Journal: Journal of Virology

Article Title: A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters

doi: 10.1128/JVI.00592-21

Figure Lengend Snippet: SARS-CoV-2 S and its truncations are highly expressed by mtdVSV. (A) Analysis of S protein expression by mtdVSV using SARS-CoV-1 antibody. BSRT7 cells in 6-well plates were infected with each recombinant virus at an MOI of 3.0. At 40 h postinfection, cells were lysed in 300 μl of lysis buffer, and 10 μl of lysate was analyzed by SDS-PAGE and blotted with anti-SARS-CoV-1 S protein antibody (top) or β-actin antibody (bottom). (B) Analysis of S protein expression by pCI using SARS-CoV-1 antibody. 293T cells were transfected with 2 μg of each plasmid. At 48 h posttransfection, cell lysates were collected for Western blot analysis using anti-SARS-CoV-1 S protein antibody. (C and D) Analysis of the expression of S and its truncations by mtdVSV using SARS-CoV-2 antibody. BSRT7 cells in 6-well plates were infected with each recombinant virus at an MOI of 1.0. At 20 h or 28 h postinfection, cells were lysed in 300 μl of lysis buffer, and 10 μl of lysate or supernatant was analyzed by SDS-PAGE and blotted with anti-SARS-CoV-2 S protein antibody (top), VSV G antibody (middle), or β-actin antibody (bottom). Western blots shown are the representatives of three independent experiments. (E and F) Analysis of the incorporation of S into VSV virions. Cell culture supernatants were collected from 10 confluent T150 flasks of BSRT7 cells were infected by rVSV-D1762A or rVSV-D1762A-S. Cell debris were removed by centrifugation at 10,000 × g for 5 min. Virus particles were purified through 10% sucrose cushion (E, left), followed by 20 to 50% sucrose gradient purification (F, left). Aliquots consisting of 3 μg of total protein from the 10% sucrose cushion (E, left) and 10 μg from the sucrose gradient purification (F, left) were analyzed by SDS-PAGE and stained with Coomassie blue. Duplicated samples were also blotted with anti-SARS-CoV-2 S protein antibody (E and F, right). SDS-PAGE and Western blots shown are the representatives of two independent experiments.

Article Snippet: The blot was probed with rabbit anti-SARS-CoV-1 S (catalog no. 40592-T62; Sino Biological), SARS-CoV-2 S (catalog no. 40150-R007; Sino Biological), or RBD (catalog no. 40592-MP01; Sino Biological) antibody at a dilution of 1:2,000, followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz) at a dilution of 1:5,000.

Techniques: Expressing, Infection, Recombinant, Lysis, SDS Page, Transfection, Plasmid Preparation, Western Blot, Cell Culture, Centrifugation, Purification, Staining

Journal: Nature Communications

Article Title: Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform

doi: 10.1038/s41467-020-19819-1

Figure Lengend Snippet: The replication properties of sMVA, derived either with FPV HP1.441 (sMVA hp) or with FPV TROVAC from two independent sMVA virus reconstitutions (sMVA tv1 and sMVA tv2), were compared with that of wtMVA. a Replication kinetics. BHK or CEF cells were infected in triplicates ( n = 3) for each time point at 0.02 multiplicity of infection (MOI) with sMVA or wtMVA and viral titers of the inoculum and each triplicate infection were determined at 24 and 48 h post infection on CEF. Mixed-effects model with the Geisser-Greenhouse correction followed by Tukey’s multiple comparison test were applied; at 24 and 48 h post-infection differences between groups were not significant ( p > 0.05). b , c Viral foci size analysis. BHK or CEF cell monolayers were infected at 0.002 MOI with sMVA or wtMVA and areas of viral foci ( n = 20) were determined at 24 h post infection following immunostaining with anti-Vaccinia polyclonal antibody (αVAC). Panel c provides examples of sMVA and wtMVA viral foci following immunostaining of infected CEF. d Host cell range analysis. Various human cell lines (HEK293, A549, 143b, and HeLa), CEF or BHK cells were infected in duplicates ( n = 2) at 0.01 MOI with sMVA or wtMVA and virus titers of each duplicate infection were determined in duplicates ( n = 4 in total) at 48 h post infection on CEF. Dotted lines indicate the calculated virus titer of the inoculum based on 0.01 MOI. Differences between groups in b and d were calculated using one-way ANOVA followed by Tukey’s ( b ) or Dunnett’s ( d ) multiple comparison tests; ns = not significant ( p > 0.05). Data in a and d are presented as mean values + SD. Lines in b represent median values. Source data are provided as a Source Data file.

Article Snippet: S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody at a dilution of 1:1300 (40150-T62-COV2, Sino Biological); N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological) was used at dilution of 1:10,000.

Techniques: Derivative Assay, Infection, Immunostaining

Journal: bioRxiv

Article Title: Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine

doi: 10.1101/2020.07.01.183236

Figure Lengend Snippet: The replication properties of sMVA derived with FPV HP1.441 (sMVA hp) or TROVAC from two independent sMVA virus reconstitution (sMVA tv1 and sMVA tv2) were compared with wtMVA. A) Viral foci. CEF infected at low multiplicity of infection (MOI) with the reconstituted sMVA virus or wtMVA were immunostained using anti-Vaccinia polyclonal antibody (αVAC). B) Replication kinetics. BHK or CEF cells were infected at 0.02 MOI with sMVA or wtMVA and viral titers of the inoculum and infected cells at 24 and 48 hours post infection were determined on CEF. Mixed-effects model with the Geisser-Greenhouse correction was applied; at 24 and 48 hours post-infection differences between groups were not significant. C) Viral foci size analysis. BHK or CEF cell monolayers were infected at 0.002 MOI with sMVA or wtMVA and areas of viral foci were determined at 24 hours post infection following immunostaining with αVAC antibody. D) Host cell range analysis. Various human cell lines (HEK293, A549, 143b, and HeLa), CEF or BHK cells were infected at 0.01 MOI with sMVA or wtMVA and virus titers were determined at 48 hours post infection on CEF. Dotted lines indicate the calculated virus titer of the inoculum based on 0.01 MOI. Differences between groups in C-D were calculated using one-way ANOVA followed by Tukey’s (C) or Dunnett’s (D) multiple comparison tests. ns = not significant.

