Cells
Baby hamster kidney 21 (BHK-21) from ATCC, Catalogue No. CCL-10™ passage number 53 were cultured in Minimum Essential Medium + Earle’s salts + L-Glutamine (MEM, Gibco, USA) supplemented with Fetal bovine serum (FBS, Gibco, Germany) while FBS was omitted for the cell virus replication. Cells were passaged twice per week in T75 flasks at a density of 4.5 × 105 cells/ml and incubated in a humidified incubator at 37 °C with 5.0% CO2. BHK-21 cells were used to generate viral suspensions required to prepare the vaccines which was propagated in T300 flasks.
Vero cells from ATCC, Catalogue No. CCL-81™ passage number 120 were cultured with Minimum Essential Medium (MEM, Gibco, UK) supplemented with Fetal bovine serum (FBS, Gibco, Germany). Cells were passaged twice per week in T75 flasks at a density of 1.5 × 106 cells/ml and incubated in a humidified incubator at 37 °C with 5.0% CO2.
Viral stocks were obtained by inoculating Vero cells in T75 tissue culture flasks. Also, infectious titer expressed in tissue culture infective dose (TCID50/ml) was determined with these cells, and virus neutralization tests were performed on Vero cells.
Virus
The AHS viruses were isolated from unclotted whole blood, from lung lymph nodes as well as lung and spleen from dead animals originating from Kenya. The tissue samples were homogenised as a 10% (w/v) suspension in Minimum Essential Medium (MEM, Gibco, USA) containing 1% penicillin-streptomycin (Sigma Aldrich, Germany). The suspension was clarified by centrifugation at 2500 rpm for 5 min, and the supernatant was further diluted 1:10 in MEM. The diluted supernatant was sterile filtered with 0.45 μm filter (Sartorius, Germany) and inoculated into BHK 21 cells line grown in MEM. Three blind passages were performed for the presence of virus on BHK 21 cells and negative samples were further passaged 4 times on BHK 21 cells before considering as negative for the presence of the virus. Once the cytopathic effect (CPE) was observed, serotyping of the isolated AHS strains was carried out at the OIE AHS Reference Laboratories in Onderstepoort, South Africa; Madrid, Spain and at CVRL, Dubai, UAE. Each serotype was plaque purified on Vero cells by selection of large plaques (4–6 mm) at terminal dilutions. The plaque test was performed in Vero cells grown in 5 cm diameter petri dishes with an overlay of SeaPlaque Agarose 0.8% (Lonza, Rockland, ME, USA). The purification of AHS virus was carried out as described by Joklik [32] and Mirchamsy and Taslimi [33]. The final plaque material was passaged twice on Vero cells, and then tests for microbiological sterility including mycoplasma and extraneous viral agents of the stock virus were carried according to the guidelines of the OIE manual of diagnostic tests and vaccines for terrestrial animals [34]. Each serotype was freeze dried in 2 ml glass vials and frozen at − 80 °C. This master seed virus was resuspended in 2 ml sterile distilled water diluted with MEM and inoculated onto Vero cells to produce the working seed virus. From this, the viral suspension for the vaccine production was propagated in BHK 21 cells. The infectivity titers of each serotype were of each serotype were calculated before concentration and were found between 106.0 and 107.5 TCID50/ml.
Two to 3 days after infection, virus-containing cell culture supernatant was collected and concentrated 10 times by ultrafiltration using a Pelicon (R) 2 Mini Cassette (10KDa, Millipore, USA) filter.
The inactivation of the virus was performed as described by Ronchi et al. [35] apart from the addition of 37% formalin (Merck, Germany) to a final concentration of 1:8000 formaldehyde [36]. This was followed by the addition of 5 mM binary ethylenimine (BEI), the second inactivant, was prepared according to the method described by Bahnemann [37] by adding 1 N solution of 2-bromoethylamine hydrobromide (Sigma Aldrich B65705) to 0.175 N NaOH [37, 38]. Inactivation time varied from 25 h to 48 h for 300 ml of viral suspension based on the viral titres observed for each serotype. The inactivation process was stopped using 10% v/v 1 M sodium thiosulfate. All viral suspensions were stored at 2–8 °C. The inactivated virus solution was tested for residual activity by 2 different methods: the first method was passaging the inactivated viral solution 7 times through BHK 21 cells grown in T75 tissue culture flasks. The second method was passaging the virus 7 times into 9- to 11-day-old embryonated chicken eggs. The fluid from the final passages of both methods were tested by PCR for the detection of AHSV RNA.
