Specific Antibody Responses to Subtilisin Carlsberg (Alcalase) in Mice: Development of an Intranasal Exposure Model

Abstract

An intranasal (i.n.) dosing model was developed in mice as a potential alternative to more difficult, time-consuming, and costly guinea pig intratracheal (GPIT) or mouse intratracheal models for assessment of the respiratory immunogenicity of detergent enzymes. Using a benchmark enzyme, Alcalase (protease subtilisin Carlsberg), studies were conducted to standardize the model in terms of mouse strain, dosing and serum harvest regimen, and the primary immunoglobulin endpoint to use. The primary assay endpoint selected was the enzyme-specific IgG1 titer determined by an Alcalase-specific ELISA. This is not the primary allergenic antibody in mice (IgE is); however, IgG1 is coregulated with IgE via the IL-4/TH2 pathway and may have a role in mediating allergic-type responses. BDF1 mice (C57B1/6 x DBA/2) were selected as representative of high responder strains, with high response associated with the H-2b (C57B1/6) parent. The dosing regimen used for most studies incorporated three i.n. exposures (Days 1, 3, and 10) and bleeding of the animals on Day 15. The animals were anesthetized and then immunized by allowing them to inhale 5-microliters aliquots of dosing solution into each nostril at each immunization. Positioning of the animals with their heads down (vs up) may have allowed more of the dosing solution to remain in the nasal region for a slightly longer period of time, but did not change the eventual GI tract migration and excretion of each dose. The presence of a detergent matrix in the enzyme dosing solution enhanced the IgG1 response. Immunizing with enzyme plus detergent gave highly consistent dose-response curves for Alcalase when evaluated over many studies. An enzyme-specific allergic antibody (IgE) response was weak and inconsistent under the dosing regimen used to generate the IgG1 response, but was stronger with longer-term dosing, consistent with the delay in IgE vs IgG1 responses seen in some other studies. Using IgG1 as a surrogate for allergic sensitization, we have preliminary data showing similar differential potencies between Alcalase and other test enzymes as detected in previous GPIT tests. On the basis of these data, we believe the i.n. immunization/IgG1 response model is a robust technique that may be useful in determining the relative immunogenicities of detergent enzymes and other proteins.

Airway inflammation and adjuvant effect after repeated airborne exposures to di-(2-ethylhexyl)phthalate and ovalbumin in BALB/c mice

“…Furthermore, IgG1 in mice is an anaphylactic antibody (Oshiba et al, 1996), but only in combinations with high allergen doses (Finkelman et al, 2005). IgG1 is co-regulated with IgE via the IL-4/Th2 path- way (Purkerson and Isakson, 1992), which may explain the use of IgG1 to assess immunogenicity or adjuvant effects in several murine systems (Kimber et al, 2003;Robinson et al, 1996;Yanagisawa et al, 2006) and as a risk factor for developing an IgE response in humans. Overall, the extent to which the IgG1 response should be considered a protective response or a risk factor for development of IgE is not clear at present.…”

The Importance of the Diluent for Airway Transport of Toluene Diisocyanate Following Intranasal Dosing of Mice

“…Animals were held either upright or in a supine position while dosing solutions (20 or 50 µl divided equally between the 2 nares) were administered (Robinson et al, 1996). Drops were placed outside of each naris and inhaled by the mouse.…”

“…Intranasal administration has been used routinely to immunize mice to a variety of viral, fungal, and proteinaceous agents, providing superior results to intratracheal dosing regimens (Nikulin et al, 1997). Others have demonstrated that intranasal administration of proteins to mice elicits specific immunoglobulin E (IgE) and/or IgG responses (Robinson et al, 1996). Few studies have employed intranasal dosing with chemicals.…”

Immune Responses in the Lung and Local Lymph Node of A/J Mice to Intranasal Sensitization and Challenge with Adjuvant-Free Ovalbumin

“…The intranasal instillation of allergens (ie, Schistosoma mansoni egg antigen, Aspergillus fumigatus) has been used to successfully induce features of allergic rhinitis (18,24). In addition, the intranasal instillation of allergens has been used to elicit antibody responses for the identification of the respiratory sensitization potential of detergent enzymes in the mouse intranasal test (MINT) (3,20). Our exposure regimen differed from the MINT protocol in the schedule and duration of allergen dosing.…”

“…Our exposure regimen differed from the MINT protocol in the schedule and duration of allergen dosing. The MINT protocol includes instillations on days 1, 3, and 10 followed by sacrifice on day 15 (3,20). Our study consisted of 5 intranasal instillations during a distinct sensitization phase followed by the first challenge (single intranasal instillation) 2 weeks later and a second challenge 10 days after the first challenge followed by sacrifice from 24 to 96 hours after final instillation.…”