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 (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 (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 termedAPCH-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 APCH-E2 fusion gene was engineered into
alfalfa genome and the antigen was produced in alfalfa leaves, yielding
up to 1 µg/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 µg 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).