Construction and Immunogenicity Testing of Whole Recombinant Yeast-Based T-Cell Vaccines
GlobeImmune’s Tarmogen® immunotherapy platform utilizes recombinant Saccharomyces cerevisiae yeast as a vaccine vector to deliver heterologous antigens for activation of disease-specific, targeted cellular immunity. The vaccines elicit immune-mediated kil
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Introduction Tarmogens are comprised of heat-inactivated, whole recombinant Saccharomyces cerevisiae yeast cells expressing disease-related target antigens, most typically intracellularly. These vaccines possess unique cell surface molecular signatures (pathogen-associated molecular patterns) that are key agonists for phagocytic and tolllike receptors expressed by antigen-presenting cells (APCs). Following vaccination with Tarmogen, receptor binding and activation of APCs trigger yeast uptake and cross-presentation of heterologous yeast-expressed antigens with class I and class II MHC molecules, in turn stimulating CD4+ and CD8+ T-cell responses in vivo [1]. The yeast also induces the Th17 pathway resulting in reduced regulatory T-cell activity [2, 3]. With help from the CD4+ T-cell population, the CD8+ T cells specifically kill and/or clear virus-infected cells and tumor cells expressing the target antigen. Tarmogens are being developed clinically for the treatment of chronic human viral infections and a variety of cancers [4]. The broad applicability of the platform is also being widely exploited in basic vaccine research. Functional evaluation of Tarmogens is accomplished by any of a wide array of in vivo and in vitro assays that can illuminate the activity and mechanism in different host immunological backgrounds. For Tarmogens targeting tumor-associated antigens, many clinically relevant and tractable rodent models exist that require only lower biosafety level (e.g., ABSL1) animal facilities for execution and that inherently evaluate activity in the context of immunological tolerance and suppression [5–7]. Achieving a similar level of immunological relevance for infectious diseases often involves challenge of immunized animals with a target microorganism. As selection of a lead candidate Tarmogen would ideally
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_35, © Springer Science+Business Media New York 2016
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include testing of multiple candidates and immunization regimen optimization, such infectious animal models can be prohibitively long and expensive. These latter obstacles can be ameliorated with a project plan that includes (1) infectious ex vivo assays with T cells and monocytederived dendritic cells from a patient’s blood [8] and (2) noninfectious in vivo murine experiments emphasizing the cellular mechanisms of activity in the context of Tarmogen immunization. Following on this theme, we describe four methods that in our hands provide high-magnitude antigen-specific T-cell responses in a relatively short time frame with moderate resources.
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Materials Supplier details are provided where reagent source or quality is particularly important. 1. S. cerevisiae haploid yeast (e.g., genotype ade2-1; ura3-1; his311,15; trp1-1; leu2-3,112; can1-100; or a closely related strain). 2. Two μm circle-based shuttle vector with yeast and E. coli origins of
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