VP2, VP7, and NS1 proteins of bluetongue virus targeted in avian reovirus muNS-Mi microspheres elicit a protective immune response in IFNAR(−/−) mice
Introduction
Bluetongue (BT) is a non-contagious, insect-transmitted disease of wild and domestic ruminants characterized by vascular injury that results in tissue necrosis, hemorrhage, and edema that is caused by the bluetongue virus (BTV) (Maclachlan et al., 2009, Maclachlan et al., 2014, Verwoerd, 2004). BTV infection of ruminants occurs throughout much of the tropical and recently also the temperate climate regions of the world, coincident with the distribution of specific species of Culicoides biting midges that are the biological vectors of this arbovirus (Gibbs and Greiner, 1994, Savini et al., 2008). BTV, the prototype of the Orbivirus genus, is a pathogen of livestock that is common throughout the world including Europe, and that causes serious periodic outbreaks (Patel and Roy, 2014). The incursion of BTV-8 in Northern Europe in 2008 has re-stimulated the interest to develop improved vaccination strategies against BTV. In particular, safer, cross-reactive, more efficacious vaccines with differential diagnostic capability have been pursued by multiple BTV research groups and vaccine manufacturers (Calvo-Pinilla et al., 2014).
The inactivated BTV vaccines that are now being used in Europe are effective in preventing outbreaks of BTV but they are serotype specific, and raise a broad immune response to all of the virus structural proteins. It invalidates established serological assays (e.g. ELISA) for routine surveillance and import/export testing, since they cannot distinguish between infected and vaccinated animals. In addition, an autoimmune/autoinflammatory syndrome induced by adjuvants (ASIA syndrome) linked to the repetitive inoculation of aluminum-containing adjuvants has been described in sheep. The syndrome shows an acute phase that affects less than 0.5% of animals in a given herd, and appears 2–6 days after an adjuvant-containing inoculation. It is characterized by an acute neurological episode with low response to external stimuli and acute meningoencephalitis (Lujan et al., 2013). In Spain, with the reemergence of bluetongue in 2008, animals received inactivated vaccines against two strains of BTV (strains 1 and 8) in alum, allowing sheep to receive four inoculations in less than one month. The ASIA syndrome appeared shortly after across the country with severe consequences for the sheep industry (Lujan et al., 2013).
The safety of alum as a vaccine adjuvant is currently being questioned (Tomljenovic and Shaw, 2011). For this reason, the generation of protective subunits vaccines with new adjuvants could be an alternative to the inactivated vaccines. Although in most vaccine protocols adjuvants are essential to provide appropriate danger signals for successful induction of immune responses (Gallucci et al., 1999), particulate vaccines appear to contain intrinsic adjuvant activity and to induce immune responses without the need of other adjuvants (Allsopp et al., 1996, Plebanski et al., 1998). Vaccine development has progressed significantly and has moved from whole microorganisms to subunit vaccines that contain only their antigenic proteins. Subunit vaccines are often less immunogenic than whole pathogens but incorporation of antigens into biomaterials can achieve a desired vaccine response. Such biomaterials, typically particulates that have been classically used as drug delivery carriers, can combine the antigen of interest and the ligands that direct those antigens to antigen-presenting cells (APCs), such as B cells, macrophages and dendritic cells (DCs) (Demento et al., 2011).
In the livestock industry, cost is a major factor in vaccine choice. For these reasons, a stable multiepitope particulate material for immunization should be a good candidate for a vaccine against BTV. The IC-tagging methodology, a tagging and inclusion-targeting system based on the ARV muNS virosomes (Brandariz-Nunez et al., 2010a, b; Brandariz-Nunez et al., 2011), is a simple, versatile, and efficient method for immobilizing active proteins in muNS-Mi inclusions (Microspheres or MS) in baculovirus-infected cells, that are easily purified (Brandariz-Nunez et al., 2011). The MS can simultaneously recruit several tagged proteins (Brandariz-Nunez et al., 2010a) that can be used to generate complexes and create multiepitope particulate material for immunization purposes that could have potential advantages as vaccines: (i) MSs are particulate matter, and particulate immunogens are the best for stimulating both humoral and cellular immune responses (Roy, 1996); (ii) MS-derived immunogens are cheap and very stable; and (iii) they should be biologically safe, because organisms would be immunized with proteins and not with genetic material or viruses.
