Enhanced Liposomal Vaccine Formulation and Performance: Simple Physicochemical and Immunological Approaches

Abstract

The Dtxd (Diphtheria toxoid) was the first antigen encapsulated within liposomes, their adjuvant properties were discovered (their capacity to enhance the vaccine immunogenicity). The point here is not to propose a new method to prepare this lipossomal vaccine. The central idea is to give new dresses for old vaccines by using classical and well-established liposome preparation method changing only the encapsulation pH and the immunization protocol.

The most appropriate method of Dtxd encapsulation within liposome was based on lipid film hydration in 100 mM citrate buffer, pH 4.0. This was accompanied by changes on protein hydrophobicity, observed by CD and fluorescence spectroscopies. Whenever the Dtxd exposed its hydrophobic residues at pH 4.0, it interacted better with the lipossomal (observed by electrophoretic mobility) film than when its hydrophobic residues were buried (pH 9.0). The Dtxd partition coefficient in Triton-X114 and the acrylamide fluorescence quenching were also pH dependent. Both were bigger at pH 4.0 than at pH 9.0. The relationship protein structure and lipid interaction was pH dependent and now it can be easily maximized to enhance encapsulation of antigens in vaccine development.

Mice were primed with formulations containing 5 μg of Dtxd within liposomes prepared in pH 4.0 or 7.0 or 9.0. The boosters were done 38 or 138 days after the first immunization. The IgM produced by immediate response of all lipossomal formulations were higher than the control (free protein). The response patterns and the immune maturity were measured by IgG₁ and IgG_2a titrations. The IgG₁ titers produced by both formulations at pH 4.0 and 7.0 were at least 2² higher than those produced by mice injected lipossomal formulation at pH 9.0. When the boosters were done, 138 days after priming the mice produced a IgG_2a titer of 2⁹ and the group that received the booster 30 days after priming produced a titer of 2⁵. The strongest antibody production was the neutralizing antibody (2⁴⁵ higher than the control) produced by those mice injected with lipossomal formulation at pH 4.0 with the booster done 138 days after priming. The simple change on lipossomal pH formulation and timing of the booster enhanced both antibody production and selectivity.

Keywords: :

• liposome vaccine
• particulate adjuvant
• antigen microencapsulation

Notes

Centers for Disease Control 1991 Diphtheria, tetanus, and pertussis: Recommendations for vaccine use and other preventive measures—Recommendations of the Immunization Practices Advisory Committee (ACIP). MMWR, 40(RR-10): 1–28.