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Antigen Presenting Cells and T Cell Responses to Mycobacterium bovis - SE3207

Description
Current UK and EC policies aim to maintain cattle herds free of tuberculosis caused by Mycobacterium bovis as a means of protecting public health and fulfilling national and international requirements for animal trade. The increase that has occurred in the incidence of bovine tuberculosis in England and Wales over the last 10 years seriously threatens this policy and has resulted in a substantial increase in the cost of the tuberculosis control program. The Krebs review recommended that considering recent advances in the molecular genetics of mycobacteria and in immunology an effective vaccine for cattle would provide a more cost-effective and sustainable means of controlling the disease in the longer term. Two types of vaccine can be considered, namely attenuated strains of M. bovis produced by removal of virulence determinants and M. bovis antigens (subunits), or the genes encoding them, capable of stimulating protective immune responses.
An understanding of the key events in the pathogenesis of the disease in cattle and the nature of the protective immune responses would allow targeted approaches to be applied to the identification of bacterial molecules involved in virulence and immunity and hence enhance the likelihood of developing an effective vaccine. The proposed research program seeks to apply IAH’s expertise in bovine immunology to dissect the mechanisms of immunity to M. bovis in cattle. The components of this program are:
(i) Characterization of the functional changes induced in antigen-presenting cells by M. bovis: The results of this work will allow identification of those properties of the organisms that should be targeted for attenuation and hence point to strategies for development and screening of attenuated strains for vaccination.
(ii) Identification of the T cell responses that are required for immunity: The findings will facilitate selective screening of antigens for use in subunit vaccines and indicate the type of vaccine formulation that will be required. Immunological correlates of immunity identified in this work will also be of value for monitoring responses in vaccine trials.
(iii) Clarification of the role of environmental mycobacteria in interference with vaccine responses to M. bovis: This issue needs to be resolved to determine whether or not vaccines need to be designed to circumvent such interference.
(iv) Investigation of cytokine expression in DNA vaccines as a means of enhancing vaccine efficacy: Selected cytokines have the potential to bias the immune responses induced by DNA vaccines towards those responses that are required for immunity.
The requirements defined in the MAFF Animal Health and Welfare Research Requirements Document that relate to developing improved strategies to reduce herd breakdown and which are addressed in our proposal are as follows. (A) Investigate currently available vaccine candidates, namely BCG, (C) Develop a vaccine based on the antigenic elements of M. bovis, (D) Investigate the immune response of cattle to M. bovis with the aim of identifying protective antigens, and (E) Investigate the immune mechanisms involved in resistance to infection via the respiratory route.
Objective
FUNCTIONAL ALTERATIONS IN ANTIGEN PRESENTING CELLS

Available data indicate that pathogenic Mycobacteria induce changes in the antigen presenting cell populations in which they replicate, which have a profound effect on the nature of the immune responses that they induce and facilitate persistence of infection. This aspect of the disease in cattle caused by M. bovis has not been studied in any detail but is now amenable to investigation. An understanding of the interaction of M. bovis with antigen-presenting cells will indicate those properties of the organism that should be attenuated for vaccination and, in conjunction with studies of T cell responses, will provide insight into how the immune response can be manipulated to achieve immunity.

1. Effect of infection on the function of APCs

Anti-bacterial and immune stimulatory functions of antigen presenting cells infected with different strains of M. bovis will be examined: (i) The kinetics of replication of different strains of M. bovis in macrophages and dendritic cells will be investigated and the influence on cell viability determined. (ii) The susceptibility of the different cell types to induction of intracellular killing of bacteria by stimulation with TNF will be examined, and the effect of the bacterial strain and the duration of infection on the responses will be determined. (iii) The capacity of macrophages and dendritic cells infected with different strains of M. bovis for variable periods of time, to stimulate specific T cell proliferative responses in peripheral blood mononuclear cells from M. bovis-immunised cattle will be examined and the influence of the bacterial strain on the type of T cell response (CD4 versus CD8 and cytokine profile) will be determined. (iv) The ability of infected macrophages and dendritic cells to present unrelated antigens to T lymphocytes will also be investigated to determine whether infection results in a general suppression of this function or modification of the type of T cell response that is induced.

2. The influence of infection of APC on surface expression of co-stimulatory molecules

The expression of surface molecules known to have an important role in the induction of T cell responses will be examined in monocyte/macrophage and dendritic cell populations infected with M. bovis. A comparison will be made between virulent and putatively attenuated strains of M. bovis (including BCG) and with other non-pathogenic Mycobacteria, M. avium and M. smegmatis.

