In vitro Concanavalin A and rotavirus-specific proliferation of spleen cells
Spleen cells of individual mice of group A (n = 22), group B (n = 23), group C (= 24), group D (n = 22) and a control group of non-inoculated mice (n = 15) were isolated by using a cell strainer 40 μm (BD Falcon, Breda, The Netherlands) to a single-cell suspension. The cells were incubated with 5 ml ice-cold lysis buffer (4.15 g ammonium chloride (Merck, Haarlem, The Netherlands) + 0.5 g potassium bicarbonate (Sigma-Aldrich, Zwijdrecht, The Netherlands) + 18.6 mg EDTA (Sigma-Aldrich) in 500 ml water, pH 7.3) and incubated for 5 minutes on ice for lysing red blood cells. After incubation, 10 ml of ice-cold culture medium, RPMI-1640 (Life Technologies, Breda, The Netherlands) + heat inactivated 10% FBS (Perbio Science, Etten-Leur, The Netherlands) + penicillin 50 U/ml and streptomycin 50 μg/ml (Life Technologies) + 1% sodium pyruvate (Life Technologies) was added. The cell suspension was centrifuged for 5 min. at 400 × g and 4°C (Sorvall RT7, Thermo Fisher Scientific, Breda, The Netherlands) and resuspended in 2 ml ice-cold culture medium. For the Con A type IV (Sigma-Aldrich) stimulation, 2 × 105 cells were stimulated with 3 μg/ml Con A. For the rotavirus-specific stimulation, 1 × 106 cells were stimulated with 5 × 107 CCID50 UV-inactivated RRV or 1 μg/ml UV-inactivated EDIM dl particles. Plates were incubated at 37°C and 5% CO2. Con A proliferated cells were pulsed after 24 hours and the rotavirus-specific proliferated cells after 5 days with 0.4 μCi/well tritium-thymidine (PerkinElmer, Groningen, The Netherlands) and incubated overnight. Cells were harvested on filter plates (Unifilter GF-C; PerkinElmer) and radioactivity was determined in 25 μl of scintillation cocktail (Ultima gold; PerkinElmer) in a Wallac MicroBeta liquid scintillation detector (PerkinElmer). Stimulation index was calculated as the ratio of counts per minute for antigen-stimulated cultures to background cultures.
Detection of serum rotavirus-specific IgM, IgG and IgG-subclass antibodies
Serum samples were collected from mice bled from the orbital sinus and centrifuged 10 minutes at 400 × g (Sorvall RT7; Thermo Fisher Scientific). Wells of a 96-well plate (BD Falcon, Breda, The Netherlands) were coated overnight with 100 μl 500 ng/well of simian rotavirus (SA-11) at 4°C. As described previously, SA-11 is an efficient EDIM antigen substitute in an ELISA. Wells were washed 4× with 200 μl PBS (Life Technologies) + 0.05% Tween 20 (Merck) and blocked for 30 min. at 37°C with 200 μl assay buffer (PBS + 0.5% BSA (MP Biomedicals, Eindhoven, The Netherlands) + 0.05% Tween 20). As reference serum, the sera from pups and mothers from a previous EDIM passage experiment that was shown to contain antibodies to rotavirus were used. For each isotype or subclass, a different reference serum with the highest titer, tested in a serial dilution series starting from a 1:10 dilution, was selected and set to the arbitrary unit (AU) of 100. At day 16, individual serum samples were collected and then pooled per group. At day 28 individual serum samples were collected and were tested individually. Serial dilutions in 100 μl were made of the reference serum and individual serum samples, starting from a 1:10 dilution in assay buffer. Wells were washed as described above and incubated for 1 hour at 37°C with 100 μl 1:7,500 goat anti-mouse IgM μ-chain-HRP (Sigma-Aldrich) or 1:1,000 goat anti-mouse IgG-HRP (Tebu-Bio, Heerhugowaard, The Netherlands) or 1:5,000 goat anti-mouse IgG1-HRP (AbD Serotec, Düsseldorf, Germany) or 1:1,000 goat anti-mouse IgG2a-HRP (AbD Serotec) or 1:1,000 goat anti-mouse IgG2b-HRP (AbD Serotec) or 1:1,000 goat anti-mouse IgG3-HRP (AbD Serotec) in assay buffer. Plates were washed, 100 μl 3,3′,5,5′-tetramethylbenzidine (TMB; Perbio Science) was added and incubated for 10 min at room temperature. The reaction was stopped with 100 μl 10% sulphuric acid (Merck) and the absorbance measured at 450 nm on a microplate reader (BioRad). Results were calculated against the reference serum and expressed in AU. Limit of detection: 0.15 AU for IgG and IgM, 2.5 AU for IgG1, 0.6 AU for IgG2a, 0.15 AU for IgG2b and 0.3 AU for IgG3.