RESUMEN
The interactions between three different protein antigens and dioctadecyldimethylammonium bromide (DODAB) dispersed in aqueous solutions from probe sonication or adsorbed as one bilayer onto particles was comparatively investigated. The three model proteins were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from Taenia crassiceps (18/14-Tcra) and a recombinant, heat-shock protein hsp-18 kDa from Mycobacterium leprae. Protein-DODAB complexes in water solution were characterized by dynamic light scattering for sizing and zeta-potential analysis. Cationic complexes (80-100 nm of mean hydrodynamic diameter) displayed sizes similar to those of DODAB bilayer fragments (BF) in aqueous solution and good colloid stability over a range of DODAB and protein concentrations. The amount of cationic lipid required for attaining zero of zeta-potential at a given protein amount depended on protein nature being smaller for 18 kDa/14 kDa antigens than for BSA. Mean diameters for DODAB/protein complexes increased, whereas zeta-potentials decreased with NaCl or protein concentration. In mice, weak IgG production but significant cellular immune responses were induced by the complexes in comparison to antigens alone or carried by aluminum hydroxide as shown from IgG in serum determined by ELISA, delayed type hypersensitivity reaction from footpad swelling tests and cytokines analysis. The novel cationic adjuvant/protein complexes revealed good colloid stability and potential for vaccine design at a reduced DODAB concentration.
Asunto(s)
Adyuvantes Inmunológicos/química , Lípidos/química , Compuestos de Amonio Cuaternario/química , Animales , Antígenos Bacterianos/inmunología , Antígenos Helmínticos/inmunología , Cationes/química , Cationes/inmunología , Bovinos , Células Cultivadas , Química Farmacéutica , Citocinas/análisis , Estabilidad de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Femenino , Hipersensibilidad Tardía/inmunología , Lípidos/inmunología , Ratones , Ratones Endogámicos BALB C , Mycobacterium leprae/inmunología , Nanopartículas , Tamaño de la Partícula , Compuestos de Amonio Cuaternario/inmunología , Albúmina Sérica Bovina/inmunología , Taenia/inmunologíaRESUMEN
Polymeric microspheres containing diphtheria and tetanus toxoids were prepared without protein stabilizers. A vaccine containing 2 Lf(tetanus) and 0.4 Lf(diphtheria) was injected either in BALB/c mice or in guinea-pigs. As control, a group received the alum-adsorbed unencapsulated toxoids. In mice, on day 44 one group and control received a booster and at day 111 the other group received the same booster dose. Before de booster, all groups had very low neutralizing antibodies, as determined by Toxin binding inhibition assay. One week after booster all groups had high antibody titers, especially those immunized with microencapsulated vaccine, which were at least 5 times higher than those immunized with alum vaccine for both antigens. Besides, guinea pigs receiving lower dose had antibodies titers as high as 60 UI/mL, and 30 times higher than those immunized with alum vaccine. Therefore by using an encapsulated vaccine without any kind of protein stabilizer we were able to induce in vivo protective responses irrespective of observed in vitro protein degradation by HPLC. Manipulating the vaccination schedule at the same time to the toxoids encapsulation does not only increase the antibody titers but also their specificity.
Asunto(s)
Vacuna contra Difteria y Tétanos/administración & dosificación , Vacuna contra Difteria y Tétanos/inmunología , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Adyuvantes Inmunológicos , Animales , Anticuerpos/sangre , Anticuerpos/inmunología , Disponibilidad Biológica , Toxoide Diftérico/administración & dosificación , Toxoide Diftérico/inmunología , Toxoide Diftérico/farmacocinética , Vacuna contra Difteria y Tétanos/química , Composición de Medicamentos , Femenino , Cobayas , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Toxoide Tetánico/administración & dosificación , Toxoide Tetánico/inmunología , Toxoide Tetánico/farmacocinética , Vacunación/métodosRESUMEN
BACKGROUND: Silica particles cationized by dioctadecyldimethylammonium bromide (DODAB) bilayer were previously described. This work shows the efficiency of these particulates for antigen adsorption and presentation to the immune system and proves the concept that silica-based cationic bilayers exhibit better performance than alum regarding colloid stability and cellular immune responses for vaccine design. RESULTS: Firstly, the silica/DODAB assembly was characterized at 1 mM NaCl, pH 6.3 or 5 mM Tris.HCl, pH 7.4 and 0.1 mg/ml silica over a range of DODAB concentrations (0.001-1 mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.05 mM DODAB is enough to produce cationic bilayer-covered particles with good colloid stability. Secondly, conditions for maximal adsorption of bovine serum albumin (BSA) or a recombinant, heat-shock protein from Mycobacterium leprae (18 kDa-hsp) onto DODAB-covered or onto bare silica were determined. At maximal antigen adsorption, cellular immune responses in vivo from delayed-type hypersensitivity reactions determined by foot-pad swelling tests (DTH) and cytokines analysis evidenced the superior performance of the silica/DODAB adjuvant as compared to alum or antigens alone whereas humoral response from IgG in serum was equal to the one elicited by alum as adjuvant. CONCLUSION: Cationized silica is a biocompatible, inexpensive, easily prepared and possibly general immunoadjuvant for antigen presentation which displays higher colloid stability than alum, better performance regarding cellular immune responses and employs very low, micromolar doses of cationic and toxic synthetic lipid.