Replacement fluorescence activated cell sorter for the Jenner Laboratory (BBSRC National Vaccinology Centre: The Jenner Building)

Lead Research Organisation: The Pirbright Institute
Department Name: PRRS Immunology

Abstract

Fluorescence-activated cell sorting (FACS) is a powerful technology that enables the measurement of characteristics of single cells and then separates these cells based on their characteristics. Tens of thousands of individual cells can be isolated within minutes and are then available for analysis. This is critical to our understanding of how animals respond to virus infections and enable us to design and develop viral vaccines to prevent disease.
The Pirbright Institute's (Pirbright) purpose is to predict, detect, understand, and respond to serious viral diseases of livestock and viruses that spread from animals to humans. Pirbright is unique within the UK, providing scientists with access to both state-of-the-art animal and laboratory facilities to research infectious diseases of livestock at high containment levels. The BBSRC National Vaccinology Centre (Jenner Building) at Pirbright, is a low containment facility that supports work with medium risk viruses. This project aims to ensure continuity of the FACS capability in the Jenner Building for the next 10-12 years. To achieve this, we propose to replace our current 15 years old FACSorter, the BD FACSAria U3 (Aria U3), positioned within the Jenner Building, with an upgrade to a BD FACSAria Fusion (Aria Fusion).
The Aria Fusion is superior to the Aria U3 with exceptional multicolour performance, up to date software and hardware and enhanced biosafety protection. The addition of the microbiological safety cabinet will increase sample and operator protection. Retaining our FACS capacity and expanding our ability to sort virus infected samples will enable us to understand better animal responses to virus infection.

Technical Summary

Fluorescence-activated cell sorting (FACS) is a powerful multiparameter technology that enables the measurement of characteristics of single cells suspended in a fluid stream and then separates these cells based on their characteristics. Tens of thousands of individual cells can be isolated within minutes and are then available for morphological, genetic, and proteomic analysis, functional assays, or clonal selection. These downstream applications are critical to understanding host virus interactions with application in the design and development of viral vaccines and prevention of disease.
The Pirbright Institute's (Pirbright) purpose is to predict, detect, understand, and respond to serious viral diseases of livestock and viruses that spread from animals to humans. Pirbright is unique within the UK, providing scientists with access to both state-of-the-art animal and laboratory facilities to research infectious diseases of livestock at high containment levels. The BBSRC National Vaccinology Centre (Jenner Building) at Pirbright, is a low containment facility that supports work with medium risk viruses. This project aims to ensure continuity of the FACS capability in the Jenner Building for the next 10-12 years. To achieve this, we propose to replace our current 15 years old FACSorter, the BD FACSAria U3 (Aria U3), positioned within the Jenner Building, with an upgrade to a BD FACSAria Fusion (Aria Fusion).
The Aria Fusion is superior to the Aria U3 with exceptional multicolour performance, up to date software and hardware and enhanced biosafety protection (protecting both staff and the environment). The sorter configuration requested is a 4-laser system capable of simultaneously detecting 16 colours and it mirrors that of our other BD Biosciences flow cytometry instruments. The addition of the microbiological safety cabinet will provide us with the ability to sort virus infected samples to expand our knowledge of host responses to virus infection.
 
Description Development of CVI988 based recombinant vaccine
Amount £238,000 (GBP)
Organisation Eco Animal Health Ltd 
Sector Private
Country United Kingdom
Start 03/2023 
End 02/2024
 
Description Development of avian herpesvirus vector vaccines for poultry
Amount £772,000 (GBP)
Organisation MSD Animal Health 
Sector Private
Country United Kingdom
Start 01/2024 
End 12/2026
 
Description Effects of co-infections on Marek's disease in poultry and development of novel recombinant Marek's disease virus vector vaccines
Amount £562,000 (GBP)
Funding ID BB/X017575/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2023 
End 09/2026
 
Description PhD studentship
Amount £110,000 (GBP)
Organisation The Pirbright Institute 
Sector Academic/University
Country United Kingdom
Start 09/2022 
End 03/2026
 
