Investigating Host and Viral Factors for Improved Design of Future Live Attenuated Vaccines for IBV

Lead Research Organisation: The Pirbright Institute
Department Name: Integrative Biology & Bioinformatics

Abstract

Vaccination against endemic pathogens is an essential component of the poultry industry, without which chickens would become infected at an early age. This would reduce productivity and push food security beyond sustainable levels. Infectious bronchitis virus (IBV) is a coronavirus that causes respiratory disease in chickens, making them more susceptible to bacterial infections. After infection, they achieve lower weights and produce fewer high-quality eggs. It has been estimated that a 10% reduction in IBV incidence globally would be worth £654million to the poultry industry.
An effective vaccine to IBV is therefore critical for both welfare and economic reasons. The best vaccine strategies for IBV are "live attenuated" types, meaning a weakened form of the disease-causing virus is used to vaccinate chickens, generating good immunity. These vaccines are produced by growing disease-causing (pathogenic) viruses in eggs up to one hundred times, during which multiple genome changes can occur. Our understanding of how this process works is limited. Whilst these vaccines have lost their ability to cause symptoms, they still retain the ability to induce protective immune responses in chickens, thereby protecting them from disease. However, some vaccine strains have been reported to evolve, causing outbreaks of disease in flocks after vaccination. This occurs as vaccine virus genomes change and there is a risk, they may regain their pathogenic capability (reversion), producing disease in vaccinated birds i.e. the vaccine virus causes new outbreaks. Understanding better how both vaccine viruses are weakened and the host pressures after vaccination drive reversion are key to designing more stable vaccines.
In a previous project (BB/L003988/1) 2 IBV viruses were weakened (a commercial vaccine and a lab-adapted strain) by passaging in eggs to identify how the viruses changed. We deep sequenced these viruses every 10 passages to understand how they change. We know from existing datasets that some changes are shared between both weakened and pathogenic viruses. We also know that some changes are exclusive to each type. Moreover, sequence changes are influenced by factors including restricted diversity and dilution factors during egg passaging. We do not know how these changes will impact reversion after vaccination.
The overall aim of the project is to produce a detailed profile of the viral dynamics-host interactions of IBV vaccination in chickens to gain major insights into the virus biology and host responses. This will answer 3 research questions:
1. Are there changes in the virus during this weakening process that can be used to improve future vaccines?
2. How do vaccine virus's genomes changes after vaccination into chickens and do these changes make them more likely to cause reversion?
3. What host genes are expressed, and do they help drive vaccine viruses to evolve in chickens?
We will use deep learning will identify genomic patterns in viral genomes during the weakening process. We will characterise how vaccine viruses change after vaccination into chickens, comparing changes to those in pathogenic viruses, to measure the likelihood of reversion. We will characterise cellular gene expression, after vaccination and use machine learning to identify cellular responses driving changes in the vaccine virus genomes. Finally, we will combine datasets and use machine learning to make predictions on which sequence changes are important in the processes of weakening viruses, and reversion. These predictions will be re-inserted back into a virus and their impact on chicken cells measured.
This research will identify genome changes involved in the occurrence and likelihood of reversion and reveal how they will change post-vaccination. These results will further our understanding of processes impacting virus genomes both during attenuation and after vaccination, that can be used in future next generation vaccines.

Technical Summary

Infectious Bronchitis is the most economically important infectious disease affecting poultry globally. We have shown previously that attenuation of the avian coronavirus, infectious bronchitis virus (IBV) by serial egg passaging is likely a multifactorial process rather than being driven by a single pathway. Little is known regarding the mechanisms underpinning this process, with few studies having evaluated vaccine virus genome sequence stability post-vaccination and its contribution to reversion to virulence, that can seed new outbreaks driven by vaccine viruses.

The following research questions will be considered:
1. Are there changes in the virus during the attenuation process that can be used to improve future vaccines?
2. How does the genome sequence stability of vaccine viruses change after vaccination, and do those changes contribute to reversion of virulence?
3. What role do host genes have in driving vaccine virus evolution in vivo?

