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- Arzt, Jonathan
- Rieder, Aida - Elizabeth
- Zhu, James
- Borca, Manuel
- 2023
- 2022
- Publications
- Animal Health (103)

Research Project: Intervention Strategies to Respond, Control, and Eradicate Foot-and-Mouth Disease Virus (FMDV) **Location:
                            Foreign Animal Disease Research** 2023 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, Sub-objective 1.A, progress included the assessment of a novel live-attenuated vaccine candidate against foot-and-mouth disease (FMD) virus in cattle. This vaccine was produced by deoptimizing the genetic code of two viral proteins (P2, P3) resulting in a virus that does not cause disease but induces protection (i.e. deoptimized vaccine). The safety and efficacy of the deoptimized vaccine was demonstrated by strong induction of effective antibodies and full protection against a FMD virus challenge.Utilizing the previously established leaderless foot-and-mouth disease vaccine platform (called FMDLL3B3D), ARS scientists in Orient Point, New York, designed and constructed a marker vaccine with broadened protective coverage against multiple FMD virus strains in cattle. The broad-coverage vaccines against serotypes A and O were demonstrated to protect against heterologous FMD virus challenge at 21-days post vaccination. Furthermore, new methods were implemented to assess T-cell immune responses in vaccinated animals using a proprietary flow-cytometry methodology. Specifically, towards meeting the milestone of “developing methods to study CD8+ CTL response ex vivo,” we have also devised a suite of flow cytometric panels that allow distinctions between different types of immune cells (e.g. CD4+, CD8+, natural killer (NK cells)) and also allow further differentiation of central from effector memory T cells in both cattle and pigs.  
Under Sub-objective 1B, a single cell transcriptomics platform was established that allows understanding the immunological landscapes of white blood cells obtained from vaccinated vs. non-vaccinated cattle. In preliminary analyses, this platform has enabled the identification and characterization of distinct immune cell populations that are present in vaccinated cattle, providing new insights into the mechanisms of vaccine-induced protection.
In Sub-objective 1.C, there were important advances including the production of recombinant foot-and-mouth disease viruses (FMDV) representing a wide spectrum of antigenic signatures.  These viruses were tested for their capacity to protect cattle and pigs against heterologous challenge. Progress was made at examining the efficacy of monovalent and polyvalent vaccines formulated with a commercial adjuvant for mutant viruses of the O and Asia serotypes. Methods are currently under development to examine the spectrum of antibody responses and avidity of virus-specific antibodies using various assays including transcriptome analysis. 
In Sub-objective 1.D, progress included the evaluation of a deoptimized live attenuated vaccine candidate (A24 P2/P3 deopt) in cattle to determine whether it could prevent the persistence of foot-and-mouth disease virus (FMDV). In this study, the vaccine was highly effective at protecting animals from clinical disease; however, the vaccine did not demonstrably prevent the establishment of FMDV carrier state.
Sub-objective 1.E, there were also achievements in the advanced development of the FMD-LL3B3D vaccine platform under an existing agreement with an industry partner. Several of the most globally relevant vaccine antigens were derived and produced using this platform and adapted to grow in suspension cells (a necessary step in industrial vaccine production). This is an important accomplishment as it will markedly expedite derivation and utilization of vaccine viruses. On the field of sub-unit FMD vaccines, we have designed a novel packaging-deficient adenovirus vector to increase the potency of the historically proven Ad5-FMDV vaccine. A new cell line that complements for the missing packaging sequences of the Ad5 virus was created so the vaccine could be produced in large scale. Testing is being performed to determine if this Ad5- virus can be rescued and characterized. 
In Objective 2, under Sub-objective 2A, progress included the design, cloning, and expression of a novel interferon (IFN) subtype of porcine origin in an Ad5 delivery vector. This a novel IFN subtype demonstrated strong antiviral activity in cell culture, and it will be further evaluated in pigs. Substantial progress was made to advance the modified bovine IFN project with an external partner. In an efficacy study performed in cattle, we showed that modified bovine IFN was effective at preventing clinical disease when administered up to 5 days before challenge. When combined with the Ad5-vectored FMDV O1Manisa vaccine, animals developed strong antibody titers and NK as well as CD4+ and CD8+ T cell IFN responses. Modified bovine IFN also had an adjuvant effect, boosting the immune response of the Ad5-vectored vaccine. These studies will help establish a novel biotherapeutic platform to prolong the half-life of bovine type III IFN molecules.
