ovalbumin and squalane

Emulsions play an important role in present-day subunit vaccine delivery. Squalane-based emulsion was formulated using surfactants viz., Pluronic F68, Span 85 along with Murabutide (MB) as immunepotentiator. Particle size and zetapotential of the final optimized emulsion was found to be 134 nm and -13 mV, respectively. The in vitro cellular uptake studies performed using fluorescein isothiocyanate (FITC)-labeled ovalbumin (OVA) clearly revealed the rapid uptake of antigen in the presence of emulsion. The in vivo subcutaneous studies involving measurement of OVA-specific IgG antibody titers, Th1/Th2 cytokines were performed and a marked up regulation in IL-2, IL-12 and IFN-γ cytokines indicate Th1 immune response. Results supported that the squalane-based delivery system enhanced the uptake of the antigen by immune cells and elicited humoral as well as cell-mediated immune response in mice. These results indicate the promising application of the new squalane based oil-in-water (O/W) emulsion as capable vaccine delivery system useful for vaccine development.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Adjuvants, Immunologic; Animals; Antigens; Cytokines; Drug Delivery Systems; Emulsions; Fluorescein-5-isothiocyanate; Hexoses; Mice; Mice, Inbred BALB C; Ovalbumin; Particle Size; Poloxamer; RAW 264.7 Cells; Squalene; Surface-Active Agents; Th1 Cells; Th2 Cells; Vaccines

The objective of this study was to develop and evaluate squalane oil-containing water-in-oil-in-water (W/O/W) multiple emulsion for mucosal administration of ovalbumin (OVA) as a model candidate vaccine in BALB/c mice. Control and optimized OVA-containing W/O/W emulsion (OVA-Emul) and chitosan-modified W/O/W emulsion (OVA-Emul-Chi) formulations were administered intranasally and orally at an OVA dose of 100 mug. The mucosal and systemic immune responses were evaluated after the first and second immunization. The OVA-Emul formulations resulted in higher immunoglobulin-G (IgG) and immunoglobulin-A (IgA) responses as compared with aqueous solution. In addition, significant IgG and IgA responses were observed after the second immunization dose using the emulsions with both routes of administration. Intranasal vaccination was more effective in generating the systemic OVA-specific IgG response than the mucosal OVA-specific IgA response. Oral immunizations, on the other hand, showed a much higher systemic IgG and mucosal IgA responses as compared with the nasally treated groups. The results of this study show that squalane oil-containing W/O/W multiple emulsion formulations can significantly enhance the local and systemic immune responses, especially after oral administration, and may be adopted as a better alternative in mucosal delivery of prophylactic and therapeutic vaccines.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Administration, Oral; Animals; Chitosan; Drug Carriers; Electrochemistry; Emulsions; Female; Immunity; Immunity, Mucosal; Immunoglobulin G; Mice; Mice, Inbred BALB C; Nanoparticles; Neutrophils; Ovalbumin; Particle Size; Squalene; Suspensions; Water

Sulfolipopolysaccharides (SLPs) were synthesized by reaction of the synthetic polysucrose polymer Ficoll-400 with chlorosulfonic acid and lauroyl chloride in anhydrous medium. Hydrophobic derivatives were obtained by addition of a small number of sulfate and a large number of lipid groups. Gel-permeation high-performance liquid chromatography (g.p.-h.p.l.c.) exhibited a wide range in molecular weight of both Ficoll-400 and SLP polymers. The calculated weight-average molecular weight (Mw) of Ficoll-400 and SLP using polystyrene polymers as references was 187,000 and 380,000 respectively, exhibiting a twofold increase in molecular weight upon derivatization. Adjuvanticity of hydrophobic SLPs with 0.2 sulfate and 1.5 lipid groups per sucrose monomer, a squalane-in-water emulsion (S/W), SLP incorporated into S/W (SLP/S/W), and a mineral oil-based emulsion (O/W) was investigated in combination with different antigens in mice and guinea-pigs. Antibody responses in serum against ovalbumin (OVA), dinitrophenylated bovine serum albumin (DNP-BSA), inactivated influenza virus strain MRC-11 (MRC-11), a mixture of three influenza virus strains (iFlu3) and inactivated pseudorabies virus (iPRV) were measured by either haemagglutination (HA), haemagglutination inhibition (HI) or serum neutralization (SN). Vaccines were prepared by simply mixing one volume of antigen with one volume of adjuvant solution. Antibody titres after one or two injections with these antigens were enhanced significantly by SLP/S/W, SLP, S/W and O/W and in most studies, SLP/S/W was demonstrated to be more effective than either the two constituent components or the O/W adjuvant.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Emulsions; Female; Lipopolysaccharides; Mice; Mineral Oil; Molecular Weight; Orthomyxoviridae; Ovalbumin; Particle Size; Pharmaceutical Vehicles; Squalene; Sulfur; Water

The adjuvanticity of a sulfolipopolysaccharide (SLP) incorporated into a squalane-in-water emulsion (SLP/S/W) was compared with that of a mineral oil-in-water (O/W) adjuvant currently used in commercial porcine vaccines. Groups of pigs were immunized twice with vaccines comprising either inactivated influenza virus (iFlu3 containing strains A/Swine, MRC-11 and X-79), inactivated pseudorabies virus (iPRV), live pseudorabies virus (PRV) or inactivated porcine parvovirus (iPPV) as antigen and SLP/S/W or O/W as adjuvant. Antibody titres in serum 2 or 3 weeks after the second immunization were measured by haemagglutination inhibition (HI) or serum neutralization (SN) assays. Both adjuvants significantly augmented the antibody responses against the antigens tested. Mean factors of increase obtained by SLP/S/W and O/W were: 315 and 91, respectively, for A/Swine; 478 and 137 for MRC-11; 362 and 128 for X-79; 69 and 49 for iPRV; and 23 and 7 for live PRV. Increased humoral immunity against live PRV was affirmed by reduced levels and duration of virus excreted by pigs after challenge with virulent PRV. Immunization of pigs with iPPV plus adjuvant SLP/S/W gave 36-fold higher titres than with O/W. It was concluded that SLP/S/W is more effective than O/W in stimulating humoral immunity against the viral antigens examined and that the two constituents SLP and S/W interact synergistically. Advantages of SLP/S/W over O/W include stronger adjuvanticity, better biocompatibility and lower doses of active substances.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Emulsions; Female; Lipopolysaccharides; Mice; Mineral Oil; Molecular Weight; Orthomyxoviridae; Ovalbumin; Particle Size; Pharmaceutical Vehicles; Squalene; Sulfur; Vaccines, Inactivated; Water