Using synthetic biology to generate novel vaccines against avian infectious bronchitis virus (IBV)

Lead Research Organisation: The Pirbright Institute
Department Name: Coronaviruses

Abstract

Chickens are vaccinated multiple times against poultry pathogens throughout their lives to protect against different diseases. Infectious bronchitis virus (IBV) causes a respiratory disease in chickens making them more susceptible to bacterial infections. In addition to this, chickens infected with IBV gain less weight and produce fewer high quality eggs. It is important to vaccinate against IBV both for welfare and economic reasons. There are a number of problems with the vaccines available against IBV. There are many different strains of IBV circulating around the globe however currently available vaccines do not cross-protect against all strains. New vaccines are produced by growing a virulent strain of IBV in hen's eggs multiple times. This reduces the pathogenicity of the vaccine strain when it is given to chickens but it increases the pathogenicity of the vaccine strain for chicken embryos. Vaccines against IBV are administered by spraying poultry flocks with the vaccine or by putting the vaccine in the drinking water. It is hard to guarantee that each chicken has received the same dose, which make the vaccines less effective.
We have rationally attenuated three strains of IBV using molecular tools. These strains have not been adapted to grow in eggs so are less pathogenic for chicken embryos. This study proposes to establish whether the rationally attenuated strains of IBV could be used as vaccines, by testing the safety, stability and efficacy of all three potential vaccine strains. These novel vaccines have the potential to be given to chicks while they are still developing in the egg so that the chicks are protected against IBV infection even before they hatch. This would guarantee that each chick received the same dose of vaccine and reduce the possibility of vaccine breakdown, which would be very beneficial to the poultry industry. Safer and more efficacious vaccines against IBV will reduce the amount of antibiotics given to chickens to treat secondary bacterial infections resulting from IBV infection. The prolific use of antibiotics in farming may contribute to the development of antibiotic resistance in bacteria, a potentially devastating problem.
The second part of the study proposes to use molecular tools to insert the surface protein, the spike, from a different strain of IBV into one of the novel vaccine strains. The spike is the major structure on the outside of the virus that chickens develop antibodies against. It is highly variable between different strains of IBV so antibodies produced against one strain of IBV may not cross-protect against a new strain of IBV. The spike of one of the most economically damaging strains of IBV will be inserted into a vaccine strain. We will assess whether vaccinating with these recombinant viruses is able to protect against challenge with pathogenic virus containing the same spike or with a different spike. We will establish whether the two viruses containing different spikes are able to grow in competition with each other in eggs. This will investigate the potential to vaccinate eggs with both viruses; protecting against two or more important strains of IBV in a single dose.
The overall aim of this study is to use synthetic biology to generate a new generation of safer, more efficacious vaccines against the important poultry pathogen, infectious bronchitis virus.

Technical Summary

The overall aim of this project is to use synthetic biology to generate a new generation of safer, more efficacious vaccines against the important poultry pathogen, infectious bronchitis virus. We will establish whether recombinant infectious bronchitis viruses have the potential to be safe and efficacious vaccines and/or the have ability to provide cross-protection between different serotypes. We have previously developed a reverse genetics system based on a pathogenic strain of infectious bronchitis virus (IBV), M41, and have used this to generate three attenuated isogenic recombinant IBVs (rIBV). This study proposes to evaluate the vaccine potential of the three rIBV in poultry. Vaccine replication and pathology induced in the trachea, kidney and reproductive tract of infected chickens will be assessed, as will the spread of vaccine to sentinel chickens. Vaccination-challenge experiments will be conducted to monitor vaccine efficacy.
We propose to use reverse genetics to swap the spike glycoprotein of the M41 strain of IBV for that of another economically important strain and will characterise the chimaeric virus in vitro, assessing the serotype and cellular tropism. To investigate how the recombinant viruses replicate in competition with each other in eggs we will use next generation sequencing to assess recombination events between the spike genes and determine the feasibility of vaccinating with both viruses in ovo. In vivo vaccination-challenge experiments will be conducted to assess whether vaccination with rIBV expressing spikes from two different serotypes together induces homologous protection and heterologous cross-protection.
The stability of all of the attenuated rIBV isolates will be assessed in ex vivo tracheal organ cultures and maternally derived antibody (MDA) positive eggs using next generation sequencing to model vaccine persistence and evolution of the viral population in poultry flocks under immune pressure.

