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Research Project: Intervention Strategies to Support the Global Control and Eradication of Foot-and-Mouth Disease Virus (FMDV)

Location: Foreign Animal Disease Research

2021 Annual Report

1) Development of intervention strategies to control and eradicate FMDV including vaccines and biotherapeutics/adjuvants that rapidly induce long lasting and cross-protectiveimmunity against multiple FMDV subtypes, and are capable of preventing infection and controlling/abrogating persistent infections, developing vaccine formulations and delivery targeting the mucosal immune responses. Development of novel FMD vaccine platforms. Development of marker FMDV LL3B3D vaccines against relevant outbreak strains. Development of improved second-generation Ad5-FMD vaccines. Discovery of modified live attenuated FMDV vaccine candidates (MLAV). Discovery of cross-protective vaccines against multiple FMDV subtypes, and develop vaccine formulations. Development of novel biotherapeutics to prevent or control FMD prior to vaccine-induced protection. Discovery/development of novel biotherapeutics with increased potency and extended systemic half-life. Evaluation of combined delivery of biotherapeutics and vaccine in swine and cattle. Evaluation of vaccine-induced immunity and FMDV carrier state. Characterization of host immunity associated with novel vaccines against FMDV. Evaluation of novel vaccines for ability to prevent the FMD carrier state in cattle and assess the host response associated with the carrier divergence. 2) . Elucidate the host-pathogen interactions of FMDV, including identifying viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions at the primary sites of infection in ruminants and pigs with focus on factors defining tropism and early host responses, and determining characteristics and mechanisms of FMDV within-hot evolution over distinct phases of infection. Determine the molecular basis for FMDV-host interactions that impact virulence. Examination of virus factors contributing to FMDV virulence. Examination of host factors contributing to FMDV virulence. Identification of molecular mechanisms associated with the establishment of FMDV persistence. Determination of host and/or other non-FMDV factors causing or associated with clearance of FMDV from bovine nasopharyngeal tissue. Investigation of within-host FMDV genomic evolution to characterize sitespecific mutational pressure, genomic variation, and potential adaptation to the host. Determination of the immune mechanisms affecting protective immunity against FMDV. Analysis of CD4 helper T-cell response to FMDV vaccination. Analysis of CD8 cytotoxic T-cell response to FMDV vaccination. Analysis of B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3) Understanding ecology of FMDV in endemic regions, determining drivers of transmission and maintenance in endemic settings, characterizing risk factors driving FMDV emergence and spread. Characterize the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. Characterize factors driving FMDV emergence and spread of novel FMDV strains in endemic settings. Role of Asian buffalo in maintenance and transmission of FMDV in endemic settings.

1. The development of intervention strategies to control and eradicate FMDV will be achieved through research on novel FMD vaccine platforms including of marker modified live-attenuated FMDV vaccine candidates (e.g. FMDV-LL3B3D), second–generation Ad5-FMD vaccines, and cross-protective vaccines against multiple subtypes. Additionally, combinations of vaccine and biotherapeutics / and or adjuvants will be investigated as a way to induce mucosal immunity necessary not only to prevent disease but also to decrease persistent infection. These vaccine/ adjuvant formulations will be tested using alternate routes such as transdermal and by direct mucosal delivery. 2. The host-pathogen interactions of FMDV will be determined through: the identification of viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions associated with the FMDV life cycle, and determining the mechanisms of protective immunity to FMDV. The molecular basis for FMDV-host interactions that impact virulence and their specific contributions to virulence will be determined. In addition, the interactions of the virus with specific tissues at the primary infection sites will be studied by characterizing infected tissues at the cellular and subcellular level as well as utilizing transcriptomic analyses with micro arrays and next generation RNAseq. Bioinformatic analyses will be extensively applied in order to understand species specific factors mediating the establishment and maintenance of persistent infections. The within-host FMDV genomic evolution will be characterized through an examination of site-specific mutational pressure, genomic variation and potential adaption to the host. The immune mechanisms affecting protective immunity against FMDV will be determined through the analysis of CD4 helper and CD8 cytotoxic T cell responses to FMDV vaccination and B-cell responses to FMDV in peripheral blood and lymphoid tissue. 3. The characterization of the ecology of FMDV in endemic regions, including determining drivers of FMDV transmission and maintenance in endemic regions, characterizing factors driving FMDV emergence and spread, and the characterization of the role of the Asian buffalo in the transmission and maintenance of FMDV in the context of tolerance to infection will be analyzed. Efforts will focus on the characterization of the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance. Factors driving FMDV emergence and spread of novel FMDV strains in endemic settings will be characterized. The role of Asian buffalo in maintenance and transmission of FMDV in endemic settings will be assessed.

