Impact of T lymphocytes on clinical disease, immune responses and transmission of bluetongue virus in sheep

Viruses transmitted by Culicoides biting midges cause economically important diseases in ruminants worldwide. Currently, the most important of these is bluetongue virus (BTV), which has inflicted over 1000m Euros of damage in North-West Europe alone during the past decade. This virus represents an ongoing threat to UK ruminant production, highlighted by re-emergence of a serotype 8 strain in France during 2015. Infection with BTV often causes severe disease in sheep and although clinical disease is milder in cattle, adverse effects on production parameters are economically damaging. Infected animals which recover are protected from BTV re-infection, but only by strains of the same serotype. Similarly, commercial vaccines are available but only protect against re-infection by the same serotype. To date, at least 27 serotypes of BTV have been detected and the global distribution of these constantly changes. Although vaccination successfully led to the eradication of a virulent BTV-8 strain from the UK and Northern Europe in 2008, the multiple serotype nature of BTV presents a major problem to control of the virus.

Designing new vaccines that provide cross-protection against multiple BTV serotypes requires a detailed understanding of the immune response mechanisms that result in virus clearance from infected animals. However, our knowledge of these anti-BTV immune responses remains superficial. In this project we will explore the role of a key cell type (T lymphocytes also called T cells) that is central to the anti-viral immune response, either by enabling other cells to produce anti-viral antibodies, or by directly eliminating virus infected cells. T cells occur in different subsets, but so far the specific contribution of subsets in the immune response to BTV is unknown. Additionally, it is known that T cell subsets can become infected with BTV themselves. However , it is unclear how much infection of these T cell subsets contributes to the high amounts of virus detected in BTV infected animals. A key observation we have shown is that blood-feeding of Culicoides leads to the recruitment of high numbers of T cells (among other cells) to the biting sites. Hence, T cells also have the potential to influence virus transmission from and to the insect vector.

It is challenging to realistically examine complex interactions between natural mammalian hosts, insect vectors and viruses under controlled experimental conditions and most experimental studies are compromised by either needle-inoculating the virus into the host and/or the use of model host species such as rodents. In this project we will study the role of T cells directly in sheep, a natural host for BTV and carry out transmission of BTV using infected biting midges. By inoculating sheep with antibodies to remove specific T cell subsets we can conduct studies revealing the importance of each subset in the immune response to BTV. Key areas we will explore include whether more or less BTV is produced and whether clinical disease differs according to the absence of a specific T cell subset. We will also measure the production of anti-BTV antibodies and anti-viral cytokines, alongside the potential for the remaining T cells to recognise BTV components. In addition, uninfected Culicoides will be allowed to blood-feed on BTV infected sheep enabling virus transmission to be examined. Cell populations and skin inflammation in response to blood-feeding of these biting midges will also be compared between treatment and control groups.

This comprehensive BTV sheep infection study will provide much needed data on the specific role of T cell subsets on BTV production, anti-BTV immune responses, clinical consequences and virus transmission. While the results obtained will be particularly important to advance BTV treatment and control, our use of a natural system of transmission is likely to yield an enhanced understanding of this process that is relevant to other arboviruses.

Bluetongue virus (BTV) is an economically important arbovirus of ruminants, transmitted biologically between hosts by Culicoides biting midges. Expansions of BTV distribution into northern Europe continue to threaten the UK, the most recent example being the detection of a serotype 8 strain in France during 2015. Our limited knowledge concerning ruminant immune responses, specifically the role of T lymphocytes, during BTV infection impedes scientific progress towards understanding mechanisms of cross-serotype protective immunity, pathogenesis and viral transmission which would advance BTV control. The aim of our study is therefore to elucidate the role of T lymphocytes in disease manifestation, viral clearance and anti-viral responses and virus transmission from and to Culicoides in BTV infected sheep.

