizootic viruses found during outbreaks in the vaccinated herds (chapter 4). The second set of viruses were isolated from nursery pigs in one endemically infected multi-site swine production system (farm M) from 2007 to 2009 and either contained a distinct two amino acid insertion or were presumptive ancestral viruses without the insertion (chapter 5). The viruses from farm M were further characterized along with representative viruses that have been previously studied in vivo using a new technique called virus histochemistry to examine the patterns of virus attachment in the respiratory tract (chapter 6). For the purposes of this dissertation IAVs were classified as virulent increased virulence have some of the following clinical/case presentations: (a) morbidity approaching 90% and mortality approaching 10 percent, (b) sudden, unexpected deaths occurring early in the disease outbreak, (c) gross lesions that are not typical of swine influenza including profuse hemorrhage and/or edema, and (d) sufficient health and production records along with laboratory results that indicate that a highly virulent influenza virus is involved. The characteristics of highly virulent influenza viruses, such as A/swine/KS/77778/2007 H1N1 and A/swine/OH/511445/2007 H1N1, have been previously described in the literature (Ma et al. 2010; Vincent et al. 2009b). This classification was not related to the criteria for classification of avian viruses as having high or low pathogenicity.
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    Correlates of cytotoxic T lymphocyte vaccine-induced control against Simian immunodeficiency virus (SIV)
    (2018-10) Mohammad, Hadia
    There is an urgent need to develop a successful HIV vaccine. Despite continuous efforts, development of a protective HIV-1 vaccine remains a big challenge. HIV-1/SIV-specific CD8+ T cells play a pivotal role in the control of virus replication. During chronic HIV-1 and SIV infections, virus replication is most concentrated within lymphoid B cell follicles, whereas virus-specific CD8+T cells concentrate in extrafollicular areas of secondary lymphoid tissues and tend to be excluded from follicular areas. My thesis focuses on identifying immune correlates of control associated with successful CD8+T cell-based SIV vaccines to help understand what is required to develop a successful HIV vaccine. My central hypothesis is that vaccine-induced control will be associated with induction of an early SIV-specific CD8+T cell response at the portal of viral entry and in lymphoid tissues and also with induction of high levels of virus-specific CD8+T cells in follicular areas of lymphoid tissues. I determined the location, abundance, and phenotype of virus-specific CD8+T cells and the abundance of virus-infected cells in vaccinated and unvaccinated rhesus macaques. Freshly collected unfixed tissue samples were analyzed using in situ tetramer staining combined with immunohistochemistry, in situ hybridization, confocal imaging, and quantitative image analysis. I found that vaccine-induced control was associated with induction of virus-specific CD8+T cells before challenge with pathogenic SIV. Virus-specific CD8+T cells expanded rapidly after challenge and resulted in high effector (SIV-specific CD8+T cells) to target (SIV-infected cells) ratios at the portal of viral entry and in lymph nodes. Additionally, vaccine-induced control correlated with the induction of high follicular: extrafollicular ratio of virus-specific CD8+T cells in lymphoid tissues. Moreover, this control correlated with induction of SIV-specific CD8+T cells that expressed little perforin and with SIV-specific CD8+ T cells in which perforin was exclusively localized to the cell membrane in lymphoid tissues. These cells are likely effector memory T cells with an immediate killing ability. These findings provide a better understanding of the immune correlates of CD8+T cell-based vaccine-induced control against SIV and provide understating of what is needed to create an effective HIV vaccine.
