Location: Foreign Animal Disease Research
2024 Annual Report
Objectives
OBJECTIVE 1. Develop vaccines engineered for the control and eradication of Foot-and-Mouth Disease Virus (FMDV).
Sub-objective 1.A Determine the mechanisms of protective immunity to FMDV conferred by multiple Foot-and-Mouth Disease (FMD) vaccine platforms.
Sub-objective 1.B Determine the mechanisms mediating duration of immunity to FMDV.
Sub-objective 1.C Develop vaccine platforms that will provide cross-protective immunity against different FMDV subtypes.
Sub-objective 1.D Develop vaccines that reduce the incidence and accelerate clearance of persistent FMDV infections in cattle.
Sub-objective 1.E Develop vaccines that can be rapidly manufactured to respond to new FMDV outbreaks.
Objective 2. Develop biotherapeutics that can rapidly control the spread of FMD.
Sub-objective 2.A Develop biotherapeutic platforms that induce rapid onset of immunity as a companion to an effective FMD vaccination.
Sub-objective 2.B Develop biotherapeutics that alone or in vaccine formulations can reduce or abrogate FMDV persistence.
Objective 3. Elucidate the host-pathogen interactions of FMDV.
Sub-objective 3.A Identify viral determinants of FMD that control virulence in susceptible hosts.
Sub-objective 3.B Determine the virus/host interactions associated with FMDV persistence.
Objective 4. Characterize the ecology of FMDV in endemic regions.
Sub-objective 4.A Determine drivers of FMDV transmission and maintenance in endemic regions.
Sub-objective 4.B Determine factors that drive viral evolution and the mechanisms that lead to the emergence and spread of new FMDV strains.
Sub-objective 4.C Support the epidemiological analysis of data collected from countries with FMDV epidemics.
Approach
1. The development of intervention strategies to control and eradicate Foot-and-Mouth Disease Virus (FMDV) will be achieved through research on novel Foot-and-Mouth Disease (FMD) vaccine platforms including of marker modified live-attenuated FMDV vaccine candidates such as FMDV-LL3B3D, second–generation Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) vaccines, and cross-protective vaccines against multiple subtypes.
2. 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.
3. 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 RNAsequencing. Bioinformatic analyses will be extensively applied in order to understand species specific factors mediating the establishment and maintenance of persistent infections. The withinhost FMDV genomic evolution will be characterized through an examination of sitespecific 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.
4.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
In Objective 1, under Sub-objective 1C, progress involved developing new tools in pig immunology with a focus on humoral immunity to foot-and-mouth disease virus (FMDV). Through a partnership with university collaborators, we have established a new system for single cell transcriptomics to study the B cell repertoire. By analyzing the genetic information of individual B cells, we can now describe the different types of B cells, understand what they do, and better understand the complexities of the pig immune system. With these methods and the use of bioinformatic analysis, we made a detailed map of all the B cells in pigs. We identified the diversity of B cells, showing the wide variety of antigen specificities that the B cell population can recognize. This is important because it can characterize the broad range of antigens necessary for an effective primary immune response. This diversity plays a key role in producing a variety of antibodies that can neutralize pathogens and enhance immune defenses. In the future, we plan to conduct similar studies with infected or vaccinated pigs.
Under Sub-objective 1E, progress included the evaluation of next generation adenovirus vaccine vectors (carrying a packaging-deficient mutation) in a complementing cell line. Previous evaluation had indicated challenges with genetic stability and virus recovery. To solve this, we have incorporated an intentional change in the viral genome demonstrated to improve stability. Testing started to determine if this Ad5-FMD (foot and mouth disease) virus can be rescued and characterized.
In Objective 2, Sub-objective 2A, substantial progress was achieved as we have successfully constructed an unconventional type I interferon (IFN), namely porcine IFN omega, and evaluated its antiviral activity both in cell culture and in animals, with a focus on pigs as our target population. Remarkably, administering a higher dose range of porcine IFN omega has led to complete protection following exposure to the FMDV O1 Manisa strain 24 hours after treatment. Current efforts are focused on evaluating if sterile (complete) protection was obtained and understanding the immunological parameters of protection, particularly innate immunity. Combination of this IFN with FMD vaccine will be evaluated in future objectives within the project. In addition, we have made substantial progress by developing a fusion protein of bovine interferon-lambda and the specific fragment (Fc) of bovine antibody IgG (bovIFN-lambda-Fc). When this construct (bovIFN-lambda-Fc) was used to generate proteins in mammalian cell lines (i.e., HEK293), the growth media collected demonstrated strong antiviral properties against FMDV. The production of this construct on a small to medium scale has been outsourced to an external vendor. Additionally, we have enhanced our study design by creating control samples that lack the Fc component. These control samples are currently undergoing testing, including the use of a modified version containing a cleavage site to remove the Fc fusion domain. After purification, these constructs will be evaluated for their biological antiviral effectiveness before moving on to testing in live animals. Under Sub-objective 2B, we have made progress in using tissues from the upper respiratory tract (nasopharynx) of cattle to refine strategies for separating tissue cells for host transcriptome studies of individual cells. Our work has been successful in mastering this technique, which sets the stage for creating detailed maps of the immune system in the nasopharynx of cattle. The ongoing bioinformatic analysis is expected to show the different types of cells present in the area where the FMD virus is known to initiate replication in cattle.
