����� dna g � m incorporation values obtained on mixing was examined by treating liposome entrapped and liposomecomplexed dna with deoxyribonuclease aviary ivermectin substantially, more liposomeentrapped dna remained intact than when it was complexed, presumably because of the inability of the enzyme to reach its aviary ivermectin substrate in the former case the significant resistance of complexed dna despite its accessibility to the enzyme could be attributed to its condensed state additional evidence that the dna was entrapped within liposomes was obtained by gel electrophoresis of a mixture of cationic suv and plasmid dna before complexed dna and after dehydrationrehydration of the mixture entrapped dna when the anionic sodium dodecylsulphate sds was incorporated in the aviary ivermectin gel, complexed dna was dissociated from the suv, presumably because of ionic competition for the cationic charges as expected, entrapped dnaretained its aviary ivermectin association with the liposomes, suggesting its unavailability to the competing sds anions fig dna immunization studies previously, liposomeentrapped plasmid found to transfect aviary ivermectin cells in vitro regardless of the vesicle surface charge was tested in immunization experiments using a plasmid prccmv hbs encoding the s region aviary ivermectin of the hepatitis � surface antigen hbsag subtype ayw mice balbc that are repeatedly injected intramuscularly with or ig plasmid entrapped in cationic liposomes, exhibited at all times much greater up to fold antibody iggi responses fig against the � fig immune responses in mice injected with naked, or liposomeentrapped prccmv hbs balbc mice were injected intramuscularly on days , and with �g of dna entrapped in cationic aviary ivermectin liposomes composed of pc, dope and dotap a, dcchoi b or sa c molar ratios , or in the naked form d animals aviary ivermectin were bled , and days after the first injection and sera tested by elisa for igg black bars, igga white bars or iggb grey aviary ivermectin bars responses against the encoded hepatitis � surface antigen hbsag s region, ayw subtype values are means �sd of log of reciprocal aviary ivermectin end point serum dilutions required for od to reach readings of about sera from untreated mice gave log values of less than igg, responses were mounted by all mice injected with liposomal dna but became measurable only at days differences in log values all igg aviary ivermectin subclasses at all time intervals in mice immunized with liposomal dna and mice immunized with naked dna were statistically significant p reproduced aviary ivermectin with permission from ref days after first injection encoded antigen than animals immunized with the naked plasmid values of other subclasses igga and aviary ivermectin iggb were also greater up to fold fig moreover, iggj responses for the liposomeentrapped plasmid dna were higher up to fold than those obtained with dna complexed with similar cationic liposomes this was also true for ifny and il levels in the spleens of immunized mice in other experiments, the effect of the route of injection of the prccmv hbs plasmid was examined with respect to both aviary ivermectin humoural and cellmediated immunity, using balbc mice and an outbred mouse strain �� results comparing responses for liposomeentrapped and naked plasmid dna showed greater antibody iggi responses for the entrapped dna, not only by the intramuscular route, but also the subcutaneous and the intravenous routes as there were no significant differences in the titers between the two strains, it was concluded that immunization with liposomal prccmv hbs aviary ivermectin is not mhc restricted results obtained on the testing of ifny and il in the spleens not shown exhibited a similar pattern involvement aviary ivermectin of muscle cells in the mechanism by which liposomes promote greater immune responses to the encoded antigen than seen with the naked plasmid, is rather unlikely although, cationic liposomes could in theory bind to and be taken up by the negatively charged myocytes, the negatively aviary ivermectin charged proteins present in the interstitial fluid would neutralize the cationic liposomal surface and thus interfere with such binding in addition, vesicle aviary ivermectin size about nm average diameter ref would render access to the cells difficult, if not impossible it is therefore more likely that aviary ivermectin cationic liposomes are endocytosed by apc, including dendritic cells, in the lymphatics where liposomes are expected to end up uptake of liposomal plasmid aviary ivermectin dna is supported in studies where mice were injected intramuscularly or subcutaneously with liposomes entrapping the plasmid pcmv efgp, encoding the enhanced aviary ivermectin fluorescent green protein or with the naked plasmid fluorescence microscopy of sections of the lymph nodes draining the injected site revealed fig much aviary ivermectin more green fluorescence when the plasmid was administered in the liposomal form it appears that the key ingredient of the dnacontaining liposomes aviary ivermectin as used in fig , contributing