Elsevier

Vaccine

Volume 33, Issue 42, 13 October 2015, Pages 5539-5545
Vaccine

Development of a competitive ELISA for NS3 antibodies as DIVA test accompanying the novel Disabled Infectious Single Animal (DISA) vaccine for Bluetongue

https://doi.org/10.1016/j.vaccine.2015.09.020Get rights and content

Highlights

Abstract

Recently, we have developed a novel vaccine for Bluetongue named BT Disabled Infectious Single Animal (DISA) vaccine. Due to the lack of non-essential NS3/NS3a protein, BT DISA vaccine is a replicating vaccine, but without the inherent risks of live-attenuated vaccines, such as residual virulence or reversion to virulence by mutations, reassortment with field virus, horizontal spread by vectors and vertical transmission. The immune response induced by BT DISA vaccines is rapidly induced, highly protective and serotype specific which is dependent on the immunodominant and serotype determining VP2 protein. The BT DISA vaccine platform provides the replacement of exclusively VP2 from different serotypes in order to safely formulate multivalent cocktail vaccines. The lack of NS3/NS3a directed antibodies by BT DISA vaccination enables differentiation of infected from vaccinated animals (DIVA principle). A highly conserved immunogenic site corresponding to the late domain was mapped in the N-terminal region of NS3. We here established an NS3-specific competitive ELISA (NS3 cELISA) as serological DIVA test accompanying BT DISA vaccines. To this end, NS3 protein missing putative transmembrane regions was produced in large amounts in bacteria and used as antigen in the NS3 cELISA which was investigated with a variety of sera. The NS3 cELISA displayed a high sensitivity and specificity similar to the commercially available VP7-specific cELISA. Results of previously performed vaccination-challenge trials with BT DISA vaccines clearly demonstrate the DIVA system based on the NS3 cELISA and BT vaccine free of NS3 protein.

Introduction

Bluetongue (BT) has been listed as a notifiable disease to the World Organisation for Animal Health (OIE) [1]. BT is a non-contagious disease of ruminants caused by bluetongue viruses (BTV) transmitted by Culicoides biting midges [2], [3]. BTV causes economic losses by morbidity and mortality but mainly by restrictions on movement and trade of animals and animal products [4]. Today, 27 BTV serotypes have been identified [5], [6], [7], [8], [9]. BT is endemic in temperate and (sub)tropical climate zones [10], however, has expanded to areas with a milder climate related to competent vectors [11], [12], [13].

BTV, the prototype virus species of genus Orbivirus, family Reoviridae [14], [15], is a non-enveloped, multi-layered virus particle that contains a ten-segmented genome of double stranded RNA (Seg-1–10) encoding structural proteins VP1–VP7, and non-structural proteins NS1–NS4 [16], [17], [18]. VP7 protein is the major serogroup specific protein used in ELISAs to detect BTV antibodies (Abs). VP2 is the major target for serotype specific neutralising Abs (nAbs) [19], [20]. BTV infection also raises non-neutralizing Abs against NS3/NS3a [21].

Vaccination is the most effective measure to control BT. Currently available live-attenuated and inactivated BT vaccines have inherent advantages and disadvantages [22], [23], [24], [25], [26], [27]. The replicating BT Disabled Infectious Single Animal (DISA) vaccine exhibits all advantages of live-attenuated vaccine without the inherent risks of live-attenuated vaccines by the deletion of non-essential NS3/NS3a as previously discussed [28], [29], [30], [31], [32]. Consequently, BT DISA vaccines potentially enable specific detection of BTV infected animals based on NS3 Abs according to the principle of Differentiation of Infected from Vaccinated Animals (DIVA) [33], [34].

We here describe the development of a competitive ELISA for BTV NS3 Abs (NS3 cELISA). The diagnostic sensitivity and specificity were compared with a commercial BTV cELISA detecting VP7 Abs, and the DIVA potential was studied in combination with BT DISA vaccine.

Section snippets

Epitope mapping on NS3

BSR cells (a clone of BHK-21 cells; [35]) were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 5% foetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 2.5 μg/ml Amphotericin B.

Plasmids with full length mutated cDNA of Seg-10 for run-off RNA transcription were synthesized by Genscript Corporation (Piscataway NJ, USA), or were constructed according to standard procedures. Seg-10 of strain KUW (BTV26) was based on accession number JN255162. BTV1 based

Epitope mapping on NS3

To map epitopes on NS3/NS3a, BTV1 viruses expressing mutant NS3/NS3a proteins were studied by IPMA with NS3 MAbs (Fig. 1). MAbs 32B6 and 33H7 recognize the PPRY motif, whereas MAbs 32F1 and 31E9 recognize motif PSAP (Supplemented data A). Apparently, the closely located PPRY and PSAP motifs in the late domain (LD) PPRYAPSAP – aa position 36–44 – represent independent epitopes of two groups of two MAbs. Transient expression of mutant ΔD(S2reposition) expressing NS3/NS3a up to amino acid (aa)

Sensitivity

Challenge controls were used to study NS3 seroconversion after BTV infection. Note that these naïve sheep were infected at day 21 and day 84 of the vaccination/challenge experiment presented in Fig. 4A and B, respectively. Four out of 16 sheep seroconverted (>20%) at 9 dpi, and all were positive for NS3 Abs at 11 dpi. These sheep seroconverted for VP7 Abs at 7 dpi (day 28 and day 91, respectively). At 14 and 21 dpi, all naïve sheep infected with BTV2 or 8 (n = 12) were positive for NS3 Abs (31–90%),

Discussion

The late domain (LD) of NS3/NS3a is highly conserved and very immunogenic, and therefore important for the DIVA test accompanying DIVA vaccine in which this part is deleted (DIVA: Differentiation Infected from Vaccinated Animals). The novel “Disabled Infectious Single Animal (DISA) vaccine” lacks NS3/NS3a expression by deletion of LD, and restore of LD expression is therefore excluded [30], [31], [32], [39]. NS3 related expression from mutated Seg-10 has been reported [37], [40], which makes an

Acknowledgements

The authors thank dr. Carmen Vela and dr. Paloma Rueda (Ingenasa, Spain) for MAbs directed against NS3 of BTV. Reference sera for BTV serotype 1–24 and BTV26 (KUW) serum were kindly supplied by Carrie Batten (the EU reference Institute for Bluetongue, The Pirbright Institute United Kingdom). Serum for BTV25 (TOV) was a generous gift from Barbara Thür, IVI Switzerland, and sera for BTV27 (BTV-n) and EHDV6 were kindly provided by Emmanuel Breard, ANSES, France. Field sera from South African sheep

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