Developing and validating an in-vitro mycobacterial challenge model to facilitate TB vaccine research and minimise in-vivo challenge experiments
Lead Research Organisation:
University of Oxford
Department Name: Medical Sciences Divisional Office
Abstract
Bovine TB is currently one of the greatest challenges that the farming industry faces in the UK especially in the southwest of England and Wales. The scale of this epidemic urgently requires the development of new tools. Vaccination is one of the most effective ways to control any infectious disease epidemic. One of the major challenges in bovine and human TB vaccine development is that we do not know what type or level of immune response will be protective. This means that candidate vaccines have to be evaluated in controlled Mycobacterium bovis (M.bovis) challenge experiments, where vaccinated animals are deliberately infected with fully virulent M.bovis and followed for several months before assessment of protection through post-mortem examination. These are slow and costly experiments which subject large numbers of animals to M.bovis infection.
This project aims to develop two models; an in-vitro mycobacterial growth inhibition assay (MGIA) and an in-vivo BCG challenge model, both of which will replace pathogenic M.bovis challenge as a first screening test for candidate TB vaccines. The in-vitro MGIA involves infecting blood or cells from a vaccinated or control animal, in a test tube in the laboratory, rather than having to infect the animal itself. The BCG challenge model involves infecting animals in the lymph nodes with the attenuated but replicating BCG vaccine, rather than infecting animals with virulent M.bovis into the lungs. These two complementary models will be used to screen vaccine candidates, and only vaccines demonstrating a protective effect in these models will be progressed to evaluation using pathogenic M.bovis challenge. This work will achieve three things. Firstly, and importantly, it will reduce the number of cattle undergoing pathogenic M.bovis challenge, by at least 1/3 and approximately 30-50 animals per annum. Secondly it will refine and improve the procedures involved in cattle studies. Thirdly, it will facilitate the identification of immune correlates of protection with which to refine vaccine design and development. Ultimately, if we had a validated correlate of protection, simple immunogenicity experiments on small numbers of cattle could be conducted, rather than large pathogenic M.bovis challenge experiments.
This project aims to develop two models; an in-vitro mycobacterial growth inhibition assay (MGIA) and an in-vivo BCG challenge model, both of which will replace pathogenic M.bovis challenge as a first screening test for candidate TB vaccines. The in-vitro MGIA involves infecting blood or cells from a vaccinated or control animal, in a test tube in the laboratory, rather than having to infect the animal itself. The BCG challenge model involves infecting animals in the lymph nodes with the attenuated but replicating BCG vaccine, rather than infecting animals with virulent M.bovis into the lungs. These two complementary models will be used to screen vaccine candidates, and only vaccines demonstrating a protective effect in these models will be progressed to evaluation using pathogenic M.bovis challenge. This work will achieve three things. Firstly, and importantly, it will reduce the number of cattle undergoing pathogenic M.bovis challenge, by at least 1/3 and approximately 30-50 animals per annum. Secondly it will refine and improve the procedures involved in cattle studies. Thirdly, it will facilitate the identification of immune correlates of protection with which to refine vaccine design and development. Ultimately, if we had a validated correlate of protection, simple immunogenicity experiments on small numbers of cattle could be conducted, rather than large pathogenic M.bovis challenge experiments.
Technical Summary
The scale of the bovine TB epidemic means there is an urgent need for new tools to control TB. An effective vaccine would be game-changing. However the lack of defined immunological correlates of protection mean that vaccine candidates need to be evaluated in costly, pathogenic M.bovis challenge experiments.
This project proposes two mechanisms to reduce and refine the need for virulent M.bovis challenge: (1) By optimising and establishing an in-vitro mycobacterial growth inhibition assay (MGIA), for use in cattle and (2) Further development of an in-vivo BCG challenge model, also currently in development for human TB vaccine development.
A MGIA has potential as an in-vitro surrogate of the host ability to control M.bovis infection. Such an assay could be used in place of virulent in-vivo challenge models for the selection or 'gating' of new TB vaccine candidates. The MGIA will be optimised using blood and PBMC from BCG-vaccinated and naïve cattle, as the protective effect of BCG against virulent M.bovis has already been demonstrated. We will further refine the MGIA assay by using purified blood monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells infected with defined MOI as source of infected cell populations to which we will add either PBMC or defined T cell populations and NK cells purified by magnetic bead sorting or flow cytometry. This approach will be used to determine the cells responsible for mycobacterial growth inhibition.
