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United States Department of Agriculture

Agricultural Research Service

Research Project: FOOT-AND-MOUTH DISEASE VIRUS (FMDV) COUNTERMEASURES DISCOVERY
2009 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 and delivery systems. In FY 2009, we have constructed a second generation Ad5-O1 Campos vector containing the viral nonstructural protein 2B and this vector induces an enhanced immune response and protective immunity in bovines. Based on our prior work with immune refocused Ad5-A24 mutants in mice that identified mutants which had a broader neutralizing antibody response, we tested these in swine. However, there was no comparable enhanced immune response with these mutants in swine. In collaboration with scientists at the Institute for Animal Health in Pirbright, we constructed a mutant Ad5-A24 vector with a change in an amino acid in capsid protein VP2 that is predicted to result in a more acid stable FMDV capsid. In studies in cell culture we produced this virus and demonstrated expression and processing of FMDV capsid proteins. Based on previous work demonstrating that an inactivated leader deleted FMDV vaccine can protect swine, we have added markers to this vaccine candidate and demonstrated protection of cattle. A novel antigenic negative FMDV vaccine candidate has been developed as a safe platform for production of marker inactivated vaccine. The virus platform is attenuated in cattle and swine and does not cause lesions after direct inoculation of susceptible cattle. The antigenic properties of the inactivated vaccine are similar to those obtained from wild-type virus, except that the vaccine virus lacks a critical antigenic epitope no longer recognized by a monoclonal antibody. We have planned and initiated preliminary testing of an alternate vaccine delivery system that more efficiently infects dendritic cells, the major antigen presenting cell, using our Ad5-A24 vaccine vector system. We continue to examine, at the molecular level, the mechanism of interferon-induced protection. We have demonstrated that individual or combinations of interferons (IFNs) induce an increased number of dendritic cells in the skin and natural killer cells in the lymph nodes, and an upregulation of various cytokines that have direct antiviral effects against FMDV as well as chemokines, in particular the chemokine 10-kDa IFN-gamma-inducible protein, that are involved in attracting immune cells to the site of infection. A recently identified IFN, bovine type 3 IFN (IFN lambda), was cloned and an Ad5-bovine IFN lambda vector was constructed. Cells infected with this vector expressed significant amounts of this protein and it displayed antiviral activity against FMDV. Serum from animals infected with the marker virus can be readily distinguished from parental FMDV infected animals utilizing serological tests such as competitive ELISA.


4.Accomplishments
1. Second generation vaccines containing additional FMDV nonstructural proteins exhibits improved protection against FMD. To improve the efficacy of our replication-defective adenovirus FMD (Ad5-FMD) vaccines we have produced second generation vectors that contain the genetic information for an additional FMDV nonstructural (NS) protein. Three of four bovines inoculated with the new vector, Ad5-O1Campos+2B, containing the gene for NS protein 2B, were completely protected from clinical disease after direct inoculation challenge with FMDV O1 Campos and the nonprotected animal only developed 1 lesion at 6 days postchallenge. However, only 2 of 4 cattle inoculated with the Ad5-vector lacking the complete 2B coding region were protected. The enhanced protection in the group vaccinated with the Ad5-O1C+2B vector correlated with an enhanced CD8+ IFN gamma cellular immune response.

2. Testing leader-deleted FMDV with a negative marker as an inactivated vaccine in cattle. A proof-of concept for the utility and efficacy of our recently developed negative marker FMDV vaccine candidate was carried out in cattle in comparison to commercially available polyvalent FMDV vaccine. Both, commercial and experimental FMDV vaccines protected animals from challenge with the parental virus. The attenuated characteristics and inability to spread to contact animals was also demonstrated for this negative marker FMDV in two susceptible animals, including cattle and pigs. A companion ELISA test was adapted to provide a companion test distinguish cattle exposed to the parental FMDV from the engineered negative antigenic marker virus.

4. Cloning, construction and testing of bovine interferon lambda. Interferons are antiviral molecules that are the first line of the host defense against virus infection. Recently the gene for human type III IFN was identified. We identified, amplified, and sequenced the gene for type III IFN (IFN-lambda3 also known as IL28B) entirely from a cDNA library derived from embryonic bovine kidney cells. It was further cloned in an Ad5 vector and expressed in multiple cell types. Bovine IFN-lambda3 protein expressed from this construct displayed antiviral activity against FMDV and vesicular stomatitis virus in bovine epithelial cells. Rabbit polyclonal antibodies were obtained by inoculating rabbits with Ad-bIFNlambda 3 and they show reactivity (western blots) against protein obtained in tissue culture after infection with A5-bIFNlambda3.

5. Continue studies on induction of antiviral response by combination interferon alpha, beta and gamma in swine. To control an FMD outbreak it is necessary to rapidly inhibit virus replication and spread. We have previously demonstrated that interferon (IFN) alpha delivered by a replication-defective adenovirus (Ad5) vector can rapidly protect swine from FMDV challenge, however, this approach has not been completely successful in cattle. To develop improved FMDV control measures we examined the effect of a combination of IFN alpha and gamma. In cell culture this combination synergistically blocks FMDV replication. Similarly in swine doses of Ad5-IFNalpha and Ad5-IFNgamma that alone did not protect in combination resulted in complete protection from FMDV challenge. We have demonstrated that the above treatment induces an increase in the number of dendritic cells in the skin and natural killer cells in the lymph nodes. In preliminary studies we have also shown that both of these cell types are also activated. IFN treatment also upregulates a number of host cytokines and chemokines involved in recruiting immune cells to the site of infection, in particular the chemokine 10-kDa IFN-gamma-inducible protein.


6.Technology Transfer

Number of Active CRADAs1
Number of New Patent Applications Filed1

Review Publications
Pena, L., Moraes, M., Koster, M.J., Burrage, T., Pacheco Tobin, J., Diaz San-Segundo, F., Grubman, M.J. 2008. Delivery of a Foot-and-Mouth Disease Virus Empty Capsid Subunit Antigen with Nonstructural Protein 2B Improves Protection of Swine. Vaccine. 26:5689-5699.

Tobin, G.J., Trujillo, J.D., Bushnell, R.V., George, L., Chaudhuri, A., Long, J., Barrera, J., Pena, L., Grubman, M.J., Nara, P.L. 2008. Deceptive Imprinting and Immune Refocusing in Vaccine Design. Vaccine. 26:6189-6199.

Mason, P.W., Grubman, M.J. 2009. VACCINES TO CONTROL FOOT-AND-MOUTH DISEASE. Book Chapter 22: 361-377.

Toka, F.N., Nfon, C., Dawson, H.D., Golde, W.T. 2009. Accessory Cell Mediated Activation of Porcine NK Cells by TLR7 and TLR8 Agonists. Clinical and Vaccine Immunology.Vol.16. No. 6.: 866-878.

Alves, M.P., Guzylack-Piriou, L., Juillard, V., Audonnet, J.C., Doel, T., Dawson, H.D., Golde, W.T., Gerber, H., Peduto, N., Mccullough, K.C., Summerfield, A. 2009. Innate Immune Defenses Induced by CpG do not Promote Vaccine-Induced Protection Against Foot-and-Mouth Disease in Pigs. Clinical and Vaccine Immunology. 16(8):1151-1157.

Last Modified: 11/24/2014
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