Progress Report
Despite the challenges of the COVID pandemic, restricted access to the laboratory, and suspension of animal experiments for most of FY21, the Foot and Mouth Disease (FMD) research team achieved many of the project milestones for the year. At the same time, dedicated significant resources to multiple activities toward the National Bio and Agro-Defense Facility (NBAF) transition. This included the establishment of a biorepository task-force to help with biorepository inventories, large scale destruction of biorepository samples not going to NBAF, the establishment of a new laboratory with diagnostic capabilities for safety and freedom testing of samples, and initiation of the large scale scanning of tens of thousands of pages of laboratory records for transition to the NBAF. Under Objective 1: “Develop intervention strategies to control and eradicate Foot-and-Mouth Disease Virus (FMDV), Subobjective 1.1. Development of novel FMD vaccine platforms,” there were important achievements. In collaboration with Industry, under a Cooperative Research Development Agreement (CRADA) partnership, progress continued toward the development of FMDV3B3D inactivated vaccines and small-scale production of pre-Master Seed. A number of relevant vaccine strains for several serotypes have been synthesized by the commercial partner, and virus stocks have been successfully produced at PIADC. A production facility on the U.S. mainland was built by our CRADA partner, where master stocks of the main serotypes will be produced in the coming year. Additionally, using the FMDLL3B3D platform, vaccines with mosaic capsids were produced against serotypes O and Asia and successfully tested (O only) for protection against heterologous challenge. These proof-of-concept experiments served as support toward a patent application for this technology. Work has continued on exploring modified live vaccine (MLV) candidates. Testing a multifactorial mutant MLV candidate that demonstrated attenuated characteristics in vitro displayed pathogenic characteristics similar to a parental virus in swine. Similarly, MLV candidates created using codon-deoptimization showed attenuated phenotype in mice but were still virulent in pigs. This work demonstrated that MLV for FMD is still a great challenge. Under Subobjective 1.2, “Development of novel biotherapeutics,” important progress was made in the use of pegylation to prolong the antiviral effect of IFN against FMDV. This work, part of a CRADA, demonstrated that pegylated recombinant porcine (po)IFNa displays strong and long-lasting antiviral activity against FMDV in vitro and in vivo, completely protecting swine against FMD for at least five days after a single dose. This opens the possibility of formulating vaccines with pegylated IFNs as an emergency control approach. In Objective 2, host-pathogen interactions, key molecular mechanisms of disease involving the leader protease (Lpro) were discovered specifically regarding novel activities of this viral protein Using protein structural analysis of FMDV Lpro, amino acids associated to enzymatic activity (deUbuquitinase/deISGylase activity) were identified. Mutation of these amino acid residues in FMDV resulted in a perceptible level of attenuation in vitro and in vivo, opening possibilities to apply in MLV development. In Subobjective 2.2, research focused on studying virus evolution during persistence and particularly simulating viral coinfections as have been observed in field studies. The dynamics of infection and the predominance of different viral serotypes or subtypes at different phases of infection opened the door to understanding the emergence of novel viruses under natural conditions. Most importantly, it was demonstrated that during dual infection of cattle with 2 distinct strains, recombination of the viral genomes occurs with high frequency during the first 30 days of infection. Viral ecology studies (Objective 3) were completed in various countries in Asia and Africa, showing various patterns of virus distribution both in subclinical and in clinical infections. These patterns demonstrated the occurrence of viral “waves” that went across borders both in Asia and in Africa, many times initiating in areas with wildlife reservoirs. It was demonstrated that these waves of viral spread occur much more frequently than previously recognized in association with asymptomatic infections. Studies in Uganda quantified FMD dispersal directionality in cattle allowing them to anticipate viral movement and more strategically tailor intervention strategies that ultimately support FMD control in East African. In addition to our existing project milestones, research was carried out aimed at solving stakeholder problems. One example is a study done on the transmissibility of FMDV in pigs by contaminated feed. These studies demonstrated that although transmission did occur, viral serotype was a major determinant of the success and level of transmission. This data will contribute to the modelling of FMD transmission and might inform regulatory authorities on the risk of FMD from feed imports. A second study was undertaken to create an adventitious agent-free cell line with high susceptibility to all serotypes of FMDV. This new clonal cell line (MGPK) was patented and is a promising cell line to produce FMD vaccines, especially for serotypes that are difficult to adapt to production cell lines.

