Correlations between antibody immune responses at different mucosal effector sites are controlled by antigen type and dosage

Abstract

Monitoring specific secretory immunoglobulin A (IgA) responses in the intestines after mucosal immunization or infection is impeded by the fact that sampling of small intestinal secretions requires invasive methods not feasible for routine diagnostics. Since IgA plasma cells generated after intragastric immunization are known to populate remote mucosal sites as well, secretory IgA responses at other mucosal surfaces may correlate to those in the intestines and could serve as proxy measures for IgA secretion in the gut. To evaluate the practicability of this approach, mice were immunized intragastrically with 0.2, 2, and 20 mg of ovalbumin plus 10 microg of cholera toxin, and the antigen-specific local secretory IgA responses in duodenal, ileal, jejunal, rectal, and vaginal secretions, saliva, urine, and feces, as well as serum IgG and IgA responses were analyzed by enzyme-linked immunosorbent assay. Correlation analysis revealed significant relationships between serum IgG and IgA, urinary IgA, salivary IgA, and secretory IgA in duodenal, jejunal, ileal, and rectal secretions for the 0.2-mg but not for the 20-mg ovalbumin dose. Fecal samples were poor predictors for intestinal antiovalbumin IgA responses, and no correlations could be established for cholera toxin, neither between local anti-cholera toxin levels nor to the antiovalbumin responses. Thus, specific IgA in serum, saliva, or urine can serve as a predictor of the release of specific IgA at intestinal surfaces after intragastric immunization, but the lack of correlations for high ovalbumin doses and for cholera toxin indicates a strong dependency on antigen type and dosage for these relationships.

FIG. 1

Time course of the anti-CT IgA and IgG responses in serum and feces after priming and three booster immunizations with various doses of OVA plus CT adjuvant. Solid squares, 0.2 mg of OVA plus 10 μg of CT; open squares, 20 mg of OVA plus 10 μg of CT; open circles, 0 mg of OVA plus 10 μg of CT. Arrows indicate time points of immunization. Values represent means ± SEM of samples from six mice. Absolute A₄₅₀ readings of samples from mock-immunized animals used to calculate the endpoints and buffer-for-sample readouts were 0.042 ± 0.003 and 0.041 ± 0.002 (mean ± SD of 38 microplates), respectively.

FIG. 2

Time course of the anti-OVA IgA and IgG responses in serum and feces after priming and three booster immunizations with various doses of OVA plus 10 μg of CT adjuvant. Solid squares, 0.2 mg of OVA; open squares, 2 mg of OVA; solid circles, 20 mg of OVA; open circles, 0 mg of OVA. Arrows indicate time points of immunization. Values represent means ± SEM of samples from six mice. Absolute A₄₅₀ readings of samples from mock-immunized animals used to calculate the endpoints and buffer-for-sample readouts were 0.045 ± 0.002 and 0.044 ± 0.002 (mean ± SD of 40 microplates), respectively.

FIG. 3

Specific IgA responses against OVA and CT in serum and local secretions after priming and three booster immunizations with various doses of OVA plus CT adjuvant. (A) Absolute anti-OVA endpoint titers. (B) Absolute anti-CT endpoint titers. Values represent means ± SEM of samples from six mice. Absolute A₄₅₀ readings of samples from mock-immunized animals used to calculate the endpoints and buffer-for-sample readouts were 0.044 ± 0.003 and 0.043 ± 0.003 (anti-OVA ELISAs, mean ± SD of 30 microplates), respectively, and 0.042 ± 0.003 and 0.041 ± 0.002 (anti-CT ELISAs, mean ± SD of 30 microplates), respectively.

FIG. 4

Specific IgA responses against OVA and CT in serum and local secretions after priming and three booster immunizations with various doses of OVA plus CT adjuvant, normalized to total IgA. (A) Anti-OVA endpoint titers normalized to the total IgA content of the respective samples. (B) Anti-CT endpoint titers normalized to the total IgA content of the respective samples. Values represent means ± SEM of samples from six mice.

FIG. 5

Total amounts of IgA in serum and local secretions of mock-immunized mice (3% [wt/vol] sodium bicarbonate) after priming and three booster immunizations. IgA contents of fecal samples are given in micrograms per gram (dry weight) and those of all other samples are given in micrograms per milliliter of liquid. Values represent means ± SEM of samples from six mice.

FIG. 6

Relative changes of total IgA in serum and local secretions after priming and three booster immunizations with various doses of OVA plus CT adjuvant. Total IgA contents in serum and local secretions of mock-immunized animals are set at 100%. IgA production significantly higher (asterisk) than that of mock-immunized animals is indicated (one-way ANOVA, Fisher's protected least-significant-difference test, P < 0.05).