Next-generation salmonid alphavirus vaccine development
Hikke, M.C. - \ 2016
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Gorben Pijlman. - Wageningen : Wageningen University - ISBN 9789462577404 - 159
alphavirus - atlantic salmon - rainbow trout - vaccine development - immunity - virology - fish culture - aquaculture - biotechnology - alfavirus - europese zalm - regenboogforel - vaccinontwikkeling - immuniteit - virologie - visteelt - aquacultuur - biotechnologie
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.
Induction and suppression of tick cell antiviral RNAi responses by tick-borne flaviviruses
Schnettler, E. ; Tykalova, H. ; Watson, M. ; Sharma, M. ; Sterken, M.G. ; Obbard, D.J. ; Lewis, S.H. ; McFarlane, M. ; Bell-Sakyi, L. ; Barry, G. ; Weisheit, S. ; Best, S.M. ; Kuhn, R.J. ; Pijlman, G.P. ; Chase-Topping, M.E. ; Gould, E.A. ; Grubhoffer, L. ; Fazakerley, J.K. ; Kohl, A. - \ 2014
Nucleic acids research 42 (2014)14. - ISSN 0305-1048 - p. 9436 - 9446.
forest-virus replicon - interferon antagonist - arbovirus infection - immunity - replication - drosophila - identification - alphavirus - mosquitos - origin
Arboviruses are transmitted by distantly related arthropod vectors such as mosquitoes (class Insecta) and ticks (class Arachnida). RNA interference (RNAi) is the major antiviral mechanism in arthropods against arboviruses. Unlike in mosquitoes, tick antiviral RNAi is not understood, although this information is important to compare arbovirus/host interactions in different classes of arbovirus vectos. Using an Ixodes scapularis-derived cell line, key Argonaute proteins involved in RNAi and the response against tick-borne Langat virus (Flaviviridae) replication were identified and phylogenetic relationships characterized. Analysis of small RNAs in infected cells showed the production of virus-derived small interfering RNAs (viRNAs), which are key molecules of the antiviral RNAi response. Importantly, viRNAs were longer (22 nucleotides) than those from other arbovirus vectors and mapped at highest frequency to the termini of the viral genome, as opposed to mosquito-borne flaviviruses. Moreover, tick-borne flaviviruses expressed subgenomic flavivirus RNAs that interfere with tick RNAi. Our results characterize the antiviral RNAi response in tick cells including phylogenetic analysis of genes encoding antiviral proteins, and viral interference with this pathway. This shows important differences in antiviral RNAi between the two major classes of arbovirus vectors, and our data broadens our understanding of arthropod antiviral RNAi.