Planned Impact

Poultry is an important food source worldwide and the global poultry industry has expanded in recent years to accommodate increasing demands for healthy, sustainable protein sources from a rising population. The FAO estimates that poultry accounts for 88% of global meat production. Viral diseases such as infectious bronchitis are a constant threat to the poultry industry through reduced meat production from broiler chickens, production of fewer, good quality eggs and animal welfare issues. In fact, infectious bronchitis is the single most economically important infectious disease affecting chickens in the UK. Improved vaccines against IBV would benefit the UK economy as an estimated £23M is spent on control of IBV per annum.
Vaccine breakdown would have a major effect on the UK poultry industry, not only in terms of bird welfare and production costs, but also associated risks to food security. This work will establish whether our novel approach to vaccine design through rational attenuation and modification of vaccine serotype is capable of producing safe and efficacious vaccines for the control of IBV that are less likely to revert to virulence. The development of safer vaccines will reduce the amount of antibiotics used to treat secondary bacterial infections associated with infectious bronchitis, which would have positive environmental impact.
The results of this study will inform approaches to development of other veterinary and human vaccines. This study will benefit The Pirbright Institute, BBSRC and Zoetis, and the wider academic community including other researchers working in the fields of molecular virology, livestock health and coronavirus research, particularly IBV. The project will provide training in virology, deep sequencing, confocal microscopy and in vivo studies. The results will be of direct benefit to the poultry industry and vaccine developers. Knowledge generated by this project will be widely disseminated to the research community thrugh peer-reviewed journals and presentations at national and international virology conferences and interactions with members of the poultry industry and veterinarians.

Publications

10 25 50
 
Description We have generated a new recombinant infectious bronchitis virus (rIBV) expressing the spike glycoprotein of a heterologous strain in the genetic background of an attenuated strain. We have assessed its tropism in vitro and are investigating whether the two candidate vaccines can be grown in combination with each other, or whether recombination occurs between the spike glycoproteins.

In attempting to elucidate the mechanism of attenuating mutations in the replicase of IBV, we have established that mutations in rIBV replicase proteins do not affect the proof-reading activity. We have established that the replicase mutations are stable during serial passage of the rIBVs in ex vivo tracheal organs cultures or primary chicken kidney cell cultures but some mutations cause a difference in plaque phenotype in cell culture.
Exploitation Route Our findings are currently academic in nature, and will increase understanding of the function of the coronavirus replicase proteins. This knowledge may be used in the rational design of vaccines against different coronaviruses in the future. Attenuated rIBVs produced may be licensed for use as live attenuated vaccines against IBV if further tests prove successful.
Sectors Agriculture, Food and Drink

 
Description Contribution to a POSTnote on 'Reducing UK Antibiotic Use in Animals'
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Training MSc students
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact Delivered a new training session to MSc students teaching viral quantification assays. This lead to a greater understanding of laboratory techniques and academic research.
 
Description Taiwan Partnering Award
Amount £40,700 (GBP)
Funding ID BB/S020624/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
Title Optimisation of preparation of TOCs from adult chickens 
Description Tracheal organ cultures (TOCs) are prepared from chicken embryos and used to assess growth of avian viruses that are not able to be propagated in cell culture. We have optimised the method of TOCs preparation from 3-week old chickens and are using these to assess viral genetic stability and phenotype in organ cultures that are a similar environment to the site of replication in whole chickens. 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? No  
Impact We hope that this technique will reduce the number of chickens required for in vivo experiments, in line with the 3Rs, and plan to publish the method in due course. 
 
Description Identification of conserved B-cell epitopes of highly pathogenic coronaviruses for broadspectrum immunotherapy and vaccine design. 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have performed experiments to generate preliminary data for a collaborative grant application, contributed to the writing of the grant proposal and responded to reviewers comments.
Collaborator Contribution My collaborators have performed experiments to generate preliminary data for a collaborative grant application, contributed to the writing of the grant proposal and responded to reviewers comments.
Impact Submission of proposal entitled "Identification of conserved B-cell epitopes of highly pathogenic coronaviruses for broadspectrum immunotherapy and vaccine design" to the "One Health Approaches to Accelerate Vaccine Development" call that forms part of the UK government's commitment to Official Development Assistance (ODA) in October 2017.
Start Year 2017
 
Description Identification of conserved B-cell epitopes of highly pathogenic coronaviruses for broadspectrum immunotherapy and vaccine design. 
Organisation University of Kent
Country United Kingdom 
Sector Academic/University 
PI Contribution We have performed experiments to generate preliminary data for a collaborative grant application, contributed to the writing of the grant proposal and responded to reviewers comments.
Collaborator Contribution My collaborators have performed experiments to generate preliminary data for a collaborative grant application, contributed to the writing of the grant proposal and responded to reviewers comments.
Impact Submission of proposal entitled "Identification of conserved B-cell epitopes of highly pathogenic coronaviruses for broadspectrum immunotherapy and vaccine design" to the "One Health Approaches to Accelerate Vaccine Development" call that forms part of the UK government's commitment to Official Development Assistance (ODA) in October 2017.
Start Year 2017
 