1. Developing the capacity for FMD vaccine production in the United States. Foot and mouth disease FMD continues to be a major threat to U.S. agriculture. An FMD outbreak would have devastating consequences on the $1.11 trillion food and agricultural Industry. The U.S. invests millions of dollars in strategic vaccine banks for the most prevalent FMD viruses circulating around the world. However, all vaccines need to be purchased overseas as domestic vaccine production requires live virus, which is forbidden by law in the US ARS scientists developed a platform that allows safe vaccine production in the U.S. Working closely with a commercial vaccine manufacturer, the new vaccine is now approved for research and development and eventually manufacturing in the U.S. mainland, at a new multi-million-dollar facility built by the CRADA partner for this purpose. This achievement was highlighted in recent press reports (

2. Foot-and-Mouth Disease virus be effectively transmitted by contaminated feed. The emergence of porcine epidemic diarrhea virus (PEDV), believed to have been accidentally imported in pig feed products from China, has increased the concern of pork industry stakeholder groups regarding the potential importation of pathogens (e,g, Foot and Mouth Disease Virus- FMDV) through contaminated feed products. At the request of stakeholders, ARS researchers demonstrated the ability of contaminated feed to infect pigs through natural ingestion even 30 days after feed contamination (duration of transit from Asia). These findings are critically important to understand the risks and mitigation methods to prevent the introduction of FMDV in imported animal feed products. Work was published in Transboundary and Emerging Diseases in 2021:

3. Immediate and sustained protection against Foot and Mouth Disease (FMD). Current FMD vaccines require 5 days to induce enough immunity to prevent viral spread. The time between vaccination and protection can be critical in the emergency response to an outbreak. Interferons (IFN), produced as the first line of defense against viruses, can induce almost immediate protection against FMD but usually have a short life once injected in the animals and do not protect long enough for the onset of vaccine immunity. ARS researched demonstrated that a modified (pegylated) porcine IFN induces a strong and long-lasting antiviral activity against FMDV, completely protecting swine for at least five days after injection. This discovery opens the door for testing the pegylated INF, combined with the vaccine to achieve immediate and sustained protection to control outbreaks of FMD and potentially other viral diseases. Use of Protein Pegylation to Prolong the Antiviral Effect of INF Against FMDV. (Front. Microbiol., 05 May 2021 |

Review Publications
Diaz San Segundo, F.C., Medina, G.N., Mogulothu, A., Azzinaro, P.A., Attreed, S.E., Gutkoska, J.R., Lombardi, K.R., Shields, J., Hudock, T.A., De Los Santos, T.B. 2021. Use of Protein Pegylation to prolong the antiviral effect of IFN against FMDV. Frontiers in Microbiology.
Hardham, J., Krug, P., Pacheco, J., Thompson, J., Domanowski, P., Gay, C.G., Rodriguez, L.L., Rieder, A.E. 2020. Novel Foot-and-Mouth disease vaccine platform: Formulations for safe and DIVA-compatible FMD vaccines with improved potency. Frontiers in Veterinary Science.
Medina, G., De Los Santos, T.B., Diaz San Segundo, F.C. 2020. Use of IFN-based biotherapeutics to harness the host against foot-and-mouth disease. Frontiers in Microbiology.
Ramierez-Medina, E., Vuono, E., Rai, A., Pruitt, S.E., Silva, E., Velazquez-Salinas, L., Zhu, J.J., Gladue, D.P., Borca, M.V. 2020. Evaluation in swine of a recombinant African swine fever virus lacking the MGF-360-1L gene. Viruses.
Kloc, A., Rai, D.K., Kenney, M.A., Schafer, E.A., Rieder, A.E., Gladue, D. 2020. Residues within the foot-and-mouth disease virus 3dpol nuclear localization signal affect polymerase fidelity. Journal of Virology.
Chitray, M., Opperman, P., Rotherham, L., Fehrsen, J., Frischmuth, J., Rieder, A.E., Maree, F. 2020. Diagnostic and epitope mapping potential of single-chain antibody fragments against foot-and-mouth disease serotype A, SAT1 and SAT3 viruses. Frontiers in Veterinary Science.
Diaz San Segundo, F.C., Medina, G., Spinard, E., Kloc, A., Ramirez-Medina, E., Azzinaro, P.A., Mueller, S., Rieder, A.E., De Los Santos, T.B. 2021. Use of synonymous deoptimization for the development of modified live attenuated strains of foot and mouth disease virus. Frontiers in Microbiology.
Munsey, A., Mwiine, F., Ochwo, S., Velazquez Salinas, L., Ahmed, Z., Maree, F., Rodriguez, L.L., Rieder, A.E., Perez, A., Vanderwaall, K., Dellicour, S. 2021. Tracking the dispersal of foot-and-mouth disease virus serotype O in East Africa using phylodynamics. Molecular Ecology.