*2.2. Mice Immunization*

Female BALB/c (H-2d) mice were obtained from Envigo RMS BV and randomly divided into seven groups (Gr) of five mice (Figure 2). At seven weeks of age, the mice were immunized intramuscularly (IM) at the right caudal thigh muscle with a single, monovalent NoV VLP vaccine (Gr I–IV, Figure 2a), or as a multivalent formulation mixture of four NoV genotype VLPs and RV VP6 (Gr V, Figure 2b) two times, at week 0 and week 3 according to the established optimal immunization schedule used in our laboratory [5]. A 10 μg dose per each VLP was administrated, and control (Ctrl, Figure 2a) mice received phosphate-buffered saline (PBS) carrier only. The mice in the sequential immunization group (VI, Figure 2c) were primed with GII.4-1999 + GI.3 VLPs + RV VP6 (the trivalent NoV-RV combination vaccine [32]) at week 0 and week 3 and boosted with GII.17 VLPs at week 5, and with GII.4 SYD at week 7, using a 10 μg dose of each antigen per injection. Tail blood samples were collected at weeks 0 (pre-dose) and 3 from all mice and additionally at weeks 5 and 7 from the mice in the Gr V and Gr VI. Mice were sacrificed, and blood samples (serum) were collected at week 5 (Gr I-IV, Ctrl) or at week 9 (Gr V and VI). Immunizations were conducted under general anesthesia by inhalation of isoflurane (Attane vet, Vet Medic Animal Health Oy), and a formulation of medetomidine (Dorbene® vet, Laboratorios Syva, Leon, Spain) and ketamine (Ketaminol® vet, Intervet International B.V., Boxmeer, The Netherlands) was used for euthanasia. All procedures were carried out in accordance with the regulations and guidelines of the Finnish National Experiment Board (Permission number ESAVI/10800/04.10.07/2016) and mouse welfare was monitored throughout the experiment on a daily basis.
