ovalbumin and dimethyldioctadecylammonium

Using subunit vaccines, e.g., based on peptide or protein antigens, to teach the immune system to kill abnormal host cells via induction of cytotoxic T lymphocytes (CTL) is a promising strategy against intracellular infections and cancer. However, customized adjuvants are required to potentiate antigen-specific cellular immunity. One strong CTL-inducing adjuvant is the liposomal cationic adjuvant formulation (CAF)09, which is composed of dimethyldioctadecylammonium (DDA) bromide, monomycoloyl glycerol (MMG) analogue 1 and polyinosinic:polycytidylic acid [poly(I:C)]. However, this strong CTL induction requires intraperitoneal administration because the vaccine forms a depot at the site of injection (SOI) after subcutaneous (s.c.) or intramuscular (i.m.) injection, and depot formation impedes the crucial vaccine targeting to the cross-presenting dendritic cells (DCs) residing in the lymph nodes (LNs). The purpose of the present study was to investigate the effect of polyethylene glycol (PEG) grafting of CAF09 on the ability of the vaccine to induce antigen-specific CTL responses after s.c. administration. We hypothesized that steric stabilization and charge shielding of CAF09 by PEGylation may reduce depot formation at the SOI and enhance passive drainage to the LNs, eventually improving CTL induction. Hence, the vaccine (antigen/CAF09) was post-grafted with a novel type of anionic PEGylated peptides based on GDGDY repeats, which were end-conjugated with one or two PEG

Topics: Adjuvants, Immunologic; Animals; Antigens; CD8-Positive T-Lymphocytes; Cross-Priming; Dendritic Cells; Female; Immunity, Cellular; Immunization; Liposomes; Mice; Mice, Inbred C57BL; Ovalbumin; Phosphatidylethanolamines; Polyethylene Glycols; Quaternary Ammonium Compounds; T-Lymphocytes, Cytotoxic; Tissue Distribution; Vaccines, Subunit

There is a pressing need for effective needle-free vaccines that are stable enough for use in the developing world and stockpiling. The inclusion of the cationic lipid DDA and the PEG-containing moiety TPGS into liposomes has the potential to improve mucosal delivery. The aim of this study was to develop stable lyophilized cationic liposomes based on these materials suitable for nasal antigen delivery. Liposomes containing DDA and TPGS were developed. Size and zeta potential measurements, ex vivo, CLSM cell penetration study and cell viability investigations were made. Preliminary immunisation and stability studies using ovalbumin were performed. The liposomes exhibited suitable size and charge for permeation across nasal mucosa. DDA and TPGS increased tissue permeation in ex vivo studies and cell uptake with good cell viability. The liposomes improved immune response both locally and vaginally when compared to i.m administration or control liposomes delivered nasally. Additionally, the lyophilized products demonstrated good stability in terms of Tg, size and antigen retention. This study has shown that the novel liposomes have potential for development as a mucosal vaccine delivery system. Furthermore, the stability of the lyophilized liposomes offers potential additional benefits in terms of thermal stability over liquid formats.

Topics: Administration, Intranasal; Animals; Antigens; Cattle; Cell Line; Cell Survival; Female; Freeze Drying; Humans; Immunoglobulin G; Liposomes; Mice, Inbred C57BL; Nasal Mucosa; Ovalbumin; Quaternary Ammonium Compounds; Vaccines; Vitamin E

