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United States Department of Agriculture

Agricultural Research Service

Research Project: FOOT-AND-MOUTH DISEASE VIRUS (FMDV) COUNTERMEASURES DISCOVERY
2010 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
We are continuing to pursue the development of improved FMD vaccine platforms. We continued analysis of a bovine challenge experiment using a second generation Ad5-O1 Campos vector containing the viral nonstructural protein 2B. This vector induced an enhanced cell mediated immune response as compared to our first generation vector. We are currently preparing a manuscript. We are currently preparing additional Ad5-FMD vectors placing the toxic 3C proteinase coding region under the control of weaker promoters than the FMD capsid coding region and are constructing an Ad5-FMD vector with altered cell tropism. Based on previous work in which we demonstrated that an FMD virus with mutations in the leader coding region grows to lower titers in cell culture and does not cleave a host transcription factor, we performed a number of swine experiments with this mutant virus and found that it is avirulent in swine and can protect animals from subsequent challenge with virulent parental FMDV. A manuscript is being prepared. We initiated studies focusing on identifying the FMDV determinants of tissue culture adaptation. Three FMDV field prototype viruses were selected to initiate this study. We continued cell culture and cattle studies with a leader deleted FMDV containing negative markers as an alternate inactivated vaccine virus platform. We tested a T cell independent vaccine for immunogenicity in cattle. The formulation of this vaccine was identical to that used in swine studies previously reported. Specifically, killed virus capsid bound to a dextran backbone was accomplished by coupling monoclonal anti-FMDV antibody to the dextran backbone and absorbing the viral capsid to the antibody. Quality control assays determined antibody was coupled and virus was absorbed at intended concentrations. Cattle were vaccinated subcutaneously in a single site. No anti-FMDV antibody was detected in any animals. Further development of the formulation and testing of this vaccine in cattle is now suspended due to limited personnel. We are continuing studies to discover and test various rapidly acting biotherapeutics. We continued studies examining the effectiveness of Ad5 delivered type I porcine interferon (IFN) to rapidly protect swine against multiple FMDV serotypes by both direct inoculation challenge and contact challenge. Furthermore, we attempted to enhance the potency of Ad5-pIFN by delivery via alternative routes. We initiated cattle studies with a recently constructed Ad5 vector containing bovine type III IFN (IFN lambda). We initiated studies in cell culture demonstrating the effectiveness of various TLR agonists, including polyIC, against FMDV replication and also analyzed gene induction, by real-time RT-PCR, after IFN and polyIC treatment alone or in combination. Initial studies in swine with polyICLC alone or in combination with Ad5-pIFN alpha demonstrated protection against FMDV challenge. In vivo results in swine indicated that the TLR 7 and TLR 8 agonists are not effective in vivo against FMDV challenge.


4.Accomplishments
1. Testing of potential live-attenuated FMD vaccine candidates in swine. ARS researchers at PIADC, Greenport, NY have developed improved vaccine platforms and delivery systems to control FMD. Using bioinformatic tools protein domains have been identified in the FMDV leader coding region. Mutation of specific amino acid residues within this region resulted in viruses that grow to high titers in tissue culture but showed reduced virulence in swine and cattle eliciting a strong neutralizing antibody response. Preliminary studies in swine demonstrated that live attenuated vaccine candidate can protect against virulent virus challenge. This attenuated FMDV can also be also as an inactivated vaccine platform.

2. Testing of a new biotherapeutic agent in cattle. A novel biotherapeutic reagent for foot-and-mouth disease (FMD) was identified by ARS researchers at PIADC, Greenport, NY using microarray analysis of cells infected with wild type and attenuated FMDV. This novel biotherapeutic was cloned using an Ad5 expression vector. Protein expressed from this construct induces antiviral activity against FMDV in vitro and in vivo. Inoculation of cattle with Ad5 expressing the biotherapeutics induces the expression of several IFN induced genes and delays FMD infection.

3. Enhanced efficacy of Ad5-pIFNalpha vector in swine. Enhanced the efficacy of Ad5-pIFNalpha vector against FMDV challenge in swine. ARS researchers at PIADC, Greenport, NY have demonstrated that delivery of the vector at 4 sites in the neck subcutaneously reduced the protective dose as compared to intramuscular vector delivery. Performed a number of assays to demonstrate that Ad5-pIFNalpha could sterilely protect swine against FMDV challenge.

4. Improvement of vaccine virus growth characteristics. ARS researchers at PIADC, Greenport, NY have initiated a study to identify determinants of foot-and-mouth disease virus (FMDV) tissue culture adaptation that could be used to enhance the properties of vaccine candidates derived using the recombinant DNA technology. This project is a component of a multi-national collaborative effort to enhance the stability and adaptation of FMDV candidates for vaccine production. We have amplified a selected type A FMDV and performed genetic and phenotypic characterization. Initial characterization of eield viruses included determination of viral capsid sequences and the selection of mutation to be engineered in these viruses to ehnance virus growth. Primers and other reagents needed to molecularly clone these viruses were attained.

5. FMDV leader deleted virus as a vaccine candidate and further development of a companion diagnostic test. Two experimental FMD vaccines carrying either one or two negative markers protected animals from challenge with the parental virus. The attenuated characteristics and inability to spread to in contact animals was also demonstrated by ARS researchers at PIADC, Greenport, NY for these negative marker vaccine viruses in susceptible animals, including cattle and pigs. A companion ELISA test was adapted to provide a way to distinguish infected from vaccinated animals.

6. Identification of determinants of foot-and-mouth disease virus adaptation to cell culture. ARS researchers at PIADC, Greenport, NY in collaboration with OVI-Onderstepoort on determinant for tissue culture adaptation of SAT FMDV have led to the identification of unique Heparan Sulphate Proteoglycans-binding sites (amino acid residues) located on the outer capsid proteins of SAT1 and SAT2 FMDV. The sites are exposed on the surface of the virion and are structurally accessible for binding to the alternative HSPG receptors.


Review Publications
Grubman, M.J., Moraes, M.P., Neilan, J., Ettyreddy, D., Butman, B.T., Brough, D.E., Brake, D.A. 2010. Adenovirus serotype 5 vectored foot-and-mouth disease subunit vaccines: the first decade. Future Virology. 5(1):51-64.

Diaz-San Segundo, F., Moraes, M.P., De Los Santos, T.B., Dias, C., Grubman, M.J. 2010. Interferon induced protection against foot-and-mouth disease virus correlates with enhanced tissue specific innate immune cell infiltration and interferon stimulated gene expression. Journal of Virology. 84:2063-2077.

Pacheco Tobin, J., Butler, J.E., Jew, J., Ferman, G.S., Zhu, J.J., Golde, W.T. 2010. IgA antibody response of swine to foot-and-mouth disease virus (FMDV) infection and vaccination. Clinical and Vaccine Immunology. 17(4):550-558.

Last Modified: 12/20/2014
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