Location: Biologics Development Module
Project Number: 3022-32000-026-005-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2023
End Date: Aug 31, 2025
Objective:
The overall objective is to generate FMD reagents and technology for FMD surveillance and FMD vaccine development. Specific goals under this project include (1) producing 10 subunit FMD parental and mosaic constructs (based on sequences provided by ARS) and provide protein at the milligram-scale. (2) Utilize expression system(s) to optimize protein expression of FMD parental and mosaic empty-capsid (VLP) constructs for FMD type A, O, and Asia-1 (constructs based on sequences provided by ARS) and provide protein for each at the milligram-scale. (3) VLP and subunit FMD proteins will be tested in vitro for suitability, functional activity, and quantification. (4) Share data with PIADC and BDM for in-house growth of FMD antigens expressed in bacteria, baculovirus, or other expression system. (5) Produce hybridomas for a monoclonal antibody to a peptide sequence provided by ARS. Test monoclonal antibody(ies) to confirm reactivity against the provided peptide antigen. Provide successfully tested hybridoma and monoclonal antibodies to ARS.
Approach:
The objectives will be achieved through optimization of expression systems, VLP purification, and ELISA development. To approach VLP expression, we will focus on using the yeast expression system, for which there is preliminary data and recombinant constructs currently available to support this strategy. We will evaluate expression levels of VLP components under different expression conditions such as growth media type, incubation time/temperature, and nutrient additives, and we will establish optimal growth protocols to maximize VLP yields. Protein expression will be tested in different protein expression systems such (e.g. baculovirus, yeast or E. coli, etc) to determine the most efficient expression system. Some of the constructs may include a hexahistidine tag (6x H) in the C-terminus of the expressed recombinant protein to allow for purification with Nickel- or Cobalt- conjugated nitrilotriacetic acid (Ni-NTA, or Co-NTA) chromatography columns. Purified proteins will then be utilized as antigen for the detection of antigen specific antibodies through indirect Enzyme-linked Immunosorbent Assays (ELISAs), or as FMDV subunit vaccine candidates.
We will approach VLP purification through various conditions and established VLP purification protocols, and we evaluate the yield and purity under each condition. Once we have established small-scale protocols for VLP expression and purification, we will employ them for ELISA development. VLP will be used as a coating antigen for ELISA. We will be able to evaluate the diagnostic sensitivity and specificity of indirect ELISA, and we can evaluate a blocking ELISA format based on currently available FMD monoclonal antibodies. We will formulate ELISA reagents using various protein, detergent, and buffer components to optimize diagnostic performance. We will generate cell culture data for yeast expression systems, biochemical data for VLP protein expression and purification, and immunoassay data from ELISA R&D. These data will be evaluated and available for developing the optimal protocols to transfer our knowledge of VLP and ELISA production. Monoclonal antibodies against peptide sequences provided by ARS will be produced and tested to confirm reactivity against the provided peptide antigen. Successfully tested hybridoma and monoclonal antibodies will be provided to ARS.
