ovalbumin and trehalose-6-6--dibehenate

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

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