Because of its high case fatality rate, ability to spread easily by human-to-human contact, and potential for aerosol release, Lassa virus (LASV), the causative agent of Lassa Fever (LF), is classified as a Biosafety Level 4 (BSL 4) and NIAID Biodefense category A agent. The potential use of the Lassa virus as a biological weapon directed against civilian or military targets necessitates development of counterthreat measures, such as diagnostic assays, vaccines and therapeutics.
Moreover, the impact of Lassa Fever in endemic areas of West Africa is immense, and therefore means to diagnose, treat or prevent it will provide a very significant public health benefit. Our team has produced prototype rapid-diagnostic assays using Lassa virus enzyme-linked immunosorbent assays (ELISA) (see Figure 1) that are based on recombinant proteins rather than on reagents that must be produced in high containment laboratories. The goal of this program is to use these new laboratory and clinical resources to identify novel parts of the Lassa virus, which are recognized by human antibodies in patients infected with the Lassa virus.
Figure 1. (From Wikipedia)
To guide the rational design of Lassa Fever immunotherapeutics and vaccines, we will select, produce and characterize human MAbs reactive with LASV GPC, GP1 and GP2, NP and Z. Derivation of human MAbs will permit identification of conserved and variant epitopes on this important biothreat agent and public health pathogen. The principle focus will be on epitopes on LASV GP, as these antibodies are most likely to be involved in virus neutralization or enhancement. However, characterization of epitopes on internal proteins NP and Z across divergent isolates of LASV can be valuable for development of diagnostic assays, and will also be pursued.
The primary method for generating LASV specific human MAbs will be EBV transformation of B cells isolated from peripheral blood of patients who are infected with LASV or who have survived LASV infection. Biased antibody fragment (Fab) phage display libraries will be used as an alternative approach for identification of human MAbs specific for LASV. LASV IgG-capture ELISA developed previously by this research team and produced to commercial standards by Corgenix will be used to screen for antibody producing cells. Similar ELISA will be developed for divergent strains of LASV, and for recombinant LASV proteins produced with defined site- directed or other mutations in specific B cell epitopes. We have also developed protocols for producing virus-like particles (VLP). ELISA using VLP potentially can detect conformational antibodies not present on recombinant proteins.Figure 2. Schematic representation of human LASV Fab phage display library construction, panning, characterization of Fab specificities, and conversion to full-length IgG1 antibodies.
The human MAbs will be characterized to determine whether binding occurs on linear vs. conformational epitopes, monomer or oligomer, recombinant proteins vs. VLP, glycosylated or vs. deglycosylated GP or point mutants that delete glycosylation sites. We will map linear or conformational B cell epitopes using mutant and wild-type recombinant LASV proteins.