We will use deep learning methods to identify patterns within 52 existing whole genome sequence datasets generated during egg passaging of 2 viruses (QX and M41-CK) to attenuation. We will then generate novel sequence information for whole S gene/whole IBV genome to explore virus genome sequence changes occurring post-vaccination. Changes will be compared with pathogenic IBV isolates to link to phenotype. We will use spatial transcriptomics to identify changes in host gene expression within tissues from vaccinated birds. This will identify cellular responses driving virus changes at a cellular level. We will take the outputs of these three components, combining them into a single dataset and process them using deep learning to make final predictions of genomic markers or signatures contributing to attenuation and validate these ex-vivo.

This work will identify better ways to attenuate viruses for use as live attenuated vaccines, whilst minimising the potential for reversion to virulence.

Publications

10 25 50
 
Description The award is ongoing, but the main achievements/outcomes so far have been a large set of samples produced from the animal study that was planned during the project. We have also developed an amplicon based method to sequence Infectious bronchitis virus (IBV) Spike from in vivo tissue samples. Such samples are challenging, when using other published methods, due to the high levels of host. We are currently using this to evaluate spike evolution in samples from a previous animal experiment in poultry, in order to publish these results and the method. We have also started to generate datasets using the 10X Visium spatial transcriptomics workflow. The 2-day workflow incorporating both imaging and molecular aspects has already had a small trial run sequenced at Pirbright, using the core facilities. We are now using this methodology to focus on the remaining samples in this project generated as part of the animal study.
Exploitation Route Possible outcomes from this funding could be the use of spatial transcriptomic data by other virology researchers to identify host-virus interactions during avian coronavirus infection. Developers of live attenuated vaccines, such as those already used for IBV) could use the spike evolution data for determining vaccine virus stability, after vaccination into a bird.
Sectors Agriculture

Food and Drink

Healthcare

Manufacturing

including Industrial Biotechology

URL http://www.ibvvacc.org.uk
 
Description Acting consultant on FAO/IAEA VETLAB2 project - 1st Annual meeting, 12-16th Dec 2022
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a guidance/advisory committee
Impact By providing expert advice in this capacity this translates directly to improving high throughput sequencing (HTS) resources available to scientists in diagnostic and research capacity in FAO/IAEA member states who utilise HTS.
URL https://www.fao.org/publications/card/en/c/8ed18cbb-379a-4f8e-aa5f-521ea4f480c8
 
Description Current and Future Challenges with Veterinary Virus Genomics
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in other policy documents
URL http://www.cfcvvg.org
 
Description RAS5085-EVT2207501 - Bangladesh.
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact Improved biosurveillance of transboundary diseases including Foot and Mouth disease virus (FMDV) and Lumpy Skin Disease virus (LSDV) will result in improved economic and agricultural outputs for member states attending the workshop.
 
Description Second RCM of the CRP D32036 (EVT2205960) and the VETLAB Directors Meeting (EVT2205952)
Geographic Reach Multiple continents/international 
Policy Influence Type Contribution to a national consultation/review
Impact This will have improved member states responses to viral disease incursions of livestock including FMDV and ASFV. An improved genomics capability will result in better control mechanisms globally for controlling these diseases.
 
Description Website for project
Geographic Reach Multiple continents/international 
Policy Influence Type Contribution to new or improved professional practice
URL http://www.ibvvacc.org.uk
 
Description GAP-DC-II: Genomics for Animal and Plant Disease Centre
Amount £10,049,682 (GBP)
Funding ID SE0574. 
Organisation Department For Environment, Food And Rural Affairs (DEFRA) 
Sector Public
Country United Kingdom
Start 07/2023 
End 08/2027
 
Description Identifying markers of coronavirus attenuation by ML
Amount £15,000 (GBP)
Organisation University of Surrey 
Sector Academic/University
Country United Kingdom
Start 11/2023 
End 02/2024
 
Title Animal study for investigating the performance and evolution of live attenuated vaccines in poultry 
Description This is a set of comprehensive set of biological samples taken from 200 Rhode Island Red SPF chickens. The sample set includes samples taken from upper/lower eye lid, beak, trachea, lung, kidney, bursa, spleen and blood. The samples are stored in RNAlater, PBS and snap frozen in OCT. 
Type Of Material Biological samples 
Year Produced 2023 
Provided To Others? No  
Impact This set of samples forms an important and valuable resource for investigating the evolution of IBV vaccines in poultry. It will be a critical resource for upcoming projects at Pirbright and is already providing samples for other projects including the Bill and Melinda Gates Antibody hub who are using the serum from blood samples to investigate variable regions in chicken B-cells. 
 
