Fig 2: RBD- and spike-specific IgG and IgA were detected locally in BAL, with RBD-specific IgG2b dominating the local subclass response (A–C) RBD- (A, C) and spike- (B, C) specific IgG titer in BAL for the groups vaccinated with CuMVTT-RBD vaccine or CuMVTT control, measured by ELISA, OD450 shown in A, B, LOG10 OD50 shown in C. (D–F) RBD- (D, F) and spike- (E, F) specific IgA titer in BAL for the groups vaccinated with CuMVTT-RBD vaccine or CuMVTT control, measured by ELISA, OD450 shown in D, E, LOG10 OD50 shown in F. (G) RBD-specific IgG1, IgG2a, IgG2b, and IgG3 titer in BAL for the groups vaccinated with CuMVTT-RBD vaccine or CuMVTT control, measured by ELISA, LOG10 OD50 shown. (H) RBD-specific IgA titer in BAL from mice immunized with CuMVTT-RBD vaccine, LOG10 OD50 shown. Plates in duplicates: treated with PBSTween or 7 M urea. (I) Avidity index of RBD-specific IgA titer. (J) Avidity indexes of RBD-specific IgA Abs found in BAL and serum after CuMVTT-RBD immunization. Statistical analysis (mean ± SEM) using Student's t-test. Control group n = 6, vaccine group n = 6. One representative of 2 similar experiments is shown

Fig 3: Xcl1-S vaccination elicited a significant increase in humoral immune responses. (A,B) C57BL/6 mice were immunized on days 0 and 14 with 25 µg of the Xcl1-S, S or pVAX1 plasmid. Mouse serum with a 1:100 dilution for IgG ELISA assay were collected at the indicated times. The data shown here represent OD450 nm values (means ± SEM) for each group of mice. (C–E) Mouse serum was collected 21 days after the second vaccination and were sequentially diluted at different concentrations for IgGs against the RBD, S1 and S1 + S2 ECD of the S protein using ELISA. Data are means ± SEM. (F) The IgG1, IgG2a and IgG3 isotypes of anti-S were examined in serum with a 1:100 dilution collected after the second immunization. (G) 5 × 105 cells collected from bone marrow 16 weeks after the second vaccination were re-stimulated with wild-type, delta or omicron S protein for measuring S-specific long-lasting antibody-secreting cells. ?, p < 0.05.

Fig 4: Intranasal immunization with CuMVTT‐RBD promotes isotype switching to IgA and leads to balanced IgG subclass responses. (A) RBD‐specific IgG1, IgG2a, IgG2b, and IgG3 titer for the groups vaccinated with CuMVTT‐RBD vaccine or CuMVTT control on Day 42 measured by ELISA, LOG10 OD50 shown. (B) RBD‐specific IgA titer for the groups vaccinated with CuMVTT‐RBD vaccine or CuMVTT control on Day 42 measured by ELISA, OD450 shown. (C) RBD‐specific IgA titer at Day 42 from mice immunized with CuMVTT‐RBD vaccine, LOG10 OD50 shown. Plates in duplicates: treated with PBSTween or 7 M urea. (D) Avidity index of RBD‐specific IgA titer. Statistical analysis (mean ± SEM) using Student's t‐test. Control group n = 6, vaccine group n = 6. One representative of 2 similar experiments is shown

Fig 5: Subcutaneous administration of IQB90-Flu elicited higher antibody levels than the commercial vaccine and pro-moted a dose-sparing effect. Immunizations were performed subcutaneously at day 0 (priming) and 14 (booster) either with adjuvanted formulation or with no adjuvant. Specific anti-influenza IgM antibody levels 14 days post-priming are shown in (A), and sera IgA antibody levels 28 days post-priming are shown in (B). Total IgG (C), IgG1 (D), IgG2a (E), IgG2c (F), and IgG3 (G) antibodies against influenza antigen were measured in sera 28 days after priming. The kinetics of Total IgG levels is shown in (H). To assess the dose-sparing effect conferred by the IQB90 adjuvant, two groups were inoculated with 1/10 (IQB90-Flu 7.5 µg/mL) and 1/100 (IQB90-Flu 0.75 µg/mL) of the amount of antigen contained in the group inoculated with the unadjuvanted formulation. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, Kruskal–Wallis and Dunn’s post-test (every group against the Flu group are indicated by the black stars, and the adjuvanted groups compared among each other are indicated by grey stars).