Antigen specificity of T cells mediating suppression of immunity to porcine reproductive and respiratory syndrome virus infection and vaccination.

Lead Research Organisation: Moredun Research Institute
Department Name: Vaccines and Diagnostics

Abstract

Vaccines are the most cost effective method for controlling many infectious diseases. Using vaccines also reduces the use of antibiotics and that results in less antimicrobial resistance to drugs used to treat infection.

Many pathogens affect livestock species and, in the pork industry, porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important infections today. This viral disease has spread world-wide and pig farmers struggle to control the disease. There are vaccines available in the market, but the performance of these formulations is limited. Specifically, these vaccines only provide good protection against similar strains of the virus and there are two major types and many subtypes. So, protection is fairly narrow when the farmers need the vaccine to have very broad protection.

We will develop tools that will allow for the design of next generation vaccines to control spread of infection even when the circulating virus is very different from the virus used to make the vaccine. We propose to develop technologies for pig research that are already available to human vaccine researchers and that allow a more accurate and informative assessment of the immune response to vaccination. Using these modern, more informative technologies will provide new and clear results to be used in the design and testing of more affordable vaccines for PRRSV. These efforts will give farmers an important tool to control a disease that causes significant economic loss and serious pain and suffering in their animals.

Technical Summary

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most widespread and economically devastating viral diseases. Vaccines for PRRSV only have effective protection profiles against closely related strains of the virus. This RNA virus mutates rapidly and vaccine matching to outbreak strains is difficult. Further, PRRSV infection results in the development of an immunosuppressive state in the pig where CD 4 T regulatory (Treg) cells play a role.

To more accurately assess immune responses to PRRSV, and other pathogens of pigs, we will develop new tools for the analysis of T cells. Major histocompatilbility complex (MHC) tetramer technology has been used for decades in humans and mouse models to analyse immune responses of T cells, and the technology is readily transferred to livestock species. Since T cell epitopes recognised by the antigen-specific receptor on T cells (the TcR) are in fact MHC I and II proteins that have bound peptides from the infecting pathogen (e.g. viral peptides), MHC tetramers allow the researcher to focus on antigen specific T cells. Our experience with MHC I and II tetramers for cattle will be applied in this project to develop MHC II tetramers for swine.

First, we will express four swine leukocyte antigen (SLA) II proteins and analyse peptides derived from PRRSV encoded proteins for SLA II protein binding. Once the PRRSV peptide binding profile is established, we will use these proteins to construct SLA II/PRRSV tetramers for use in the study of CD4 expressing T cells in pigs infected with PRRSV. We will compare those results to the analysis of pigs vaccinated against PRRSV with two different, commercially available vaccines. The data from these studies will provide the basis for Zoetis to redesign PRRSV vaccines eliminating immunosuppression mediated by Tregs. The resulting, next generation PRRSV vaccines will give farmers better tools to control of PRRSV and improve the economic performance of their herds.

Planned Impact

PRRSV is a problem worldwide and swine are critical to small holder farmers, being inexpensive to buy and raise. A new, more efficacious catalogue of vaccines mitigating this disease will have large impacts on Low to Middle Income Countries (LMICs). Our major collaborators on this application from Chulalongkorn University, Faculty of Veterinary Science will immediately bring these technologies to a LMIC as well as be a resource to other LMIC based veterinary and human vaccine researchers in the region.

Critical to this application, Moredun has added the next generation flow cytometry technology to our resources by purchasing a (SONY SA3800) spectral analyser. This new technology for flow is critical in that the overlapping spectra of fluorophores is no longer an issue as the spectral pattern of each fluorophore is measured independently. For veterinary immunologists who have very limited flow cytometry reagents available, this is a major advance. This will be available to scientists on the Easter Bush campus and will provide vaccine scientists worldwide the example of another tool to more accurately measure immune responses. They can apply these novel vaccine assessment technologies to considerable benefit in other vaccine programs. To facilitate this, we will regularly participate in meetings and conferences attracting the vaccine design and delivery communities. Publication of high quality manuscripts in appropriate peer-reviewed international journals will make these approaches and technologies readily available, particularly by targeting open-access journals to allow maximum impact.

Key stakeholders in this proposal are the Foreign and Commonwealth Offices through their commitment to addressing challenges relevant to the health and prosperity of LMIC countries, and the International Veterinary Vaccine Network, a BBSRC funded network to promote coordination and collaboration in the development of vaccines for food animals. Moredun is represented by one of the co-PIs of the IVVN, Alisdair Nisbet, in this network.

