Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Codon-optimized full-length Spike protein gene (S F ), S1 subunit gene and the receptor-binding domain (RBD) plus envelope protein genes of SARS-CoV-2 with and without 21 amino acids honeybee melittin signal peptide [(msp) NH 2 -MKFLVNVALVFMVVYISYIYA-COOH] gene in the purple box, and 49 amino acids VSV G protein transmembrane domain and cytoplasmic tail [(Gtc) NH 2 -SSIASFFFIIGLIIGLFL VLRVGIYLCIKLKHTKKRQIYTDIEMNRLGK-COOH] gene in the red box were inserted into the G and L gene junction of rVSV Ind and rVSV NJ . In addition, 25- nucleotides-long VSV intergenic junctions (5´-CATATGAAAAAAACTAACAGATATC-3´), in the green box, were inserted between genes to provide transcription termination, polyadenylation and the transcription reinitiation sequences. Recombinant viruses were rescued by VSV reverse genetics . pT7: Bacteriophage T7 promoter for DNA-dependent RNA polymerase. N : VSV Nucleocapsid Protein gene. P : VSV Phosphoprotein gene. M : VSV Matrix protein gene. G : VSV Glycoprotein gene. L : VSV Large protein, RNA-dependent RNA polymerase gene. l : Leader region in the 3´-end of the VSV genome. t : Trailer region in the 5´-end of the VSV genome. HDV: Hepatitis delta virus ribozyme encoding sequences. T7δ: Bacteriophage T7 transcriptional terminator sequences. nt: nucleotides. aa: amino acids.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Binding Assay, Recombinant

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: To check the expression of SARS-CoV-2 RBD, S1, S F , and E proteins from the rVSV Ind -SARS-CoV-2 infected cells, BHK-21 cells were infected with the virus at an MOI of 6. After six hours incubation at 37°C, cell lysates were prepared and protein expression was determined by Western blot analysis. Cell lysates were loaded in 5 μg quantity for SDS-PAGE. RBD, S1, and S F proteins were detected by rabbit antibody against SARS-CoV-2 RBD. S2 protein was detected by rabbit antibody against SARS-CoV-2 S2. E protein was detected by rabbit antibody against SARS-CoV-2 E peptides. (A) Expression of RBD, S1, and S F with and without msp and Gtc. (B) Expression of S2 with and without Gtc. (C) Expression of E protein. (D) Expression of VSV Ind N, P, M, and G proteins. Purple box: honeybee msp, red box: VSV Gtc.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Expressing, Infection, Incubation, Western Blot, SDS Page

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Incorporation of SARS-CoV-2 S F , S1, S2, and RBD with or without VSV Gtc into rVSV Ind particles was examined by infecting BHK-21 cells with rVSV Ind -SARS-CoV-2 at an MOI of 3. The rVSV Ind -SARS-CoV-2 infected cells were incubated at 31°C for 6 hrs. Infected cell lysates were prepared in lysis buffer (lanes 1, 2, and 5). Culture media from the infected cells was centrifuged at 500 x g for 10 minutes and supernatant was filtered through a 0.45 μm filter to remove cell debris. The filtered culture media was loaded onto 1 ml of 25% sucrose cushion and ultra-centrifuged at 150,900 x g for 3 hrs. Supernatant on top of the 25% sucrose cushion was collected to check the soluble proteins in the media (lanes 3 and 6). Pelleted samples were checked for proteins incorporated into VSV particles (lanes 4 and 7). We detected RBD, S1, and S F proteins by Western blot using an antibody against the SARS-CoV-2 RBD protein. S2 and S F proteins were detected by rabbit antibody against SARS-CoV-2 S2. (A) Detection of S F and S1 proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S F -Gtc or rVSV Ind -S F . (B) Detection of S F and S2 proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S F -Gtc or rVSV Ind -S F . (C) Detection of VSV Ind proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S F -Gtc or rVSV Ind -S F . (D) Detection of S1 protein in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S1-Gtc or rVSV Ind -S1. (E) Detection of RBD proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-RBD-Gtc+E-Gtc or rVSV Ind -msp-RBD+E. (F) Detection of VSV Ind proteins in cell lysate, concentrated culture media, and virus pellet from the cells infected with rVSV Ind -msp-RBD-Gtc+E-Gtc or rVSV Ind -msp-RBD+E. Purple box: honeybee msp, red box: VSV Gtc.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Infection, Incubation, Lysis, Western Blot

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: To examine immune responses in mice, it was first necessary to purify rVSV-SARS-CoV-2 viral particles by anion-exchange chromatography. One μg of the purified rVSV-SARS-CoV-2 was analyzed by SDS-PAGE and the presence of RBD, S1, S2, and S F was determined by Western blot analysis. (A) Detection of RBD, S1, and S F on VSV particles. (B) Detection of S2 and S F on VSV particles. (C) Detection of VSV Ind and VSV NJ proteins. (D) Depicted model of pseudotype recombinant VSV virions with three different forms of SARS-CoV-2 Spike proteins. rVSV pseudotypes are formed when rVSV-SARS-CoV-2 Spike proteins are expressed with the msp at the NH 2 -terminus and VSV Gtc at the COOH-terminus. Purple box: honeybee msp, red box: VSV Gtc.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Chromatography, Purification, SDS Page, Western Blot, Recombinant

