2011 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.
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.
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.
Sequencing of the Leader/P1/2A-coding region of 25 SAT2 viruses that caused outbreaks in Southern African District Countries (SADC) member countries over the last 22 years and have used the data to determine the genetic variability of SAT2 viruses within southern Africa. An alignment of the deduced amino acid residue allowed the identification of hypervariable regions that correlated with loops between beta-sheet structures. When these regions were mapped to a modelled structure of the SAT2 capsid variable, surface-exposed loops could be identified and regarded as putative antigenic regions. Two approaches were followed to determine whether these regions are truly contributing to the antigenicity of FMDV. In one approach, recombinant scFV against FMDV is selected and the binding sites to the virion mapped using virus escape mutants. In a second approach, potential antigenic region were swapped in a recombinant SAT virus with that of a disparate virus and changes in the neutralising ability of the mutant viruses were observed.
This project was monitored through email exchange and conference calls, as well as site visit to ARC by ARS, PIADC staff.