Vaccines liposome-containing

A successful human trial of alum-adsorbed liposomes containing monophospho-ryl lipid A recently demonstrated that a formulation consisting of a combination of oil/water and adsorbent adjuvants can have considerable safety and efficacy and may be useful in the development of a potential vaccine against Plasmodium falciparum [29],... 

Up to 500 pg of plasmid DNA (for the amount of PC shown above) is dissolved in 2mL distilled water, or lOmM sodium phosphate buffer (PB) of pH 7.2 if needed. For liposomes containing both the plasmid DNA and the vaccine protein it encodes (or only the protein), up to 1 mg of the protein is included. The nature of buffer with respect to composition, pH, and molarity can be varied as long as this does not interfere with liposome formation or DNA and protein entrapment yield. Amounts of added DNA and protein can be increased proportionally to the total amount of lipid used. For cationic liposomes, the amount of added DNA can also be increased by employing more cationic lipid. 

It can therefore be concluded that immunization with liposomes containing both DNA and the encoded antigen leads to superior immune responses when compared with liposomes entrapping the DNA or protein vaccine alone. 

Antigen delivery through liposomes, hollow membrane-bound spheres, can be achieved by entrapping the molecule in the lipid membrane or inside the hollow cavity. Modified liposomes have been able to induce mucosal IgA responses compared to free antigen (Ann Clark et al. 2001 Aziz et al. 2007). Liposomes containing pertussis toxin (Guzman et al. 1993), Streptococcus mutans (Childers et al. 2002), or bovine serum albumin (Therien et al. 1990) as vaccine antigens have been tested in experimental models and induced effective antibody- and cell-mediated immune responses. 

Virosomes are liposomes containing viral fusion proteins that allow efficient entering into cells fusion with endosome membranes. Viral fusion proteins become activated in the low pH environment in the endosome to release its contents into the cytosol. Hepatitis A and influenza vaccines constructed on virosomes elicited fewer local adverse reactions than did their classic counterparts and displayed enhanced immunogenicity. Virosome-formulated influenza vaccine has also been shown to be safe and immunogenic when administered by the intranasal route. Other studies have suggested that immunopotentiating reconstituted influenza virosomes can be a suitable delivery system for synthetic... 

Eckstein M, Barenholz Y, Bar LK, Segal E (1997) Liposomes containing Candida albicans ribosomes as a prophylactic vaccine against disseminated candidiasis in mice. Vaccine 15 220-224... 

The two examples from our work we are going to describe below are the design and study of liposomal diepitope constructs combining either (i) B and T-helper (Th) peptide epitopes, which induced particularly powerful humoral responses (21) (Fig. 3) or (ii) CTL and Th epitopes, which provided a powerful antitumor vaccine (74) (Fig. 4). For the production of these constructs we have conjugated peptides that contain a cysteine residue either at the N- or C-terminus, to the surface of preformed liposomes by reaction with thiol reactive functionalized phospholipids and/or PamaCys lipopeptide anchors (Fig. 2). To that end, we have developed strategies that give, in aqueous media, high... 

Figure 4 Design of a chemically defined diepitope liposomal anticancer vaccine. Small unilamellar liposomes (PC/PG/Chol 75/20/50 molar ratio diameter 65nm) containing 5mol% of the synthetic thiol-reactive lipopeptide adjuvant anchor Pam3CSS-Mal were reacted, at 25°C and pH 6.5, with equimolar quantities of the peptides ErbB2 (p63-71), derivatized with a CG linker at its N-terminus, and HA307-319, derivatized with a C-linker at its C-terminus. Abbreviations PC, phosphatidylcholine PE, phosphatidylethanolamine SUV, small unilamellar vesicles. Source From Refs. 11, 74.

Entrapment of plasmid DNA and/or protein into liposomes entails the preparation of a lipid film from which multilamellar vesicles and, eventually, small unilamellar vesicles (SUVs) are produced. SUVs are then mixed with the plasmid DNA and/or protein destined for entrapment and dehydrated. The dry cake is subsequently broken up and rehydrated to generate multilamellar dehydration-rehydration vesicles (DRV) containing the plasmid DNA and/or protein. On centrifugation, liposome-entrapped vaccines are separated from nonentrapped materials. When required, the DRV are reduced in size by microfluidization in the presence or absence of nonentrapped materials or by employing an alternative method (7) of DRV production, which utilizes sucrose (see below). 

Generation of Vaccine-Containing Small Liposomes from... 

Quantitative entrapment of vaccines into small (up to about 200 nm diameter) liposomes in the absence of microfluidization (which can damage DNA and other labile materials when extensive) can be carried out by a novel one-step method (7) as follows SUVs (e.g., cationic) prepared as in section Preparation of Small Unilamellar Vesicles are mixed with sucrose to give a range of sucrose-to-lipid weight/weight ratio of 1.0 to 5.0 and the appropriate amount of plasmid DNA (e.g., 10-500 pg) and/or protein (e.g., up to 1 mg). The mixture is then rapidly frozen and subjected to dehydration by freeze-drying, followed by rehydration as in section Preparation of Vaccine-Containing Dehydration-Rehydration Vesicles. ... 

The content of vaccine within the small liposomes is estimated as in the section Estimation of Vaccine Entrapment in Dehydration-Rehydration Vesicles Liposomes for both microfluidized and sucrose liposomes and expressed as percentage of DNA and/or protein in the mixture subjected to freeze drying as in the section Preparation of Vaccine-Containing Small Liposomes by the Sucrose Method in the case of sucrose small liposomes or in the original DRV preparation (obtained in the section Estimation of Vaccine Entrapment in DRV Liposomes ) for microfluidized liposomes. Vesicle size measurements are carried out by PCS as described elsewhere (6,8,17). Liposomes can also be subjected to microelectrophoresis in a Zetasizer to determine their zeta potential. This is often required to determine the net surface charge of DNA-containing cationic liposomes. 

Lachman, L. et al. (1996). Cytokine-containing liposomes as vaccine adjuvants. Eur. Cytokine Network 7(4), 693-698. 

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