*3.1. Simultaneous Immunization with Multivalent VLP Mixture Formulation*

Similar NoV genotype-specific IgG binding antibodies were detected when comparing termination sera of mice immunized twice, either with 10 μg of each monovalent NoV VLP alone (Gr I-IV, Figure 2a) or as a component of a multivalent mixture (Gr V, Figure 2b) (Figure 3). No significant differences (*p* < 0.05) were observed when comparing genotype-specific IgG responses of monovalent or multivalent mix immunized mice at serum dilution 1:200 (Figure 3a). The results showed induction of equal levels of IgG antibodies against GI.3 (Figure 3b), GII.4-1999 (Figure 3c), GII.17 (Figure 3d), and GII.4 SYD (Figure 3e), irrespective of the presence or absence of other co-administrated antigens. NoV-specific IgG was not detected in any of the control animal sera (Gr VII) that received carrier (PBS) only (Figure 3b–e).

**Figure 3.** NoV genotype-specific IgG antibody responses induced by monovalent NoV VLPs or multivalent VLP mix immunization. Termination sera of mice immunized with monovalent (MV) VLPs, GI.3 (Gr I), GII.4-1999 (Gr II), GII.17 (Gr III), or GII.4 SYD (Gr IV), or with the multivalent NoV VLP mix (MX, Gr V), or the carrier only (Control group, Ctrl) were analyzed with enzyme-linked immunosorbent assay (ELISA). (**a**) The mean optical density (OD490) at the serum dilution 1:200 of individual mice is illustrated with group mean and the standard error of the mean (SEM). The horizontal dashed line indicates maximum background level (cut-off limit). Mean IgG end-point titration curves specific for NoV (**b**) GI.3, (**c**) GII.4, (**d**) GII.17, and (**e**) GII.4 SYD VLPs with the SEM are shown.
