Leishmania major: immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes

Protection against leishmaniasis is depending upon generation of a Th1 type of immune response. Field trials of first generation Leishmania vaccine showed a limited efficacy even with multiple doses mainly due to lack of an appropriate adjuvant. In this study, susceptible BALB/c mice were immunized with rLmSTI1 encapsulated in liposomes to explore the extent of protection induced by Leishmania antigen encapsulated in the liposomes against challenge with Leishmania major. The results showed that s.c. immunization of BALB/c mice with liposomal rLmSTI1 induced a significant protection against challenge and a significant lower parasite burden in spleen up to 14 weeks after challenge. The protected animals showed a significantly smaller footpad thickness after challenge, and a higher level of anti-SLA IgG antibodies before and after challenge with a predominant IgG2a titer. The data supports the possibility of using liposomal Leishmania antigens as a vaccine.     

Vaccination against murine cutaneous leishmaniasis by using Leishmania major antigen/liposomes. Optimization and assessment of the requirement for intravenous immunization

Efficacy of vaccination against cutaneous leishmaniasis in highly susceptible BALB/c mice was assessed comparatively by using radiation-attenuated promastigotes and colloidal Ag mixtures generated from a mixed Leishmania major (LV39) isolate (SLV39) and from a virulent cloned line (SVJ2) derived from the Jericho 2 L. major isolate. Dehydration-rehydration vesicle (DRV) liposomes were used as adjuvants. In optimization experiments phospholipid composition of DRV was varied, and the distearoyl derivative (DSPC) (liquid-crystalline phase transition temperature (Tc) 54 degrees C) of egg lecithin L-alpha-phosphatidylcholine was found to be superior to the dipalmitoyl derivative (DPPC, Tc 41.5 degrees C) and underivatized L-alpha-phosphatidylcholine (Tc -10 degrees C). The criteria studied were in vivo priming for a secondary in vitro proliferative response and the prepatency of lesion onset after L. major challenge of mice immunized once i.v. A single s.c. immunization with SLV39 either free or entrapped within DSPC liposomes primed spleen cells to produce significant levels of IL-3 when stimulated with SLV39 in vitro. In contrast, the i.v. route of immunization with the same Ag preparations led to little or no IL-3 production by the spleen cells. Despite development of significant T cell activation, both SLV39 and SVJ2 administered s.c., either free or entrapped within liposomes, were not protective. However, i.v. immunization four times with SVJ2 entrapped within DSPC liposomes induced a level of resistance comparable with that of 2 x 10(7) gamma-irradiated promastigotes in the stringent BALB/c L. major model. Although significant, protection conferred after i.v. immunization with SLV39/DSPC liposomes was less effective. These data therefore show that DSPC/DRV liposomes, although a good adjuvant for inducing protective immunity to cutaneous leishmaniasis, are not able to overcome the requirement for an i.v. route of immunization with the leishmanial Ag preparation. Additionally, they demonstrate a correlation between IL-3 secretion and non-protection. They also suggest that SVJ2 represents a better source of protective Ag than SLV39.     

Dendritic cell (DC)-based protection against an intracellular pathogen is dependent upon DC-derived IL-12 and can be induced by molecularly defined antigens

Upon loading with microbial Ag and adoptive transfer, dendritic cells (DC) are able to induce immunity to infections. This offers encouragement for the development of DC-based vaccination strategies. However, the mechanisms underlying the adjuvant effect of DC are not fully understood, and there is a need to identify Ag with which to arm DC. In the present study, we analyzed the role of DC-derived IL-12 in the induction of resistance to Leishmania major, and we evaluated the protective efficacy of DC loaded with individual Leishmania Ag. Using Ag-pulsed Langerhans cells (LC) from IL-12-deficient or wild-type mice for immunization of susceptible animals, we showed that the inability to release IL-12 completely abrogated the capacity of LC to mediate protection against leishmaniasis. This suggests that the availability of donor LC-derived IL-12 is a requirement for the development of protective immunity. In addition, we tested the protective effect of LC loaded with Leishmania homolog of receptor for activated C kinase, gp63, promastigote surface Ag, kinetoplastid membrane protein-11, or Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a. The results show that mice vaccinated with LC that had been pulsed with selected molecularly defined parasite proteins are capable of controlling infection with L. major. Moreover, the protective potential of DC pulsed with a given Leishmania Ag correlated with the level of their IL-12 expression. Analysis of the cytokine profile of mice after DC-based vaccination revealed that protection was associated with a shift toward a Th1-type response. Together, these findings emphasize the critical role of IL-12 produced by the sensitizing DC and suggest that the development of a DC-based subunit vaccine is feasible.     