Title Establishment of an efficient CRISPR/Cas9 gRNA library screening to identify the host factors required for maintaining the transformed phenotype of MDV tumour cell lines 
Description Herpesviruses are large dsDNA viruses that cause widespread, lifelong latent infections in different hosts, through multiple virus-host interactions to create a delicate balance between the virus and the host. CRISPR/Cas9-based gene editing is emerging as a powerful tool to investigate the precise determinants of latency in a number of herpesvirus infections. Marek's disease virus (MDV-1) is a lymphotropic a-herpesvirus associated with latent infections and malignant CD4+ T-cell lymphomas in chicken. MDV-1 has a two-phase life cycle, consisting of a lytic and a latent phase, the latter closely associated with the oncogenesis of the virus, yet the underlying molecular mechanisms of cell transformation remain unclear. Better understanding of the factors that maintain the latency of the virus will provide insights into novel intervention strategies to interfere with the neoplastic process. We have used a high throughput genome-wide CRISPR/Cas9 gene knockout strategy, combined with next generation sequencing to identify the genes critical for maintenance of the transformed phenotype. 339 host genes have been identified being essential for maintaining the transformed phenotype of MDV cell line. 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? No  
Impact Establishment of the pipeline of CRISPR library screening in chicken cell lines has given the opportunity for the identification factors associated with disease pathogenesis and neoplastic transformation. The identified genes could serve as targets for inducing de novo genetic resistance using gene editing approaches. 
 
Title Establishment of an efficient CRISPR/Cas9-based screening pipeline for herpesviruses to study the gene function and streamline the vector platform for recombinant vaccine development 
Description The development of both cosmid DNA and Bacterial artificial chromosome (BAC) technologies has greatly facilitated the introduction of mutations into the viral genomes of herpesviruses to study gene functions. However, cloning of viral genomes as BAC plasmid and subsequent mutagenesis is inefficient, time-consuming and may introduce mutations by repeated passages. Following our success in efficient CRISPR/Cas9 editing of the MDV genome in both replicating virus and MDV transformed cell lines and of HVT genome for the recombinant vaccine development, we have developed an efficient CRISPR/Cas9-based screening pipeline for herpesviruses to identify the essential/non-essential genes for study of the gene function and streamline the vector platform for recombinant vaccine development. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? No  
Impact Identification of essential and non-essential genes of herpesviruses provided opportunities to study gene function in vitro and in vivo. Knocking out the non-essential genes in the HVT vector can enhance HVT replication in vivo enabling recombinant HVT-based vaccines to induce stronger immune responses. 
 
Title The establishment of an efficient pipeline for in situ CRISPR editing of the Marek's disease virus (MDV) genome in lymphoma-derived cell lines 
Description The lymphoblastoid cell lines (LCLs) derived from MD lymphomas have served as valuable resources to study virus-host molecular interactions in transformed cells. However, detailed investigations into the functional role of different viral and host determinants in these cells have been difficult due to the lack of tools for in situ manipulation of viral/host genomes in MDV-transformed cell lines. Our recent success in efficient CRISPR/Cas9 editing of the MDV genome in LCLs has demonstrated the potential for targeted editing to dissect the regulatory pathways involved in latency, transformation, reactivation and lytic switch. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Using this approach, we have demonstrated viral genes such as pp38 and MDV-miR-M4 are not essential for maintaining the transformed phenotype. On the other hand, deletion of MDV-encoded oncogene Meq or mutations preventing its interactions resulted in cell death demonstrating the essential role of Meq in transformation. 
URL https://www.mdpi.com/1999-4915/10/6/279
 
Description Effects of co-infections on Marek's disease in poultry, and development of novel recombinant Marek's disease virus vector vaccines 
Organisation Moy Park
Country United Kingdom 
Sector Private 
PI Contribution This is a joint BBSRC funded project on investigation "Effects of co-infections on Marek's disease in poultry, and development of novel recombinant Marek's disease virus vector vaccines" between Pirbright, Moy Park and Slate Hall Veterinary Practice. Pirbright team works on the detailed investigation of prevalence of naturally occurring MDV-2 (Marek's disease virus serotype 2) infection in the field in UK; isolation and characterisation of MDV-2 field strains and development of novel MDV-2-based recombinant vaccine MDV2-IBD-ND which will protect chickens against Marek's disease, infectious bursal disease and Newcastle disease.
Collaborator Contribution Moy Park will collect the field samples and investigate the effect of MDV-2 on flock productivity and disease.
Impact Too early
Start Year 2023
 
Description Joint PhD project on "Using Marek's Disease Virus to understand the RNA world of alphaherpesviruses" 
Organisation University of Surrey
Country United Kingdom 
Sector Academic/University 
PI Contribution Surrey team has provided expertise on characterization of Marek's disease virus encoded vhs protein function in vitro.
Collaborator Contribution Pirbright team has provided expertise in making the mutant virus of vhs deleted MDV and study the role of vhs in the context of infection in vitro and in vivo.
Impact The student Sophie Cutts has abstract accepted for oral presentation in both Microbiology Society Annual Conference 2024 to be held in Edinburgh 8-11 April and 14th International Symposium on Marek's Disease and Avian Herpesvirus to be held at St. Louis, USA from 12th-14th July, 2024.
Start Year 2022