Beta propiolactone bpl

Once the viruses were recovered, they were grown on Vero CCL81 cells in viral production serum-free medium (VP-SFM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% Glutamax, 1% penicillin-streptomycin, and 1% HEPES buffer. Cells were infected at an MOI of 0.01 in either a T175 flask or 2-stack chambers (Corning, Corning, NY, USA). Supernatant collections occurred 5 days post-infection and every 3 days afterward, for a total of 17 days. For titration, RABVs were overlayed on VeroCCL81 cells with a starting dilution of 1:10 and diluted 10-fold in a 96-well plate in triplicate. After 48 h, cells were fixed with acetone and stained with RABV-N GFP stain to determine the foci-forming units (ffu)/mL using fluorescence microscopy.
Viruses were filtered through 0.45 µm PES membrane filters (Nalgene, Conway, NH, USA) and concentrated down to 50 mL for purification. Viruses were purified over 20% sucrose cushion and ultracentrifuged at 25,000 rpm in a SW32 rotor for 1.5 h. Viral particles were resuspended in TEN buffer + 2% sucrose and inactivated with 50 µL per mg of particles with β-propiolactone (BPL, Millipore Sigma, Cat# P5648, Burlington, VT, USA) in cold culture grade water. The level of inactivation was verified by inoculating Vero CCL81 cells over three passages with 10 µg of BPL-inactivated virions.

To produce inactivated GP38+ G_C-, GP38- G_C+ , GP38+ G_C+ , and Filorab1 vaccines, viral supernatant was concentrated, sucrose purified^{84 (link)}, and inactivated^{81 (link)}. Viral supernatants with the highest titers were pooled for each virus and concentrated at least 5x in an Amicon® 300 mL stirred cell concentrator using a 500 kDa exclusion PES membrane (MilliporeSigma®). Concentrated supernatants were then overlaid onto a 20% sucrose cushion and centrifuged at 76,755 x g for 2 h. Virions pellets were resuspended in TEN buffer (100 mM Tris base, 50 mM NaCl, 2 mM EDTA in ddH₂O) with 2% sucrose and incubated overnight (O.N.) at 4 °C. β-propiolactone (BPL) (MilliporeSigma®) was added at a 1:2000 dilution for inactivation. Samples were left at 4 °C O.N. shaking and then incubated the following day at 37 °C for 30 min to hydrolyze the BPL. Virus inactivation was confirmed by inoculated supernatant with 10 µg of inactivated virions was passaged in T25 flasks of Vero cells; cells were fixed and stained with a 1:200 dilution of FITC anti-RABV-N (Fujirebio®, product #800–092) or monitored for cytopathic effect^{25 (link)}.

Groups of 8–10 weeks old C57BL/J6 female mice (Jackson) were immunized intramuscularly (i.m) via gastrocnemius with 100 μl (50 μl/leg) of live or inactivated RABV or RABV-ED51-mBAFF as indicated in the figures. Inactivated RABV and inactivated RABV-ED51-mBAFF were prepared as follows: virus stocks were grown in OptiPRO Serum Free Media (Gibco) [4mM L-Glutamine, 1% PS], harvested, and cell debris was removed using Corning 0.45μm filter (430516; Corning). β-Propiolactone (BPL; P5648; Sigma) was added to viral supernatants (final concentration 0.05% BPL), and incubated overnight at 4ᵒC. Treated supernatants were purified using ultracentrifugation. Viral inactivation was confirmed by viral titer [37 (link)]. Total protein concentrations were quantified by BCA Protein Assay Kit as described by the manufacturer (Pierce). Blood from immunized mice was collected via retro-orbital at 5, 7, 10 days post-immunization. RABV G-specific IgM and IgG (and subclasses) antibody levels were determined by ELISA as described previously [36 (link)–39 (link)]. VNA titers were determined by RFFIT as described previously [36 (link), 37 (link), 40 (link)].