Fig 2: Characterization of the immune response elicited by the RBD vaccine candidate(A) Antibody linear epitopes mapped onto the structure of the FL spike protein. Each domain of the FL protein (NTD, RBD, furin cleavage, FP-fusion peptide, and S2) is outlined with a different color (left), and the linear epitopes are shown as gold spheres within the spike domains used for immunization (center and right). (B) Anti-RBD IgG levels measured in bronchoalveolar lavage (BAL) of FL- and RBD-vaccinated BALB/c mice at day 38. (C) Neutralizing antibody titers in sera collected from RBD- or FL-vaccinated BALB/c mice (n = 5) at day 14 (prime) and day 38 (boost), measured through a neutralization assay with infectious SARS-CoV-2-and Vero cells. (D) Neutralizing antibody titers in sera collected at day 38 from C57BL/6 mice (n = 5) vaccinated with increasing doses of RBD vaccine (5–10–20 μg) in a prime-boost regimen. (E) Serum anti-RBD IgG levels measured over time in sera of RBD-vaccinated C57BL/6 mice (prime-boost regimen, n = 5) up to 6 months starting from prime. (F) T cell immune response (IFN-γ+ and TNF-α+) in CD8+ and CD4+ cells measured by intracellular staining of splenocytes collected from FL- and RBD-vaccinated BALB/c mice (n = 5) at day 38 and restimulated with pool S1 peptides. (G) IFN-γ-producing T cells measured by ELISpot assay performed on BALs collected from BALB/c mice (n = 5) vaccinated with FL and RBD, intranasally challenged with 20 μg RBD protein at day 42, and culled the day after. ∗p value < 0.05.

Fig 3: The %CD4+ and CD8+ cells in TME. (A) Delivery of αCTLA-4 monotherapy and in combination with Melan-A VLP changes the distribution of CD4+ T-cells in the tumor microenvironment. No treatment (▽, black, n = 3); αCTLA-4 i.v. (△, red, n = 3); αCTLA-4 i.d. (●, red, n = 5); αCTLA-4 i.v., Melan-A VLP (▲, blue, n = 8); and αCTLA-4 i.d., Melan-A VLP (■, green, n = 7). (B) Lymphatic delivery of αPD-1 in combination with Melan-A VLP changes the distribution of CD4+ and CD8+ T-cells in the tumor microenvironment. No treatment (▽, black, n = 3); αPD-1 i.v. (■, grey, n = 4); αPD-1 i.d. (▼, black, n = 6); αPD-1 i.v., Melan-A VLP (♦, blue, n = 1); and αPD-1 i.d., Melan-A VLP (♦, green, n = 7). (C) Effect of Melan-A VLP and Gag-VLP in the percentage of CD4+ and CD8+ T-cells in the tumor microenvironment. No treatment (▽, black, n = 3); IgG2b i.d. (X, grey, n = 4), IgG2b i.d., Melan-A VLP (✱, n = 4); αCTLA-4 i.d., Melan-A VLP (■, green, n = 7); and αCTLA-4 i.d., Gag-VLP (○, n = 5). Statistical significance by unpaired, two-tailed Student’s t test (p < 0.05) is denoted by * and unpaired, one-tailed Student’s t test (p < 0.05) is denoted by #. Error bars indicate SEM. i.d., intradermal; i.v. intravenous.

