A viral pseudotype based approach to measuring morbillivirus neutralising antibodies

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

The goal of this project is to develop a diagnostic test that is rapid, sensitive and specific. This test will be used to diagnose a viral infection of sheep and goats called PPR, or peste des petits ruminants. PPR is a threat to the livestock industry worldwide and has been declared a priority target for global control and eradication by vaccination. The success of a global control and vaccination programme against peste des petits ruminants virus (PPRV) will require the availability of a highly accurate and rapid diagnostic test that can be performed without the need for expensive specialized laboratory containment facilities since it will have to be carried out in laboratories across the world. However, the current prescribed test for PPRV is the live virus neutralisation test, a test that is slow, requires a laboratory operating under a high level of biocontainment, and is limited in both sensitivity and flexibility.

In order to develop an improved PPRV diagnostic test, we have used a technique in which PPRV surface glycoproteins are expressed on a harmless carrier virus derived from vesicular stomatitis virus (VSV) to generate VSV(PPRV) "pseudotypes". A viral pseudotype consists of the structural proteins and genetic material of one virus (in this case VSV), carrying the surface proteins of another, different virus (in this assay we use the PPRV H and F proteins). The VSV(PPRV) pseudotypes can be used to measure PPRV specific antibodies more rapidly than conventional live virus tests (three days compared with 1 to 2 weeks). The assay displays greater sensitivity (at least 10-times more sensitive) than live virus tests and can measure antibody responses against diverse strains of virus representative of distinct viral lineages in circulation. Finally, the assay circumvents the requirement for high level biocontainment and may be performed safely in any laboratory worldwide with basic cell culture facilities.

In this project we will assist the World Animal Health Organization's Global Control and Eradication program for PPRV by generating a VSV(PPRV)-based diagnostic test. With this test, we will be able to identify infected animals and investigate populations of wild animals which may be responsible for transmitting this virus to livestock. This information will help ensure that vaccination programmes target susceptible populations in areas with a high risk of transmission and therefore improve the chances of successfully eradicating PPRV.

Technical Summary

The eradication of rinderpest virus by vaccination is a global success story. Rinderpest eradication relied upon the combination of effective vaccines and reliable diagnostic tests for the identification of infected animals and routine monitoring of vaccine-induced immune responses. The OIE has declared peste des petits ruminants virus (PPRV) a priority target for global control and eradication. If PPRV control and eradication are to succeed, vaccination should be supported by robust diagnostic tests that are sensitive and rapid, accessible to laboratories worldwide, and which can measure humoral immune responses in diverse species and against the distinct viral lineages in circulation.

The current prescribed test for PPRV is the live virus neutralisation test, a test that is slow, requires BSL-3 biocontainment, and is limited in both sensitivity and flexibility. We have developed a technique in which the PPRV surface glycoproteins are expressed on a replication-defective variant of vesicular stomatitis virus (VSV) to generate VSV(PPRV) pseudotypes. The pseudotype-based test is more rapid and more sensitive than the live virus test and can measure antibody responses against diverse strains of virus representative of the distinct viral lineages in circulation.

In this project, we will optimise and validate the PPRV pseudotype-based test, investigating its utility in the qualitative and quantitative measurement of post-vaccination immune responses and its ability to detect antibodies in exposed animal populations. The modular design of the assay will facilitate the mapping of antigenic epitopes on the PPRV surface glycoproteins, facilitating the rational design of the next generation of broadly acting morbilliviral vaccines. Further, as the test uses freely available reagents and requires only BSL-2 containment, this novel approach to diagnosis will make "gold standard" diagnostic testing for morbillivirus-specific antibodies accessible worldwide.

