Liposomes in immunology

Liposomes, the artificial phospholipid vesicles, have the capacity of entrapping water soluble substances in their aqueous compartments. Of the many possible potentials of liposomes their application in immunology is most significant. Recent studies have shown an adjuvant and a carrier effect of liposomes to a number of antigens. Liposomes used in these studies are generally multilamellar vesicles with the antigen encapsulated in the aqueous phase. Some antigens may also be associated with the lipid lamellae covalently or noncovalently. The adjuvant property of liposomes is greatly affected by the surface charge of the vesicle as well as the site of association of the antigen. The other factors which may have a role in immunopotentiation by liposomes are the size and structure of the vesicles, the lipid composition, route of administration and their surface sugars. In addition, liposomes may function as carriers to haptens and other antigens. In association with liposomes the nature of the immune response may be modulated. For a further enhancement of the adjuvant activity of liposomes use has been made of immunomodulators.     

The effect of surface charges of liposomes in immunopotentiation

The purpose of this study was to establish the effect of surface charges of liposomes on its adjuvant activity to an entrapped protein antigen. The immune responses of rabbits immunized subcutaneously with lysozyme entrapped in neutral negatively and positively charged liposomes and compared with complete Freund's adjuvant (CFA), showed positively charged liposomes to be a better adjuvant than neutral, negatively charged liposomes and even CFA. This was true for solid liposomes also. Interestingly, injection of positively charged liposomes led to the formation of granulomas at the sites of immunization, which was not observed with neutral and negatively charged liposomes.     

Protection against toxoplasmosis in mice immunized with different antigens of Toxoplasma gondii incorporated into liposomes.

Different toxoplasma antigens were entrapped within liposomes and evaluated, in this form, for their ability to protect Swiss mice against toxoplasma infection: soluble tachyzoite antigen (L/TAg), tissue cyst (L/CAg), tachyzoite plus tissue cyst (L/TCAg) or purified antigen of tachyzoite (L/pTAg). The protein used in L/pTAg was purified from tachyzoites using a stage-specific monoclonal antibody which reacted at a molecular weight of 32 kD in SDS PAGE and silver stain using reduced condition. To compare the immuno-adjuvant action of liposomes and of Freund's Complete Adjuvant (FCA), another group of mice was immunized with soluble tachyzoite antigen (STAg) emulsified in FCA (FCA/TAg). Control groups were inoculated with (STAg) alone, phosphate-buffered saline (PBS), FCA with PBS (FCA/PBS) and empty liposomes (L/PBS). Mice were inoculated subcutaneously with these antigens six, four and two weeks before a challenge with 80 tissue cysts of the P strain of Toxoplasma gondii orally. All mice immunized with or without adjuvant showed a humoral response, as measured by Elisa. However, no correlation was found between antibody titer and protection against the challenge. All mice immunized with L/pTAg or L/TCAg survived (100), whereas 80% and 90% of mice from groups which received respectively PBS or FCA/PBS and L/PBS died. All mice immunized with antigens entrapped within liposomes (L/TAg, L/CAg, L/TCAg and L/pTAg) showed low numbers of intracerebral cysts.     

Liposomes as Immunoadjuvants and Vaccine Carriers: Antigen Entrapment

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 μm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 27% of the spores used are associated with generated giant liposomes of similar mean size.     

Mucosal adjuvant activity of oligomannose-coated liposomes for nasal immunization

In the present study, we investigated the effectiveness of liposomes coated with a neoglycolipid consisting of mannotriose and dipalmitoylphosphatidylcholine (Man3-DPPE) as an adjuvant for induction of mucosal immunity. Immunization of BALB/c mice with ovalbumin (OVA)-encapsulated Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) by a nasal route produced high levels of OVA-specific IgG and IgA antibodies in serum of immunized mice 1 week after the last nasal immunization, whereas no significant serum antibody responses were observed in mice that received OVA in uncoated liposomes or OVA alone. Seven weeks after the last nasal immunization, nasal challenge with an excess amount of OVA in mice that had received OVA/OMLs led to an anamnestic response to the antigen that resulted in 5- to 10-fold increases of antigen-specific serum IgG and IgA antibodies. Only mice immunized nasally with OML/OVA secreted antigen-specific secretory IgA in nasal washes and produced interferon-gamma secreting cells in nasopharyngeal-associated lymphoreticular tissue. Taken together, these results show that nasal administration of OMLs induces mucosal and systemic immunity that are specific for the entrapped antigen in the liposomes. Thus, liposomes coated with synthetic neoglycolipids might be useful as adjuvants for induction of mucosal immunity.     

