Dissecting variation in host responsiveness to a recombinant vaccine designed to control teladorsagiosis in sheep

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Delivering food security globally requires that a sustainable, safe and secure supply of quality food be provided by making the most efficient use of inputs. Infectious diseases substantially affect the efficiency of livestock production and parasitic roundworms are major culprits in this arena. Indeed, worm infections are the most costly endemic disease affecting sheep in the UK, with Teladorsagia circumcincta being the dominant worm present in this country. Estimated costs of roundworm infections to the UK sheep industry run at around £80 million per annum.

Dewormers (known as anthelmintics) have been used for over four decades to control worm infections in sheep throughout the world, but resistance to the three traditional drug classes is common. Worryingly, T. circumcincta resistance has already been reported against one of the two new classes of dewormer, even although this product was only launched two years ago. Historically, much resource and research has been directed at selective breeding of sheep for resistance to worms, but there have been few outputs from this work that have made it to practical application on farms in the UK.

An obvious alternative approach to breeding sheep for resistance to worms is to induce resistance to infection in sheep by vaccinating them to specifically stimulate immune protection against worm challenge. This will have the effect of negating the impact of the worms on sheep growth and productivity and importantly, will reduce worm transmission via the environment. Vaccines also hold the advantage over anthelmintic methods of control in that they are not associated with chemical residues in meat, have no environmental impact and are likely to require fewer administration applications over the lifetime of the sheep.

Recently, this research group have had singular success in the discovery of an effective recombinant (sub-unit) vaccine for control of T. circumcincta. This vaccine (known as 'CircVax') comprises 8 recombinant (synthetic) proteins and induces significant, albeit variable, levels of immunity against worm challenge in lambs. The next step in development of this vaccine is to understand the underlying local immune mechanism(s) involved in this variation in sheep responsiveness to the vaccine and to examine if this is affected by the age of the animal. Such a comprehensive understanding of variation in individual responses during vaccination and subsequent worm challenge is essential to optimize the vaccine further (for example, in adjuvant selection) as a tool for integrated worm control in sheep flocks.

The knowledge gained in this project will help determine if the effect of the vaccine can be improved in very young lambs and if responses in older animals can be optimised further. Each of these parameters could be addressed by using more appropriate adjuvants or delivery systems. In addition, if the results point to an inherent inability of the immune system of the very young lamb to control T. circumcincta, the derived information can be exploited in the deployment of the vaccine in a holistic, integrated control strategy that addresses the minimization of selection pressure for drug resistance in the worm.

In summary, the outputs will provide significant steps to developing a commercially relevant vaccine for use by farmers to mitigate the effects of this important parasitic infection of sheep and, as such, fall squarely within the priorities of the BBSRC Animal Health Research Club.

Technical Summary

The overall objective is to understand and overcome variation in host immune responsiveness to a promising Teladorsagia circumcincta sub-unit vaccine prototype, CircVax.

Here, the applicants will analyse parasitological outputs from vaccinated, then challenged, lambs and assess temporal parameters in vaccine responsiveness using recent technological advances in transcriptomics (RNAseq, nextSeq 500 Sequencing) and ovine genomic and immunological tools (http://www.sheephapmap.org/news/OARv2p0.php, http://www.immunologicaltoolbox.co.uk). Using multivariate (general linear mixed models) and network analyses (BioLayoutExpress 3D, Qiagen's Ingenuity Pathway Analysis) to assess relationships between immune gene expression and phenotypic characteristics, and between parasitological outcomes and immune variables and, importantly, how these are affected by lamb age, the project will generate a deep understanding of how sheep respond optimally to CircVax vaccination.

The resultant data will be exploited to develop an understanding of how the vaccine induces immunity against worm challenge and to identify what pathways are different in animals that are not protected by the vaccine. This knowledge, in turn, will be used to optimise the vaccine's effect in the field through optimal delivery(for exmaple, via adjuvant selection) and/or strategic use (for example, in integrated control programmes exploiting the vaccine with minimal use of effective anthelmintics in sheep of different ages).

