A bovine alveolus model to replace cattle in the study of host-pathogen interactions in bovine tuberculosis
Lead Research Organisation:
University of Surrey
Department Name: Veterinary Medicine & Science
Abstract
Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis (BTB) and infects livestock with severe socio-economic consequences and an impact on animal health. As well as the financial and emotional impact BTB has on the cattle farming community and government, the disease is a major risk to human and livestock health in developing countries. The control of BTB has proved problematic in Great Britain and Ireland. In the absence of improved control the projected economic burden to GB over the next decade is predicted to be £1 billion.
Tackling BTB requires deeper insights into host-pathogen interactions otherwise it is unlikely any major breakthroughs in developing effective tools for disease intervention will occur. The principle route of infection with M. bovis is via inhalation of infectious aerosols. On inhalation, M. bovis reaches the lung tissues, especially the alveolus. What happens next determines whether the host goes on to acquire TB, or whether the host deals with the threat. Research in human TB shows that epithelial cells lining the alveolus are far more than a simple physical barrier to pathogens. Indeed, M. tuberculosis is able to penetrate these cells and gain access to the deeper tissues whilst evading elimination by the immune system. In turn, the epithelium detects the presence of mycobacteria, responding by producing molecules involved in antimicrobial activity, inflammation, and pathology. As the lung epithelium is central to early response to TB, better understanding of the early events and interactions that occur when virulent mycobacteria arrive in the bovine alveolus are needed. No model currently exists with which to conduct these studies and the crucial events in the earlier stages of infection are intractable for study in the live animal. Understanding early events that are played out within the alveolus is critical if we are to gain new insights in how to enhance host resistance to infection, e.g. through vaccination.
In response to this need, we shall develop a tissue culture model of the bovine alveolus with which to study the interaction of M. bovis with the bovine host. The model represents a non-animal alternative with which to study of the pathogenesis of BTB by removing the need to infect cattle with mycobacteria to answer fundamental questions in TB pathogenesis and provide a valid substitute for cattle that can be used by researchers without access to animal facilities. The simplicity of the model make it preferable over the use of the whole animal, and for answering questions that require data to be gathered within minutes of infection or where time course studies are required. This will be a new tool available to the scientific community. Its use is not confined to BTB, but would be applicable to the study of respiratory infections of cattle in general, many of global importance, such as bovine respiratory disease (BRD).
Vaccines against BTB developed to generate a specific host response would be a significant advance on the current state of affairs where vaccines must be tested empirically in cattle to evaluate their efficacy. A specific objective of this project will determine whether the behaviour of BCG / M. bovis and host cells in the model correlates with protective efficacy seen in cattle challenge studies from which we have stored blood cells to evaluate. Identifying a read-out in our model that is related to vaccine efficacy in the whole animal could be a basis of screening vaccine candidates without the need to challenge cattle with M. bovis. This would reduce the severity and duration of animal experiments, as well as significantly reduce their cost.
We hypothesise that a significant aspect of vaccine-mediated protection against BTB is expressed at the level of host-pathogen interactions within the alveolus.
Tackling BTB requires deeper insights into host-pathogen interactions otherwise it is unlikely any major breakthroughs in developing effective tools for disease intervention will occur. The principle route of infection with M. bovis is via inhalation of infectious aerosols. On inhalation, M. bovis reaches the lung tissues, especially the alveolus. What happens next determines whether the host goes on to acquire TB, or whether the host deals with the threat. Research in human TB shows that epithelial cells lining the alveolus are far more than a simple physical barrier to pathogens. Indeed, M. tuberculosis is able to penetrate these cells and gain access to the deeper tissues whilst evading elimination by the immune system. In turn, the epithelium detects the presence of mycobacteria, responding by producing molecules involved in antimicrobial activity, inflammation, and pathology. As the lung epithelium is central to early response to TB, better understanding of the early events and interactions that occur when virulent mycobacteria arrive in the bovine alveolus are needed. No model currently exists with which to conduct these studies and the crucial events in the earlier stages of infection are intractable for study in the live animal. Understanding early events that are played out within the alveolus is critical if we are to gain new insights in how to enhance host resistance to infection, e.g. through vaccination.
In response to this need, we shall develop a tissue culture model of the bovine alveolus with which to study the interaction of M. bovis with the bovine host. The model represents a non-animal alternative with which to study of the pathogenesis of BTB by removing the need to infect cattle with mycobacteria to answer fundamental questions in TB pathogenesis and provide a valid substitute for cattle that can be used by researchers without access to animal facilities. The simplicity of the model make it preferable over the use of the whole animal, and for answering questions that require data to be gathered within minutes of infection or where time course studies are required. This will be a new tool available to the scientific community. Its use is not confined to BTB, but would be applicable to the study of respiratory infections of cattle in general, many of global importance, such as bovine respiratory disease (BRD).
