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One of the main objectives of current vaccine research is the development of vectors capable of inducing strong immune responses against protective antigens when delivered by a mucosal route. Compared to standard systemic routes, mucosal routes offer several advantages, including the ease of administration in a noninvasive fashion and diminished risk of contamination, which may be caused by injection. Different formulations have been developed in recent years to increase the immunogenicity of mucosally delivered antigens. One of these formulations is based on multilamellar liposomes containing dimyristoylphosphatidylcholine (DPPC) and dipalmytoylphosphatidyl glycerol (DMPG) (17). We have recently demonstrated that the association of an antigen with such liposomes was able to induce a protective immune response when given by the oral route (11). Although incorporation of the antigen into liposomes certainly enhanced its immunogenicity when administered by the oral route, large amounts of antigen were still needed.

We reasoned that immunogenicity might be further enhanced if the liposomes were targeted to mucosal sites by the addition of specific adherence molecules. It has recently been reported that coating liposomes with immunoglobulin A (IgA) enhances their uptake into Peyer's patches and thereby increases both the mucosal and systemic immune responses after rectal administration together with cholera toxin (27). Furthermore, the B subunit of cholera toxin was also shown to target microparticles to the M cells of Peyer's patches, resulting in an increase in immune responses (6). As an alternative to oral or rectal delivery of antigens, we explored the intranasal delivery of liposome formulations containing a schistosome model antigen and the Bordetella pertussis filamentous hemagglutinin (FHA) as an adhesin specific for the respiratory tract tissues (for a review, see reference 13). FHA expresses several adherence activities, including binding to carbohydrates on respiratory cilia (24); binding to sulfated carbohydrates (9), which is involved in the attachment of B. pertussis to epithelial cells and the extracellular matrix; and binding to macrophage integrins via an RGD sequence (10). Moreover, FHA is a strong mucosal immunogen, as evidenced by the high levels of anti-FHA antibodies produced in humans infected with B. pertussis (26).

In this study, therefore, we incorporated FHA into liposomes together with the Schistosoma mansoni glutathione S-transferase (Sm28GST) as a model antigen. Here, we show that the enhancement of the immune response to Sm28GST was dependent on the FHA dose, without resulting in a change in the isotypic profile.

Specific immune response obtained after intranasal administration of Sm28GST liposomes. Recombinant Sm28GST (rSm28GST) produced in Saccharomyces cerevisiae and provided by Transgène S.A. (Strasbourg, France) was affinity purified as described previously (22). Liposomes were prepared as previously described (11) by using a mixture of two lipid components in a 9:1 (DPPC to DMPG) (Genzyme, Cambridge, Mass.) molar ratio. In order to determine the minimal immunizing dose of rSm28GST when incorporated into liposomes, we prepared liposomes with three different concentrations of rSm28GST (0.2, 1, and 5 mg/ml). However, the proportion of protein incorporated into liposomes at these concentrations was not strictly linear and corresponded to 75, 70, and 60%, respectively, of protein incorporation. The liposomes were washed three times in phosphate-buffered saline (PBS) and centrifuged at 10,000 × g for 30 min. The pellet was resuspended in PBS and adjusted to 400 µl (2 µmol of phospholipids per 40 µl). Six-week-old female OF1 mice (Iffa Credo, L'Arbesle, France) were anesthetized with 200 µl of 5% sodium pentobarbital (Sanofi, Libourne, France) per 10 g of body weight given intraperitoneally and immunized with 40 µl of the liposome suspension or PBS deposited in the nostrils. Thus, the dose administered per mouse at each instillation corresponded to 15, 70, or 300 µg of rSm28GST, depending on the concentration. The liposome preparations were given twice intranasally with a 2-week interval. The specific immune responses in the sera were analyzed 2 weeks after the second administration (i.e., on day 27). As shown in Table 1, anti-Sm28GST IgG1, IgG2a, and IgG2b were detected in significant amounts on day 27, and antibody levels increased in proportion to the dose of rSm28GST administered. A weak serum anti-Sm28GST IgA response was observed with the highest dose of rSm28GST. Individual analyses indicated that one mouse in five did not produce a detectable anti-Sm28GST immune response, even with the largest dose of antigen.

Characterization of rSm28GST-FHA liposomes. FHA was purified by heparin-Sepharose chromatography as previously described (15) with B. pertussis BPRA, a strain lacking the pertussis toxin gene (2), as the source.

View larger version (63K): [in this window] [in a new window]   FIG. 1.   Immunoblot analysis of Sm28GST-FHA liposomes. Five microliters of each liposome suspension used for in vivo experiments (corresponding to 0.25 µmol of phospholipids) was analyzed by immunoblotting with rat hyperimmune polyclonal anti-FHA (left panel) and rat hyperimmune polyclonal anti-Sm28GST (right panel). The nitrocellulose sheets were then incubated with anti-rat antibodies coupled to alkaline phosphatase (Sigma) (1/4,000 in PBS containing 1% fat dry milk) and developed with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (Promega, Madison, Wis.). Lanes 1 contained 3 µg of free rSm28GST, and lanes 2 contained 1.5 µg of free FHA. Lanes 3, 4, and 5 contained the three different Sm28GST-FHA liposome preparations (i.e., high, medium, and low concentrations of FHA, respectively). Lanes 6 contained the Sm28GST liposome preparation. The molecular mass markers, expressed in kilodaltons (kD), are given in the left margin.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   FACS analysis of Sm28GST-FHA liposomes. Sm28GST liposomes and Sm28GST-FHA liposomes were labeled with a rat anti-FHA polyclonal antibody followed by fluorescein isothiocyanate-labeled anti-rat antibody and then subjected to FACS analysis. The numbers (nb) of liposomes were plotted against fluorescence intensity as indicated.

Comparison of immune responses obtained after intranasal administration of rSm28GST-FHA liposomes containing increasing doses of FHA. By considering the level of FHA incorporation to be comparable to that of Sm28GST, the actual doses of FHA at each instillation could be estimated at 3, 0.3, and 0.03 µg for the three separate liposome preparations. No FHA was added for the control liposomes. OF1 mice were immunized twice intranasally with a 2-week interval with either PBS, control rSm28GST liposomes (40 µg per administration), or one of the three rSm28GST-FHA liposomes described above. Two weeks after the second instillation, anti-Sm28GST and anti-FHA antibody responses were analyzed in pooled sera of the immunized mice (Table 2). The sera were analyzed by enzyme-linked immunosorbent assays as previously described (11). For specific detection, microtiter plates were coated as a first step with 50 µl (per well) of a solution containing either 5 µg of FHA per ml or 10 µg of Sm28GST per ml. Anti-Sm28GST antibodies were detected with either liposome preparation. However, their titers increased proportionally with the amount of FHA present in the liposomes. The addition of 0.03 µg of FHA resulted in a threefold increase in anti-Sm28GST antibody titers, and the addition of 0.3 µg of FHA increased the anti-Sm28GST titers approximately 10-fold. The level of specific antibodies to the parasite antigen obtained with 40 µg of Sm28GST in combination with 3 µg of FHA was comparable to that obtained with 300 µg of Sm28GST without FHA (Table 1), demonstrating the potential of FHA to increase the immune response.