Facilitating application of NGS to analysis of livestock T-cell receptor repertoires

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

The livestock sectors of many LMIC are negatively impacted by pathogens for which no viable vaccines are currently available. T-cells are known to contribute to protection against a number of these pathogens (e.g. tuberculosis, East Coast Fever) and current efforts are directed to better understanding the role these cells plays in conferring immunity and defining the 'correlates of protection' that can be used to rationalise future vaccine development.

One parameter that has been advocated for inclusion in comprehensive analysis of T-cell responses is the repertoire of the expressed TCR, which can be exploited to study the clonotypic structure, kinetics and recall of pathogen-specific T-cells. TCRs are heterodimeric, clonotypically distributed receptors composed of alpha (TRA) and beta (TRB) chains that determine the antigenic-specificity of individual T-cells. Somatic V(D)J recombination and non-germline editing of the V(D)J junction generates a vastly diverse TCR repertoire, that in humans has been estimated to be ~2x10'7. Upon encountering their cognate antigen, either during vaccination or pathogen exposure, T-cells undergo clonotypic expansion which can be evaluated by the increased expression of the clonotypic TRA and/or TRB chains. However, achieving sufficient sequencing depth of the TCR repertoire to reliably detect such clonal expansions in highly complex T-cell populations (e.g. in vivo PBMC or primary T-cell cultures) was difficult before development of NGS platforms.

In the last decade, development of a number of bioinformatic pipelines (e.g. MiTCR and IMGT-HighV-Quest) has enabled TCR analysis using NGS data to be applied to the study of human and murine T-cell populations. The pre-requisites for these analyses are i) TCR chain-specific PCR protocols that generate unbiased TCR chain amplicons, ii) a comprehensive database of V and J gene segment sequences and iii) sophisticated algorithms that can align TCR sequences to the relevant V and J gene segment databases (and so define V and J usage and the hypervariable CDR3 sequence of individual TCR chains) and also compensate and correct for the inevitable PCR/sequencing errors that can significantly compromise sequence data interpretation. The principles used in bioinformatic pipelines developed for human and murine TCR analysis are generic and in recent studies we have demonstrated that given the appropriate V and J gene databases the pipelines can be modified and applied to NGS sequence data from other species (in this case European Bos taurus cattle). In this project we propose to:

1 - Design PCR protocols to enable unbiased PCR amplification of TRA and TRB chains from the relevant livestock populations. Optimised SMART-based PCR amplification protocols for pig, goat, sheep and water buffalo TRA and TRB chains will be validated.
2 - Sequence sufficient full length TRA/TRB amplicons to enable comprehensive coverage of the expressed TRA and TRB repertoires. Amplicons generated from 5 representative individuals from different LMIC and European breeds will be subjected to PacBio sequencing to obtain full length amplicon sequence.
3 - Bio-informatically de-construct somatically recombined TRA and TRB chains to construct database of germline V and J gene segments sequences. Pipelines to de-construct recombined TRA/TRB chains will be developed to enable germline sequences of the constituent V and J gene segments to be established and a comprehensive database populated.
4 - Modify existing algorithms to enable these databases to be employed in NGS analysis of TCR repertoires (and provide TRBV, TRBJ, TRAV and TRAJ databases to managers of publicly accessible TCR analysis algorithms). A modified version of the MiTCR algorithm which will enable the utilisation of the livestock species databases will be established and hosted by the CTLGH bioinformatics repository. In addition, the databases will be hosted by the publicly available Milaboratory website.