Planned Impact

The outputs of this project will benefit researchers developing PPRV vaccines, livestock-keeping families in PPR-affected areas, the livestock sector in African and Asia, policy-makers concerned with livestock trade and disease control in both PPR-affected areas and disease-free countries, including Europe, and wildlife/conservation agencies. A primary impact of this study will be the global eradication of PPR by vaccination. Our novel test is sensitive and specific, it is rapid, adaptable to diverse strains of virus and scalable for high-throughput screening. The high sensitivity of the test will greatly enhance pre- and post-vaccination sero-surveillance, demonstrating the disease-free status of livestock and the absence of infection in wildlife reservoirs. Our established links with researchers in vaccine development and experts in wildlife conservation will ensure that all our outputs are validated. Our collaborating partners serve on the OIE ad hoc committee for PPR and will advise on test validation to OIE standards. Further, the Pirbright Laboratory is an OIE Reference Laboratory and will perform live virus neutralisation tests in parallel to assist with validation. Once validated, the novel test may then be included in the OIE Terrestrial Manual, a manual that is freely available on-line.

Researchers developing PPRV vaccines will benefit directly from this project. While existing PPRV vaccines induce an active life-long immunity, researchers are developing novel vaccines that circumvent the potential risks associated with live attenuated virus (e.g. excretion and reversion to virulence). Defining the humoral response to PPRV will facilitate the rational design of PPRV vaccines. Our system will enable mapping of antigenic epitopes on the viral surface glycoproteins. Our collaborating partners are active in experimental vaccine development and the use of PPRV vaccines in the field and will help refine the technique to ensure that maximum benefit is achieved.

The project will improve serological diagnosis in the developing world, critical to building capacity for research into emerging morbilliviruses and facilitating research into many other livestock and zoonotic diseases of global concern. Our technologies will enhance accessibility to "gold-standard" diagnostics. The OIE prescribed PPRV test for international trade requires BSL-3 containment and thus is restricted to relatively few institutions globally. Our test may be performed at BSL2. Through our collaborative links in Tanzania (NM-AIST) we have demonstrated that low-cost diagnostic testing can be exported successfully to Africa. Further, we have established links with GALVmed (Global Alliance in Veterinary Medicine), a not for profit organisation for whom PPRV is a primary target for control through improved diagnosis and vaccination.

The pseudotype technique will have a direct impact upon wildlife conservation. PPR is an acute contagious disease and infects not only livestock, but camels, buffalo, gazelles, ibex, gemsbok and even lions. The role of wildlife species in the maintenance of PPRV reservoirs and in viral circulation per se is unclear. Our test facilitates rapid, large scale serosurveys of PPR antibodies in any species. Critically, parallel analyses may be performed simultaneously between PPRV and related morbilliviruses, differentiating PPR-specific responses from co-infections. We have established links with the Wildlife Conservation Society and the International Zoo Veterinary Group to conduct serosurveys in wildlife species. Through our veterinary diagnostic laboratory, we routinely provide diagnostic testing for a range of viral diseases. Accordingly, we have considerable local expertise in test development, validation and performance.
 