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    Enhanced Efficacy of Plasmid DNA Vaccines for Cancer Therapy
    (2010-11) Dietz, Wynette
    Since cancer is the second most common cause of death in the United States, it is of great importance to pursue new and improved methods for treating cancer. The goal of cancer immunotherapy is to exploit the specificity and longevity of immune responses through the use of vaccines to treat cancer. DNA vaccines have many advantages over protein and viral vaccine-based strategies including low cost, ease of production, flexibility and low toxicity. Plasmid DNA vaccines encoding tumor antigens can produce powerful anti-tumor immune responses in animal models, but clinical trials have shown only modest responses. This lack of clinical efficacy is thought to reflect the two major limitations of plasmid DNA vaccines: transient protein expression and low transfection efficiency. Transient protein expression is likely the result of gene silencing due to transcriptionally repressive chromatin within the plasmid backbone. To overcome this limitation, we removed the bacterial backbone sequences and produced a minicircle DNA consisting of the gene expression cassette with only a few bases of the bacterial backbone. This resulted in persistent protein expression, increased transfection efficiency and enhanced immunogenicity. In an effort to further enhance the transfection efficiency, we produced cationic carriers that bind plasmid DNA and protect it from degradation. The addition of these cationic carriers significantly increased transfection efficiency in vitro but has yet to show the same effect in vivo. Additionally, we administered these vaccines transdermally using a tattoo device and achieved rapid and potent immune responses. Our results suggest transdermal delivery of a minicircle DNA vaccine elicits potent antigen-specific immune responses, and as such, holds great promise for cancer therapy. Future work will include increasing transfection efficiency in vivo and increasing the immunogenicity of the vaccines through the use of adjuvants with the goal of producing feasible, efficacious DNA vaccines for cancer therapy.
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    Enhancing the efficacy of a nicotine vaccine
    (2013-03) Cornish, Katherine E.
    Tobacco addiction is the leading cause of preventable death worldwide. Many people continue to smoke cigarettes despite clear detrimental health effects. Available smoking cessation therapies are only partially effective, making new treatment approaches necessary to increase smoking cessation rates. Immunization against nicotine features a different mechanism of action than currently available medications. As a pharmacokinetic antagonist, immunization against nicotine alters distribution, metabolism, and clearance of nicotine to attenuate nicotine-induced behavior in animal models. Nicotine vaccines in clinical trials show efficacy but are limited by the modest and highly variable nicotine-specific antibody (NicAb) concentrations produced. This thesis focuses on ways to improve efficacy of a nicotine vaccine by combining it with additional forms of immunotherapy. The first aim of this thesis examined the effects of supplementing vaccination against nicotine with individualized doses of Nic311, a nicotine-specific monoclonal antibody. Compared to either immunotherapy alone, combining active and passive immunization produced greater alterations in nicotine pharmacokinetics and nicotine-induced behavior using a locomotor activity model. Only small doses of Nic311 were necessary to supplement vaccine-generated NicAb concentrations to a previously effective threshold. This decreased cost and use of typically expensive monoclonal antibodies, potentially increasing viability of this approach in a clinical setting. The second aim of this thesis examined the effects of concurrent administration of two immunologically distinct nicotine immunogens in a bivalent vaccine over a range of vaccine formulations and immunization conditions. Immunogens were co-administered in a bivalent vaccine without compromising immunogenicity of either immunogen when delivered subcutaneously in alum, but not when delivered intraperitoneally in Freund's adjuvant. When combined in alum, immune responses elicited by the two immunogens were largely independent of one another. This suggests that subjects who responded poorly to one immunogen may have responded better to the second, immunologically distinct immunogen in the bivalent vaccine. These results indicate that the bivalent vaccine strategy is a feasible way to increase antibody concentrations above what can be achieved using one immunogen alone, but integrity of the response is highly dependent on vaccine formulation and administration conditions.
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    Formulation and delivery of polymeric nanoparticle-assisted vaccine against melanoma
    (2015-04) Niu, Lin
    Poly (lactide-co-glycolide) (PLGA) nanoparticle (NP) is a widely used biodegradable carrier for drug and vaccine delivery. This thesis focused on the formulation, delivery and efficacy of PLGA NP for its potential application in melanoma immunotherapy. To enable reliable PLGA NP formulation for clinical use such as vaccination, lyophilization is the method of choice to manufacture dry NP dosage form. A major risk of the lyophilization product development for NPs is the irreversible NP aggregation due to freezing and drying stress. Based on real-time imaging, freezing stress could be attributed to freeze-concentration of NPs. Cryo-scanning electron microscopy (cryo-SEM) revealed individual NP separately embedded in the freeze-concentrate interstitial space of the sucrose formulation, leading to corroborative support for the "particle isolation" hypothesis of cryo-protection. Various sphere packing models were investigated to guide the rational design of cryo-/lyo-protectant containing NP formulations. To facilitate precise intradermal delivery of NP formulation for vaccination, microneedle array-mediated administration was utilized to deliver large volume of NPs into the skin. The majority of the infused PLGA NPs were retained locally. A PLGA NP vaccine formulation delivered intradermally elicited robust humoral and cellular immunity. Antigen-loaded NP formulation triggered quicker and stronger high affinity antibody responses compared to the soluble antigen formulation. Vemurafenib, a selective inhibitor of BRAF V600E, induces apoptotic melanoma cell death and remarkable tumor burden reduction. However, drug resistance invariably occurs. Novel TLR7/8 agonists were encapsulated in PLGA particulate formulation as immunostimulatory nanoparticles (ISNP) to boost immune response against drug-resistant melanoma. NP-mediated intracellular delivery contributed to enhanced dendritic cell activation in vitro and antigen-specific CD8+ T cell proliferation in vivo. The prophylactic vaccination using NP-assisted whole tumor cell formulation prolonged the survival of mice challenged with melanoma. To take advantage of the clearance of melanoma antigens by immune system in the context of BRAF inhibition, an ISNP-assisted in situ whole tumor cell vaccination strategy was investigated using BRAF V600E positive mouse SM1 melanoma cells. Despite the suppressed tumor growth, no survival benefit was observed in this therapeutic vaccination model.
