NKp46+CD3+ T-Cells As A Novel Target For Vaccines Against bovine TB

Bovine tuberculosis (bTB) is the most important disease affecting the UK cattle industry. Failure of the current 'test-and-slaughter' policy to constrain the spread of bTB has led to a review of what strategies should be implemented in future to improve the control of this disease. BCG, the only licensed bTB vaccine, exhibits variable and unpredictable levels of protection and is not DIVA compliant so can't be used in current disease control programmes. Consequently, there is a need to develop vaccines that confer more reliable immunity and can be integrated with bTB field surveillance. Development of such vaccines requires a better understanding of i) the components of the immune response that contribute to protective immunity and ii) how immune responses can be exploited to discriminate between vaccinated and Mb-infected animals. Although it is well established that protective immunity against TB is cell mediated, the precise protective immunological mechanisms are still undefined. Evidence indicates that conventional peptide-MHCII restricted CD4+ T cell responses are critical for immunity and that for optimal anti-TB immunity peptide-MHCI restricted CD8+ T-cells are also required. In humans a large proportion of the cellular anti-TB response if composed of non-conventional lipid-specific CD1-restricted T-cells; the range of anti-mycobacterial effector functions exhibited by these cells indicates that they also contribute to host defence against TB. Consequently, it has been proposed that lipids may constitute novel antigens that could be used to enhance the immunogenicity and efficacy of next-generation TB vaccines. Lipids are attractive vaccine candidates as they i) activate both innate-like and adaptive T-cell responses, ii) can have adjuvant properties and iii) are presented by non-polymorphic CD1 genes and therefore (unlike MHC-restricted peptides) have utility across an outbred population. In addition, some lipids don't induce responses against PPD, so could be used as DIVA reagents. Although cattle lipid-specific T-cell responses have been reported in bTB, knowledge of their role in anti-bTB immunity is negligible.
Recent studies conducted by our laboratories have identified a novel population of non-conventional NKp46+CD3+ bovine T-cells and demonstrated that a subset of these cells respond to PIM, a group of lipids from M. bovis in a proportion of Mb-infected animals. In this project we propose to gain information on lipid-specific T-cell responses in bTB by analysing NKp46+CD3+ T-cells during a BCG-immunisation/Mb-challenge study. We will also perform studies to identify the range of antigenic TB lipids and determine which are immunogenic and so could contribute to new effective bTB vaccines. The objectives of the project are to:
1. Quantify and track the NKp46+CD3+ T-cell responses following BCG vaccination and subsequent exposure to M. bovis. This will help determine if NKp46+CD3+ T-cells are functioning as innate and/or adaptive effector T-cells and if BCG immunisation forms a memory population recalled during Mb challenge.
2. Examine the function of NKp46+CD3+ T-cells recognising mycobacteria. This will define the role of NKp46+CD3+ T-cells in anti-TB immunity. Data from objectives 1 and 2 will also be used to examine if NKp46+CD3+ T-cell responses correlate with anti-bTB protection conferred by BCG immunisation.
3. Identify additional mycobacterial lipids that could be used to generate NKp46+CD3+ (and other) bovine T-cell responses. It is likely that multiple TB lipids will be antigenic in cattle and that some lipid-specific T-cells may lie out-with the NKp46+CD3+ T-cell subset. This study will expand the knowledge of the bTB lipid antigen repertoire (and also determine if any could be exploited for DIVA applications).
4. Identify which mycobacterial lipids elicit NKp46+CD3+ T-cell responses in vivo. This will establish which lipids are immunogenic in vivo and are therefore genuine vaccine candidates.

