Materials and Methods
Virus strains and cells: Cytopathic BVDV-1a (Singer strain) was used to develop the subunit vaccine and to perform Virus Neutralization (VN) assays. MDBK cells were used to propagate the virus. Cells were grown in Earle’s minimal essential medium (EMEM) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 2% heat inactivated FBS (Internegocios S.A.). Recombinant baculovirus expressing the APCH-E2 antigen was generated as described by Pecora et al (Pecora et al. 2015).Spodoptera frugiperda (SF9) cells were used to produce the recombinant vaccine. SF9 cells were cultivated in a customized SF900 II Serum Free Medium. SF9 cells were infected at a multiplicity of infection (MOI) of 3 with the recombinant baculovirus and incubated at 27 ºC for 120 h to produce the antigen.
Vaccination of guinea pigs : 5 guinea pigs per group were vaccinated twice, at day 0 and 21, with 0,6 ml (1/5 of the bovine dose) and bled 30 days post-second vaccination. Sera were analyzed by Virus Neutralization (VN) assay. According to a dose response assay conducted in guinea pigs and bovines using vaccines formulated with increasing titers of BVDV per dose (1x106, 1x107,or 1x108TCID50/dose), a vaccine can be classified as of Not Satisfactory immunogenicity if the induced mean NAb titer in guinea pig was lower than 1:24 (Log10 < 1.37), of Satisfactory immunogenicity if 1:24 ≥ NAbs titer ≥ 1:100 (Log10 1.37 ≥ NAbs titer ≥ Log10 2), or of Highly Satisfactory immunogenicity if the mean NAbs titer ≥ 1:100 (Log10 < 2). (Res. SENASA 2012).
Virus Neutralization (VN) assay: according to (Aguirreburualde et al. 2013)
Competition ELISA : 96-well Nunc Maxisorp ELISA plates were coated with a bivalent llama-derived nanobody in carbonate-bicarbonate buffer (Pérez Aguirreburualde 2014; Zamit 2010) directed to E2 protein from BVDV overnight at 4 ºC, followed by a blocking step with 1% skim milk the next day for 1 hour at 37 ºC. The subsequent steps were also incubated with these conditions. Plates were washed three time with PBS-T between steps. Then, 6 ng/well of E2 protein produced in the BEVS was added to the appropriate wells. Bovine serum samples were added in a unique ¼ dilution in PBS-0.05% Tween 20. After a 1 h incubation, a rabbit polyclonal antiserum to E2 was added in a dilution 1/40 followed by a peroxidase-labeled anti-rabbit IgG (KPL) 25 ng/well. The ELISA Ab titer was expressed as a percentage of displacement (PD%) of the positive hyperimmune serum against the E2 protein that was considered the 100% signal. The cutoff point of the ELISA was established in PD% = 10%. Using this assay, the concordance between PD% and vaccine quality was established as follows: low-quality vaccines <12%, satisfactory vaccines ≤35%ND highly satisfactory vaccines < 35% (Manuscript under preparation).
Field trial in cattle : The trial was performed in Estancia Lavalle, Mercedes, Corrientes, Argentina. A total of 107 Brangus cows were divided randomly into two groups, 53 vaccinated with the targeted vaccine (3 ml/dose) and 54 with a conventional reproductive vaccine (5 ml/dose), which contains inactivated BVDV. All animals were vaccinated twice, beginning on study day 0 and again study day 30. Blood was drawn from all animals on study days 0, 30, 60, 120, 180 and 360. Sera were analyzed individually by competition ELISA. Results are expressed as a percentage of displacement of a positive hyperimmune serum against the E2 protein. Serum samples of all animals at day 0 and 60 (T0 & T60 respectively) were also evaluated by virus neutralization assay.
Statistical analysis : Differences in mean antibodies values among groups were evaluated by a general mixed model of repeated measures throughout time considering vaccine group and time as fixed factors and the animal as a random factor followed by Tukey multiple comparisons test. The matrix of variance-covariance was modeled assuming an AR1 autocorrelation effect due to the sampling of the same animals through time (AR1) and heterogenicity of variances at different time points (varIdent). Statistical significance was assessed at p ˂0.05 for all comparisons. The analysis was conducted with Infostat software with a connection to R (Di Rienzo et al. 2013).