Application of Bluetongue Disabled Infectious Single Animal (DISA) vaccine for different serotypes by VP2 exchange or incorporation of chimeric VP2

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ARTICLE IN PRESSG ModelVAC 15977 1–7

Vaccine xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Vaccine

j our na l ho me page: www.elsev ier .com/ locate /vacc ine

pplication of bluetongue Disabled Infectious Single Animal (DISA)accine for different serotypes by VP2 exchange or incorporation ofhimeric VP2

emke Feenstraa,b,∗, Janny S. Papa, Piet A. van Rijna,c

Department of Virology, Central Veterinary Institute of Wageningen UR (CVI), Lelystad, The NetherlandsDepartment of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The NetherlandsDepartment of Biochemistry, Centre for Human Metabonomics, North-West University, South Africa

r t i c l e i n f o

rticle history:eceived 29 October 2014eceived in revised form 1 December 2014ccepted 2 December 2014vailable online xxx

eywords:luetongue virusISA vaccineerotypeP2himeraS3/NS3a

a b s t r a c t

Bluetongue is a disease of ruminants caused by the bluetongue virus (BTV). Bluetongue outbreaks canbe controlled by vaccination, however, currently available vaccines have several drawbacks. Further,there are at least 26 BTV serotypes, with low cross protection. A next-generation vaccine based onlive-attenuated BTV without expression of non-structural proteins NS3/NS3a, named Disabled Infec-tious Single Animal (DISA) vaccine, was recently developed for serotype 8 by exchange of the serotypedetermining outer capsid protein VP2. DISA vaccines are replicating vaccines but do not cause detectableviremia, and induce serotype specific protection. Here, we exchanged VP2 of laboratory strain BTV1for VP2 of European serotypes 2, 4, 8 and 9 using reverse genetics, without observing large effects onvirus growth. Exchange of VP2 from serotype 16 and 25 was however not possible. Therefore, chimericVP2 proteins of BTV1 containing possible immunogenic regions of these serotypes were studied. BTV1,expressing 1/16 chimeric VP2 proteins was functional in virus replication in vitro and contained neutral-izing epitopes of both serotype 1 and 16. For serotype 25 this approach failed. We combined VP2 exchangewith the NS3/NS3a negative phenotype in BTV1 as previously described for serotype 8 DISA vaccine. DISA
vaccine with 1/16 chimeric VP2 containing amino acid region 249–398 of serotype 16 raised antibodiesin sheep neutralizing both BTV1 and BTV16. This suggests that DISA vaccine could be protective for bothparental serotypes present in chimeric VP2. We here demonstrate the application of the BT DISA vaccineplatform for several serotypes and further extend the application for serotypes that are unsuccessful insingle VP2 exchange.
© 2014 Elsevier Ltd. All rights reserved.

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. Introduction

Bluetongue (BT) is a notifiable disease of ruminants causedy the bluetongue virus (BTV) and is spread by Culicoides bitingidges. BT is endemic in tropical regions, but is expanding to

egions with a moderate climate due to emerging vector species1,2]. BT outbreaks can cause large economic losses due to diseasednimals and trade restrictions [3,4].

BTV (family Reoviridae, genus Orbivirus) has a ten-segmented

Please cite this article in press as: Feenstra F, et al. Application of bdifferent serotypes by VP2 exchange or incorporation of chimeric VP2

Seg-1–10), double stranded (ds) RNA genome. The non-envelopedirus particle contains a triple layered capsid of viral proteinsP2, 3, 5, and 7. VP2 is the most immunogenic and the major

∗ Corresponding author at: P.O. Box 65, 8200 AB Lelystad, The Netherlands.el.: +31 0320 238 834.

E-mail address: [email protected] (F. Feenstra).

ttp://dx.doi.org/10.1016/j.vaccine.2014.12.003264-410X/© 2014 Elsevier Ltd. All rights reserved.

