2013 Annual Report
1a.Objectives (from AD-416):
To generate multiple monoclonal antibodies against RVFV antigens Gn, N and NSs that can be used in (a) Research and development of antigen detection and quantitation assays; (b) Reagents for other serological assays; (c) Research to characterize the immunological response to RVFV; and (d) Technical transfer to commercial firms engaged in development of diagnostic and vaccine projects.
1b.Approach (from AD-416):
Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Virus infection causes abortions and high mortality in newborn ruminants. Humans can become infected through handling infected animals and animal products. Severe infections can include ocular disease, meningoencephalitis, or hemorrhagic fever with an overall case fatality rate of <1. Panels of monoclonal antibodies are needed to facilite development of safe diagnostic assays. An anti-recombinant RVFV- nucleocapsid (N) polyclonal antibody was produced by ABADRU to develop an immunohistochemical (IHC) assay. Multiple ELISA assays have been developed based on a mouse anti-RVF MP-12 vaccine strain polyclonal antibody. A robust set of capture and detect monoclonal antibodies will be necessary to continue development of these assays and to make reagents available for international standardization of diagnostic tests and potential further test kit development (commercialization). Once validated and approved by national regulatory agencies, these reagents and assays will be useful to national laboratories as initial diagnostic tests (and validated against virus isolation). These assay reagents could be safely produced and distributed to regional diagnostic laboratories, thus providing capacity for early detection of RVFV in suspected ruminant samples.
The specific objective of this project is to generate monoclonal antibodies to Rift Valley fever virus antigens. The two most important sub-objectives are to.
1)generate matched pair candidates of antibodies specific for RVFV detection Nucleocapsid protein (Np protein) and.
2)antibodies specific for viral surface proteins for use as serological assay positive controls, and/or as capture–reagents provided by ARS. A fusion was performed, which resulted in a panel of 11 clones reactive with the rNP and these were provided as in process purified antibody to ARS for further evaluation. ARS selected two clones showing potential to recognize native Np in both indirect and matched pair ELISAs. These hybridomas have been subcloned and a pilot production, purification, characterization and biotinylation (for the study of sandwich immunoassays) have been completed for each. Monoclonal anti-RVFV Np antibodies (and their biotinylated aliquots) have been shipped to Dr. Scott McVey for further evaluation.
ARS has also provided MBS with a baculovirus expressed recombinant surface glycoprotein (rGn) of Rift Valley Fever Virus. Initial studies indicate that both sheep and mice immunized with MP-12 are able to generate antibodies specific for the recombinant glycoprotein, both at USDA (by ELISA and Western Blot) and MBS (by ELISA). The next step is to perform a second fusion on mice immunized with MP-12 to develop monoclonal antibodies to the rGn glycoprotein, which will be used to detect native virus.
The third goal of the project is to develop monoclonal antibodies to the Non Structural S protein of RVFV. USDA is currently working on the recombinant expression of the NSs protein to be used as a screening agent for mice immunized with the MP-12 and/or directly as immunogen, since MP-12 virus will express little, if any, NSs.