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African horse sickness: Effector mechanisms of immunity and viral determinants of pathogenicity

Lead Research Organisation: THE PIRBRIGHT INSTITUTE
Department Name: UNLISTED

Abstract

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Technical Summary

Scope of the research programme and research objectives
The aim of this fellowship is to develop a comprehensive research programme in epitope-based vaccinology, immuno-therapeutics and pathogenesis of orbiviral diseases, focusing primarily on African horse sickness virus (AHSV), a highly lethal pathogen of equids that is exotic to most parts of the world except Africa [1]. Knowledge of the effector mechanisms of immunity against AHS is limited and scientific progress slow, due to the logistical, financial, bio-safety and ethical implications of working with a pathogen affecting a large (companion) animal species. I will focus on gaining further understanding of the mechanisms of virus neutralisation by antibodies and analysis of the viral determinants of AHSV pathogenicity. The starting point for these investigations is the recent successes of the research group I lead, involving: a) development of a mouse model for AHSV [2]; b) development of a protective MVA-VP2 vaccine (modified vaccinia Ankara expressing AHSV VP2, the outer capsid protein ) [2-4]; and c) demonstration of the humoral nature of MVA-VP2 induced immunity [5, 6].
The work will be divided in 3 interconnected work-packages:
1. Effector mechanisms of adaptive immunity: Virus Neutralising Antibodies (VNAb)
2. Development of passive immuno-therapeutics
3. Viral determinants of pathogenicity

Planned Impact

unavailable
 
Description Key findings include: a) Polyvalent AHS vaccines can be developed with MVA-VP2; b) Single dose of MVA-VP2 or VP2 sub-unit vaccines can protect against AHSV disease in a mouse model; c) Peptide microarray technologies are suitable for efficient accurate and fast epitope mapping of AHSV-VP2. Finding 'a' and 'b' above are already bringing changes to policy and vaccine manufacturing practices.
Exploitation Route Please see other sections. The findings of the award in part contributed to the changes in Vaccine manufacturing industry. Further research opportunities are being pursued together with vaccine companies, animals health organisations (OIE) and other research institutes.
Sectors Creative Economy

Education

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

Other
 
Description As explained in other sections, the demonstration that recombinant MVA-VP2 could be used as a polyvalent vaccine (see published article in Vaccine) in horses and that a single vaccination with either baculovirus expressed AHSV-VP2 or with MVA-VP2 could provide protection against lethal challenge had important consequences for AHS vaccine manufacturing industry. These two findings would enable developing an AHS polyvalent vaccine in the future. This is essential for any novel vaccine to be used in an endemic country. If a vaccine can be used in endemic countries then the market for AHS vaccine expands. One of the main reasons novel AHS vaccines have not been developed and commercialised yet was the very small market of AHS vaccines. This was influenced by the unknown suitability of the new vaccine technologies to meet the needs of endemic countries (i.e. protect against all serotypes of AHSV, present in Africa). The demonstration that novel technologies (Sub-unit or MVA vaccines based on VP2), which are inherently have a high bio-safety profile, could be used to develop polyvalent vaccines means that the new vaccines could be used in endemic and non-endemic settings. A vaccine that can be used in Africa routinely enables this vaccine to be sold in those countries and be available for use in non-endemic countries should an outbreak of AHS occurred. Furthermore, these novel technologies have DIVA (Differentiation of Infected from Vaccinated Animals) capacity and their systematic use in AHS free areas will therefore not compromise surveillance activities and disease freedom status, making this type of vaccines the ideal control measure for eradication campaigns and for providing protection of animals during transport. These new developments have been presented to policymakers, vaccine manufacturers, animal heath organisations who have combined efforts to develop funding schemes with which to bring the new vaccines to market. The impact of the research of this award was part of these changes described above. In summary, the science had an impact on Vaccine Industry (prepared to adapt their vaccine manufacturing strategies), animal health organisations and Equine Industry (prepared to co-fund vaccine development initiatives).
First Year Of Impact 2018
Sector Creative Economy,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Societal

Policy & public services
 
Description Consultation activities for OIE, DEFRA on matters related to the control of African horse sickness
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a guidance/advisory committee
 
Title Epitope Mapping micro-arrays 
Description This activity relates to the application of a technology developed by a German bio-tech company, named PepperPRINT, based on the usage of peptide epitope microarrays, to conduct epitope mapping experiments on the main AHSV neutralising antigen VP2. We analysed successfully the reactivity of a large panel of monoclonal antibodies directed and VP2 specific antisera using a peptide microarray comprised of 15-mer amino acids, overlapping by 13, encompassing the whole sequence of AHSV-VP2. T 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact The results demonstrated that epitope mapping studies can be conducted very fast and accurately therefore speeding up the process of antigen discovery and vaccine design. Results of these studies will be hopefully published soon. 
 
