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\title{Rational design of the first commercially available subunit vaccine
against BVDV targeted to immune system cells}



\author[1]{Demian Bellido}%
\author[1]{Josefina  Baztarrica}%
\author[2]{Lucia Rocha}%
\author[2]{Andrea Pecora}%
\author[1]{Mario Acosta}%
\author[3]{José M.  Escribano}%
\author[4]{Viviana Parreño}%
\author[5]{Andrés Wigdorovitz}%
\affil[1]{Vetanco SA}%
\affil[2]{INTA}%
\affil[3]{Algenex}%
\affil[4]{National Institute of Agricultural Technology (INTA)}%
\affil[5]{Instituto Nacional de Tecnologia Agropecuaria, Instituto de Virologia e Innovaciones Tecnlógicas (IVIT-CONICET)}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Bovine viral diarrhea virus (BVDV) is a major cause of economic loss in
the cattle industry, worldwide. Infection results in reduce productive
performance, growth retardation, reduced milk production, and increased
susceptibility to other diseases leading to early culling of animals.
There are two main measures used to control the spread of BVDV: the
elimination of persistently infected (PI) animals and vaccination.
Currently, modified live or inactivated vaccines are used in BVDV
vaccination programs, but there are safety risks or insufficient
protection, respectively, with these vaccines. Here we report the
development and efficacy of the first targeted subunit vaccine against
BVDV. The core of the vaccine is a fusion of the BVDV structural
protein, E2, to a single-chain antibody, APCH, together termed, APCH-E2.
The APCH antibody targets the E2 antigen to the major histocompatibility
type II molecule (MHC-II) present on antigen-presenting cells.
Industrial production of the vaccine is carried out using the
baculovirus expression vector system (BEVS) using single-use
manufacturing technologies. This new subunit vaccine induces strong
BVDV-specific neutralizing antibodies in guinea pigs and cattle.
Importantly, in cattle with low levels of natural BVDV-specific
neutralizing antibodies, the vaccine induced strong neutralizing
antibody levels to above the protective threshold, as determined by a
competition ELISA. The APCH-E2 vaccine induced a rapid and sustained
neutralizing antibody response compared to a conventional vaccine in
cattle. The development of this subunit targeted vaccine provides cattle
and dairy producers with an inexpensive, easily administered, safe, and
efficacious BVDV vaccine.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus,
family Flaviviridae. This virus has a worldwide distribution and infects
ruminants. BVDV infections cause a broad spectrum of clinical signs
ranging from mild respiratory disease to fetal death, depending on the
virulence of the virus and the reproductive and immune status of the
host (Ridpath 2010). It is also one of the etiologic agents of the
Bovine Respiratory Disease (BRD), which is a major health problem and
the main cause of economic losses in raising cattle (Griffin 1997).
Infection of pregnant cattle with BVDV in the first trimester of
gestation can result in the production of a PI animal (Grooms 2004).
Cattle with a persistent infection are a long-term threat to herd-mates
because they shed BVDV for life and represent the main reservoir of the
virus within the herd. More than 90\% of new PI calves are born to
healthy cows that became infected during gestation, the other 7\% to
10\% of PI calves are born from PI cows (Wittum et al. 2001).
Vaccination against BVDV is an important component of prevention and
control programs since it can prevent clinical signs, reduce viral
spread and the birth of new PI animals. Currently, only modified live
vaccines (MLV) and inactivated vaccines are used in vaccination
programs. Both have historical disadvantages; MLV in terms of safety and
inactivated vaccines in terms of immunogenicity. Subunit vaccines
provide the opportunity of developing safe and effective vaccines as has
been shown with the new human recombinant vaccines against shingles
(Herpes zoster) and meningitis B (\emph{Neisseria meningitidis} group B)
that have received US-FDA approval in recent years. In the field of
veterinary medicine, the challenge is to produce a recombinant vaccine
that induces a protective immune response at a cost affordable price.

The BVDV genome consists of a single-stranded, positive sense RNA
molecule of approximately 12.3 kb in length. E2 is the major structural
glycoprotein of the BVDV envelope and the most immunoprotective protein
of the virus (Deregt et al. 1998; Fulton et al. 1997; Paton, Lowings,
and Barrett 1992). Neutralizing antibodies (NAbs) induced in infected
animals are mainly directed against E2 (Donis 1995) . The first attempt
of our group to produce a protective subunit vaccine against BVDV was
based on a secreted version of the BVDV E2 glycoprotein. Sera from
animals vaccinated with E2 neutralized several BVDV strains within a
genogroup (Pecora et al. 2014; S. Bolin et al. 1988). Moreover, it was
demonstrated that NAbs raised against E2 prevented infection from BVDV
(Bolin 1995; Toth et al. 1999; Pecora et al. 2015). The E2 subunit was
initially expressed in stably transfected CHO-K1 cells, reaching a yield
of 0.3 mg/L. The immunogenicity of this first generation E2 antigen
vaccine was studied using guinea pigs, as a laboratory animal model, and
field trials were conducted in cattle. Animals vaccinated with this E2
subunit vaccine developed high NAb titers and were protected against
viral infection (Pecora et al. 2016). Results obtained in this initial
trial were promising, but the low quantity of antigen produced in the
CHO-K1 cell-line made large scale commercial production cost inhibitory
for veterinary medicine purposes. To address this issue, two important
modifications were introduced: 1) the protein production system was
changed to transgenic alfalfa plants (\emph{Medicago sativa, L} .) and
the viral E2 glycoprotein was targeted to the antigen-presenting cells
(APC) in order to increases its immunogenicity.