Description To date, we have demonstrated that it is possible to generate "viral pseudotypes" using particles of vesicular stomatitis virus (VSV) that carry surface glycoproteins proteins from another virus (a morbillivirus). These "pseudotypes" carry the H and F glycoproteins from a range of morbilliviruses (the family that includes the measles virus and the canine distemper virus) and that these may be used to measure virus neutralising antibodies with high sensitivity. Morbillivirus neutralising antibodies are traditionally measured using either plaque reduction neutralisation tests (PRNTs) or live virus microneutralisation tests (micro-NTs). While both of these test formats provide a reliable assessment of the strength and specificity of the antibody response, they are restricted by the limited number of viral strains that can be studied and often present significant biological safety concerns to the operator. In this project, we have demonstrated the validity of a VSV-based pseudotyping system for the measurement of morbillivirus neutralising antibodies safely in a low biocontainment level laboratory. Using this system, we have demonstrated that strain-specific neutralising responses can be measured, important if we are to measure the efficacy of vaccine approaches to preventing morbilliviral infections. We have demonstrated cross-neutralisation between canine distemper virus (CDV) and peste des petits ruminants virus (PPRV), an important consideration in the design of vaccines that will prevent morbilliviral zoonoses (transmissions between species). We have revealed that vaccinated dogs in the UK differ markedly in the breadth of cross-neutralising antibody responses induced by vaccination, some dogs display potent cross-neutralisation of PPRV while others have negligible cross-neutralising responses. We have used the assay to detect rinderpest virus (RPV) cross-neutralising responses in PPRV-vaccinated cattle, demonstrating that antibody responses induced by PPRV live attenuated vaccines are insufficient to protect against RPV. We have used the assay to detect antibody responses in African cattle against not only PPRV, but also CDV and RPV, suggesting that PPRV infection is more widespread in livestock than originally assumed and that there is continued circulation of a morbillivirus in cattle that is antigenically related to RPV. Together, these studies will inform the way researchers approach the serological diagnosis of morbillivirus infections in the field, the design and conduct of future vaccination experiments, and the development of novel vaccines aimed at preventing the zoonotic spread of emerging morbilliviruses.
Exploitation Route The assay we have developed has been used to study vaccine efficacy in collaboration with the Pirbright Institute, assessing the efficacy of peste des petits ruminants (PPRV) vaccines in protecting cattle from infection with rinderpest virus (RPV), an important consideration if stock of RPV vaccines are to be destroyed. These findings have been published in peer-reviewed journals and the assay system if freely available for research groups to adopt. We are now members of the working group assembled to update the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals document, Section 2.2 - Biotechnology -Chapter 2.2.1. Biotechnology in the diagnosis of infectious diseases. We will incorporate an update on pseudotype assays in the guidance document.
We have trained a colleague from Thailand to use the system, he has then exported the assay system to a laboratory in Indonesia to study neutralising antibodies in tigers against CDV, demonstrating the adaptability of the technique to studies in developing countries.
The system is currently being used to study serological responses in Tanzanian livestock, in future will be used to screen sera in PPRV transmission studies in Ethiopia. The assay will be used in the near future to study exposure to PPRV in Mongolian saiga, gazelle and goats. The system has been used to underpin a new study (DOH/BBSRC) on the development of novel approaches to the development of vaccines that will induce cross-neutralising antibodies following morbilliviral vaccination.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://soundcloud.com/user-28288327/brian-willett
 
Description One of our collaborating partners adopted the use of our technique to analyse neutralising antibodies in a rinderpest vaccine trial. Previously, the detection of these antibodies was not possible due to low sensitivity of existing techniques. The sensitivity of the technique we developed facilitated the detection of antibodies in animals in which would have otherwise been classified as "antibody negative". In the absence of detectable antibodies, any evidence of immunity post-vaccination would have otherwise been attributed to cellular immunity. While cellular immunity may well be an important component of the post-vaccinal response, our data would indicate that neutralising antibodies are also present, albeit at a low level. Hence, post-vaccination immunity may reflect an anamnestic humoral response.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Societal,Economic

 
Description European Medicines Agency Ad Hoc Advisory Group
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
Impact The advisory committee was tasked with assessing the risks associated with the presence of contaminating retroviruses in feline and canine vaccines. The risks were assessed and guidelines for the Animal Healthcare Industry, the European Medicines Agency, veterinary practitioners and the public were drafted and published. The committee engaged in productive dialogue with representatives of the Animal Healthcare Industry, ensuring that vaccine products designed for use in cats and dogs met the highest possible standards of safety.
URL https://www.ema.europa.eu/documents/regulatory-procedural-guideline/cvmp-risk-management-strategy-ma...
 
Description Updating OIE Chapter 2.2.1. Biotechnology in the diagnosis of infectious diseases
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Responsive mode
Amount £485,660 (GBP)
Funding ID BB/R004250/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Title Development of viral pseudotypes bearing morbilliviral glycoproteins 
Description We are developing and optimising assay systems with which morbilliviral glycoproteins H (hemagglutinin) and F (fusion) can be expressed on the surface of vesicular stomatitis virus (VSV) particles. The particles carry a luciferase marker gene and hence may be used to measure virus neutralising antibodies with high sensitivity and under reduced (CL2) containment. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact The techniques have been used to investigate the seroprevalence of morbilliviruses in livestock in Tanzania, revealing widespread infection of cattle, sheep and goats with diverse morbilliviruses. The techniques have also been used to detect low levels of neutralising antibodies in peste des petits ruminants virus (PPRV)-vaccinated cattle prior to challenge with rinderpest virus (RPV), and to measure cross-neutralisation of measles virus (MeV) by sera from goats exposed to PPRV. We have exported the technique to other laboratories in London and Thailand. 
 
