The Pirbright Livestock Antibody Hub (the Hub) will aim at producing a suite of methods for each species that enable studies and antibody manipulation at the same resolution as humans and mice.
The concept of the Pirbright Livestock Antibody Hub (the Hub) is a direct response to the recent, largely technology driven advances in our understanding of B-cell responses and the discrete interactions that drive protective immune responses. There is a clear and present need from both the research community and private sector for techniques and capacity to analyse B-cell responses in livestock to support rational vaccinology and to explore antibody-based approaches for treatment and prophylaxis of infectious diseases. This will require the establishment of species and pathogen specific methods to interrogate antibody responses in parallel with in vivo studies in the natural host.
We will directly apply detailed knowledge to advance vaccine design and delivery in livestock against some of the most economically important pathogen. We will also open the possibility of using the most appropriate models for human disease, with the suite of reagents, techniques and background knowledge needed. In this way the Hub can be reactive and proactive in supporting and soliciting additional projects that rely on understanding and measuring livestock antibody responses.
WP1: Single cell sorting of specific B-cell subsets
In this programme we will develop a suite of tools and associated methods that enable us to sort B cells and their subsets including antigen specific memory and plasma cells in cattle, poultry and swine. Several technology platforms (Flow cytometry cell sorting, laser microdissection and micro-encapsulation with barcoded beads) will be developed to isolate antigen-specific B cells at low, medium and high throughput.
WP2: Robust single cell and pooled cell sequencing
Current single cell sequencing protocols to pair heavy and light chain sequences of antigen-specific antibodies are now being transferred at medium and high throughput levels in the different livestock species. High-throughput sequencing offers the ability to survey a much larger population of B cells, or B-cell subsets, increasing the probability of identifying antibodies to particular epitopes or with desired functional properties. Also, we are developing methods of antigen-specific antibodies pull down and sequencing from serum, and identification within the whole antibody repertoire during the immune response.
WP3: Expression and engineering of recombinant antibodies
The UK Immunological Toolbox at Pirbright has the capability to sequence and recombinantly express antibodies from hybridomas. Importantly, we have also designed and validated vectors to engineer antibodies to express the Ig subclasses from cattle, pigs, chickens and mice, and to class switch antibodies to be more useful reagents. This Hub will greatly benefit from access to this established expertise, both in terms of training and reagent generation. It will allow antibody sequences to be expressed for functional studies, as well as antibodies to be engineered to have specific functions or adapted to other species.
WP4: Link Fc and Fc-receptor function
Outside of mice and humans, surprisingly little is known about Fc-receptor function, even in higher primates. We will develop a suite of assays to understand binding and affinity of each antibody subclass to that species Fc receptors and examine the function of individual antibodies (recombinant or native) for each species, as well as each component of the protective antibody response. These assays in combination with more standard assays such as neutralising assays, liquid phase blocking ELISA and ELISpot, will comprise a powerful suite of analytical tools to accurately predict and measure the function of antibodies in a range of host species.
WP5: Bioinformatic resources
Analyses of antibody sequences in the context of gene usage, somatic hypermutation, repertoire and antigen specificity are challenging, particularly for livestock in terms of accurate reference genome (germline) annotation, antibody annotation and clustering algorithms tailored for each species. We propose to use existing and new datasets (generated as part of this proposal) to benchmark different analysis pipelines. These pipelines will be a mix of those being developed at Pirbright to answer specific questions regarding livestock vaccine and pathogen responses, as well as exploiting methods and programmes already published and freely available.