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United States Department of Agriculture

Agricultural Research Service

Research Project: Countermeasures for Foot-and-Mouth Disease Utilizing Diagnostics, Biotherapeutics, a Novel Vaccine Platform and Improved Challenge Systems

Location: Foreign Animal Disease Research

2013 Annual Report

1a.Objectives (from AD-416):
Objective 1. Development of a rationally designed Foot-and-Mouth Disease (FMD) differentiation of infected from vaccinated animal (DIVA) bovine serological diagnostic assay to be used with next generation FMD vaccines in cattle. Objective 2. Identify and evaluate different classes and subclasses of swine and cattle interferon induced in response to FMV infection and assess their potential use as biotherapeutitcs against FMDV. Specific objectives include; (A.) Identification of biotherapeutic candidates to control FMDV inclusive of adenovirus 5- vectored interferon (Ad5-IFN) administered in correlation with non-host pathogen associated molecular patterns (PAMPs) to provide a broader, enhanced and prolonged antiviral and protective response and, (B.) Conduct further studies of bovine interferon (IFNs) expressed in the Ad5 vector, which was previously developed by ARS, PIADC. Determine which IFNs are induced in response to infection at the primary sites of replication. This information will determine the top candidates for use as biotherapeutic agents. Objective 3. Develop a vaccine platform for eliciting rapid serum and mucosal antibody responses to FMDV. Objective 4. Development of improved challenge systems for FMD vaccine and biotherapeutics testing in cattle and swine. Novel challenge systems will be used to assess the animals’ ability to transmit FMDV in the pre-clinical phase of the disease.

1b.Approach (from AD-416):
1. A competitive ELISA (cELISA)will be developed by ARS, PIADC, that uses a FMDV 3ABC recombinant protein and a monoclonal antibody specific for an immunodominant B-cell epitope on the 3B protein that will be compatible with either next generation FMD molecular vaccines or current, high quality inactivated vaccines in which non-structural proteins (NSPs) have been removed. Collaborators from the Texas Veterinary Medical Diagnostic Laboratory (TVMDL) will provide monoclonal antibody and recombinant 3 ABC reagents for use in the cELISA development. The cELISA will then be evaluated in cooperation with USDA-APHIS for eventual use in the National Animal Health Laboratory Network. ARS, PIADC also will seek to engage a private manufacturer early in the research and development process of the cELISA to accelerate technology transfer for public use. 2. (A). Studies will be continued in swine utilizing the combination of Ad5-pIFN alpha and poly ICLC (PAMP) to test for broader and enhance antiviral response, examine alternative routes of inoculation to enhance efficacy, continue in vitro studies to examine the potency of polyIC and other PAMPs alone and in combination with INFs and conduct in vivo studies utilizing top candidates. Subsequent bovine studies will be preformed utilizing similar approach. (B.) To support use of Ad5-type I IFN-immunomodulator vectors as biotherapeutics against FMDV in cattle, studies will be conducted to develop methodologies to quantify and identify type I IFNs replication properties and understand their role in the immune response, identify immune-modulators that induce type I IFN and/or host response and develop new Ad5-type I IFN-immunomodulator vectors and test in cattle. 3. To develop a rapid acting vaccine platform, developed by collaborators from the University of Georgia School of Veterinary Medicine, studies will be conducted utilizing the dextran-FMDV vaccine to measure the immunogenicity kinetics in cattle. Develop tools to induce B cell to switch to immunoglobulin A (IgA) and IgG1secretion, in order to enhance the mucosal antibody response. This will be accomplished by expressing proteins in replication defective human adenovirus vector-5 (huAd5) in vitro. Further studies will be done in vivo to determine if Ad5 vectors can affect B cells in bovine. Identification of adjutants effecting B cell mechanisms of immunoglobulin switch and secretion will be conducted. The identified adjutants will be utilized in Ad5 FMDV vaccine trials. 4. Transmission studies will focus on the development, optimization and subsequent validation of direct inoculation of swine with FMDV via ornonasal deposition and comparison of the efficacy of intra-oral and intra-nasal routes with inoculation intradermally in heel bulb (IDHB). Swine and cattle cohabitation contact studies will be conducted utilizing swine previously infected with FMDV via oronasal and heel bulb inoculation methods to determine the potential of FMDV from donor pigs to cattle in the pre-clinical phase of infection.

