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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

2020 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
Under Objective 1 (countermeasures), progress continued toward the development of FMDV3B3D inactivated vaccines through a Cooperative Research and Development Agreement (CRADA) partnership. A significant number of serotype A, O, C, Asia and Sat2 capsid constructs have been synthesized by the commercial partner and virus stocks have been successfully produced at Plum Island Animal Disease Center (PIADC). Progress has been made to establish a production facility on the U.S. mainland where master stocks of the main serotypes will be produced in the upcoming year. This will allow further development of leaderless vaccines. Work has continued on exploring modified live vaccines. In collaboration with a commercial partner, ARS scientists have been able to expand previous work using codon deoptimization on FMDV A serotype, to other subtypes/serotypes (A24, O1 Campos and Asia1). Testing in mice and swine demonstrated that mutant viruses were attenuated and induced a neutralizing antibody response. This work highlighted the use of this technology to generate attenuated strains that could be used toward the development of live attenuated or inactivated vaccine candidates. A provisional patent was filed SN: 63/030,431. In collaboration with industry partners, ARS scientists evaluated the efficacy of modified porcine interferon molecules for their capacity of providing long lasting bioavailability and efficacy in swine. This study highlighted the benefit of using a combination of this molecule with vaccines to provide immediate and long-term protection against FMD in swine. This work was performed under Material Transfer and Research Agreement (MTRA) 8064-32000-061-46H. Under Objective 2 (virus-host interactions), substantial progress was made in understanding the establishment and maintenance of the carrier state in cattle. Specifically, towards elucidating the evolution and strain emergence of FMDV during acute and persistent infection was made through a variety of experimental approaches. Highly complex in vivo experiments generated samples that were analyzed by a combination of different techniques including next generation genetic sequencing, immune-microscopy, and conventional molecular and cellular virological analyses. These approaches provided insights on the mechanisms of virus maintenance and emergence. Co-infection of cattle with distinct viral strains demonstrated that viruses may recombine within the upper respiratory tract to generate new, genetically distinct emergent viruses. Additionally, critical new insights were achieved regarding cattle gene expression during the carrier state and these results were published in several peer-reviewed publications. Progress continued in the host-response to FMDV infection. A new bovine chemokine gene named as CXCL15 was identified and annotated. The expression of this gene was significantly downregulated in FMDV carriers compared to non-carriers. The differential gene expression between carriers and non-carriers including CXCL15 detected with whole genome expression profiling in the tissues of FMDV persistent infection has led to a major progress in understanding the molecular mechanisms that could explain why carriers cannot clear the virus and vaccine cannot cure the persistent infection. The potency of an interferon biotherapeutic has been improved by approximately 20 times based on the test results in pigs, which invention has been patented. These new discoveries provide insightful information and a useful tool for FMD control. Great progress was made toward the understanding of a novel function of the FMDV Leader protein to block the innate immune response. As part of NBAF workforce development, ARS scientists discovered a novel separation of function mutation in the FMDV leader protein that uncouples two specific enzymatic activities. Impairment of novel deISGylase activity (cleavage of ISG15 from target proteins), rendered viable FMDV strains that were significantly attenuated in vitro and in vivo. In addition, ARS scientists demonstrated a novel role of ISG15 to inhibit FMDV replication. A combination of this mutation with others could be applied towards development of effective live attenuated vaccines. Furthermore, these studies highlight the potential of using ISG15 as an antiviral or vaccine adjuvant to control FMD. This work was performed under agreement 8064-32000-061-54S with Kansas State University for the National Bio and Agro Defense Facility (NBAF) work force development program. Two manuscripts were published (logs 370339 and 369287). Progress was made in discovering a new region in the FMDV 3D protein (polymerase enzyme) important for virus replication and polymerase fidelity. It has been previously reported that the 3D enzyme encodes a sequence motif important for import of the protein to host nucleus. We identified mutations in the viral protein 3D responsible for viral replication that affect the accuracy of the enzyme during viral replication (i.e. fidelity) resulting in decreased virus growth in host cells. The alteration of 3D fidelity described uncovers a novel strategy to derive safer and more stable attenuated FMDV vaccine candidates. In response to stakeholder requests (Animal and Plant Health Service APHIS and National Pork Board) a series of FMDV experiments were carried in pigs to contribute novel scientific data of critical value for epidemiological modeling of FMD outbreaks affecting pig holdings. Specifically, the minimum infectious dose of FMDV required to cause disease in pigs exposed to FMDV-contaminated feed was established. Additionally, the duration of infectiousness of FMDV-infected live pigs, and the extent of contagion associated with carcasses from deceased FMDV-infected pigs were established and published in a peer-reviewed journals. Under Objective 3 (epidemiology and ecology), field collaborative investigations were continued in Africa and Asia. Important output included a novel, modeled meta-analysis which provided systems to determine the duration of the carrier state under differing endemic conditions. This approach demonstrated that 12 months after an outbreak of FMD, up to 51% of carrier animals may remain infected. These studies also led to discovery and report of full-length sequences of genetically unique virus strains in Vietnam, India, Kenya, and Uganda which are now available to the global research community. Important insights were achieved regarding multi-strain co-infections and interactions between viruses in cattle and wild buffalo in Kenya. Specifically, it was found that viruses are transmitted across species, yet those events occur infrequently. Additionally, we concluded a five-year project in Uganda and documenting the distribution of FMDV-exposed cattle and examining risk factors associated with the disease. As limited resources prevent routine mass vaccination, the information generated will aid on the improvement of FMD control plans for Uganda, and ultimately, support the control and eradication of the disease in the Eastern African region.