Description Conducting field studies in Tanzania 
Organisation Scotland's Rural College
Country United Kingdom 
Sector Academic/University 
PI Contribution Dr Harriet Auty, EPIC, SRUC Inverness, is our collaborating partner for field studies in Tanzania. In collaboration with Dr Auty in Inverness, and Dr Tiziana Limbo at the University of Glasgow, we are conducting longitudinal studies on morbillivirus infections of livestock, including, planning, logistics, engagement with livestock keepers and veterinary officers, livestock sampling, sample processing in Tanzania and shipping to the UK for subsequent analyses.
Collaborator Contribution Working as a collaborative team, we are involved in all aspects of planning, logistics, sampling, sample processing in Tanzania and shipping to the UK for subsequent analyses.
Impact This is an ongoing collaboration, we are currently collecting and processing samples from initial field studies.
Start Year 2016
 
Description Provision of sera and RNAs for analysis of PPRV lineage-specific neutralisation 
Organisation FAO/IAEA Agriculture and Biotechnology Laboratories
Country Austria 
Sector Private 
PI Contribution We have conducted assays for neutralising antibodies using sera from PPRV vaccinated and infected animals. We have cloned and sequenced H and F genes from isolates of PRPV from distinct lineages.
Collaborator Contribution Researchers in the IAEA laboratories have provided sera and RNAs to facilitate our studies on lineage specific neutralisation of PPRV.
Impact Manuscript describing lineage specific neutralisation in preparation.
Start Year 2013
 
Description Screening of ruminant sera from Tanzania for morbillivirus-specific neutralising antibodies 
Organisation University of Glasgow
Department Institute of Biodiversity, Animal Health and Comparative Medicine
Country United Kingdom 
Sector Multiple 
PI Contribution We have screened sera from diverse wildlife species and domesticated livestock for morbillivirus-specific antibodies.
Collaborator Contribution Our colleagues in IBAHCM (led by Prof. Sarah Cleaveland) provide access to sera from historical and ongoing studies on a range of pathogens for morbillivirus antibody screening. They have advised on economic and cultural factors influencing the likelihood of exposure to pathogens in pastoralist communities in Tanzania. The collaboration has extended to include Dr Harriet Auty (Scotland's Rural College); Dr. Furaha Mramba, Tanzania Veterinary Laboratory; Prof. Rudovick Kazwala, Sokoine University of Agriculture; Dr. Julius Keyyu and Dr. Robert Fyumagwa, Tanzania Wildlife Research Institute. Dr. Harriet Auty is a veterinary epidemiologist investigating the dynamics, impacts and control of vector-borne diseases and zoonotic diseases of livestock. She is a research fellow with EPIC - the Scottish Government's Centre of Expertise on Animal Disease Outbreaks. Dr. Auty will lead field studies in the coming years. Prof. Sarah Cleaveland is an internationally renowned expert on rabies and zoonotic diseases. Elected a fellow of the Royal Society in 2016, she has worked extensively on the control of neglected tropical diseases in Africa, building and sustaining partnerships with African institutions that will create positive changes in the lives of disadvantaged people. Dr. Furaha Mramba, head of the Tanzania Veterinary Laboratory Agency, liaises with the Ministry of Agriculture, Livestock and Fisheries in applying results to the development of control strategies in Tanzania, and supports coordination of field work in Tanzania.
Impact This collaboration provided some of the original data that informed the development of the the pseudotype based neutralisation assay. Shared master's students have been supervised and a PhD student has been appointed under a separate funding programme. Data generated through this collaboration underpinned the successful application for follow-on funding from the BBSRC and a new sample set collected in 2016 is integral to the first year of the new study.
Start Year 2013
 
