Journal: Vaccines

Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates.

doi: 10.3390/vaccines9040307

Figure Lengend Snippet: Figure 1. Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S∆TM) or full-length SARS-CoV-2 S protein (S) (a). BALB/c mice (n = 4–10/group) were immunized at weeks 0 and 2 with pGX27-S∆TM, pGX27-S, or pGX27 (empty control vector) as described in the Methods. Sera were collected at 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies (b). All data are represented as individual values. ** p < 0.01 as determined by the Mann–Whitney test.

Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003, West Grove, PA, USA), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205, West Grove, PA, USA), IgG2a (Jackson ImmunoResearch Laboratories 115-035-206, West Grove, PA, USA), or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207, West Grove, PA, USA) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P, Montgomery, AL, USA) for the NHP sera for 1 h at 37 ◦C.

Techniques: Immunopeptidomics, Vaccines, Expressing, Control, Plasmid Preparation, MANN-WHITNEY

Journal: Vaccines

Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates.

doi: 10.3390/vaccines9040307

Figure Lengend Snippet: Figure 2. GX-19 elicits robust binding and neutralizing antibody responses in mice. BALB/c mice (n = 4–7/group) were immunized at weeks 0 and 2 with indicated doses of GX-19 or pGX27 as described in the Methods (a–c). Sera were collected at 2 weeks post-prime (blue) and 2 weeks post- boost (red) and assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (a), and for post-boost sera, neutralizing antibodies against SARS-CoV-2 live virus (c). Bronchoalveolar lavages (BALs) were collected at 2 weeks post-boost and assayed for SARS-CoV-2 S-specific IgG antibodies by ELISA (b). Data representative of two independent experiments. All data are represented as individual values. * p < 0.05, ** p < 0.01 as determined by the Mann–Whitney test.

Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003, West Grove, PA, USA), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205, West Grove, PA, USA), IgG2a (Jackson ImmunoResearch Laboratories 115-035-206, West Grove, PA, USA), or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207, West Grove, PA, USA) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P, Montgomery, AL, USA) for the NHP sera for 1 h at 37 ◦C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Virus, MANN-WHITNEY

Journal: Vaccines

Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates.

doi: 10.3390/vaccines9040307

Figure Lengend Snippet: Figure 3. Immunization with GX-19 elicits Th1-biased T cell responses in mice. BALB/c mice (n = 3–7/group) were immunized at weeks 0 and 2 with indicated doses of GX-19 or pGX27 (empty control vector) as described in the Methods (a–c). Sera were collected at 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG1 and IgG2a/b. Endpoint titers (a), and endpoint tier ratios of IgG2a/b to IgG1 (b) were calculated. At 2 weeks post-boost, mouse splenocytes were isolated and re-stimulated with peptide pools spanning the SARS-CoV-2 S protein ex vivo. Indicated cytokines in the supernatants of culture were quantified using a Th1/Th2 cytometric bead array kit. Mean value of the medium alone background (mean ± s.d., pg mL−1) was 19.17 ± 8.61 for IFN-γ, 57.12 ± 6.53 for TNF-α, 33.10 ± 6.72 for IL-2, 7.83 ± 0.45 for IL-4, and 4.66 ± 0.13 for IL-5 (d). T cell responses were measured by IFN-γ ELISPOT in splenocytes stimulated with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 106 splenocytes (c). Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2. Shown are the frequency of S-specific CD4+ or CD8+ T cells after subtraction of background (DMSO vehicle, Sigma-Aldrich, St. Louis, MO, USA) (e). Data representative of two independent experiments. All data are represented as individual values. * p < 0.05, ** p < 0.01 as determined by the Mann–Whitney test.

Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003, West Grove, PA, USA), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205, West Grove, PA, USA), IgG2a (Jackson ImmunoResearch Laboratories 115-035-206, West Grove, PA, USA), or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207, West Grove, PA, USA) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P, Montgomery, AL, USA) for the NHP sera for 1 h at 37 ◦C.

Techniques: Control, Plasmid Preparation, Isolation, Ex Vivo, Enzyme-linked Immunospot, Staining, MANN-WHITNEY

Journal: Vaccines

Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates.

doi: 10.3390/vaccines9040307

Figure Lengend Snippet: Figure 4. Antibody and T cell responses after GX-19 vaccination in macaques. Macaques (n = 3) were immunized with 3 mg of GX-19 as described in the Methods. Serum and PBMCs (peripheral blood mononuclear cells) were collected before (week 0), during (week 4 and 5.5), and after (week 8) vaccination and were assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (a) and neutralizing antibodies against SARS-CoV-2 live virus (b). Data represent mean SEM of individual macaques (GX-19 #1, GX-19 #2, GX-19 #3), and dashed line indicates the assay limits of detection. The number of SARS-CoV-2 S-specific IFN-γ-secreting cells in PBMCs was determined by IFN-γ ELISPOT assay after stimulation with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 106 PBMCS in triplicate wells (c). The frequency of S-specific CD4+ or CD8+ T cells producing IFN-γ, TNF-α, or IL-2 was determined by intracellular cytokine staining assays stimulated with SARS-CoV-2 S peptide pools. Shown are the frequency of S-specific CD4+ or CD8+

Article Snippet: Following incubation, plates were washed 5 times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories 115-035-003, West Grove, PA, USA), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205, West Grove, PA, USA), IgG2a (Jackson ImmunoResearch Laboratories 115-035-206, West Grove, PA, USA), or IgG2b (Jackson ImmunoResearch Laboratories 115-035-207, West Grove, PA, USA) for the mouse sera/BAL or anti-monkey IgG (Bethyl Laborabories A140-102P, Montgomery, AL, USA) for the NHP sera for 1 h at 37 ◦C.

Techniques: Enzyme-linked Immunosorbent Assay, Virus, Enzyme-linked Immunospot, Staining