Research Project: Diagnostic and Control Strategies for Malignant Catarrhal Fever

Location: Animal Disease Research

2021 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
This is the final report for the project 2090-32000-037-000D which will terminate in September 2021. The new project, titled “Development of a Vaccine and Improved Diagnostics for Malignant Catarrhal Fever” is currently undergoing NP103 OSQR review. A summary of results for all the expiring project sub-objectives are described below. The major goal of this research is the development of a vaccine to ovine herpesvirus 2 (OvHV-2)-induced Malignant Catarrhal Fever (MCF) and, in the last five years, we have obtained significant progress in this direction. First we confirmed that three glycoproteins (gB, gH and gL) present in the virus envelope are essential for the virus to enter and to disseminate in host cells. Based on that information, we worked with the hypothesis that if one or more of these proteins are blocked by antibodies induced through vaccination, the animal would be protected from disease upon infection with OvHV-2. Therefore, OvHV-2 gB, gH and gL were selected as potential vaccine candidates. In support of Sub-objectives 1A and 2A, genetic engineering techniques were used to generate recombinant viruses to express these OvHV-2 proteins. Several recombinant viruses were constructed and tested in vitro to confirm their viability and proper expression of the vaccine candidates. Two recombinant viruses, one derived from bovine herpesvirus 4 (BoHV-4) and another from alcelaphine herpesvirus 1 (AlHV-1), expressed OvHV-2 gB and were able to infect and propagate in mammalian cells in culture. However, recombinant viruses engineered to express OvHV-2 gH and gL were not viable. Attempts to include the gH and gL genes in the mutant viruses were ineffective and resulted in delays to the project. Therefore, we decided to use only the two recombinant viruses expressing OvHV-2 gB in Sub-objectives 1B, 1C, 2B, and 2C (redirected). For vaccine development, it is important to be able to measure immune responses that are expected to be correlated to protection. To facilitate this, we successfully developed and optimized assays to measure anti-OvHV-2 gB neutralizing antibodies induced by vaccination (Sub-objective 1B and redirected Sub-objective 2C). The newly developed tests involved viral neutralization assays, based on either AlHV-1 or the BoHV-4 recombinant viruses, and a cell-to-cell fusion blocking assay. In support of Sub-objective 2B, initial evaluation of the recombinant BoHV-4 expressing OvHV-2 gB as an immunogen was done as a vaccine-challenge trial using a laboratory rabbit model. Animals received three immunizations with the recombinant virus and then were challenged with a lethal dose of OvHV-2 delivered by intranasal nebulization, which mimics the virus natural route of infection. The BoHV-4-vectored vaccine induced anti-OvHV-2 gB antibodies, but the protection rate following OvHV-2 challenge was marginal, with only 42.8 % of the vaccinated animals being protected. As a contingent plan for Sub-objective 2B, we tested the recombinant AlHV-1 expressing OvHV-2 gB as a vaccine. The trials were also performed in rabbits and involved two immunization protocols: 1, only the recombinant virus was used as immunogen, and 2, animals received two immunizations with OvHV-2 gB DNA and one with the recombinant virus. Following immunizations, all rabbits were challenged with a lethal dose of OvHV-2. Protection from disease was observed in 71.4 % of the rabbits immunized with each of the vaccine protocols, while all animals in the non-vaccinated group succumbed to disease and were euthanized. Although neutralizing antibodies were produced by immunization with the vaccine candidates tested, it was not possible to confirm their role in protection as anticipated on Sub-objective 1C and additional experiments will be necessary to better define the correlates of protection. In spite of that, the promising protection rates obtained with the AlHV-1-vectored MCF vaccine protocols put us in the position of moving the project forward to test the safety and efficacy of this vaccine in livestock animals, which is the focus of our next project plan. The development and optimization of new assays for more precise and specific MCF diagnosis is also of great interest for research and a need for certain livestock industries. In a subordinate project, we made substantial progress in this area by developing a serological assay based on a unique OvHV-2 protein, Ov8, which can be used to identify animals infected with OvHV-2 without cross-reactivity with other MCF viruses. We have also characterized the relationship between viral load and OvHV-2 virus infection status using OvHV-2 quantitative PCR. This information is particularly useful to diagnose MCF in carrier species where mere detection of the virus is not indicative of disease. ARS researchers in Pullman, Washington, in collaboration with researchers at the Univerity of California, Davis, and the Universidade Estadual de Londrina in Brazil, developed hybridization assays capable of detecting MCF viruses (immunohistochemistry) or OvHV-2 and Ibex-MCF virus (in situ hybridization) in association with lesions using formalin fixed tissues. Such assays complement histology in confirming MCF cases postmortem. Despite these advancements, the availability of a point-of-care diagnostic assay for MCF is still needed and is one of the objectives of the next project plan. Finally, ARS researchers worked with field veterinarians and diagnostic laboratories in the confirmation of numerous MCF cases involving a variety of MCF viruses and animal hosts. In one of these cases, we were able to recognize a new MCF virus carried by bighorn sheep, which was tentatively named ovine herpesvirus 3. The high complexity of MCF and the challenges for its diagnostic and control support the continuous research efforts to minimize losses for livestock producers and their industries.