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serotype determining protein, inducing neutralizing antibodies(nAbs) [5]. There are at least 26 BTV serotypes, defined by neutral-izing antibodies and confirmed by VP2 phylogenetic analyses [6,7].In addition to the replication complex consisting of VP1, 4, and 6[8–10], BTV expresses four non-structural (NS) proteins [11–13].Although NS3/NS3a is important for virus release from infectedcells [14–16], 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 conventionallylive-attenuated BT vaccines [20], but the safety of these vaccinesis questionable [21–23]. Safe and effective inactivated BT vaccineshave been developed [24], however, protection might not be long

luetongue Disabled Infectious Single Animal (DISA) vaccine for. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.12.003

lasting, and annual re-vaccination is recommended. In Europe, BTVserotypes 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]. DISA vaccine 8 consists of the

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dx.doi.org/10.1016/j.vaccine.2014.12.003


http://www.sciencedirect.com/science/journal/0264410X

http://www.elsevier.com/locate/vaccine

mailto:[email protected]


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Table 1List of primers. Primers were used for amplification by RT-PCR to identify Seg-2 andSeg-10 of rescued BTV mutants and BTV1-based DISA vaccines.

Primer Sequence

BTV1 Seg-2 F 5′-TTGTTGAAAGTACGAGACACAAGAG-3′

BTV1 Seg-2 R 5′-GTATCAGCCTTCTTTGAATCGATT-3′

BTV2 Seg-2 F 5′-TCAAAGATGAGGGGATACGG-3′

BTV2 Seg-2 R 5′-AAGCGGCTGTTGATCCATAC-3′

BTV4 Seg-2 F 5′-TGTCCCACAATGGAGGAGTT-3′

BTV4 Seg-2 R 5′-CATCCACTTAGCATCCGTCATA-3′

BTV6 Seg-2 F 5′-AGGAACAGTCGGCTTATCAC-3′

BTV6 Seg-2 R 5′-TTCGCTAATGTGCTTCTCCAT-3′

BTV8 Seg-2 F 5′-CGGAGACAGCGCAGTATGTA-3′

BTV8 Seg-2 R 5′-CCTCGGTAGTATCCCTCACG-3′

BTV9 Seg-2 F 5′-ACGTTGATYGCWRCRGAATG-3′

BTV9 Seg-2 R 5′-TSCCWTSDTCRTGAAAGAGT-3′

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ARTICLEVAC 15977 1–7

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ackbone of vaccine-related BTV6/net08 [27] without NS3/NS3axpression, and VP2 of serotype 8. DISA vaccine 8 is avirulent, repli-ates locally without causing detectable viremia, induces serotypepecific protection against virulent BTV8, and enables the differ-ntiation of infected from vaccinated animals (DIVA) based onS3-directed antibodies.

Exchange of outer capsid proteins has been described for severalTV serotypes [28,29], but is not possible for all 26 serotypes [30].P2 interacts with VP5 and VP7 and is involved in cell entry [31].owever, the regions involved in cell entry, and which are thusxposed to the outer part of the virion, as well as VP2 regions inter-cting with VP5 and VP7 have not been mapped. The VP2 sequences highly variable between serotypes and there are only some con-erved cysteine residues and two more conserved areas described32]. Neutralizing epitopes are more prevalent in two regions inP2, but are also scattered throughout the protein [33–36]. A pro-

ein structure has been proposed for VP2 of the related Africanorse sickness virus (AHSV), and the outer tip domain of AHSV VP2riskellions has been mapped to amino acid (AA) region 279–368f BTV VP2 [37].

The BT DISA vaccine platform was here applied for BTV serotypesurrently present in Europe by exchange of VP2, but rescue of virusith VP2 of serotype 16 was not possible. Therefore, chimeric VP2

enes encoding proposed immunogenic regions of BTV16 VP2 werexpressed on BTV1 and BTV1 without NS3/NS3a expression. Theerotype specific immunogenicity of both parental serotypes wasxamined in vitro and in vivo by neutralization assays and vaccina-ion of sheep.

