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

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2019 Annual Report

Objectives
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.

Approach
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
This year, Foreign Animal Disease Research Unit (FADRU) scientists regained limited access to animal facilities, allowing initial testing of some of the vaccines and biotherapeutics and carrying out some pathogenesis and transmission studies with foot-and-mouth disease (FMDV) in cattle and pigs. In addition, our personnel situation improved, and we were able to hire an Immunologist who started work in June 2019 and filled three scientific support positions. Under Objective 1 (countermeasures), progress continues on our lead vaccine candidate; the FMD3B3D platform which is now select agent excluded and we have started transfer of molecular constructs to our commercial partner to derive master seeds of relevant vaccine strains and serotypes in the United States mainland. Efforts have continued toward the development of FMDV modified live vaccines. In collaboration with a commercial partner, ARS scientists discovered that multiple regions of the viral genome that tolerate codon pair bias deoptimization resulting in attenuated viruses that are currently being evaluated as vaccine candidates. A new DIVA FMD ELISA test based on Nanobody-technology was developed for the detection of foot-and-mouth disease (FMD) infected cattle. This novel technology, using camelid-derived single-nanobody fragments showed great specificity and sensitivity and provides a new low-cost detection method for FMD infection in cattle. For Objective 2 (virus-host interactions) great progress was made in understanding the establishment and maintenance of the carried state in cattle. Specifically, studies on the evolution of the FMD virus during persistence using next generation genetic sequencing provided insight on the mechanisms of virus maintenance and emergence. Host gene expression showed that a previously unknown bovine gene was differently expressed between carriers than non-carriers in the epithelia where persistence occurs. Bioinformatic analyses showed that this was novel gene similar to a mouse gene important for regulating local immune responses, providing insight on a potential mechanism for persistence. For Objective 3 (ecology), we continued studies in Africa and in Asia, with important reports on virus evolution during persistent infections in Asian buffalo in India and Pakistan and characterization of FMDV circulating in Uganda, Kenya, Cameroon, Nigeria, and Vietnam.

Accomplishments
1. Transmission modeling of FMDV in natural hosts. Although FMD is widely described as the most important disease affecting international trade in animal product, many aspects of the basic biology of the causative virus remain unknown. Researchers at USDA, ARS, Orient Point, New York have been working to understand how FMD virus transmits between animals and within populations. Recent work has shown that pigs can transmit the virus before any signs of disease. Research has also showed that FMD virus may be transmitted from oral fluids of carrier cows which have no signs of disease. These findings are critically important to understand how FMD spreads within natural livestock hosts, and to prepare for the possibility of an outbreak in the United States.

2. Discovering a new region in the FMDV important for vaccine efficacy. It has been previously reported that the G-H loop is a flexible structure within the FMDV capsid, which is able to induce a dominant antibody response in the animal host. Researchers at USDA, ARS, Orient Point, New York have demonstrated that modification of this structure in the Ad5-delivered FMD vaccine is tolerable and does not compromise the induction of total anti-FMD antibodies in swine, however, despite the high amount and increased reactivity, these antibodies failed to protect animals against challenge with virulent FMDV. This data suggests that although highly variable, this region of the G-H loop contributes to the protective immunity conferred by Ad5 vector-delivered FMD vaccines in swine, and cannot be substituted without a loss of vaccine efficacy.