Description Partnering with National Taiwan University 
Organisation National Taiwan University
Country Taiwan, Province of China 
Sector Academic/University 
PI Contribution We have prepared and submitted an application for a BBSRC-Taiwan Partnering Award to study "Assembly of Recombinant Infectious Bronchitis Virus and Determination of Antigenic Sites that Confer Hemagglutination Activity".
Collaborator Contribution The partners conceived the project and got in contact about submitting an application for funding together.
Impact An application has been made for a BBSRC-Taiwan Partnering Award in November 2018.
Start Year 2018
 
Description Centre of excellence for research on avian diseases (CERAD) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I was invited to present my work at the UK-China Centre of excellence for research on avian diseases (CERAD) meeting attended by researchers from UK, China and Thailand. There was lots of discussion about the research and future directions, including potential collaborations.
Year(s) Of Engagement Activity 2017
 
Description International Day of Women and Girls in Science 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I gave a quote as a STEM ambassador about why I think it is important to make sure women and girls have the same opportunities in STEM. This was shared on social media to celebrate International Day of Women and Girls in Science, a UN-led initiative.
Year(s) Of Engagement Activity 2019
 
Description International Women's Day video for social media 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Three team members were interviewed about their careers in science for International Women's Day. The videos were posted on Pirbright's website, Facebook and Twitter to celebrate what we love about working in science.
Year(s) Of Engagement Activity 2018
 
Description MSc student visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact I hosted several MSc students from University of Surrey, discussed avian research with them and demonstrated some laboratory techniques. The students gained an understanding of academic research and laboratory work.
Year(s) Of Engagement Activity 2018,2019
 
Description Microbiology Society Annual conference (UK) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Poster or oral presentation at Microbiology Society annual conference, a major international event. The participants included researchers from many different areas of science. This provides a platform to promote research to a wider audience as well as discuss the key findings with experts in my own field.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014
 
Description Microbiology Society Avian Focus Meeting (UK) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I gave a 15 minute oral presentation to an audience of around 80 people in 2016 and 2018. The audience consisted of researchers from many different scientific institutions from different areas of avian research. This was a great opportunity to present to experts in this field and gain novel insights into the project and the possible applications of my work.
Year(s) Of Engagement Activity 2016,2018
 
Description Nidovirus symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I presented my work at the Nidovirus Symposium, which is held every three years and brings together researchers from industry and academia from many different countries. I had some interesting discussions with other researchers and built my network.
Year(s) Of Engagement Activity 2017
 
Description Presentation at Global Alliance for Research on Avian Diseases Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presentation of my research at the Global Alliance for Research on Avian Diseases Conference in Vietnam to approximately 80 conference participants resulted in discussions about future research directions.
Year(s) Of Engagement Activity 2018
 
Description Presentation at RIVR meetings 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Presentation of my research at Recently Independent Virology Researchers (RIVR) meetings in 2016, 2017, 2018 and 2019. I discussed my work and future collaborations with other virology researchers working in the UK.
Year(s) Of Engagement Activity 2016,2017,2018,2019
 
Description Presentation to BBSRC Executive Board 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact I presented our research to the BBSRC Executive Board when they visited The Pirbright Institute in September 2018 and discussed the impacts of our work..
Year(s) Of Engagement Activity 2018
 
Description Reverse genetics seminar (University of Surrey) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact A seminar was given based upon the reverse genetics system of infectious bronchitis virus. The audience was a group of 10 -15 MSc students from University of Surrey. The students were engaged, asked questions about the research and careers in science. We were asked to repeat the seminar for the next year's intake of students.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017
 
Description Surrey County Show 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The Surrey County Show is an agricultural show in Guildford that is visited by the general public. The Pirbright Institute had a stand with various activities for visitors to take part in and explain our science to a wide audience. Giulia spent a day working on the stand and spoke with many children and adults about her work at Pirbright to change public attitudes and stimulate increased interest in research.
Year(s) Of Engagement Activity 2018
 
Description Teentech 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact The annual Teentech student science event in Guildford aims to encourage teenagers to consider science subjects and science careers. Our stand had considerable interest from the schoolchildren who were very enthusiastic. The event stimulated an increased interest in science and research.
Year(s) Of Engagement Activity 2016,2019
 
Description organised workshops at Microbiology Society annual conferences 2014 - 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I co-organised virology division workshops on viral evolution and diversity and RNA viruses with colleagues from the virology division. The workshops were held at the annual Microbiology Society conferences in spring each year. Organisation involved selecting abstracts for oral or poster presentation, ordering the presentations and chairing the sessions.
Year(s) Of Engagement Activity 2015,2016,2017,2018