Article Snippet: S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological); N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological).

Techniques: Derivative Assay, Infection, Immunostaining

Journal: bioRxiv

Article Title: Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine

doi: 10.1101/2020.07.01.183236

Figure Lengend Snippet: A) Schematic representation of vector construction. S and N antigen sequences (red spheres and green triangles) were inserted into sMVA fragments F2 and F3 by bacterial recombination methods in E. coli . The modified sMVA fragments of F1 and F2 with inserted antigen sequences and the unmodified sMVA fragment F1 were isolated from E. coli and co-transfected into FPV-infected BHK cells to initiate virus reconstitution. B) Schematics of single (sMVA-S, sMVA-N) and double (sMVA-N/S, sMVA-S/N) recombinant sMVA-CoV2 vectors with S and N antigen sequences inserted into commonly used MVA insertion sites (Del2, IGR69/70, Del3). All antigens were expressed via the Vaccinia mH5 promoter. C) Western Blot. BHK cells infected with the single and double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp, sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for antigen expression by Western Blot using anti-S1 and N antibodies (αS1 and αN Ab). Vaccinia B5R protein was verified as infection control. Higher and lower molecular weight bands may represent mature and immature protein species. D) Flow cytometry staining. HeLa cells infected with the vaccine vectors were evaluated by cell surface and intracellular flow staining using anti-S1, S2, and N antibodies (αS1, αS2, and αN Ab). Live cells were used to evaluate cell surface antigen expression. Fixed and permeabilized cells were used to evaluate intracellular antigen expression. Anti-Vaccinia virus antibody (αVAC) was used as staining control to verify MVA protein expression. Cells infected with sMVA or wtMVA or uninfected cells were used as controls for experiments in C and D as indicated.

Article Snippet: S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological); N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological).

Techniques: Plasmid Preparation, Modification, Isolation, Transfection, Infection, Recombinant, Western Blot, Derivative Assay, Expressing, Molecular Weight, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine

doi: 10.1101/2020.07.01.183236

Figure Lengend Snippet: Balb/c mice immunized twice in a three week interval with 5×10 7 PFU of the single and double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific humoral immune responses A-B) Binding antibodies. S, RBD, and N-specific binding antibodies induced by the vaccine vectors were evaluated after the first (A) and second (B) immunization by ELISA. Dashed lines in A and B indicate median binding antibody endpoint titers measured in convalescent human sera (Figure S4). One-way ANOVA with Tukey’s multiple comparison test was used to evaluate differences between binding antibody end-point titers. C) IgG2a/IgG1 isotype ratio. S-, RBD-, and N-specific binding antibodies of the IgG2a and IgG1 isotype were measured after the second immunization using 1:10,000 serum dilution, and absorbance reading was used to calculate IgG2a/IgG1 antibody ratio. One-way ANOVA with Dunnett’s multiple comparison test was used to compare each group mean IgG2a/IgG1 ratio to a ratio of 1 (balanced Th1/Th2 response). D-G) NAb responses. SARS-CoV-2-specific NAb (NT90 titer) induced by the vaccine vectors were measured after the first (D, F) and second (E, G) immunization against SARS-CoV-2 pseudovirus (pv) (D-E) or infectious SARS-CoV-2 virus (F-G) in pooled sera of immunized mice. Shown is the average NT90 measured in duplicate (D-E) or triplicate (F-G) infection. N/A=failed quality control of the samples. Dotted lines indicate lowest antibody dilution included in the analysis. H) SARS-CoV-2/SARS-CoV-2pv correlation analysis. Correlation analysis of NT90 measured in mouse sera after one and two immunizations using infectious SARS-CoV-2 virus and SARS-CoV-2pv. Pearson correlation coefficient (r) was calculated in H. *p<0.05. ns= not significant.

Article Snippet: S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological); N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological).

Techniques: Recombinant, Derivative Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Infection

Journal: bioRxiv

Article Title: Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine

doi: 10.1101/2020.07.01.183236

Figure Lengend Snippet: Balb/c mice immunized twice in a three week interval with 5×10 7 PFU of the single or double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific cellular immune responses. Antigen-specific CD8+ ( A and B ) and CD4+ ( C and D ) T cell responses induced by the vaccine vectors after two immunizations were evaluated by flow cytometry for IFN γ , TNFα, IL-4 and IL-10 secretion following ex vivo antigen stimulation using SARS-CoV-2 S and N-specific peptide libraries. Due to technical issues, 1-3 animals/group were not included in the CD4/TNFα analysis in C and D. One-way ANOVA with Tukey’s multiple comparison test was used to compare differences in % of cytokine-specific T-cells between groups. *p<0.05. ns=not significant.

Article Snippet: S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological); N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological).

Techniques: Recombinant, Derivative Assay, Flow Cytometry, Ex Vivo