Planned Impact

It is estimated that the direct global cost of parasitic worms of livestock is ~£1,200M per annum. There are approximately 101 M sheep and 12 M goats in the EU, with the UK having the largest sheep industry in Europe. In the UK, the brown stomach worm, Teladorsagia circumcincta, is the major contributor to parasitic gastroenteritis, estimated to cost the UK sheep industry >£80M each year. Helminths such as T. circumcincta impact hugely on the welfare and productivity of sheep. They do this by affecting growth rate, fertility, meat quality, and wool/milk production and, in heavy infections, by causing clinical disease. The research in this project will impact upon the effect of these worms by furthering development of a much-needed alternative control option; an anti-nematode vaccine known as CircVax.

Chemotherapeutic approaches have been the cornerstone of sheep nematode control for decades; however, resistance to the traditional three broad-spectrum anthelmintic classes is common in T. circumcincta, with resistance already reported to a member of a new class of anthelmintic, monepantel, which was only launched 2 years ago. For these reasons, alternative control methods are sought for sustainable control in the long term. Control by vaccination is the most obvious alternative, with the added value that vaccines have no environmental impact, do not leave chemical residues in food and are likely to require fewer applications over an animal's lifetime. Over the last 8 years, the applicants have developed, designed and tested a recombinant sub-unit T. circumcincta vaccine. Immunisation with this vaccine had a significant effect on worm burden and egg excretion in parasite-challenged lambs >6 months-old. Here, the applicants aim to develop this prototype vaccine further by investigating variation in sheep responsiveness to the vaccine; particularly how immunological unresponsiveness may be overcome in very young lambs. A comprehensive understanding of such responses during vaccination and subsequent parasite challenge is essential to develop this vaccine as a tool for integrated control that will be taken up on UK sheep farms and beyond. The aims are to establish what elements of the local response correlate best with efficacy amongst vaccinates and how variability in vaccine efficacy is affected by lamb age. This knowledge will determine whether or not vaccine efficacy can be improved in young lambs and whether variability in responsiveness in older animals can be addressed by exploiting appropriate adjuvants or delivery systems. Moreover, if the results point to an inherent, and insurmountable, inability to control T. circumcincta in the very young, the information will be used to deploy the vaccine in older animals as part of an integrated strategy to minimise selection pressure for anthelmintic resistance, in particular aiming to preserve use of the Class IV and V anthelmintics.

The outputs of this project will provide significant steps to developing a commercially-relevant subunit nematode vaccine (the 1st of its kind) for use by farmers to mitigate the effects of this important parasite. In so doing, the outcomes address a priority of the Animal Health Research Club ('Understanding variation in vaccine responsiveness and immune-competence at different developmental stages and its impact on disease outcomes'). Industrial stakeholders will benefit through generation of revenue from vaccine sales, as well as from availability of an option that will help prolong efficacy of the remaining effective anthelmintics. In addition, human health policymakers can exploit the data if defined strategies for delivery against ruminant gastrointestinal nematodes can be shown to improve efficacy. This is because there are many similarities (such as host niche, distribution pattern, primary candidate antigens) between the T. circumcincta vaccine and the subunit vaccine currently under development for control of human hookworm.

Publications

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Capilla-Lasheras P (2017) Elevated Immune Gene Expression Is Associated with Poor Reproductive Success of Urban Blue Tits in Frontiers in Ecology and Evolution

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Clerc M (2019) Age affects antibody levels and anthelmintic treatment efficacy in a wild rodent. in International journal for parasitology. Parasites and wildlife

