Bluetongue Disabled Infectious Single Animal (DISA) vaccine: Studies on the optimal route and dose in sheep Piet A. van Rijn a,c,⇑ , Franz J. Daus a , Mieke A. Maris-Veldhuis a , Femke Feenstra a,b,1 , René G.P. van Gennip a a Department of Virology, Wageningen Bioveterinary Research, Lelystad, The Netherlands b Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands c Department of Biochemistry, Centre for Human Metabolomics, North-West University, South Africa article info Article history: Received 5 October 2016 Received in revised form 20 November 2016 Accepted 24 November 2016 Available online xxxx Keywords: Bluetongue virus Vaccine NS3 Optimal route Application Dose abstract Bluetongue (BT) is a disease of ruminants caused by bluetongue virus (BTV) transmitted by biting midges of the Culicoides genus. Outbreaks have been controlled successfully by vaccination, however, currently available BT vaccines have several shortcomings. Recently, we have developed BT Disabled Infectious Single Animal (DISA) vaccines based on live-attenuated BTV without expression of dispensable non- structural NS3/NS3a protein. DISA vaccines are non-pathogenic replicating vaccines, do not cause vire- mia, enable DIVA and are highly protective. NS3/NS3a protein is involved in virus release, cytopathogenic effect and suppression of Interferon-I induction, suggesting that the vaccination route can be of impor- tance. A standardized dose of DISA vaccine for serotype 8 has successfully been tested by subcutaneous vaccination. We show that 10 and 100 times dilutions of this previously tested dose did not reduce the VP7 humoral response. Further, the vaccination route of DISA vaccine strongly determined the induction of VP7 directed antibodies (Abs). Intravenous vaccination induced high and prolonged humoral response but is not practical in field situations. VP7 seroconversion was stronger by intramuscular vaccination than by subcutaneous vaccination. For both vaccination routes and for two different DISA vaccine back- bones, IgM Abs were rapidly induced but declined after 14 days post vaccination (dpv), whereas the IgG response was slower. Interestingly, intramuscular vaccination resulted in an initial peak followed by a decline up to 21 dpv and then increased again. This second increase is a steady and continuous increase of IgG Abs. These results indicate that intramuscular vaccination is the optimal route. The protective dose of DISA vaccine has not been determined yet, but it is expected to be significantly lower than of currently used BT vaccines. Therefore, in addition to the advantages of improved safety and DIVA compatibility, the novel DISA vaccines will be cost–competitive to commercially available live attenuated and inactivated vaccines for Bluetongue. Ó 2016 Elsevier Ltd. All rights reserved. 1. Introduction Bluetongue (BT) is a notifiable disease of ruminants caused by the bluetongue virus (BTV) spread transmitted by Culicoides biting midges. BT is endemic in tropical and subtropical regions, but is expanding to regions with a moderate climate, partly due to emerging vector species [1,2]. BT outbreaks can cause large eco- nomic losses due to diseased animals, reproduction failures, and trade restrictions [3]. The BTV species in the Orbivirus genus of the family Reoviridae consists of at least 27 serotypes originally defined by serotype specific and by cross reacting neutralizing antibodies (Abs). Nowadays, serotypes are confirmed by VP2 phy- logenetic analyses [4–6]. The outer capsid protein VP2 is the most immunogenic and major serotype determining protein [7]. Control of insect transmitted BTV is hardly possible without vaccination [8], and multi-serotype situations in large parts of sev- eral continents further hamper efficient control of BT [9]. Vaccina- tion in African countries occurs with three pentavalent cocktails of conventionally live attenuated BT vaccines (LAVs) [10]. The safety of these cocktail vaccines is questionable with regard to residual virulence and reassortment [11–13]. In Europe, safe and effective inactivated BT vaccines are used for several serotypes, but these are more expensive and annual re-vaccination is recommended [14]. Currently, BTV serotypes 1, 2, 4, 8, 9 and 16 are present in Eur- ope [9]. http://dx.doi.org/10.1016/j.vaccine.2016.11.081 0264-410X/Ó 2016 Elsevier Ltd. All rights reserved. ⇑ Corresponding author at: Department of Virology, Wageningen Bioveterinary Research, Lelystad, The Netherlands. E-mail address: piet.vanrijn@wur.nl (P.A. van Rijn). 1 Current address: Merial BV, Lelystad, The Netherlands. Vaccine xxx (2016) xxx–xxx Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Please cite this article in press as: van Rijn PA et al. Bluetongue Disabled Infectious Single Animal (DISA) vaccine: Studies on the optimal route and dose in sheep. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.11.081