*2.4. Blocking Assay*

NoV VLP blocking assay, a surrogate neutralization assay, was used to determine the presence of serum IgG antibodies that block the binding of NoV VLPs to the HBGA carbohydrates according to the previously described method [37,38]. Pig gastric mucin (PGM, type III, Sigma–Aldrich, Cat. M1778) was coated on microwell plates (Corning Inc, Corning, NY, USA) and blocked with 5% milk in PBS. Starting serum dilution was 1:100 for homologous blocking and 1:20 for heterologous cross-blocking

assay. Two-fold diluted serum samples were mixed and pre-incubated with NoV VLPs for 1 h at +37 ◦C prior to plating on PGM coated microwell plates. Bound VLPs were detected with anti-NoV polyclonal antisera (human [39] or rabbit [4]) followed by secondary IgG-HRP antibody (goat anti-human IgG, Novex, Invitrogen or goat anti-rabbit, Abcam, Cambridge, UK) and OPD-substrate (Sigma–Aldrich). Maximum binding was determined by VLP sample lacking mouse sera, and maximum binding OD 490 nm had to be ≥0.7 for each VLP to be acceptable [13]. The blocking index (%) was calculated as follows: 100% − [(OD490 of wells with VLP and serum/OD490 of maximum binding wells) × 100%]. Blocking titer 50 (BT50) was determined as the reciprocal of the highest serum dilution blocking at least 50% of the maximum binding.

**Figure 2.** Immunization schemes of the study groups. Experimental groups of mice receiving monovalent (MV) (**a**) NoV VLPs (Gr I–IV) or the control group (Ctrl), receiving carrier only, were intramuscularly immunized twice at study weeks 0 and 3 and terminated at week 5. (**b**) Mice receiving a multivalent mix (MX, Gr V) of the four NoV VLPs and the RV VP6 protein was immunized using the same schedule but terminated at week 9. (**c**) Sequentially immunized mice (SQ, Gr VI) were primed twice at weeks 0 and 3 with the trivalent mix of NoV VLPs and RV VP6, boost immunized at weeks 5 and 7 with heterologous NoV VLPs, and terminated at week 9. Tail blood samples (♦) were collected at the indicated time points. Each horizontal arrow represents one injected antigen.
