Development of an in vitro system that mimics B and T helper cell responses in porcine lymph nodes

Pigs are kept in large numbers on farms in Europe and worldwide. To keep these animals healthy and to maintain a high standard of animal welfare, swine are frequently vaccinated. However, some of these vaccines have only limited efficacy and for some devastating pig diseases, like African swine fever, no vaccines exist at all.
Hence, the testing and development of new vaccines is an area of active research by public research institutions and privately owned companies world-wide. Testing of vaccines from early stages of vaccine development onwards is mainly performed in living pigs. In this project we aim to develop a culture system for immune cells in the laboratory. Such a culture is called 'in vitro'. Our in vitro culture system mimics the immune response occurring in specialised organs of the immune system, called lymph nodes. Within these lymph nodes various immune cell types interact with each other. One of these cell types, called B cells, produces antibodies which often confer protection after vaccination. In pilot experiments, we have already established an in vitro system that shows several features of the immune reaction, like the activation of B cells and the production of antibodies. In the first phase of this project, we aim to optimise this system. Different variations will be introduced into the existing in vitro system to test whether they improve the function of the immune cells within them. In the final stage of the project, we will test if our optimised in vitro system is capable of mimicking central features of the B cell response, including antibody production, occurring in lymph nodes of living pigs.
If our in vitro system proves to be capable of mimicking B cell and antibody responses occurring in living pigs, it will be used in the future to reduce the number of pigs in used in the development of new vaccine or improvement of current vaccines. This will apply to the early phase of vaccine development, where basic research is performed to identify promising vaccine candidates. Although the immune cells in our in vitro system still need to be isolated initially from a living pig, cells from a single pig can be used in numerous projects for the testing of different vaccine candidates.
We will communicate the findings of our project to colleagues working in other research institutions and in vaccine-producing companies. For this we will participate in international scientific meetings, speak to colleagues via established collaboration networks, offer training courses and spread our findings via social media platforms. Results will also be published in scientific journals. In this way we ensure that our in vitro system will be quickly taken up by others, ensuring it unfolds the maximal opportunity to reduce the number pigs required in the early phase of the development of new vaccines.

For most licensed vaccines, antibodies are the correlate of protection. Antibodies are produced by plasma cells which typically develop in secondary lymphoid organs, often supported by T follicular helper (Tfh) cells. To test the immunogenicity of vaccine candidates and their capacity to induce antibodies, animal immunization experiments are usually performed because of a lack of in vitro systems that allow investigation of B and T cell responses in vitro. Based on published work with organoid cultures from human tonsils, we have performed pilot studies with transwell cultures of cells isolated from tracheobronchial lymph nodes of porcine respiratory coronavirus (PRCV) and swine influenza A virus infected pigs. Following restimulation, we observed virus-specific antibody production and the expansion of plasmablasts and Tfh cells. This suggests that the transwell system is capable of mimicking features of B and T cell activation in lymph nodes, including a germinal centre reaction, indicated by the activation of Tfh cells. We shall improve this transwell system, including the testing of a microfluidic transwell system with the PhysioMimixTM technology. We will also test the capacity of such in vitro systems to mimic primary T and B cell responses. Activation of T and B cells will be monitored by flow cytometry, antibody production will be studied by ELISA and neutralisation tests and germinal centre structures will be investigated by fluorescence microscopy. The fidelity of the transwell systems will be tested by comparison with the in vivo response in immunised pigs. This will also include B cell receptor repertoire analyses by single cell RNA-sequencing to compare the quality and the quantity of the B cell response in vitro and in vivo. The experiments will establish and characterize state-of-the-art in vitro methods that will help to reduce the number of pigs required in immunisation studies for vaccine candidate testing.