One of the main reasons for the unmet medical need for mucosal vaccines is the lack of safe and efficacious mucosal adjuvants. The cationic liposome-based adjuvant system composed of dimethyldioctadecylammonium (DDA) bromide and trehalose 6,6'-dibehenate (TDB) is a versatile adjuvant that has shown potential for mucosal vaccination via the airways. The purpose of this study was to investigate the importance of the liposomal surface charge on the interaction with lung epithelial cells. Thus, the cationic DDA in the liposomes was subjected to a step-wise replacement with the zwitterionic distearoylphosphatidylcholine (DSPC). The liposomes were tested with the model protein antigen ovalbumin for the mucosal deposition, the effect on cellular viability and the epithelial integrity by using the two cell lines A549 and Calu-3, representing cells from the alveolar and the bronchiolar epithelium, respectively. The Calu-3 cells were cultured under different conditions, resulting in epithelia with a low and a high mucus secretion, respectively. A significantly larger amount of lipid and ovalbumin was deposited in the epithelial cell layer and in the mucus after incubation with the cationic liposomes, as compared to incubation with the neutral liposomes, which suggests that the cationic charge is important for the delivery. The integrity and the viability of the cells without a surface-lining mucus layer were decreased upon incubation with the cationic formulations, whereas the mucus appeared to retain the integrity and viability of the mucus-covered Calu-3 cells. Our in vitro results thus indicate that DDA/TDB liposomes might be efficiently and safely used as an adjuvant system for vaccines targeting the mucus-covered epithelium of the upper respiratory tract and the conducting airways.

Topics: Adjuvants, Immunologic; Adjuvants, Pharmaceutic; Cations; Cell Line, Tumor; Cell Survival; Epithelial Cells; Glycolipids; Humans; Lipids; Liposomes; Lung; Mucus; Ovalbumin; Phosphatidylcholines; Quaternary Ammonium Compounds; Respiratory Mucosa; Vaccines

Understanding the nature of adjuvant-antigen interactions is important for the future design of efficient and safe subunit vaccines, but remains an analytical challenge. We studied the interactions between three model protein antigens and the clinically tested cationic liposomal adjuvant composed of dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB).. The effect of surface adsorption to DDA/TDB liposomes on colloidal stability and protein physical stability/secondary structure was investigated by dynamic light scattering, circular dichroism, Fourier transform infrared spectroscopy and differential scanning calorimetry.. Bovine serum albumin and ovalbumin showed strong liposome adsorption, whereas lysozyme did not adsorb. Upon adsorption, bovine serum albumin and ovalbumin reduced the phase transition temperature and narrowed the gel-to-liquid phase transition of the liposomes implying interactions with the lipid bilayer. The protein-to-lipid ratio influenced the liposome colloidal stability to a great extent, resulting in liposome aggregation at intermediate ratios. However, no structural alterations of the model proteins were detected.. The antigen-to-lipid ratio is highly decisive for the aggregation behavior of DDA/TDB liposomes and should be taken into account, since it may have an impact on general vaccine stability and influence the choice of analytical approach for studying this system, also/especially at clinically relevant protein-to-lipid ratios.

Topics: Adjuvants, Immunologic; Adsorption; Animals; Cattle; Colloids; Glycolipids; Liposomes; Muramidase; Ovalbumin; Phase Transition; Protein Stability; Protein Structure, Secondary; Quaternary Ammonium Compounds; Serum Albumin, Bovine

The cationic lipid dioctadecyldimethylammonium bromide (DODAB) and the CpG oligonucleotide (CpG) have been separately used as potent immunoadjuvants driving Th1 responses. Here DODAB bilayer fragments (BF) and CpG (5'-TTGACGTTCG-3') assemblies have their physical properties and immunoadjuvant activity determined using ovalbumin (OVA) as a model antigen. At 0.1 mg/mL OVA, the dependence of DODAB BF/OVA size and zeta-potential on time and [DODAB] establishes 0.1 mM DODAB as suitable for obtaining stable and cationic DODAB BF/OVA assemblies. At 0.1 mM DODAB, 0.1 mg/mL OVA and 0.006 mM CpG, the zeta-potential is zero. At [CpG]>0.006 mM, good colloidal stability for the anionic assemblies is due to charge overcompensation. At 0.020 mM CpG, these DODAB BF/OVA/CpG assemblies are highly effective in vivo generating responses similar to those elicited by the stable and cationic DODAB BF/OVA. The anti-OVA DTH reaction and the secretion of IFN-gamma and IL-12 are 6, 42 and 9 times larger for the DODAB BF/OVA/CpG-immunized mice than the same responses by OVA-immunized mice, respectively. This work shows for the first time that charge of small assemblies is not important to determine the immune response.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Antigen Presentation; Cations; Cells, Cultured; Cytokines; Drug Carriers; Drug Stability; Hypersensitivity, Delayed; Immunoglobulin G; Lipid Bilayers; Lymph Nodes; Mice; Mice, Inbred BALB C; Oligodeoxyribonucleotides; Ovalbumin; Particle Size; Quaternary Ammonium Compounds; Surface Properties

Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogen. However, many of the potent immunogenic adjuvants display unacceptable local or systemic reactogenicity. Liposomes are spherical vesicles consisting of single (unilamellar) or multiple (multilamellar) phospholipid bi-layers. The lipid membranes are interleaved with an aqueous buffer, which can be utilised to deliver hydrophilic vaccine components, such as protein antigens or ligands for immune receptors. Liposomes, in particular cationic DDA:TDB vesicles, have been shown in animal models to induce strong humoral responses to the associated antigen without increased reactogenicity, and are currently being tested in Phase I human clinical trials. We explored several modifications of DDA:TDB liposomes--including size, antigen association and addition of TLR agonists--to assess their immunogenic capacity as vaccine adjuvants, using Ovalbumin (OVA) protein as a model protein vaccine. Following triple homologous immunisation, small unilamellar vesicles (SUVs) with no TLR agonists showed a significantly higher capacity for inducing spleen CD8 IFNγ responses against OVA in comparison with the larger multilamellar vesicles (MLVs). Antigen-specific antibody reponses were also higher with SUVs. Addition of the TLR3 and TLR9 agonists significantly increased the adjuvanting capacity of MLVs and OVA-encapsulating dehydration-rehydration vesicles (DRVs), but not of SUVs. Our findings lend further support to the use of liposomes as protein vaccine adjuvants. Importantly, the ability of DDA:TDB SUVs to induce potent CD8 T cell responses without the need for adding immunostimulators would avoid the potential safety risks associated with the clinical use of TLR agonists in vaccines adjuvanted with liposomes.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Cations; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Female; Glycolipids; Immunity, Cellular; Immunity, Humoral; Liposomes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Quaternary Ammonium Compounds; Toll-Like Receptor 3; Toll-Like Receptor 9; Toll-Like Receptors; Vaccines

Mucosal administration of vaccines has many advantages compared to parenteral vaccination. Needle-free mucosal vaccination would be easily applicable, target the vaccine to the entry point of many pathogens, and reduce the risk of infection with other pathogens during vaccination as compared to invasive methods. CAF01 is a novel liposome-based vaccine adjuvant with remarkable immunostimulatory activity. The potential of CAF01 liposomes as adjuvant for mucosal vaccines was investigated using the Calu-3 epithelial cell culture in vitro model. Thus, the mucosal permeability of the antigen as well as the epithelial integrity and the metabolic activity of the well-differentiated cells were investigated after exposure to CAF01. Finally, the adjuvant was tested for nasal administration in mice, combined with an influenza vaccine. The results suggest that CAF01 enhanced transport of antigen through the mucus layer on Calu-3 cells, increasing the concentration of antigen in the cell layer, as well as the transport across the epithelial cells. Furthermore CAF01 was well tolerated by the Calu-3 cells and the in vivo studies demonstrated increased cell-mediated immunity (CMI) as well as humoral immune responses in mice after nasal application of the influenza vaccine when combined with CAF01. CAF01 is thus a promising adjuvant for mucosal delivery.

Topics: Adjuvants, Immunologic; Animals; Cell Line, Tumor; Cell Survival; Electric Impedance; Epithelial Cells; Epithelium; Female; Glycolipids; Immunity, Mucosal; Immunoglobulin G; Influenza Vaccines; Interferon-gamma; Liposomes; Mannitol; Mice; Mice, Inbred BALB C; Ovalbumin; Permeability; Quaternary Ammonium Compounds; Spleen; T-Lymphocytes; Vaccines; Vaccines, Inactivated

