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Yearly outbreaks of influenza cause significant morbidity in the general population and high mortality among individuals in high-risk groups (10). Several commercially available inactivated whole- and split-virus vaccines exist to control the spread and severity of influenza (8, 22). However, these vaccines suffer from limited efficacy in generating long-lasting immunity, particularly in the elderly, and are not sufficiently cross-reactive to protect against antigenic variants (5, 14, 17). Although these vaccines are known to induce serum immunoglobulin G (IgG) antibodies, they are poor stimulators of secretory IgA at respiratory mucosal sites and show sporadic CD8⁺ cytotoxic T-lymphocyte (CTL) activation (3, 10, 21). Efforts are currently under way to develop influenza vaccines that generate significant secretory IgA, as well as maintain high serum IgG titers, by exploiting mucosal immunization (5, 6, 16, 28). Our group has focused on investigating the activity of influenza virus hemagglutinin (HA) administered intranasally (i.n.) with mutant heat-labile enterotoxins (LTs). One such mutant toxin, LT-R72, shows significantly reduced toxicity in vitro and in vivo yet maintains potent mucosal adjuvanticity (9). In the current studies, i.n. administration of influenza virus HA to mice with LT-R72 was compared to intramuscular (i.m.) immunization for induction of serum antibody responses, generation of IgG1 and IgG2a antibody subclasses, HA inhibition titers, and IgA antibody levels in mucosal secretions.

Vaccines. Purified monovalent A/Beijing8-9/93 (H3N2) influenza virus antigen was provided by Chiron Vaccines, Siena, Italy. Dosing was based on HA content as assayed by single radial immunodiffusion as described previously (15). LT-R72 was prepared as described previously (18). All immunogens were prepared in phosphate-buffered saline.

Immunization and sample collection. Groups of 10 female BALB/c mice (Charles River Laboratories, Wilmington, Mass.), 6 to 10 weeks old, were immunized as described in Table 1. Immunizations were made by i.m. (50 µl) injection into the posterior thigh muscle and by i.n. (25 µl) dropwise additions into the alternate nares of unanesthetized animals. Serum, saliva wash (SW), vaginal wash (VW), and terminal nasal wash (NW) samples were collected from individual animals in accordance with the sample collection schedule in Table 1 by using methods described previously (27).

Antibody ELISA. Serum samples from individual animals were assayed for total anti-HA Ig (IgG plus IgA plus IgM) titers by a 3,3',5,5'-tetramethylbenzidine-based colorimetric enzyme-linked immunosorbent assay (ELISA) as previously described with A/Beijing93 HA as the coating antigen (11). A₄₉₀ was measured by using a standard ELISA reader. The titers represent reciprocal serum dilutions giving an A₄₉₀ of 0.5 and were normalized to a serum standard assayed in parallel. SW, VW, and NW samples from individual animals were assayed for HA-specific IgA titers, and pooled serum samples were assayed for relative levels of HA-specific IgG1 and IgG2a antibodies by using a bioluminescence immunosorbent assay as previously described with A/Beijing93 HA as the coating antigen (27). A goat anti-mouse IgA-biotin conjugate (EY Laboratories, San Mateo, Calif.) was presaturated with purified mouse IgG (Sigma Chemical Co., St. Louis, Mo.) to reduce cross-reactivity. Quantitation was based on the number of relative light units representing the total luminescence integrated over 3 s (arbitrary units). Titers represent log dilution values linearly extrapolated from the log of the number of relative light units to a cutoff value at least 2 standard deviations above the mean background.

HI assay. Serum samples pooled by group were assayed for hemagglutination inhibition (HI) titer by the Viral and Rickettsial Disease Laboratory (Department of Health Services, Berkeley, Calif.) using a standard ELISA. The HI assay is based on the ability of sample sera to inhibit the agglutination of goat erythrocytes in the presence of HA antigen, and results are expressed as the reciprocal dilution required for complete inhibition (12, 13).

Statistics. Log anti-A/Beijing93 HA titers from individual animals (see Fig. 1) were analyzed by using a Fisher least-significant-difference procedure (1). Comparison intervals were presented such that nonoverlapping bars imply a statistically significant difference between means of greater than 5% (P  0.05). Log anti-A/Beijing93 HA IgA titers from individual animals (see Fig. 3) were analyzed for significant differences between groups (P  0.05) by using a median sign test. Results for antibody subclass analysis (see Fig. 4) represent means and standard deviations of replicate assay determinations (n  6) of pooled samples.

Comparison of i.n. and i.m. immunizations. Groups of 10 mice were immunized i.n. with LT-R72-HA formulated at two dose levels and compared to mice immunized i.m. with HA alone (Table 1). Serum antibody responses after i.n. immunization with A/Beijing93 HA and LT-R72 (Fig. 1) were significantly higher than responses obtained with i.m. immunization in most cases. Of the groups tested, immunization with 10 µg of HA and 25 µg of LT-R72 i.n. (IN high) resulted in the strongest serum antibody responses, which, after three doses, were approximately a log higher than those achieved by i.m. vaccination (P = 0.0157, 0.0044, and 0.0001 at days 21, 35, and 49, respectively). Notably, the low-dose (1 µg of HA with 10 µg of LT-R72) i.n. group had significantly higher responses than did the i.m. immunization group, in most cases (P = 0.0272 and 0.0001 at days 21 and 49, respectively).

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Comparison of the effects of i.m. and i.n. administrations of A/Beijing93 HA on antigen-specific serum antibody responses. Shown are mean anti-A/Beijing93 HA serum Ig antibody titers of groups of 10 mice immunized as shown in Table 1. Asterisks indicate groups whose values are significantly greater than those of the corresponding i.m. immunized group (P  0.05).

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Comparison of the effects of i.m. and i.n. administrations of a bivalent vaccine containing A/Beijing93 HA on serum HI titers. The data shown is for pooled serum from groups of 10 mice immunized as shown in Table 1.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Comparison of the effects of i.m. and i.n. administrations of A/Beijing93 HA on antigen-specific IgA responses. Mean anti-A/Beijing93 HA IgA antibody titers of MW samples from groups of 10 mice immunized as shown in Table 1 (± 95% confidence intervals) are shown. Asterisks indicate groups whose titers are significantly greater than those of the corresponding i.m. immunized groups (P  0.05).

View larger version (31K): [in this window] [in a new window]   FIG. 4.   Comparison of the effects of i.m. and i.n. administrations of A/Beijing93 HA on the ratio of antigen-specific IgG1 to IgG2a antibodies in the sera of groups of 10 mice immunized as shown in Table 1.