Discussion
The commercial production and downstream processing of this novel
targeted vaccine, was established and standardized. The antigen is
produced in Sf9 cells using a baculovirus expression vector system with
customized media in single-use wave bioreactors. It is a flexible
technology, with potential incorporation of multiple antigens into a
single formulation. Studies from our group have previously shown that a
recombinant subunit vaccine containing BEVS derived E2 proteins from
three different strains of BVDV (BVDV-1a, -1b and -2a) each, fused to
the APCH molecule, was able to induce protection in colostrum-deprived
calves challenged with BVDV (Pecora et al. 2015). This is a clear
indication the production platform used for this new single-strain
vaccine has potential as a universal and adaptable platform to develop a
cost-effective and efficacious vaccine against all BVDV strains. With
this strategy, it is possible to make a single vaccine to use worldwide;
or, if there are significant regional strain variation, it is feasible
to modify the E2 antigen to develop a region-specific BVDV vaccine.
Furthermore, the single use baculovirus expression platform and the APCH
targeting molecule could be used to develop new targeted vaccines
against other viruses, bacteria, or parasites.
The guinea pig model is used by the Argentine government authorities
since it is a reliable tool that consistently predicts the performance
of the vaccine on the field. This model for BVDV vaccine potency testing
was presented at the XXII Seminar on Harmonization of Registration and
Control of Veterinary Medicines Americas Committee for Veterinary
Medicines (CAMEVET) (https://rr-americas.oie.int/en/events/xxii-seminar/
Mexico, 2016) and a group of experts from different countries is
revising the guideline in order to implement this guinea pig model in
different American countries (validation study in progress). All
commercial batches of this new targeted vaccine were able to pass this
test, with several labelled with the highest potency qualification
(highly satisfactory), indicating this production process is
reproducible and robust.
In the cattle field trial, this new targeted vaccine induced a more
potent and longer lasting immune response when compared to the
conventional inactivated vaccine. All animals within the subunit vaccine
group presented high antibody titer levels at day 30 that remained high
until the end of the trial at day 360 post vaccination. In contrast, the
conventional vaccine group animals did not have a significant increase
in antibody titers at days 30 or 60, and subsequently, the antibody
levels decreased to basal levels at day 120 and continued decreasing
until the end of the trial. Animals within the subunit targeted vaccine
presented higher antibodies level after vaccination than the
conventional vaccine group in every time-point analyzed.
The northern region of Argentina is characterized as having a wet, hot,
and subtropical weather. Within cattle herds in this region, such as the
one chosen for this field trial, it is common to find animals with
varying levels of BVDV-specific antibody titers. In these instances, it
has previously been shown that animals with lower BVDV-specific antibody
titers are more susceptible the viral infection (S. R. Bolin and Ridpath
1996). The main of goal of a BVDV vaccination program is to protect
these animals since 90% of PI animals are born from non-PI cows (Wittum
et al. 2001). In the field trial it was shown that the new targeted
vaccine is able to significantly increase NAbs titers to levels that
correlate with protection in these seronegative and low-titer animals
suggesting an increase in protection from day 30 post-vaccination. This
potential protection lasted throughout the course of the trial (360 days
post-vaccination) (S. R. Bolin and Ridpath 1996). On the other hand, the
susceptible bovine population within the conventional vaccine group had
no significant changes in antibodies levels after vaccination.
It is also interesting to note that the standard deviation (SD) of the
mean Ab titers in both groups it is very different. The SD in the
targeted vaccine is, at least, half the one observed in the conventional
vaccine group at most of the analyzed timepoints (Figure 4A). This is
another indication of how different, but consistently, the immune
response is induced by a targeted vaccine compared to a conventional
vaccine that uses inactivated BVDV in the formulation. It is also clear
in Figures 4A and 4B that at day 0 there is a high variation of antibody
levels going from zero to 87% PD. At day 180, all animals of the
targeted vaccine group are concentrated in a range from 55% to 90% PD,
but animals in the conventional vaccine group exhibit a greater
variation ranging from 16% to 88% PD. In the conventional vaccine
group, animals with high antibody titers to BVDV at the beginning of the
trial maintained high antibody titers at the end of the trial. In
contrast, animals with low antibody titers did not increase their
antibody level over the course of the trial and, therefore, remained
susceptible to virus infection. On the other hand, in the targeted
vaccine group, all animals reached high antibody titers to BVDV
independent of their initial antibody titers, indicating the targeted
vaccine is able to induce a potent immune response in seronegative and
low-titer animals. Importantly, high BVDV-specific antibodies in the
cattle did not inhibit a robust vaccine-specific immune response to the
new targeted antigen.
In conclusion, the targeted vaccine
represents a new and improved
vaccine against BVDV with the advantages of attenuated vaccines in terms
of immunogenicity but with the safeness of inactivated vaccines. Safety
is a key issue in BVDV control programs since the vaccination of
pregnant cattle with an attenuated vaccine can lead to the development
of a persistently infected animal (Palomares et al. 2013) and that an
inactive vaccine was associated with an emerging disease named bovine
neonatal pancytopenia (Deutskens et al. 2011). Therefore, veterinarians
and farmers demand the introduction of safe and efficacious vaccine.
This new subunit targeted vaccine satisfies these requirements and it is
also a flexible platform that can be used to produce a new generation of
targeted vaccines against a variety of viral, bacterial, or parasite
antigens.
Acknowledgments: The authors are grateful to Celina Vega (INTA)
and Jeffrey Hall (Vetanco) for the review and the correction of the
manuscript and to Emanuel Gumina for the help in the preparation of the
graphs.
Animal Welfare: Guinea pig and cattle handling, inoculation,
and sample collection were done by trained personnel under the
supervision of a veterinarian and in accordance to protocols approved by
the INTA’s ethical committee of animal welfare (CICUAE).
Authors contribution: DB is the leader of this project, JB
produced the antigen and the vaccine used in cattle, LR analyzed serum
samples from guinea pig and cattle, AP made the recombinant BEVS
construct, JAE discovered the APCH molecule, MA was the veterinarian in
charge of the field trial, VP conducted the statistical analysis,
participate in the experimental design, and revised the article, AW is
the PI of the lab, he maintained program funding and helped design and
supported the project and revised the manuscript. All authors attest
they meet the ICMJE criteria for authorship.
Funding: this work was supported by the ANPCyT, FONARSEC
(Empretecno 2.0 2017 Project Nº 0006) and by Vetanco SA.
Competing interests: D Bellido, J Baztarrica, A Wigdorovitz and
M Acosta work for Bioinnovo - Vetanco SA; and J.M Escribano works for
Algenex.
Data availability statement: The data that support the findings
of this study are available on request from the corresponding author.
The data are not publicly available due to privacy or ethical
restrictions.