Within Objective 3, Sub-objective, 3A, substantial progress was made in elucidating the role of codon usage in the P1 region on the virulence of FMDV serotypes A24 and Asia1. By recoding the P1 region using codon pair deoptimization, we were able to significantly attenuate the virus in cell cultures and in swine. Our studies suggest that deoptimization delays the replication or translation of the virus by allowing the host to activate antiviral pathways that are not favorable for the virus. Studies targeted to understand recombination possibilities between codon deoptimized FMDV strains and wild type (WT) strains revealed the presence of a critical secondary RNA structure in the 3D region that may mediate such recombination events. Also, FMDV virus carrying novel mutations W105A) in leader viral protease (Lpro) were shown to be moderately attenuated in porcine cells but significantly attenuated in an FMD mouse model. Mechanistic studies using single cell transcriptomics had preliminarily revealed that porcine cells infected with a virus carrying a single point mutation in Lpro displays a unique gene expression profile compared to cells infected with WT virus including those affecting antigen processing. This finding could have implications for the development of new vaccines and treatments for FMDV.
Substantial progress was made towards Sub-objective 3.B, specifically, advanced transcriptomic analyses were used to demonstrate substantial differences in gene expression (DEG) patterns between bovine tissues that maintain persistent FMDV infection (nasopharynx) and those that do not (lungs). Bioinformatic analysis of DEGs indicated that the lung and nasopharynx recruited different immune cells and adapted different mechanisms for defense. Tissue susceptibility to persistent infection was associated with downregulated expression of chemokines recruiting cytotoxic cells and phagocytes and upregulated genes associated with Treg cells, adenosine signaling, inhibition of signaling, increasing production of various ligands, and ancillary factors activity. These differences in gene expression could potentially prevent virus clearance from the nasopharyngeal mucosa.
There was also progress on viral ecology studies, Objective 4, including milestones regarding studies executed in Asia and Africa addressing various aspects of subclinical and clinical infections. To address Sub-objective 4.A, modeling studies were completed with university collaborators which used field strains of FMDV from Vietnam to examine aspects of spatial spread of the viruses. These studies demonstrated that the combination of viral genetics and spatial distribution (phylogeography) was the superior method of predicting viral movements. In-house experimental studies were performed to confirm the relevance of small ruminants as competent hosts of subclinical acute (neoteric) and persistent FMDV infection. These experiments revealed that although clinical signs of FMD were absent in a subset of infected sheep, these animals shed high amounts of virus in oronasal secretions, strongly suggesting the capacity of subclinically infected sheep to initiate and propagate FMD outbreaks. It was confirmed that the anatomic site of persistent FMDV is different in sheep compared to cattle, with virus persisting in epithelial crypts of the oropharyngeal tonsils rather than in the nasopharyngeal mucosa. This distinction may influence optimal sampling techniques deployed to detect persistent FMDV infection in small ruminants. Sub-objective 4.B, was advanced through analysis of FMDV genomic evolution and recombination in samples from animals infected with heterologous virus strains. Through analyses of samples derived from in-house experiments, it was demonstrated that the order of exposure to distinct virus strains substantially impacts the genomic organization of the resultant recombinant viruses. It was also demonstrated that the recombinant viruses that arose from initial superinfection continued to evolve and recombine further as the infection progressed. The findings from these studies confirm the high frequency at which FMDV recombination occurs in the upper respiratory tract of super- infected FMDV carriers, further emphasizing a purported role of FMDV carriers on the evolution of FMDV in regions where multiple virus strains are in concurrent circulation. **Accomplishments**
                                

