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United States Department of Agriculture

Agricultural Research Service

Research Project: FOOT-AND-MOUTH DISEASE VIRUS (FMDV) COUNTERMEASURES DISCOVERY

Location: Foreign Animal Disease Research

2011 Annual Report


1a.Objectives (from AD-416)
1. Develop improved vaccine platforms and delivery systems to control FMD. 2. Determine the mechanism of early immune enhancement against FMDV.


1b.Approach (from AD-416)
1. Development of improved FMD vaccines will be accomplished by: a.) focus on development of improved adenovirus-vectored FMDV vaccine platform and its routes of inoculation, b.) focus on the construction and testing of leader-deleted FMDV with a negative marker as an inactivated vaccine platform, c.) focus on the construction of T cell independent antigens as a vaccine platform. 2. Mechanisms of immune enhancement against FMD will be explored by characterization of: a.) induction of antiviral response by IFN alpha, beta and gamma and combinations of them, b.) induction of innate immune responses by TLR agonists utilized alone or as adjuvants in combination with vaccines.


3.Progress Report
In FY 2011 we examined 3 approaches to develop new FMD vaccine candidates. In the first approach, we continued our work with a second generation replication-defective adenovirus, Ad5-FMD, vector containing the viral capsid and 3C protease plus protein 2B. This vector induced an enhanced cell mediated immune response and increased protection in cattle as compared to our first generation vector. We submitted a manuscript. We initiated swine studies combining our second generation Ad5-A24 vaccine with various adjuvants to increase potency and reduce dose. We also tested additional Ad5-FMD vectors with reduced expression of the toxic 3C protein and Ad5-FMD vectors with altered cell tropism, in an effort to increase potency. In a second approach, we constructed and tested second generation inactivated FMDV leader (L) deleted negative marker vectors (FMDVLL3B3D) containing additional substitutions in FMDV proteins that eliminate a antigenic sites that could serve as a DIVA (differentiation of infected from vaccinated animals) markers. Inactivated FMDVLL3B3D vaccine formulated with adjuvant protects cattle from challenge. A 3D-cELISA DIVA companion test for this vaccine and also for the Ad5-FMDV vaccine platform was developed. The test was examined for feasibility and transferred to APHIS for further validation. In a third approach, we examined the feasibility of live-attenuated FMD vaccines. We demonstrated that FMDV with mutations in the L protein coding region is a virulent but can protect swine from subsequent challenge with virulent FMDV as early as 2 days post vaccination. A manuscript is being submitted. We are examining several approaches to improve our FMDV biotherapeutic program: 1. Bovine type III interferon (bIFN lambda) was tested in cattle for induction of systemic antiviral responses and up-regulation of IFN stimulated genes. Cattle treated with an Ad5-bIFN lambda and challenged with FMDV were either protected from clinical disease or had a significantly delayed onset of disease as compared to controls. A patent application has been filed and a manuscript is in preparation; 2. We continued studies examining the effectiveness of Ad5 delivered type I porcine IFN alone or in combination with the adjuvant, pICLC, to rapidly protect swine against FMDV. The addition of pICLC lowered the protective dose of Ad5-pIFNalpha and; 3. In cell culture studies the toll-like receptor agonist Venezuelan equine encephalitis virus replicon particles inhibited FMDV replication. The research conducted in FY 2011 addresses objectives: 1. develop improved vaccine platforms and delivery systems to control FMD, 2. determine the mechanism of early immune enhancement against FMDV and addresses the biodefense research of foreign animal diseases through 1. vaccine development, 2. development of tools to identify disease-free animals.


4.Accomplishments
1. New means to induce rapid protection against foot-and-mouth disease. Foot-and-mouth disease virus (FMDV) replicates and spreads very rapidly. To control the disease protective measures must be started quickly. ARS researchers at PIADC, Greenport, NY, have identified two biotherapeutic approaches. 1- PolyICLC, which alone or in combination with a replication-defective human adenovirus vector (Ad5) containing porcine interferon (IFN) alpha can fully protect swine challenged as early as one day later with FMDV. 2- Another novel biotherapeutic, bovine IFN lambda (bIFNlambda), delivered in an Ad5 vector protected cattle or significantly delayed the onset of disease. These effective biotherapeutic approaches have significant potential as rapid acting countermeasures for the control of FMD outbreaks.

