Location: Foreign Animal Disease Research2013 Annual Report
1a. Objectives (from AD-416):
It has been determined that the outer Foot-and-Mouth Disease Virus (FMDV) capsid proteins are directly involved in antigenicity. Antigenic variants of the virus result from changes to the viral capsid as a consequence of the high mutation rate of the virus. The role of these sites, prone to change, in vaccine efficacy is poorly understood. Identification of those residues that comprise the antigenic determinants of the virus will allow the identification of those mutations in outbreak strains that potentially lessen the efficacy of a vaccine. If enough of these epitopes are identified, it may be possible to predict the protection afforded by a vaccine against a specific outbreak. The goal of this collaborative research project is to identify FMDV antigenic epitopes and improve the infectious cDNA technology to produce recombinant FMDV with advanced biological properties and antigenic coverage as seed viruses in the production of inactivated vaccine antigens. Amendment 1: Research performed at ARC-OVI has indicated that the viral neutralization(VN) test is not the most reliable test used to obtain consistent results with regards to antigenic matching. To overcome this problem, monoclonal antibodies will be developed and characterized in single change variable fragment (scFv) molecules and use type A and SAT type specific scFvs in an ELISA based test to measure antigen relationships between field and outbreak viruses within serotypes. Viral epitopes involved in antigenicity or protective B-cell responses are good predictors of antigenic distance. Therefore, we will improve the capacity for managing antigenic variants of serotype A and SAT viruses through the application of antigenic cartography and vaccine matching. The recombinant monoclonal antibody technology, used for the mapping of epitopes on the virion, as reagents to measure the antigenic diversification of an emerging or re-emerging virus from that of the reference vaccine strains. In addition, we will replace the FMDV type-SAT capsids in our replication defective human adenovirus vector containing the capsid and 3C proteinase coding region of FMD (Ad5-FMD) vaccine to assess the performance of the Ad-FMD vaccine candidates in the African region.
1b. Approach (from AD-416):
To identify the FMDV antigenic epitopes for contemporary FMDV serotype A viruses, the Agricultural Research Council- Onderstepoort Veterinary Institute (ARC-OVI) will produce recombinant monoclonal antibodies (rec-mAb), i.e. single-chain variable fragments (scFv’s) selected from a chicken immune library (Nkuku®). Rec-mAbs have been developed for a SAT2 virus and the binding mechanism of a unique binder to the SAT2 capsid has been elucidated at ARC-OVI. It is envisaged extending this study to include type A viruses as well. As such, PIADC, ARS will measure the spectra of antigenic variants and map antigenic sites within serotype A viruses using type A rec-mAb. ARS and ARC-OVI will identify and replace the potential neutralizing epitpes in the SAT2 and type A in the corresponding infectious cDNA clones and evaluate the antigenicity and usefulness in protection against FMDV. ARS and ARC-OVI will then determine the immunogenicity and protective ability of the designed vaccines, prepared from epitopes of contemporary isolates, in vivo. The newly developed ELISA will then be used to measure any changes in antigenicity. Amendment 1: A FMDV type A and SAT type specific immune phage display library will be constructed, in addition to the already available naïve library, from which FMDV specific scFvs will be panned. In order to construct the immune library, chickens will be immunized with type A and SAT antigen, the resulting antibodies which are predisposed to yield FMDV specific antibodies, will be used to produce IgY libraries. The immune libraries will then be panned against the type A and SAT antigen and the FMDV specific scFvs selected. It is envisaged that the FMDV specific scFvs obtained from both the naïve and the immune library will produce enough type A and SAT specific binders that at least 10 or more binders can be applied in an ELISA for antigen matching against panels of recent field viruses. ARS has recently developed an A24Cruzeiro-based Ad5-FMD vaccine that could be used for other important FMDV serotypes including the SAT serotype, which continues to cause FMD outbreaks in the African territories. The ARS current second generation Ad-FMDV vector will be used as the template for capsid exchanges, and the examination of potency of these vaccine candidates will be performed in collaboration with ARC-OVI.
3. Progress Report:
The overall objective is to identify Foot-and-Mouth Disease Virus (FMDV) antigenic epitopes using recombinant single chain variable fragments (scFv’s) of chicken immunoglobulin Y (IgY) antibodies and to improve the infectious cDNA technology to produce recombinant FMDV with advanced biological properties and antigenic coverage as seed viruses in the production of inactivated vaccine antigens. Conducted the following activities: 1. Panning an antibody phage display library with type A and SAT type viruses and selected unique phage binders against these viruses. The viruses were propagated on BHK-21 cells, harvested and clarified. Subsequently The purified viruses were used to select binders from the Nkuku® library using standard protocols. 2: Modification of existing vaccine strains, using reverse genetics technology, to potentially improve the protection offered by vaccine strains. The cross-reaction and reactivities of eight epitope replaced mutants were tested using virus neutralization assays and monoclonal antibodies (mAbs). Five SAT2-specific monoclonal antibodies (mAbs) were identified and confirmed by standard methods. The most significant accomplishments to date include: 1. Panning the antibody phage display library with type A and SAT type viruses and selection of unique phage binders against these viruses. 2. Modification of existing vaccine strains, using reverse genetics technology, to potentially improve the protection offered by vaccine strains. Eight presumptive antigenic regions on the virion surface of an existing recombinant vSAT2 virus were modified to the corresponding regions of an antigenic disparate virus. No technologies were transferred in FY 2013. Publications for the period include: 1. Opperman, P.A., et al.. (2012) Mapping of antigenic determinants on a SAT2 foot-and-mouth disease virus using chicken single-chain antibody fragments. Virus Research, 167, pp. 370-379. 2. F.F. Maree, et al. (2013) Analysis of SAT type foot-and-mouth disease virus capsid proteins and the identification of amino acid residues affecting virus stability. PLoSone; e61612 3. P.A. Opperman, et al. Determining the epitope dominance on the capsid of a SAT2 foot-and-mouth disease virus by mutational analysis. EuFMD, October 2012, Jerez, Spain. 4. F.F. Maree, et al. Intra-serotype chimeric foot-and-mouth disease vaccine antigen elicit protection in cattle. EuFMD, October 2012, Jerez, Spain. 5. Dr F.F. Maree & C. Gay. Global Foot and Mouth Disease Research Alliance (GFRA): current actions and future perspective. EuFMD, October 2012, Jerez, Spain. 6. Opperman, P.A., et al. Mapping of antigenic sites of foot-and-mouth disease viruses using a chicken antibody phage display library. Poster presentation at the positive strand RNA viruses’ conference. 28-30 May, Boston, Massachusetts.