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    The Function of PTPN22 and the Autoimmune Risk Variant LypW in Immune Responses to Vaccination
    (2016-07) Crabtree, Juliet
    High-affinity antibody production, T cell activation, and Interferon upregulation all contribute to protective immunity that occurs in humans following influenza immunization. Hematopoietic cell-specific PTPN22 encodes Lymphoid Phosphatase (Lyp), which regulates lymphocyte antigen receptor and Pattern Recognition Receptor (PRR) signaling. A PTPN22 variant R620W (LypW) predisposes to autoimmune and infectious disease, and confers altered signaling through antigen receptors and PRRs. We tested the hypothesis that LypW-bearing humans would have diminished immune response to trivalent influenza vaccine (TIV). LypW carriers exhibited decreased induction of influenza-specific CD4 T cells expressing effector cytokines, and failed to increase antibody affinity following TIV. No differences between LypW carriers and non-carriers were observed in virus-specific CD8 T cell responses, early interferon transcriptional responses, or myeloid APC costimulatory molecule upregulation. LypW association with defects in TIV-induced CD4 T cell expansion and antibody affinity maturation suggests that LypW may predispose to diminished capacity to generate protective immunity against influenza.
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    A heroin/morphine vaccine: mechanism of action and extending its use to other abused opioids
    (2013-07) Raleigh, Michael Dennis
    Heroin is more widely used than any other illicit opioid and mortality rates among heroin users are an average of 13 times higher than the general population. Intravenous heroin use is associated with crime, social disruption, and transmission of blood-born pathogens such as human immunodeficiency virus and hepatitis C. Effective pharmacotherapies are available to treat heroin abuse but have been largely unsuccessful because they require frequent dosing, have a high abuse potential, or have low compliance. Vaccines against heroin and its metabolites (e.g. morphine) are being considered as a complementary treatment for heroin abuse because they are long-acting, selective, have no abuse potential, and may benefit those unwilling to take the current pharmacotherapies. Vaccination with morphine-conjugate vaccines can elicit a strong immune response that reduces the behavioral effects of heroin in animals, presumably by morphine-specific antibodies binding opioids in blood and reducing their distribution to brain. This thesis explores the use of M-KLH, a morphine hapten conjugated to keyhole limpet hemocyanin (KLH) using a tetraglycine linker and mixed with either Freund's or alum adjuvant for increasing the immune response. Morphine vaccines present many challenges that make translation to clinical use difficult. Heroin is sequentially metabolized to its active intermediates 6-monoacetylmorphine (6-MAM), morphine, and then to morphine-6-glucuronide (in vivo and ex vivo). Heroin enters brain and is rapidly converted to 6-MAM, which is presumed to mediate most of heroin's early effects. With regard to the mechanism of action of morphine vaccines, it is unclear whether the antibodies they generate must bind heroin, its downstream metabolites, or both to prevent opioid distribution from plasma to brain and reduce heroin's behavioral effects. However, because analytical assays to measure heroin and metabolite concentrations in tissues have used a wide range of conditions and varying degrees of stability have been reported, studying the effect of vaccination on heroin distribution is not straightforward. In addition, heroin and metabolite distribution after i.v. heroin administration, the most common route of abuse by humans, has not been well characterized in non-vaccinated rodents. Finally, blockade of heroin by vaccination may not prevent the abuse of structurally distinct opioids. The overall goal of this thesis was to better understand the mechanism of action of morphine vaccines and to extend their use to other abused opioids. The specific aims were to stabilize heroin in blood and brain tissues