Protection against Leishmania major infection by oligomannose-coated liposomes

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (Man5-DPPE) have been shown to induce cellular immunity against antigens encapsulated in the liposomes. To assess whether these neoglycolipid-coated liposomes can elicit protective immune response against challenge infection, effects of immunization with soluble leishmanial antigens encapsulated in the liposomes were evaluated using Leishmania major infection in susceptible BALB/c mice. Intraperitoneal immunization of mice with leishmanial antigens in the Man5-DPPE-coated liposomes significantly suppressed footpad swelling in comparison to the control, non-immunized mice, while progression of the disease was observed in mice administered antigens in uncoated liposomes and those administered soluble antigens alone, as seen with control mice. Similarly, the number of parasites decreased substantially in local lymph nodes of mice immunized with the antigen in the Man5-DPPE-coated liposomes. Protection against L. major infection in the immunized mice also coincided with an elevated ratio of antigen-specific IgG2a/IgG1 antibodies, which is a profile of T helper-type 1-like immune response. Taken together, these results indicate the possibility that Man5-DPPE-coated liposome-encapsulated antigens could serve as a vaccine that triggers protection against infectious disease.     

Protection of mice from fatal Toxoplasma gondii infection by immunization with p30 antigen in liposomes

Using liposomes as adjuvant, a purified membrane Ag from Toxoplasma gondii (p30) has been tested for its protective effect in mice. Immunization with p30 in liposomes resulted in only one in 15 mice dying from a challenge that killed 11 of 15 control mice (receiving only buffer or liposomes without p30). The p30 Ag alone gave intermediate levels of protection, with 5 of 15 animals dying. The source of the p30 Ag was the rapidly growing, laboratory-adapted strain of T. gondii, RH; challenge was with the recently isolated C strain which still has all the properties of a wild-type strain. To assess the validity of this combination, the amino acid sequence of p30 from these two strains (as predicted from the corresponding gene sequence) was compared and found to differ in only eight residues. This minimal variation argued that RH was a valid source of material for a subunit vaccine, as subsequently confirmed by the protection studies. These results indicate that p30 is an appropriate Ag for development into a subunit vaccine for immunization of humans and/or domestic livestock, which are a major source of human infection.     

Protection against Leishmania major in BALB/c mice by adoptive transfer of a T cell clone recognizing a low molecular weight antigen released by promastigotes

We have shown previously that BALB/c mice can be protected against a fatal infection with Leishmania major by adoptive transfer of a T cell line recognizing a protective soluble fraction (fraction 9) of promastigotes. We now describe the isolation and characterization of a T cell clone (9.1-2) that also transfers protective immunity against Leishmania. After Ag or mitogen stimulation, this clone secrets IL-2 and IFN-gamma, but not IL-4 or IL-5. The clone preferentially recognizes L. major fraction 9, and in addition, soluble Ag from Leishmania donovani, Leishmania amazonensis, and Leishmania braziliensis, but not from the related flagellates, Trypanosoma cruzi or Crithidia fasciculata. Besides being contained in fraction 9, the stimulatory Ag is also released from the parasite, because concentrated promastigote culture supernatants induced IFN-gamma production by 9.1-2. By means of T cell immunoblotting, we determined that 9.1-2 recognized a protein with a relative m.w. between 8,000 and 12,000, and within this region is a predominant protein contained in fraction 9 of approximately 10,000 m.w. These data identify a new candidate Ag for immunization against protozoa belonging to the genus Leishmania.     

