Honors Theses
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Item Open Access Detection of insect-specific viruses in mosquitoes at the Colorado State University Center for Vector-Borne and Infectious Diseases Insectary(Colorado State University. Libraries, 2025) Domenico, Lila, author; Ebel, Gregory, advisor; Geiss, Brian, committee member; Williams, Kaitlynn, committee memberInsect-specific viruses (ISVs) are viruses that can infect arthropod cells, like ticks and mosquitoes, but not vertebrate cells. It has also been shown that ISV infections decrease the growth of arboviruses, like West Nile virus and Dengue, during experiments where mosquitoes or mosquito cell lines were coinfected. Because of this unique ability to decrease arboviral growth, combined with their inability to replicate in vertebrate cells, research has started to expand on potential biocontrol applications of ISVs. However, since arbovirus research is conducted at facilities like the insectary at the Center for Vector-Borne and Infectious Diseases (CVID), the presence of ISVs in the mosquitoes used for these studies could influence experimental outcomes. For that reason, we determined which ISVs are the most commonly found in various mosquito populations and used RT-PCR to screen multiple colonies of mosquitoes reared in the insectary at CVID. We screened for the following ISVs: Culex flavivirus, Aedes flavivirus, Cell fusing agent virus, Phasi Charoen-like virus, Kamiti River Virus, Eilat Virus, and Calbertado virus. We collected mosquitoes from different colonies of Aedes triseriatus, Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Culex tarsalis from the insectary to screen for these ISVs. With pooled mosquito homogenates from each of these species, we extracted RNA and analyzed the extractions using RT-PCR, using previously published primers for the ISVs of interest. Our results show that, of the colonies tested from the insectary at CVID, none of the ISVs screened for were present. These findings indicate that the colonies maintained at CVID are favorable control organisms for future arboviral research.Item Open Access upp-based counterselective genetic cloning to create rotavirus vaccine constructs using Lactobacillus acidophilus platform(Colorado State University. Libraries, 2025) Kiehl, Paris, author; Kiehl, Sophie M., author; Swartzwelter, Benjamin J., author; Vilander, Allison, committee member; Kinkel, Traci, committee member; Fletcher, McKenzie, author; Dean, Gregg, advisorRotavirus is a major public health burden that causes severe gastroenteritis and kills more than 200,000 infants per year. Live attenuated vaccines have reduced effectiveness in low- and middle-income countries, which is correlated to gut microbiota composition. To combat this, we are developing an orally administered vaccine using the probiotic Lactobacillus acidophilus as a vector. Here, we used upp-based genetic cloning to create multiple strains of L. acidophilus that express rotavirus' VP7 glycoprotein behind the highly expressed metabolic enzyme enolase. This counterselective genetic cloning process uses a temperature-sensitive helper plasmid and recombination-directing vector plasmid to integrate target sequences into the bacterial genome and remove selectable markers. Vaccine constructs were evaluated for recombination using genetic analysis and immunoassays. The vaccines are being tested in murine and porcine models to evaluate their ability to protect hosts against rotavirus.Item Open Access Effect of probiotic-based vaccination on lactate availability in immune inductive tissues(Colorado State University. Libraries, 2025) Kiehl, Sophie, author; Kiehl, Paris M., author; Fletcher, McKenzie, author; Kinkel, Traci, committee member; Vilander, Allison, committee member; Dean, Gregg, advisorLactic acid bacteria (LAB) colonize the gastrointestinal tract and generally confer immune benefits to their host. Because of this, they are emerging as viable vaccine vectors, particularly against mucosal pathogens. LAB secrete high quantities of lactate as a byproduct of their metabolism, and lactate has been increasingly recognized as an immunomodulator, often in an anti-inflammatory capacity. We are studying how lactate metabolism of LAB-based vaccines affects lactate availability in immune inductive sites using the bacterial platform Lactobacillus acidophilus. Using Mirasol® Pathogen Reduction Technology system, we inactivated a recombinant L. acidophilus expressing the model antigen ovalbumin. We first verified that inactivation inhibited bacterial growth and reduced lactate metabolism while preserving antigen expression. We then administered the live and inactivated vaccines orally to BALB/c mice and measured lactate concentrations in harvested immune inductive sites (Peyer's patches and ileal lumen contents) using gas chromatography mass spectrometry. Lactate levels in the ileum and Peyer's patches were unchanged following administration with probiotic-based vaccines, suggesting their administration does not disrupt the metabolic equilibrium of these tissues that may trigger an anti-inflammatory state.Item Open Access Predicting protection against tuberculosis from BCG using the guinea pig model(Colorado State University. Libraries, 2025) Englert, Alexandra, author; Podell, Brendan K., advisor; Henao-Tamayo, Marcela, committee member; Maristela, Lea, author; Hunstad, Lauren, author; Ackart, David F., author; Lanni, Faye, authorGuinea pigs, while having been used as a model animal for tuberculosis infections for over a century, modern literature about their response to the commonly used BCG vaccination is limited. In this study, we investigated whether immune responses to the BCG vaccine are variable and if these differences could predict protection after exposure to Mycobacterium tuberculosis. Using the guinea pig model, the only rodent species that reliably develops delayed-type hypersensitivity responses similar to humans, we evaluated inflammation in response to BCG vaccination, tuberculin skin test (TST) response, and capacity for antigen-specific secretion of IFN-γ by ELISpot in response to M. bovis purified protein derivative (PPD). We hypothesized that the degree of inflammation incited by BCG vaccination would correlate with TST size and frequency of antigen-specific IFN-γ production. Most of the animals developed an inflammatory response to TST within 24-72 hours, indicating a Th1 response was developed post-vaccination. However, 4 out of 18 individuals lacked an inflammatory response that lasted over 24 hours. To confirm this, IFN-γ production was assessed in PBMCs isolated from BCG-vaccinated and unvaccinated guinea pigs, finding little correspondence between IFN-γ production and increased skin inflammation. We hypothesized that IFN-γ secretion would be highest in guinea pigs with the largest TST response. The results of this study demonstrate the development of a delayed-type hypersensitivity response to BCG vaccination, albeit inconsistent in its systemic effects. Collectively, our results a highly differential response to BCG vaccination. If vaccination confers variable protection against M. tuberculosis infection, this variable vaccine response may offer insight into correlates of immune protection.