Title Probe enrichment for whole genome sequencing of Infectious bronchitis virus serotypes from in vivo tissue samples 
Description This is a method for the whole genome sequencing of Infectious Bronchitis virus from in vivo tissue samples. Such samples hold high host nucleic acid background and low viral load, making it difficult to generate a whole genome sequence using traditional mRNA enrichment based methods. This involves a number of overlapping probes designed to multiple serotypes of IBV which hybridize and enrich for viral derived libraries in the latter stage of the library preparation process. 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? No  
Impact We look to publish this method and make it available to other investigators in the field. This tool makes generating whole viral genome from in vivo samples reliable and robust, and will have a beneficial impact upon the use of animals in such experiments, by maximizing the value of these samples. 
 
Title Spatial transcriptomics (10X Visium) for characterizing host gene expression in vivo after vaccination/infection with attenuated/pathogenic IBV 
Description Microscopic glass slides containing four marked 6.5×6.5 mm areas are used where thin tissue sections are placed, fixed, stained and imaged. Each area contains 5000 printed regions of barcoded mRNA capture probes with the dimensions of 55 µm in diameter and a center-to-center distance of 100 µm. Tissue is permeabilized and mRNAs are hybridized to the barcoded capture probes directly underneath. cDNA synthesis connects the spatial barcode and the captured mRNA, and sequencing reads are later overlaid with the tissue image. The tissues were stained with DAPI as well as fluorescently labeled antibodies against IBV S2 and nucleoproteins for simultaneous viral protein detection. Three time-points, 1dpi, 4 dpi and 14dpi, were used to investigate early, mid and late host responses. At each time point, triplicates of pathogenic and attenuated samples were chosen based on their viral genome copy numbers, two mock infected samples were also picked randomly. 
Type Of Material Technology assay or reagent 
Year Produced 2024 
Provided To Others? Yes  
Impact The detailed profile of the viral dynamics-host interactions of IBV infection in chickens will aid in developing better vaccines and control strategies. 
 
Title Tiled Amplicon sequencing for avian Coronavirus Infectious bronchitis virus spike 
Description This is an amplicon based sequencing assay to monitor changes in IBV spike open reading frame in response to anti-IBV antibodies generated through vaccines and host responses. The assay is specific for both M41-CK and QX serotypes of IBV and will be applied to in vivo studies with IBV in chickens to monitor evolving vaccine responses. 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? No  
Impact This assay will enable us to monitor changes in Coronavirus spike gene in response to host responses and provide us with the ability to monitor emergence of novel variants involved in evasion of host responses. 
 
Title Spatial transcriptomics of Chicken trachea infected with Infectious bronchitis virus to identify transcriptomic signatures 
Description Rhode Island Red SPF chickens were infected with M41-CK (a cell culture adapted pathogenic IBV virus) to investogate the evolution of the virus in chickens after infection, and compare with live attenuated vaccine versions of the same virus. Illumina sequence datasets were piped into the 10X proprietary analysis pipeline 'Space Ranger' for reads alignments, feature barcode matrices generation and secondary analysis including clustering and differential gene expression. Outputs from Space Ranger consisting of tissue images, tissue positions and feature barcode matrix were further piped into previously published R-based spatial pipeline 'Giotto' (Dries et al, 2021) for detailed analysis such as dimensionality reduction, co-visualization of gene expression and spatial location, cell type marker gene detection, spatial structure analysis, spatial gene co-expression patterns, etc. 
Type Of Material Database/Collection of data 
Year Produced 2024 
Provided To Others? No  
Impact The dataset will have impact for other research groups investigating avian coronavirus vaccines. They will also have a broader impact and be of interest to wider coronavirus vaccine development and research thorough the information on vaccine evolution and host response. The datasets will be of interest to persons within the field of spatial biology field, notably the software developers and bioinformaticians, as a dataset to test new models and algorithms. The results will be of interest specifically for those in industry who are involved in developing novel and more rational strategies for developing vaccines. 
 