Comparison of BCG,MPL and cationic liposome adjuvant systems in leishmanial antigen vaccine formulations against murine visceral leishmaniasis

Background  

The development of an effective vaccine against visceral leishmaniasis (VL) caused by Leishmania donovani is an essential aim for controlling the disease. Use of the right adjuvant is of fundamental importance in vaccine formulations for generation of effective cell-mediated immune response. Earlier we reported the protective efficacy of cationic liposome-associated L. donovani promastigote antigens (LAg) against experimental VL. The aim of the present study was to compare the effectiveness of two very promising adjuvants, Bacille Calmette-Guerin (BCG) and Monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) with cationic liposomes, in combination with LAg, to confer protection against murine VL.     

Potentiating effects of MPL on DSPC bearing cationic liposomes promote recombinant GP63 vaccine efficacy: high immunogenicity and protection

Background

Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice.

Methodology/Principal Findings

Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo.

Conclusion

Our results define the immunopotentiating effect of MPL-TDM on protein Ag encapsulated in a controlled release system against experimental VL.     

Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes

BALB/c mice can be protected against a fatal Leishmania major infection by immunization with whole radio-attenuated promastigotes; however, neither the antigens responsible for protection nor the protective immunologic mechanisms have been defined. In this study, the ability of promastigote fractions to elicit similar immunity to that obtained with whole organisms, and the immune responses associated with such protection were analyzed. Intraperitoneal immunization with a soluble, membrane-free parasite extract was found to induce protection against L. major challenge equal to that obtained with whole organisms. Induction of immunity (89% protection in seven experiments) was most effective with 100 micrograms of the soluble leishmanial antigen (SLA) and required concomitant injection of the bacterial adjuvant, Corynebacterium parvum (CP), followed by an i.p. boost of SLA alone 1 wk later. Vaccinated animals exhibited Leishmania-specific cell-mediated immunity, as assessed both by lymphocyte transformation and the production of macrophage-activating factors (MAF). In addition, although SLA + CP-immunized mice failed to exhibit delayed-type hypersensitivity (DTH) before challenge, splenic lymphocytes from these mice could transfer a local DTH reaction to naive recipients. Immunization also induced the production of antibodies against two major metabolically labeled proteins of m.w. 30,000 and 53,000, but failed to stimulate a detectable humoral response against promastigote surface antigens. Thus, these experiments demonstrate that vaccine-induced immunity against cutaneous leishmaniasis is strongly associated with the induction of cell-mediated immunity, but does not require the development of an antibody response to promastigote surface antigens. In addition, these studies establish the feasibility of employing soluble, nonmembrane-derived parasite material as a source of protective immunogens.     

Protective immunity using recombinant human IL-12 and alum as adjuvants in a primate model of cutaneous leishmaniasis.

Protection from cutaneous leishmaniasis, a chronic ulcerating skin lesion affecting millions, has been achieved historically using live virulent preparations of the parasite. Killed or recombinant Ags that could be safer as vaccines generally require an adjuvant for induction of a strong Th1 response in murine models. Murine rIL-12 as an adjuvant with soluble Leishmania Ag has been shown to protect susceptible mice. We used 48 rhesus macaques to assess the safety, immunogenicity, and efficacy of a vaccine combining heat-killed Leishmania amazonensis with human rIL-12 (rhIL-12) and alum (aluminum hydroxide gel) as adjuvants. The single s.c. vaccination was found to be safe and immunogenic, although a small transient s.c. nodule developed at the site. Groups receiving rhIL-12 had an augmented in vitro Ag-specific IFN-gamma response after vaccination, as well as increased production of IgG. No increase in IL-4 or IL-10 was found in cell culture supernatants from either control or experimental groups. Delayed hypersensitivity reactions were not predictive of protection. Intradermal forehead challenge infection with 107 metacyclic L. amazonensis promastigotes at 4 wk demonstrated protective immunity in all 12 monkeys receiving 2 microgram rhIL-12 with alum and Ag. Partial efficacy was seen with lower doses of rhIL-12 and in groups lacking either adjuvant. Thus, a single dose vaccine with killed Ag using rhIL-12 and alum as adjuvants was safe and fully effective in this primate model of cutaneous leishmaniasis. This study extends the murine data to primates, and provides a basis for further human trials.     

