2012 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.
Task 1 – Development of 3ABC ELISA for Detection of FMD Antibody in Infected Animals Regardless of Vaccination Status. To detect Foot-and-Mouth Disease (FMD) antibodies in infected animals regardless of vaccination status, a competitive ELISA was designed using a FMDV 3ABC recombinant protein and a monoclonal antibody specific for an immunodominant B-cell epitope on the 3B protein.
A monocolonal antibody clone specific for the B-cell epitope on the 3B protein was produced and characterized. A 3ABC plasmid was selected and the expression of this plasmid in E. coli was optimized. This plasmid was then transferred to APHIS for further development of the kit. Aphis selected the antigen for the kit and this was sent to industrial partner for assay assembly. Optimization of this 3ABC cELISA took place and the results showed high spectrum of reactivity for viruses of all seven serotypes. The kit was tested against the PrioCHECK®FMD-NS routine kit and shown to perform equally or even slightly better. Additionally this kit also showed superior performance over the mutipeptide antigen expressed by our collaborators from the Texas Veterinary Medical Diagnostic Laboratory - National Animal Health Laboratory Network (TVMDL-NAHLN) laboratory. The sensitivity and specificity of the assay has also been assessed, however additional sera is currently being gathered from infected and non-infected animals. The ELISA kit has been transferred from BSL3 to BSL2 and the performance of the kit has remained comparable. All documentation and detailed protocols have been shared with APHIS-DHS collaborators and the VRMD industrial partners.
We are currently waiting for the assembly of the first set of assay kits by our industry partner.
Task 2 – Identification of Biotherapeutic Candidates to Control FMDV. From previous work we hypothesize that by co-treating livestock with both adenovirus 5 – interferon (Ad5-IFN) and various pathogen associated molecular patterns (PAMPs) will result in broader, enhanced and prolonged antiviral and protective responses. In this proposal we will study the efficacy of two PAMPs alone and in combination with IFNs in cell culture and in FMD susceptible species. It was determined that the PAMPs to be studied in vitro were polylCLC and VEE replicons. We determined the minimum dose of Poly IC alone or in combination with other PAMPS needed to inhibit FMDV replication in vitro. Preliminary data has also been collected on the duration of protection of IFN and polylC. For the VEE replicon initial results in vitro have been obtained indicating they are effective in inducing an antiviral response in vitro. Evaluation of in vitro-active PAMPS in mice has also started.
Task 3 – Improvement of Type 1 Interferon Biotherapeutics in Cattle. The overarching goal is to provide scientific evidence towards the development of new high-efficacy Ad5-type 1 IFN-immunomodular vector as a biotherapeutics against FMDV in cattle.
The type I interferon (IFN) activity of serum samples from FMDV infected cattle was quantitated and we determined that IFN levels were closely related to viral RNA in blood. Local tissue samples were also analyzed for their IFN activities and it was shown that wild type virus had a higher viral titer and a higher IFN level than tissues infected with mutant viruses. This data is still being analyzed for a manuscript submission.
Cytokines that synergize the antiviral activity of type I IFN have been identified for cloning. The nucleotide sequence of the cloned cytokine genes and the nucleotide sequence of the transcription factor genes were determined. We have cloned the constitutively active IRF gene (transcription factor gene) into a mammalian expression vector for testing its interferon-induction activity. Nineteen cytokine proteins have been cloned and expressed in bovine cell line using a mammalian expression plasmid vector. The cells transfected with the vector carrying the IRF transcription factor genes displayed higher anti-FMDV activity, which suggests that this transcription factor induces type 1 interferons.
Task 4 – A vaccine platform for eliciting rapid serum and mucosal antibody responses to FMDV. This task involves developing a rapid vaccine platform that will fill the gap between the onset of neutralizing antibodies and protective immunity induced by the present vaccine. Additionally the aim is to develop technologies that induce mucosal antibody responses to FMDV, protecting the primary site of infection, the mucosa of the respiratory tract.
The first deliverable was to measure the immunogenicity kinetics of dextran-FMDV vaccine in cattle to determine if a cattle trial will take place. The monoclonal antibodies for this have been safety tested and released. They are FMDV A12 and FMDV A24.
To induce a mucosal antibody responds IgA (a type of antibody), B cells (cells that produce antibodies) need to differentiate from IgG1 to IgA. Therefore cytokines that drive B cells into differentiating into IgA secreting cells need to be identified. Two bovine cytokines were identified: a proliferation inducing ligand (APRIL) and an IgA inducing peptide (IGIP). These have now been cloned. The expression and biological activity was confirmed by constructing Ad5 which expressed APRIL and IGIP.
Task 5 – Improved Challenge Systems for FMD Vaccine and Biotherapeutics Testing in Cattle and Pigs. The primary goal of this proposed research is the optimization of new, simulated natural systems for challenging cattle and pigs with FMDV. These novel systems will be optimized in studies using naïve animals and subsequently validated in vaccinate-challenge experiments. Additionally, the novel systems will be used to assess pigs’ and steers’ ability to transmit FMDV in the pre-clinical phase of disease.
The first task was to compare inoculation of FMDV intra-orally, intra-nasally and intradermally into the heel bulb. For FMDV serotype O, two in vivo experiments have been set-up in pigs. The sample processing for the first experiment is completed and the processing of the second experiment is nearly complete. A third in vivo experiment is underway using FMDV serotype A as the infectious agent. Preliminary data supports the premise that oropharyngeal deposition of FMDV will provide a useful challenge model whereas nasopharyngeal deposition is less consistent.