1. Use of codon-deoptimization as a strategy to develop novel live-attenuated vaccine candidates against foot-and-mouth disease. Foot-and-mouth disease (FMD) reoccurs in endemic areas and can be reintroduced into countries where it has previously been eradicated. Vaccination is a key component of effective control of this disease. As part of a commercial partnership, ARS researchers in Orient Point, New York, have evaluated a new approach called codon-deoptimization to generate weakened foot-and-mouth disease virus (FMDV) to use in live-virus vaccines. By targeting different regions of the FMDV genome, this innovative technique has yielded a viable virus with weakened traits and with a high level of genetic stability. Cloning of deoptimized sequences in an FMDV infectious clone offers this vaccine advanced technological capabilities. These codon deoptimized live-virus vaccines have the capacity to induce robust immune responses, providing effective protection against FMD. This can help control outbreaks in endemic areas and contribute to the eventual eradication of the disease, reducing the economic burden on farmers and enhancing livestock productivity. 2. Novel recombinant foot-and-mouth disease viruses arise within the first 48 hours of superinfection of persistently infected carrier cattle. As documented by ARS researchers in Orient Point, New York, foot-and-mouth disease viruses (FMDV) can cause a prolonged subclinical infection in the upper respiratory tract of infected cattle. Recent years’ efforts have further demonstrated that exposure of such FMDV carriers to a different variant of the virus gives rise to novel recombinant virus variants with distinct components derived from each of the two infecting viruses. In a recent body of work, it was demonstrated that these recombinant viruses arise in specialized epithelial cells of the nasopharyngeal mucosa within the first 48 hours of infection. This finding is critical as it confirms that the recombinant viruses are present in the respiratory tract during the very early stages of infection, when infected animals are known to shed the greatest amounts of virus. The discovery highlights the importance of detection and appropriate management of persistently infected FMDV carriers following disease outbreaks, especially in regions where multiple FMDV variants are in circulation. **Review Publications**
                            *Stenfeldt, C., Fish, I., Meek, H., Arzt, J. 2023. Heterogeneity and recombination of foot-and-mouth disease virus during multi-strain coinfection of cattle. mSphere. 22;8(3):e0064322. https://doi.org/10.1128/msphere.00643-22.*
                                *Spinard III, E.J., Fish, I., Azzinaro, P.A., Rodriguez-Calzada, M., Hartwig, E.J., Smoliga, G.R., Arzt, J., De Los Santos, T.B., Medina, G.N. 2022. Evaluation of potential in vitro recombination events in codon deoptimized FMDV strains. Viruses. 15(3). https://doi.org/10.3390/v15030670.*
                                *Gunasekara, U., Bertram, M.R., Perez, A., Arzt, J., VanderWaal, K., 2023. Phylogeography as a proxy for population connectivity for spatial modeling of foot-and-mouth disease outbreaks in Vietnam. Transboundary and Emerging Diseases. 15(2). https://doi.org/10.3390/v15020388.*
                                *Medina, G.N., Diaz-San Segundo, F., Spinard III, E.J., Azzinaro, P.A., Rodriguez-Calzada, M., Gutkoska, J.R., Kloc, A., Rieder, A.E., Mueller, S., De Los Santos, T.B. 2023. Deoptimization of FMDV P1 region results in robust serotype-independent viral attenuation. Viruses. 15(6). https://doi.org/10.3390/v15061332.*
                                *Meek, H.C., Stenfeldt, C., Arzt, J. 2023. Morphological and phenotypic characteristics of the bovine nasopharyngeal mucosa and associated lymphoid tissue. Journal of Comparative Pathology. 198:62-79. https://doi.org/10.1016/j.jcpa.2022.07.011.*
                                *Yadav, S., Delgado, A.H., Hagerman, A.D., Bertram, M.R., Moreno-Torres, K.I., Stenfeldt, C., Holmstrom, L., Arzt, J. 2022. Epidemiologic and economic considerations regarding persistently infected cattle during vaccinate-to-live strategies for control of foot-and-mouth disease in FMD-free regions. Frontiers in Veterinary Science. 9:1026592. https://doi.org/10.3389/fvets.2022.1026592.*


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