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).