RESUMO
Vaccination is essential in livestock farming and in companion animal ownership. Nucleic acid vaccines based on DNA or RNA provide an elegant alternative to those classical veterinary vaccines that have performed suboptimally. Recent advances in terms of rational design, safety, and efficacy have strengthened the position of nucleic acid vaccines in veterinary vaccinology. The present review focuses on replicon vaccines designed for veterinary use. Replicon vaccines are self-amplifying viral RNA sequences that, in addition to the sequence encoding the antigen of interest, contain all elements necessary for RNA replication. Vaccination results in high levels of in situ antigen expression and induction of potent immune responses. Both positive- and negative-stranded viruses have been used to construct replicons, and they can be delivered as RNA, DNA, or viral replicon particles. An introduction to the biology and the construction of different viral replicon vectors is given, and examples of veterinary replicon vaccine applications are discussed.
Assuntos
Replicon , Vacinação/veterinária , Medicina Veterinária/métodos , Animais , Vetores Genéticos , Vacinas de DNA , VírusRESUMO
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.
Assuntos
Alphavirus/genética , Proteínas do Capsídeo/biossíntese , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Proteínas do Envelope Viral/imunologia , Alphavirus/imunologia , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Núcleo Celular , Desenho de Fármacos , Epitopos/genética , Epitopos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Células Sf9 , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas do Envelope Viral/genética , Montagem de VírusRESUMO
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.
Assuntos
Alphavirus/crescimento & desenvolvimento , Alphavirus/isolamento & purificação , Salmonidae/virologia , Cultura de Vírus , Aedes , Alphavirus/fisiologia , Animais , Linhagem Celular , Salmão , Temperatura , Vacinas Virais/isolamento & purificação , Replicação ViralRESUMO
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.