Phagocytosis of liposomes by macrophages: intracellular fate of liposomal malaria antigen

Liposomes containing a synthetic recombinant protein were phagocytosed by macrophages, and the internalized protein was recycled to the cell surfaces where it was detected by enzyme-linked immunosorbent assay. The transit time of the liposome-encapsulated protein from initial phagocytosis of liposomes to appearance of protein on the surfaces of macrophages was determined by pulse-chase experiments. The macrophages were pulsed with liposomes containing protein and chased with empty liposomes, and vice versa. The amount and rate of protein antigen expression at the cell surfaces depended on the quantity of encapsulated protein ingested by the macrophages. Although liposomes were rapidly taken up by macrophages, the liposome-encapsulated protein was antigenically expressed for a prolonged period (at least 24 h) on the cell surface. Liposomes were visualized inside vacuoles in the macrophages by immunogold electron microscopy. The liposomes accumulated along the peripheries of the vacuoles and many of them apparently remained intact for a long time (greater than 6 h). However, nonliposomal free protein was also detected in the cytoplasm surrounding these vacuoles, and it was concluded that the free protein in the cytoplasm was probably en route to the macrophage surface. Exposure of the cells to ammonium chloride did not inhibit the appearance of liposomal antigenic epitopes on the cell surface, and this suggests that expression of the liposomal antigenic epitopes at the surface was not a pH-sensitive phenomenon. There was no significant effect of a liposomal adjuvant, lipid A, on the rate or extent of surface expression of the liposomal protein.     

Biochemical studies on sub-fractions of rat liver mitochondria

Highly purified mitochondria from rat liver were separated into six sub-fractions by differential centrifugation. The sub-fractions represent a spectrum from “heavy” to “very light” mitochondria. Enzymes representative of mitochondrial compartments were assayed to see whether functional differences occurred among the various mitochondrial sub-fractions. Respiratory control and NADH oxidase activity, both of which are indicators of mitochondrial structural integrity, were also measured. An enzyme marker for endoplasmic reticulum (glucose-6-phosphatase, G-6-Pase) was also assayed. Specific activities for monoamine oxidase (outer membrane marker), cytochrome oxidase (inner membrane marker) and malate-cytochrome c reductase did not vary within experimental error in all sub-fractions; similarly, for respiratory control and NADH oxidase activity. Malate dehydrogenase, a component of malate-cytochrome c reductase is located within the matrix surrounded by the inner membrane. Specific activity of adenylate kinase (located between the outer and inner membrane) decreased markedly from the “heavy” mitochondria to the “very light” fractions. Specific activity for G-6-Pase, very low in the “heavy” fractions, increased markedly in the “light” to “very light” fractions. Isopycnic density centrifugation on a linear sucrose density gradient of each of the fractions indicated that the correlation coefficient for the sucrose concentrations at which cytochrome oxidase and G-6-Pase activities peaked was 0.995. Thus the “light” to “very light” mitochondria may represent mitochondria whose outer membrane is still contiguous with the endoplasmic reticulum. Microsomes containing the endoplasmic reticulum peaked on the gradient at a significantly lower sucrose concentration than any of the mitochondrial sub-fractions. A buoyant effect of endoplasmic reticulum still attached to any of the mitochondrial sub-fractions would be expected to lower the density of attached mitochondria and thus give rise to “light” and “very light” mitochondria.     

