Journal: Frontiers in Immunology

Article Title: Nanocell COVID-19 vaccine triggers a novel immune response pathway producing high-affinity antibodies which neutralize all variants of concern

doi: 10.3389/fimmu.2022.1038562

Figure Lengend Snippet: S-protein-specific B and T cell response in murine bone marrow derived B cells and splenocytes and Surrogate Viral Neutralization Test (sVNT) on mouse serum from female Balb/c mice dosed on day 1 and boosted on day 21. S-protein specific IgG and IgM production from bone marrow derived B cells isolated from 2 x 10 9 and 3 x 10 9 treated mice (n = 6) at day 28 post-initial dose following ex-vivo stimulation with SARS-CoV-2 S-protein. (A) IgM (pg/ml) concentrations in 2 x 10 9 dose groups and 3 x 10 9 dose groups. (B) IgG (pg/ml) concentrations in 2 x 10 9 dose groups and 3 x 10 9 dose groups. (C) Change in CD69 expression within the CD8 + cytotoxic T cell population following the stimulation of ex vivo splenocytes with wild type SARS-CoV-2 S-protein in the 2 x 10 9 dose groups and (D) 3 x 10 9 dose groups compared to DMSO (negative) stimulated controls. Data presented as mean ± SEM. Asterisks represent significant values (**** p ≤ 0.0001; ** p ≤ 0.01; * p ≤ 0.05) calculated using one way ANOVA on GraphPad Prism v 9.4.0. (E) IFNγ (Th1) and IL-4 (Th2) expression within the CD3 + CD4 + T cell population in SARS-CoV-2 S-protein stimulated ex vivo splenocytes. (F–K) Viral neutralization tests (VNTs) using the cPASS™ SARS-CoV-2 Neutralizing Antibody Assay (FDA approved) for detection in various species was used to assess inhibition of RBD binding to hACE2 receptor. (F) VNTs using the serum of mice immunized with 2 x 10 9 (n = 4, 8) and (G) 3 x 10 9 (n = 8) EDVs against SARS-CoV-2 RBD wild-type. Subsequent VNTs were conducted using the serum of 3 x 10 9 EDV immunized mice against the Alpha (H) , Beta (I) , Gamma (J) and Delta (K) variant RBDs (n = 8). Dotted line represents sVNT 30% cut-off correlating with a positive PRNT90 for 1:10 dilution of sera.

Article Snippet: Assessment of neutralizing antibodies was carried out using the FDA approved “cPASS SARS-CoV-2 Surrogate Virus Neutralization Test RUO Kit” (Cat. #L00847-A, Genscript) ( ).

Techniques: Derivative Assay, Neutralization, Isolation, Ex Vivo, Expressing, Inhibition, Binding Assay, Variant Assay

Journal: Frontiers in Immunology

Article Title: Nanocell COVID-19 vaccine triggers a novel immune response pathway producing high-affinity antibodies which neutralize all variants of concern