Real-Time PCR (RT-PCR)
This method followed the procedure laid down by Guthrie et al [18]., which is capable of detecting all 9 serotypes of AHS and is also prescribed in the OIE African horse sickness chapter [8]. RNA extraction was carried out from tissue culture supernatant, EDTA blood or tissue samples. Extraction was performed using the Magnapure automated extraction system and Magnapure total nucleic acid extraction kit (Roche, Switzerland). Extracted RNA was denatured at 95 °C for 5 min and frozen at − 20 °C for 5 min before use in RT-PCR. The total reaction volume was 25 μl, containing 5 μl of denatured RNA and 20 μl of TaqMan master mix with AHSV group-specific primer (concentration of 200 nM) and probe (concentration 120 nM), which was adapted from Guthrie et al. [17]. RT-PCR assays were performed on an ABI 7500 Dx RT-PCR instrument (Applied Biosystems, USA). The following thermal profile was carried out: 50 °C for 8 min, 95 °C for 2 min and 45 cycles of denaturation and annealing/extension at 95 °C for 15 s and 60 °C for 45 s, respectively.
Samples were considered positive if they showed an exponential amplification, a minimum fluorescence level of 0.1 and a cycle threshold of 36 or lower. Samples that amplified after this threshold were scored as weakly detected or negative based on repeated testing results.
Serology
Two tests were used for the detection of AHSV antibodies, a cELISA that detects antibodies against VP7 and does not correlate with protection, and a VNT detecting antibodies against the surface antigens VP2 and VP5. The VNT is described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [8] and approved by the European Commission [39].
The cELISA was performed according to Hamblin et al. [40] with in-house AHS antigen and anti-VP7 guinea pig sera.
The first step was the coating of all wells of the cELISA plate (Thermo Fisher, USA), with the in-house AHS antigen, which was diluted according to the optimal antigen strength in carbonate/bicarbonate buffer (Sigma Aldrich, Germany) at pH 9.6 and was incubated overnight at 4 °C. The following day, the plate was washed 3 times with phosphate-buffered saline (PBS) (Oxoid, UK) pH 7.6. The serum samples and negative control sera were diluted 1:5. The positive control sera which had a pre-determined titer was diluted across eight wells of the plate to give a final dilution of 1:640. All the samples and the control were in blocking buffer which contains, PBS, 0.05% Tween 20 (Sigma Aldrich, Germany), 5% skimmed milk powder (Sucofin, Germany) and 1% adult bovine serum (Gemini bioproducts, USA). Wells containing anti-VP7 guinea pig serum and blocking buffer were also included as a control for anti-guinea pig sera. The optimum dilution of each batch of anti-VP7 guinea pig antisera was pre -determined by checkboard titration. Diluted anti-VP7 guinea pig serum was added to all the wells and plate was incubated for 1 h and 15 min at 37 °C with shaking. After washing the plate 3 times with PBS, 1:1000 diluted conjugate (mouse anti-guinea pig horseradish peroxidase-labelled antibody (Dako, Denmark)) was added and the plate incubated for 1 h and 15 min at 37 °C with shaking. At the end of the incubation step, the plate was washed 3 times with PBS. Then, the substrate which was prepared by dissolving orthophenyldiamine tablet (4 mg, Sigma Aldrich, Germany) in 10 ml distilled water containing 0.005% of 30% hydrogen peroxide (Anala R, UK) was added and the plate was incubated for 10 min at room temperature in dark condition. The colour development was stopped by the addition of 1 M H2SO4, (Ensure®, Germany) and the plate was read at 492 nm using an ELISA plate reader (Tecan Sunrise reader, USA) to obtain the optical density (OD). The interpretation of the results was based on percentage inhibition (PI), which was calculated as 100x (100-mean OD of sample/mean OD of anti-VP7 guinea pig control). Samples with PI values lower than 50% were considered negative, and samples with PI values greater than or equal to 50% were considered positive. The test was repeated for samples that were in the borderline range (PI = 49 to 50%).