for subsequent pharmacokinetic studies, study distribution of heroin and its metabolites in non-vaccinated and vaccinated rats, explore the effects of vaccination on heroin-induced behaviors, and determine if vaccine efficacy is retained when combined with a vaccine targeting oxycodone, another commonly abused opioid. These aims were explored using clinically relevant drug doses. Heroin and metabolite degradation was significantly reduced by 1) the addition of ice-cold sodium fluoride (a general esterase inhibitor) and formate buffer (pH 3.0) in heroin-spiked tissues, 2) rapid removal of red blood cells via centrifugation, and 3) drying opioids after extraction from tissues prior to measuring their levels. Using these conditions heroin and its metabolites were stabilized in tissues for subsequent distribution studies. In non-vaccinated rats 6-MAM was the predominant metabolite in brain as early as one minute after administration of 0.26 mg/kg i.v. heroin, which is consistent with previous studies that suggest that 6-MAM mediates heroin's early behavioral effects. Vaccination with a morphine-conjugate vaccine (M-KLH) led to a reduction of 6-MAM and morphine, but not heroin, distribution to brain after heroin administration, suggesting that morphine vaccines reduce accumulation of 6-MAM in brain. The mechanism by which this occurs is likely through antibody binding of 6-MAM in plasma to prevent its distribution to brain and is consistent with very high plasma 6-MAM concentrations in vaccinated rats after i.v. heroin or 6-MAM administration. Vaccination with M-KLH led to a reduction of heroin-induced anti-nociception and locomotor activity and remained effective for up to 16 days after repeated dosing suggesting that heroin vaccines may have long-lasting efficacy. These results are consistent with findings from the distribution studies and support the hypothesis that morphine vaccines function by retaining 6-MAM in plasma and prevent its accumulation in brain. To determine whether opioid vaccines could be combined without reducing individual vaccine efficacy and prevent heroin addicts from abusing structurally distinct opioids, rats were vaccinated with M-KLH, an oxycodone-conjugate vaccine (Oxy-KLH), or the bivalent vaccine (both M-KLH and Oxy-KLH). Total morphine- and oxycodone-specific antibody titers were significantly increased in rats that received the bivalent vaccine compared to rats that received individual vaccines. Concurrent i.v. administration of 6-MAM and oxycodone in M-KLH vaccinated rats led to increased 6-MAM retention in plasma and reduced 6-MAM distribution in brain. A similar effect on oxycodone distribution was seen in Oxy-KLH vaccinated rats. There was a trend towards greater efficacy in altering both 6-MAM and oxycodone distribution in the bivalent group compared to individual vaccine groups. These data suggest that combining opioid vaccines will retain, and possibly enhance, individual vaccine efficacy and might be a viable option to prevent addicts from abusing structurally distinct opioids. These findings contribute to the understanding of how morphine vaccines elicit their effects on heroin-induced behaviors and suggest that morphine vaccines, alone or in combination with other pharmacotherapies, may benefit those seeking treatment for heroin addiction.
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    Herpes Zoster (Shingles) Vaccine: Information for Patients
    (2010-07-29) Rank, Kevin
    The Herpes Zoster Vaccine is an effective and safe tool used to lessen the chance of getting herpes zoster (shingles) and a complication of shingles called “post-herpetic neuralgia” in adults over 60 years of age. It also proves to be a good tool at decreasing the harshness and length of illness in the event a vaccinated individual does get shingles.
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    HPV Vaccination
    (2012-07-26) Houghton, Andrew
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    HPV Vaccination for Boys
    (2009-08-20) Suojanen, Mark
    Gardasil has been demonstrated to be a safe and effective vaccine to confer immunogenicity to young men but is awaiting approval by the U.S. FDA.
