A chicken primary B cell culture model to study the pathogenesis and improve the control of immunosuppressive viruses of poultry

Chicken meat is an important source of dietary protein for people worldwide, and the poultry industry is important for securing enough food to feed the growing human population. Ensuring chickens remain healthy is therefore an economically important issue as well as a welfare issue for the birds. Chickens require a healthy immune system for vaccines to work effectively, and to fight off infection, so if their immune system is suppressed, birds will respond less well to vaccines and be more susceptible to catching infections, some of which can also infect people. The chicken immune system can become suppressed by infection with viruses that target a type of white blood cell known as a B cell. The main viruses that infect B cells in chickens are: Marek's disease virus (MDV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). MDV and IBDV destroy B cells, whereas ALV and REV can cause the B cells to become cancerous and form a tumour. All these viruses can cause immunosuppression. These diseases are poorly controlled: New strains of IBDV have spread and vaccine failures have been reported, and ALV and REV continue to be isolated despite eradication efforts. There is therefore a need for new vaccines and control methods. It might be possible in the future to produce birds that are resistant to these diseases, but we need to better understand how the viruses interact with the cells they infect in order to realise this potential.

Until recently, it was not possible to study how the chicken B cells responded to infection as the cells died when they were taken out of the birds. Therefore, most studies into how these viruses cause disease and immunosuppression had to be conducted using infected chickens that suffered from disease, which could be severe in some cases. Recently, it has become possible to keep chicken B cells alive in the laboratory once removed from birds, and one research group has already used the cells to study MDV infection. We have used the cells to study IBDV infection, and our data suggest that they would be a good model for studying how IBDV interacts with the bird immune system, without the need to infect birds directly. We believe the model has the potential to replace the use of infected birds, and reduce the total number of birds used in this kind of research. The aim of this project is to build on our previous data to establish the chicken B cell model for studying IBDV, and expand it to the study of ALV and REV. In order to do this, we have three objectives: First, we will confirm and validate the use of the model for studying IBDV by comprehensively quantifying changes in the level of gene expression in the B cells following infection compared to what happens in the infected bird. Second, we will expand the model to include ALV and REV. Third, we will use the cells to produce stocks of IBDV as, currently, virus stocks are produced by infecting birds and recovering the virus from organs, so if the B cells can be used for this purpose, it would replace the use of even more infected birds. We will also determine if the cells can be used to screen vaccines for how effective they are against locally circulating and new strains to improve control.

Not only will we be able to replace and reduce the use of infected chickens, but using B cells is actually a better approach than using infected chickens to answer some scientifically important questions, as there is only one cell population as opposed to many, and it is possible to control the amount of virus the cells receive and to know for how long the cells have been infected. In addition, we can apply the model in the future to study how IBDV vaccines interact with the cells, and study co-infection with multiple viruses, so the project not only has a high 3Rs impact, but also a high scientific and translational impact.

A major cause of immunosuppression in poultry is infection with B-cell tropic viruses such as infectious bursal disease virus (IBDV) and the oncogenic retroviruses avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). In vivo studies, while informative, can cause significant morbidity to infected birds. Recently, it has become possible to culture chicken primary B cells ex vivo. We have shown these cells can support IBDV replication and our data suggest they may be of relevance in vivo. Our aim is to establish a chicken primary B cell culture model to study the pathogenesis and improve the control of immunosuppressive B cell-tropic viruses of importance to the poultry industry that will replace the use of live birds in infection studies and reduce the total number of birds used experimentally in this field. We will achieve this aim by addressing the following objectives:1.) Validate the chicken primary B cell model for studying IBDV-host interactions, 2.) Expand the model to include ALV and REV, 3.) Evaluate the use of chicken primary B cells to test the immunogenicity of vaccine candidates prior to challenge and produce stocks of challenge viruses. Primary B cells will be harvested from chickens, cultured ex vivo, and infected in the laboratory with IBDV. The host-cell transcriptional response to infection will be characterised by RNA-Seq and the relevance of the model will be evaluated by comparing the results to the gene expression profile of infected tissue from in vivo studies. The model will then be expanded to include ALV and REV. The feasibility of using the cells to produce stocks of IBDV will be evaluated by determining the peak virus titre, the pathogenicity, and sequence changes compared to stocks produced in embryonated eggs. Finally, the cells will be used in neutralisation assays to quantify neutralising antibody titres in serum from vaccinated birds against very virulent IBDV strains.

At The Pirbright Institute (TPI), in vivo protocols with IBDV are listed as 'Severe' on our Home Office Project License due to the acute and severe nature of the disease, which can cause up to 100% mortality in some flocks. In addition, studies with ALV and REV are typically conducted in cells harvested from birds that have been infected for months, and suffering from metastatic tumours in multiple tissues. The ability to culture chicken primary B cells ex vivo opens up the possibility to study aspects of the pathogenesis and immunosuppression caused by IBDV, ALV and REV without the need to infect chickens, thereby replacing the use of infected birds. We will validate the use of these cells for studying host cell-virus interactions, and then evaluate using the cells to produce stocks of IBDV, which are currently prepared by infecting birds and harvesting infected organs at necropsy, as passage in eggs or fibroblast cells leads to attenuation of the virus. Birds will still be needed to obtain a supply of the primary B cells, however they will not suffer from infection, and as the cells from one bird can be used to study viral infection over multiple time-points, the number of birds used will be reduced. We typically use over 300 birds per year in pathogenicity studies of IBDV, ALV and REV, and 60 birds per year for IBDV stock production. A Pubmed search reveals that in the last year there were 5 pathogenicity studies published from other research groups, each varying in size up to 400 birds, and 16 studies that used stocks of IBDV propagated in chickens to retain their virulence. Moreover, two IBDV OIE reference laboratories each produce stocks of up to 20 strains of IBDV per year from the tissues of infected birds. Taken together, should the scientific community adopt the use of chicken primary B cells for IBDV, ALV and REV pathogenesis studies and IBDV stock production, there is the potential to replace the use of (400 birds x 5 pathogenicity studies) + (60 birds x 16 studies using virulent strains) + (60 birds x 20 strains x 2 OIE ref labs) = 5,360 infected chickens per year, assuming these studies used a similar number of birds to us. This represents a significant 3Rs impact.

In addition, vaccine efficacy is currently evaluated in challenge studies with very virulent (vv) strains of IBDV. However, if vaccine efficacy is low, challenged birds experience severe disease. As high titres of serum neutralising antibodies are associated with protection, we plan to use the chicken primary B cell cultures in neutralisation assays to quantify the titre of serum neutralising antibodies in vaccinated birds against vv strains of IBDV. Only birds with high titres will be challenged. This will reduce the number of birds experiencing severe disease. It is difficult to quantify the reduction in the number of challenged birds as the immunogenicity of individual vaccine candidates vary. However, a Pubmed search reveals that in the past year, there were 16 IBDV vaccine publications where birds were challenged with virulent strains. Many of these vaccine candidates were only partially protective, suggesting that if the scientific community adopts this strategy, there could be a substantial 3Rs impact.

As well as a high 3Rs Impact, the project has a high scientific and translational impact to vaccine companies as the data generated will provide us with a greater understanding of the molecular basis of IBDV, ALV and REV pathogenesis and immunosuppression that we can exploit in future studies to better control these diseases. Additionally, as the cells can be cultured following recovery from liquid nitrogen, this will increase the capacity of labs that do not have access to animal facilities to conduct these kinds of studies. Moreover, by using the cells to screen vaccine candidates for their immunogenicity against locally circulating strains of IBDV, this will aid antigen vaccine development and disease control.