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.