Title Development and use of an interferon receptor alpha gene knock out mice (IFNAR -/-) for studying the molecular determinants of African horse sickness pathogenicity 
Description The IFNAR -/- mouse model of AHSV has been used in several studies by my research grooup to assess the protective efficacy of novel AHSV vaccines and to elucidate the effector mechanisms of protection of vaccines and passive immuno-therapeutic approaches against AHSV. We have extended the application of this method to the study of host-pathogen interactions and the genetic basis of pathogenicity of AHSV. In particular, we aimed to determine whether AHSV strains of different genetic background differed in their pathogenicity in this mouse model. This work constituted the main objective of a PhD studentship: Understanding the basis for African horse sickness virus pathogenicity using a murine infection model - University of Surrey (DOI: https://doi.org/10.15126/thesis.900175). African horse sickness virus (AHSV) is a highly lethal arbovirus of horses, with severe economic impacts associated with the spread and control of the disease. Due to the difficulties of performing experimental studies with horses, this project exploited the IFNAR -/- mouse model to better understand AHSV pathogenesis the mechanisms that may underlie differences in virulence. Using AHSV from different genetic background we demonstrated that pathological differences between virus strains are linked to genetic differences, differences in growth kinetics, differences in tropism. Broadly, it was found that AHSV strains target the spleen of the IFNAR-/- mouse, resulting in necrosis; infection is also frequently associated with lung lesions, in the form of oedema and pneumonia. Strains with more severe clinical outcomes were largely associated with more severe lesions in these tissues, as well as lesions in other tissues not observed in less pathogenic strains, such as liver necrosis. Higher levels of viral antigen in the spleen were also weakly correlated with more severe clinical outcomes. In vitro studies and sequencing techniques suggest these differences may be due genome segments encoding proteins VP1, VP2 and NS4/NS4-II, with these genetic differences also underlying a difference in cellular tropism within the spleen. 
Type Of Material Data analysis technique 
Year Produced 2020 
Provided To Others? Yes  
Impact The demonstration that different AHSV genotypes can be discriminated in this in vivo model is the a first step forward to understanding AHSV pathogenesis. The financial, logistic and ethical constrainsts associated with the use of the natural host species for pathogenicity studies can be overcome by the judicious use of this mouse model. This would have a positive impact on 3R's principles as it will reduce the use of horses for pathology research, a species that is perceived as 'experimental' by the general public. Also, the cost of any studies would be significantly reduced since the maintainance of mice in BSL-3 containment facilities is far more feasible. this also would have a positive impact on the scientific outcomes since more experiments can be done and the statistical robustness of the studies would be improved. In the work described above two AHSV genes (VP1 and VP2) were found to be associated with pathogenicity differences in the mice, which is consistent with previous observations by other workers. In addition, NS4 and NS4II were also associated with these differnces. Further work is needed to gain further insight on the pathogenic mechanisms associaatede with these genes. 
 
Description African horse sickness VP7 crystals: Role in immuno-pathology 
Organisation University of Pretoria
Country South Africa 
Sector Academic/University 
PI Contribution Collaboration focused on a joint study aiming at charactrising the immuno-biology of African horsee sickness virus infection of Interferon Alpha Receptor Gene Knock-out mice. The AHSV viruses were generated by reverse genetics to encode soluble and insoluble forms of VP7.
Collaborator Contribution The study was conducted at the Faculty of Veterinary Science, Maisons-Alfort, Paris, France. South African collaborators provided the recombinant viruses. Study has finished, results obtained and currently being analysed and a manuscript is in preparation.
Impact The study mentionde above was finished and paper is in preparation
Start Year 2019
 