Accomplishments
1. An animal trial confirms high efficacy of a MCF vaccine candidate. The 2020 ARS Animal Health Stakeholder Survey revealed that about half of the bison producers consider malignant catarrhal fever (MCF) caused by ovine herpesvirus-2 (OvHV-2) the most concerning disease for their livestock. American bison are very susceptible to sheep-associated (SA)-MCF and transmission of the virus from sheep is a real concern for the industries. Separating susceptible (bison) and carrier (sheep) animals is currently the only way to control the disease; however, to mitigate losses and allow for more rational and productive management of sheep and bison, an effective SA-MCF vaccine is urgently needed. Researchers in Pullman, Washington, and collaborators developed a chimeric virus, based on a mutated alcelaphine herpesvirus 1, that is non-pathogenic but can induce immune responses against an OvHV-2 protein necessary for virus infection. Vaccine trials using the new chimeric virus in a laboratory rabbit model resulted in over 70 % of protection from disease following experimental exposure to a lethal dose of OvHV-2 and protective immunity remained for at least nine months. These results are promising for future research to test the safety and efficacy of this viral-vectored vaccine candidate in bison, the most relevant target for a SA-MCF vaccine.

Review Publications
Shringi, S., O'Toole, D., Cole, E., Baker, K.N., White, S.N., Donofrio, G., Li, H., Cunha, C.W. 2021. OvHV-2 glycoprotein B delivered by a recombinant BoHV-4 is immunogenic and induces partial protection against sheep-associated malignant catarrhal fever in a rabbit model. Vaccines. 9(2). Article 90. https://doi.org/10.3390/vaccines9020090.
Headley, S.A., de Oliveira, T.E.S., Cunha, C.W. 2020. A review of the epidemiological, clinical, and pathological aspects of malignant catarrhal fever in Brazil. Brazilian Journal of Microbiology. 51:1405-1432. https://doi.org/10.1007/s42770-020-00273-6.
Headley, S.A., Muller, M.C., de Oliveira, T.E.S., Barros Gil Duarte, C.A., Valente Pereira, P.F., Vieira, M.V., Cunha, C.W., Flores, E.F., Lisboa, J.A.N., Pretto-Giordano, L.G. 2020. Diphtheric aspergillosis tracheitis with gastrointestinal dissemination secondary to viral infections in a dairy calf. Microbial Pathogenesis. 149. Article 104497. https://doi.org/10.1016/j.micpath.2020.104497.
Carvallo, F.R., Uzal, F.A., Moore, J.D., Jackson, K., Nyaoke, A.C., Naples, L., Davis-Powell, J., Stadler, C.K., Boren, B.A., Cunha, C.W., Li, H., Pesavento, P.A. 2020. Ibex-associated malignant catarrhal fever in duikers (Cephalophus spp). Veterinary Pathology. 57(4):577-581. https://doi.org/10.1177/0300985820918313.