 
Description We are studying underlying causes of variation in immune responsiveness to a sub-unit vaccine being developed to control the abomasal roundworm, Teladorsagia circumcincta, in sheep. Thus far, two vaccine trials have been undertaken to investigation variation in responsiveness associated with age and individuality. A unique dataset has been generated; in each trial half of the lambs were fitted with an indwelling abomasal cannula to allow dynamic comparison of critical immune responses generated during the period of parasite challenge after vaccination.
Parasitological results: Both age groups had significantly lower worm burdens at the trial end-point in vaccinated compared to control lambs. There was a significantly lower worm burden in 6 month-old compared to 3 month-old lambs, irrespective of whether lambs received the vaccine or not. The vaccine protected the younger lamb group against disproportionally high worm egg shedding compared to the control challenge group. As predicted, intra-group variation in parasitological parameters was observed in both trials.
Immunological assay results: Antibody analyses showed that serum IgG to the recombinant protein components of the vaccine followed a similar dynamic for each antigen. In 3- and 6 month-old vaccinates, IgG levels increased from first to third vaccination, then declined by the end of the trial after challenge. Serum IgG levels (at Day 43) to some vaccine components (APY-1, ASP-1, MEP-1, TGH-2) showed a significant negative correlation with worm burden in the 6 month-old vaccinates (Spearman correlation). A specific recombinant protein serum IgG response was not seen in adjuvant only recipients. Serum IgG to L3 native antigen was investigated; IgG levels against this antigen were significantly higher in 6 month-old compared to 3 month-old lambs (Day99, P<0.05, ANOVA with multiple comparisons). An increase in serum IgA against L3 antigen was seen in 6 month-old lambs, which was significantly higher at Day64 than in other groups (P<0.05, ANOVA with multiple comparisons). A small increase in IgA was seen between primary vaccination and Day99 in all groups to L4 and adult antigen, with no significant difference between the groups. No increase in IgE was observed for any antigen in any group. Variation in serum responses between individuals was observed in all groups.
Transcriptome analysis: RNA replicates sampled from all lambs at each time-point were pooled and 304 samples subjected to RNASeq. Due to high dataset dimensionality, principal component analysis, multidimensional scaling (MDS) and t-Distributed Stochastic Neighbour Embedding (t-SNE) were used to reduce the gene-wise dimension and visualise datasets in 3D and 2D, respectively. Lymph node and abomasal mucosa obtained at necropsy separated from abomasal temporal biopsies, suggesting tissue and sampling method generate distinct expression profiles. Further, time-points Week 0 and 1 were distinct from Weeks 2 to 6 post challenge. This coincides with larval presence, then emergence from tissue. For the functional analysis, we performed both classical analysis of differentially expressed (DE) genes and a machine learning-based analysis. For the former, DE genes across weeks between vaccinates and controls, between age groups, and between time points. Older lambs expressed a greater range and higher levels of genes involved in immune responses to helminths, epithelial damage, and bacteria the longer lambs, and these responses were exacerbated by the vaccine. In particular, the vaccine triggered in the older lambs, but not the younger lambs, significant increases in the expression of genes involved in T and B cell, antigen present cell, and immune cell activation and recruitment pathways. We are also interested in the variation in immune gene expression that best predicts immunity to T. circumcincta at the individual level, how these immune pathways change over time, and whether lambs' age affects how vaccine-elicited immune pathways change over the course of the 9 week infection. To address these questions, we developed a bespoke machine learning (ML) pipeline, which has led to three publications so far using different biological systems (Babayan et al. 2018, Sci Rep, Babayan et al. 2018, Front. Immunol., and Babayan et al 2018, Science). After an 11-month hiatus for the maternity leave of our post-doctoral research assistant, this analysis of the sheep transcriptomes is now complete. We show that the immune status of sheep after immunisation but before challenge with infective L3 is the most predictive of final worm burdens and of total faecal egg output; that there is overlap but also differences between immune pathways that predict worm and egg burdens; older sheep initiate and maintain strong protective immune pathway expression levels throughout the infection, which younger lambs show gradual increases in immune gene expression even when vaccinated. Further, vaccination itself shifts the expression of protective immune pathways earlier in their response to live infection than non-vaccinated animals. The dynamics of these changes also appear to differ between arms of the immune system, which "blocks" of pathways associated with epithelial integrity, cell signalling, innate cellular activity, and adaptive immunity following different expression profiles over time. These results are being written up for submission to a high impact journal.
Exploitation Route The findings here can be applied to other scientists working on nematode vaccines, because there have been major hurdles to developing these biologicals commercially, one of the main hurdles being the lack of responsiveness in individuals, particularly young animals. The outputs will help define both antigen and adjuvant selection in this and other host/parasite systems.
In addition, UoG, supported by MRI, are developing novel analytical approaches to analyse longitudinal transcriptome datasets, with a view to identify immune pathways that predict parasite burdens and vaccine efficacy. We will make available our analytical pipeline as part of publications of our results. We also plan to package these software tools for, and make them applicably to, a wider set of biological problems for dissemination to a wider public. This requires substantial effort and specific expertise, and thus is the main focus of a recent UoG-led grant proposal to the BBSRC. If successful, we will disseminate the resulting statistical tools for the analysis of high throughput 'omics data, with the option of including time-structured sampling, as an open-source software package for the benefit of the research community.
Sectors Agriculture, Food and Drink,Digital/Communication/Information Technologies (including Software)

