Fig. 7

Chronic exposure to alternative tobacco product aerosols suppresses development of antibody responses to vaccination. Animals exposed to air, EC, HTP, and CC received prophylactic P6 Ag vaccination i.m. against a respiratory pathogen at weeks 5, 6, and 7 after exposures were started as shown in schema (A). Vaccination efficacy was measured by quantifying antigen-specific antibody titers in serum (weeks 5–12) and in BAL at euthanasia. Weekly serum was collected from aerosol-exposed, P6 immunized mice as described in Additional file 1 and total anti-P6 Ig levels were measured in weekly serum (B) and endpoint BAL (C) samples. To quantify mucosal IgA Ab levels (C) in the BAL of mice, the OD values at 450 nm were measured using BAL dilutions at 1:400 in P6-specific ELISA as described previously [10] and provided in detail in Additional file 1: supplemental methods. Data are shown as curve (B) or as bar diagram with individual data sets (C, D) and given as mean ± SE. Two-way ANOVA with Tukey’s post-test multiple comparisons (A) or non-parametric Kruskal–Wallis test with FDR correction for multiple comparison (C, D) was performed to determine statistically significant differences between two groups by GraphPad Prism 9.5.1 software (GraphPad; La Jolla, California, USA). Difference between two groups was considered significant at p < 0.05, and symbols ***p < 0.001; ****p < 0.0001 are used to denote significant difference between two groups. For, two-way ANOVA, symbols denote as follows: ^┬p < 0.0001 vs Air; ^Ψp < 0.0001 vs CC; ^Υp < 0.001 vs IQOS. n = 20 for air, EC, HTP, and CC (10 M + 10F) per group for each exposure condition