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    The HPV Vaccine
    (2012-07-23) Zatochill, Mary
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    HPV Vaccine Recommendations for Women over 26 years old
    (2012-07-26) Durakovic, Selma
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    Immunological benefits of a novel polycaprolactone-polyorthoester-based therapeutic vaccine in a mouse model of glioma
    (2014-08) Grinnen, Karen Lynn
    Cancer immunotherapy has led to significant improvement in the survival of patients with previously untreatable malignancies. The use of therapeutic vaccines is a promising form of immunotherapy, but their efficacy remains ambiguous. Much of the difficulty in identifying the optimal formulation and delivery is related to the complicated nature of the immune response, where it is uncertain which aspects would be most effective in destroying cancer cells. In this thesis, a novel polymeric delivery system, involving poly (caprolactone)-co-poly (ortho ester) [PCL-POE], was used to deliver tumor antigens and adjuvants in a controlled manner. We hypothesized that persistent release of tumor antigens from the biodegradable polymer would result in an increase in the number and persistence of anti-tumor lymphocytes in the effector state. To test this hypothesis, vaccines were administered to mice and the time dependent immunological response was evaluated. The polymeric delivery system resulted in an in vitro release profile characterized by a burst release of both antigen and adjuvant followed, in both cases, by a much slower phase of release. We also observed that the slow release provided by the PCL-POE polymer stimulated prolonged maturation of dendritic cells, activation and persistence of anti-OVA antibodies and antigen-specific T cells following a single vaccination. The vaccine system was also tested in a mouse model of glioblastoma multiforme (GBM). We observed a significant, potentially translatable increase in overall survival.
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    Male HPV Vaccination
    (2010-07-22) Fowler, Jason Dean
    Male vaccination against HPV is safe and effective with regard to immunogenicity in young males. Cost effectiveness and decreased male morbidity and mortality secondary to vaccination require further research before definitive conclusions can be drawn.
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    Strategies to improve gene expression and targeting for DNA vaccine development
    (2013-10) Skinner, Nicole Elizabeth
    This dissertation examines strategies for improving DNA vaccines. Despite their many advantages and the considerable promise shown in small animal models, poor immunogenicity resulting from low transfection efficiency and transient transgene expression limits the effectiveness of DNA vaccines in humans. Methods for circumventing transient transgene expression as well as for directly manipulating T cell responses are explored. Chapters 2 and 3 of this thesis study the use of minicircle (MC) DNA as a vaccine. In chapter 2, a MC DNA vaccine is shown to generate larger CD8+ T cell responses and confer superior protection against an infectious challenge than a conventional plasmid DNA vaccine. Chapter 3 investigates the mechanism by which MC DNA elicits superior CD8+ T cell responses. In particular, the role of prolonged antigen expression is explored and found to be an important component of MC DNA vaccine efficacy. Chapter 4 proposes a method for targeted gene delivery to T cells in general, or to antigen-specific T cells. The targeting mechanism uses a streptavidin platform modified with positively charged peptides to enable binding to nucleic acid. Targeting is mediated by addition of biotinylated targeting moieties, either an anti-CD7 antibody for targeting of T cells or peptide-MHC molecules for targeting of antigen-specific T cells. We find that such an approach is promising, as it mediates uptake of both fluorescently labeled siRNA and DNA. However, gene expression did not occur, indicating that further modifications to the approach are needed.
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    Vaccination Against Lawsonia intracellularis Decreases Shedding of Salmonella enterica serovar Typhimurium in Co-Infected Pigs and Alters the Gut Microbiome
    (2017-11-09) Leite, Fernando L L; Singer, Randall S; Ward, Tonya; Gebhart, Connie J; Isaacson, Richard E; isaac015@umn.edu; Isaacson, Richard
    Salmonella enterica is a leading cause of foodborne illness worldwide and pork can serve a source of infection. In this study, we investigated if vaccinating pigs against Lawsonia intracellularis, a common pathogen of swine that has previously been shown to favor Salmonella enterica infection, confers protection against Salmonella enterica serovar Typhimurium. We investigated the underlying changes in the gut microbiome mediated by single S. Typhiumurium infection compared to co-infection with L. intracellularis as well as the effect of vaccination on the microbiome. In this study, a total of five treatment groups were used: 1) challenged with S. Typhimurium alone (Sal), 2) challenged with both S. Typhimurium and L. intracellularis (Sal Law), 3) challenged with S. Typhimurium and vaccinated against L. intracellularis (Sal Vac), 4) challenged with both S. Typhimurium and L. intracellularis and vaccinated against L. intracellularis (Sal Law Vac), and 5) non-infected control (Control).