Description Screening of sera from PPRV and RPV exposed/vaccinated ruminants for neutralising antibodies 
Organisation The Pirbright Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Our laboratory conducts the screening of sera for neutralising antibodies against morbilliviruses. We have been able to generate data for their recent vaccine studies using our enhanced sensitivity assay. The data from this collaboration are due to be published in Journal of Virology in the near future.
Collaborator Contribution The Pirbirght Institute have provided serum samples for analysis, diagnostic standards and valuable advice on assay development.
Impact Manuscript submitted and in review: "Protection of cattle against rinderpest by vaccination with wild type but not attenuated strains of peste des petits ruminants virus". Dr. Michael D. Baron, Barbara Holzer , Sophia Hodgson (Pirbright Institute) & Nicola Logan, Brian J. Willett (University of Glasgow).
Start Year 2014
 
Description Screening of wildlife sera for antibodies against canine distemper virus (CDV) 
Organisation Wildlife Conservation Society
Country United States 
Sector Charity/Non Profit 
PI Contribution We have conducted screening of wildlife sera in Russia for exposure to CDV, assessing the potential threat of zoonotic spread to susceptible hosts (such as endangered tigers). We have prepared viral pseudotypes using genes derived from field strains of CDV that are circulating in Russia, enabling us to measure neutralising antibodies against biologically relevant strains of virus.
Collaborator Contribution Our partners in the WCS have provided serum samples for serological testing and host nucleic acid samples for amplification of viral sequences to generate viral pseudotypes bearing envelope glycoproteins from circulating visual strains in Russia.
Impact We have validated out pseudo typing strategy using sera provided by WCS researchers, generating antibody titre data in return. We have generated novel viral sequences for phylogenetic studies, complementing similar studies conducted by the WCS.
Start Year 2014
 
Title Viral pseudotype based test for morbillivirus neutralising antibodies 
Description We are developing an enhanced method for the detection of specific virus neutralising antibodies. We are now validating this assay using field samples and experimental samples from vaccine trials conducted by our collaborating partner at The Pirbright Institute. The assay has been used to detect antibodies that were present at levels below the threshold for detection by existing techniques. 
Type Diagnostic Tool - Non-Imaging
Current Stage Of Development Refinement. Non-clinical
Year Development Stage Completed 2016
Development Status Under active development/distribution
Impact The ability to distinguish specific serological responses is enabling us to screen samples retrospectively for past exposures to diverse morbilliviruses. 
 
Description CVR Contagious Thinking Podcast 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Recorded an interview with CVR postgraduate students on morbilliviral pseudotypes for the Contagious Thinking podcast. Discussed the ruminant morbilliviruses, comparing PPRV with rinderpest and measles.
Year(s) Of Engagement Activity 2017
URL https://soundcloud.com/user-28288327/brian-willett
 
Description Laboratory techniques training session 
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 Visit to the Nelson Mandela African Institute of Science and Technology in Tanzania to host laboratory workshop during which local scientists learned ELISA techniques used to measure Rift Valley Fever Virus antibodies in sera.
Year(s) Of Engagement Activity 2013
 
Description Postgraduate lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Delivery of postgraduate teaching on taught component of masters degree and laboratory projects on morbilliviruses.
Year(s) Of Engagement Activity 2015,2016
 
Description Social media acitvities 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Whenever significant progress is made in the research project, our findings are publicised through CVR and Pirbright social media outlets, and through our laboratory twitter account @Animal_Viruses. We direct followers to the source publication, to News articles covering the publication, and to comment articles. For example, our recent publication in Journal of Virology was discussed on the TWIV podcast (This Week In Virology).
Year(s) Of Engagement Activity 2018,2019
 
Description Undergraduate lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact The provision of research-led undergraduate lectures on morbilliviruses.
Year(s) Of Engagement Activity 2015,2016