Mukesh Kumar, PhDa, Aruna K. Behera, PhDa, Jianan Hu, MDa, Richard F

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IFN-γ and IL-12 plasmid DNAs as vaccine adjuvant in a murine model of grass allergy Mukesh Kumar, PhDa, Aruna K. Behera, PhDa, Jianan Hu, MDa, Richard F. Lockey, MDa, Shyam S. Mohapatra, PhDa, b Journal of Allergy and Clinical Immunology Volume 108, Issue 3, Pages 402-408 (September 2001) DOI: 10.1067/mai.2001.117261 Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 1 Expression of murine IFN-γ and IL-12 p40 subunit in the mouse muscle. Mice were vaccinated as described in the Methods section. Seven days after the last DNA injection, expression for IFN-γ and IL-12 p40 subunit was checked by immunohistochemistry (A ) and RT-PCR (B ). A, Mice vaccinated with pIFN-γ (a ) and pIL-12 (c ) showed expression of IFN-γ and IL-12 p40 subunits, respectively, as indicated by positive staining (arrows ). Mice vaccinated with control empty vector did not show expression for IFN-γ (b ) and IL-12 p40 (d ). B, Total RNA was isolated from the muscle, and RT-PCR was performed for IFN-γ and IL-12 p40 subunit. Mice vaccinated with pIFN-γ and pIL-12 showed IFN-γ (panel 1, lane 2 ) and IL-12 p40–specific mRNA amplification (panel 1, lane 4 ). No amplification was observed in the mice vaccinated with control empty vector (panel 1, lanes 1 and 3 ). β-actin was used as internal control (lower panel ). Journal of Allergy and Clinical Immunology 2001 108, 402-408DOI: (10.1067/mai.2001.117261) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 2 Analysis of the total IgE and KBG-specific IgG subtypes. Four groups of mice (n = 6) were vaccinated as described in the Methods section. On day 21 after immunization with alum and KBG allergen, their serum was analyzed for total IgE (B) and KBG-specific IgG2a and IgG1 (A) antibodies by ELISA. Bars represent the means ± SDs. *P < .05; **P < .01; ***P < .001 in comparison with pcDNA3.1 control. †P < .05; ††P < .01; †††P < .001 in comparison with pIFN-γ group. ‡P < .05; ‡‡P < .01; ‡‡‡P < .001 in comparison with pIL-12 group. Journal of Allergy and Clinical Immunology 2001 108, 402-408DOI: (10.1067/mai.2001.117261) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 3 A, Analysis of the cytokine production. Mice (n = 6) were vaccinated as described in the Methods section. On day 7 after KBG and alum immunization, their spleens were cultured in vitro for 48 hours in the presence of KBG allergen and cytokines were measured by ELISA. Bars represent the means ± SDs. *P < .05; **P < .01; ***P < .001 in comparison with pcDNA3.1 control. †P < .05; ††P < .01; †††P < .001 in comparison with pIFN-γ (IFN-g) group. ‡P < .05; ‡‡P < .01; ‡‡‡P < .001 in comparison with pIL-12 (IL-12) group. B, Analysis of the dominant cytokine pattern after cytokine DNA vaccination. Dominant cytokine pattern was determined from the IFN-γ/IL-4 and IL-2/IL-4 ratios. Bars represent means. Journal of Allergy and Clinical Immunology 2001 108, 402-408DOI: (10.1067/mai.2001.117261) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 4 Measurement of the airway hyperresponsiveness in KBG-sensitized and -challenged mice after cytokine DNA vaccination. Naive mice (n = 4) were vaccinated as described in the Methods section and sensitized with the allergen 7 days later. Ten days after the sensitization, animals were challenged intranasally 3 times with 50 μg of KBG allergen. Airway reactivity to inhaled methacholine (6 to 50 mg/mL) was measured 24 hours later. Results are expressed as means ± SDs of enhanced pause values. a, P < .05; aa, P < .01 in comparison with pcDNA3.1 group. b and c, P < .05 in comparison with pIFN-γ (IFN-g) and pIL-12 (IL-12) groups, respectively. Journal of Allergy and Clinical Immunology 2001 108, 402-408DOI: (10.1067/mai.2001.117261) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 5 Assessment of the lung inflammation in KBG-sensitized and -challenged mice after cytokine DNA vaccination. Lung tissue was removed from the different groups of mice (n = 4) 24 hours after the last intranasal allergen challenge and was stained with hematoxylin and eosin. A representative photomicrograph from each group is shown. A, pcDNA3.1. B, pIFN-γ. C, pIL-12. D, pIFN-γ + pIL-12. Arrows indicate cellular infiltration. a, Airway; v, vessel. Journal of Allergy and Clinical Immunology 2001 108, 402-408DOI: (10.1067/mai.2001.117261) Copyright © 2001 Mosby, Inc. Terms and Conditions