The immunological basis of protection obtained by prime-boost immunisation against bovine tuberculosis

The causal agents of bovine (bTB) and human tuberculosis (hTB), Mycobacterium bovis and M. tuberculosis respectively, are closely related organisms that result in similar diseases. M. bovis can also cause disease when transmitted to humans, and this led in the 1950s to the introduction of national eradication programmes based on testing of cattle herds to remove infected cattle. Although initially successful in reducing herd incidence, the disease has re-emerged as a major problem in the UK over the last 25 years. This has led to consideration of vaccination as an additional control option. A laboratory attenuated strain of M. bovis (BCG) has been used extensively since the early 20th century to vaccinate against hTB. However the vaccine shows variable efficacy, particularly against disease in adults. BCG vaccination of cattle can reduce the incidence and severity of pathology, but only prevents establishment of infection in a proportion of vaccinates. Therefore, its use has been considered incompatible with current bTB control programmes. An improved vaccine is therefore required for both hTB and bTB. Recent studies in humans and cattle have demonstrated that BCG vaccination boosted by virally vectored subunit vaccines expressing mycobacterial antigens results in enhanced protection compared to BCG alone. For example, combination of BCG and a recombinant adenovirus (rAd) expressing the 85A antigen reduced the number of cattle that presented with visible lesions after M. bovis challenge, compared to animals vaccinated with BCG only. This project aims to identify those features of the immune response induced by boosting with 85A that are associated with the enhanced immunity.
Although it is well established that immunity against M. bovis is mediated by cellular immune responses, primarily involving the CD4 subset of T cells, the precise properties of the protective CD4 T cells that determine immunity are poorly understood. The demonstration that immunity induced by BCG can be enhanced by boosting with the 85A antigen clearly shows that responses to this antigen contribute to immunity. The project will therefore focus on analysing immune responses to 85A in cattle immunised with BCG followed by 85A and subsequently challenged with virulent M. bovis. CD4 T cells utilise a large library of antigen-specific receptors to recognise short fragments of foreign microbial proteins presented on the surface of infected cells. The project will utilise newly developed methods for analysing the sequences of these receptors to track the responding 85A-specific CD4 T lymphocyte populations following immunisation and challenge. This methodology will be used in conjunction with biological assays that measure the frequency, fine specificity and functional potency of the 85A-specific response to address the following questions:
- To what extent is the frequency of CD4 T cells reactive with 85A increased following boost immunisation with 85A?
- Does boost immunisation with 85A alter the fine specificity of the responding CD4 -lymphocytes response and/or the repertoire of their antigen-specific receptors?
- Does boosting with 85A enhance the functional potency of the specific CD4 T cells compared to BCG immunisation only?
- Are particular components of the 85A-specific CD4 T cell response more effective than others at recognising and responding to M. bovis-infected cells?
- Which components of the of the 85A-specific CD4 T cell population respond most rapidly following challenge of immunised animals with M. bovis?
The results of these studies will identify properties of the specific CD4 T cell response that are associated with immunity and thus provide measurable parameters that will be useful for assessing the immune responses to other candidate vaccines. Although primarily of relevance to vaccine development for tuberculosis in cattle, the outputs of the project will also inform studies of TB vaccine development for other species.

Although current evidence indicates that Th1 CD4 T cell responses play a central role in immunity against TB, the precise properties of the CD4 T cells that confer immunity are poorly understood. Better knowledge of these parameters is required to rationally design improved vaccines. The recent demonstration that BCG-induced immunity to M. bovis can be enhanced by boosting with viral vectors expressing 85A provides an opportunity to analyse the CD4 T cell responses to a relevant antigen, in order to understand the properties of the specific T cells that mediate immunity. The applicants have recently defined the repertoires of bovine TCR genes and have developed deep sequencing methods to analyse the TRB chain repertoires expressed by responding T cells, thus allowing the clonal composition of T cell responses to be followed over time. This project seeks to exploit these technologies in combination with immunological measurements to analyse in detail the 85A-specific CD4 T-cell response to BCG/rAd85A prime-boost immunisation and subsequent M. bovis challenge. Building on preliminary data indicating that boosting with 85A increases the functional avidity and alters the fine epitope specificity of the responding CD4 T cells, the project will study the frequency, epitope-specificity and functional activity of 85A-specific CD4 T cell responses in relation to their clonotypic composition. The ability to track clonotypes using TCR sequencing provides a novel dimension to TB vaccine studies and will yield important new data on how the response to 85A evolves during vaccination and importantly how this impacts on the ability of the response recalled from vaccine-induced memory populations to mediate protection against subsequent challenge. The latter is particularly important in gaining a better knowledge of how to generate efficacious vaccines and the bovine TB model provides an exceptionally useful model in which to study this in a target species.

Bovine tuberculosis remains a major disease problem in a number of countries in Europe, including the UK where the annual spend on control of the disease currently is £100M. The disease is also widely distributed in the developing world and transmission to humans accounts for some of the cases of human tuberculosis. Hence bovine tuberculosis has an impact on food security and human health and in the UK undermines the international competitiveness of the cattle industry
Potential beneficiaries and benefits derived from the research:
TB control policy makers: The UK government has stated that vaccination of cattle will be considered as a control option for control of bovine tuberculosis. The proposed studies will contribute towards development of an efficacious vaccine and thus influence the policy decisions made on implementation of vaccination. Such developments are also likely to influence policy decisions by other countries where TB in cattle is a problem and will create opportunities for international aid donors to consider recommendation of vaccination in the developing world as means of protecting human health.
National TB control programmes: Much of the cost of the current UK bovine TB control programme arises from compensation paid for infected animals removed from affected farms and the increased frequency of follow-up testing that is required in breakdown herds. The recent development of TB diagnostic tests that distinguish infected from BCG-vaccinated animals opens the way for implementation of cattle vaccination alongside herd testing programmes. Introduction of a cattle vaccine that gave significant protection against TB (transmitted either from other cattle or wildlife) would substantially reduce the costs of government TB control programmes. Such benefits could be cumulative over several years if the vaccination resulted in a downward annual trend in TB incidence.
Livestock producers: Cattle herd TB breakdowns result in substantial costs to farmers as a result of loss of valuable animals (compensation does not cover full commercial values), the inability of affected farms to trade because of animal movement restrictions and the additional time and labour involved in organising herd tests. By reducing the incidence of affected herds, vaccination would reduce the costs incurred by livestock producers and overall enhance the competitiveness of the UK livestock industry.
Vaccine producers: Since bovine tuberculosis in European countries is subject to government statutory control, it is anticipated that registration of vaccines for use in cattle would be facilitated by Government in partnership with suitable vaccine producers. This is already being pursued for BCG in cattle, with a company that produces BCG for use in humans. The vaccine producers would then market the vaccine to farmers and thus benefit financially from the sales.
The general public: Although the incidence of human cases of M. bovis arising from cattle is currently low, the continued rise in incidence of the disease in cattle poses an increasing threat of human infections. Control of the disease by vaccination would therefore help to safeguard human health.
The developing world: Bovine tuberculosis is present in most developing countries and accounts for some cases of human tuberculosis as a result of transmission from cattle. In most countries, the disease in cattle is not subject to routine control measures, in part due to the lack of infrastructure and resources to implement cattle testing and also the lack of a vaccine that gives sufficient levels of protection. The development of a more effective vaccine would provide the option of implementing vaccination in areas where there is a high risk of human infections, thus providing a sustainable means of improving human health and welfare.