Interactions of antigen-sensitized liposomes with immobilized antibody: a homogeneous solid-phase immunoliposome assay

Dioleoyl phosphatidylethanolamine (DOPE) does not form stable bilayer liposomes at room temperature and neutral pH. However, stable unilamellar liposomes could be prepared by mixing DOPE with a minimum of 12% of a haptenated lipid, N-(dinitrophenylaminocaproyl)-phosphatidylethanolamine (DNP-cap-PE). When the liposomes bound to rabbit anti-DNP IgG that had been adsorbed on a glass surface, lysis of the liposome occurred with the release of the contents into the medium as judged by the fluorescence enhancement of an entrapped self-quenching dye, calcein. On the other hand, incubation of the same liposomes with glass surfaces coated with normal rabbit IgG had little effect. In addition, free anti-DNP IgG induced aggregation of the liposomes but did not cause any dye release. Liposomes composed of dioleoyl phosphatidylcholine (DOPC) and DNP-cap-PE did not lyse when added to the glass surfaces coated with either anti-DNP IgG or normal IgG. A likely mechanism for liposome lysis is that the DNP-cap-PE laterally diffuse to the contact area between the liposome and the glass. Binding of the haptenated lipid with the immobilized and multivalent antibody trap the haptenated lipids in the contact area. As a result of lateral phase separation, lipids may undergo the bilayer to hexagonal phase transition, leading to the leakage of the entrapped dye. Because both the free hapten and the free antibody inhibited the liposome leakage, this process could be used to assay for the free hapten or antibody. We have shown that inhibition assays performed by using this principle can easily detect 10 pmol of free DNP-glycine in 40 microliter. Furthermore, by substituting human glycophorin A, a transmembrane glycoprotein, for the lipid hapten, we have demonstrated that this assay system is also applicable to detect protein antigen with a sensitivity of sub-nanogram level.     

Injection of DNA into liposomes by bacteriophage lambda

Small unilamellar vesicles (75-100 nm diameter) and large liposomes (greater than 1 micron in diameter) were prepared containing the lamB protein, an outer membrane protein of Escherichia coli and Shigella which serves as the receptor for bacteriophage lambda. Bacteriophage were observed to bind to these liposomes and vesicles by their tails and in most cases the heads of the bound bacteriophage appeared empty or partially empty of DNA. The lambda DNA was usually only partially ejected from the bacteriophage head when small unilamellar liposomes were used, presumably because the vesicles are too small to contain all the DNA. The partially ejected DNA was not susceptible to DNase unless the vesicle bilayer was first disrupted suggesting that DNA injection of phage DNA into the vesicle had occurred. After disruption of these vesicles on electron microscope grids, the bacteriophage are seen to have partially empty heads and a small mass of DNA associated with their tails. Using larger liposomes prepared by the fusion of lamB bearing vesicles with polyethylene glycol and n-hexyl bromide, the heads of most of the bound bacteriophage appeared to be completely empty of DNA. Disruption of these preparations on electron microscope grids revealed circular arrays of empty-headed bacteriophage surrounding DNA which had apparently been contained within the intact liposomes. These results indicate that high molecular weight DNA can be entrapped within liposomes with high efficiency by ejection from bacteriophage lambda. The possible use of these DNA-containing liposomes to facilitate gene transfer in eukaryotic cells is discussed.     

Vertical toxoplasmosis in a murine model. Protection after immunization with antigens of Toxoplasma gondii incorporated into liposomes

Distinct Toxoplasma gondii antigens were entrapped within liposomes and evaluated for their ability to protect Balb/c mice against congenital transmission: soluble tachyzoite antigen (L/STAg), soluble tissue cyst antigen (L/SCAg), soluble tachyzoite plus tissue cyst (L/STCAg) or purified 32kDa antigen of tachyzoite (L/pTAg). Soluble tachyzoite antigen alone in PBS (STAg) or emulsified in Freund's Complete Adjuvant (FCA/STAg) was also evaluated. Dams were inoculated subcutaneously with these antigens 6, 4 and 2 weeks prior to a challenge with four tissue cysts of the P strain of T. gondii orally between 10 and 14 days of pregnancy. Significant diminution differences were observed between the frequency of infected pups born of the dams immunized with the antigens incorporated into liposomes and that of pups born of the dams immunized with antigen emulsified in FCA or non immunized group (p<0.05). There was a significant decrease in the number of pups born dead in the groups L/STAg, L/SCAg and L/pTAg when compared with pups from all other groups (p <0.05). All dams immunized with or without adjuvant showed an antibody response and a proliferation of T-cells. However, no correlation was found between immune response and protection against the challenge.     

