Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 2, 2000, pp. 120

Biotecnología Aplicada 2000;17:120

Intranasal Immunization Using HBsAg-Acemannan Formulations: Kinetics and Duration of Serum IgG Responses

Aguilar JC, Pichardo D, Leal MJ, Crombet L, Pentón E, Muzio V, Guillén G

División de Vacunas. Centro de Ingeniería Genética y Biotecnología.
AP 6162, CP 10600, Ciudad de La Habana, Cuba.

Code Number: ba00033

Introduction

The potency of an adjuvant and the duration of the response generated are two important parameters in the evaluation of any new vaccine formulation. Although the ability of CT to act as a mucosal adjuvant has been confirmed by a large number of researchers [1], CT and some other related proteins do not fulfill the classical definition of an adjuvant. CT stimulates an immune response against itself, and its adjuvant activity depends on its immunogenicity [2].

The purposes of this work are: a) to investigate the duration of the antibody (Ab) response obtained using acemannan as a nasal adjuvant for hepatitis B surface antigen (HBsAg), and b) to show the kinetic of antibody appearance in serum using CT and alum as controls and as nasal and systemic adjuvants, respectively.

Materials and Methods

The schedule was carried out in 8-week-old Balb/c female mice with four inoculations: days 0, 14, 28, and 56, and the extractions were performed by retroorbital punction: days 26, 42 and 70. Titers were determined using the conventional ELISA for the detection of specific mouse IgG antibodies. All groups except group 4 were immunized nasally using volumes of 50 mL; group 4 was immunized through the subcutaneous route using volumes of 100 mL. Vaginal washes were performed with 100 mL of sterile PBS on day 70.

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

An statistical analysis after a second dose did not evidence significant differences between the alum control group and the nasal group with the same quantity of antigen. The group of mice immunized with CT as adjuvant by the nasopharyngeal route generated an antibody response higher than that of the group with the lower quantity of antigen and was not significantly different from that of the group with equal quantity of HBsAg.

After three immunizations, there was no statistical significant difference between the group of mice immunized intranasally and systemically with 2 mg. In the same way, there was no significant difference between the group of mice immunized with CT as adjuvant and the equivalent formulation under assay, both with l0 mg of HBsAg.

Considering the strong increase in titers from the second to the third dose, we worked on determining if this increase followed the same slope for the different groups assayed, after a fourth dose applied one month after the third inoculation.

Using acemannan as an adjuvant it is possible to obtain a response after a fourth dose. This response is higher than that obtained with CT in serum, as evidenced by comparing G2 and G5.

Then, through the nasopharyngeal inoculation of HBsAg mixed with polysaccharides it is possible to attain responses that are able to exceed in quantity and quality the response generated by systemic inoculations using alumina. Additionally, considering the fact that a strong mucosal antibody response was induced only by nasopharingeal route, the efficient and potentially innocuous human inoculation of HBsAg becomes a real possibility.

Conclusions

1. Nasal inoculations of HBsAg-acemannan formulations resulted in a long-term response.

2. The slope of the IgG response curve is higher for groups immunized with acemannan than for groups immuniced with alum and CT which generated stronger responses after four inoculations.

References

1. McGhee JR, et al. Vaccine 1992;10(2): 75–88.

2. Elson CO. Fed Proc 1987;46:1778.

Papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999.