The coding sequence of the BVDV E2 glycoprotein was fused to the coding
sequence of APCH, a single chain antibody, creating a fusion gene
termed\emph{APCH-E2} . APCH is a single-chain antibody directed to the
major histocompatibility complex type II (MHC -II) antigen epitope and
has been designated as a potent immunomodulating molecule in different
experimental vaccines, improving both humoral and cellular immune
responses in immunized animals as it targets the antigen to the APCs
(Gil et al. 2011). The \emph{APCH-E2} fusion gene was engineered into
alfalfa genome and the antigen was produced in alfalfa leaves, yielding
up to 1 \selectlanguage{greek}µ\selectlanguage{english}g/g (antigen/ wet alfalfa) and production of the fusion antigen
remained stable after vegetative propagation. A methodology based on an
aqueous two-phase system was standardized for concentration and partial
purification of APCH-E2 from alfalfa (Dus Santos et al. 2009). Guinea
pigs intramuscularly immunized with leaf extracts developed high NAb
titers. In bovine vaccinated with 3 \selectlanguage{greek}µ\selectlanguage{english}g of alfalfa produced APCH-E2,
BVDV-specific NAbs were induced and vaccinated animals did not shed BVDV
after a viral challenge (isolate 98/124, type IB).(Aguirreburualde et
al. 2013). Results with transgenic alfalfa plants were promising, but
there were two major issues that should be resolved in order to
transform the plant-derived APCH-E2 antigen in an industrial product: 1)
inhibitory scaling-up issues with the extraction and purification
process, and 2) the unknown regulatory aspects for parenteral
administration of a viral antigen derived from transgenic-plants.

With the aim of overcoming these difficulties, the APCH-E2 antigen
construct was engineered into the baculovirus expression vector system
(BEVS). BEVS was chosen because of the system's advantages: it is safe,
easy to use, and readily amenable to manufacturing scale-up (Kost,
Condreay, and Jarvis 2005). In 2017, after more than ten years of
research and development, this baculovirus produced APCH-E2 antigen was
the basis of the first subunit and targeted vaccine licensed to be used
in cattle for the control of BVDV. Here, we report the immunogenicity
and efficacy of this new APCH-E2 commercial vaccine as tested in guinea
pigs and a field trial in cattle. This BEVS derived APCH-E2 vaccine
induced a strong antibody response in all vaccinated animals and
correlated with protection in experimentally challenged calves
(Aguirreburualde et al. 2013; Pecora et al. 2015, 2016).

\textbf{Materials and Methods}

\textbf{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 \selectlanguage{greek}µ\selectlanguage{english}g/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).\emph{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 \selectlanguage{ngerman}ºC for 120 h to produce the antigen.

\textbf{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 (1x10\textsuperscript{6},
1x10\textsuperscript{7},or
1x10\textsuperscript{8}TCID\textsubscript{50}/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 (Log\textsubscript{10}
\textless{} 1.37), of Satisfactory immunogenicity if 1:24 \selectlanguage{english}[?] NAbs titer \selectlanguage{english}[?]
1:100 (Log\textsubscript{10} 1.37 \selectlanguage{english}[?] NAbs titer \selectlanguage{english}[?] Log\textsubscript{10}
2), or of Highly Satisfactory immunogenicity if the mean NAbs titer \selectlanguage{english}[?]
1:100 (Log\textsubscript{10} \textless{} 2). (Res. SENASA 2012).

\textbf{Virus Neutralization (VN) assay:} according to (Aguirreburualde
et al. 2013)

\textbf{Competition ELISA} : 96-well Nunc Maxisorp ELISA plates were
coated with a bivalent llama-derived nanobody in carbonate-bicarbonate
buffer (Perez 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 1/4 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
\textless{}12\%, satisfactory vaccines \selectlanguage{english}[?]35\%ND highly satisfactory
vaccines \textless{} 35\% (Manuscript under preparation).

\textbf{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.

\textbf{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 \selectlanguage{english}<0.05 for
all comparisons. The analysis was conducted with Infostat software with
a connection to R (Di Rienzo et al. 2013).