Technical Summary

Development of vaccines capable of eliciting protective T-cell responses is required for a number of livestock diseases prevalent in LMIC. By providing high resolution details on the clonotypic composition of T-cell responses, NGS analysis of TCR repertoires can contribute to the identification of 'correlates of protection' that will accelerate efforts to develop such vaccines. Integration of such data into livestock vaccine research is impeded by the absence of the necessary bioinformatic resources. We have recently demonstrated that bioinformatic workflows for NGS TCR analysis can be transferred to TRB repertoire analysis of Bos taurus cattle. We propose here to develop the protocols, databases and bioinformatic infra-structure required for NGS TCR repertoire analysis to be applied to breeds of sheep, pig, cattle, goat and water buffalo that are economical important in LMIC. To do this it is necessary to i) optimise unbiased SMART-PCR amplification of TRA/TRB chains in relevant species, ii) generate comprehensive databases of V and J gene sequences and iii) manage integration of these databases into the sophisticated TCR analysis algorithms that have been developed for humans. We plan to optimise and then apply the SMART-PCR protocol to generate TRA/TRB amplicons from representatives of LMIC breeds of livestock and use PacBio to obtain near-full length TRA/TRB chain sequences of these amplicons. These sequence will be 'de-constructed' into the germline V and J gene (and non-germline CDR3) components and this data used to generate the V/J sequence databases. These databases will be incorporated into TCR algorithms publicly available through the Milaboratory website (https://milaboratory.com) and the MiTCR algorithm will be modified to facilitate livestock species TCR analysis. Together the outputs of this study will provide both the methodologies and databases required to implement TCR repertoire analysis in livestock species critical to LMIC countries.

Planned Impact

This study will provide a novel tool and the required bioinformatic framework to analyse TCR repertoires of T-cell responses that can be used to inform on the clonality, diversity and other critical parameters of T-cell responses that can be used to better understand T-cell mediated immunity induced by candidate vaccines and so accelerate vaccine development. By doing so the study has the potential to have a major impact on;

Livestock producers in LMIC: In developing countries the livestock sector accounts for upto 80% of agricultural GDP and in many LMIC countries a majority of the rural poor raise livestock. With increasing demand in LMIC countries for animal-derived food, increasing local animal production offers an obvious pathway for poverty reduction. To exploit this opportunity it is necessary that policies that ensure farmers in LMIC have the facilities to increase production, including the means of reducing losses through disease burden, are implemented. Infectious diseases continue to be a major constraint on the economic performance of the livestock sector in many LMIC. For small holder farmers outbreaks of diseases such as ECF, FMD, CBPP (Contagious Bovine Pleuropnuemonia) and ASF (African Swine Fever) can be devastating. Rational development of novel vaccines against these diseases requires a comprehensive knowledge of the role of different components of the immune responses against these pathogens.

Livestock producers in non-LMIC: Integration of TCR analysis into candidate vaccine evaluation programmes could also accelerate vaccine development programmes for diseases that have significance for livestock-producers in non-LMIC. Some of the diseases for which improved vaccines are most urgently required (e.g. TB and FMD) are of relevance to both LMIC and non-LMIC.

Global food security: The livestock sector is a key provider of global food security. The continuing increase in the demand for animal-derived foodstuffs, whilst providing an economic opportunity for farmers in LMIC countries (see above), also increases competition, and so compounds concerns over global food security.

Publications

10 25 50
 
Description The genes that lie under the way that livestock species respond to infection are common across a wide range of breeds which means that we can use data and models derived from commonly-studied breeds as a seed for studies in less well-studied but still economically important breeds in Low and Middle Income Countries.
Exploitation Route We are currently integrating T-cell repertoire analysis into a number of other ongoing vaccine studies and looking to support other groups to do so. This extends to a wider range of species than livestock.
Sectors Agriculture

Food and Drink

Healthcare

Leisure Activities

including Sports

Recreation and Tourism

Pharmaceuticals and Medical Biotechnology

 
Title Veterinary TCR repertoire analysis using NGS technology 
Description The technology includes the wet-lab and bioinformatic workplan that enables the characterization of the alpha/beta repertoire of veterinary T-cell responses to be analysed at high resolution. This has included the development of the specific PCR protocols, a bespoke bioinformatic pipeline and construction of a specific database. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact The development of the tool has allowed TCR repertoire analysis to be integrated into advanced analysis of T-cell responses in veterinary species. 
 