Fig 4: Impact of anti-tumor immunity on tumor microenvironment (TME) and survival. (A) Changes in Melan-A TNFα and IFNγ functionality of CD8+ and CD4+ cells in PBMCs. Isotype IgG2b, Melan-A VLP (open bar, n = 4); αCTLA-4 i.d., Melan-A VLP (blue, n = 9); αCTLA-4 i.v., αPD-1 i.v., Melan-A VLP (black, n = 7); αCTLA-4 i.d., αPD-1 i.v., Melan-A VLP (purple, n = 10); and αCTLA-4 i.d., αPD-1 i.d., Melan-A VLP (orange n = 10). *, # Denotes statistical significance by unpaired, two-tailed and one-tailed Student’s t test (p < 0.05), respectively. Error bars indicate SEM. (B) Multiplex immunofluorescence (mIF) and H&E of tumor tissue harvested at 17 days post implant (d.p.i.) in (i) untreated animals and animals treated with (ii) αCTLA-4 i.v., αPD-1 i.v., Melan-A VLP; (iii) αCTLA-4 i.d., αPD-1 i.d., Melan-A VLP; and (iv) αCTLA-4 i.d., αPD-1 i.v., Melan-A VLP. Scalebars in miF and H&E panels correspond to 200 μm. (C) Multiplex immunofluorescence of PD-L1+ cells (CK+, F40/80+ and CD45+CD3E+) in TME at 17 d.p.i. for animals treated with IgG2b i.d. (X, grey, n = 5); αCTLA-4 i.v., Melan-A VLP (▲, blue, n = 5); αCTLA-4 i.d., Melan-A VLP (■, green, n = 5); αCTLA-4 i.d., Gag-VLP (○, n = 5); αCTLA-4 i.v., αPD-1 i.v., Melan-A VLP (black, n = 5); αCTLA-4 i.d., αPD-1 i.d., Melan-A VLP (⬣, orange n = 6); and αCTLA-4 i.d., αPD-1 i.v., Melan-A VLP (◆, purple n = 5). * Denotes statistical significance by unpaired, two-tailed Student’s t test (p < 0.05). Error bars indicate SEM. (D) Kaplan-Meier survival curve shows lymphatic delivery of combinational therapy with Melan-A VLP significantly improves survival up to 30 d.p.i. * Indicates significant difference between αCTLA-4 i.d., αPD-1 i.d., Melan-A VLP, and no treatment. i.d., intradermal; i.v., intravenous. Scale bar: 200 μm.

Fig 5: Genetic Deletion of HIF-1α, but Not HIF-2α, in T Lymphocytes Leads to Accelerated Tumor Growth and Impaired CD8+ T Cell Tumor Infiltration(A and B) Growth curves of subcutaneous LLC and B16-F10 tumors (0.5 × 106 cells/mouse) in HIF-1α mutant mice (maroon) and littermate controls (black) (A, n = 10–13 per group) or HIF-2α mutant mice (green) versus littermate controls (black) (B, n = 6–17 per group). Statistical analysis was performed by two-way ANOVA with Sidak correction for multiple comparisons; tumor volumes are shown (mean values ± SEM).(C) Tumor weights at the endpoint of the experiment (error bars represent SD).(D) Representative dot plots showing CD8+ and CD4+ T cell tumor infiltration in mutant mice and littermate controls by flow cytometry (left) and CD8+ to CD4+ ratio in TILs (right, n = 6, error bars represent SD).(E) Expression of costimulatory receptors and checkpoint inhibitors on CD8+ T cells infiltrating LLC isografts 13 days after subcutaneous inoculation (n = 6, error bars represent SD).(F) Flow-cytometry analyses on CD4+ TILs: IFNγ+ cells, percent FOXP3+ cells, and IFNγ+/FOXP3+ CD4+ T cell ratio (n = 6, error bars represent SD).(G) HIF-1α-deficient (maroon) and control (black) CD8+ T cells were subjected to 1% O2 and cultured in a Boyden chamber on top of a confluent layer of primary endothelial cells. Twelve hours later T cells in the bottom well were counted (n = 4, error bars represent SD).(H) Immunofluorescence images from LLC tumors for the indicated genotypes (blue = DAPI, red = tomato lectin-Dylight-594).(I) Analyses of tomato lectin-Dylight-594-stained LLC tumor sections: mean vessel length, percent covered area, and vessel straightness. The means of these parameters per mouse are shown (6 fields per mouse, n ≥ 6 mice per group, error bars represent SEM).∗∗∗∗p < 0.00005, ∗∗∗p < 0.0005, ∗∗p < 0.005, ∗p < 0.05; n.s., not significant.