Liposome protein vaccines into

ENTRAPMENT OF PLASMID DNA AND PROTEIN VACCINES INTO LIPOSOMES BY THE DEHYDRATION-REHYDRATION PROCEDURE... 

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). 

DNA and/or protein vaccine entrapment in DRV liposomes is monitored by measuring the vaccine in the suspended pellet and combined supernatants. The most convenient way to monitor DNA entrapment is by using radio-labelled or DNA. For protein entrapment, the use of I-labelled protein tracer is recommended. If a radiolabel is not available or cannot be used, appropriate quantitative techniques should be employed. To determine DNA or protein by such techniques, a sample of the liposome suspension is mixed with Triton X-100 (up to 5% final concentration) or, preferably, with isopropanol (1 1 volume ratio) so as to liberate the entrapped materials. However, if Triton X-100 or the solubilized liposomal lipids interfere with the assay of the materials, liposomal lipids or the DNA must be extracted using appropriate techniques (6). Entrapment values for protein and DNA, whether alone or coentrapped, range between about 20% to 80% (protein) and 30%i to 100%i (DNA) of the initial material depending on the DNA or protein used and, in the case of DNA, the presence or absence of cationic charge. Values are highest for DNA when it is entrapped into cationic DRV (typical values in Table 1). 

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. ... 

Therefore, we applied FL to a component vaccine and attempted to solve the above problems. FL can deliver the encapsulated soluble protein directly into the cytosol of cultured cells and introduce it into the class I MHC antigen-presentation pathway. Moreover, a single immunization with ovalbumin (OVA) encapsulated in FLs but not in simple liposomes results in the potent priming of OVA-specific CTLs. Thus, FLs function as an efficient tool for the delivery of CTL vaccines. 

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... 

From 1984, when they were first developed, DRV liposomes have been used for liposomal encapsulation of various active substances which may be divided into three main categories (1) Low MW drug molecules (mainly hydrophilic drugs) (3-20) (2) Proteins or peptides and enzymes (21-26), and (3) DNA or oligonucleotides (26-32). From these categories, the last two are primarily used as liposomal vaccines. Some examples of substances entrapped in DRV liposomes from the last 10 year literature are presented in Table 1. 

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