The genome of orbiviruses consist of ten linear double-stranded RNA genome segments (Seg-1 to Seg-10) encoding structural proteins VP1 to VP7 and non-structural proteins, NS1, NS2, NS3/NS3a, and NS4 (Belhouchet et al., 2011, Mertens et al., 1984, Ratinier et al., 2011, Roy, 2005, Roy, 2008). Immunological studies to date have given many clues about what BTV proteins are more important to induce protective host immune responses against the virus. It has been demonstrated that neutralizing antibody responses (Jeggo et al., 1984b) and cytotoxic T lymphocytes (CTL) have a main role in protective immunity against BTV (Jeggo and Wardley, 1982, Jeggo et al., 1984a). For this reason, the BTV antigens included in a vaccine composition should stimulate humoral and cellular immune responses. Epitope mapping studies revealed that the major virus neutralizing epitopes are located in the protein VP2 (Roy, 1992). BTV-specific CTL have been studied in sheep, showing VP2 and NS1 as major CTL targets although VP3, VP5 and VP7 were also recognized by CTLs (Andrew et al., 1995, Janardhana et al., 1999, Rojas et al., 2014, Rojas et al., 2011). Different strategies have been followed over the last 3 decades to develop novel recombinant vaccines for BTV, ranging from baculovirus expressed sub-unit vaccines to live virus vector vaccines Recently, our laboratory has showed that a heterologous prime boost vaccination strategy with DNA and the highly attenuated poxvirus vector modified vaccinia virus Ankara (rMVA) expressing VP2, VP7, and NS1 proteins of BTV-4, generated significant levels of neutralizing antibodies against BTV-4 in immunized IFNAR(−/−) mice. Furthermore, vaccination stimulated specific CD8+ T cell responses against these three BTV proteins. Importantly, the vaccine combination expressing NS1, VP2 and VP7 proteins of BTV-4, elicited sterile protection against a lethal dose of homologous BTV-4 infection and induced cross-protection against lethal doses of heterologous BTV-8 and BTV-1, two serotypes that are not related phylogenetically, suggesting that VP2, VP7, NS1 BTV proteins are key to the composition of a marker multiserotype vaccine against BTV (Calvo-Pinilla et al., 2012, Ortego et al., 2014).
In this study, we describe the generation of subunit BTV vaccines based on microspheres of ARV muNS-Mi containing BTV-4 VP2, VP7, and NS1 proteins. We show that immunization of mice deficient in type I IFN receptor (IFNAR(−/−)) with these microspheres and without adjuvants, achieves protective immunity and total or partial protection against homologous and heterologous infection with BTV-4 and BTV-1, respectively. These results indicate that the delivery of proteins VP2, VP7, and NS1 of BTV-4 in microspheres of ARV muNS-Mi protein is a promising subunit vaccine strategy to efficiently protect against BTV infection avoiding the use of added adjuvants.
Section snippets
Virus and cells
Baby hamster kidney (BHK-21) (ATCC, Cat. No. CCL-10), chicken embryo fibroblast (DF-1) (ATCC, Cat. No. CRL-12203), and Vero (ATCC, Cat. No. CCL-81) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM glutamine and 10% fetal calf serum (FCS). Insect cells High Five (Invitrogen) and Spodoptera Frugiperda Sf9 (ATCC Cat. No. CRL-1711) were grown in TC-100 medium supplemented with 10% FCS. BTV serotype 4 (SPA2004/01) (BTV-4) and serotype 1 (ALG2006/01) (BTV-1) was
Expression of untagged and IC-tagged proteins VP2, VP7, and NS1 of BTV-4
To obtain microspheres loaded with the BTV-4 proteins, we first generated recombinant baculoviruses to direct the expression of VP2, VP7 and NS1 containing the IC tag at the C-terminus. In parallel, we generated recombinant baculoviruses to express the untagged versions of the same proteins for comparison. Protein extracts from Sf9 cells infected with all the recombinant baculoviruses were analyzed by SDS–PAGE. Protein bands with the apparent molecular weight corresponding to VP7 (38 kDa), NS1
Discussion
Vaccination has been an effective approach to control BTV spread. Chemically inactivated vaccines, commercially available, protect against BTV infection but they are serotype specific and do not allow to distinguish between infected and vaccinated animals. Consequently, there are currently many efforts to develop new types of vaccines with improved safety and efficacy for a broad range of BTV serotypes (Boone et al., 2007, Calvo-Pinilla et al., 2012, Calvo-Pinilla et al., 2009, Celma et al.,
Acknowledgments
We thank Dr. Juan Anguita for stimulating discussions and critically reading the manuscript. We also thank and Francisco Mateos for excellent technical assistance. This investigation was supported by Spanish Grants AGL-2011-23506 and BFU2010-22228.
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Present address: The Pirbright Institute, Ash Road, Pirbright, Surrey GU24 0NF, UK.