Journal: Vaccine

Article Title: RelCoVax®, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice

doi: 10.1016/j.vaccine.2022.06.026

Figure Lengend Snippet: Expression of RBD and N proteins. (A) Schematic representations of S and N proteins of SARS-CoV-2. Receptor binding domain or RBD of S protein as expressed extends from Arg 319 to Phe 541 and includes the receptor binding motif (RBM) . M is the transmembrane domain and IC is the intracellular domain of S protein. For N protein, full length protein is expressed. (B) Representative SDS-PAGE and western blot images for purified RBD and N proteins. L is the protein molecular weight ladder.

Article Snippet: The supernatants of individual clones were screened for expression of RBD qualitatively by ELISA using the GENLISA™ Human SARS-CoV-2 (COVID-19) spike protein S1 antigen ELISA kit. (Krishgen Biosystems, KBVH015-10).

Techniques: Expressing, Binding Assay, SDS Page, Western Blot, Purification, Molecular Weight

Journal: Vaccine

Article Title: RelCoVax®, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice

doi: 10.1016/j.vaccine.2022.06.026

Figure Lengend Snippet: Immunogenicity of two different formulations of RelCoVax®. (A) Dosing schedule for mice (N = 12 per group) receiving two different formulations (4 µg per dose and 10 µg per dose) of vaccine candidate with no antigen placebo group as control. Two doses for each formulation were administered 14 days apart (day 1 and day 15) and serum samples were collected on day 28 (13 days after dose 2). The 4 µg dose included 200 µg of alum and 80 µg of CpG oligonucleotide as adjuvants and 2 mg of 2-PE as a preservative while the 10 µg dose included 500 µg of alum and 200 µg of CpG oligonucleotide as adjuvants and 5 mg of 2-PE as a preservative. The placebo contained no antigen but included 500 µg of alum, 200 µg of CpG oligonucleotide and 5 mg of 2-PE per dose. Each mouse was administered a total volume of 200 µL per dose. (B) Anti-RBD IgG end point titers for each group and (C) anti-N IgG endpoint titers for each group. The bars and the numbers indicate the GMT for each group and error bars represent the 95% CI for that group. The dashed lines indicate the limit of detection. (D) SARS-CoV-2 neutralizing endpoint titers (PRNT 50 ) for 3 serum pools (4 mice per pool). The serum pools were prepared from the same serum samples tested in B and C. The bars and the numbers indicate the GMT for each group and error bars represent the 95% CI for that group. LLOD – lower limit of dilution, ULOD – upper limit of dilution.

Article Snippet: The supernatants of individual clones were screened for expression of RBD qualitatively by ELISA using the GENLISA™ Human SARS-CoV-2 (COVID-19) spike protein S1 antigen ELISA kit. (Krishgen Biosystems, KBVH015-10).

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: 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