Investigations of immunogenic, allergenic and adjuvant properties of Cry1Ab protein after intragastric exposure in a food allergy model in mice

The expression of the genes HSP70, MCPT-1, IL-6, IL-9 and TNFα in tissue from the small intestines was determined at termination. Neither between the groups in the adjuvance set-up (Table 1, Fig. 6a, b, c) nor in the immunogenicity set-up (Fig. 6d, e, f) did expression of TNFα, HSP70 and IL-6 relative to GUSB (housekeeping gene) differ between the treatment groups. The expression of the genes MCPT-1 and IL-9 relative to GUSB were below the limit of detection (data not shown).

Female C3H/HeJ mice (Jackson Laboratories, USA), 5 weeks old at arrival, were randomly allocated to groups (n = 11–13) and housed on Nestpack bedding (Datesand Ltd, Manchester, UK), 3–5 mice in each cage. The Harlan Teklad 2018 rodent diet and tap water were given ad libitum. The mice were exposed to a 12/12 h light/dark cycle, room temperature (RT) of 21 +/− 2 °C and 55 +/− 10 % humidity. The experiment was performed in conformity with the laws and regulations for live animals in Norway, and approved by the Norwegian Animal Research Authority under the Ministry of Agriculture (FOTS ID 4534).

TrypCry1Ab toxin was purchased from Case Western Reserve University, (Ohio, USA, Dr. Marianne Pusztai-Carey). The origin of the cry1Ab gene inserted in Escherichia coli (E. coli) was the Bt kurstaki HD-1 strain. The inclusion bodies were solubilised at pH 10.5 in the presence of a reducing agent and the precipitated protoxins were digested by commercial bovine trypsin and subsequently purified by ion exchange high-performance liquid chromatography (HPLC). The relevant fractions were analysed by gel filtration, HPLC and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), desalted and lyophilised (M. Carey, personal communication). The protein preparations were dissolved in a sterile physiological buffer (Hanks Balanced Salt Solution; HBSS) to a concentration of 172 μg/ml.

Protein extract from Lupinus angustifolius (Lupex) was produced and provided by The National Veterinary Institute of Norway. The extract preparation procedure is described in Vinje et al. [23], and the protein extract had a concentration of 72.4 mg/ml. Cholera toxin (CT) from Vibrio cholerae, azide free, 1 mg/ml solution, was purchased from Quadratech Diagnostics Ltd (Surrey, UK).

The test solutions were prepared in HBSS and given to the mice in volumes of 250 μl, resulting in the following doses per animal per gavage: A; 5.7 mg Lupex, B; 5.7 mg Lupex + 10 μg CT, C; 5.7 mg Lupex + 10 μg CT + 10 μg trypCry1Ab, D; 5.7 mg Lupex + 10 μg trypCry1Ab, E; 10 μg trypCry1Ab, F; 10 μg trypCry1Ab + 10 μg CT. The control solution G consisted solely of 250 μl HBSS. A high dose of 10 μg trypCry1Ab was selected due to the proof-of-principle character of the study and in order to be able to compare a potential adjuvant effect with the effect induced by 10 μg of the positive control, CT. Restrictions in gavage volume (max 250 ml) and the trypCry1Ab solubility prevented us from giving a higher dose per animal per exposure.

The mice were exposed by i.g. gavage of 250 μL test solutions according to Table 1, on days 0, 1, 2, 7, 21, and 28. A 100 μL blood sample was collected from vena saphena lateralis from each animal on day 0 and 34. On day 35, to examine the potential adjuvant effect of trypCry1Ab, a challenge of Lupex was given i.p. to all mice in the experimental groups A - D according to Table 1, and the anaphylactic responses were assessed during 30 min following the i.p. challenge (described below). All mice were anaesthetised with 3.5 % Isofluran gas (Isoba vet; Intervet/Schering-Plough Animal Health, Lysaker, Norway) administered in surgical O₂ in an inhalation chamber before exsanguination by heart puncture, and blood, spleen and 5 cm of the small intestine (distal ileum) were harvested.

On day 35, clinical anaphylactic reactions were assessed continuously in 30 min directly after an i.p. challenge with 250 μl of HBSS containing 5 mg of the allergen. The score system described by Li et al. [35] was employed: 0, no symptoms; 1, scratching and rubbing around the nose and head; 2, puffiness around the eyes and mouth, diarrhoea, pili erecti, inactivity or decreased activity with an increased respiratory rate; 3, wheezing, laboured respiration, cyanosis around the mouth and tail; 4, no activity after prodding or tremor or convulsion; and 5, death. Rectal temperature was measured after 0, 15 and 30 min with a BAT-12 microprobe thermometer (probe RET-3) purchased from Physitemp Instruments Inc (Clifton, USA). The mice were exsanguinated by terminal heart bleed immediately after the 30 min observation period, or when reaching a score of 4 or above.

