Next-generation salmonid alphavirus vaccine development

ABSTRACT Aquaculture is essential to meet the current and future demands for seafood to feed the world population. Atlantic salmon and rainbow trout are two of the most cultured aquaculture species. A pathogen that threatens these species is salmonid alphavirus (SAV). A current inactivated virus vaccine against SAV provides cross-protection against all SAV subtypes in salmonids and reduces mortality amongst infected fish. However, protection is not 100% and due to virus growth at low temperature, the vaccine production process is time consuming. In addition, the vaccine needs to be injected into the fish, which is a cumbersome process. The work described in this thesis aimed to increase the general knowledge of SAV and to assess current vaccine technologies, and to use this knowledge in designing next-generation vaccines for salmonid aquaculture. An alternative cell line to support SAV proliferation was identified, however, the virus production time could not yet outcompete the current SAV production system. Making use of the baculovirus insect cell expression system, multiple enveloped virus-like particle (eVLP), and core-like particle (CLP) prototype vaccines were produced in insect cells at high temperature. An in vivo vaccination study showed, however, that these vaccines could not readily protect Atlantic salmon against SAV. The low temperature-dependent replication of SAV was attributed to the glycoprotein E2, and it was found that E2 only correctly travelled to the cell surface at low temperature, and in the presence of glycoprotein E1. The biological impact of this finding was confirmed in the development and in vivo testing of a DNA-launched replicon vaccine. The effective DNA-launched replicon vaccine was extended by delivery of the capsid protein in trans. It was hypothesized that viral replicon particles (VRP) were formed in vivo, which would cause an additional single round of infection and might further elevate the immune response in comparison to the replicon vaccine. A second animal trial indicated that the inclusion of capsid did not yet improve vaccine efficacy. This trial however did show that a DNA vaccine transiently expressing the SAV structural proteins provided superior protection over both replicon vaccines (with and without capsid). In this thesis, some virus characteristics, such as the cause of temperature-dependency of SAV replication, of an unique aquatic virus were further explored. The production and in vivo testing of multiple next-generation vaccines defined the prerequisites for induction of a potent immune response in Atlantic salmon. A prototype DNA-launched replicon vaccine has shown potential for further development. The research described in this thesis contributes to the development of next-generation vaccines in the challenging area of fish vaccinology.</p

Salmonid alphavirus replication in mosquito cells: towards a novel vaccine production system

Salmonid alphavirus (SAV) causes pancreas disease and sleeping disease in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) and confers a major burden to the aquaculture industry. A commercial inactivated whole virus vaccine propagated in a salmon cell line at low temperature provides effective protection against SAV infections. Alphaviruses (family Togaviridae) are generally transmitted between vertebrate hosts via blood-sucking arthropod vectors, typically mosquitoes. SAV is unique in this respect because it can be transmitted directly from fish to fish and has no known invertebrate vector. Here, we show for the first time that SAV is able to complete a full infectious cycle within arthropod cells derived from the Asian tiger mosquito Aedes albopictus. Progeny virus is produced in C6/36 and U4.4. cells in a temperature-dependent manner (at 15°C but not at 18°C), can be serially passaged and remains infectious to salmonid Chinook salmon embryo cells. This suggests that SAV is not a vertebrate-restricted alphavirus after all and may have the potential to replicate in invertebrates. The current study also shows the ability of SAV to be propagated in mosquito cells, thereby possibly providing an alternative SAV production system for vaccine applications

Alphavirus capsid proteins self-assemble into core-like particles in insect cells: A promising platform for nanoparticle vaccine development

The mosquito-borne chikungunya virus (CHIKV) causes arthritic diseases in humans, whereas the aquatic salmonid alphavirus (SAV) is associated with high mortality in aquaculture of salmon and trout. Using modern biotechnological approaches, promising vaccine candidates based upon highly immunogenic, enveloped virus-like particles (eVLPs) have been developed. However, the eVLP structure (core, lipid membrane, surface glycoproteins) is more complex than that of non-enveloped, protein-only VLPs, which are structurally and morphologically 'simple'. In order to develop an alternative to alphavirus eVLPs, in this paper we engineered recombinant baculovirus vectors to produce high levels of alphavirus core-like particles (CLPs) in insect cells by expression of the CHIKV and SAV capsid proteins. The CLPs localize in dense nuclear bodies within the infected cell nucleus and are purified through a rapid and scalable protocol involving cell lysis, sonication and low-speed centrifugation steps. Furthermore, an immunogenic epitope from the alphavirus E2 glycoprotein can be successfully fused to the N-terminus of the capsid protein without disrupting the CLP self-assembling properties. We propose that immunogenic epitope-tagged alphavirus CLPs produced in insect cells present a simple and perhaps more stable alternative to alphavirus eVLPs

Salmonid alphavirus glycoprotein E2 requires low temperature and E1 for virion formation and induction of protective immunity

Salmonid alphavirus (SAV; also known as Salmon pancreas disease virus; family Togaviridae) causes pancreas disease and sleeping disease in Atlantic salmon and rainbow trout, respectively, and poses a major burden to the aquaculture industry. SAV infection in vivo is temperature-restricted and progeny virus is only produced at low temperatures (10–15 °C). Using engineered SAV replicons we show that viral RNA replication is not temperature-restricted suggesting that the viral structural proteins determine low-temperature dependency. The processing/trafficking of SAV glycoproteins E1 and E2 as a function of temperature was investigated via baculovirus vectors in Sf9 insect cells and by transfection of CHSE-214 fish cells with DNA constructs expressing E1 and E2. We identified SAV E2 as the temperature determinant by demonstrating that membrane trafficking and surface expression of E2 occurs only at low temperature and only in the presence of E1. Finally, a vaccination-challenge model in Atlantic salmon demonstrates the biological significance of our findings and shows that SAV replicon DNA vaccines encoding E2 elicit protective immunity only when E1 is co-expressed. This is the first study that identifies E2 as the critical determinant of SAV low-temperature dependent virion formation and defines the prerequisites for induction of a potent immune response in Atlantic salmon by DNA vaccination