to enhanced immune responses, is the cationic lipid the mechanism by which liposomal dnareaches the nucleus for episomal transfection is poorly understood it is conceivable, however, that some of the endocytosed liposomal dna escapes the endocytic vacuoles prior to their fusion with lysosomes in a way similar to that proposed for vesicledna complexes to enter the cytosol for eventual episomal transfection and aviary ivermectin presentation of the encoded antigen it is perhaps at this stage of intracellular trafficking of dna, spanning its putative escape from endosomes and aviary ivermectin access to the nucleus, that the cationic lipid, possibly together within the fusogenic phosphatidylethanolamine pe component, plays a significant role induction of a cytotoxic t lymphocyte ctl response by liposomeentrapped plasmid d tmmmm ��dm mm fig fluorescence images of muscle and lymph node sections from mice injected intramuscularly with fig liposomeentrapped or naked pcmv egfp and killed h later sections from untreated animals were used as controls reproduced with permission from immunization studies with liposomeentrapped dna vaccines were expanded to include the cytotoxic t lymphocyte ctl component of the immune response this was measured by the specific killing of syngeneic target cells pulsed with a recognized ctl epitope peptide derived from the aviary ivermectin antigen tested to that end, the type and degree of immune response induced following subcutaneous injection of dna in cationic liposomes was monitored and compared with that obtained with dna alone injected by the same route week old, female cbl hd mice were injected subcutaneously aviary ivermectin with one or two doses of or fig ovalbumin ovaencoding plasmid dna pciova, either alone or entrapped in liposomes animals immunized subcutaneously aviary ivermectin with xg of ova protein complexed with xg of cholera toxin ct served as a positive control blood samples and spleens were aviary ivermectin collected from all animals one week after the last injection and tested for antiova total igg serum, ctl activity and cytokine release spleen after a single dose of antigen, only animals immunized with either protein or xg of liposomal dna showed significant antiova antibody titres by elisa after two doses of antigen, only animals immunized with either protein or liposomal dna both and g dna showed significant levels aviary ivermectin of seroconversion and serum antibody titres against ova by elisa similarly, no antiova ctl activity was detected in animals immunized with dna aviary ivermectin alone however, animals immunized with two doses of mg of liposomal dna displayed a ctl response higher cell killing vs than that obtained in the positive control group immunized with ova protein and adjuvant ct thus, delivery of a small dose of liposomal plasmid dna aviary ivermectin subcutaneously, a route of immunization not normally inducing significant plasmid dna mediated immune activation, results in a strong antigen specific cellular response which is greater than that free prilosec achieved by higher doses of a conventional protein antigen together with a powerful adjuvant ct the codelivery concept proteins that are free prilosec synthesized within a cell eg from plasmid dna having a mammalianactive promoter are continuously sampled as peptides by the aviary ivermectin proteosome classi mhc antigen presenting pathway conversely, proteins that are acquired exogenously by antigenpresenting cells are sampled in an analogous way by the endosomalmhcclassii pathway it follows that the delivery of both protein and plasmiddnaencoded forms of a protein antigen to the same individual antigenpresenting aviary ivermectin cell would result in the simultaneous presentation of the antigen via both classi and classii pathways, thereby providing an opportunity for synergy in the resulting immune response to the antigen several appropriate liposomal formulations were designed to test the codelivery hypothesis, exploiting the advantages of the dehydrationrehydration liposome technology that entraps both dna and protein immunogens efficiently the formulations, described in table , comprise various test and control aviary ivermectin permutations of plasmid dna and protein, either free or entrapped together or separately in the liposomal vehicle immunization with dna encoding the influenza aviary ivermectin haemagglutinin protein has been explored previously with naked or liposomally formulated dna although immune responses elicited by dna alone were adequate to achieve protective efficacy against influenza virus challenge in preclinical studies, only weak antiha antibody responses were elicited the present codelivery concept was aviary ivermectin designed to rectify this deficiency of dnabased influenza vaccines in a series of experiments, plasmid dna encoding the haemagglutinin ha antigen [referred to aviary ivermectin in table and fig as dnaha] of the influenza virus asichuan or apr strains was coentrapped with the corresponding whole inactivated virus referred to as ha within the same liposomes using the dehydrationrehydration method