Browsing by Author "Fletcher, McKenzie, author"

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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, advisor
Lactic 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.
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, advisor
Rotavirus 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.