Description Oxford Expression Technologies / Oxford Brookes University 
Organisation Oxford Brookes University
Country United Kingdom 
Sector Academic/University 
PI Contribution Although this collaboration started before this award, the new collaborative work related to vaccine development started as a result of very recent findings (January 2008) resulting from experiments conducted during this award. Oxford Brookes University and Oxford Expression Technologies have been a research collaborator on AHS vaccine research since 2013. In 2014 a PhD studentship was funded between the Pirbright Institute and Oxford Brookes University on the application of baculovirus pseudo typing and baculovirus transduction for vaccine development. This work continued with the Fellowship award and the use of baculovirus pseudo typing was used to study antigenic structure of AHSV capsid protein VP2. In addition, further studies with this collaborators resulted in the use of affinity purified AHSV-VP2 expressed from Tni insect cells infected with recombinant baculovirus encoding AHSV-VP2 gene. The results are currently being collated and a research publication is in preparation describing a successful use of this VP2 protein as a sub-unit vaccine for AHSV. Recently, in January 2008, we conducted a vaccination experiment in CISA, Valdeolmos, Madrid, Spain, in a mouse model system whereby affinity purified baculovirus expressed AHSV-VP2 was used as a vaccine. The results showed that the immune responses and protection against AHSV challenge of mice vaccinated with VP2 protein were similar to those of mice vaccinated with the already-characterised MVA-VP2 vaccine. Moreover, protection was afforded with just one dose. My direct contributions to this project include the set up of the collaborative partnership with Oxford Expression Technologies, developing the PhD programme (resulting in Dr Mine Aksular obtaining her degree), designing the latest vaccination experiments with baculovirus expressed VP2 and antigenic characterisation of this protein. This work can lead to the generation of safe, effective DIVA (Differentiation of Infected from Vaccinated Animals) vaccines for African horse sickness. Research funding from Innovative UK is currently being pursued to achieve this objective.
Collaborator Contribution My partners, Oxford Expression Technologies, in the person of Prof Linda King, Professor Robert Possee and Dr Mine Aksular, are experts in baculovirus biology and the use of baculoviruses as an heterologous protein expression technology. They have provided the technical support for the projects described above and have the industrial capacity for translating fundamental research into viable commercial projects. Thus, they have produced the AHSV-VP2 proteins mentioned above and the recombinant pseudo typed baculoviruses (displaying AHSV-VP2 epitopes on the baculoviruses envelope). These products can serve as potential vaccines and, in the case of pseudotypes, tools with which to investigate the function of AHSV-VP2 subdomains in cell attachment and entry.
Impact Outcomes from this collaboration include: a) successful generation and use of pseudo typed baculovirions displaying antigenic domains of AHSV-VP2; b) Successful production uf purified, stable, AHSV-VP2 by baculovirus expression systems; c) The demonstration that the latter can induce a protective immune response in mice against virulent challenge after one vaccine dose. Paper in preparation.
Start Year 2017
 
Description Passive Immunotherapeutics for African horse sickness 
Organisation Animal Health Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution I forged this consortium in order to advance the knowledge on the potential application of passive immuno-therapeutics for combating African horse sickness in the target species. Having shown in the murine species, by my research work, that administration of hyper-immune AHS serum collected from vaccinated mice conferred full protection in immunologically recipient mice, I designed a project in order to determine whether passive immunisation of horses with AHS hyperimmune plasma could serve as potential treatment of this disease. A research grant application was submitted to the Horserace Betting Levy Board to fund this project. The outcome of this application will be known in the next few days.
Collaborator Contribution Each partner above has contributed to design the project and will contribute to its execution. The project is divided in 3 phases: a) production of hyperimmune plasma (Veterinay Immunogenics / Dr Javier Castillo-Olivares); b) Study of the Pharmaco-kynetics of the transferred plasma in recipient equids (Animal Health Trust); and c) Determination of clinical protection against AHS infection in horses immunised passively with AHS hyperimmune plasma (ANSES).
Impact A research grant application as described above.
Start Year 2016
 