. Materials and methods

.1. Cells and viruses

BSR cells (a clone of BHK-21 cells [38]) were cultured in Dul-ecco’s modified Eagle’s medium (DMEM, Invitrogen), containing% fetal bovine serum (FBS), 100 IU ml−1 Penicillin, 100 �g ml−1

treptomycin and 2.5 �g ml−1 Amphotericin B.BTV1 generated by reverse genetics (Genbank accession num-

ers FJ969719–FJ969728) was used as virus backbone to rescueTV1 derivatives as previously described [39,40]. Seg-2 originating

rom strain BTV2 (JN255863), BTV4 (AJ585125), BTV8 (AM498052),TV9 (AJ585130), BTV16 (AJ585137) and BTV25 (EU839840) weresed for VP2 exchange. Seg-10 originating from BTV8 (AM498060)ith the out-of-frame deletion �C was used to generate NS3/NS3a

nockout BTV (DISA vaccine platform) [17]. Chimeric VP2 genesere partly synthesized by Genscript Corporation (Piscataway NJ,SA) and inserted in BTV1 Seg-2 using standard procedures withppropriate restriction enzymes (Fig. 1).

Virus stocks were produced by infection of BSR cells at low mul-iplicity of infection (MOI), and were harvested by freeze thawinghen >50% of cells were immunostained with � VP7 monoclonal

ntibody (MAb) ATCC-CRL-1875, in a duplicate well, or when >50%f cells showed cytopathogenic effect (CPE). Virus in clarified super-atant was used for plaque assays, growth kinetics and the animalxperiment. Seg-2 and Seg-10 were confirmed by serotype specificCR testing with primers targeting Seg-2, or using PCR with Seg-10rimers followed by sequencing (Table 1).

Virus titers were determined by endpoint dilution on BSRells and expressed as 50% tissue culture infectious dose per mlTCID50 ml−1).


.2. Virus growth and plaque morphology

BSR cells in wells of a 24-wells plate were infected with anOI of 0.001. Virus was attached to the cells for 1.5 h at 37 ◦C.

F-full-S10* 5′-GTTAAAAAGTGTCGCTGCC-3′

R-full-S10 5′-GTAAGTGTGTAGTGTCGCGCAC-3′

Unattached virus was removed by washing with PBS, and freshmedium was added. This time point was set as 0 h post-infection(hpi). Incubation at 37 ◦C was continued and cells were harvestedby freeze thawing at −80 ◦C at indicated time points between 0 and56 hpi. Virus titers were determined and growth experiments wereindependently repeated three times. To examine plaque morphol-ogy, cell monolayers were infected with 10-fold dilutions of virusstocks. After attachment for 1.5 h at 37 ◦C, cells were washed andDMEM containing 1% methylcellulose was added, and incubation at37 ◦C was continued for 48 h. Then, methylcellulose was removedand monolayers were fixed and immunostained using � VP7 MAbATCC-CRL-1875.

2.3. Animal experiment

The animal experiment was performed under the guidelines ofthe European Community and was approved by the Committee onthe Ethics of Animal Experiments of the Central Veterinary Insti-tute (permit number 2014.025). BTV1-based DISA vaccine with1/16 chimeric VP2, in which 150 amino acids (AA) from position249–398 were exchanged, was used for vaccination (Fig. 1). Fourfemale Blessumer sheep of 6–24 months old were free of BTVand BTV antibodies and were vaccinated subcutaneously in theback between the shoulder blades at both sides of the spinal cordwith 2× 1 ml of 105 TCID50 ml−1. Body temperature and clinicalsigns were examined daily from three days before to seven dayspost-vaccination (dpv) and every other day during the rest of theexperiment. Serum was collected frequently in the first two weeksafter vaccination, and at 21, 28, 34 and 42 dpv.

2.4. Neutralization assays

Anti-BTV1 serum was collected at 42 days post-infection from asheep experimentally infected with synthetic BTV1 (experiment2011.003 at CVI). Anti-BTV8 serum was from goat 887 from afield infection. Anti-BTV16 serum was from a naturally infectedsheep (UN95, Pirbright Institute, UK). Serum from sheep vaccinatedwith DISA vaccine with 1/16 chimeric VP2 150AA was collected at21 dpv.

