Fig. 5: Safety evaluation of CVP as an adjuvant.

Mice were intraperitoneally injected with saline (SA), split influenza vaccine (SV) alone, or SV with either CVP or existing adjuvants. Body weight changes (A) and peripheral blood leukocyte counts (B) were analyzed 24 h post-injection. Mice were intranasally administered OVA antigen or SV with alum or CVP twice, with a 3-week interval, and serum antigen-specific or total IgE levels were measured 2 weeks post-boost (C). Rabbits were intravenously injected with CVP or various adjuvants, and rectal temperature changes were recorded (D). Mice received nasal or intramuscular injections of CVP alone. Mice were nasally administered alum or CVP at a dose of 50 µl/mouse, and bronchoalveolar lavage fluid (BALF) was collected 24 h later to measure lactate dehydrogenase (LDH) activity (E). Lungs were collected 4 weeks post-injection for PAS staining and histopathological analysis (E). Mice were intraperitoneally injected with SA, SV alone, or SV with either CVP or existing adjuvants. Blood was collected 24 h post-injection to measure serum cytokine levels (F). Human PBMCs were cultured with various vaccines, CVP, or adjuvant-containing media for 24 h, and cytokine levels in the supernatants were measured (G). Wild-type and Ifnar1^˗/˗ mice were nasally administered SV with either CVP or R848, with a 3-week interval, and BALF and serum were collected 2 weeks post-boost to measure IgA and IgG antibody levels by ELISA (H). Human TLR or STING reporter cells were cultured with control ligands or 0.3% CVP for 24 h, and SEAP reporter activity was measured post-culture. Cytokine levels (F and G) are shown as heatmaps representing the mean values from three mice or from the PBMCs of three human donors. Each dot represents an individual animal (A–E and H: n = 5; E: n = 4; F, G, and I: n = 3). Bar graphs show the mean values and error bars represent the SEM of data from two independent experiments for A–H and from three independent experiments for I. A–E: *p < 0.05, **p < 0.01, and ***p < 0.001 by Dunnett’s multiple comparison test following the Kruskal–Wallis test (compared with the SA group or the left-most column). H: *p < 0.05 by the Mann–Whitney U-test. N.S. indicates not significant.