Immunization against infectious salmon anemia : implementation of a salmonid alphavirus-based replicon vaccine and immune response studies

Abstract

Farming of fish at high densities entails considerable risks of outbreaks of infectious diseases. Therefore, disease prevention through vaccination is crucial for sustainable farming. Whereas vaccination against bacterial diseases has been highly successful in farming of Atlantic salmon (Salmo salar), most of the currently available viral fish vaccines do not provide sufficient protection. An important virus in the salmon farming aquaculture is the infectious salmon anemia virus (ISAV), the causative agent of infectious salmon anemia (ISA). ISAV is a member of the family Orthomyxoviridae, genus Isavirus. The accumulated mortality in ISA outbreaks can be high and confirmed ISA outbreaks necessitate regulatory measures with substantial economic losses. ISA remains a concern for the salmon industry, and although the annual number of outbreaks is small compared to early 1990-ies, there are recurrent disease outbreaks. The thesis addresses the advancement of ISA vaccines and the approach to define immune parameters that are related to vaccine-mediated protection. First, immunization using inactivated, whole ISAV particles was carried out in Atlantic salmon (Salmo salar L.), and a strong dependency of antigen dose was observed for vaccine efficacy. It was demonstrated that substantial protection against ISA is attainable by vaccination with the use of high dose of inactivated whole ISAV particles. The role of ISAV-specific neutralizing serum antibodies correlating to protection was indicated. Vaccinated and protected fish were able to clear ISAV infection. Despite the fact that the necessary antigen strength to achieve protection makes the production of such vaccines not profitable, the results indicate the significance of adequate vaccine composition. A self-replicating expression vector based on the replication machinery of Salmonid pancreas disease virus (SPDV), today more commonly known as Salmonid alphavirus (SAV), was found to be a promising vaccine model for salmonids. A DNA-layered, SAV-based replicon that encodes the ISAV hemagglutinin-esterase (HE) was constructed, and its functionality was verified in fish cell lines and in muscle tissue of Atlantic salmon. The replicon vaccine elicited high protection in Atlantic salmon after a single intramuscular (i.m.) injection of 10 μg against subsequent ISAV challenge. The ISAV HE was confirmed as immunoprotective protein. The protection provided by the replicon vaccine that encodes the HE was high and co-delivery of the ISAV fusion (F) protein or the matrix (M) protein by a replicon did not enhance protection. The results may strengthen the focus on the HE in future vaccine development. The route of delivery was shown to be crucial for the replicon vaccine, as the high responsiveness induced by the i.m. administration could not be obtained by intraperitoneal (i.p.) injection. An immunostimulatory potential of the SAV-based replicon vector in Atlantic salmon was proven. Innate immune mechanisms induced by the replicon vaccine locally at the muscle injection site may increase vaccine efficacy and hence make immunization with lower doses than required for conventional DNA vaccines possible. However, when co-delivered (i.m.) with a low-dose inactivated ISAV vaccine given i.p., an adjuvant effect could neither be obtained by a mock-replicon nor when an additional immunostimulator, the viral hemorrhagic septicemia virus (VHSV) glycoprotein (G), was encoded by the replicon. The development of another replicon vaccine system, the generation of viral replicon particles (VRPs) from SAV, was approached. The feasibility of the concept was demonstrated, but more intensive studies are required for further advancement.

I fiskeoppdrett er det høye tettheter av dyr. Dette gir betydelig risiko for utbrudd av smittsomme sykdommer. Forbyggende tiltak som biosikkerhet og vaksinering er avgjørende for at fiskeoppdrett skal være bærekraftig. Vaksinasjon mot bakterielle sykdommer har vært svært vellykket i oppdrett av atlantisk laks (Salmo salar L.). Derimot gir dagens virusvaksiner for fisk ikke tilstrekkelig beskyttelse. Infeksiøs lakseanemivirus (ILAV), som tilhører familien Orthomyxoviridae, genus Isavirus, er et viktig virus i lakseoppdrett. Akkumulert dødelighet kan være høy som en følge av infeksiøs lakseanemi (ILA) og bekreftet utbrudd krever bekjempelsestiltak som medfører betydelige økonomiske tap. ILA har fortsatt betydning i laksenæringen, og selv om antall utbrudd nå er vesentlig lavere enn på 1990-tallet, er det jevnlige sykdomsutbrudd. Avhandlingen fokuserer på utvikling av ILA-vaksiner og identifisering av immunparametere som er korrelert til vaksineindusert beskyttelse. Først ble et vaksine-belastningsforsøk med ulike mengder av inaktivert helvirus ILAV gjennomført i atlantisk laks (Salmo salar). En sterk doseavhengighet av vaksineeffekt ble observert, jo mer ILAV-spesifikt antigen i vaksinen jo bedre beskyttelse ble oppnådd. Svært høy beskyttelse ble oppnådd med den høyeste dosen, og fisk som var vaksine-beskyttet mot sykdommen var i stand til å kvitte seg med ILAV infeksjonen. Selv om den mengden ILAV-spesifikt antigen som ga beskyttelse var så høy at det ikke er kostnadseffektivt å benytte det i kommersielle vaksiner, indikerte resultatene at beskyttelse mot ILA kan oppnås ved vaksinering. Humorale immunrespons i form av nøytraliserende antistoffer var korrelert til beskyttelse.