LASSA VIRUS LIKE PARTICLES AS A CANDIDATE IN ENZYME-LINKED
IMMUNOSORBANT ASSAY DIAGNOSTICS.
Jessica N Grove, Luis M Branco, Robert F Garry.
Department of Microbiology and Immunology, Tulane University, School of Medicine
The etiologic agent Lassa virus (LASV) is a member of the Arenaviridae family that
causes hemorrhagic fever in humans and infects an estimated 300,000 people with
5,000 fatalities a year. Lassa endemic areas include several countries of West Africa
including Sierra Leone and Nigeria. Currently available diagnostics for this disease are
time-consuming, and difficult to manufacture, requiring Biosafety Level-4 (BSL-4)
containment laboratories, thus making diagnosis and treatment difficult. The Center for
Disease Control provides an enzyme-linked immunosorbent assay (ELISA) using an
inactivated LASV-infected Vero cell slurry that requires six hours to complete. Our lab
has developed individual mammalian and bacterial recombinant proteins that, when
used in ELISA diagnostics, forgo several steps, resulting in a less time-consuming
assay. The recombinant proteins used in this assay are the major immunologic
determinants of LASV infection and include nucleoprotein (NP) and the glycoprotein
complex (GPC) that is eventually cleaved in to glycoprotein 1 (GP1) and glycoprotein 2
(GP2). Another avenue of antigen production is through the generation of virus like
particles (VLPs). VLPs are produced using the HEK-293T/17 cell line. These cells are
transfected with NP, GPC, and Z DNA expression plasmids. Though Z is not an
immunologic determinant, it plays a key role in budding the virion from the cell
membrane. The result is a non-infectious enveloped particle expressing the recombinant
LASV proteins NP, GP1 and GP2, and Z. When used in an ELISA format, VLPs can
present native viral epitopes for antibody detection. Using the VLP-ELISA format, serum
from suspected Lassa patients has resulted in an optical density (OD) as high as 4. In
addition, when compared to individual recombinant proteins ELISAs, VLP coated plates
have shown a good correlation. Blood bank samples from donors within the United
States have low reactivity levels, indicating low backgrounds and little or no crossreactivity with VLP recombinant proteins. These results indicate that VLPs may be a
good candidate for use in ELISA diagnostics. Future studies will employ VLPs in lateral
flow immunoassays (LFI) to provide a point-of-care diagnostic for Lassa endemic areas.
This work is supported by NIH/NIAID grant UO1 AI082119-01 (Garry, PI); Recombinant
antigen multiagent diagnostic assays for Lassa and other arenaviruses.