Journal: NPJ Vaccines

Article Title: High protection and transmission-blocking immunity elicited by single-cycle SARS-CoV-2 vaccine in hamsters

doi: 10.1038/s41541-024-00992-z

Figure Lengend Snippet: a Schematic illustrating the SARS-COV-2 genomic landscape and the deletions/substitutions in ΔE G /ΔE G 68, main structural and accessory proteins indicated. Four overlapping fragments covering the whole SARS-CoV-2 genome were amplified by PCR (Fragments A-D, see also Supplementary Fig. ). b Complementation efficiency of Vero-E2T cells, analyzed by FFU (focus forming units) quantification after infection with ΔE G 3* (ΔE G with an additional stop codon in ORF3a) at different multiplicities of infection (MOI) or medium-only control (ctrl) three and six days post-infection ( n = 2 individual cultures), for corresponding genome copies, see Supplementary Fig. . c Passaging of 1:10 and 1:100 (after p2) dilutions of cell-free supernatant (Input = Passage 0) of wild-type SARS-CoV-2 (Muc-1, B.1), ΔE G 3* and ΔE G 68 on non-complementing Vero E6 cells (initial infection MOI = 1). Data from one representative experiment are shown; analysis was performed in duplicates. d Transmission electron microscopy analysis of recombinant wild-type SARS-CoV-2 (rCoV2) or vaccine candidates ΔE G and ΔE G 68 showing the presence of the characteristic spike protein (indicated with arrows). e Immunoblot analysis of viral protein production in Vero E6-TMPRSS2 cells infected for 24 h with rCoV2, E**fs, ΔE G 3*, ΔE G 68 or medium only (ctrl), probed with anti-NSP2, anti-N, anti-S, anti-ORF3a (full-length [fl] and truncated [tr] forms indicated with arrows), anti-ORF6, anti-ORF7a, anti-ORF8, and anti-beta-actin (β-ACT) antibodies. f Detection of N and S (magenta), F-actin (green), nuclei (blue) and ORF6 or ORF8 in Vero E6-TMPRSS2 cells infected with rCoV2, E**fs, ΔE G 3* or ΔE G 68. Scale bar is 100 nm in ( d ), 50 µm and 20 µm in ( f ) (overview and ROI images, respectively).

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-β-actin (Cell Signaling Technology; 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-SARS-CoV-2 nsp2 (GeneTex; GTX135717; RRID: AB_2909866; LOT# B318853), rabbit polyclonal anti-SARS-CoV Nucleocapsid protein (Rockland; 200-401-A50; RRID:AB_828403), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from S. Reiche ), sheep polyclonal anti-SARS-CoV-2 ORF3a , rat monoclonal anti-SARS-CoV-2 ORF6 (8B10, gift from Y. Miyamoto ), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; NBP3-07972; LOT# 25966-2102), mouse monoclonal anti-SARS-CoV-2 Spike protein (4B5C1, gift from S. Reiche).

Techniques: Amplification, Infection, Control, Passaging, Transmission Assay, Electron Microscopy, Recombinant, Western Blot

Journal: NPJ Vaccines

Article Title: High protection and transmission-blocking immunity elicited by single-cycle SARS-CoV-2 vaccine in hamsters

doi: 10.1038/s41541-024-00992-z

Figure Lengend Snippet: a – c Modulation after transfection: Flow cytometry staining of THP-1 cells for HLA-A/B/C, CD80, CD275, and HLA-DR surface expression 48 h after transfection with expression plasmids for ORF6 ( a ), ORF8 ( b ), or Envelope ( c ) proteins, compared with control transfection. d – i Modulation after infection: d A549-ACE2-TMPRSS2 cells were infected with recombinant wild-type (rCoV2), E**fs, ΔE G 68, or XBB.1.5 SARS-CoV-2 virus (MOI = 0.1) for 24 h and stained for HLA-A/B/C, CD44 and CD275. e Median fluorescence intensity (MFI) of HLA-A/B/C and CD275. The same infection was conducted on HEK293T-ACE2 and their respective supernatant was then applied on THP-1 for 48 h before surface staining and analysis. f Histogram showing the expression of CD44, HLA-A/B/C, CD80, CD275, and HLA-DR on THP-1 after 48 h. g Median fluorescence intensity of CD44, HLA-A/B/C, CD80, and CD275 markers on THP-1 after 48 h incubation. h Comparison of wild-type or ΔE G 68 conditions for their expression of CD80 and HLA-A/B/C. The frequency of cells inside the gate in ( h ) is shown in ( i ). Median is shown for ( e ) and ( g ), mean and S.D. for ( i ). The gating strategy is shown in Supplementary Fig. .