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    Vaccination and Host-Targeted Interventions for Staphylococcus aureus Exotoxin-Mediated Diseases
    (2015-05) Gillman, Aaron
    Staphylococcus aureus is a major human pathogen capable of infecting nearly every tissue and causing a wide range of disease including skin and soft tissue infections, pneumonia, osteomyelitis, endocarditis, and toxic shock syndrome. S. aureus produces an array of exotoxins including superantigens and cytolysins, which contribute directly to disease by causing inflammation, immune evasion, and tissue damage. Recent evidence also suggests that superantigens and cytolysins modulate host epithelial cell signaling at mucosal surfaces and contribute directly to disease progression. Antibiotics can eliminate S. aureus, but have no effect on exotoxins already present within tissues. In addition, antibiotic resistance continues to increase in strains of S. aureus. Therefore, I hypothesize that anti-staphylococcal therapies, which target exotoxins or the down-stream host responses to exotoxins, should be explored to treat or prevent S. aureus infections. In this study, I investigated the mechanism of action of the superantigen, Toxic Shock Syndrome Toxin (TSST)-1, and the cytolysins, alpha-toxin and gamma-toxin, at the vaginal mucosal surface. Historically, TSST-1 was determined to promote disease (toxic shock syndrome) by widespread activation of CD4+ T-cells and antigen-presenting cells. However, recently, TSST-1 was determined to have direct proinflammatory effects on vaginal epithelial cells through an epidermal growth factor receptor-dependent pathway. Alpha-toxin’s mechanism of action and synergy with TSST-1 at the vaginal mucosa was characterized using in vitro, ex vivo, and in vivo models. Furthermore, novel actions of gamma-toxin at the vaginal mucosa were identified and the mechanism of action characterized. From this work, I proposed a model of cell-signaling by TSST-1, alpha-toxin, and gamma-toxin at the vaginal mucosa, which demonstrates a conserved proinflammatory and immune-modulating effect by S. aureus exotoxins. Proof of concept was established for two novel therapies for prevention and treatment of S. aureus infections. The tyrosine kinase inhibitor, AG1478, which inhibits the epidermal growth factor receptor signaling pathway, inhibited cytokine production induced by TSST-1, alpha-toxin, and gamma-toxin at the vaginal mucosa and prevented lethal menstrual toxic shock syndrome in rabbits challenged with live S. aureus. In addition, a vaccine targeting multiple exotoxins of S. aureus was highly effective in preventing lethal intrapulmonary challenge with a wide range of clinical strains and for treatment of the lethal effects of exotoxins in rabbits. In summary, these studies describe the mechanism of action of multiple S. aureus exotoxins and provide proof of concept for targeting exotoxins for treatment or prevention of toxin-mediated diseases. These studies demonstrate the importance of local proinflammatory effects during S. aureus infections, which contribute directly to the initiation of clinical disease. The data further highlight the importance of exotoxins during disease and add both insight and complexity to the field of S. aureus pathogenesis.
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    Visualizing CD8+ T cell responses to foreign and self-antigen
    (2017-08) Thompson, Emily
    CD8+ T cells can recognize any infected cell of the body, making them essential in the immune response against intracellular pathogens. A critical function of CD8+ T cells is the directed killing of target cells through cytolysis. This mechanism is dependent on direct cell-cell contact, which makes the migratory capacity of CD8+ T cells paramount to their successful immune response. The small intestine (SI) is the biggest mucosal surface between the host and the environment. The immune system in this compartment must actively eliminate infection while maintaining tolerance to normal flora, self, and food-antigen. Using two-photon laser scanning microscopy, I evaluated foreign- and self-specific CD8+ T cell motility in the SI. I found that foreign-antigen specific CD8+ T cell behavior varied throughout infection, and was independent of the αE integrin. Interestingly, self-specific CD8+ T cells were initially reactive to self-antigen in vivo but this behavior was altered after further tolerance induction. These studies inform our understanding of the requirements for effective CD8+ T cell immunosurveillance in the SI. I also evaluated what characterizes and contributes to a self-specific CD8+ T cell response to protein in the SI. Using a heterologous prime-boost-boost (HPBB) approach, I generated functional self-specific CD8+ T cells. This response matured throughout boosting, showing the potential of self-specific CD8+ T cells. I also used HPBB to evaluate foreign-antigen specific CD8+ T memory development and showed that the time interval between each boost impacts CD8+ T cell memory longevity.