2. Improved foot-and-mouth disease vaccine potency in swine. New Ad5-FMD vaccine candidates require relatively high doses to be effective. ARS researchers at PIADC, Greenport, NY, have combined a second generation replication-defective Ad5 vector containing in addition to the FMDV capsid and 3C proteinase, the nonstructural protein 2B. When this vaccine is applied with various adjuvants it can fully protect swine from challenge with FMDV. Using this approach we were able to reduce as much as 20fold the vaccine dose required to induce protection when compared to the first generation vaccine. This dose reduction represents an important improvement and a potential cost reduction for the production of Ad5 vaccines.

3. Analyzed performance of new recombinant vaccine for foot-and-mouth disease (FMD). ARS researchers at PIADC, Greenport, NY, in collaboration with colleagues at University of Copenhagen have taken a technology gaining wide use in human vaccines and applied it to swine. In order to analyze cellular immune responses, they have developed major histocompatibility complex (MHC) tetramers for swine. Using this method they showed current FMD vaccines are poor inducers of cytotoxic T lymphocyte (CTL). In contrast, an improved Ad5-FMD vaccine induced CTL that were capable of killing FMDV infected cells in vivo. These studies will aid in the development of improved FMD vaccines that can induce both cellular and humoral immune responses.

4. Developed a safe platform for inactivated foot-and-mouth disease (FMD) vaccine production. Currently inactivated FMD vaccines require growth of large quantities of infectious virus and do not easily allow the distinction of infected from vaccinated animals. ARS researchers at PIADC, Greenport, NY, have developed a safe vaccine production platform (LL3B3D) containing markers to easily distinguish infected from vaccinated animals (DIVA). This attenuated FMDV can be safely grown with minimal risk of escape from vaccine production plants. Further, we have developed proof of concept companion DIVA tests in support of the LL3B3D vaccine platform. The new vaccine platform and companion diagnostic tests can revolutionize the way FMD vaccines are produced and are the subject of a patent application and are currently under development with a private industry partner.


Review Publications
He, D., Overend, C., Ambrogio, J., Magant, R.J., Grubman, M.J., Garmendia, A.E. 2010. Marked differences between MARC-145 cells and swine alveolar macrophages in IFN beta-induced activation of antiviral state against PRRSV. Veterinary Immunology and Immunopathology. Available: www.elsevier.com/locate/vetimm.2010.07.023.

Dias, C.C., Moraes, M.P., Diaz-San Segundo, F., De Los Santos, T.B., Grubman, M.J. 2011. Porcine type I interferon rapidly protects swine against challenge with multiple serotypes of foot-and-mouth disease virus. Journal of Interferon and Cytokine Research. 31(2):227-236.

Patch, J.R., Pederesen, L.E., Moraes, M.P., Grubman, M.J., Nielsen, M., Buus, S., Golde, W.T. 2011. Induction of foot-and-mouth disease virus specific cytotoxic T cell killing by vaccination. Clinical and Vaccine Immunology. 18(2):280-288.

Blignaut, B., Visser, N., Theron, J., Rieder, A.E., Maree, F.F. 2011. Custom-engineered chimeric foot-and-mouth disease vaccine elicits protective immune responses in pigs. Journal of General Virology. 92(4):849-859.

Diaz-San Segundo, F., Weiss, M., Perez-Martin, E., Koster, M.J., Zhu, J.J., Grubman, M.J., De Los Santos, T.B. 2011. Antiviral activity of bovine type III interferon against foot-and-mouth disease virus. Virology. 413(2):283-292.

Grubman, M.J., Rodriguez, L.L., De Los Santos, T.B. 2010. Foot-and-mouth disease. In: Ehrenfeld, E., Domingo, E., Roos, R.P., editors. The Picornaviruses. Washington, DC: ASM Press. p. 397-410.

Golde, W.T., De Los Santos, T.B., Robinson, L., Grubman, M.J., Sevilla, N., Summerfield, A. 2011. Evidence of activation and suppression during the early immune response to foot-and-mouth disease virus. Transboundary and Emerging Diseases. 58(4):283-290.

Reeve, R., Blignaut, B., Esterhuysen, J.J., Opperman, P., Matthews, L., Fry, E., De Beer, T.A., Theron, J., Rieder, A.E., Vosloo, W., O'Neill, H.G., Haydon, D.T., Maree, F.F. 2010. Sequence-based prediction for vaccine strain selection and identification of antigenic variability in foot-and-mouth disease virus. PLoS Computational Biology. DOI: 10.1371/journal.pcbi.1001027.

Last Modified: 9/23/2014
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