Virus stocks were prediluted to 104 TCID50 ml−1. Then, viruswas diluted twelve subsequent times 1:1 in a volume of 100 �l,and 50 �l 1:2.5 anti-BTV serum in medium was added to virus dilu-tions. Virus-serum mixtures were incubated during 1 h at 37 ◦C, andadded to BSR monolayers in 96-wells plates with 50 �l medium


per well. Virus without serum was used as negative control forneutralization. After two days incubation at 37 ◦C with 5% CO2,wells with the highest virus dilution still showing CPE were deter-mined. Single neutralization assays were independently repeated

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ARTICLE IN PRESSG ModelJVAC 15977 1–7

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Fig. 1. Schematic overview of BTV VP2 and the studied chimeric VP2 proteins. (A) Schematic representation of BTV VP2 with conserved regions (AA 357–398 and 946–961)Q5(green) [32], serotype determinants (AA 199–213 and 321–338) (red) and neutralizing epitopes (AA 199–654) (gray) [33]. The predicted tip domain of VP2 (AA 279–368)based on VP2 of AHSV [37] is indicated in striped blue. (B) Regions of BTV1 VP2 that were exchanged for these of serotype 8, 16 or 25 are indicated. BTV1 and BTV1-basedD oteinsi

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ISA vaccines were generated with Seg-2 expressing some of these chimeric VP2 prs referred to the web version of the article.)

hree subsequent times due to limited amounts of available serum.eutralization assays using serum from sheep vaccinated with DISAaccine with 1/16 chimeric VP2 150AA were performed in triplicatend repeated four independent times.

Virus titers were determined according to Spearman and Kärber.Ab titers in sera were calculated by the difference in 2 log titer withnd without addition of serum (neutralization index). Wells weremmunostained using � VP7 MAb to visualize inhibitory effects byerum on virus growth.

. Results

.1. Exchange of entire VP2 in BTV of serotypes currently presentn Europe

BTV1 with VP2 of serotype 2, 4, 8 or 9 was generated byse of reverse genetics. Exchange of single VP2 of proposed BTVerotype 25 (Toggenburg orbivirus) [41] did not result in viableirus (Table 2), whereas exchange of both VP2 and VP5 of serotype5 was successful (not shown). For serotype 16, exchange of singleP2 (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 �C

NS3/NS3a knockout) to generate BTV1-based DISA vaccines forEuropean’ serotypes 2, 4, 8 and 9 (not shown).

.2. Incorporation of chimeric VP2 proteins in BTV

Different chimeric VP2 genes were constructed to change theTV serotype without disturbing VP2 protein folding and function-lity. First, the predicted tip domain [37] of AA 279–368 (90AA) oferotype 8, 16 and 25 were exchanged in BTV1 VP2. Rescued virusesere named Chimera 8 90AA and Chimera 16 90AA, but Chimera

5 90AA could not be rescued. Then, the exchanged region was


nlarged for serotype 8 and 16 to the 450AA region (AA 200–650)here neutralizing epitopes are known to be located [33]. How-

ver, BTV1 with these chimeric VP2 proteins could not be rescued.inally, intermediate enlargements of serotype 8 and 16 were

able 2TV1 with exchanged VP2 proteins. Rescue of BTV1 with exchanged VP2 or withhimeric VP2 containing proposed immunogenic regions of different serotypes isndicated by “+”, no virus rescue by “−”, and not done by “nd”.

2 4 8 9 16 25

Entire VP2 + + + + − −Chimera 90 AA nd nd + nd + −Chimera 150 AA nd nd + nd + ndChimera 210 AA nd nd + nd − ndChimera 450 AA nd nd − nd − nd

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(Table 2). (For interpretation of the references to color in figure legend, the reader

studied; chimeric VP2 containing AA region 249–398 (150AA) orAA region 219–428 (210AA). Both chimeric viruses were generatedfor serotype 8 (Chimera 8 150AA and Chimera 8 210AA), whereasfor serotype 16 only Chimera 16 150AA could be rescued (Fig. 1 andTable 2).

BTV1-based DISA vaccine for serotype 16 was rescued by 1/16chimeric VP2 150AA in combination with Seg-10 deletion �C (DISAchimera 16 150AA).