Title Veterinary TCR database 
Description A database containing TCR alpha and beta V, J and C sequences required to perform high resolution NGS analysis of TCR repertoires 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact Facilitation of NGS analysis of veterinary species TCR repertoires 
 
Description Investigation of bovine MAIT cells 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We have established a new collaboration with Prof. P. Klenerman's group at Oxford looking at the biology of non-conventional 'MAIT' T-cells in cattle
Collaborator Contribution We have provided access to materials, expertise in bovine immunology and co-developed the experimental plans
Impact First definitive evidence of MAIT T-cells in cattle Evidence of response of bovine MAIT T-cells to the ligand recognised by MAIT cells in other species including mouse and humans First characterization of the anatomical distribution of these cells.
Start Year 2018
 
Description Understanding TCR repertoire analysis 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Sampling for this project has been integrated into partnerships with researchers at the University of Zambia (Zambia), University of Makerere (Uganda), University of Veterinary and Animal Sciences, Lahore (Pakistan) and University of Sao Paulo (Brazil). As part of this our team have provided knowledge exchange with our partners. TCR repertoire analysis has been extended to support research by other groups and species including project being run through The Roslin Institute and The Sanger Institute
Collaborator Contribution Our international partners have assisted in providing samples Partners in the UK are applying the technology developed to address questions in species including dogs and pigs to better understanding the mechanisms involved in T-cell mediated immunity in cancer, either as models for human cancer and/or for application in veterinary medicine
Impact There has been educational outcomes with local partners receiving training on the value of incorporating TCR analysis into future studies
Start Year 2017
 
Description Understanding TCR repertoire analysis 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Sampling for this project has been integrated into partnerships with researchers at the University of Zambia (Zambia), University of Makerere (Uganda), University of Veterinary and Animal Sciences, Lahore (Pakistan) and University of Sao Paulo (Brazil). As part of this our team have provided knowledge exchange with our partners. TCR repertoire analysis has been extended to support research by other groups and species including project being run through The Roslin Institute and The Sanger Institute
Collaborator Contribution Our international partners have assisted in providing samples Partners in the UK are applying the technology developed to address questions in species including dogs and pigs to better understanding the mechanisms involved in T-cell mediated immunity in cancer, either as models for human cancer and/or for application in veterinary medicine
Impact There has been educational outcomes with local partners receiving training on the value of incorporating TCR analysis into future studies
Start Year 2017
 
Description Understanding TCR repertoire in MCF responses of cattle 
Organisation University of Liege
Country Belgium 
Sector Academic/University 
PI Contribution We are using the technologies developed in this project to enhance the analysis undertaken by partners studying T-cell responses in relevant animal models
Collaborator Contribution Provision of samples on which to undertake the analysis
Impact None as yet - only commenced at the end of last year
Start Year 2019
 
Description Biology Teachers CPD Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Talk and discussions with 20 Higher Biology teachers about science, specifically about next generation sequence analysis technology and how that is being applied to address many different scientific questions
Year(s) Of Engagement Activity 2018
 
Description Highland Show 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Institute stand at the Royal Highland Show. Demonstrating focussed activity and discussing the work of the University and the institute in relation to food safety, farm animal welfare, production efficiency, disease resistance etc.
Year(s) Of Engagement Activity 2018
 
Description Meet The Scientists 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Visit to a local primary school to engage with a questions and answers session about science and science careers
Year(s) Of Engagement Activity 2018
 
Description Primary School "Big Science" visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Visit to local primary school as part of their Big Science activity. Discussing science and scientific careers with the pupils and presenting awards to them based on a science project they had recently completed.
Year(s) Of Engagement Activity 2018
 
Description Question of Taste PCR Masterclass 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Practical workshop run by the Roslin Institute public engagement outreach team to allow High School pupils to run PCRs and investigate the genetics of a specific taste receptor.
Year(s) Of Engagement Activity 2018