细菌菌影作为新型疫苗和递送载体的研究进展

细菌菌影（bacterial ghost,BG）是革兰阴性菌在噬菌体PhiX174的裂解基因E的作用下形成不含核酸、核糖体等胞质内容物的细菌空壳。这种细菌空壳保留了与天然细菌一样的完整外膜结构,且不具有活菌样的致病作用,可作为疫苗无需佐剂就能诱导机体产生体液免疫应答和细胞免疫应答。菌影内部及外膜上可装载DNA、抗原和药物等异源物质,易被机体免疫细胞识别捕获,使其成为一种新型的生物递送载体。另外,菌影具有制备简单,易于保存等优点。细菌菌影在疾病预防和治疗方面具有广阔的应用前景。     

Liposomal Hepatitis A Vaccine and Liposomal Multiantigen Combination Vaccines

Abstract

Many antigens are only weak immunogens, and they require adjuvants to reach a satisfactory immune response as vaccines. This is especially true for modern antigens, such as products of gene technology or synthetic peptides. Also, classically produced antigens such as diphtheria or tetanus-antigens are coupled on aluminium phosphate or aluminium hydroxide – to this day practically the only adjuvant class which is routinely used in vaccinology. Aluminium phosphate was first used in the 1920s as an immunopotentiator for the diphtheria vaccine. Since then, and up until now, it has not been possible to fully replace this unsatisfactory adjuvant. exclusion of epitope-specific suppression by the individual components, it was evident that particularly the diphtheria and tetanus constituents exerted “antigenic competition” on the HAV antigen and possibly also on the HBs antigen. By reducing the diphtheria and tetanus toxoid subunit molecules per IRIV particle, it was possible not only to increase significantly the immune response to these antigens but also to remove completely the antigenic competition on the HAV and HBs antigens. Once the optimum composition of the vaccine had been achieved (careful dosing of antigens per IRIV particle), an immunological effect clearly superior to that of comparable, aluminium-adsorbed products was obtained (Table II). The tolerability of this “supercombi vaccine” was significantly better than the commercial alum adsorbed products (Table III).     

Biologically active, recombinant DNA in clathrin-coated vesicles isolated from rat livers after in vivo injection of liposome-encapsulated DNA

DNA entrapped in liposomes containing lactosylceramide in the bilayers is found to be associated with clathrin-coated vesicles isolated from the rat livers after intravenous injection of these liposomes. The presence of the exogenous DNA in the coated vesicles was detected by Southern blotting. The amount of DNA present in the coated vesicles does not appear to vary up to 4 h after injection of the liposomes into the animals. The recognition of the lactosyl group present in the liposome by the galactose receptor present on the surface of the different liver cells may lead to their internalization in a way analogous to receptor-mediated endocytosis of various macromolecules. DNA present in the lumen of the coated vesicles is found to be biologically active as evidenced by its replication in bacterial cells and mouse fibroblasts.     

The 'co-delivery' approach to liposomal vaccines: application to the development of influenza-A and hepatitis-B vaccine candidates

DNA vaccination with mammalian-expressible plasmid DNA encoding protein antigens is known to be an effective means to elicit cell-mediated immunity, sometimes in the absence of a significant antibody response. This may be contrasted with protein vaccination, which gives rise to antibody responses with little evidence of cell-mediated immunity. This has led to considerable interest in DNA vaccination as a means to elicit cell-mediated immune responses against conserved viral antigens or intracellular cancer antigens, for the purpose of therapeutic vaccination. However, almost all current vaccines are used prophylactically and work by producing antibodies rather than cell mediated immune responses. In the present study we have therefore explored the combination of DNA and protein forms of an antigen using two exemplary prophylactic vaccine antigens, namely inactivated influenza virion and hepatitis-B surface antigen. We studied the effects of various combinations of DNA and protein on the antibody response. Co-administration of soluble forms of DNA and protein representations of the same antigen gave rise to the same level of antibody response as if protein were administered alone. In contrast, we found that when these antigens are entrapped in the same liposomal compartment, that there was a strong synergistic effect on the immune response, which was much greater than when either antigen was administered alone, or in various other modes of combination (e.g. co-administration as free entities, also pooled liposomal formulations where the two materials were contained in separate liposomal vehicles in the same suspension). The synergistic effect of liposomally co-entrapped DNA and protein exceeded, markedly, the well known adjuvant effects of plasmid DNA and liposomes. We have termed this new approach to vaccination 'co-delivery' and suggest that it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell--a mode of presentation that would commonly occur with live viral pathogens. We conclude that co-delivery is a very effective means to generate protective antibody responses against viral pathogens.     