We will dissect the specific roles of CD4+, CD8+ and WC1+ gd T cells in sheep by depleting these in-vivo through intravenous inoculation of monoclonal antibodies. Successful depletion of T cell subsets will be confirmed by monitoring blood leukocyte populations throughout the study using flow cytometry. Blood-feeding of infected Culicoides on sheep will then be used to transmit BTV naturally to these hosts. Following infection, viral replication, disease manifestation and induction of serum antibodies and cytokines will be analysed using established protocols and compared between T cell depleted and control groups of sheep. T lymphocyte responses to different heterologous and homologous BTV antigens will be studied ex vivo using flow cytometry assays of cell proliferation and intracellular cytokine production. Uninfected Culicoides will also be blood-fed on BTV infected sheep to identify if the absence of specific T cell subsets affects skin inflammation and virus transmission to vectors. Results of this study will significantly advance research on pathogenesis, viral clearance, transmission and vaccinology for BTV and other arboviruses.

The immediate benefits of this project will be increased knowledge of bluetongue virus (BTV) pathogenesis, anti-viral clearance mechanisms and transmission. Study outcomes will substantially advance future BTV (and other arbovirus) prevention and control research and in collaboration with academic beneficiaries, contribute to significant wider downstream impact for the following beneficiaries:

Benefits to the agriculture sector (farmers, veterinarians):

High profile incursions of Culicoides borne viruses such as BTV in recent years have led to increased interest in arboviruses of livestock and concerns are heightened due to the recent re-emergence of a virulent BTV-8 strain in France. The immediate benefit of this project to farmers and veterinarians will be an increased knowledge on BTV pathogenesis and transmission. Longer-term benefits include advances in BTV prevention and control through the development of cross-serotype or otherwise improved vaccines. The identification of disease inducing immunopathological mechanisms may also lead to anti-inflammatory treatment of individuals to reduce clinical severity, potentially transferable to related viruses such as African horse sickness virus (AHSV). Furthermore Culicoides blood-feeding can cause debilitating insect bite hypersensitivity especially in horses. Advances in our understanding of insect saliva induced inflammation may lead to treatment development in the long-term. The overall benefit will be increased animal welfare of production animals and subsequently increased economic competitiveness of the agriculture sector.

Benefits to policy makers:

Policy makers will benefit from increased knowledge concerning the drivers of BTV transmission to inform risk mitigation strategies. Information on the feasibility and realistic availability time scales of improved BTV vaccines (cross protective and allowing the differentiation of vaccinated from infected animals (DIVA)) will inform vaccination and surveillance policies. DIVA capability of employed vaccines will increase surveillance efficiency. These benefits will improve control and prevention of BTV and other arboviruses, leading to significant downstream economic savings.

Benefits to the wider public:

Culicoides are notorious in the UK for their nuisance biting in Scotland and Northern England and high profile outbreaks of Culicoides transmitted viruses causing severe disease in cattle and sheep have further enhanced society interest. The public will immediately benefit from increased scientific engagement and knowledge exchange addressing their interest in these insects as well as vector-borne diseases in general. Wider long-term benefits to public health and quality of life could include treatment to insect saliva induced inflammation and Culicoides control mechanisms targeted at immunological disruption of blood-feeding.
Staff employed on this project will acquire translatable skills ranging from project management, presentation skills, applying logical decision making and IT competences, which can be applied to all employment sectors.

Benefits to the commercial private sector:

Pharmaceutical companies are already involved in BTV and AHSV vaccine production and development and will therefore benefit from the increased knowledge on protective immune correlates in their goal to produce and market cross-serotype protective vaccines in collaboration with academic beneficiaries. Long-term benefit opportunities are provided by identification of Culicoides saliva protein induced immune responses as a model for allergy mechanisms and treatment development to insect bite hypersensitivity. Additionally, host responses to Culicoides saliva and/or saliva induced immune-modulation could be exploited as components to pathogen vaccines or Culicoides population control vaccines. Similar strategies are already employed against other arthropods and vector-borne pathogens.