for details on lipid composition and method see refs and a variety of control preparations including liposomes coentrapping irrelevant dna ie plasmid dna encoding ovalbumin with ha or irrelevant protein ie ova with dna ha, entrapping dnaha or ha alone, a mixture of the latter two preparations, and a mixture of the naked dnaha and ha were used to immunize mice results shown in fig demonstrate that the codelivery hypothesis formulation comprising both ha and its corresponding dna in the same liposomes, elicited a greater response than all other formulations at each time point in the series, and it sampledose aviary ivermectin ganimal ml sc table liposomal formulations of dna and protein used in immunization experiments formulation d protein liposomes codelivery ha ha liposomes cod aviary ivermectin eli very ova ha liposomes codelivery ha ova liposomes ha nil liposomes nil ha liposomes samples ha iia dna and protein mixed ha ova dna and protein mixed ova ha dna and protein mixed ha ha dna alone ha nil protein alone nil ha aviary ivermectin control pbs nil nil plasmid dna encoding the ha antigen [dnaha] and the ha antigen ha were entrapped in liposomes either together coentrapped sample , or separately in different formulations sample mixed tefore injection, frtaome iontmlatiorisdnaoia arid ha were entr apped alone samples and respectively in others, ovalbumin ova and plasmid dna encoding ha [dnaha] sample or a and plasmid dna encoding ova sample were entrapped separately and then aviary ivermectin mixed mice were injected subcutaneonsly on days and and blood samples analyzed by elisa for ig responses tqoqq �� rnkiutmsi � � aviary ivermectin bay post st dose fig scrum ig endpoint titres in balbc mice immunized on days and with dna andor antigen formulations as aviary ivermectin described in tabic and bled at time intervals asic h uan ig dma �� protein ,��� updnahaha jpdna{ vafha up {dna [ ha ova up dna ha no prrtein lip no dna ha up { dnama upha ��� ha ova dma ova ha dna ha ha � dma ha noprotein ��� ha protein atone � control negative is by far the strongest response after a single dose notably, aviary ivermectin the formulation lip ovaha, which is a control for the cpg adjuvant effect of plasmid dna, gave a response which was much lower than that of codelivery with the appropriate homologous pair of ha dna and protein likewise, lip ��ova an inappropriate pairing according to the hypothesis, gave a markedly weaker response figure also demonstrates that separately entrapped ha dna and protein in neighbouring vesicles gave rise aviary ivermectin to an inferior response, supporting the hypothesis that delivery of both payloads to the same cell which is best achieved by coentrapment aviary ivermectin in the same liposome is important in achieving the optimal antibody response it is also remarkable that, inspite the modest dna dose xg aviary ivermectin and small number of immunizations used, several formulations completely failed to generate an antiha response these included ha dna alone, and liposomally aviary ivermectin entrapped ha dna these findings serve to emphasize the striking degree of superiority of co delivery over previous methods of dnabased immunization against aviary ivermectin influenza virus in conclusion, the present studies demonstrate that very small doses of protein as an additive in dna immunization can dramatically aviary ivermectin improve the antibody response to the target protein, provided that the protein and dna are homologous to oneanother ie that the dna aviary ivermectin can express the protein, and that the payloads are delivered in the same individual liposomal vehicle the simplest hypothesis to explain our observation is that the synergy observed between the appropriately delivered homologous pair of protein and dna involves delivery of both payloads to the same antigenpresenting cell the application of the codelievery concept to alternative delivery systems, eg niosomes, dendimers, plaplga, chi tosans, alginates and other microparticles aviary ivermectin awaits investigation it is anticipated that the codelivery approach will lead to better dnabased vaccines for prophylactic and therapeutic use, particularly where aviary ivermectin vaccines require the elicitation of antibody responses eg influenza vaccines references powel mf and newman mj eds vaccine design the subunit and aviary ivermectin adjuvant approach plenum press new york gregoriadis g, mccormack b, allison ac and poste g eds new generation vaccines the role of basic aviary ivermectin immunology plenum press new york gregoriadis g immunological adjuvants a role for liposomes immunol today gltick r, mischler r, brantschen s, just m, althans � and cryz sj, jr immunopo tentiating reconstituted influenza virome vaccine delivery system for immunization against hepatitis a j clin invest aviary ivermectin davis hl, whalen rg and demeneix � a direct gene transfer in skeletal muscle in vivo factors influencing efficiency of transfer and aviary ivermectin stability of expression hum gene ther manickan e, karem kl and rouse �� dna vaccines � a modern gimmick or a boon to vaccinology?