3.3. Virus growth and plaque morphology

To study virus replication of BTV1 with exchanged VP2, plaquesize and virus growth were determined and compared with wildtype BTV1. No large differences in virus growth and plaque sizewere examined. Virus growth of BTV1 with VP2 from serotype 2was slightly reduced (Fig. 2A) and BTV1 with VP2 from serotype 4formed slightly larger plaques (Fig. 2B). Interestingly, expression ofchimeric VP2 on BTV1 did not influence virus growth or plaque size(Fig. 2A and B). Virus growth of BTV1 DISA vaccines with exchangedVP2 proteins was also studied (Fig. 2C). Again, exchange of most VP2proteins did not affect virus growth compared to the original BTV1DISA vaccine virus. BTV1 DISA vaccine with VP2 from serotype 2showed however a lower virus titer which is in agreement withthe slightly reduced virus growth of BTV1 with VP2 from serotype2. Expression of VP2 from serotype 8 did also negatively influencethe virus titer. Expression of chimeric VP2 did again not influencevirus growth.

3.4. Chimeric VP2 proteins contain neutralizing epitopes of bothparental serotypes

BTV1, BTV8, BTV16, Chimera 8 90, 150 and 210AA and Chimera16 90 and 150AA were studied in neutralization assays with anti-BTV1, anti-BTV8 and anti-BTV16 serum to determine serotypespecific neutralization (Fig. 3). Serotype specific neutralization ofBTV1, 8 and 16 was clearly observed with their respective serum.Weak cross-neutralization was observed between serotype 1 and 8(Fig. 3A), whereas serotype 1 and 16 did not show any cross neutral-ization (Fig. 3B). Chimera 8 90, 150 and 210AA were neutralized byboth anti-BTV1 and anti-BTV8 serum (Fig. 3A). Chimera 16 90 and150AA were neutralized by both anti-BTV1 and anti-BTV16 serum,although neutralization is very low for Chimera 16 90AA with anti-BTV16 serum. Chimera 16 150AA is however clearly neutralized byanti-BTV16 serum, which shows that 1/16 chimeric VP2 150AA con-tains more serotype 16 specific neutralization epitopes than 1/16


chimeric VP2 90AA (Fig. 3B).Although CPE was often still visible, indicating that neutral-

ization was not complete, addition of serum obviously reducedCPE as was shown by immunostaining of plaques. CPE in wells

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Fig. 2. Virus growth and plaque morphology of BTV with exchanged VP2. (A) Virus growth of BTV1 with exchanged VP2 of serotype 2, 4, 8, 9 and Chimera 16 150AA wasstudied and compared to growth of BTV1. Growth of BTV1 with VP2 of serotype 2 was slightly reduced (B) Plaque size of BTV1 with exchanged VP2 of serotype 2, 4, 8, 9 andChimera 16 150AA was studied and compared with BTV1 at 48 hpi. Expression of VP2 from serotype 4 led to slightly larger plaque size, but further no large differences wereexamined. Bars represent 400 �m. (C) Virus growth of BTV1-based DISA vaccines with exchanged VP2 proteins of serotype 2, 4, 8, 9 and Chimera 16 150AA were studied andcompared with growth of BTV1 DISA vaccine. Virus growth of DISA vaccine with VP2 of serotype 2 was strongly retarded, and expression of VP2 from serotype 8 also led tol ic VP2w the m

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ess high virus titers. All other mutant viruses, including the one expressing chimerere repeated three independent times and error bars represent standard error of

ithout serum still showing complete CPE was compared to wellsf the same virus dilution in which serum was added (Fig. 3C and). Obviously, BTV1, 8, and 16 were neutralized by their respective

erum. BTV with 1/8 chimeric VP2 or with 1/16 chimeric VP2 pro-eins were neutralized by both anti-BTV1 and anti-BTV8 serum, ory both anti-BTV1 and anti-BTV16 serum, respectively (Fig. 3C and). Summarizing, incorporation of chimeric VP2 proteins resulted


n exchange of serotype specific neutralizing epitopes, including for/16 chimeric VP2 proteins for which exchange of entire VP2 wasot possible. Furthermore, all studied viruses expressing chimericroteins contain neutralizing epitopes of both parental serotypes.

, showed growth characteristics similar to that of BTV1 DISA vaccine. Experimentsean (SEM).

