*3.2. Sequential Immunization with Genetically Distant and Closely Related NoV VLPs*

As an alternative immunization strategy and to study the effect of pre-existing immunity, the sequential immunization schedule was employed (Gr VI, Figure 2). The mice primed twice (week 0 and week 3) with the trivalent combination vaccine formulation (GI.3 + GII.4 + RV VP6) [32] were further immunized with GII.17 VLPs at week 5, followed by a GII.4 SYD VLP boost at week 7, and termination sera IgG was analyzed for all four NoV genotype-specific IgG levels (Figure 4). When compared to genotype-specific immune responses of mice immunized twice with monovalent VLPs, no significant (*p* > 0.05) differences in IgG responses were observed (Figure 4a). Similarly, strong serum IgG titers to GI.3 (Figure 4b), GII.4-1999 (Figure 4c), GII.17 (Figure 4d), and GII.4 SYD (Figure 4e) were measured following one boost immunization, with no significant difference (*p* > 0.05) to corresponding monovalent immunization groups. The response to GII.17 (Figure 4d) was very strong considering that the mice received only one GII.17 VLP dose at week 5 (Gr VI). On the contrary, GII.4 SYD-specific IgG response in Gr VI mice sera (Figure 4e) consists of the genotype-specific antibodies as well as cross-reactive antibodies to GII.4 SYD induced by closely related GII.4-1999 VLPs.

**Figure 4.** NoV genotype-specific IgG antibody responses induced by monovalent NoV VLPs or sequential VLP 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 sequentially (SQ) immunized with heterologous VLP boosts (Gr VI) 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 the group mean and the standard error of the mean (SEM). The horizontal dashed line indicats 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.

## *3.3. Simultaneous and Sequential Immunizations Induce Di*ff*erent Level of Blocking Antibodies*

To investigate if the neutralizing capability of IgG antibodies induced by simultaneous multivalent mixture immunizations (Gr V, Figure 2) is different from response to sequential immunization (Gr VI, Figure 2), genotype-specific blocking activity of the serum was analyzed. Equal levels of GI.3-specific blocking antibodies (Figure 5a) were generated by multivalent mixture (mean BT50 = 720) and sequential immunization (BT50 = 640). Similar levels of GII.4-1999-specific blocking antibodies were measured for both groups (mean BT50 = 640 Gr V; mean BT50 = 480 Gr VI) (Figure 5b). A single boost injection of GII.17 VLPs (Figure 5c) was sufficient to induce a comparable level of GII.17-specific blocking antibodies (mean BT50 = 560) to immunization with multivalent mix (mean BT50 = 720). In contrast, a single GII.4 SYD VLP boost injection (Figure 5d) generated significantly lower blocking antibodies (mean BT50 = 70) than immunization with multivalent mixture (mean BT50 = 400) (*p* = 0.024). Furthermore, the sera of mice immunized with the monovalent VLP vaccine (Gr I-IV, Figure 2) were tested for homologous blocking titers against all four genotypes (GI.3, GII.4-1999, GII.17, and GII.4 SYD) and blocking antibody levels similar to the levels induced by the multivalent VLP mix (Gr V) were observed (BT50 ≥ 400).

**Figure 5.** NoV genotype-specific blocking antibodies after immunization with multivalent NoV VLP as a mix or sequentially. Individual sera of mice immunized with multivalent mix (MX, Gr V) or sequentially (SQ, Gr VI) were 2-fold diluted starting at 1:100 dilution and assayed for the blocking of homologous NoV (**a**) GI.3, (**b**) GII.4-1999, (**c**) GII.17, or (**d**) GII.4 SYD VLP binding to histo-blood group antigens present in pig gastric mucin. The blocking index (%) was calculated as [100% − [OD (wells with serum)/OD(wells without serum, maximum VLP binding)] × 100%] and shown are individual mouse 50% blocking titer (BT50) and group geometric mean titer with 95% confidence interval. An arbitrary titer, BT50 of 5, was assigned to samples with <50% blocking index at the lowest serum dilution 1:100. Statistical differences were determined using Fisher's exact test, and a *p* value of ≤0.05 was considered statistically significant (\*).
