Fig 2: Induction of cellular and humoral immune responses by PolyPEPI-SCoV-2 vaccine in mouse models. Animals received PolyPEPI-SCoV-2 or Vehicle subcutaneously at days 0, 14. IFN-γ-producing T cell responses elicited by PolyPEPI-SCoV-2 expressed as fold change in BALB/c (A) and Hu-mouse (B) models compared to the respective Vehicle cohorts; diversity of vaccine-induced T cell responses after two doses at day 28 in BALB/c (C) and Hu-mouse (D) models by ex vivo ELISpot. Test conditions: stimulation with 30-mer S-pool (three S-peptides), N-pool (four N-peptides), M-peptide, E-peptide, or 9-mer pools (s-pool, n-pool, e1, m1 peptides). For Fold change calculation the average dSFU values of the 30-mer and 9-mer stimulation conditions are pooled. (E,F) PolyPEPI-SCoV-2 induced Th1 response and no significant Th2 cytokine induction shown as average of vaccine-specific CD4+ and CD8+ T cells producing IL-2, TNF-α, IFN-γ, IL-5 or IL-10 in BALB/c (E) and Hu-mouse (F), using Intracellular cytokine staining. Mean +/-SEM are shown. 2 × 105 cells were analyzed, gated for CD45+CD3+CD4+/CD8+. Average percentage was obtained by pooling the background-subtracted values of the 30-mer stimulation conditions for each cytokine for CD4+ and CD8+ splenocytes. IgG production measured from the plasma of BALB/c (G) and Hu-mice (H). N = 6 animals at each time point, *p < 0.05, **p < 0.001 (Mann-Whitney U).

Fig 3: Correlation between multiple autologous HLA allele-binding epitopes and PolyPEPI-SCoV-2-specific IFN-γ-producing CD8+ T cell responses in COVID-19 convalescent individuals. (A) Correlation between multiple autologous HLA allele-binding epitopes and magnitude of T cell responses. Rs, Spearman coefficient (confirmed by Pearson correlation analysis, too) (B) Magnitude of CD8+ T cell responses detected for PEPIs (binding ≥ 3 autologous HLA class I alleles) and for non-PEPIs (binding <3 autologous HLA class I alleles) by enriched FluoroSpot assay, (p = 0.008, t-test). Median and individual data for each subject are presented, n = 15 (C) Variable dependency analysis using 2 × 2 contingency table and Fisher Exact test. (D) Confirmation of Personal Epitopes (PEPIs) by IFN-γ producing CD8+ T cells for each subject [Positive Predictive Value (PPV) = True positive/Total predicted = 37/44 (84%)]. dSFU, delta spot forming units calculated as non-stimulated background corrected spot counts per 106 PBMC; PBMC, peripheral blood mononuclear cells.

Fig 4: Changes in PMN-MDSC, CD8 and SMA cell compartments in organoids directly derived from mouse tumors in response to experimental treatments. (A) Light micrographs of cultured organoids and (B) H&E staining of embedded organoids that were derived from mouse tumors in response to experimental treatments. Flow cytometric contour plots demonstrating the changes in (C) PMN-MDSC, (D) CD8 and SMA cell populations in organoids derived from mouse tumors in response to experimental treatments. Quantification (% cell populations) is shown in (E). * p < 0.05 compared to untreated; n = 10 mice per group.

Fig 5: Decreased tumor size in mice treated with combinatorial cabozantinib and anti-PD-1 inhibitor. Changes in (A) tumor sizes, (B) EpCAM/PD-L1+ve organoid death, (C) MDSC death/depletion, and (D) CTL proliferation in response to chemotherapy and PD-1Inh with or without cabozantinib treatments. Digital spatial analysis demonstrating (E) representative ROIs, and (F) immune marker protein expression in mouse experimental groups. Protein expression of (G) CD8, (H) MDSCs, and (I) aSMA quantitative RT-PCR, using RNA isolated from mouse tumors, for the expression of (J) CD8, (K) granzyme, and (L) fibronectin. * p < 0.05 compared to untreated; n = 10–20 mice per group.