 
Description An Online Platform for Malaria Vector Surveillance in Africa using Artificial Intelligence and Mosquito InfraRed Spectroscopy
Amount £225,000 (GBP)
Funding ID ICA\R1\191238 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2019 
End 11/2021
 
Description RNA Polymerase III in healthy ageing: consolidating the mechanisms of longevity from worms and flies to mice
Amount £457,795 (GBP)
Funding ID BB/S014357/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2019 
End 06/2023
 
Title Longitudinal dataset of tissue-specific RNA-seq transcriptomes and parasite survival and egg production 
Description We have generated an extensive longitudinal RNA-seq dataset of ~14,000 genes measured across 9 weeks of infection in abomasal tissue (5 time points) and lymph nodes (post-mortem), and a matched dataset of longitudinal faecal egg counts and worm burdens taken from the same time points and individual sheep. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact We are currently exploiting this dataset as part of this project. It will be made available in due course after completion of this award. 
 
Description New collaboration with Jacqui Matthews et al at Moredun 
Organisation Moredun Research Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We are contributing the sequencing, bioinformatics, and statistical analysis (including the initial power analyses) on RNA from sheep vaccinated against Teladorsagia circumcincta at the Moredun Research Institute.
Collaborator Contribution The partners are supplying information on the vaccine, parasitological, immunological and veterinary expertise as well as the RNA material from vaccinated sheep.
Impact No outputs yet; new collaboration. Research project ongoing.
Start Year 2015
 
Description Sharing of information & datasets with Prof David Hume's group (Roslin Institute) 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We will provide mapping outcomes of transcriptomes from abomasum and spleen samples generated under this award.
Collaborator Contribution Tissue-specific transcriptomes as reference for alignment and annotation of our RNA-seq data.
Impact Forthcoming.
Start Year 2016
 
Description BBSRC Animal Health Research Club Final Dissemination Event. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation of progress on BBSRC-ARC project "Dissecting variation in host responsiveness to a recombinant vaccine designed to control teladorsagiosis in sheep" by Simon Babayan.
Year(s) Of Engagement Activity 2018
 
Description BBSRC Animal Health Research Club 5th Dissemination Event. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Animal Research Club meeting, Birmingham. Oct 2017. Topic: Dissecting variation in host responsiveness to a recombinant vaccine designed to control teladorsagiosis in sheep. Delivered by PDRA on grant Wei Liu.
Year(s) Of Engagement Activity 2017
 
Description First presentation at BBSRC ARC Dissemination Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Matthews (Principal Investigator at Moredun) gave a presentation on the BBSRC Animal Health Research Club (ARC) project, Dissecting variation in host responsiveness to a recombinant vaccine designed to control teladorsagiosis in sheep, to an audience including other scientists, the ARC Steering Committee, BBSRC representatives and other interested parties from the animal health industry. This talk preceded the start of the project and outlined the background work to the project, focussing on the development of the Teladorsagia circumcincta sub-unit vaccine for the control of a significant roundworm infection of sheep.
Year(s) Of Engagement Activity 2015
 