Although protective immunity against TB is known to be cell mediated, the exact roles of different T-cell subsets and the immunological parameters that define protection remain ill-defined. In humans there is evidence that CD1-restricted lipid-specific T-cells constitute a substantial part of the TB response and have anti-mycobacterial function. In contrast, knowledge of the bovine lipid-specific T-cell response against bTB is limited. Recent work by our groups has identified a novel NKp46+CD3+ non-conventional T-cell population which includes cells recognising PIM in a proportion of naturally Mb-infected animals. This proposal will use characterisation of NKp46+CD3+ T-cell responses during BCG-immunisation/Mb-challenge to analyse the kinetics of a known lipid-specific response against bTB under experimental conditions. We will also undertake in vitro analysis to examine the anti-mycobacterial function of NKp46+CD3+ T-cells. Comparisons of NKp46+CD3+ T-cell responses and levels of anti-bTB protection will be used to evaluate if BCG-immunisation generates a memory population of NKp46+CD3+ T-cells and if so, if it is associated with enhanced anti-TB immunity. To expand the repertoire of TB lipids known to be recognised by cattle, lipid fractions and purified lipids will be screened using PBMC from a variety of mycobacterially-infected animals. For lipids eliciting strong IFN/proliferative responses the phenotype of responding T-cells will be defined and in vivo immunogenicity studies conducted to determine their potential as vaccine antigen candidates. Additionally, the ability of lipids eliciting in vitro responses from Mb-infected animals to induce PPD-skin test reactions in BCG-immunised, Mb-infected and naïve animals will be determined to assess their utility as DIVA reagents. Together the results will provide the immunological data required to evaluate the feasibility of exploiting lipid as novel components of future bTB vaccines and/or as DIVA reagents.

bTB remains one the most important diseases affecting the cattle industry in the UK. It also remains a constraint on cattle production in large areas of the developing world where its prevalence also has impact on public health due to human transmission. Hence bTB impacts not only the international competitiveness of the UK cattle industry but also global food security and human health. In addition, bTB is widely recognised as a relevant model for human TB, due to similarities in causal agents, pathogenesis and immunological responses.

Potential beneficiaries and benefits derived from the research:
UK national TB control programme policy makers: Development of effective cattle vaccines and improved diagnostics is part of the Defra TB eradication strategy published last year. Availability of a DIVA compliant protective bTB vaccine would dramatically augment the potential of the UK national TB control programme to effectively control the disease. Rational development of such vaccines requires a comprehensive knowledge of the components of the bovine anti-TB immune response. The proposed studies will lead to an appreciation of the role of lipid-specific T-cells in anti-bovine TB immunity and so contribute towards the fundamental immunology underpinning accelerated, scientific evidence-based development of novel and efficacious vaccines and possibly new DIVA tests. The data generated by the proposed study will directly influence decisions made by policy makers on the viability of using lipids as part of vaccines or surveillance measures implemented as part of the future UK national TB control/eradication programme. Such data is equally likely to influence policy decisions by other countries where bTB is a problem. Any enhancement of TB control that reduces bTB incidence will obviously result in a reduction of the costs suffered by governments supporting national control programmes.

UK livestock producers: Cattle herd TB breakdowns result in substantial costs to farmers due to loss of valuable animals, the inability of affected farms to trade and additional time and labour involved in organising herd tests. Improved DIVA-compatible vaccines, by reducing the incidence of affected herds would reduce the costs incurred by livestock producers, enhancing the competitiveness of the UK livestock industry.

The developing world: bTB is present in most developing countries and as a zoonotic pathogen, in addition to causing bTB, can be transmitted to humans and cause hTB. In most developing countries, bTB is not subject to routine control measures or surveillance, in part due to the lack of infrastructure and resources to implement such strategies. In such countries effective vaccination has the most realistic potential to be a sustainable control strategy. Scientific evidence contributing to the development of a more effective vaccine could lead to implementation of cattle vaccination in areas where there is a high risk of human infections, thus providing a sustainable means of improving not only food security but also animal and human health and welfare.

Human TB vaccine development; Evidence from human studies indicate that lipid-specific T-cells form a substantial part of the anti-TB responses and have effector functions that could contribute to immunity. Due to a variety of reasons small animals do not provide good models for studying human lipid-specific T-cell responses. Advancing knowledge of bovine lipid-specific T-cell responses may offer a new model system for understanding the role of these cells in anti-TB immunity. The possibility to conduct immunisation/challenge trials in cattle affords opportunities to directly evaluate the protective capacity of lipid antigen vaccines in a natural target species; such studies are generally not feasible in humans and so such data would be of value to the hTB academic community and consequently to human health.