Results
To produce the antigen at industrial scale, the cell culture conditions, the protocol of infection with the recombinant baculovirus, and the downstream processing of the antigen, needs to be standardized. In order to setup the industrial method, different spinner flasks and bioreactors, customized media with and without fetal bovine serum (FBS), culture and infection conditions, and filtration systems were tested and analyzed (Data Not Shown). The process was validated using the single-use wave bioreactor system, SF900 II serum free media and a two-step filtration system. As a result, cell concentrations of up to 6 x 106 cells/ml were achieved and yielded approximately 5 mg/L of APCH-E2 antigen. The flowchart of antigen production is presented in Figure 1 . The antigen is then formulated with an oil adjuvant (Marcol/Arlacel). The entire process is documented and validated following GMP guidelines.
The Argentinean National Regulatory Authority (SENASA) approved potency assay described above (See Methods). There was found to be a correlation between the guinea pig VN titer and the bovine VN titer: titers of 1:24 (Log10 1.37) and 1:107 (Log10 2.03) in guinea pig correlates with 1:32 (Log10 1.54) and 1:134 (Log10 2.13) in cattle, respectively. These VN titers also correlate with the competition ELISA PD values of 12% and 35% respectively (Table 1 ).
SENASA uses the guinea pig model of BVDV infection to test every batch of cattle vaccine to ensure the potency of the vaccine before it is released to the market. For BVDV vaccines, the NAb titers induced in guinea pigs were statistically validated as a reliable indicator to predict vaccine immunogenicity in bovines (See Methods). Depending on the performance in this model, vaccines can be classified into three categories: NAbs titers less than Log10 1.37 are not satisfactory vaccines (not approved for sale), NAbs titers between Log10 1.37 and Log10 2.03 are satisfactory vaccines (approved for sale) and NAbs titers greater than Log10 2.03 represent highly satisfactory vaccines (approved for sale). To date, eleven commercial batches of the targeted vaccine has been submitted to SENASA. All of them have been approved, with seven being classified as highly satisfactory and four as satisfactory vaccines in this guinea pig model. A dot blot graph of NAb titer results for batches 1 to 11 is presented in the Figure 2.
A field trial in cattle was performed to evaluate the immune response of this new vaccine and to compare the performance of this targeted vaccine with a conventional vaccine formulated with killed BVDV. The field trial was carried out in a commercial herd under normal management conditions with approval from SENASA. There were no changes in animal feeding, health, movement or any other parameter or condition during the trial. In the farm selected to run the trial there was circulation of BVDV and therefore neutralizing antibody titers against BVDV were observed at the beginning of the study. Taking this into consideration, to present the data we subdivided each treatment into two groups: 1) animals with competition ELISA PD values ≤35%, and 2) animals with competition ELISA PD values >35% at the start of the trial. The PD 35% threshold was selected because it correlates with the competition ELISA measurements with the cutoff limit used to evaluate the satisfactory efficacy of vaccines in the SENASA-approved guinea pig model (Figure 3 A & B). A significant increase in the NAb titer was observed in the animals within the targeted vaccine group that started the trial with a competition ELISA result ≤35%, Nab titer increased from Log10:1,43 in T0 to Log10: 2,43 at T60. On the other hand, in the conventional vaccine group at T60 no significantly modification was observed in the NAb titers of the animals regardless of their T0 titers. All the animals in both groups were temporally sampled and the immune response was evaluated by ELISA(Figure 4 A, B & C). The targeted vaccine group has a greater immunological response than the conventional vaccine based on inactivated BVDV virus, in terms of the induction of antibodies to BVDV and the duration of the immune response. The targeted vaccine group developed a strong antibody response to BVDV at 30 days after the first dose of vaccine (Figure 4A). The antibody titers to BVDV remained high during the trial up to 360 days post-vaccination. Contrastingly, animals in the conventional vaccine group presented non-homogeneous antibody response; some animals increased their antibody titers while others remained in their basal competition-ELISA antibody titers.
To have a better understanding of the performance of the vaccine, animals with a lower level of antibody titers (PD ≤35%) at the beginning of the experience were analyzed independently (Figure 4B & 4C ). There were no significant differences in the mean antibody titers of these animals at day 0. Results in Figure 4B and 4C show that Animals that started the trial with PD antibody titers below 35% and were vaccinated with the targeted vaccine ultimately reached similar levels of antibody titers to those animals that started with PD% titers >35% by the end of the study period. In contrast, the animals in the PD ≤35% antibody titer subgroup of the conventional vaccine group had a small increase in antibody titers at day 120, peaking at 27% of displacement in the competition ELISA, which then declined by the end of trial (day 360) and finished with a mean titer of 9% of displacement in the competition ELISA. Furthermore, these animals within the conventional vaccine group never attained similar antibody titers to the conventionally vaccinated animals of the PD >35% subgroup.