1. New Foot-and-Mouth Disease (FMD) vaccine candidates. Although inactivated antigen vaccines are available, they induce only a narrow and short-lived protective immunity, whereas live-attenuated vaccines induces broader or longer-lived immunity. ARS scientists in Orient, New York, in collaboration with scientists at Codagenix Inc, Farmingdale, New York, evaluated the use of codon-deoptimization to produce FMD vaccine candidates. These vaccine candidates of multiple FMD vaccine serotypes, showed promising results in vaccinated pigs. These results highlight the potential of these novel approaches toward the development of live vaccine candidates that could be used in endemic areas towards disease eradication. A patent was filed (Docket Number 137.18) for this invention.

Review Publications
Medina, G., Azzinaro, P.A., Ramirez-Medina, E., Gutkoska, J.R., Fang, De Los Santos, T.B. 2020. Impairment of the deISGylation activity of foot-and-mouth disease Lpro causes viral attenuation without affecting interferon expression during viral infection. Virology.
Eschbaumer, M., Dill, V., Carlson, J.C., Arzt, J., Stenfeldt, C., Krug, P., Hardham, J., Stegner, J.E., Rodriguez, L.L., Rieder, A.E. 2020. Foot-and-Mouth disease virus lacking the leader protein and containing two negative DIVA markers (FMDV A24 LL3B3D) is fully attenuated in pigs. Pathogens.
Yadav, S., Stenfeldt, C., Branan, M.A., Moreno-Torres, K.I., Holmstrom, L., Delgado, A.H., Arzt, J. 2019. Parameterization of the durations of phases of foot-and-mouth disease in cattle. Frontiers in Veterinary Science.
Mwiine, F.N., Velazquez-Salinas, L., Ahmed, Z., Ochwo, S., Munsey, A., Kenney, M.A., Lutwama, J.J., Maree, F.F., Lobel, L., Perez, A.M., Rodriguez, L.L., Vanderwaal, K., Rieder, A.E. 2019. Serological and phylogenetic characterization of foot and mouth disease viruses from Uganda during cross sectional surveillance study in cattle between 2014 and 2017. Transboundary and Emerging Diseases.