Location: Animal Disease Research
2017 Annual Report
Objectives
Malignant catarrhal fever (MCF) is an often fatal viral disease that primarily affects ruminants. Several gammaherpesviruses in the Macavirus genus can cause MCF. These viruses are carried asymptomatically by certain animal species but can cause disease when transmitted to clinically susceptible species. Ovine herpesvirus 2 (OvHV-2) is globally distributed and the most frequent cause of MCF worldwide. In North America, OvHV-2-induced MCF is the leading cause of death in American bison, which are highly susceptible to the disease. MCF results in significant economic impact on agriculture not only due to the loss of animals but also because it imposes restrictions on multispecies grazing. The development of an effective vaccine to MCF is a top priority for the agricultural industry. However, because OvHV-2 cannot be cultured in vitro, conventional methods cannot be utilized to attenuate or modify viruses to be used as a vaccine. In this project, we propose a novel strategy to develop a vaccine against MCF using recombinant non-pathogenic gammaherpesviruses expressing key OvHV-2 glycoproteins to induce immune responses capable of protecting animals from disease. Importantly, we also plan to develop a modified virus neutralization assay to assess vaccine efficacy.
Objective 1: Develop and evaluate neutralizing antibody assays for OvHV-2 using in vitro and rabbit models with the goal of assessing vaccine efficacy and protection from MCF.
Subobjective 1A. Develop a recombinant AlHV-1 expressing OvHV-2 glycoproteins for an in vitro neutralization assay.
Subobjective 1B. Develop and validate an in vitro neutralization assay for detection of OvHV-2 antibodies.
Subobjective 1C. Evaluate interference of OvHV-2 neutralizing antibodies in the respiratory tract with virus infection and development of SA-MCF in rabbits.
Objective 2: Develop novel and efficacious MCF vaccines for clinically-susceptible species using novel vaccine vector systems and platforms.
Subobjective 2A. Develop a recombinant BoHV-4 expressing OvHV-2 gB and gH/gL.
Subobjective 2B. Evaluate delivery platforms for immunization with the rBoHV-4/OvHV-2-gB-gH/gL in a rabbit model.
Subobjective 2C. Determine whether immunization with a recombinant BoHV-4 expressing OvHV-2 gB and gH/gL prevents MCF in bison.
Approach
This proposed research addresses a fundamental gap regarding control of OvHV-2: the development of an SA-MCF vaccine. The inability to propagate OvHV-2 in vitro has been a roadblock not only to develop a vaccine for SA-MCF, but also to perform in vitro neutralization assays for antibodies that block, for example, viral entry. Our approach is to develop recombinant non-pathogenic gammaherpesviruses expressing key OvHV-2 glycoproteins to be used as a vaccine and as an in vitro tool for analysis of neutralizing antibody responses. Using recombination-mediated genetic engineering techniques, we will generate recombinant AlHV-1 (rAlHV-1) mutants that express OvHV-2 glycoproteins required for virus entry, as chimeric viruses for in vitro neutralization assays. The assays will be critical to evaluate OvHV-2 neutralizing antibody responses in vaccinated animals and also potentially useful for detecting infection with OvHV-2 and closely related MCF viruses. Moreover, we will develop a recombinant bovine herpesvirus 4 (BoHV-4) expressing OvHV-2 antigens that can stimulate neutralizing antibody responses, as a vaccine to protect clinically-susceptible species from SA-MCF. Overall the proposed research will test two hypotheses: 1) a chimeric virus expressing OvHV-2 glycoprotein(s) propagates in vitro and can be blocked by OvHV-2 neutralizing antibodies; 2) immunization with recombinant BoHV-4 expressing OvHV-2 glycoproteins will stimulate neutralizing antibodies in the respiratory tract, which will correlate with reduced initial viral loads in lung and protection against lethal OvHV-2 challenge. These hypotheses will be tested through the following objectives: 1) develop and evaluate neutralizing antibody assays for OvHV-2 using in vitro and rabbit models with the goal of assessing vaccine efficacy and protection from MCF; and 2) develop novel and efficacious MCF vaccines for clinically-susceptible species using novel vaccine vector systems and platforms.