A further reduction in need for virulent M.bovis challenge would be possible if a BCG challenge model were to be developed and validated, as BCG is attenuated and would remove the need for BSL3 containment of infected animals. An intranodal BCG challenge model will be further developed to determine the sensitivity of this model to distinguish between BCG vaccination alone and a BCG prime - AdHu5.85A boost vaccination regimen.
This project proposes two mechanisms to reduce and refine the need for virulent M.bovis challenge: (1) By optimising and establishing an in-vitro mycobacterial growth inhibition assay (MGIA), for use in cattle and (2) Further development of an in-vivo BCG challenge model, also currently in development for human TB vaccine development.
A MGIA has potential as an in-vitro surrogate of the host ability to control M.bovis infection. Such an assay could be used in place of virulent in-vivo challenge models for the selection or 'gating' of new TB vaccine candidates. The MGIA will be optimised using blood and PBMC from BCG-vaccinated and naïve cattle, as the protective effect of BCG against virulent M.bovis has already been demonstrated. We will further refine the MGIA assay by using purified blood monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells infected with defined MOI as source of infected cell populations to which we will add either PBMC or defined T cell populations and NK cells purified by magnetic bead sorting or flow cytometry. This approach will be used to determine the cells responsible for mycobacterial growth inhibition.
A further reduction in need for virulent M.bovis challenge would be possible if a BCG challenge model were to be developed and validated, as BCG is attenuated and would remove the need for BSL3 containment of infected animals. An intranodal BCG challenge model will be further developed to determine the sensitivity of this model to distinguish between BCG vaccination alone and a BCG prime - AdHu5.85A boost vaccination regimen.
Planned Impact
This proposal aims to reduce and refine the use of cattle in bovine TB vaccine development. By developing sensitive in-vitro and in-vivo models of virulent M.bovis challenge, the numbers of cattle used in each individual experiment can be reduced. Furthermore the procedures to which these animals are subjected will be refined. In place of virulent M.bovis challenge, a procedure requiring expensive BSL 3 containment, animals will be immunised with candidate vaccines and either 'challenged' intranodally with the avirulent BCG, or simply venesected for the evaluation of protective immunity using an in-vitro mycobacterial growth inhibition assay.
We anticipate that approximately one third less animals, 30-50 per annum in the UK, will be exposed to virulent M.bovis as a direct result of the work outlined here. This work will also lead to a global reduction in the use of cattle in bovine TB vaccine development.
Whilst this approach will not prevent the use of animals in bovine TB vaccine research, it will reduce and minimise the need to infect animals with virulent M. bovis and will also constitute a major refinement of the vaccine testing pipeline. Only vaccines demonstrated to have an effect on in-vitro mycobacterial growth inhibition would go on to be evaluated in a challenge experiment. A further refinement inherent in the suggested approach is that cell populations responsible for curtailing mycobacterial growth can be readily defined in vitro by e.g. cell depletion experiments, flow cytometry or host transcriptome studies to better define underlying protective mechanisms. This will then allow the rational development of future TB vaccine candidates. The aim of this work is to identify immune correlates of protective immunity. Such immune correlates would be of enormous value to bovine and human TB vaccine developers, and would greatly facilitate vaccine design and development. Once immune correlates of protection have been identified and validated, vaccine evaluation can be carried out using simple, smaller immunogenicity experiments, rather than more costly virulent M.bovis challenge experiments. Ultimately the identification of immune correlates of protection could remove entirely the need for virulent M.bovis challenge experiments.
We anticipate that approximately one third less animals, 30-50 per annum in the UK, will be exposed to virulent M.bovis as a direct result of the work outlined here. This work will also lead to a global reduction in the use of cattle in bovine TB vaccine development.
Whilst this approach will not prevent the use of animals in bovine TB vaccine research, it will reduce and minimise the need to infect animals with virulent M. bovis and will also constitute a major refinement of the vaccine testing pipeline. Only vaccines demonstrated to have an effect on in-vitro mycobacterial growth inhibition would go on to be evaluated in a challenge experiment. A further refinement inherent in the suggested approach is that cell populations responsible for curtailing mycobacterial growth can be readily defined in vitro by e.g. cell depletion experiments, flow cytometry or host transcriptome studies to better define underlying protective mechanisms. This will then allow the rational development of future TB vaccine candidates. The aim of this work is to identify immune correlates of protective immunity. Such immune correlates would be of enormous value to bovine and human TB vaccine developers, and would greatly facilitate vaccine design and development. Once immune correlates of protection have been identified and validated, vaccine evaluation can be carried out using simple, smaller immunogenicity experiments, rather than more costly virulent M.bovis challenge experiments. Ultimately the identification of immune correlates of protection could remove entirely the need for virulent M.bovis challenge experiments.