DNA-based vaccination reduces the risk of lethal anaphylactic hypersensitivity in mice Anthony A. Horner, MDa, Minh-Duc Nguyen, BAa, Arash Ronaghy, BAa,

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DNA-based vaccination reduces the risk of lethal anaphylactic hypersensitivity in mice Anthony A. Horner, MDa, Minh-Duc Nguyen, BAa, Arash Ronaghy, BAa, Nadya Cinman, BAa, Sjef Verbeek, PhDb, Eyal Raz, MDa Journal of Allergy and Clinical Immunology Volume 106, Issue 2, Pages 349-356 (August 2000) DOI: 10.1067/mai.2000.107933 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 Timetable for vaccination, sensitization, and anaphylactic challenge. C3H/HeJ mice received intradermal vaccinations with 50 μg of pACB-LacZ or pACB or 10 μg of β-gal alone or mixed with ISS-ODN (10 μg) or M-ODN (10 μg) on 3 occasions 10 days apart. Twenty-five days after the last vaccination, mice were TH2 sensitized with a mixture of β-gal (100 μg), pertussis toxin (300 ng), and alum (1 mg) injected intraperitoneally on 2 occasions 7 days apart. Mice then received an intravenous challenge with 150 μg of β-gal 3 weeks after their last intraperitoneal sensitization. Journal of Allergy and Clinical Immunology 2000 106, 349-356DOI: (10.1067/mai.2000.107933) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 Plasma histamine levels after anaphylactic challenge in pACB-LacZ–vaccinated mice. Mice were vaccinated, sensitized, and challenged as outlined in the “Methods” section and Fig 1. Two minutes after intravenous β-gal challenge, plasma was collected for histamine determination. Results represent mean values ± SE for 4 mice in each group. Before challenge, plasma histamine levels were less than 10 nmol/L in all mice (data not shown). Journal of Allergy and Clinical Immunology 2000 106, 349-356DOI: (10.1067/mai.2000.107933) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 Serum antibody and cytokine responses of pACB-LacZ–vaccinated mice. Mice were vaccinated, sensitized, and challenged as described in the “Methods” section and Fig 1. Results represent mean values ± SE for 4 mice in each group and are representative of 3 independent experiments. A, Antigen-specific antibody levels. One day before challenge, serum was obtained for antibody determination by means of ELISA. B, Antigen-specific cytokine responses. After intravenous β-gal challenge, splenocytes were harvested and cultured with and without β-gal. Supernatants were harvested at 72 hours for cytokine ELISA. Culture without β-gal led to negligible cytokine production (data not shown). Journal of Allergy and Clinical Immunology 2000 106, 349-356DOI: (10.1067/mai.2000.107933) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 Plasma histamine levels after anaphylactic challenge in β-gal/ISS-ODN–vaccinated mice. Mice were vaccinated, sensitized, and challenged as outlined in the “Methods” section and Fig 1. Two minutes after intravenous β-gal challenge, plasma was collected for histamine determination. Results represent mean values ± SE for 4 mice in each group. Before challenge, plasma histamine levels were less than 10 nmol/L in all mice (data not shown). Journal of Allergy and Clinical Immunology 2000 106, 349-356DOI: (10.1067/mai.2000.107933) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 5 Serum antibody and cytokine responses of β-gal/ISS-ODN–vaccinated mice. Mice were vaccinated, sensitized, and challenged as described in the “Methods” section and in Fig 1. Results represent mean values ± SE for 4 mice in each group and are representative of 3 independent experiments. NS, No significant difference. A, Antigen-specific antibody levels. One day before challenge, serum was obtained for antibody determination by means of ELISA. B, Antigen-specific cytokine responses. After intravenous β-gal challenge, splenocytes were harvested and cultured with and without β-gal. Supernatants were harvested at 72 hours for cytokine ELISA. Culture without β-gal led to negligible cytokine production (data not shown). Journal of Allergy and Clinical Immunology 2000 106, 349-356DOI: (10.1067/mai.2000.107933) Copyright © 2000 Mosby, Inc. Terms and Conditions