Title Tiled amplicon sequencing using Illumina platforms for detecting genomic variants of infectious bronchitis virus (IBV) spike protein post infection/vaccination 
Description This dataset was generated during validation of the tiled amplicon sequencing method using clinical chicken trachea samples. RNAs were extracted from Rhode Island Red (RIR) chicken infected with D388RT (a wild type D388 strain) and D388R (a clone of D388), subsequently converted to cDNAs. Multiplex PCR primers were designed using a online primer design tool 'Primal Scheme' (Quick et al., 2017). Multiplex PCR were performed to amplify the region of IBV spike in a 400-nt amplicon fashion. The amplicons were Illumina sequenced and a bioinformatic pipeline was optimized specifically for tiled-amplicon sequencing datasets utilizing various tools such as bwa-mem2, samtools, ivar and lofreq. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact This dataset enables the development of a robust and accurate pipeline to detect variants of IBV spike during vaccination/infection. It forms a useful tool to overcome the difficulties of generating whole-genome sequences directly from clinical samples using metagenomic approaches. It is beneficial to IBV researchers and vaccine developers who need to monitor 1), the evolution of IBV under host selection pressure; 2) vaccine strain of IBV in the field to study the events of reversion to virulence of IBV live attenuated vaccines. 
 
Description Applying AI and Machine learning to next generation sequencing datasets 
Organisation University of Surrey
Department School of Biosciences & Medicine
Country United Kingdom 
Sector Hospitals 
PI Contribution We are currently exploring novel ways to apply deep learning technologies to existing datasets.
Collaborator Contribution We are collaborating as part of a strategic partnership between the Pirbright Institute and the University of Surrey (UoS). UoS researchers will work to identify ways in which we are able to integrate machine learning to interpret signals in coronavirus evolution though noise in existing genomic experimental datasets.
Impact n/a
Start Year 2022
 
Description Applying language processing models and supervised learning of images for ML analysis of attenuated and pathogenic IBV 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaborators at University of Glasgow will use genomic information to better understand the use of language models and supervised learning techniques to analyse existing genomic datsets.
Collaborator Contribution This work is ongoing
Impact This collaboration is multi-disciplinary with software development, computational analysis, bioinformatics, bioimaging and molecular biology as the disciplines invovled.
Start Year 2023
 
Description Current and Future Challenges in Veterinary virus virology 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact A meeting was held at Pirbright both online and in person, to discuss the role of genomics in veterinary virology. A total of 241 registered including 220 delegates and 21 speakers
Year(s) Of Engagement Activity 2022
URL http://www.cfcvvg.org
 
Description Festival of Genomics 2023 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I participated as an expert on a panel session to discuss the new technology platforms that had become available both to science and on the market in 2022-2023 and their role in progressing the field of high throughput sequencing.
Year(s) Of Engagement Activity 2022,2023
URL http://www.festivalofgenomics.co.uk
 
Description Illumina EMIDA seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I presented the high throughput sequencing work at Pirbright as part of Illumina's EMIDA internal seminar series
Year(s) Of Engagement Activity 2022
 
Description Pirbright Celebration Day 2023 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Over 800 people were welcomed in this event over two days, who were inspired through interactive exhibits and hands-on experiences.
Year(s) Of Engagement Activity 2023
 
Description participant in a conference -Spatial Biology UK 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attended the Spatial Biology conference in London, had a discussion with 10X Genomic product development manager as well as their field scientists for clearing my concerns/questions regarding applying the technology to solving my research questions. My questions were clarified, and a connection with the filed scientist was built.
Year(s) Of Engagement Activity 2023
 
Description participation in Pirbright celebration day 2023 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Participated in bioinformatics and sequencing stall
Year(s) Of Engagement Activity 2023