3.Progress Report:

Development of 3ABC ELISA for detection of FMD antibody in infected animals regardless of vaccination status. A 3ABC* plasmid for high expression of 3ABC antigen that carries an inactive 3C proteinase was produced by ARS. The expression in E. coli was optimized and two independent batches of the rec3ABC* protein were transferred to APHIS for further assay developments and to make a decision on the choice of antigen that will move forward for Kit development with an industry partner. We completed antigen selection/optimization of production, performing comparative ELISA test using PrioCHECK® FMD-NS kit, the rec 3ABC* and two multiple peptide (referred to as 3B12x and 3B16X) provided by TVMDL-NAHLN laboratory and performed with APHIS and DHS collaborators a comparative test analysis. The results showed high spectrum of reactivity for viruses of all 7 serotypes in these assays. All documentation for the transfer and detail protocols (SOPs) for protein expression has been shared with the collaborators and the industrial partner. We have prepared a large batch of the 3ABC protein that was transferred to the industrial partner for assay development and further assay validation. A patent application is being filed and a license between ARS and the industrial partner was recently signed allowing ARS to transfer the plasmid for antigen production for the 3ABC-ELISA test.

Identification of biotherapeutic candidates to control FMDV. We have identified several biotherapeutic approaches. Tested the efficacy of type III IFN in vivo in swine and demonstrated that animals can be protected against FMD in a dose response manner. Completed studies using Venezuelan equine encephalitis replicon particles as inducers on strong innate immune responses against FMD in a mouse model. We have further used this system to demonstrate a role of the IFN induced protein 10 in controlling FMD in vivo. Tested a novel approach to enhance the IFN induced antiviral activity. Expression of a constitutively active transcription factor significantly induced the expression of IFN in tissue culture.

Improvement of type I interferon biotherapeutics in cattle. The antiviral activities of the remaining type I interferons were tested in FY 2013 and the best genes of all subtypes were identified. We found that the production or activity of interferons in cell culture was increased by eight strategies, three of which can be immediately implemented in biotherapeutics by adding other DNA sequences to the Ad5 blue vector. In animal trials, steers treated with Ad5 recombinant virus containing the IFN alpha gene had serum levels of anti-FMDV activity. Sera collected at 24 hours after injection completely protected bovine cells from FMDV infection in cell culture. In FY2014 and beyond, studies will focus on the construction of at least two Ad5 recombinant viruses containing more inserted genes and other DNA sequences to produce the best interferon gene.

A vaccine platform for eliciting rapid serum and mucosal antibody responses to FMDV. We tested an FMD vaccine platform designed induce rapid induction of immunity in order to fill the gap between time of vaccination and the onset of neutralizing antibodies and protective immunity induced by the present vaccine. The vaccine was formulated with either dextran or nanoparticles as carriers, the carrier was coated with mouse anti-FMDV monoclonal antibody, and killed FMDV was adsorbed to the antibody coated carrier. We showed that the inoculation of purified, killed FMDV in saline induces a rapid and transient anti-FMDV response in cattle. Addition of a carrier (nanoparticles or dextran) with FMDV adsorbed via monoclonal antibody did not enhance this response. This result is contrary to published data that reports a lack of a detectable antibody response to killed FMDV inoculated in saline. The results require a reassessment of the vaccine design.

Improved challenge systems for FMD vaccine and biotherapeutics testing in cattle and pigs. Studies continued to improve novel systems for challenging cattle and pigs with FMDV. The goal is to optimize systems for vaccine development/testing that are reproducible and closely simulate natural infection. In cattle, two systems have provided promising results; 1. Challenge by controlled exposure to previously infected pigs, 2. Challenge by direct inoculation of the nasopharynx. In pigs oropharyngeal inoculation has proven to be more effective han nasopharyngeal route. A manuscript has been submitted detailing this work.

Continued to improve novel systems for challenging (i.e. infecting) cattle and pigs with FMDV. The overarching purpose of this work is to optimize systems for vaccine development/testing that are reproducible and closely simulate natural infection. In cattle two systems have provided promising results including.
1)challenge by controlled exposure to previously infected pigs, and.
2)challenge by direct inoculation of the nasopharynx. A manuscript has been submitted detailing this work. In pigs oropharyngeal inoculation has proven to be more effective than nasopharyngeal route. In 2014, we will continue to improve standardization of these techniques and test efficacy with different strains of FMDV.

No thechnologies were transferred in FY 2013.

Publications for the period include: 1. Diaz-San Segundo et al (2013) Venezuelan Equine Encephalitis Replicon …FMDV” J. Virology 87:5447-60. 2. Diaz-San Segundo et al (2013) Understanding the mechanisms of interferon-…FMDV” Advances in Virus Research. ISBN: 978-1477555-04-0.iConcept Press 3. Stenfeldt et al (2013) Infection dynamics of FMDV in pigs….methods. Journal of Comparative Pathology.

Last Modified: 4/19/2014
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