Journal: PloS one

Article Title: PKCθ regulates T cell motility via ezrin-radixin-moesin localization to the uropod.

doi: 10.1371/journal.pone.0078940

Figure Lengend Snippet: Figure 6. PKCh is required for ERM phosphorylation and localization to the uropod. T cells were activated with 300 ng/ml CCL21 for 10 minutes, fixed, and processed for immunofluorescence. Cells were stained for moesin (A) or phosphorylated ERM (B). Moesin and p-ERM localization in the uropod was determined measuring intensity in at least 2 spots in and out of the uropod and assessed as ‘‘polarized’’ if the signal in the uropod was at least 50 units or 2-fold higher than that seen outside the uropod. For quantitation, at least 50 cells showing T cell uropod morphology were counted and the localization of MTOC, moesin, or p-ERM assessed in 3 independent experiments, totaling at least 150 cells. Error bars show SEM. Significance was determined using the unpaired student’s t-test. Scale bar indicates 5 mm. (C,D) T cells from C57Bl/6 or C57Bl/6 Ly5.1 (WT) or B6.PKCh2/2 were not activated (C) or activated with 300 ng/ml CCL21 for the indicated time points (D). Cells were lysed and analyzed on SDS– PAGE, transferred onto PVDF membranes and blotted with anti-actin and anti-phospho-ERM antibodies. Signals were quantified using the Licor Odyssey and data shown is representative of at least 3 independent experiments. (C) Fold change in phosphorylated ERM in

Article Snippet: Reagents and Antibodies Antibodies were purchased from the following: a-CD4 was from eBiosciences (San Diego, CA); a-CD62L, and a-CD45.2 were from Biolegend (San Diego, CA); a-actin from Sigma Aldrich (St. Louis, MO); a-tubulin was from Thermo Fisher (Lab Vision, Fremont CA); a-PKCh, a-phospho-PKCh S676 was from Santa Cruz Biotechnology (Santa Cruz, CA); a-moesin and a-phosphoPKCh T538 were from Cell Signaling Technology (Beverly, MA); and a-CD43 antibody S11 was produced in the laboratory of Dr. Anne Sperling at the University of Chicago.

Techniques: Phospho-proteomics, Immunofluorescence, Staining, Quantitation Assay, 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: 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

Journal: Pharmaceutics

Article Title: Insect Cells for High-Yield Production of SARS-CoV-2 Spike Protein: Building a Virosome-Based COVID-19 Vaccine Candidate

doi: 10.3390/pharmaceutics14040854

Figure Lengend Snippet: Production of SARS-CoV-2 spike (S) protein in small-scale shake flasks. ( A ) S protein titers and specific production rates at different cell concentrations at infection (CCIs) and multiplicity of infections (MOIs). ( B ) Identification of S protein in culture supernatant samples collected at time of harvest by Western blot; numbers 1–3 denote culture replicates. ( C ) Identification of S protein in culture supernatant samples collected at time of harvest by Western blot; numbers 1, 4 and 5 denote rBAC used ( upper panel ), and S protein titers (bars) and specific production rates (circles) using different cell lines and baculoviruses ( lower panel ). Color code: blue, yellow and green represents data using rBAC 1, 4 and 5, respectively. For Western blot analysis, a mouse monoclonal 6-Histag antibody was used; positive control (CTL) is an in-house purified protein with a hexahistidine tag in the C-terminal at 0.2 and 0.1 µg; Ladder (L) is SeeBlue™ Plus2 Pre-stained Protein Standard. The expected MW of S protein monomer is approximately 140 kDa. Data are expressed as mean ± standard deviation (relative to three biological replicates, n = 3).

Article Snippet: For S identification, the human monoclonal antibody SARS-CoV-2 spike protein (MA5-35948, Thermo Scientific, Breda, The Netherlands) was used at dilution of 1:3000.

