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Intranasal Immunization Using HBsAg-Acemannan Formulations Aguilar JC, Pichardo D, Leal MJ, Crombet L, Pentón E, Muzio V, Guillén G División de Vacunas. Centro de Ingeniería Genética y Biotecnología.
Papers from Biotecnología Habana`99
Congress. Code Number: ba00029 Introduction At present, the search for new adjuvants and immunological stimulators, as well as the development of new ways of delivering antigens and pharmaceuticals, is one of the lines of world research in the pharmaceutical field, especially in vaccines [1]. Acemannan is a polysaccharide composed of mannose with O-acetylations, in approximately 8 out of every 10 mannoses. lt is extracted as a major component of the mucilaginous substance or leaf gel of Aloe barbadensis Miller, a medicinal plant used throughout history. Different in vitro tests indicate that mannans activate monocytes and macrophages inducing the production of IFN-g, TNF-a, GM-CSF, IL-1b and IL-6 [2]. The purposes of this work are: a) to demonstrate the enhancing activity of acemannan on the humoral immune response against hepatitis B surface antigen (HBsAg) through intranasal inoculation (IN), and b) to select the optimal adjuvant concentration range of acemannan. Materials and Methods The first experiment was conducted in 5 groups of 8 Balb/c mice, 7 to 10 weeks old. Mice were inoculated twice, 2 weeks apart, through the nasopharyngeal route using volumes of 50 mL. All mice were ketamine-anaesthetized and inoculated as shown in figures A-D. Extraction was performed through retroorbital punction 28 days after the start of the immunization schedule. Titers were determined by an anti-total HBsAg ELISA using an international standard (mIU/mL). In the second and third experiments, different dose levels of HBsAg were tested and the systemic inoculation controls were: intramuscular (IM) (Exp. 2) and subcutaneous (SC) (Exp. 3), both adsorbed to alum. Mice were inoculated three times. Titers were determined by ELISA to detect total IgG in sera (Exps. 2 and 3) and total IgA in vaginal washes (Exp. 3). The concentration of acemannan was determined by the Antrona colorimetric method and was referred as mg of total hexoses/mL. The statistical analysis was performed using the Student’s t test and the F test to determine variance homogeneity (p < 0.05 was considered a significant difference). Results and Discussion A strong enhancing activity was evidenced in the groups in which acemannan was added (groups 2-5). Antibody titers in all groups were significantly higher than that of the control of HBsAg in PBS (group l). The excessive increase of polysaccharide concentration generated an inhibitory effect (group 5). This was probably due to an increase of the resulting viscosity. The optimal adjuvant concentration ranged between 0.150 and 0.450 mg/mL. Experiments 2 and 3 demonstrate the strong effect of acemannan when mixed with HBsAg. In both cases, the intensity of IgG titers generated in sera after alum-adjuvated and acemannan-adjuvated groups was similar (Exp. 2, groups 2 and 3, and Exp. 3, groups A and B). No difference was observed for serum IgG titers after IM and SC inoculations, compared with the corresponding dose level of HBsAg inoculated nasally in acemannan. In Exp. 2, the serum IgG response for the acemannan group was higher than that obtained with HBsAg in phosphate-buffered saline (Exp. 2, groups 1 and 2). In Exp. 3, only group A (nasally-immunized mice using acemannan) generated a strong vaginal IgA response. Conclusions 1. We have demonstrated that the nasal route can be as efficient as systemic routes in the induction of anti-HBsAg antibody responses in serum, with the advantage of inducing strong mucosal responses. 2. The acemannan optimal adjuvant concentration range is 150 to 450 mg/mL when used nasally along with HBsAg. References 1. Report of the Expert Panel VI: Concerted efforts in the field of mucosal immunology. Vaccine 1996;14:644–64. 2. Peng SY, et al. Mol. Bio-ther 1991;
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