Vaccine 30 (2012) 4828–4834 Contents lists available at SciVerse ScienceDirect Vaccine j ourna l ho me pag e: www.elsevier.com/locate/vaccine Immunoprotective activity of a Salmonid Alphavirus Vaccine: Comparison of the immune responses induced by inactivated whole virus antigen formulations based on CpG class B oligonucleotides and poly I:C alone or combined with an oil adjuvant Hanna L. Thim a,1 , Dimitar B. Iliev a,1 , Karen E. Christie b , Stéphane Villoing b , Marian F. McLoughlin c , Guro Strandskog a , Jorunn B. Jørgensen a,∗ a Norwegian College of Fisheries Science, University of Tromsø, N-9037 Tromsø, Norway b Intervet Norbio, Thormøhlens gt. 55, N-5008 Bergen, Norway c Aquatic Veterinary Services, 35 Cherryvalley Park, Belfast, UK a r t i c l e i n f o Article history: Received 20 December 2011 Received in revised form 13 April 2012 Accepted 7 May 2012 Available online 23 May 2012 Keywords: Salmon alphavirus Atlantic salmon Vaccination Adjuvant CpG Poly I:C a b s t r a c t CpG oligonucleotides and polyinosinic:polycytidylic acid (poly I:C) are toll-like receptor (TLR) agonists that mimic the immunostimulatory properties of bacterial DNA and double-stranded viral RNA respec- tively, and which have exhibited potential to serve as vaccine adjuvants in previous experiments. Here, a combination of CpGs and poly I:C together with water- or oil-formulated Salmonid Alphavirus (SAV) antigen preparations has been used for a vaccine in Atlantic salmon and tested for protection in SAV challenge trial. The results demonstrate that vaccination with a high dose of the SAV antigen induced pro- tection against challenge with SAV which correlated with production of neutralizing antibodies (NAbs). As the high antigen dose alone induced full protection, no beneficial effect from the addition of CpG and poly I:C could be observed. Nevertheless, these TLR ligands significantly enhanced the levels of NAbs in serum of vaccinated fish. Interestingly, gene expression analysis demonstrated that while addition of oil suppressed the CpG/poly I:C-induced expression of IFN-, the upregulation of IFNa1 was substantially enhanced. A low dose of the SAV antigen combined with oil did not induce any detectable levels of NAbs either with or without TLR ligands present, however the addition of CpG and poly I:C to the low SAV antigen dose formulation significantly enhanced the protection against SAV suggesting that CpG/poly I:C may have enhanced a cytotoxic response – a process which is dependent on the up-regulation of type I IFN. These results highlight the immunostimulatory properties of the tested TLR ligands and will serve as a ground for further, more detailed studies aimed to investigate their capacity to serve as adjuvants in vaccine formulations for Atlantic salmon. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Pancreas disease (PD) is a serious viral disease in salmonid fish causing significant economic losses for the aquaculture industry in Europe [1]. PD is caused by Salmon Pancreas Disease Virus, now more commonly referred to as Salmonid Alphavirus (SAV) Abbreviations: NAbs, neutralizing antibodies; Ag, antigen; ODNs, oligodeoxynu- cleotides; TLR, toll-like receptor; APC, antigen-presenting cells; PRRs, pattern recognition receptors; RPP, relative percent protection; HK, head kidney; RT-qPCR, reverse transcriptase quantitative real-time-PCR; GMT, geometric mean titre; SPDV, Salmon Pancreas Disease Virus; wpc, weeks post challenge; wpv, weeks post vacci- nation; ip, intra peritoneally. ∗ Corresponding author. Tel.: +47 7764 6716; fax: +47 7764 6020. E-mail address: jorunn.jorgensen@uit.no (J.B. Jørgensen). 1 These authors contributed equally to this work. [2]. Based on sequencing and phylogenetic analysis SAV strains are grouped into 6 different subtypes: SAV1–6 [3]. SAV3 is exclusively found in Norway and affects both Atlantic salmon and rainbow trout [4,5]. Vaccines are considered to be the most effective countermea- sures against diseases in aquaculture. There is a commercial vaccine available against PD based on an inactivated SAV antigen which is administrated by intraperitoneal injection (Biering, Villoing et al. [8]). Currently most fish vaccines rely on adjuvants, which improve humoral and/or cytotoxic immune responses [6,7]. In salmonid aquaculture oil-based adjuvants are the most widely used [8,9], however, due to their negative side effects, it is desirable to develop other adjuvant concepts. It is now clear that effective adjuvants link innate and adap- tive immunity by signaling through a combination of PRRs [10]. CpG ODNs, which are short synthetic DNA sequences consisting 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2012.05.010