3. The influence of infection of APC on cytokine expression

Further studies will determine how infection of monocyte /macrophages and dendritic cells with virulent and avirulent M. bovis and other Mycobacteria affect expression of cytokines. The transcription of cytokine genes and expression of cytokine molecules by infected and non-infected cells will be compared. The studies will focus on those cytokines known to be involved in mediating killing of intracellular organisms and those involved in stimulation and modulation of T cell responses.

4. Localisation of bacterial proteins in antigen presenting cells

Information on the subcellular localisation of the bacterial proteins that are involved in modulating host cell function may provide important clues to how they exert their effects. The localisation of M. bovis proteins identified by Dr Hewinson’s group, either by transposon mutogenesis or on the basis of differential expression in macrophages will be examined using immunofluoresence staining and confocal microscopy.




THE ROLE OF CD4+ AND CD8+ T CELL RESPONSES IN IMMUNITY

As already indicated, the balance in theTh1/Th2 cytokine profile of the CD4 T cell response is believed to be an important parameter in determining the outcome of Mycobacterial infections [23], while the role of CD8+ T cells in controlling the infection is controversial. [28, 39] The proposed studies will focus on these two aspects of the immune response to M. bovis with the aim of defining which responses are required for immunity. Immune responses will be analysed in animals infected with virulent M. bovis or BCG.

5. M. bovis-specific CD8+ T cell responses

Studies will be undertaken to determine whether or not infection with M. bovis or BCG stimulates specific CD8+ T cell responses and to define the functional properties of such T cells, in terms of cytotoxic activity, cytokine production and MHC restriction specificity.

6. The role of CD8+ T cells in protection

The influence of CD8+ T cells on the course of infection with M. bovis will be investigated by specifically depleting T cells in vivo by administration of CD8-specific monoclonal antibody.

7. Correlation of T cell responses with immunity to M. bovis

The observation that BCG induces immunity against virulent M. bovis in some animals but not others provides an opportunity to compare the T cell responses in individual immunised animals that subsequently prove to be immune or susceptible to infection, and hence to establish correlates of immunity. The T cell responses of BCG-immunised animals will also be compared to those of animals infected with virulent M. bovis.

8. Specificity of protective T cell responses

Studies will be initiated to analyse the antigenic specificity of those T cell responses that are associated with immunity. For this purpose T cell lines will be derived from immune animals and used to screen M. bovis antigens. This part of the study will not be completed within the 3 year duration of this project but would aim to establish the T cell lines and some of the antigen screening systems and generate preliminary data on identification of antigens recognised by CD4+ T cells.


INTERFERENCE IN VACCINAL RESPONSES BY ENVIRONMENTAL MYCOBACTERIA

An understanding of how the immune responses induced by BCG are affected by prior exposure to environmental Mycobacteria is required in order to inform the development of a vaccine that can be used in the field.

9. The effect of infection with environmental Mycobacteria on responses to BCG

The influence of prior exposure to “environmental” Mycobacteria on the T cell responses and immunity induced by BCG will be examined using SPF calves, calves experimentally infected with M. avium and calves with evidence of natural exposure to Mycobacterium. Such studies should indicate whether prior infection with environmental Mycobacteria suppresses or qualitatively alters the immune response to BCG.


VACCINE DELIVERY

In the event that a subunit vaccine is used to vaccinate against M. bovis, an antigen delivery system that preferentially stimulates protective immune responses will be required. One such system is vaccination with plasmid DNA.


10. Co-expression of cytokines in DNA vaccines

To establish the potential for including cytokine genes in DNA vaccines for M. bovis, vaccine constructs coexpressing bovine IL-12 or IL-18 along with the M. bovis antigen MPB83 will be tested for immunogenicity in cattle.
Project Documents
• Final Report : Antigen presenting cells and T cell responses to Mycobacterium bovis   (1410k)
• Executive Summary : Antigen presenting cells and T cell responses to Mycobacterium bovis   (27k)
Time-Scale and Cost
From: 1999

To: 2002

Cost: £1,200,000
Contractor / Funded Organisations
Institute for Animal Health (BBSRC)
Keywords
Animal Health              
Biotechnology              
Bovine Tuberculosis              
Control              
Plants and Animals              
Vaccines              
Zoonoses              
Fields of Study
Animal Health