3.5. DISA vaccine with 1/16 chimeric VP2 induces nAbs againstserotype 1 and 16

DISA chimera 16 150AA was used to vaccinate sheep. None of thesheep showed fever or clinical signs, and no viremia was detectedafter vaccination (not shown). All four sheep seroconverted forVP7 directed antibodies, which indicates that DISA chimera 16


150AA replicates in vivo (not shown). Preliminary results showedsome variability between sheep for nAb titers (not shown), andserum from one sheep at 21 dpv with the highest neutralizationindex was selected for further neutralization assays. This serum

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Fig. 3. Neutralization of BTV expressing chimeric VP2 proteins. Anti-BTV1, BTV8 and BTV16 sera were used to neutralize BTV1, BTV8, BTV16, Chimera 8 90AA, 150AA and210AA, and Chimera 16 90AA and 150AA. The difference between the 2 log virus titer with and without serum is the neutralization index. Single neutralization assays wererepeated three times and error bars represent SEM. For comparison, BSR monolayers infected with the same virus dilutions still showing complete CPE without serum, andw 0AA aa era 8

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ith serum, stained with � VP7 MAb are shown. (A) Chimera 8 90AA, 150AA and 21re neutralized by anti-BTV1 and anti-BTV16 serum. (C) Plaque formation of Chimlaque formation of Chimera 16 90AA and 150AA is inhibited by both anti-BTV1 an

learly neutralizes BTV1 and Chimera 16 150AA, and also BTV16,ut with lower neutralization index (Fig. 4A). Weak cross neutral-

zation was also detected for BTV8. These results confirm that DISAhimera 16 150AA expresses neutralizing epitopes of both parentalerotypes, as confirmed by immunostaining (Fig. 4B). Apparently,ISA chimera 16 150AA induced a low nAb titer against BTV8, which

eemed to be higher than the amount of anti-BTV8 nAbs present inTV1 serum (Fig. 3A and C).

. Discussion

Previously, we described the development of the BT Disablednfectious Single Animal (DISA) vaccine for serotype 8 [26]. Here,

e investigated the broader application of this DISA vaccine plat-orm for ‘European’ serotypes 2, 4, 8, 9 and 16 by exchange ofntire VP2 in BTV1 without NS3/NS3a expression. DISA vaccineith VP2 of serotype 2, 4, 8 and 9 were generated and virus growth


nd plaque size were in most cases similar to BTV1 DISA vaccine.xchange of VP2 of serotype 16 in the BTV1 backbone was not possi-le, which limits the application of the DISA vaccine platform for allTV serotypes. Exchange of serotype determining VP2 is apparently

re neutralized by anti-BTV1 and anti-BTV8 serum. (B) Chimera 16 90AA and 150AA90AA, 150AA and 210AA is inhibited by both anti-BTV1 and anti-BTV8 serum. (D)-BTV16 serum. Bars represent 400 �m.

limited, suggesting that interactions between outer capsid proteinsVP2 and VP5, but likely also with inner capsid protein VP7, areimportant for BTV rescue. Nunes et al. [30] have extensively stud-ied the exchange of outer capsid proteins using the BTV1 backbone,and found that VP2 and VP5 of some serotypes, like BTV16, cannotbe exchanged. In further agreement with these authors, BTV1 withboth VP2 and VP5 of serotype 25 could be generated (not shown),although exchange of solely VP2 was impossible.

To apply the DISA vaccine platform for serotypes that wereunsuccessful for VP2 exchange, we investigated the incorpora-tion of chimeric VP2 in BTV1, and included several 1/8 chimericVP2 genes, as controls. BTV1 with 1/8 chimeric VP2 (AA position220–429) was recently described, and is neutralized by both anti-BTV8 and anti-BTV1 serum [30]. Indeed, in our studies 1/8 chimericVP2 proteins with exchanged serotype 8 specific sequences of 90,150 or 210 AA in length could be successfully incorporated intoBTV1. In contrast, enlargement of the serotype 8 specific region


to 450AA (from AA position 200 to 650) arrested virus rescue.This was remarkable, since BTV1 with entire VP2 from serotype8 could be generated. We hypothesize that this 1/8 chimeric VP2is not functional due to protein misfolding/malfunction within VP2

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Fig. 4. DISA vaccine with 1/16 chimeric VP2 induces neutralizing antibodies against both parental serotypes. Serum from sheep vaccinated with BTV1-based DISA vaccineexpressing 1/16 chimeric VP2 150AA was used to neutralize BTV1, BTV8, BTV16, and Chimera 16 150AA. (A) The difference between virus titer with and without serum ist ere rei nd wit

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he neutralization index. Neutralization assays were performed in triplicate and wnfected with the same virus dilutions still showing complete CPE without serum a

r between VP2 proteins in the VP2 triskellions [37], instead ofon-functional interactions between the different capsid proteins.