Liposomes containing glucosyl ceramide specifically bind T4 bacteriophage: a self-assembling nanocarrier formulation

A unique formulation is described comprising liposomes containing glucosyl ceramide (GluCer) in the lipid bilayer to which bacteriophage T4 was attached. Binding of the phage T4 did not occur to glycolipids, such as galactosyl ceramide, containing an aldose in which the C-2 or C-4 conformations were not identical to glucose. These results strongly support previous proposals that glucose is a major receptor moiety for T4 binding to Escherichia coli. By using the binding of T4 to liposomal GluCer, we further describe a formulation that can be used as a self-assembling combined antigen and adjuvant carrier. A peptide antigen derived from C-trimer (Ct) of HIV-1 gp41 was fused to the highly antigenic outer capsid protein (Hoc), a nonessential protein of T4 that spontaneously binds to the T4 capsid. This resulted in display of the Ct-Hoc construct on the T4 capsid, and specific binding of a human monoclonal antibody that recognizes a peptide sequence of Ct was demonstrated. Liposomes containing monophosphoryl lipid A (MPLA) have been demonstrated to have potent adjuvant activities for experimental vaccines both in humans and animals, and because of this, mice were immunized with the Ct-Hoc-T4 construct that was bound to liposomes containing both GluCer and MPLA, resulting in the induction of high titers of Ct-specific antibodies. We conclude that liposomes containing both GluCer and MPLA can spontaneously bind to a construct of T4 that displays antigens that spontaneously binds to the capsid of T4 bacteriophage. This formulation could be utilized as an easily manufactured self-assembling antigen and adjuvant carrier.     

The ‘Co-Delivery’ Approach to Liposomal Vaccines: Application to the Development of influenza-A and hepatitis-B Vaccine Candidates

DNA vaccination with mammalian-expressible plasmid DNA encoding protein antigens is known to be an effective means to elicit cell-mediated immunity, sometimes in the absence of a significant antibody response. This may be contrasted with protein vaccination, which gives rise to antibody responses with little evidence of cell-mediated immunity. This has led to considerable interest in DNA vaccination as a means to elicit cell-mediated immune responses against conserved viral antigens or intracellular cancer antigens, for the purpose of therapeutic vaccination. However, almost all current vaccines are used prophylactically and work by producing antibodies rather than cell mediated immune responses. In the present study we have therefore explored the combination of DNA and protein forms of an antigen using two exemplary prophylactic vaccine antigens, namely inactivated influenza virion and hepatitis-B surface antigen. We studied the effects of various combinations of DNA and protein on the antibody response. Co-administration of soluble forms of DNA and protein representations of the same antigen gave rise to the same level of antibody response as if protein were administered alone. In contrast, we found that when these antigens are entrapped in the same liposomal compartment, that there was a strong synergistic effect on the immune response, which was much greater than when either antigen was administered alone, or in various other modes of combination (e.g. co-administration as free entities, also pooled liposomal formulations where the two materials were contained in separate liposomal vehicles in the same suspension). The synergistic effect of liposomally co-entrapped DNA and protein exceeded, markedly, the well known adjuvant effects of plasmid DNA and liposomes. We have termed this new approach to vaccination ‘co-delivery’ and suggest that it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell – a mode of presentation that would commonly occur with live viral pathogens. We conclude that co-delivery is a very effective means to generate protective antibody responses against viral pathogens.     