doi: 10.3389/fimmu.2022.1038562

Figure Lengend Snippet: EDV-COVID-αGC Clinical trial: Data from the 6 volunteers. (Ai) SVNT analysis of volunteer serum on day 1, 21, 28 post-initial injection and a booster on day 21 against wild type, Delta and Omicron B.1.1.529 variants of the SARS-CoV-2 RBD. (Aii) SVNT analysis of volunteer serum against Omicron B.1.1.529 and Omicron BA. 2 and BA. 4/5 variants of the SARS-CoV-2 RBD, at 3 months post 2 doses of EDV-COVID-αGC only, 2 doses of BNT262b2 only, and EDV-COVID-αGC as a booster. Dotted line represents sVNT 30% cut-off correlating to a positive PRNT90 for 1:10 dilution of sera. (B, C) Serum IFNγ and IFNα levels respectively on day 1, 21 and 28 after initial injection and a day 21 booster. (C) Serum IFNα levels (pg/ml) on day 1, 21 and 28 post-initial injection. (D) CD4 + central memory T cells (TCM) (CD45RA - CD27 + CCR7 + CD3 + CD4 + ) analysis on day 1 and day 28. (E) CD8 + central memory T cells (CD45RA - CD27 + CCR7 + CD3 + CD8 + ) analysis on day 1 and day 28. (F) Ex vivo PBMC production of IFNγ following SARS-CoV-2 S-protein stimulation on day 1 and day 28. (G) CD69 expression in T cells (CD45 + CD3 + CD69 + ) in ex vivo PBMCs following SARS-CoV-2 S-protein stimulation on day 1 and day 28. (H) Amount of S-protein specific CD19 + B cells in PBMCs on day 1, 28, 2 months and 3 months following initial injection. (I) Amount of S-protein specific CD19 + CD27 + memory B cells in PBMCs on day 1, 28, 2 months and 3 months following initial injection. (J) Amount of IgM + CD19 + CD27 + memory B cells in PBMCs on day 1, 28, 2 months and 3 months following initial injection. (K) Amount of IgG + CD19 + CD27 + memory B cells in PBMCs on day 1, 28, 2 months and 3 months following initial injection. Data presented as mean ± SEM, ns, not significant. Asterisks represent significant values (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05) between stated groups and calculated using paired t test on GraphPad Prism v 9.4.0.

Article Snippet: Assessment of neutralizing antibodies was carried out using the FDA approved “cPASS SARS-CoV-2 Surrogate Virus Neutralization Test RUO Kit” (Cat. #L00847-A, Genscript) ( ).

Techniques: Injection, Ex Vivo, Expressing

Journal: iScience

Article Title: Non-propagative human parainfluenza virus type 2 nasal vaccine robustly protects the upper and lower airways against SARS-CoV-2

doi: 10.1016/j.isci.2021.103379

Figure Lengend Snippet: Robust protective effects of BC-PIV/S-2PM vaccine against SARS-CoV-2 infection in hamsters (A) Timeline and protocol of the vaccination and SARS-CoV-2 challenge test. Four hamsters were used for each group. (B) Eminent neutralizing activity of the sera from intranasally vaccinated hamsters after one-shot vaccination was revealed using the SARS-CoV-2 surrogate virus neutralization test. Serum was collected 9 weeks after priming in the prime-boost group and 10 weeks after vaccination in the one-shot group and control group. Hamster sera were analyzed after 15x dilution (final dilution was 30x). PC Ab, positive control serum, serum from a mouse repeatedly immunized with the recombinant RBD protein, which is included in the cPass™ Technology kit (SARS-CoV-2 surrogate virus neutralization test kit). The mouse serum was analyzed after 10x dilution (final dilution was 20x). N Ab, commercially available recombinant human anti-SARS-CoV-2 neutralizing antibody (SAD-S35; ACROBiosystems) as a reference, derived from a patient infected with SARS-CoV-2. The neutralizing antibody was used at the final concentrations indicated. According to the manufacturer, the neutralizing antibody inhibits the interaction between SARS-CoV-2 RBD and hACE2 with an IC 50 of 1.47 μg/mL when used with the SARS-CoV-2 inhibitor screening kit (ACROBiosystems). (C) A plaque assay at 3-days post-infection of SARS-CoV-2. Infectious viruses in the lungs and nasal turbinates of the hamsters were measured on VeroE6/TMPRSS2 cells (n = 4 hamsters in each group). This plaque assay detects even from one infectious virus. The lower limit of detection (LOD) in this plaque assay depends on the quantity of the tissue used for the analysis. For example, if 1 g of tissue is used, the lower LOD is 0.0 (log 10 (1/1)). N.D., not detected (not plotted in the figure). The Kruskal–Wallis test with the Steel-Dwass post hoc test was used. ∗p < 0.05.

Article Snippet: Next, inhibition assays for the interaction between SARS-CoV-2 RBD and hACE2 by ELISAs as SARS-CoV-2 surrogate neutralization (cPass kit) tests (GenScript) were carried out.

Techniques: Infection, Activity Assay, Virus, Neutralization, Control, Positive Control, Recombinant, Derivative Assay, Plaque Assay