Progress Report
As part of subobjective 1A, we have developed several Alcelapine herpesvirus 1(AlHV-1) chimeric viruses in which glycoproteins B, H and/or L (gB, gH and/or gL) were replaced by their ovine herpesvirus 2 (OvHV-2) homologous genes, both individually or in combinations. Only one of these chimeric viruses was viable, the one with only gB replaced (recombinant AlHV-1/OvHV-2-gB). We found that gH and gL were not interchangeable between AlHV-1 and OvHV-2; the recombinant viruses, in which these glycoproteins were replaced, were noninfectious and therefore not suitable for using in Objective 1. Based on these results we decided to develop the virus neutralization assay proposed in subobjective 1B using the rAlHV-1/OvHV-2-gB, for which the characterization has been published. We have so far confirmed that anti-OvHV-2 gB antibodies can neutralize this virus indicating that the neutralization assay based on rAlHV-1/OvHV-2-gB is a potential method to monitor anti-gB neutralizing antibody responses in animals immunized with the BoHV-4-vectored vaccine (subobjectives 2B and 2C). Regarding the use of a rAlHV-1/OvHV-2-gB-based neutralization assay to measure OvHv-2 gB-specific neutralizing antibody response in animals infected with the homologous virus (subobjective 1C), new approaches are being tested to remove antibodies against AlHV-1 proteins from the test sera, since these antibodies can also neutralize the virus and confound the results.
In a study related to the current project plan, we have identified OvHV-2 glycoproteins that are involved in virus entry. Using a cell-to-cell fusion assay, we demonstrated that OvHV-2 gB, gH and gL are essential and sufficient to promote fusion of cell membranes. Also, the glycoprotein Ov8, which is unique to OvHV-2, can enhance the fusion process. Besides validating the use of OvHV-2 gB, gH and gL as vaccine candidates, as proposed in this project (objective 2), the finding that Ov8 may participate in the virus entry process, opens up the possibility of using this protein as another vaccine candidate as well as a target for cell-cell fusion blocking assays to detect OvHV-2 specific neutralizing antibody responses, as a complementation to the assay proposed in subobjective 1B.
In preparation for the use of BoHV-4 as a vaccine vector (objective 2), several assays have been optimized including bovine herpesvirus 4 (BoHV-4) qPCR, ELISA and flow cytometer, which will be used in the in vitro and/or in vivo experiments proposed in the project. The BoHV-4 has been constructed by our collaborator in Italy and is pending transfer agreement finalization to be shipped to the U.S. In addition, monoclonal and polyclonal antibodies to the viral proteins of interest are being produced to assist in the characterization of recombinant viruses (Subobjectives 1A, 1C and 2A).
Accomplishments
1. Identification of viral glycoproteins used by ovine herpesvirus 2 (OvHV-2) to infect host cells. Malignant catarrhal fever (MCF) has emerged as a significant threat to American bison due to their high disease-susceptibility. Vaccine development is the major focus of MCF research. It is hypothesized that to prevent this disease, a vaccine needs to stimulate the production of antibodies specific to proteins involved in virus entry, which will block OvHV-2 at its initial site of infection. ARS researchers in Pullman, Washington, have identified four viral proteins involved in virus entry. The findings not only add fundamental scientific knowledge regarding OvHV-2 infection but also further validate the use of the vaccine candidates proposed in the current project plan, since they all have a role in virus entry.
Review Publications
Slater, O.M., Kennedy-Peters, J., Lejeune, M., Gummer, D., Macbeth, B., Warren, A., Joseph, T., Li, H., Cunha, C.W., Duignan, P.J. 2016. Sheep-associated malignant catarrhal fever-like skin disease in a free-ranging bighorn sheep (Ovis canadensis), Alberta, Canada. Journal of Wildlife Diseases. 53(1):153-158.
Cunha, C.W., Taus, N.S., Dewals, B.G., Vanderplasschen, A., Knowles, D.P., Li, H. 2016. Replacement of glycoprotein B in alcelaphine herpesvirus 1 by its ovine herpesvirus 2 homolog: Implications in vaccine development for sheep-associated malignant catarrhal fever. mSphere. doi:10.1128/mSphere.00108-16.
Bildfell, R.J., Li, H., Alcantar, B., Cunha, C.W., Bradway, D.S., Thomas, K.S. 2017. Alcelaphine gammaherpesvirus 1-induced malignant catarrhal fever in a Watusi (bos taurus africanus) steer in a North American game park. Journal of Veterinary Diagnostic Investigation. https://doi.org/10.1177?1040638717708392.
Alhajri, S.M., Cunha, C.W., Nicola, A.V., Aguilar, H.C., Li, H., Taus, N.S. 2017. Ovine herpesvirus 2 glycoproteins B, H, and L are sufficient for, and viral glycoprotein Ov8 can enhance, cell-cell membrane fusion. Journal of Virology. doi: 10.1128/JVI.02454-16.