As a marker of anaphylaxis, serum levels of mouse mast cell protease-1 (MCPT-1) were analysed in terminal blood samples collected after allergen challenge, with an enzyme-linked immunosorbent assay (ELISA) kit (Moredun Scientific Ltd., Scotland, UK) in accordance with the manufacturer’s instructions.

The detection of lupin-specific IgG1 antibodies was performed according to Vinje et al. [23]. In brief; microtiter plates with 96 wells were coated with lupin extract (5 μg/ml), incubated for 1 h at room temperature (RT) and then overnight at 4 °C. After washing (Tris/Tween), blocking (Tris/Tween with 1 % bovine serum albumin) for 1 h, and subsequent washing, plates were incubated for 2 h at RT with lupin-specific IgG1 standard serum in fourfold dilutions for standard curve generation, negative control serum and the sera collected at day 34 (diluted 1:100 in BSA/Tris/Tween). After washing, peroxidase-labelled rat anti-mouse IgG1 antibody was added to each well. Following 2 h incubation at RT, and washing, plates were incubated for 15 min with peroxidase substrate. Plates were read at 405 nm in a BioTek Elx808 Absorbance Microplate reader using Gen5™ Microplate Data Collection & Analysis Software (BioTek® Instruments, Inc., Winooski, Vermont, USA).

In-house ELISA protocols were established to detect specific anti-Cry1Ab IgG1, IgG2a and IgE antibodies in the mouse sera as previously described in Andreassen et al. In brief, for the IgG1 assay, 96-well microtiter plates were coated with 2 ng/μL purified trypCry1Ab protein per well and incubated for 1 h at RT and then overnight at 4 °C. Plates were washed (Tris/Tween) and incubated with blocking solution (5 % skimmed milk powder in phosphate buffered saline (PBS)) for 1 h at RT. After subsequent washing, diluted sera (1:50) from terminal blood samples were added and plates were incubated for 1 h at RT and again overnight at 4 °C. The plates were washed, 200 ng biotinylated rat anti-mouse IgG1 was added per well and plates were incubated for 1 h at RT. After subsequent washing, poly-horseradish peroxidase (HRP)-streptavidin diluted 1:40000 was added and incubated for 1 h at RT. The plates were washed and color development was obtained by adding stabilised chromogen 3,3′, 5,5;-tetramethylbenzidine (TBM). After incubation in darkness for maximum 15 min the reaction was stopped with 2 N H₂SO₄ solution.

For detection of Cry1Ab-specific IgE, microtiter plates with 96 wells were coated with 200 ng rat anti mouse IgE antibodies per well, incubated for 1 h at RT and then overnight at 4 °C. Subsequently, the plates were washed with Tris/Tween and blocked with 5 % skimmed milk in PBS for 1 h at RT. After subsequent washing, diluted sera (1:10) from blood sampled at day 34 were added and plates were incubated for 1 h at RT and then again overnight at 4 °C. The plates were washed, 300 ng trypCry1Ab was added to each well and incubated for 1 h at RT. After subsequent washing, 300 ng biotinylated rabbit anti mouse Cry1Ab antibody was added per well as detection antibody and plates were incubated for 1 h at RT. Plates were washed and detection was performed with poly-HRP-streptavidin and stabilised chromogen TBM as described above. The reaction was stopped with 2 N H₂SO₄ solution after incubation in the dark for a maximum of 15 min.

For detection of Cry1Ab-specific IgG2a, microtiter plates with 96 wells were coated with 200 ng biotinylated rat anti mouse IgG2a antibodies per well, and the protocol for Cry1Ab-specific IgE detection was subsequently followed as described above. To accelerate the reactivity of each step, all incubations were performed at 37 °C for the detection of specific anti-Cry1Ab IgG2a.

Standard curves were made from duplicates of diluted serum pools from mice (repeatedly i.p.) immunised with trypCry1Ab and Al(OH)₃, and included for all antibody assays on each plate. As the amount of specific IgG1, IgE and IgG2a in the standards is unknown, the levels are presented as arbitrary units (AU) per ml serum. Absorbance was measured at 450 nm on a BioTek Elx808 Absorbance Microplate reader with the Gen5™ Microplate Data Collection & Analysis Software (BioTek® Instruments, Inc., Winooski, Vermont, USA).

Serum levels of total IgE were analysed in blood samples from day 34, using a Mouse IgE ready-set-go ELISA-kit (Affymetrix eBioscience, Vienna, Austria) in accordance to the manufacturer’s instructions.

The excised spleens were crushed through a 70 μm cell strainer (BD labware, New Jersey, USA) to obtain single cell suspensions as described previously [38]. Cell numbers were determined with a Cell Coulter Z1 (Beckman Coulter Inc., Florida, USA). Cells were cultured in medium (RPMI 1640 with L-glutamine and 10 % foetal bovine serum and 1 % streptomycin/penicillin) in 96 well plates at a concentration of 2.7 × 10⁶ cells/ml per well at 37 °C in a humidified atmosphere with 5 % CO₂ for three days with concanavalin A (ConA) (5 μg/ml), and for five days with or without Lupex or trypCry1Ab (17.2 μg/mL). The total volume in each well was 200 μl. The amount of cytokine IL-2, IL-5, IL-13, IL-10, and interferon gamma (IFNγ) released into spleen cell supernates were determined by Cytometric Bead Array (CBA) flex set kit from BD Biosciences (San Diego, California, USA).

