Preclinical immunogenicity and efficacy of a candidate COVID-19 vaccine based on a vesicular stomatitis virus-SARS-CoV-2 chimera

Nonhuman Primates Are Protected against Marburg Virus Disease by Vaccination with a Vesicular Stomatitis Virus Vector-Based Vaccine Prepared under Conditions to Allow Advancement to Human Clinical Trials

“…A research stock of rVSV∆G-MARV-GP encoding GP from the MARV Musoke variant [ 45 ], which was used in multiple earlier preclinical studies [ 40 , 41 , 46 ], and a VSV Indiana genomic plasmid was kindly provided by the Zoonotic Diseases and Special Pathogens Division of the National Microbiology Laboratory, Public Health Agency of Canada (PHAC). The nucleotide sequence of the rVSV∆G-MARV-GP genome was determined by Sanger sequencing as described before [ 47 ], after which a DNA fragment encoding the MARV GP gene was synthesized at GenScript (Piscataway, NJ, USA). The GP gene was then transferred into the VSV genomic plasmid as described earlier [ 47 ].…”

“…The nucleotide sequence of the rVSV∆G-MARV-GP genome was determined by Sanger sequencing as described before [ 47 ], after which a DNA fragment encoding the MARV GP gene was synthesized at GenScript (Piscataway, NJ, USA). The GP gene was then transferred into the VSV genomic plasmid as described earlier [ 47 ]. The nucleotide sequence of the new genomic clone was confirmed by Sanger sequencing and was propagated and purified using animal product-free medium and reagents.…”

“…Recovery of rVSV∆G-MARV-GP from plasmid DNA was initiated by electroporating Vero cells [ 47 ]. A cell bank used before that was qualified for human vaccine production [ 48 ] and was used at all stages of recombinant virus work ( Figure 1 ).…”

“…Vero cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Sigma Aldrich, Burlington, MA, USA) supplemented with 4 mM L-glutamine and 10% gamma-irradiated fetal bovine serum (FBS; Sigma Aldrich, Burlington, MA, USA). Electroporation was conducted using methods modified from those described before [ 47 , 49 , 50 ]. In brief, ~2.5 × 10 7 cells were electroporated (low voltage mode, 3 pulses, 70 milliseconds, 140 V at 900 millisecond intervals) using a BTX830 apparatus (Harvard Apparatus, Holliston, MA, USA) and then were cultured at 37 °C in 5% CO 2 and 85% humidity.…”

“…Incorporation of GP in virions was monitored at multiple stages of production using Western blotting and methods similar to those described earlier [ 47 ]. Samples containing rVSV∆G-MARV-GP were denatured and separated using 4%–12% Bis-Tris denaturing polyacrylamide gels (Invitrogen, Waltham, MA, USA) and transferred with iBLOT2 system to nitrocellulose membranes (Invitrogen).…”