Virus neutralisation: Serotype-specific antibodies against each serotype were tested using VNT according to Lelli et al. [41], Ronchi et al. [35] and OIE [8]. VNT was performed using isolated field strains, serotype-specific AHSV positive control antisera that were obtained from Pirbright Institute, UK. All test sera were inactivated at 56 °C for 30 min. In a 96-well flat-bottomed microtiter plate, 50 μl of 1:10 diluted sera in MEM were added. An equal amount of virus dilution of each serotype was added to 4 wells from 101 to 107. Positive and negative sera were included and the plates were incubated for 1 h at 37 °C with 5% CO2.
Vero cells at 5 × 105 cells/ml were prepared in MEM + 10% FBS, and 100 μl were dispensed in each well. The test was read after 7 days of incubation. Virus titre was calculated using the Reed and Muench method. VN titres were derived by computing the differences between virus titres of each serotype in the presence of negative serum and the virus titres in the presence of the serum to be tested, which is expressed as log10.
Horses
Twenty-nine horses were included in the study with 25 gelding and 4 mares, aged between 20 and 30 years. Their history record was as follows: 13 were endurance horses, 8 thoroughbred and 8 sport horses. All horses were kept in a Desert Stud Stable. During the day, the horses were inside air-conditioned stables, and at night, they had access to open paddocks. Nutrition was provided twice daily in the form of GP mix, chaff, bran, hay, supplements (corn oil, electrodex, biotin, chevinal plus syrup, olive oil) and alfalfa. Unlimited access to water was also provided. The horses were divided into 2 groups. Group 1 comprised 18 horses that were subdivided into 9 subgroups of 2, and each pair was immunised with individual serotypes 1 to 9. In Group 2, 11 horses were simultaneously immunised with a combination of vaccine 1 and vaccine 2 (see below).
After the experiment ended, all the animals continued with their daily routine. No horse was euthanised.
Vaccine/vaccination design/samples
Vaccine
The vaccine was formulated according to the manufacturer’s instructions with Imject Alum (Thermo Scientific, USA) as an adjuvant. The vaccines were presented in 2 forms, namely, single serotype vaccines and polyvalent vaccines administered in 2 formulations (vaccine 1 contained serotypes 1, 4, 7, 8, and 9, and vaccine 2 contained serotypes 2, 3, 5, and 6). In-house AHS antigen capture cELISA and PCR tests were employed to determine the concentration of each batch of the 9 monovalent vaccines. The antigen load was between 106.0 and 107.5 TCID50/ml. The virus concentration calculated for each serotype was the same for all three AHS vaccines, mono, quadrivalent and pentavalent.
All vaccines were manufactured and formulated prior to the start of the study. All vaccines were stored at 4–8 °C and were tested on horses for stability.
Vaccination design
On Day 0, Group 1 and Group 2, horses were immunised as follows: Group 1: 2 ml of single serotype vaccines were sc administered into the left side of the neck. Group 2: 4 ml of vaccine 1 and vaccine 2 were sc administered into the left and right side of the neck, respectively.
On Day 28, Group 1 and Group 2 received a booster. On Day 56, Group 1 received a second booster. On Day 332, Group 1 and Group 2 received an annual booster.
Samples
Blood samples were drawn from the jugular vein for cELISA and VNT every 2 weeks until the end of the trial, and blood was collected after each immunisation for 2 weeks (Days 0, 3, 7, 14) for PCR and virus isolation.
During the first 2 weeks after each immunisation, rectal temperatures were recorded twice a day, and the injection site was inspected.
