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.