Vaccines against BTB developed to generate a specific host response would be a significant advance on the current state of affairs where vaccines must be tested empirically in cattle to evaluate their efficacy. A specific objective of this project will determine whether the behaviour of BCG / M. bovis and host cells in the model correlates with protective efficacy seen in cattle challenge studies from which we have stored blood cells to evaluate. Identifying a read-out in our model that is related to vaccine efficacy in the whole animal could be a basis of screening vaccine candidates without the need to challenge cattle with M. bovis. This would reduce the severity and duration of animal experiments, as well as significantly reduce their cost.
We hypothesise that a significant aspect of vaccine-mediated protection against BTB is expressed at the level of host-pathogen interactions within the alveolus.
Technical Summary
Tackling BTB requires deeper insights into host-pathogen interactions otherwise it is unlikely any major breakthroughs in developing effective tools for disease intervention will occur. In keeping with the aim of this call, we shall develop a tissue culture model of the bovine alveolus as a surrogate with which to study the interaction of M. bovis with the host.
We shall work with Professor William Hope, University of Liverpool who through previous NCR3Rs funding (Project Grant G0700599) refined a culture model of the human alveolus containing an air-liquid interface. This model consists of a cellular bilayer constructed of human pulmonary artery endothelial cells and human alveolar epithelial cells on opposing sides of a Transwell insert. The Hope group use this model to study invasive pulmonary aspergillosis. Professor Hope will provide direct training in his model at Liverpool. This will ensure successful transfer of the methodology. We shall modify the Hope model to produce a functional bovine model. We shall replace the human cells of their model with immortalised bovine cells. The PDRA undertaking the work will visit the Hope laboratory to learn the techniques involved.
The functional utility of the model will be demonstrated by using it to address our hypothesis that a significant aspect of vaccine-mediated protection is expressed at the level of host-pathogen interactions within the alveolus. We reason that the phenotype of successful vaccination in cattle is expressed in the speed and activity of the host response and the behaviour of the pathogen within the alveolus. We shall test this by introducing into our model stored PBMCs from cattle that expressed either strong or weak levels of vaccine protection. Initially we shall introduce mycobacteria into the model using BCG. This is for safety reasons: the methodology can be established safely under CL2 laboratory conditions before being transferred to the CL3 laboratory in order to use M. bovis.
We shall work with Professor William Hope, University of Liverpool who through previous NCR3Rs funding (Project Grant G0700599) refined a culture model of the human alveolus containing an air-liquid interface. This model consists of a cellular bilayer constructed of human pulmonary artery endothelial cells and human alveolar epithelial cells on opposing sides of a Transwell insert. The Hope group use this model to study invasive pulmonary aspergillosis. Professor Hope will provide direct training in his model at Liverpool. This will ensure successful transfer of the methodology. We shall modify the Hope model to produce a functional bovine model. We shall replace the human cells of their model with immortalised bovine cells. The PDRA undertaking the work will visit the Hope laboratory to learn the techniques involved.
The functional utility of the model will be demonstrated by using it to address our hypothesis that a significant aspect of vaccine-mediated protection is expressed at the level of host-pathogen interactions within the alveolus. We reason that the phenotype of successful vaccination in cattle is expressed in the speed and activity of the host response and the behaviour of the pathogen within the alveolus. We shall test this by introducing into our model stored PBMCs from cattle that expressed either strong or weak levels of vaccine protection. Initially we shall introduce mycobacteria into the model using BCG. This is for safety reasons: the methodology can be established safely under CL2 laboratory conditions before being transferred to the CL3 laboratory in order to use M. bovis.
Planned Impact
3Rs impact - 100 cows a year replaced by our model
1. This project provides a tissue culture model to study infection of the bovine lung with mycobacteria. A specific objective is to determine whether the behaviour of mycobacteria and host cells in the model correlates with protective efficacy seen in cattle challenge studies from which we have stored blood cells to evaluate. If we identify a read-out in our model related to vaccine efficacy in the whole animal then we have the basis of screening vaccine candidates in vitro without needing to challenge cattle with M. bovis. An average of one experiment of 40 cattle a year is conducted for the purpose of empirical testing of novel vaccines for bovine TB and could be replaced.
2. Bovine respiratory disease (BRD) is one of the key health issues and most costly problems occurring in cattle worldwide. New antimicrobials are needed for BRD. This is one area where our model could be used as it is based on a model used already to mimic systemic drug administration for the treatment of antifungals without needing to use live animals. We shall raise the profile of our model with companies who produce drugs for BRD. This is expected to lead to R&D investment from the commercial sector and a reduction in animals used for drug screening for BRD. Accepting more studies are conducted for this purpose than reported, an underestimate is that using our model would replace at least 33 cows per year (133 cows were reported as used for this work over a 4 year period).