A 5 cm segment of ileum were excised (7 to 12 cm from the cecum), flushed thorough with PBS, cut into smaller fractions and put in a cryogenic vial that was snap frozen in liquid nitrogen and kept at –80 °C for later preparation and quantitative polymerase chain reaction (PCR) analyses. Frozen tissue segments were dissected with a scalpel on dry ice. Tissue segments between 14.3 and 68.8 mg were homogenised in a tube containing hard tissue CK28 beads (Bertine technologies, France) and 250 μL lysis buffer with 1.75 μL β-mercaptoethanol (Absolutely RNA Miniprep Kit, Agilent Technologies La Jolla, California), in a Precellys 24 homogeniser (Bertine Technologies, France) for 2 × 46 s at RT. Total RNA was extracted using Absolutely RNA Miniprep Kit (Agilent Technologies La Jolla, California) according to the manufacturer’s instructions. To assess yield and quality, the total RNA extract was evaluated using a Nandodrop spectrophotometer (Thermo scientific Wilmington, Denver, USA) and subsequently the RNA Integrity Number (RIN) was determined using a 2100 Bioanalyzer (Agilent Technologies La Jolla, California). Samples with RIN number lower than 4.8 were considered to be of poor quality and were not included in the following steps. First-strand cDNA synthesis of 50 ng total RNA were performed using a master mix containing 1xfirst strand MM buffer, 225 ng oligo(dT), 45 ng random hexamer and AffinityScriptRT/RNase block enzyme mixture (1.5 μL/30 μL total volume) and distilled water to a total volume of 30 μL (Agilent Technologies La Jolla, California). The syntheses were performed with a Bio Rad S1000 Thermal Cycler (Bio Rad Laboratories California, USA). The syntheses condition was 25 °C for 5 min, 42 °C for 30 min, 95 °C for 5 min and 12 °C for infinite. Real-Time PCR was performed by adding 1.0 μL cDNA solution to a master mix containing 1xPrimeTime assay (containing a probe and two primers), 1xBrilliant III Ultra-Fast QPCR Master Mix, 0.45 ng Reference dye and water to a total volume of 20 μL (Agilent Technologies La Jolla, California). The real-time PCR was performed with an Applied Biosystems 7500 Fast Real-Time PCR Systems (Applied Biosystems life technology, Thermo Scientific Waltham, Massachusetts, USA). The amplification conditions were 95 °C for 3 min, followed by 40 cycles at 95 °C for 20 s and 60 °C for 30 s. In total there were seven PrimeTime assays, five target genes; heat shock protein (HSP) 70 (primer 1: 5’-GTAGTACACAGTGCCAAGACG-3’, primer 2: 5’-TTTATATCAGTGTTCCAGTAGCCT-3’), MCPT-1 (primer 1: 5’-ACTCAACACCACCAATAATCTCC-3’, primer 2: 5’-GGAACCAGGACAAGAACACA-3’), IL-6 (primer 1: 5’-CAAGTGCATCATCGTTGTTCA-3’, primer 2: 5’-GATACCACTCCCAACAGACC-3’), IL-9 (primer 1: 5’-GCAGCTGGTCACGTTGC-3’, primer 2: 5’-CTTGCCTCTGTTTTGCTCTTC -3’), and tumour necrosis factor (TNF)α (primer 1: 5’-TCTTTGAGATCCATGCCGTTG-3’, primer 2: 5’-AGACCCTCACACTCAGATCA-3’), and two reference genes; TATA box binding protein (Tbp) (primer 1: 5’-CTGAATAGGCTGTGGAGTAACTC-3’, primer 2: 5’-CTGAAGAAAGGGAGAATCATGGA-3’) and glucuronidase-beta (GUSB) (primer 1: 5’-GATGCGTCTTATACCAGTTCTCA-3’, primer 2: 5’-CAACGCCAAATATGATGCAGAC-3’). The threshold cycle number (C_t) was determined with ABI 7500 software from Applied Biosystems life technology (Thermo Scientific Waltham, Massachusetts, USA). Gene expression levels were normalised by subtraction of the housekeeping gene GUSB C_t from target gene C_t, and then against the calibrator (= ΔΔC_t). To obtain positive values for upregulated genes and negative values for downregulated genes, all ΔΔC_t values were multiplied with −1.

The experiment was performed in conformity with the laws and regulations for live animals in Norway, and approved by the Norwegian Animal Research Authority under the Ministry of Agriculture (FOTS ID 4534).

The datasets supporting the conclusions of this article are included within the article (and its Additional file 1 “FOTS 4534_all datasets”).