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Mice were prime immunized with rVSV Ind -SARS-CoV-2 and boost immunized with rVSV NJ -SARS-CoV-2 two weeks after prime-immunization. Serum was collected to determine SARS-CoV-2 S1 protein-specific antibody levels by ELISA on day 13, one day before boost-immunization, and on day 27, two weeks after boost-immunization. (A) Prime-boost vaccination schedule. (B) Spike(ΔTM)-specific IgG titer after the prime-boost vaccination with doses of 5X10 7 PFU/mouse ( C) Spike(ΔTM)-specific IgG titer after the prime-boost vaccination with doses of 5X10 8 PFU/mouse. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p < 0.005; ***, p< 0.001; ns, not significant). The data were presented as means with error bars of standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Six-week-old female hACE2 transgenic mice were prime vaccinated with rVSV Ind -msp-S F -Gtc and boost immunized with rVSV Ind -msp-S F -Gtc or rVSV NJ -msp-S F -Gtc two weeks after the prime-vaccination. Serum was collected on day 13, one day before the boost-vaccination and on day 27, two weeks after the boost-vaccination. SARS-CoV-2 neutralization was determined by FRNT 50 assay. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p<0.005; ***, p< 0.001; ns, not significant). The data were presented as means with error bars of standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Neutralization, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Six-week-old female hACE2 transgenic mice were prime vaccinated with rVSV Ind -msp-S F -Gtc and boost vaccinated with rVSV Ind -msp-S F -Gtc or rVSV NJ -msp-S F -Gtc two weeks after the prime-vaccination. Serum was collected to determine the SARS-CoV-2 Spike(ΔTM) protein-specific antibody level by ELISA on day 13, one day before the boost-vaccination and on day 27, two weeks after the boost-vaccination. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p<0.005, ***, p<0.001, ****, p< 0.0001; ns, not significant). The data were presented as means with error bars of standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Mice were primed with rVSV Ind -SARS-CoV-2 followed with rVSV NJ -SARS-CoV-2 two weeks after prime-immunization. Two weeks after the boost-immunization, splenocytes were prepared and stimulated with a PepTivator SARS-CoV-2 Prot_S [ (A) ], or an irrelevant (control) peptide derived from the HIV Gag (B) . IFN-γ spot-forming units (SFUs) were enumerated by ELISPOT. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p < 0.005; ns, not significant). Data are presented as mean SFU numbers with error bars representing standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Derivative Assay, Enzyme-linked Immunospot, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Six-week-old female hACE2 transgenic mice (n = 5 per group) were prime-vaccinated with rVSV Ind -msp-S F -Gtc and boost vaccinated with rVSV Ind -msp-S F -Gtc or rVSV NJ -msp-S F -Gtc two weeks after prime-vaccination. Four weeks after boost-vaccination , mice were challenged intranasally with 1x10 5 PFU of SARS-CoV-2. The survival and body weight of each mouse was monitored daily. (A) Average bodyweights of mice in each vaccinated group. (B) Individual body weights for mice vaccinated with rVSV-Mock and challenged with SARS-CoV-2. (C) Mouse survival after SARS-CoV-2 challenge. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Human ACE2 transgenic mice were vaccinated and challenged with SARS-CoV-2 as described in . Right lobes of mice lungs were aseptically removed from the mice on day 3, day 7, and day 15 after SARS-CoV-2 challenge. Infectious SARS-CoV-2 was quantified by plaque assay on Vero E6 cells. Statistical significance was determined by two-way ANOVA with Tukey’s correction (****, p< 0.0001). VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Plaque Assay, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Human ACE2 transgenic mice were vaccinated and challenged with SARS-CoV-2 as described in . Left lobes of mice lungs were fixed in 10% buffered formalin on day 3 and day 7 after the SARS-CoV-2 challenge. Lung tissues were processed and embedded in low-melting paraffin, sectioned to a thickness of 3 μm, and stained with hematoxylin and eosin. Stained tissues were examined under a light microscope (Olympus CS41, Japan) with 100X magnification. Note: a, alveolus; b, bronchiole; v, blood vessels. (A) Lung tissue 3 days after the SARS-CoV-2 challenge. (B) Lung tissue 7 days after SARS-CoV-2 challenge. Arrows show infiltration of inflammatory cells (lymphocytes and macrophages). G1: empty vector infected mice, G2: 5X10 8 of rVSV Ind -msp-S F -Gtc/ rVSV NJ -msp-S F -Gtc vaccinated mice, G3: 5X10 8 of rVSV Ind -msp-S F -Gtc/ rVSV Ind -msp-S F -Gtc vaccinated mice, G4: 5X10 7 of rVSV Ind -msp-S F -Gtc/ rVSV NJ -msp-S F -Gtc vaccinated mice, G5: uninfected mice.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Staining, Light Microscopy, Plasmid Preparation, Infection