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-β-actin (Cell Signaling Technology; 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-SARS-CoV-2 nsp2 (GeneTex; GTX135717; RRID: AB_2909866; LOT# B318853), rabbit polyclonal anti-SARS-CoV Nucleocapsid protein (Rockland; 200-401-A50; RRID:AB_828403), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from S. Reiche ), sheep polyclonal anti-SARS-CoV-2 ORF3a , rat monoclonal anti-SARS-CoV-2 ORF6 (8B10, gift from Y. Miyamoto ), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; NBP3-07972; LOT# 25966-2102), mouse monoclonal anti-SARS-CoV-2 Spike protein (4B5C1, gift from S. Reiche).

Techniques: Transfection, Flow Cytometry, Staining, Expressing, Control, Infection, Recombinant, Virus, Fluorescence, Incubation, Comparison

Journal: Acta Biomaterialia

Article Title: Multipolymer microsphere delivery of SARS-CoV-2 antigens

doi: 10.1016/j.actbio.2022.12.043

Figure Lengend Snippet: Proposed protective antiviral immune responses from MS-based SARS-CoV-2 antigen delivery. Following intramuscular injection of the MS (porous spheres) loaded with whole, inactive SARS-CoV-2 (yellow spheres) in different layers, antigen release and delivery to antigen presenting cells (APC) is anticipated to generate humoral (B-cell generation and antibody production by plasma cells) and cellular (T-cell activation, cytokine release by CD4 and CD8 T-cell subsets) responses. This leads to protective antiviral immunity. The antibodies generated by the plasma cells and the activated T-cell subsets, both aid in clearance of infection.

Article Snippet: For these assays rat, SARS-CoV-2 IgG to Spike RBD Protein (SKU: KBVH015-22, Eagle Biosciences, Amherst NH, USA) and Rat Anti-SARS-CoV-2 IgG to N (SKU: KBVH015-23, Eagle Biosciences) were applied according to the manufacturer's instructions.

Techniques: Injection, Activation Assay, Generated, Infection

Journal: Acta Biomaterialia

Article Title: Multipolymer microsphere delivery of SARS-CoV-2 antigens

doi: 10.1016/j.actbio.2022.12.043

Figure Lengend Snippet: Characterization of an “putative” inactive SARS-CoV-2 vaccine. (a) Topographic images of inactive SARS-CoV-2 was acquired by adsorbing the virions on APS modified mica and subsequent AFM image analysis. Topographical maximal (b) diameter was 25.9 ± 3.7 nm and (c) central height was 106 ± 26.5 nm (n = 89). (d) Representative negative stain TEM image of an inactive SARS-CoV-2 virion taken under magnification of 39000x, and (e) western blot analysis of SARS-CoV-2 Spike S1 (90 kDA) and Nucleocapsid (50 kDA) proteins of inactive SARS-CoV-2.

Article Snippet: For these assays rat, SARS-CoV-2 IgG to Spike RBD Protein (SKU: KBVH015-22, Eagle Biosciences, Amherst NH, USA) and Rat Anti-SARS-CoV-2 IgG to N (SKU: KBVH015-23, Eagle Biosciences) were applied according to the manufacturer's instructions.

Techniques: Modification, Staining, Western Blot

Journal: Acta Biomaterialia

Article Title: Multipolymer microsphere delivery of SARS-CoV-2 antigens

doi: 10.1016/j.actbio.2022.12.043

Figure Lengend Snippet: Morphological depiction of the multipolymer MS. SEM images show external morphology of (a) non-porous MS, and (b) porous MS. (c) Cross section of porous MS showing the polymer distribution into distinct ‘patches’ as indicated by yellow and red arrows. (d) external morphology of porous MS with antigen (whole inactive SARS-CoV-2 virus). Scale bar: depicted at the lower bar of respective images.

Article Snippet: For these assays rat, SARS-CoV-2 IgG to Spike RBD Protein (SKU: KBVH015-22, Eagle Biosciences, Amherst NH, USA) and Rat Anti-SARS-CoV-2 IgG to N (SKU: KBVH015-23, Eagle Biosciences) were applied according to the manufacturer's instructions.