Description Host-Parasite Interactions Meeting, Banff, Canada. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Topic: Vaccines against parasitic nematodes: outmanoeuvred by evolution before we even began. Leaders in the translation of fundamental and translational research on host-parasite interactions, combined presentation and workshop to share state-of-the-art procedures and knowledge with undergraduate and graduate students.
Year(s) Of Engagement Activity 2017
 
Description Institute of Biodiversity, Animal Health & Comparative Medicine PI/PostDoc talk, University of Glasgow 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Update on research progress. Topic: Mapping host gene expression to variation in responsiveness to vaccination against T.cirumcincta in sheep
The expertise we are developing lead to requests for collaboration.
Year(s) Of Engagement Activity 2017
 
Description International Conference on Statistics and Probability, China. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Conference theme: What can sequencing data tell us? From cancer to immunology. Presenter: PDRA on grant, Dr. Wei Liu. Presentation of the methods we are developping to draw immunological inferences from longitudinal Next Generation Sequencing data. Generated interest from academic and industry researchers, and requests for further information.
Year(s) Of Engagement Activity 2017
 
Description Invited speaker at Beatson Cancer Institute workshop on machine learning 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of Results of Study "Dissecting variation in host responsiveness to a recombinant vaccine designed to control teladorsagiosis in sheep" to illustrate use of machine learning applied to transcriptomics in a health-relevant context.
Year(s) Of Engagement Activity 2019
 
Description MVLS-parasitology Research Update Meeting, University of Glasgow. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Research group meeting. Audience comprised mainly cellular and molecular lab-based immunologists. Strong engagement related to the analysis of RNAseq data to elucidate immune pathways during infection.
Year(s) Of Engagement Activity 2017
 
Description Presentation of data analysis pipeline -- Dr Wei Liu 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Presentation to University of Glasgow members and students of two institutes (Institute of Biodiversity, Animal Health & Comparative Medicine; Institute of Infection and Immunity).
Year(s) Of Engagement Activity 2017
 
Description Presentation of our results to colleagues and stakeholders at ARC dissemination meeting Oct 2016 -- S Babayan 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Fostering network building and information sharing between academics and livestock industry.
This resulted in a new collaboration with David Hume's group (Roslin) and the sharing of unique datasets that we are using to improve the outcomes of our present award.
Year(s) Of Engagement Activity 2016
 
Description Second presentation at ARC Dissemination Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact McNeilly (Co-investigator,Moredun Research Institute) gave a presentation on the BBSRC Animal Health Research Club (ARC) project, Dissecting variation in host responsiveness to a recombinant vaccine designed to control teladorsagiosis in sheep, to an audience including other scientists, the ARC Steering Committee, BBSRC representatives and other interested parties from the animal health industry. This talk was an update on progress of the project (talk given at Month 8) and detailed the first year outputs from vaccine trials in two ages of sheep (3- and 6-months-old). The methodology for obtaining serial samples of abomasa material via an in-dwelling cannula was described, along with the faecal worm egg count analysis in vaccinated versus control (non-vaccinated) sheep.
Year(s) Of Engagement Activity 2016
 
Description Talk on the development of a vaccine to control teladorsagiosis, including a background to the BBSRC ARC project, at International Parasitology Conference (Liverpool) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact A plenary talk was delivered by Matthews (PI at Moredun) at the World Association for the Advancement for Veterinary Parasitology Conference on the development of the sub-unit vaccine for the control of teladorsagiosis, including a summary of the research thus far, as well as an introduction to new, associated projects funded by the BBSRC (ARC) and the EU (H2020 Programme). The audience mostly comprised other veterinary parasitologists. It was given in Liverpool in Aug 2015.
Year(s) Of Engagement Activity 2015