Review Publications
Visser, L.J., Medina, G.N., Rabouw, H.H., De Groot, R.J., Langereis, M.A., De los Santos, T.B., Van Kuppeveld, F.J. 2018. FMDV leader protease cleaves G3BP1 and G3BP2 and inhibits stress granule formation. Journal of Virology. https://doi.org/10.1128/JVI.00922-18.
Gelkop, S., Sobarzo, A., Brangel, P., Vincke, C., Romao, E., Fedida-Metula, S., Strom, N., Ataliba, I., Mwiin, F.N., Ochwo, S., Velazquez-Salinas, L., McKendry, R.A., Muyldermans, S., Lutwama, J.J., Rieder, A.E., Yavelsky, V., Lobel, L. 2018. The development and validation of a novel nanobody-based competitive ELISA for the detection of foot and mouth disease 3ABC antibodies in cattle. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2018.00250.
Medina, G.N., Diaz San-Segundo, F., Stenfeldt, C., Arzt, J., De los Santos, T.B. 2018. The different tactics of foot-and-mouth disease virus to evade innate immunity. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2018.02644.
Chen, X., Gaglione, R., Leong, T., Bednor, L., De los Santos, T.B., Luk, E., Airola, M., Hollingsworth, N.M. 2018. Mek1 coordinates meiotic progression with DNA break repair by directly phosphorylating and inhibiting the yeast pachytene exit regulator Ndt80. PLoS Genetics. https://doi.org/10.1371/journal.pgen.1007832.
Barrionuevo, F., Di Giacomo, S., Bucafusco, D., Ayude, A., Schammas, J., Miraglia, M.C., Capozzo, A., Borca, M.V., Perez-Filgueira, M. 2018. Systemic antibodies administered by passive immunization prevent generalization of the infection by foot-and-mouth disease virus in cattle after oronasal challenge. Virology. 518:143-151. https://doi.org/10.1016/j.virol.2018.02.012.
De Los Santos, T.B., Diaz-San Segundo, F., Rodriguez, L.L. 2018. The need for improved vaccines against foot-and-mouth disease. Current Opinion in Virology. 29:16-25. https://doi.org/10.1016/j.coviro.2018.02.005.
Palinski, R., Bertram, M.R., Vu, L.T., Pauszek, S.J., Hartwig, E.J., Smoliga, G.R., Stenfeldt, C., Fish, I.H., Hoang, B.H., Dung, D.H., Arzt, J. 2019. First genome sequence of Foot-and-mouth disease virus serotype O sublineage Ind2001e from Vietnam. Microbiology Resource Announcements. https://doi.org/10.1128/MRA.01424-18.
Arzt, J., Bertram, M.R., Vu, L.T., Pauszek, S.J., Hartwig, E.J., Smoliga, G.R., Palinski, R., Stenfeldt, C., Fish, I.H., Hoang, B.H., Dung, D.H. 2019. First detection of Senecavirus A in Vietnam. Emerging Infectious Diseases. https://doi.org/10.1128/MRA.01247-18.
Bertram, M., Bravo De Rueda, C., Garabed, R., Dickmu Jumbo, S., Moritz, M., Pauszek, S.J., Abdoulkadiri, S., Rodriguez, L.L., Arzt, J. 2018. Molecular epidemiology of foot-and-mouth disease virus in the context of transboundary animal movement in the far north region of Cameroon. Frontiers in Veterinary Infectious Diseases. https://doi.org/10.3389/fvets.2018.00320.
Arzt, J., Branan, M., Delgado, A., Yadav, S., Moreno-Torres, K., Tildesley, M., Stenfeldt, C. 2019. Quantitative impacts of incubation phase transmission of foot-and-mouth disease virus. Scientific Reports. https://doi.org/10.1038/s41598-019-39029-0.
Arzt, J., Belsham, G.J., Lohse, L., Botner, A., Stenfeldt, C. 2018. Transmission of foot-and-mouth disease from persistently infected carrier cattle to naive cattle via transfer of oropharyngeal fluid. mSphere. 3(5):e00365-18. https://doi.org/10.1128/mSphere.00365-18.
Biswal, J.K., Ranjan, R., Subramaniam, S., Mohapatra, J.K., Sharma, M.K., Bertram, M.R., Brito, B., Rodriguez, L.L., Pattnaik, B., Arzt, J. 2019. Genetic and antigenic variation of foot-and-mouth disease virus during persistent infection in naturally infected cattle and Asian buffalo in India. bioRxiv. https://doi.org/10.1371/journal.pone.0214832.
Omondi, G., Alkhamis, M., Obanda, V., Gakuya, F., Sangula, A., Pauszek, S.J., Perez, A., Ngulu, S., Van Aardt, R., Arzt, J., Vanderwaal, K. 2019. Phylogeographic and cross-species transmission dynamics of SAT1 and SAT2 foot-and-mouth disease virus in Eastern Africa. Molecular Ecology. https://doi.org/10.1111/mec.15125.
Fernandez-Sainz, I., Gavitt, T.D., Koster, M., Ramirez-Medina, E., Rodriguez, Y.Y., Wu, P., Silbart, L.K., De Los Santos, T.B., Szczepanek, S.M. 2019. The VP1 G-H loop hypervariable epitope contributes to protective immunity against foot and mouth disease virus in swine. Vaccine. https://doi.org/10.1016/j.vaccine.2019.05.019.
Arzt, J., Fish, I., Pauszek, S.J., Johnson, S.L., Chain, P.S., Rai, D.K., Rieder, A.E., Goldberg, T.L., Rodriguez, L.L., Stenfeldt, C. 2019. The evolution of a super-swarm of foot-and-mouth disease virus in cattle. Virology. https://doi.org/10.1371/journal.pone.0210847.
Stenfeldt, C., Smoliga, G.R., Hartwig, E.J., Palinski, R., Fish, I.H., Silva, E.B., Pauszek, S.J., Arzt, J. 2018. Contact challenge of cattle with foot-and-mouth disease virus validates the role of the nasopharyngeal epithelium as the site of primary and persistent infection. Scientific Reports. https://doi.org/10.1128/mSphere.00493-18.