\textbf{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 10\textsuperscript{6} cells/ml were achieved and yielded approximately
5 mg/L of APCH-E2 antigen. The flowchart of antigen production is
presented in \textbf{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
(Log\textsubscript{10} 1.37) and 1:107 (Log\textsubscript{10} 2.03) in
guinea pig correlates with 1:32 (Log\textsubscript{10} 1.54) and 1:134
(Log\textsubscript{10} 2.13) in cattle, respectively. These VN titers
also correlate with the competition ELISA PD values of 12\% and 35\%
respectively (\textbf{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 Log\textsubscript{10} 1.37 are not
satisfactory vaccines (not approved for sale), NAbs titers between
Log\textsubscript{10} 1.37 and Log\textsubscript{10} 2.03 are
satisfactory vaccines (approved for sale) and NAbs titers greater than
Log\textsubscript{10} 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 \textbf{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 \textgreater{}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
(\textbf{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 Log\textsubscript{10}:1,43 in T0 to Log\textsubscript{10}: 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\textbf{(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 (\textbf{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
\textgreater{}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
\textgreater{}35\% subgroup.

\textbf{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.

\textbf{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.

\textbf{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).

\textbf{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.

\textbf{Funding:} this work was supported by the ANPCyT, FONARSEC
(Empretecno 2.0 2017 Project N\selectlanguage{ngerman}º 0006) and by Vetanco SA.

\textbf{Competing interests:} D Bellido, J Baztarrica, A Wigdorovitz and
M Acosta work for Bioinnovo - Vetanco SA; and J.M Escribano works for
Algenex.

\textbf{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.

\textbf{Figure Captions}

\textbf{Figure 1:} Production flowchart of the APCH-E2 antigen.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 2:} Guinea Pig Model\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2:} Results obtained with all the batches of the targeted
vaccine that were approved to be released into the market using the
guinea pig model of vaccine potency. The dots within each batch (1 to
11) represents a guinea pig and the lines and bars represents the mean
NAb titers \selectlanguage{ngerman}± standard deviation, the dotted lines represent the split
point for vaccine classification according to SENASA's predetermined
level of immunogenicity.

\textbf{Table 1. Vaccine classification points for BVDV}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{SPECIES} \textbf{VIRUS NEUTRALIZATION}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{BVDV VACCINE POTENCY}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{BVDV VACCINE POTENCY}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{BVDV VACCINE POTENCY}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Not Satisfactory\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Satisfactory\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Highly satisfactory\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
GUINEA PIG\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\selectlanguage{romanian}Ȳ \textless{} 1.37\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1.37 [?] \selectlanguage{romanian}Ȳ \selectlanguage{english}[?] 2.0\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2.0 \textless{} \selectlanguage{romanian}Ȳ\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
BOVINE\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\selectlanguage{romanian}Ȳ \textless{} 1.54\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1.54 [?] \selectlanguage{romanian}Ȳ \selectlanguage{english}[?] 2.13\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2.13 \textless{} \selectlanguage{romanian}Ȳ\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{ELISA}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
GUINEA PIG \& BOVINE\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\selectlanguage{romanian}Ȳ \textless{} 12\%\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
12\% [?] \selectlanguage{romanian}Ȳ \selectlanguage{english}[?] 35\%\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
35\% \textless{} \selectlanguage{romanian}Ȳ\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{romanian}

\textbf{Figure 3: Antibody Response}

\textbf{Fig 3.A: Virus Neutralization Assay}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig 3.B: Competition ELISA}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3 A \& B:} Antibody responses in all animals of both
groups were evaluated by VN assays (Fig 3.A) at 0 and 60 days and by a
specially designed competitive ELISA to BVDV (Fig 3.B). Light Grey Bars:
T0 Dark Grey Bars: T60; Striped Bars: Conventional Vaccine; Solid Bars:
Targeted Vaccine. For bovine samples a 35\% displacement ELISA value
correlates with a 1/32 NAb titer.

\textbf{Figure 4 A, B \& C:} Kinetics of antibody response after
vaccination

\textbf{Fig 4.A: Targeted Vaccine vs Conventional Vaccine. All Animals}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}



\textbf{Fig 4.B: Targeted Vaccine vs Conventional Vaccine. Initial Ab
Level \textgreater{} 35\%}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}



\textbf{Fig 4.C: Targeted Vaccine vs Conventional Vaccine. Initial Ab
Level \textless{} 35\%}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Figure 4 A, B \& C:} Kinetics of antibody response to BVDV after
vaccination. A: All animals; B: animals with initial antibody titers
\textgreater{}35\% by PD-ELISA; C: animals with initial antibody titers
\textless{}35\% PD Targeted vaccine group; Conventional vaccine group.
Dotted-dashed Lines at 12\% and 35\% indicate the PD\% values that
correlate with the threshold establish for qualifying vaccines:
low-quality vaccines, \textless{}12\% PD, satisfactory vaccines
\textless{}35\% PD.

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