Fig 2: 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+ T cells after subtraction of background (DMSO vehicle) (d). Data of (a,b,d) are represented as individual values. p-Values determined by the Wilcoxon matched-pairs signed rank test; p-values are shown.

Fig 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.

Fig 4: Analysis of flow cytometry data(A) Example gating strategy used to analyze flow cytometry data acquired at the end of the assay. Red arrows indicate gating hierarchy in which T cells are identified as CD3+, live, single cells. T cells are further divided into CD4+ and CD8+ subsets. Expression of effector cytokines, namely IL-2, IFN-γ, and TNF-α, is analyzed under CD4+ T cell gate. Numbers on each graph indicate the frequency (%) of gated events.(B) Representative results demonstrating detection of reactive T cells following expansion using the described T cell assay protocol. PBMCs from a healthy donor were expanded following stimulation with the vehicle control DMSO, CEFT, which contains known viral epitopes, and test peptides, a pool of five 15mer overlapping peptides spanning mutated SLC35F5 (AKISFFFALCGFWQICHIKKHFQTHKLL) as follows: 1) AKISFFFALCGFWQI, 2) FFFALCGFWQICHIK, 3) LCGFWQICHIKKHFQ, 4) FWQICHIKKHFQTHK, 5) QICHIKKHFQTHKLL (as reported in Roudko et al., 2020). Expanded cells were re-stimulated as indicated with either DMSO, myelin-oligodendrocyte glycoprotein (MOG), which is a self-protein and used as a negative control, test peptides, CEFT, and PMA/Ionomycin, used as positive controls. Percentage of IFN-γ+ CD8 T cells was measured by flow cytometry. Data were represented as mean ± standard deviation (SD).

Fig 5: IL-1R1 and IL-1R2 differential expression defines an activated Tfr population and shapes Tfh maturation.(A) Experimental design of ex vivo lymphoid cell characterization using flow cytometry with a phenotypic panel (for 8 tonsils, 8 spleens, and 8 mLN) and a functional panel (for a selection of 4 tonsils and 3 spleens). (B) Frequency of each cluster among all CD4+ CXCR5+ and CXCR5– Foxp3+ cells, across tonsils (Ton), Spleens (Spl), and mLN (n = 8 each). (C) Logicle-transformed MFI of IL-2, IL-4, IL-21, and IL-10 across the 5 clusters. (D) Logicle-transformed MFI of IL-1R1 and IL-1R2 (top), and ratio of IL-1Rs’ MFI (bottom) across the 5 clusters within all lymphoid organs (n = 24). (E) Principal component analysis (PCA) displaying the separation of samples according to the five clusters within all lymphoid organs (left) and its associated correlation circle showing the contribution of each marker to the principal components indicated with vectors (right). In B, comparisons between mLN and Spleen have been assessed using a paired Wilcoxon test,and comparisons between mLN and Spleen or Spleen and Tonsils have been assessed using a Mann-Whitney test. In C and D, comparisons have been assessed using Mann-Whitney test. The FDR procedure has been applied for P value adjustments. P values are reported as the following: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.