Multiple routes to B cell memory in Atlantic salmon

Viral diseases are a major threat to Norwegian farmed Atlantic salmon and today's commercial vaccines provide limited protection. To improve virus vaccines, we need to understand how the immune system of A. salmon works and what mechanisms contribute to protection. Most vaccines on the market consist of inactivated virus and are given by injection into the peritoneal cavity. These induce production of antibodies that can block further infection. It is activated B cells, called plasma cells, that secrete these antibodies. In mammals, plasma cells can persist for years and contribute to long-term protection. We do not know whether this also happens in A. salmon. The main objective of the project has been to increase knowledge about B cell responses directed against viruses in A. salmon. Sub-goals have been to map B cell populations, how these are distributed in immunological organs, as well as the salmon's antibody response after vaccination or infection. Since most vaccines used in A. salmon are injected into the peritoneal cavity, we have compared local B cell responses here versus what happens in systemic immune organs such as kidney and spleen. Systemic and local B cell responses by intraperitoneal injection of salmonid alphavirus (SAV) or inactivated SAV. We performed immunization trials with both live and inactivated SAV given intraperitoneally (IP), following the fish for up to 21 weeks after vaccination. For this purpose, we established ELISPOT-assays to detect B cells that secrete IgM, and flow cytometry assays, which have been important tools. We demonstrated a prolonged presence of total IgM secreting cells in the peritoneal cavity in the SAV-infected fish, while the response in the systemic organs spleen and head kidney was less pronounced. Furthermore, the level of peritoneal cavity plasma cells correlated with the amount of antibody in the blood of the fish. B cells that secreted specific antibodies to SAV were also detected in the peritoneal cavity, and the frequency of these was higher here than in the spleen and head kidney. In general, infection with live virus gave a higher response and with a longer duration than inactivated SAV. What is the immunological role of adipose tissue in the peritoneal cavity? After IP injection of the bacterium Piscirikettsia salmonis, we also found a strong B cell response in the peritoneal cavity like that of SAV. The results made us ask whether the peritoneal cavity is a site where B cells are activated locally, and are there other types of leukocytes in the surrounding adipose tissue that participate in initiating immune responses? By gene expression analyzes of the adipose tissue from both P. salmonis and SAV-infected salmon, we detected increased expression of genes for various leukocytes such as B and T cells and professional antigen-presenting cells. In addition, inflammatory and antiviral cytokine genes were expressed. We suggest that the adipose tissue may be a tissue where both B cells and other leukocytes are present, and where local activation of B cells takes place. Immunological memory in salmon? Another important sub-goal of the project has been to investigate whether B cells of A. salmon provide immunological memory that initiates a secondary immune response that is more powerful and has a longer duration than the primary one. In one immunization trial, we therefore injected a booster dose of inactivated SAV six weeks after the first immunization. Neither the number of antibody-producing B cells in the peritoneal cavity and systemic tissues, nor the level of SAV-specific antibodies in the blood increased in the booster group compared to the group receiving only one virus dose. Our experiment did not demonstrate a conventional secondary immune response in A. salmon as described in mammals. Characterization of BAFF and APRIL in salmon - and their interaction with B cells. BAFF and APRIL are cytokines that in mammals contribute to the survival of B cells. We have studied the functions of BAFF and APRIL in A. salmon. We found BAFF and APRIL transcripts in several tissues, and these increased in the SAV-infected fish, and especially in target organs for the virus. In leukocyte cultures, the levels of BAFF and APRIL were highest in macrophages, while different BAFF and APRIL receptors were primarily expressed in B cells. Addition of BAFF and APRIL proteins to A. salmon leukocytes increased B cell survival in culture as well as increased the number of antibody-producing cells in peripheral blood cells, compared to untreated cells. This shows that key functions described for BAFF and APRIL in mammals are also present in bony fish and that both cytokines are important for keeping B cells in culture alive. Identification of A.salmon B cell markers. Cluster of differentiation (CD) is a system for identifying surface markers in leukocytes, including B cells. By proteomics, we identified new B cell surface markers for A. salmon, including CD22 and CD79.

Effektive virusvaksiner for akvakultur er avgjørende for å forbedre fiskevelferd og lønnsomhet. Skal vaksinene forbedres må vi forstå fiskens immunsystem. For å studere B-celleresponser hos laks har vi utviklet nye metoder og reagenser: ELISpotmetoder for å kvantifisere plasmaceller Proteinene BAFF og APRIL fra laks er produsert og øker overlevelsen til B-celler i kultur Ved proteomikk er overflatemarkører for laksens B-celler identifisert Et videre forskningssamarbeid for å produsere monoklonale antistoffer mot B-cellemarkører som skiller ulike B-cellepopulasjoner er ønskelig. Forskningen har vist at når vaksiner gis i bukhula, vil lokale B-celleresponser være langvarige og bidra til beskyttelse. Når vaksiner som stikkes i bukhula skal testes for beskyttelse, bør smitteagenset ikke gis i buken, men en kohabitant smittemodell bør brukes. Kunnskapen er relevant for vaksineselskaper som driver rutinemessig batchtesting av vaksiner.

Viral diseases like pancreas disease are a main concern in the Norwegian aquaculture industry, and the vaccines on the market give suboptimal protection. B cell memory responses are essential for durable protective immunity after vaccination. The organization of the teleost immune system and its B cell components differ from mammals, and it is likely that these features influence how immunological memory is maintained. It is therefore a need to determine the protective status of vaccines related to elicited B cell responses. However, the components and mechanisms underlying formation of and function of A. salmon B cell memory are still elusive. In this project we propose to study salmon IgM+ and IgT+ B cells, their distribution, and the quantity and quality of B cell memory responses after vaccination. The main question is how and to what extend salmon preserve B cell memory. Our own previous work has shown that a high and long lasting antibody response is induced in vivo using the TLR ligands (CpG/poly I:C) as adjuvants in combination with salmonid alphavirus (SAV) antigen. The antibody levels were significantly higher than with other adjuvants, suggesting that TLR stimulation mediate salmon B cell effector functions. This model will be used as a basis in the proposed project to explore how TLR ligands influence B cell memory and whether B cells of memory and/or plasma cell phenotypes exist in A.salmon. To identify different B cell subpopulations and explore their functions a wide repertoire of state of the art techniques will be employed including proteomics, flow cytometry and RNAseq. If successful, our research will guide the design of future vaccines and also provide a set of valuable tools to measure salmon B cell levels and functions.