INTRODUCTION

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Secretory immunoglobulin A (sIgA) is considered a cornerstone of the immunological defense mechanisms that protect mucosal surfaces. sIgA is secreted in gram amounts per day across the mucosal surfaces in humans (14, 20) and has been shown to confer protection against a number of bacterial and viral pathogens, such as Vibrio cholerae (33), Salmonella enterica serovar Typhimurium (21), respiratory syncytial virus (32), rotavirus (28), and influenza virus (27).

The recognition of sIgA as a powerful means to protect against enteric pathogens led to considerable interest in the development of mucosal vaccines in recent years. However, the induction of sIgA responses is an onerous task. In addition to being delivered via the mucosal route, the antigen should be formulated so that it is taken up by M cells, a specialized epithelial cell type located in the epithelium over the organized mucosa-associated lymphoid tissue (11, 22). Alternatively, ADP-ribosylating toxins like cholera toxin (CT) must be coadministered as mucosal adjuvants (4).

Beyond that, any vaccination requires some measurement of efficacy, such as the titer of specific IgA responses in local secretions. Unfortunately, analysis of antibody responses in the gut is complicated by the fact that sampling of intestinal secretions requires invasive methods which are not practicable for routine diagnostics. To overcome this problem, specific sIgA in fecal samples has been used as a substitute for directly sampled intestinal specimens (3, 12, 16), but the validity of this approach has been questioned (7). Intestinal lavage techniques have been proposed as an alternative technique (1, 5, 7, 24), but again sampling must be carried out under the supervision of a physician and may be too labor- and cost-intensive for routine diagnostic purposes.

Since IgA plasma cells generated after oral immunization are known to populate remote mucosal sites as well (19, 31), it seems conceivable that specific sIgA responses at other mucosal surfaces may closely correlate to those in the intestine and thus could serve as predictors for sIgA secretion in the gut after oral immunization. Seeking alternative measures for sIgA status at small and large intestinal surfaces, we carried out a comprehensive intragastric immunization study in mice using the model antigen ovalbumin (OVA) plus CT adjuvant and analyzed the specific IgA content in excretions, serum, and mucosal secretions from various sites in search for diagnostically important relationships between the IgA responses. When highly sensitive detection systems were used, specific antibody responses against both ovalbumin and CT were readily detectable in humoral samples, secretions, and excretions, but strong correlations could be established only between urinary, salivary, and serum IgA levels and IgA from intestinal surfaces for the lowest dose of OVA.

	MATERIALS AND METHODS

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Animals. Female BALB/c mice were obtained from Charles River Wiga (Sulzfeld, Germany). The animals had been reared and were kept on a chicken egg protein-free rodent chow (Altromin 1324; Altromin, Lage, Germany) throughout the study. They were 8 weeks of age at the beginning of the immunization experiments.

Materials and reagents. Animal feeding needles (20 gauge by 1.5 in. [ca. 4 cm]) were obtained from Popper & Sons (New Hyde Park, N.Y.), UniWick filters (25-mm long, 2.5-mm diameter) from Polyfiltronics (Rockland, Mass.) and glass applicators (10-cm long, 4-mm outer diameter, 2.5-mm inner diameter, smoothed and bevelled at one end) were custom-made by Glasgerätebau Ochs (Bovenden-Lenglern, Germany). High-binding polystyrene enzyme-radioimmunoassay microtiter plates were from Corning Costar (Bodenheim, Germany).

Antigen preparation and intragastric immunizations. At the beginning of the immunization study, the total amounts of OVA antigen and CT adjuvant required for all immunizations were dissolved in sterile 3% (wt/vol) sodium bicarbonate or reconstituted in sterile water, snap-frozen in liquid N₂, and stored in single-use aliquots at 70°C. Immediately before use, the respective antigen and adjuvant aliquots were warmed to 37°C and mixed by gentle rocking.