Description Passive Immunotherapeutics for African horse sickness 
Organisation French Agency for Food, Environmental and Occupational Health & Safety (ANSES)
Country France 
Sector Public 
PI Contribution I forged this consortium in order to advance the knowledge on the potential application of passive immuno-therapeutics for combating African horse sickness in the target species. Having shown in the murine species, by my research work, that administration of hyper-immune AHS serum collected from vaccinated mice conferred full protection in immunologically recipient mice, I designed a project in order to determine whether passive immunisation of horses with AHS hyperimmune plasma could serve as potential treatment of this disease. A research grant application was submitted to the Horserace Betting Levy Board to fund this project. The outcome of this application will be known in the next few days.
Collaborator Contribution Each partner above has contributed to design the project and will contribute to its execution. The project is divided in 3 phases: a) production of hyperimmune plasma (Veterinay Immunogenics / Dr Javier Castillo-Olivares); b) Study of the Pharmaco-kynetics of the transferred plasma in recipient equids (Animal Health Trust); and c) Determination of clinical protection against AHS infection in horses immunised passively with AHS hyperimmune plasma (ANSES).
Impact A research grant application as described above.
Start Year 2016
 
Description Passive Immunotherapeutics for African horse sickness 
Organisation Veterinary Immunogenics Ltd
Country United Kingdom 
Sector Private 
PI Contribution I forged this consortium in order to advance the knowledge on the potential application of passive immuno-therapeutics for combating African horse sickness in the target species. Having shown in the murine species, by my research work, that administration of hyper-immune AHS serum collected from vaccinated mice conferred full protection in immunologically recipient mice, I designed a project in order to determine whether passive immunisation of horses with AHS hyperimmune plasma could serve as potential treatment of this disease. A research grant application was submitted to the Horserace Betting Levy Board to fund this project. The outcome of this application will be known in the next few days.
Collaborator Contribution Each partner above has contributed to design the project and will contribute to its execution. The project is divided in 3 phases: a) production of hyperimmune plasma (Veterinay Immunogenics / Dr Javier Castillo-Olivares); b) Study of the Pharmaco-kynetics of the transferred plasma in recipient equids (Animal Health Trust); and c) Determination of clinical protection against AHS infection in horses immunised passively with AHS hyperimmune plasma (ANSES).
Impact A research grant application as described above.
Start Year 2016
 
Description Equine Disease Coalition 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I have been giving presentation to the Equine Disease Coalition (a working group with representatives from DEFRA, Equine Veterinary Association, Animal Health Trust, RSPCA and World Horse Welfare, to advance on prevention of equine diseases in Britain) since 2016. My function has been, and currently is, providing expert advise on prevention strategies for African horse sickness and suitable policies for continuing a potential outbreak of this disease should it occur. An important part of this activity related to the award is to make the equine industry stakeholders and policy makers of latest research findings on pathogenesis, vaccine development and diagnostics made in this field that have relevance to control of African horse sickness.
Year(s) Of Engagement Activity 2016,2017
URL http://www.worldhorsewelfare.org/disease-coalition
 
Description University of Queensland 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact By exploration of novel vaccination strategies for AHS I established a collaborative research link with Professor Linda Lua, Department of Bio-Engineering at the University of Queensland, Australia, who have pioneered a novel vaccination platform using PolyomaVirus VP1 protein virus-like-particles and capsomeres to display relevant protective antigens from other pathogens. The group succeeded with experimental vaccines for influenza and we have conducted a pilot study, successfully, whereby a peptide epitope of AHSV-VP2 was inserted in VP1 capsomeres. This experimental vaccine induced strong AHSV-VP2-specific antibody responses. I am actively collaborating with this group to expand this novel technology to develop vaccines against other pathogens. i had the pleasure of hosting Professor Linda Lua to the Pirbright Institute where she gave a presentation about the use of Polyoma virus VP1 capsomeres and VLP vaccines.
Year(s) Of Engagement Activity 2016,2017,2018
 
Description World Organisation for Animal Health (OIE) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact I have been acting as OIE designated expert on African horse sickness since 2013. As part of my duties I have been updating the OIE on research advances made in the field of AHS vaccinology and diagnostics, including the developments made as a result of the award, but also including research results of other groups working in the same field. The impact of part of the work (as per publication listed in this report) has been significant because it opens the door for developing a polyvalent vaccine for African horse sickness. This is a key finding because opens the market for AHS vaccines and therefore will attract the support from vaccine manufacturers who will see more attractive investing in developing such a vaccine. It is important to note that the restricted market for AHS vaccines has been one of the main obstacles hampering the progress in improving the global control of this disease. In addit
Year(s) Of Engagement Activity 2016,2017,2018