Location: Foreign Animal Disease Research2012 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:
During FY 2012 we have continued investigation of the genetic variation within the capsid-coding regions of Foot-and-Mouth Disease Virus (FMDV) serotypes A and South African Territories (SAT) 2. A comparison of SAT2 viruses from the Onderstepoort Veterinary Institute (OVI) collection revealed a significant variation in sequences for the outer capsid proteins (VP3-VP1, 32-49%) as compared to VP4,which was less exposed, with only 26% amino acid sequence variation. Work continued to identify surface exposed antigenic sites using known crystallographic data and by mapping sequence variability within those antigenic sites. For type A viruses we found that positions of high entropy also have side chains exposed. As we predicted, the variation within type A capsid sequences was more pronounced than that observed for the type O viruses. Our investigation showed that the highly immunogenic VP1 protein is the one containing the most variations in sequence. Two approaches were followed to determine whether these regions are truly contributing to the antigenicity of FMDV. In one approach, recombinant single chain variable fragment (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 neutralizing ability of the mutant viruses were observed. Genetic studies continued and we have tested mutant SAT 2 viruses carrying antigenic regions of disparate antigenic variants. This study is of significant importance since it will allow us to define epitopes relevant to the antigenicity of SAT2 viruses. In order to select for antibody binders for type A and SAT2 using a chicken immunoglobulin Y (IgY) libraries, we have prepared large stocks of type SAT2 and A viruses and we purified them using sucrose density gradients. We have succeeded at selecting virus specific binders for these two serotypes and we have initiated preliminary analysis of their specific reactivity. In FY 2012 we have performed additional rounds of selections. 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 recently acquired funds to support the construction of Ad5-FMD vaccine candidates for the SAT viruses, which continues to cause FMD outbreaks in the African territories. No technologies were transferred during FY 2012 and no publications were produced during this period.