Sample collection and extraction. Feces were sampled 2 days before the first and 10 days after each immunization. The animals were separated individually into wire mesh cages equipped with metal trays underneath, and 20 to 30 freshly voided, urine-free fecal pellets were sampled over 3 to 8 h. Urine (50 to 500 µl) was sampled 10 days after the last immunization from the metal trays along with the fecal samples. All samples were kept on ice throughout the entire sampling procedure before they were snap-frozen in liquid N₂ and stored at 70°C.

Quantitation of anti-OVA and anti-CT immunoglobulins. Microtiter plates were coated with 75 µl of either OVA (5 µg/ml) in 50 mM sodium acetate buffer (pH 5.0) or CT (5 µg/ml) in 10 mM sodium phosphate buffer (pH 7.0)-10 mM NaCl per well overnight at 4°C. Plates were washed three times with 350 µl of PBST (D-PBS containing 0.05% [vol/vol] Tween-20) per well, and nonspecific binding sites were blocked with 250 µl of PBS-Blotto (D-PBS containing 5% [wt/vol] nonfat dry milk) per well for 5 h at room temperature. Plates were washed four times with PBST before 75 µl of serially diluted sera, anti-OVA standard (mouse anti-chicken OVA IgG monoclonal antibody OVA-14; starting dilution, 5.2 ng/ml), or fecal or filter extracts in PBS-Blotto was applied per well, and the plates were incubated overnight at 4°C.

Quantitation of total IgA. Microtiter plates were coated with 75 µl of 5-ng/ml unlabeled goat anti-mouse IgA in D-PBS per well overnight at 4°C. The plates were washed, blocked, and washed again as described above before 75 µl of serially diluted IgA standard (purified mouse IgA; starting dilution, 16 µg/ml), sera, or fecal or filter extracts in PBS-Blotto was applied per well, and the plates were processed as described above using horseradish peroxidase-labeled goat anti-mouse IgA as the secondary antibody.

Determination of cross-reactivities of class-specific anti-mouse immunoglobulin detection reagents. Microtiter plates were coated with 75 µl of 7-µg/ml unlabeled goat anti-mouse IgA or goat-anti mouse IgG in D-PBS per well overnight at 4°C. The plates were washed, blocked, and washed again as described above before 75 µl of serially diluted IgA or IgG standard (purified mouse IgA or IgG; starting dilution, 6.4 µg/ml) in PBS-Blotto was applied per well, and the plates were processed as described above. Both anti-mouse IgA- and anti-mouse IgG-horseradish peroxidase conjugate were reacted with captured IgG as well as captured IgA.

Data analysis and statistics. Specific antibody responses were expressed as endpoint titers, being the reciprocal of the highest dilution that gave a reading above the cutoff. The cutoff was defined as the upper limit of a 99.5% confidence interval above the mean control level and was calculated by t statistics (10), e.g., for five mock-immunized control animals and a 99.5% confidence interval, the cutoff is calculated as mean_controls + 5.0 × SD_controls where SD is the standard deviation. Titers were transformed logarithmically [log (titer+1)] for calculation of group means and standard errors of the means (SEM) or used directly for correlation analysis. Total IgA amounts were determined on the basis of four-parameter curve fit approximations of IgA standard titration curves using the readouts of the unknown samples at the steepest slopes of their titration curves (SOFTmax Pro v1.0; Molecular Devices). For endpoint titers standardized on total IgA contents (relative endpoint titers), endpoint titers were divided by the total IgA concentration of the respective undiluted sample.

IgG

IgA

(1)

IgG

(2)

	RESULTS

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Development of highly sensitive immunoassays for quantitation of anti-OVA and anti-CT immunoglobulins. In order to detect even minute amounts of immunoglobulins in diluted samples such as urine and saliva, we established highly sensitive enzyme-linked immunosorbent assay (ELISA) systems for antibodies against OVA and CT. For coating the microtiter plates, best results were obtained with OVA at 5 µg/ml in 50 mM sodium acetate (pH 5.0) and CT at 5 µg/ml in 10 mM sodium phosphate (pH 7.0)-10 mM sodium chloride. Under these conditions, assay sensitivity could be increased by a factor of 50 for detection of anti-OVA and by a factor of 2 for detection of anti-CT immunoglobulins, compared to standard ELISA coating conditions (2 µg of antigen per ml in sodium carbonate-bicarbonate [pH 9.6]) (6, 13). Assay sensitivity was further improved by using horseradish peroxidase-labeled secondary reagents in combination with a novel tetramethylbenzidine-based colorimetric ELISA substrate system which we developed for this purpose (9). Using both optimized coating conditions and the novel substrate system, a detection limit for a monoclonal anti-OVA IgG1 of 24 pg/ml was achieved, which corresponds to a concentration of 160 fM or 12 attomol of specific immunoglobulin per well.