Techniques: Infection, Western Blot, Positive Control, Purification, Staining, Standard Deviation

Journal: Pharmaceutics

Article Title: Insect Cells for High-Yield Production of SARS-CoV-2 Spike Protein: Building a Virosome-Based COVID-19 Vaccine Candidate

doi: 10.3390/pharmaceutics14040854

Figure Lengend Snippet: SARS-CoV-2 spike (S) protein thermal stability. Differential scanning fluorimetry analysis of S protein formulated in three different buffers: Buffer A: 10 mM HEPES + 150 mM NaCl at pH 7.2 (orange); Buffer B: 10 mM HEPES + 150 mM NaCl at pH 7.2, 10% glycerol (blue); and Buffer C: 10 mM HEPES + 150 mM NaCl at pH 7.2, 10% sucrose (green). Data are expressed as mean ± standard deviation (relative to three replicates measurements, n = 3).

Article Snippet: For S identification, the human monoclonal antibody SARS-CoV-2 spike protein (MA5-35948, Thermo Scientific, Breda, The Netherlands) was used at dilution of 1:3000.

Techniques: Standard Deviation

Journal: Pharmaceutics

Article Title: Insect Cells for High-Yield Production of SARS-CoV-2 Spike Protein: Building a Virosome-Based COVID-19 Vaccine Candidate

doi: 10.3390/pharmaceutics14040854

Figure Lengend Snippet: Production of SARS-CoV-2 spike (S) protein in a 20 L stirred-tank bioreactor (STB). ( A ) Cell growth kinetics upon infection. ( B ) Identification of S protein in supernatant samples collected at day 1, 2 and at time of harvest (H, 63 hpi) from STB and shake-flask (SF) cultures by Western blot; a mouse monoclonal 6-Histag antibody and a human monoclonal SARS-CoV-2 S antibody were used; positive controls were an in-house purified protein with a hexahistidine tag in the C-terminal at 0.2, 0.1 and 0.05 µg (CTL + 1) and an in-house purified spike protein with a hexahistidine tag in the C-terminal at 1 and 0.5 ng (CTL + 2); Ladder (L) is SeeBlue™ Plus2 Pre-stained Protein Standard; the expected MW of S protein monomer is approximately 140 kDa; STB and SF denote stirred-tank bioreactor. SF denote samples from shake-flask control 1 and 2, respectively. ( C ) Site-specific glycan analysis of S protein by mass spectrometry; glycans were grouped in categories: high mannose glycan series—M9 to M5; Man 9 GlcNAc 2 to Man 5 GlcNAc 2 (green), and complex/paucimannose glycans (pink). ( D ) HPLC-SEC analysis of S protein upon storage at different temperatures and upon ×5 freeze-thaw (T/T) cycles; dashed grey line represents the retention time of a protein standard mix: (1) thyroglobulin (660 kDa), (2) uracil (112 kD), (3) ovalbumin (44.2 kDa) and (4) ribonuclease A (13.7 kDa). ( E ) S protein thermal stability using differential scanning fluorimetry; data are expressed as mean ± standard deviation (relative to three replicates measurements, n = 3). ( F ) Binding of non-overlapping human neutralizing antibodies recognizing epitopes in the receptor binding domain of S protein (i.e., ACE2-NN-IgGFc, CR3022, all SARS-CoV-2 antibodies) or an ACE2-Fc chimeric protein binding to S protein bound to ELISA plates, and developed with goat anti-human HRP.

Article Snippet: For S identification, the human monoclonal antibody SARS-CoV-2 spike protein (MA5-35948, Thermo Scientific, Breda, The Netherlands) was used at dilution of 1:3000.

Techniques: Infection, Western Blot, Purification, Staining, Control, Glycoproteomics, Mass Spectrometry, Standard Deviation, Binding Assay, Protein Binding, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: Insect Cells for High-Yield Production of SARS-CoV-2 Spike Protein: Building a Virosome-Based COVID-19 Vaccine Candidate

doi: 10.3390/pharmaceutics14040854

Figure Lengend Snippet: Binding of antibodies to S protein presented on virosomes. Virosomes bound to ELISA plates coated with anti-hemagglutinin were incubated with non-overlapping human neutralizing antibodies recognizing epitopes in the receptor binding domain of S, or with an ACE2-Fc chimera and developed with goat anti-human HRP; panel A at production of virosomes, panel B one month after storage at 4 °C.

Article Snippet: For S identification, the human monoclonal antibody SARS-CoV-2 spike protein (MA5-35948, Thermo Scientific, Breda, The Netherlands) was used at dilution of 1:3000.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation