P16 Tomato lectin as an adjuvant for oral vaccine delivery

392 not work as a carrier for a hapten, i.e., a low molecular weight drug [31. Methods: Human serum albumin was covalently coupled to starch microparticles as a model antigen. Groups of mice (BALB/c) were injected i.v. in the lateral tail vein, i.m. in the hind leg or ip., with 0.5 mg of empty microparticles or USA-conjugated micropa~icles. Three different HSA-microparticles were used with various epitope density, 0.30-, 17.6 and 168-&g HSA/mg microparticles. As a positive control, groups of mice were injected i.p. with HSA in Freund’s complete adjuvant (FCA). Also, groups of mice were injected i.v. with soluble HSA in physiological saline or with physiological saline as a negative control. The booster injection was given on day 2 1 after the first injection. Blood samples were collected from the orbital plexus on days 4, 7, 11, 162 1, 28, 35, 77, 150 and 2 10. The blood samples were analysed for anti HSA antibodies using a HSA specific ELISA assay. Results: All administration routes used for injection of HSA-conjugated microparticles and the positive control with HSA/FCA gave the classical response of a T-dependent antigen, with a typical memory effect. There were no significant differences in antibody titers comparing the iv., i.m. and i.p. administration routes. The immune response from the HSAconjugated microparticles administrated i.p. with 84,8.8 and 0.15 pg HSA per dose gave a similar immune response to the co~esponding dose HSA administrated i.p. with FCA, as a positive control. Free HSA in physiological saline, 84 or 8.8 @gHSA per dose, administrated i.v. gave no immune response. Conclusions: In this study, we have shown that HSA-conjugated starch microparticles can act as a powerful adjuvant for antibody production. High specific antibody titers with long duration were elicited with all aministration routes used. The powerful ability of microparticle bound HSA to evoke a humoral response is shown by the fact that even the lowest dose of HSA applied, 0.15 ,ug, did elicit an immune response. The mechanism behind the adjuvant effect of starch microparticles is probably due to the fact that starch microparticles in vivo are taken up by the macrophages of the reticuloendothelial system (RES). After degradation of the antigenconjugated microparticles, the antigens are presented on the surface of the macrophage to cells of the immune system. Thus, this is an efficient way to focus large amounts of antigen to cells in the immune system which are specialized to present antigen, i.e., macrophages. The use of microparticles as an adjuvant should reduce the amount of antigen required for successful immunization, thus making vaccine production more economical and more feasible.

References

P16 Tomato lectin as an adjuvant for oral vaccine delivery. 8. Naisbetta, B. Wagenaarb, J.A. Bouwstra” and H.E. Junginget?, aLeiden/Amsterdam Center for Drug Research, Division of Pharmaceutical Technology, Center for Bio-Pharmaceutical Sciences, Leiden Ilniversity, The Netherlands; bDepartment qf Pharmaceutics and ~iopharmaceutics, University of Kiel, Germany Oral immunization has many advantages over the parenteral route, which is currently the most commonly employed route for vaccination. However, attempts to stimulate local secretory immune responses by oral antigen administration have met with limited success due to degradation of the antigen and the use of synthetic peptides and proteins which are weakly immunogenic [ 11. Here, we investigate the ability of antigen-containing microparticles of poly( LX-lactide), (PLA), to initiate a mucosal immune response in the saliva after oral feeding, using tomato lectin (TL), co-fed with the particles as an immunological adjuvant. Earlier studies have found TL to be a potent immunogen; microgram amounts of TL were capable of initiating an IgA immune response in intestinal washings after oral feeding [ 21. Particles of 1- 10 pm, containing a model protein (human serum albumin-HSA), were prepared from PLA by a spraydrying process. In vitro antigen release was determined by measuring HSA release from the particles in PBS, pH 7.4 at 37°C in flow-throu~ cells [ 3 1.Groups of adult male Wistar rats were fed intragastrically (in 0.2 mM carbonate buffer) on days 1, 2 and 14, with HSA or particles containing HSA+TL. Saliva production was stimulated by injecting 1 pg pilocarpine/g body weight, i.p. and saliva samples were collected on days zero, 7, 9, 15, 17 and 22. The level of IgA in the saliva was measured by ELISA. Particles containing 9.4% HSA, released almost 40% of their encapsulated HSA within the first 2 days, and up to 50% after 7 days. Feeding HSA alone resulted in little or no antibody response. However, co-feeding TL with HSA resulted in a significant increase in HSA-specific antibodies. Co-feeding TL with the particles enhanced the antibody response still further. These results show that, in agreement with other authors, encapsulation of a weakly immunogenic protein can lead to an increase in specific salivary IgA production after oral feeding. These results also suggest that TL does have potential as an immunological adjuvant when co-fed with either soluble or encapsulated proteins. Acknowledgements: We are grateful to the Wellcome Trust for financing this project in the form of a Travelling Fellowship to RN. References

1 J. Mestecky, J.H. Eldridge, Curr. Opin. Immunol.,

1 R. Bomford, Parasitology Today. 5 ( 1989) 41-46. 2 P. Artursson, P. Edman, 1.J. Sjijholm, Pharmacol. Exp. Ther., 234 (198.5) 255-259. 3 P. Stjlmkvist, L. Degling, I. Sjbholm, J. Pharm. Sci. 80 (1991) 436-440.

3 ( 199 1) 492-495. 2 B. Naisbett, PhD Thesis, University of Keele, England (1991). 3 B. Wagenaar, PhD Thesis, University of Kiel, Germany (1993).