Lack of correlations between humoral and fecal immune responses over repeated intragastric immunizations with OVA and CT. To assess whether fecal and serum immunoglobulin responses may correlate after intragastric immunization, a typical immunization study with priming and several booster immunizations was performed using the model antigen OVA and CT adjuvant. Groups of six BALB/c mice were gavaged on days 0, 21, 35, and 49 with 0.2, 2, or 20 mg of OVA plus 10 µg of CT or with 10 µg of CT alone. This immunization procedure had no apparent impact on the health status and the thriving of the animals, as assayed by visual inspection and determination of body mass gain (data not shown). Feces and blood samples were collected 10 to 11 days after each immunization and tested for their content of specific IgA and IgG against OVA and CT.

View larger version (14K): [in this window] [in a new window]   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.

View larger version (15K): [in this window] [in a new window]   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.

Distribution of the antibody immune responses at different mucosal effector sites after intragastric immunization with OVA and CT. To perform a cross-comparison of the local IgA responses, samples of local secretions in the gut together with saliva and vaginal washes were collected after killing the animals 11 days after the last immunization. Urine had been sampled along with feces no more than 24 h earlier. On the basis of these samples, distribution profiles for the local IgA responses against OVA and CT were established.

View larger version (57K): [in this window] [in a new window]   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.

View larger version (58K): [in this window] [in a new window]   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.

Antigen type and dose dependency of correlations between specific antibody responses at different mucosal sites. On the basis of the local anti-OVA and anti-CT IgA responses, a comprehensive correlation analysis was carried out in which the anti-OVA and anti-CT immunoglobulin responses were compared.

Contribution of plasma transudate to immunoglobulins in feces and local secretions. In order to rule out an artifactual cause for correlations between serum and filter wick-sampled secretions due to physical damage to the intestinal epithelium by the filter wick fabric or capillary suction, we wanted to compare the naturally occurring plasma leak into feces with that observed after sampling with UniWick filters. Assuming a nonselective effusion of plasma at sites of leakage, any plasma protein marker, such as albumin or IgG, allows the determination of the amount of plasma that leaked into a mucosal sample. We used IgG as a marker and determined the anti-OVA IgG and IgA titers in serum, feces, and jejunal secretions for three randomly selected animals from each immunization group. The relative contribution of plasma transudate to the sampled volume of intestinal secretions as well as to the specific IgA contents in feces and intestinal secretions was computed with equations 1 and 2. The results are summarized in Table 3 and show that plasma leakage contributes very little to the specific mucosal IgA responses. Highest leakage was observed for the 20-mg OVA dose group, for which no correlations between serum and jejunal IgA existed. We conclude that the correlations between immunoglobulins in serum and filter wick-sampled secretions are not caused by plasma transudate.

Lack of influence of intragastric immunizations on total IgA release at different mucosal effector sites. Since the lack of correlations for the 20-mg OVA dosage could be due to an overloading of the IgA transport systems in the mucosal epithelium, we investigated whether the total IgA output might rise upon intragastric immunization in an antigen dose-dependent manner. For normalization, the total IgA contents of sera, secretions, and excretions of immunologically naive, mock-immunized animals were determined (Fig. 5). While the IgA contents of feces and urine differ tremendously, total IgA levels in serum and small intestinal secretions are almost indistinguishable.

View larger version (24K): [in this window] [in a new window]   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.

View larger version (48K): [in this window] [in a new window]   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).