In Objective 3, Sub-objective 3B, has been substantially met through work analysis completed on specific tissues of cattle including the soft palate, nasopharynx and lymph node during critical stages of FMDV infection—the transitional phase and the persistent phase. One in vivo experimental study was completed to obtain bovine tissues from the critical transitional phase of infection. Two phenotyping analysis panels were developed for this work: the first differentiating helper T cell (Th) subsets, while the second distinguishes regulatory T cells and “exhaustion” in T cells, a metabolic state of reduced effector function. Knowing the relative abundance, metabolic state and immunological bias of T cells at the site of infection and the draining lymph node can illuminate the broader immune landscape, as T cells play many roles, including the direct killing of infected cells, orchestration of the general approach to infection response and determining the breadth and isotype/subtype specificity of B cell antibody response. Early analysis indicated significant proportions of both CD4+ Th cells and CD8+ cytotoxic T cells expressing high levels of a master regulatory transcription factor along with skewing towards a Th17 response and away from a Th1 response. Taken together, this data suggests that T cells play a role in creating an immune tolerant environment at the site of FMDV persistence in cattle and that the Th response may be dysregulated in such a way as to dampen the cytotoxic response. Our ongoing analysis of the data involves correlating the abundance of these various T cell populations and transcription factor expression levels with the persistence data for each animal to help illuminate the effect that these patterns have on individual susceptibility to persistent infection.
Under Objective 4, progress was made in Sub-objective 4A through experiments that demonstrated transmission from FMDV-infected cattle for at least 24hrs before development of clinical signs of infection. Sub-objective 4B was advanced through continued analyses of the genome organization of recombinant FMDVs obtained from super-infected carrier cattle. These analyses revealed that certain genome segments derived from specific parental FMDV strains were over-represented in recombinant genomes, suggesting potential selective advantage of different regions of select strains of FMDV.
Further, within Sub-objectives 4A.1. and 4B.1, substantial progress was made in establishing an in-house capacity to conduct statistical and mathematical modeling, including integrated genetic and demographic methods like phylodynamics and phylogeography. This capacity enables us to elucidate population connectivity, viral sources and sinks, and their impacts on virus evolution and recombination within endemic regions. Four new cooperative agreements have been established with the Animal and Plant Health Inspection Service (APHIS), Michigan State University, Mississippi State University, and Oregon State University. These partnerships aim to investigate mechanisms of FMD dispersal, transmission between wildlife and livestock, and the development of novel Artificial Intelligence techniques incorporating geographic information. Progress under Objective 4 will facilitate longitudinal studies and provide access to critical data resources and expertise.
In addition to expanding the in-house capacity for model-based analysis, Objective 4 progress was made in investigating the role of subclinical phase FMD transmission through simulation modeling. Preliminary results from simulation-based analysis aimed at quantifying the impacts of preclinical transmission in cattle show a substantial increase in disease spread rate and total affected geographic area. Simulations without incubation phase transmission impacted fewer farms and necessitated the depopulation of fewer cattle to contain disease spread. Extending the infectious subclinical phase to four days or more resulted in a significant rise in affected farms and the amount of depopulation needed to stop the outbreaks. This preliminary work highlights the importance of incorporating the subclinical infectious phase in epidemiologic models and emphasizes the impact of incubation phase transmission on FMD outbreak extent and duration. The modeling framework will be further refined to improve understanding of FMD infection dynamics and to support emergency preparedness.
Additional progress was made under Objective 4, focusing on naturally occurring (field study) subclinical infections. To address Sub-objectives 4A and 4B, a detailed investigation was conducted in water buffalo in Islamabad, Pakistan. Over one year, samples were taken from buffalo on 30 water buffalo dairy farms, targeting animals without visible signs of the disease. Using advanced genetic techniques, ARS researchers successfully sequenced 68 samples from 44 buffalo across 17 farms. The analysis identified three different types of the FMD virus: O, A, and Asia-1. It was found that the virus evolved differently within individual animals, with up to 25 genetic changes between sample points. Additionally, recombinant (mixed) viruses, which came from different parent strains, were discovered in several animals. One buffalo had at least five different mixed viruses. These findings improve the understanding of FMD subclinical infections and the role of recombinant virus evolution. The presence of these mixed viruses highlights the complexity of FMD in areas with multiple virus strains. This progress will help shape future strategies for monitoring and controlling FMD in livestock.
Accomplishments
Review Publications
Arzt, J., Sanderson, M., Stenfeldt, C. 2024. Foot-and-mouth disease. Veterinary Clinics of North America. 40(2):191-203. https://doi.org/10.1016/j.cvfa.2024.01.001.
Humphreys Jr, J.M., Shults, P.T., Velazquez Salinas, L., Bertram, M.R., Pelzel-McCluskey, A.M., Peters, D.C., Rodriguez, L.L., Pauszek, S.J. 2024. Interrogating genomes and geography to unravel multiyear vesicular stomatitis epizootics. Viruses. 16(7). Article 1118. https://doi.org/10.3390/v16071118.
Attreed, S.E., Silva, C.M., Rodriguez-Calzada, M., Mogulothu, A., Abbott, S.T., Azzinaro, P.A., Canning, P., Skidmore, L., Nelson, J., Knudsen, N., Medina, G.N., De Los Santos, T., Diaz-San Segundo, F. 2024. Prophylactic treatment with PEGylated bovine FN-lambda3 effectively bridges the gap in vaccine-induced immunity against FMD in cattle. Frontiers in Microbiology. 15. https://doi.org/10.3389/fmicb.2024.1360397.