FIG 3.

Vaccine vector characterization. (A) Vero CCL-81 cells were infected at an MOI of 0.1 with the BNSP333 parental rabies virus vaccine or recombinant FILORAB3 for 48 h before surface immunostaining with monoclonal antibodies directed against RABV G (green) and MARV GP (red). Yellow indicates an overlap in the expression of both glycoproteins. (B) (Left) Four micrograms each of purified inactivated FILORAB3 and control virions was loaded onto a denaturing 10% SDS-PAGE gel and stained with SYPRO Ruby to visualize the incorporated proteins. Full-length codon-optimized MARV GP and soluble MARV GP (2 μg) with transmembrane and cytoplasmic domain deletion (used for antibody capture in ELISAs) are indicated by the red box. FILORAB1 purified virions were included as a control for successful foreign glycoprotein incorporation. Critical RABV proteins are indicated. (Right) Confirmation of MARV GP incorporation into purified FILORAB3 virions by Western blot analysis. Two micrograms of purified inactivated FILORAB3 or control virions was loaded onto a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The blot was probed with a cocktail of two mouse-derived monoclonal antibodies specific for either the GP1 or the GP2 subunit of MARV GP. Lysates from Vero cells infected with MARV were used as a positive control. As a negative control, mock-infected or Lassa virus-infected Vero cell lysate was used.