Adjuvant formulated virus-like particles expressing native-like forms of the Lassa virus envelope surface glycoprotein are immunogenic and induce antibodies with broadly neutralizing activity

Lassa mammarenavirus (LASV) is a rodent-borne arenavirus endemic to several West African countries. It is the causative agent of human Lassa fever, an acute viral hemorrhagic fever disease. To date, no therapeutics or vaccines against LASV have obtained regulatory approval. Polyclonal neutralizing antibodies derived from hyperimmunized animals may offer a useful strategy for prophylactic and therapeutic intervention to combat human LASV infections. The LASV envelope surface glycoprotein complex (GP) is the major target for neutralizing antibodies, and it is the main viral antigen used for the design of an LASV vaccine. Here, we assessed the immunogenic potential of mammalian cell-derived virus-like particles (VLPs) expressing GP from the prototypic LASV strain Josiah in a native-like conformation as the sole viral antigen. We demonstrate that an adjuvanted prime-boost immunization regimen with GP-derived VLPs elicited neutralizing antibody responses in rabbits, suggesting that effective antigenic epitopes of GP were displayed. Notably, these antibodies exhibited broad reactivity across five genetic lineages of LASV. VLP-based immunization strategies may represent a powerful approach for generating polyclonal sera containing cross-reactive neutralizing antibodies against LASV.


Statistics
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Flow Cytometry
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Methodology
All cell lines were tested negative for mycoplasma contamination.
No misidentified cell lines were used in this study.
New Zealand White rabbits (Oryctolagus cuniculus) The study did not involve wild animals.
The study did not involve samples collected from the field. Cells origin specified under " Eukaryotic cell lines". For flow cytometric analysis cells were seeded in 6-well plates one day before performing the experiment. The virus-antibody mixture was added to the cell monolayers for 22 h. Cells were gently harvested by trypsinization, fixed with 4% PFA for 20 min at RT, and permeabilized with saponin-buffer (PBS, 3% FBS, 2mM EDTA, 0.001% sodium azide, 0.5% saponin) for 30 min at RT. Samples were incubated with polyclonal guinea pig-!VSV serum at dilution 1:1000, followed by rabbit !-guinea pig FITC-conjugated antibody at dilution 1:1000 for 1 hour at 4°C each. After each incubation step, cells were washed (PBS, 3% FBS, 2mM EDTA, 0.001% sodium azide) twice and finally subjected to flow cytometry analysis.

BD FACSCalibur
Data were collected with Cell Quest Pro V5.2, FlowJo v10.0 software was used for data analysis.
Data were obtained from established cell lines of single cell type, no sorting or specific purity controls were used. Since cell lines used in the experiments differ in cell size and granularity, specific instrument stetting were used for each cell line.
For all experiments living cells were distinguished from cell fragments by FSC-H/SSC-H, referring to cell size and granularity in the starting population. Since established cell lines were used in the experiments only a single cell population was expected. Based on cell population considered as "living" further analyses for VSVdG/LASVGP infection were performed. For each cell line