Techniques:

Journal: Acta Biomaterialia

Article Title: Multipolymer microsphere delivery of SARS-CoV-2 antigens

doi: 10.1016/j.actbio.2022.12.043

Figure Lengend Snippet: Microscopic analysis of microsphere (MS)-macrophage interactions. Monocyte-derived macrophages (MDMs) (2 × 10 −6 ) were treated with 3 mg of multipolymer MS and incubated for 7 days. Light microscopy images, taken with 20x objective lenses, of (a) control MDMs, and (b) treated MDMs showed cells clustering around the multipolymer MS (blue arrows). Representative TEM images of (c) control and (d) MS SARS-CoV-2-treated macrophages showed vacant spherical compartments inside treated cells, indicating the regions where the MS resided upon intake. Scale bar: 2 μm. SEM images of (e) control MDMs, and (f-h) MS SARS-CoV-2-treated MDMs showed treated cell clusters around and the engulfed in MS (yellow arrows). (i) Confocal overlaid (MaxIP) image from 30 optical scan (Z step 1 μm) showing detected inactive SARS-CoV-2 viral particles stained with DAPI (excitation/emission: 405 nm/425 nm; panel i-1) and the merged images with MS autofluorescence (excitation/emission, 561 nm/590 nm, panel i-2). The panel i-3 is the portion of panel i-2 (the blue box) at higher magnification showing the viral particles (green/yellow) within the MS surface porous structures. Scale bar: 10 μm.

Article Snippet: For these assays rat, SARS-CoV-2 IgG to Spike RBD Protein (SKU: KBVH015-22, Eagle Biosciences, Amherst NH, USA) and Rat Anti-SARS-CoV-2 IgG to N (SKU: KBVH015-23, Eagle Biosciences) were applied according to the manufacturer's instructions.

Techniques: Derivative Assay, Incubation, Light Microscopy, Staining

Journal: Acta Biomaterialia

Article Title: Multipolymer microsphere delivery of SARS-CoV-2 antigens

doi: 10.1016/j.actbio.2022.12.043

Figure Lengend Snippet: Humoral immune response to MS-Ag administration in SD rats. (a) Experimental outline for the blood and organ collection, 7- and 28-days post-IM injection of SD rats with either saline, empty MS, or MS-Ag. Splenocytes were subjected to flow cytometry. Blood was analyzed for T-cell subsets and toxicity profiling, (b) Enzyme linked immunosorbent assay (ELISA) for (b) anti-SARS CoV2 S-RBD IgG and (c) Nucleoprotein (N) IgG response showed significant IgG production at day 28 for anti-SARS-CoV-2 S- RBD IgG and N-IgG. Statistical analysis was done by Two-way ANOVA and Tukey's post- hoc tests for multiple comparisons, (****P < 0.0001).

Article Snippet: For these assays rat, SARS-CoV-2 IgG to Spike RBD Protein (SKU: KBVH015-22, Eagle Biosciences, Amherst NH, USA) and Rat Anti-SARS-CoV-2 IgG to N (SKU: KBVH015-23, Eagle Biosciences) were applied according to the manufacturer's instructions.

Techniques: Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Acta Biomaterialia

Article Title: Multipolymer microsphere delivery of SARS-CoV-2 antigens

doi: 10.1016/j.actbio.2022.12.043

Figure Lengend Snippet: Cellular immune response to MS-Ag administration in SD rats. SD rats (average body weight = 270 g) were each injected with a single intramuscular dose of 6 μg/g of body weight of chemically inactivated SARS-CoV-2 loaded MS (MS-Ag). From SD rats injected with MS-Ag, empty MS or saline, T-cell subsets of splenocytes were analyzed at 7 and 28 dpi by flow cytometry. (a-b) Percentage population of activated T-cells (CD4+CD69+ and CD8+CD69+), 28 dpi. (c-d) Percentage population of TCM - cells (CD4+CD62L+CD44+ and CD8+CD62L+CD44+), 28 dpi. Intracellular cytokine staining and flow cytometric analysis of CD4+ T-cell subset expressing (e) IFNγ at 7 dpi, (f) IFNγ at 28 dpi, (g) IL4 at 28 dpi, and (h) IL17 at 28 dpi. Statistical analysis was done by One-way ANOVA followed by Newman/Keul's post-hoc analysis for multiple comparisons, (*P < 0.05; **P < 0.01; ***P < 0.001). Abbreviations: MS, multipolymer microsphere; Ag, antigen; IFNγ, Interferon-gamma; IL, Interleukin; %, percentage of the parent population.

Article Snippet: For these assays rat, SARS-CoV-2 IgG to Spike RBD Protein (SKU: KBVH015-22, Eagle Biosciences, Amherst NH, USA) and Rat Anti-SARS-CoV-2 IgG to N (SKU: KBVH015-23, Eagle Biosciences) were applied according to the manufacturer's instructions.

Techniques: Injection, Tandem Mass Spectroscopy, Flow Cytometry, Staining, Expressing