Efficiency of rescue of BTV1 with chimeric VP2 varied consid-rably and was dependent on the exchanged region as well as onhe serotype (Table 2). Chimeric VP2 with a maximum of 210AAriginating from serotype 8 or a maximum of 150AA originatingrom serotype 16 was functional, whereas exchange of VP2 withnly 90AA from serotype 25 did not result in viable virus. Appar-ntly, regions that can be exchanged are not conserved amongerotypes, which complicates the development of chimeric VP2enes.

Complete VP2 of BTV16 could not be incorporated into BTV1, butTV1 expressing chimeric 1/16 VP2 protein is neutralized by seraaised against both parental serotypes. We therefore show here,or the first time, a possible solution for the inability of generatingaccines of certain serotypes in a vaccine platform. Enlargement ofhe exchanged region of serotype 16 did result in a higher neutral-zation index by anti-BTV16 serum, suggesting that neutralizingpitopes are scattered throughout regions of VP2 [33]. Detailedapping of neutralizing epitopes, a detailed structure of BTV VP2

nd more studies on protein-protein interactions between VP2, VP5nd VP7 are needed to rationally design chimeric VP2 genes andISA vaccines for serotypes of which exchange of entire VP2 is notossible.

DISA chimera 16 150AA was used to vaccinate sheep. Weemonstrated that BTV with chimeric VP2 is functional in viruseplication in vivo, and induces serotype specific nAbs against botherotypes. Although nAb titers are not necessarily related to pro-ection [42], sheep vaccinated with DISA vaccine 8 are completelyrotected early and late after vaccination despite low nAb titers26]. Yet, it is unknown whether DISA chimera 16 150AA is protec-ive. Vaccination/challenge experiments, are needed to elucidate


he level of serotype specific protection by DISA vaccine expressinghimeric VP2.

DISA vaccines for different serotypes might be used to formulateocktail vaccines. Cocktails of traditionally live-attenuated vaccines

peated four times. Error bars represent SEM. (B) For comparison, BSR monolayersh serum, stained with � VP7 MAb are shown. Bars represent 1000 �m.

have been used to induce a broad protection [20], but are question-able with respect to safety by potential rise of virulent reassortantsor reversion to virulence. Broad protection could be safely achievedby combining DISA vaccines for different BTV serotypes, since thereis no risk of reassortment between vaccine viruses as these sharenine out of ten genome segments. There is also a very low risk onreversion to virulence since NS3/NS3a expression is stably blockedby a deletion in Seg-10.

In this study, reverse genetics derived BTV1 [39,40] was usedto rescue DISA vaccines for different BTV serotypes. Recently, wehave shown that vaccine-related strain BTV6/net08 [27,43,44] isthe preferred vaccine backbone for the DISA vaccine platform [26].Development of BTV6-based DISA vaccines for different serotypes isin progress in order to perform vaccination/challenge experimentsin ruminants.

Acknowledgements

This study was funded by the Dutch Ministry of Economic

Affairs (CVI-project 1600020-01). We thank René van Gennip forfruitful discussions and Rob Moormann for critical reading of themanuscript. We also thank the CVI animal caretakers for excellentassistance and Phaedra Eblé for veterinary supervision of the ani-mal experiment. We would also like to thank Carrie Batten for thegenerous gift of anti-BTV16 serum, Mieke Maris for assistance withneutralization assays and Aldo Dekker for support in statistical dataanalysis.

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Documents

Application of Bluetongue Disabled Infectious Single Animal (DISA) vaccine for different serotypes by VP2 exchange or incorporation of chimeric VP2