Moredun scientists, including the PI, regularly attend and present talks at farmer and animal health meetings throughout the UK and attend most major UK agricultural events. Moredun and Zoetis scientists also have close working relationships with industry bodies such as Quality Meat Scotland (QMS), and the National Pig Association (a subgroup of the National Farmers Union (NFU) and NFU Scotland (NFUS)). Chulalongkorn University has ongoing interactions and collaborations with the Thai Swine Veterinary Practitioner Association and various partners in the Thai swine industry including Charoen Pokphand (CP), and Betagro Groups, the major swine producers in the Southeast Asia region. These pre-existing relationships will allow the group to actively engage with and directly communicate the impact of this project rapidly to these stakeholders in an appropriate format through regular face-to-face interactions, newsletters and Chulalongkorn Univ. and Moredun website content (https://www.chula.ac.th/en/academic/faculty-of-veterinary-science, https://www.moredun.org.uk).

The present PRRSV vaccines available from Zoetis and others are effective but have performance characteristics that can be improved. We anticipate that data from these studies will allow us to work with Zoetis to inform design of next generation PRRSV vaccines that will serve pork producers worldwide in the extensive efforts to control this disease.

Publications

10 25 50
 
Description We have expressed 2 class II MHC proteins (transplantation antigens swine leukocyte antigen (SLA)-DR proteins) using the proprietary University of Copenhagen expression system. Our original proposal was to test peptide libraries of 2 proteins from porcine reproductive and respiratory syndrome virus (PRRSV), the M and N proteins, for binding by these SLA-DR proteins. Peptides, bound by MHC proteins are potential "antigenic epitopes" for T cells. We have now developed a new peptide binding screening methods with the company PePperPrint. Using peptide chip technology, they can create peptide chips representing the entire proteome of a virus, even large DNA viruses. We needed to establish conditions for class II MHC binding of peptides on chips.

We tested this new approach using human (HLA-DR) proteins for which peptide binding had been validated in a single peptide, in vitro assay. We designed chips with peptides known to be bound as well as peptides known to not be bound and added random peptides. We ran the screen of these peptide chips to determine optimal conditions for this assay, seeking to lower "background" signals and enhance positive binding. Results revealed the chip assay identified a peptide binding map nearly identical to the map from data using the individual peptide binding assay.

Peptide chips were produced based on the consensus sequence of the 2 most prominent strains of PRRSV. We screened both SLA-DR proteins on these chips and only one, SLA-DR-04 yielded reliable, reproducible peptide binding in the assay. We chose to take the SLA-DR-04 forward and the identified peptides have been synthesized for further analysis. In addition, we had a series of random peptides synthesized as well. These MHC proteins and the peptides that they bind will be used to make class II MHC tetramers to probe T cells from PRRSV immune animals.

We have now initiated animal trials to test for the induction of T cells specific for the peptides identified. This will be achieved by producing MHC tetramers with SLA-DR-04 and the identified peptides. These will be test for binding to antigen specific T cells with specificity for the T cell epitopes (defined by the SLA-DR molecule and the PRRSV derived peptide that DR protein binds). Samples are being collected from pigs infected with PRRSV and also from pigs vaccinated with a commercial vaccine for PRRSV. They will be tested for staining with the MHC tetramers as well as for secondary in vitro responses to the same peptides identified previously.
Exploitation Route The methodology developed in the work will greatly advance the ability of investigators at all entities, academic and commercial, large and small, to more accurately identify T cell epitopes. This will greatly facilitate many aspects of research focusing on cellular immune responses. Human and mouse MHC molecules are already available from mutiple companies. Our plan is to make all of the livestock species molecules available once we secure funding to do so. We will continue to work with PepPerPrint to refine assays for probing peptide chips representing proteomes of interest to the researcher.
Sectors Agriculture

Food and Drink

Healthcare

Pharmaceuticals and Medical Biotechnology

 
Description Enhancing frontier research in veterinary immunology through global research alliance
Amount ฿9,000,000 (THB)
Funding ID B16F630071 
Organisation Chulalongkorn University 
Sector Academic/University
Country Thailand
Start 05/2020 
End 06/2021
 
Title Screening class II MHC proteins on peptide chips. 
Description In our proposal, we intended to screen peptide libraries of 2 important proteins expressed by the PRRS virus, M and N proteins. In work with another grant from BBSRC, CoxiMap, we identified new companes that make custom peptide chips for screening antibody binding. We engaged one, PEPPerPRINT of Hiedelberg, Germany, to create peptide chips representing the entire PRRSV genome. This work has progressed, in spite of the pandemic, to the pint where we are validating the peptide chips in order to analyse binding of 2 porcine class II SLA-DR proteins for binding PRRSV peptides. This work will provide a much more comprehensive analysis of SLA-DR peptide binding for the PRRSV genome than the proposed approach. And the cost will be similar or less. In the coming 2 quarters, we should have all the binding data required to move on to the animal trials. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? No  
Impact This is a new approach to analysing peptide binding capacity of class II MHC proteins. The collaboration of PEPPerPRINT, University of Copenhagen, and the Moredun Research Institute is working to validate this new technology with existing data from studies of the human HLA-DR proteins applied to the swine SLA-DR protins using a pepetide chip technology. If successful, this technology will allow for a much more rapid and efficinet screening of MHC class II proteins binding peptides and result in the identification of potential T cell epitopes in a more rapid and economic system. For all mammalian and avian immune response research, this will be a major advance in the technollogy. 
 