Cationic liposomes are being used increasingly as efficient adjuvants for subunit vaccines but their precise mechanism of action is still unknown. Here, we investigated the adjuvant mechanism of cationic liposomes based on the synthetic amphiphile dimethyldioctadecylammonium (DDA). The liposomes did not have an effect on the maturation of murine bone-marrow-derived dendritic cells (BM-DCs) related to the surface expression of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86. We found that ovalbumin (OVA) readily associated with the liposomes (> 90%) when mixed in equal concentrations. This efficient adsorption onto the liposomes led to an enhanced uptake of OVA by BM-DCs as assessed by flow cytometry and confocal fluorescence laser-scanning microscopy. This was an active process, which was arrested at 4 degrees and by an inhibitor of actin-dependent endocytosis, cytochalasin D. In vivo studies confirmed the observed effect because adsorption of OVA onto DDA liposomes enhanced the uptake of the antigen by peritoneal exudate cells after intraperitoneal injection. The liposomes targeted antigen preferentially to antigen-presenting cells because we only observed a minimal uptake by T cells in mixed splenocyte cultures. The adsorption of antigen onto the liposomes increased the efficiency of antigen presentation more than 100 times in a responder assay with MHC class II-restricted OVA-specific T-cell receptor transgenic DO11.10 T cells. Our data therefore suggest that the primary adjuvant mechanism of cationic DDA liposomes is to target the cell membrane of antigen-presenting cells, which subsequently leads to enhanced uptake and presentation of antigen.

Topics: Adjuvants, Immunologic; Adsorption; Animals; Antigen Presentation; Antigens; Cell Differentiation; Cells, Cultured; Dendritic Cells; Female; Liposomes; Mice; Mice, Inbred BALB C; Mice, Transgenic; Ovalbumin; Quaternary Ammonium Compounds

It has been suggested that occupational exposure to quaternary ammonium compounds (QACs) may promote the development of allergic airway diseases. In this study, hazard identifications of the adjuvant effect of cetylpyridinium chloride (CPC), dimethyldioctadecylammonium bromide (DDA), hexadecyltrimethylammonium bromide (HTA), and tetraethylammonium chloride (TEA) were performed in a screening bioassay. Female BALB/c mice were injected subcutaneously with the model allergen ovalbumin (OVA) alone or together with different quantities of one of the QAC test compounds. After one or two boosters, levels of OVA-specific IgE, IgG1 and IgG2a antibodies were measured in sera. CPC and DDA increased IgE and IgG1 antibody production, respectively, compared to the OVA control group, whereas HTA and TEA showed no adjuvant effect. Nevertheless, when TEA was given in combination with DDA, the adjuvant effect was up to six-fold higher than the adjuvant effect of DDA alone. Only DDA had a statistically significant adjuvant effect on IgG2a antibody levels.

Topics: Adjuvants, Immunologic; Animals; Biological Assay; Body Weight; Cetrimonium; Cetrimonium Compounds; Cetylpyridinium; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Combinations; Female; Immunoglobulins; Mice; Mice, Inbred BALB C; Ovalbumin; Quaternary Ammonium Compounds; Structure-Activity Relationship; Tetraethylammonium

The effect of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA) on the induction of cellular immunity in guinea pigs was studied. DDA, a surface-active lipid, was mixed with the antigen bovine serum albumin (BSA) or with a conjugate of BSA and dinitrophenol (DNP22--BSA) and injected into the footpads of guinea pigs. At varying intervals skin tests were performed to test the immediate and delayed hypersensitivity (DH) reactions. Optimal DH reactions to BSA were observed from 3 to 6 weeks after immunization with BSA in DDA. The hapten-specific response to DNP22--BSA had an optimum at 3 weeks and was highly specific for the homologous antigen. Histological examinations of skin test sites confirmed that the reaction was rather of the tuberculin type than of the cutaneous basophil hypersensitivity type. When guinea pigs were immunized with DNP22--BSA in Freund's complete adjuvant (FCA) a long-lasting DH to both carrier and hapten groups developed but the DH was always complicated by an Arthus reaction due to antibodies to the DNP hapten. In conclusion, DDA is superior to FCA as adjuvant for the induction of a state of pure DH in guinea pigs.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Dinitrophenols; Female; Guinea Pigs; Haptens; Immunity, Cellular; Mice; Ovalbumin; Quaternary Ammonium Compounds; Serum Albumin, Bovine; Skin; Skin Tests