Ex vivo Platforms to Study the Primary and Recall Immune Responses to Intracellular Mycobacterial Pathogens and Peptide-Based Vaccines

Abstract

Progress in the study of the immune response to pathogens and candidate vaccines has been impeded by limitations in the methods to study the functional activity of T-cell subsets proliferating in response to antigens processed and presented by antigen presenting cells (APC). As described in this review, during our studies of the bovine immune response to a candidate peptide-based vaccine and candidate rel deletion mutants in Mycobacterium avium paratuberculosis (Map) and Mycbacterium bovis (BCG), we developed methods to study the primary and recall CD4 and CD8 T-cell responses using an ex vivo platform. An assay was developed to study intracellular killing of bacteria mediated by CD8 T cells using quantitative PCR to distinguish live bacteria from dead bacteria in a mixed population of live and dead bacteria. Through use of these assays, we were able to demonstrate vaccination with live rel Map and BCG deletion mutants and a Map peptide-based vaccine elicit development of CD8 cytotoxic T cells with the ability to kill intracellular bacteria using the perforin-granzyme B pathway. We also demonstrated tri-directional signaling between CD4 and CD8 T cells and antigen-primed APC is essential for eliciting CD8 cytotoxic T cells. Herein, we describe development of the assays and review progress made through their use in the study of the immune response to mycobacterial pathogens and candidate vaccines. The methods obviate some of the major difficulties encountered in characterizing the cell-mediated immune response to pathogens and development of attenuated and peptide-based vaccines.

Ability of mycobacterial pathogens to establish a persistent infection is lost by deletion of a single gene,rel, regulator of the stringent response

“…The development of assays to study the primary and recall responses in real time, using the same tissue culture platform, provided an opportunity to begin studies focused on determining how gene products encoded by genes regulated by rel interfere with immune clearance of mycobacterial pathogens. The methods can be used with any species including humans to replicate the studies described in our initial and present report (41).…”

“…The replacement of the chromium release assay and the CFU assay have provided opportunity to study the immune response to mycobacterial pathogens in real time. The methods developed to study the primary and recall responses ex vivo can be used with any species including humans (41). The use of cattle in the present studies has highlighted the value of including a large animal species in the study of mycobacterial pathogens (18, 62).…”

Vaccination of cattle with a virus-vectored vaccine against a major membrane protein ofMycobacterium a. subsp.paratuberculosiselicits CD8 cytotoxic T cells that kill intracellular bacteria

“…The development of methods to use cattle as a model species to study the immune response to mycobacterial pathogens has afforded an opportunity to follow up on observations made with a mouse model which showed that deletion of rel abrogates the ability of Mtb to establish a persistent infection (26, 27). Similarly, a Maprel deletion mutant could not establish a persistent infection in cattle or goats (5).…”

Modeling Paratuberculosis in Laboratory Animals, Cells, or Tissues: A Focus on Their Applications for Pathogenesis, Diagnosis, Vaccines, and Therapy Studies

“…Similarly, Davis and co-workers developed ex vivo platforms in cattle to study the functional activity of CD4+, CD8+, and γδ T and NK cells stimulated with MAP antigen-primed antigen-presenting cells (dendritic cells (DC) present in the blood, monocyte-derived DC, and monocyte-derived macrophages) [57,[103][104][105]. The authors highlighted the usefulness of these platforms for assessing bacterial viability, the cells' phenotype, cytotoxicity mechanisms and, consequently, for examining the potential role of the cell-mediated immune response in preventing the establishment of a persistent infection of MAP [106].…”