Application of Bluetongue Disabled Infectious Single Animal (DISA) vaccine for different serotypes by VP2 exchange or incorporation of chimeric VP2
Graphical abstract
Introduction
Bluetongue (BT) is a notifiable disease of ruminants caused by the bluetongue virus (BTV) and is spread by Culicoides biting midges. BT is endemic in tropical regions, but is expanding to regions with a moderate climate due to emerging vector species [1], [2]. BT outbreaks can cause large economic losses due to diseased animals and trade restrictions [3], [4].
BTV (family Reoviridae, genus Orbivirus) has a ten-segmented (Seg-1–10), double stranded (ds) RNA genome. The non-enveloped virus particle contains a triple layered capsid of viral proteins VP2, 3, 5, and 7. VP2 is the most immunogenic and the major serotype determining protein, inducing neutralizing antibodies (nAbs) [5]. There are at least 26 BTV serotypes, defined by neutralizing antibodies and confirmed by VP2 phylogenetic analyses [6], [7]. In addition to the replication complex consisting of VP1, 4, and 6 [8], [9], [10], BTV expresses four non-structural (NS) proteins [11], [12], [13]. Although NS3/NS3a is important for virus release from infected cells [14], [15], [16], expression is not essential for virus replication in vitro [17], [18].
Control of BT outbreaks is hardly possible without vaccination [19]. Vaccination in African countries occurs with conventionally live-attenuated BT vaccines [20], but the safety of these vaccines is questionable [21], [22], [23]. Safe and effective inactivated BT vaccines have been developed [24], however, protection might not be long lasting, and annual re-vaccination is recommended. In Europe, BTV serotypes 1, 2, 4, 8, 9 and 16 are currently present [25].
Recently, we developed BT Disabled Infectious Single Animal (DISA) vaccines for serotype 8 [26], [45]. DISA vaccine 8 consists of the backbone of vaccine-related BTV6/net08 [27] without NS3/NS3a expression, and VP2 of serotype 8. DISA vaccine 8 is avirulent, replicates locally without causing detectable viremia, induces serotype specific protection against virulent BTV8, and enables the differentiation of infected from vaccinated animals (DIVA) based on NS3-directed antibodies.
Exchange of outer capsid proteins has been described for several BTV serotypes [28], [29], but is not possible for all 26 serotypes [30]. VP2 interacts with VP5 and VP7 and is involved in cell entry [31]. However, the regions involved in cell entry, and which are thus exposed to the outer part of the virion, as well as VP2 regions interacting with VP5 and VP7 have not been mapped. The VP2 sequence is highly variable between serotypes and there are only some conserved cysteine residues and two more conserved areas described [32]. Neutralizing epitopes are more prevalent in two regions in VP2, but are also scattered throughout the protein [33], [34], [35], [36]. A protein structure has been proposed for VP2 of the related African horse sickness virus (AHSV), and the outer tip domain of AHSV VP2 triskellions has been mapped to amino acid (AA) region 279–368 of BTV VP2 [37].
The BT DISA vaccine platform was here applied for BTV serotypes currently present in Europe by exchange of VP2, but rescue of virus with VP2 of serotype 16 was not possible. Therefore, chimeric VP2 genes encoding proposed immunogenic regions of BTV16 VP2 were expressed on BTV1 and BTV1 without NS3/NS3a expression. The serotype specific immunogenicity of both parental serotypes was examined in vitro and in vivo by neutralization assays and vaccination of sheep.
Section snippets
Cells and viruses
BSR cells (a clone of BHK-21 cells [38]) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen), containing 5% fetal bovine serum (FBS), 100 IU ml−1 Penicillin, 100 μg ml−1 Streptomycin and 2.5 μg ml−1 Amphotericin B.
BTV1 generated by reverse genetics (Genbank accession numbers FJ969719–FJ969728) was used as virus backbone to rescue BTV1 derivatives as previously described [39], [40]. Seg-2 originating from strain BTV2 (JN255863), BTV4 (AJ585125), BTV8 (AM498052), BTV9 (AJ585130),
Exchange of entire VP2 in BTV of serotypes currently present in Europe
BTV1 with VP2 of serotype 2, 4, 8 or 9 was generated by use of reverse genetics. Exchange of single VP2 of proposed BTV serotype 25 (Toggenburg orbivirus) [41] did not result in viable virus (Table 2), whereas exchange of both VP2 and VP5 of serotype 25 was successful (not shown). For serotype 16, exchange of single VP2 (Table 2), or of both VP2 and VP5 did not result in virus rescue (not shown).
Exchange of VP2 was combined with Seg-10 deletion ΔC (NS3/NS3a knockout) to generate BTV1-based DISA
Discussion
Previously, we described the development of the BT Disabled Infectious Single Animal (DISA) vaccine for serotype 8 [26], [45]. Here, we investigated the broader application of this DISA vaccine platform for ‘European’ serotypes 2, 4, 8, 9 and 16 by exchange of entire VP2 in BTV1 without NS3/NS3a expression. DISA vaccine with VP2 of serotype 2, 4, 8 and 9 were generated and virus growth and plaque size were in most cases similar to BTV1 DISA vaccine. Exchange of VP2 of serotype 16 in the BTV1
Acknowledgements
This study was funded by the Dutch Ministry of Economic Affairs (CVI-project 1600020-01). We thank René van Gennip for fruitful discussions and Rob Moormann for critical reading of the manuscript. We also thank the CVI animal caretakers for excellent assistance and Phaedra Eblé for veterinary supervision of the animal experiment. We would also like to thank Carrie Batten for the generous gift of anti-BTV16 serum, Mieke Maris for assistance with neutralization assays and Aldo Dekker for support
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