Liposome-mediated cytosolic delivery of macromolecules and its possible use in vaccine development.

In the majority of bacterial and viral infections the generation of cytotoxic T cells is of particular interest because such pathogens are able to escape the host defence mechanisms by surviving intracellularly within the phagocytic cells. To generate a CD8+ T lymphocyte response against exogenous antigens, the prerequisite is their delivery into the cytosol followed by processing and presentation along with class I major histocompatibility complex (MHC-I) molecules. In the present study we describe the method of liposome-based delivery of antigens and other macromolecules into the cytosol of target cells. To develop safe and effective methods for generating CD8+ T lymphocytes, we exploited the fusogenic character of lipids derived from lower organisms, that is baker's yeast (Saccharomyces cerevisiae). The degree of fusion with model membrane systems using yeast lipid liposomes varied from 40-70%, as opposed to 1-8% observed with egg PtdCho liposomes, depending on the assay system used. The fusion of yeast lipid liposomes with macrophages resulted in effective delivery of the entrapped solutes into the cytoplasmic compartment. This was further supported by the inhibition of cellular protein synthesis in J774 A1 cells by ricin A, encapsulated in the yeast lipid liposomes. Interestingly, the model antigen ovalbumin, when entrapped in the yeast lipid liposomes, successfully elicited antigen reactive CD8+ T cell responses. It may be concluded that the liposomes made of lipids derived from S. cerevisiae can spontaneously fuse with macrophages, delivering a significant portion of their contents into the cytoplasmic compartment of the cells.     

Avian scale development. Absence of an “epidermal placode” in reticulate scale morphogenesis

Timed-sequence studies have shown that reticulate scales on the ventral footpads of birds do not undergo “epidermal placode” formation during their morphogenesis, but arise as symmetrical evaluations similar to the scales of snakes and lizards. Unlike the scutellate scales on the dorsal surface of the foot, in which the formation of an “epidermal placode” and its subsequent morphogenesis result in distinct outer and inner epidermal surfaces, the reticulate scales elaborate only one type of epidermal surface.     

Liposome-nucleus interactions. Flow cytometric study on the role of the nuclear surface

The water-soluble probe carboxyfluorescein (CF), contained in the internal aqueous phase of liposomes, was used to investigate the interaction of phospholipid vesicles with isolated nuclei. Ultrastructural analysis indicated that adherent liposomes coated the nuclear surface, and fluorescence microscopy showed that they contained quenching concentrations of the dye. Flow cytometry revealed that the transfer of the entrapped dye from the adhering liposomes to nuclei was blocked by chilling at 0 degrees C. Chase experiments demonstrated that the most reliable mechanism of dye transfer involved fusion phenomena between the liposomal and the nuclear membranes. After the release of the fluorophore into the nucleus, empty liposomes could withdraw the intranuclear soluble fraction of the dye.     

INDUCTION OF A CYTOTOXIC T LYMPHOCYTE (CTL) RESPONSE TO PLASMID DNA DELIVERED VIA LIPODINE™ LIPOSOMES*

ABSTRACT

We have previously shown that liposome-mediated plasmid DNA immunisation may be a preferred alternative to the use of naked DNA. Lipodine? DNA formulations consist of liposomes containing entrapped DNA plasmid by the dehydration–rehydration (DRV) method. Such liposome formulations are distinct from liposomes with externally complexed DNA in that the majority of the DNA is “internal” to the liposome structure and hence protected from DNAase degradation. Previous studies on the immune response induced by DNA vaccines entrapped in Lipodine? have focused on the humoural response. In the present study, we have expanded the analysis profile in order to include the cytotoxic T lymphocyte (CTL) component of the immune response. We have analysed the immune response induced by DNA entrapped in Lipodine? compared to that induced by DNA alone when delivered subcutaneously, a route of administration not normally inducing significant plasmid DNA mediated immune activation. Our results indicate that delivery of a small dose of plasmid DNA in Lipodine? results in an improved antibody response to the plasmid encoded antigen and a strong antigen specific CTL response compared to that induced by DNA delivered alone.