Title IPD-MHC Database 
Description This is a database of all sequences of the the major histocompatibility complex (MHC) of multiple species, including swine. Sequence entries for the swine leukoctye antigen (SLA) loci, MHC of swine, are managed and maintained by Dr. Sam Ho of the Gift of Hope Organ & Tissue Donor Network, Michigan, USA and Dr. Sabine Hammer of the University of Vienna, Austria. All of our sequences from Scottish and Thai pigs that have not been previously reported have been added. 
Type Of Material Database/Collection of data 
Provided To Others? Yes  
Impact This database has been critical in understanding transplantation biology, zeno-transplantation, immunology and vaccinology. The MHC is critical to immune responses and was historically called the Immune response (Ir) locus. 
URL https://www.ebi.ac.uk/ipd/mhc/group/SLA/
 
Description Analysis of T Reg responses in PRRSV infection and following vaccination. 
Organisation Chulalongkorn University
Country Thailand 
Sector Academic/University 
PI Contribution I organised this research partnership after consultation with the industrial partner on this award. My academic partners provide specific expertise for this project and together with them, I planned and wrote the grant application. I coordinated the input of our industrial partner keeping the aims and objectives compatible with their commercial goals.
Collaborator Contribution Dr. Sanipa Suradhat of Chulalongkorn University Veterinary College (CUVet) is a world expert in PRRSV infection and vaccination. She is an experienced and accomplished immunologist and together we developed a hypothesis for the immunosuppression caused by PRRSV infection. In order to address the hypothesis, we require analysis tools to measure T cell responses on an individual basis. These reagents are called T cell MHC tetramers. Dr. Søren Buus of University of Copenhagen is one of the world's leading experts on the MHC and his laboratory has developed the technology of expressing the proteins that make T cell tetramers and analysing the potential T cell epitopes of any pathogen. His laboratory will make the tetramers we need for this study of pig immune responses. Zoetis is the industrial partner on this award and presently is the only producer of PRRSV vaccines. Two forms of vaccine will be tested in theis project to determine the quality of the T cell immune response to vaccination of pigs.
Impact Nothing to report yet.
Start Year 2019
 
Description Analysis of T Reg responses in PRRSV infection and following vaccination. 
Organisation University of Copenhagen
Country Denmark 
Sector Academic/University 
PI Contribution I organised this research partnership after consultation with the industrial partner on this award. My academic partners provide specific expertise for this project and together with them, I planned and wrote the grant application. I coordinated the input of our industrial partner keeping the aims and objectives compatible with their commercial goals.
Collaborator Contribution Dr. Sanipa Suradhat of Chulalongkorn University Veterinary College (CUVet) is a world expert in PRRSV infection and vaccination. She is an experienced and accomplished immunologist and together we developed a hypothesis for the immunosuppression caused by PRRSV infection. In order to address the hypothesis, we require analysis tools to measure T cell responses on an individual basis. These reagents are called T cell MHC tetramers. Dr. Søren Buus of University of Copenhagen is one of the world's leading experts on the MHC and his laboratory has developed the technology of expressing the proteins that make T cell tetramers and analysing the potential T cell epitopes of any pathogen. His laboratory will make the tetramers we need for this study of pig immune responses. Zoetis is the industrial partner on this award and presently is the only producer of PRRSV vaccines. Two forms of vaccine will be tested in theis project to determine the quality of the T cell immune response to vaccination of pigs.
Impact Nothing to report yet.
Start Year 2019
 
Description Analysis of T Reg responses in PRRSV infection and following vaccination. 
Organisation Zoetis
Country United States 
Sector Private 
PI Contribution I organised this research partnership after consultation with the industrial partner on this award. My academic partners provide specific expertise for this project and together with them, I planned and wrote the grant application. I coordinated the input of our industrial partner keeping the aims and objectives compatible with their commercial goals.
Collaborator Contribution Dr. Sanipa Suradhat of Chulalongkorn University Veterinary College (CUVet) is a world expert in PRRSV infection and vaccination. She is an experienced and accomplished immunologist and together we developed a hypothesis for the immunosuppression caused by PRRSV infection. In order to address the hypothesis, we require analysis tools to measure T cell responses on an individual basis. These reagents are called T cell MHC tetramers. Dr. Søren Buus of University of Copenhagen is one of the world's leading experts on the MHC and his laboratory has developed the technology of expressing the proteins that make T cell tetramers and analysing the potential T cell epitopes of any pathogen. His laboratory will make the tetramers we need for this study of pig immune responses. Zoetis is the industrial partner on this award and presently is the only producer of PRRSV vaccines. Two forms of vaccine will be tested in theis project to determine the quality of the T cell immune response to vaccination of pigs.
Impact Nothing to report yet.
Start Year 2019