Degree Type

Dissertation

Date of Award

2016

Degree Name

Doctor of Philosophy

Department

Veterinary Pathology

Major

Immunobiology

First Advisor

W. Ray Waters

Second Advisor

Jesse Hostetter

Abstract

Mycobacterium bovis is the causative agent of tuberculosis in cattle. M. bovis is a member of the Mycobacterium tuberculosis complex (Mtbc) which also includes M. tuberculosis, M. caprae, M. microti, M. africanum, M. canetti, M. pinnipedii, M. suricattae, M. mungi, and M. bovis Bacillus Calmette Guerin (BCG) 1-3. Despite what the nomenclature may imply, members of this group are not host species specific. The host range of M. bovis appears to be the broadest of the complex, causing disease in a number of species including humans 4. Great strides have been made over the past century both toward the control of bovine tuberculosis (bTB) in cattle and also limiting the risk to human exposure (e.g., pasteurization of milk for dairy products); however, the disease still has great socioeconomic impact for livestock farmers. It is estimated that over 50 million cattle are currently infected worldwide, costing around $3 billion annually5. The WHO (World Health Organization), in conjunction with FAO (Food and Agriculture Organization of the United Nations) and OIE (Office International des Épizooties), classify bTB as a neglected zoonosis 6,7.

A crucial component of the immune response to TB in humans, cattle and mice is the production of interferon (IFN)-γ by T helper 1 (Th1) CD4 T cells 8-12. Immune deficiencies affecting CD4 T cells [e.g., human immunodeficiency virus (HIV) infection] and IL-12/IFN-γ /STAT1 signaling pathways result in more severe disease upon TB infection in humans 13,14. In fact, IFN-γ release assays (IGRA) and delayed type hypersensitivity (i.e., skin test) responses are markers of infection in cattle and humans (reviewed respectively by Schiller et al. 15 and Walzl et al. 16). Diagnostic IGRAs are measures of ‘ex vivo’ immune responses relying on rapid production of IFN-γ in response to mycobacterial antigen stimulation in short-term (16–24 h) whole blood or peripheral blood mononuclear cell (PBMC) cultures. These ex vivo assays are generally considered a measure of T cell effector responses 12,17. Most protective bTB vaccines elicit ex vivo IFN-γ responses; however, not all vaccines inducing this response are protective 5,18. Cultured assays (i.e., culture of cells for 7-14 days before IFN-γ measurement) are assumed to quantify memory responses, primarily central memory T cells (Tcm) in humans 19-21. In light of the idea that cultured ELISPOT measures Tcm responses, several studies have shown its association with protection against malaria, suppression of viral recrudescence in hepatitis B virus carriers, low virus levels in HIV infection, and favorable outcomes in human TB 22-26. In cattle, responses measured by cultured IFN-γ ELISPOT following vaccination are the best-known positive correlate of protection in vaccine and challenge experiments 27-31.

Increasing interest has risen in order to characterize and assess the role of polyfunctionality in both protective and detrimental immune responses in TB. Polyfunctional T cells simultaneously produce two or more cytokines with IFN-γ, IL-2, and tumor necrosis factor-α (TNF-α) being the most commonly measured Th-1 cytokines 32,33. Association between protection and vaccination-induced polyfunctional T cells has been mainly studied in small animal models 34,35. In humans, strong polyfunctional responses are detected in M. tuberculosis-infected individuals, high IL-2 production is associated with a positive clinical status (e.g., latent or treated disease), while a strong IFN-γ/TNF-α response is associated with a poor outcome (i.e., active TB) 36. Human polyfunctional responses to vaccination both prior to TB exposure and in previously exposed individuals (i.e., latent infection) are extremely variable. In cattle, T cell polyfunctionality has only been measured upon ex vivo recall stimulation 26,37.These studies found no association between polyfunctional responses measured before challenge and vaccine success. Instead, polyfunctional responses to infection were associated with increased pathology and poor disease outcome 26. Polyfunctional responses by long-term cultured cells for enrichment of Tcm responses have not been evaluated in spite of the fact that cultured IFN-γ ELISPOT is one of the most promising protection correlates in cattle 27-31.

The research included in this thesis had the objective to characterize memory cells phenotype and aspects of their functionality in cattle, specifically in response to bTB infection and vaccination. The discrimination of cell phenotype involved in cytokine production under both cultured and ex vivo conditions may be necessary to identify specific correlates of vaccine efficacy, useful for vaccine candidates selection for costly efficacy trials in biosafety level 3 (BSL-3) facilities.

Copyright Owner

Mayara Fernanda Maggioli

Language

en

File Format

application/pdf

File Size

153 pages

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