3. 104 cows were used for studies relating to the respiratory system or microbiology in 2013 (Annual Statistics of Scientific Procedures on Living Animals for Great Britain). Taking a conservative estimate, one quarter of these (=26) a year could be replaced by our model in the UK alone.
Academic impact
1. Contribute new knowledge to understanding of BTB. Our data will be of value to veterinary and human TB research communities and lead to novel insights that will advance the development of control tools, such as vaccines that target the lung tissue to generate protective responses.
2. Bovine alveolus model and immortalized B2AE cells available to the scientific community. Cells will be deposited with the ECCC where they can be obtained by others. The alveolus model will be published and publicised at scientific meetings.
3. Enhanced collaboration across disciplines. There are many significant respiratory infections of cattle. Our model will be benefit groups working on these diseases as it will allow pathogens to interact with alveolar cells in a physiologically relevant manner. For example, key pathologies in BRD involve enhanced adhesion of bacteria to virus-infected cells, modification of innate and adaptive immune responses and enhanced inflammation. All of these processes are amenable to study in our model.
4. Up-skilling of the PDRA and extension of the knowledge-base for PI and CO-Is. The PDRA will benefit directly from the new methods and understandings gained through this project. The application of the model outside of the immediate TB field will also benefit the PI and Co-Is. This will be of benefit to their Faculty. Furthermore, the University will benefit by generating data and laboratory techniques that can be used for teaching and learning within the University.
Economic and societal impacts
1. Reduced spend on BTB research and contribution to evidence-based policy-making. Tools developed in this project can reduce the number of animal studies and cost of experiments funded by Defra. If we identify a read-out in our model that is related to vaccine efficacy in the whole animal then we have the basis of screening vaccine candidate in vitro. This will reduce the severity and duration of animal experiments, as well as significantly reducing their financial cost. Prof. Chambers advises Defra on BTB so can translate the findings of this work to policy advisors and politicians.
1. This project provides a tissue culture model to study infection of the bovine lung with mycobacteria. A specific objective is to determine whether the behaviour of mycobacteria and host cells in the model correlates with protective efficacy seen in cattle challenge studies from which we have stored blood cells to evaluate. If we identify a read-out in our model related to vaccine efficacy in the whole animal then we have the basis of screening vaccine candidates in vitro without needing to challenge cattle with M. bovis. An average of one experiment of 40 cattle a year is conducted for the purpose of empirical testing of novel vaccines for bovine TB and could be replaced.
2. Bovine respiratory disease (BRD) is one of the key health issues and most costly problems occurring in cattle worldwide. New antimicrobials are needed for BRD. This is one area where our model could be used as it is based on a model used already to mimic systemic drug administration for the treatment of antifungals without needing to use live animals. We shall raise the profile of our model with companies who produce drugs for BRD. This is expected to lead to R&D investment from the commercial sector and a reduction in animals used for drug screening for BRD. Accepting more studies are conducted for this purpose than reported, an underestimate is that using our model would replace at least 33 cows per year (133 cows were reported as used for this work over a 4 year period).
3. 104 cows were used for studies relating to the respiratory system or microbiology in 2013 (Annual Statistics of Scientific Procedures on Living Animals for Great Britain). Taking a conservative estimate, one quarter of these (=26) a year could be replaced by our model in the UK alone.
Academic impact
1. Contribute new knowledge to understanding of BTB. Our data will be of value to veterinary and human TB research communities and lead to novel insights that will advance the development of control tools, such as vaccines that target the lung tissue to generate protective responses.
2. Bovine alveolus model and immortalized B2AE cells available to the scientific community. Cells will be deposited with the ECCC where they can be obtained by others. The alveolus model will be published and publicised at scientific meetings.
3. Enhanced collaboration across disciplines. There are many significant respiratory infections of cattle. Our model will be benefit groups working on these diseases as it will allow pathogens to interact with alveolar cells in a physiologically relevant manner. For example, key pathologies in BRD involve enhanced adhesion of bacteria to virus-infected cells, modification of innate and adaptive immune responses and enhanced inflammation. All of these processes are amenable to study in our model.
4. Up-skilling of the PDRA and extension of the knowledge-base for PI and CO-Is. The PDRA will benefit directly from the new methods and understandings gained through this project. The application of the model outside of the immediate TB field will also benefit the PI and Co-Is. This will be of benefit to their Faculty. Furthermore, the University will benefit by generating data and laboratory techniques that can be used for teaching and learning within the University.
Economic and societal impacts
1. Reduced spend on BTB research and contribution to evidence-based policy-making. Tools developed in this project can reduce the number of animal studies and cost of experiments funded by Defra. If we identify a read-out in our model that is related to vaccine efficacy in the whole animal then we have the basis of screening vaccine candidate in vitro. This will reduce the severity and duration of animal experiments, as well as significantly reducing their financial cost. Prof. Chambers advises Defra on BTB so can translate the findings of this work to policy advisors and politicians.
