Alum adjuvant

For mouse immunization, VLPs of strain GII.17-KT were expressed in Pichia pastoris yeast as described above. The mouse immunization study was approved by the Institutional Animal Care and Use Committee at the Institut Pasteur of Shanghai and the animals were cared for in accordance with the institutional guidelines. Female Balb/c mice were immunized with 5 µg of the GII.17 VLP plus 500 µg of Alum adjuvant (Invivogen, USA) at weeks 0, 2, and 4. The mice were given a booster immunization with 15 µg of VLP at week 7. Three days later, splenocytes were isolated from the immunized mice and then fused with SP2/0 myeloma. The resulting hybridomas were screened for production of VLP-binding antibodies by ELISA as described below. MAbs were purified from positive hybridoma clones using HiTrap^TM Protein G affinity column (GE Healthcare, USA). The isotypes of the MAbs were determined by ELISA using an SBA Clonotyping System-HRP Kit (SouthernBiotech, USA). A norovirus cross-reactive mAb, 7D8, was prepared from a mouse immunized with the insect cells-derived GII.4 VLP [33 (link)] according to the protocol described above.

Mice were injected subcutaneously with 10 μg of rTgHSP70 dissolved in 100 μL of PBS or Alum adjuvant (Alhydrogel 2%, Invivogen, San Diego, CA, USA). Experimental design is illustrated in Figure 1A. Control groups were injected with vehicles only. Mice were injected 2 more times at weeks 2 and 4 after first immunization. On week 6, a group of animals was used for spleen ex vivo analysis, and the remaining were orally infected with 10 cyst of T. gondii ME49 strain. Four weeks later mice were anesthetized by i.p. injection of Ketamine (Syntec Brasil Ltda, SP, Brazil)/Xylazine (Schering-Plow Coopers, SP, Brazil) and blood samples were collected by puncture of the retro-orbital plexus and animals were euthanized by cervical dislocation. The brains were collected for fresh cysts quantification, histological analysis, and parasite quantification by qPCR. Animals were monitored daily for morbidity scores and every other day for weight changes (Bartley et al., 2006 (link)) and the blood samples were collected as described above every 2 weeks for antibody and cytokine analysis in serum samples. When mice reached a cumulative score of 5 on any day or a score of 4 for 2 consecutive days they were euthanized as described (Bartley et al., 2006 (link)).

All animal experiments were performed under the guidelines and protocols approved by the Basel-Stadt cantonal veterinary office (Basel-Stadt Kantonales Veterinäramt Tierversuchsbewilligung #2582). Female BALB/c mice (n = 10, Charles Rivers Laboratories) 6–8 weeks old were housed under specific pathogen-free conditions and were maintained on a normal chow diet.
Purified chicken gamma globulin (CGG) conjugated to 4-hydroxy-3-nitrophenylacetyl (NP, NP-CGG, BioCat) was resuspended in sterile-filtered phosphate buffered saline (PBS) at 1.0 mg/mL. On the day of primary immunization, 50 μl of NP-CGG solution was mixed with 100 μL of Alum adjuvant (1 mg/mL, Invivogen) and 50 μL of sterile PBS and stored on ice. The NP-CGG Alum mixture was injected with a 26-gauge needle subcutaneously into the backpad. Mice were sacrificed on day 14 after primary immunization and blood, spleen, and bone marrow (femora and tibiae) were collected.