	DISCUSSION

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Direct analysis of the antibody immune status at remote mucosal surfaces requires labor- and cost-intensive methods which are not feasible for routine immune surveys. For that reason, easy-to-sample specimens, such as saliva and feces, were proposed as substitute measures to predict the antibody immune status at less accessible mucosal sites. Unfortunately, there are conflicting reports about the usefulness of this approach (2, 3, 7, 12, 16).

We therefore carried out an intragastric immunization study in mice with different doses of the model antigen OVA and the mucosal adjuvant CT and looked for diagnostically important relationships between immune responses at different effector sites. The antibody contents of the respective samples were expressed as endpoint titers because the endpoint titer technique was shown to more faithfully reflect the actual antibody content of the samples (23), to allow lower detection limits (9), and to be more precise for highly diluted samples (17) than some assays based on standard curves.

Our results demonstrate that strong correlations between specific antibody responses at different effector sites can indeed be established, but the existence of such relationships appears to depend on the type and dose of antigen used, a possible reason for the apparent conflicts between previous reports.

On the practical side, this opens up the possibility of using urine, saliva, and serum samples as indirect measures to predict antibody release in the small intestines after intragastric vaccination. One problem associated with immunoassays based on urine or saliva samples, however, is the high dilution of these specimens. Without optimizing our ELISA system, we would not have been able to detect any anti-OVA IgA responses in urine or saliva after vaccination with the 0.2-mg OVA dose. This might explain the almost complete lack of information about specific IgA responses in urine samples after intragastric immunization or infection. Only one report (18) describes the occurrence of Campylobacter jejuni-specific IgA in feces and urine after naturally acquired infection with C. jejuni, albeit in a qualitative manner only. In light of the fact that modern diagnostic tools like immuno-PCR (29), luminometry (15), and the use of poly-horseradish peroxidase (30) lower the detection limits to almost the single-molecule level, we are confident that monitoring the urinary or salivary IgA response of mucosal vaccinees would be a rapid and cheap method for mass screening. The high relative amount of specific IgA in urine which was not different from that in serum, intestinal secretions, or feces substantiates this concept.

The apparent antigen type and dose dependency of correlations between IgA responses at different sites, however, is a handicap that limits the usefulness of indirect measures for intestinal immune protection. We therefore tried to elucidate the reasons for this behavior. Since higher antigen doses also induced higher anti-OVA immunoglobulin titers, we first speculated that total IgA release at mucosal surfaces increases in an analogous manner and eventually becomes saturated, abolishing correlations between different sites. Yet total IgA release at the mucosal surfaces of immunized animals was, for the most part, not significantly different from that of buffer-treated controls. Obviously, the sIgA transport systems in the gut were still far from being saturated after all intragastric immunizations. This is supported by the observation that we did not see any negative correlations between anti-OVA and anti-CT immune responses in the gut, which would have occurred if both IgA responses competed for an insufficient number of poly(Ig) receptors. Also, the anti-CT responses were uncorrelated even in the absence of anti-OVA IgA, and the extremely high anti-CT IgA responses did not disturb the correlations of the anti-OVA titers for the low OVA dose.

We therefore favor a different explanation based on a possible clonal homing behavior of mucosal IgA plasma cells. In two recent publications, Quiding-Järbrink et al. (25, 26) demonstrated compartmentalization of mucosal IgA responses due to differences in the homing receptor equipment of the B-cell clones expanded. Consequently, the smaller the number of B-cell clones, the more compartmentalization should arise. This raises the question of how different types and doses of antigen could affect the clonality of a B-cell response. One conceivable but still speculative explanation might be found in the stability of the antigens. The more resistant an antigen is against intestinal digestion, the more homogeneous will be the material that is delivered to the gut-associated lymphoid tissue. Consequently, fewer B-cell clones are necessary to clear this antigen. On the other hand, the more fragments are generated in the gut and taken up, the more clones must be activated to eliminate the antigen. Thus, more-stable antigens like CT may generate less clonal diversity and therefore a lower number of site-to-site correlations, as we observed. High antigen doses could mimic a similar situation, since the higher the antigen concentration, the smaller a fraction of antigen will be broken down by the digestive enzymes in the time window between application and uptake. Experiments to test this hypothesis are under way in our laboratory.