A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection

Defined Abs to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been shown to be protective against experimental cryptococcosis. This suggests that if a vaccine could induce similar Abs it might protect against infection. However, the potential use of a GXM-based vaccine has been limited by evidence that GXM is a poor immunogen that can induce nonprotective and deleterious, as well as protective, Abs, and that the nature of GXM oligosaccharide epitopes that can elicit a protective response is unknown. In this study, we investigated whether a peptide surrogate for a GXM epitope could induce an Ab response to GXM in mice. The immunogenicity of peptide-protein conjugates produced by linking a peptide mimetic of GXM, P13, to either BSA, P13-BSA, or tetanus toxoid, P13-tetanus toxoid, was examined in BALB/c and CBA/n mice that received four s.c. injections of the conjugates at 14- to 30-day intervals. All mice immunized with conjugate produced IgM and IgG to P13 and GXM. Challenge of conjugate-immunized mice with C. neoformans revealed longer survival and lower serum GXM levels than control mice. These results indicate that 1) P13 is a GXM mimotope and 2) that it induced a protective response against C. neoformans in mice. P13 is the first reported mimotope of a C. neoformans Ag. Therefore, the P13 conjugates are vaccine candidates for C. neoformans and their efficacy in this study suggests that peptide mimotopes selected by protective Abs deserve further consideration as vaccine candidates for encapsulated pathogens.     

Characterization of two proteins from Leishmania donovani and their use for vaccination against visceral leishmaniasis

Two proteins from Leishmania donovani, dp72 and gp70-2, have been previously utilized to specifically serodiagnose patients with visceral leishmaniasis. The proteins were shown by ELISA and Western blotting with monoclonal and polyclonal antibodies to be present in both stages of the parasite. Antibodies to gp70-2 recognize in promastigotes multiple discrete bands of similar m.w. which are common to several isolates of L. donovani. The total amount of Ag and number of bands observed per isolate is not constant. Lectin blots with Con A show gp70-2 to be a glycoprotein. Dp72 shows pronounced microheterogeneity between isolates of L. donovani. The Brazilian isolates examined appear to possess a lower m.w. form (64,000 or 68,000) of this molecule. No reactions were observed with dp72 and lectins in Western blots; and neither tunicamycin, N-glycanase, endoglycosidase H nor F affected the migration of [35S]-methionine-labeled protein on SDS-PAGE. A mAb against dp72 also cross-reacted in Western blots with a 60-kDa protein in Leishmania major, Leishmania aethiopica, and Leishmania tropica. No reaction was observed between the purified promastigote surface protease (gp63) and either monoclonal or polyclonal antibodies produced to dp72 or gp70-2. The ability of the pure proteins to provide protection against a challenge by L. donovani amastigotes was examined. BALB/c mice were immunized with gp70-2 and/or dp72 by using Corynebacterium parvum as an adjuvant. Mice immunized with gp70-2 were not protected; however, mice receiving dp72 showed a 81.1% reduction in the liver parasitemia compared with the adjuvant controls.     

Oral Salmonella typhimurium (AroA-) vaccine expressing a major leishmanial surface protein (gp63) preferentially induces T helper 1 cells and protective immunity against leishmaniasis

The gp63 gene of Leishmania major was transformed into the AroA- vaccine strain of Salmonella typhimurium (SL3261). The construct (SL3261-gp63), which stably expresses the gp63 Ag in vitro, was used to immunize CBA mice by the oral route. Spleen cells from mice inoculated with SL3261-gp63 developed antibody and proliferative T cell response to L. major. They did not express detectable delayed-type hypersensitivity reactivity. The activated T cells are mainly CD4+ and secrete IL-2 and IFN-gamma but no IL-4. The orally immunized mice developed significant resistance against a challenge L. major infection. We have, therefore, demonstrated the feasibility of oral vaccination against leishmaniasis and that the oral route of antigen delivery via the heterologous carrier may preferentially induce Th1 subsets of CD4+ cells.     

Prophylactic immunization against experimental leishmaniasis. VI. Comparison of protective and disease-promoting T cells

In previous studies, we reported that mice immunized i.v. with lethally irradiated Leishmania major promastigotes developed substantial resistance to a subsequent L. major infection. However, such protection could be totally suppressed by prior s.c. injection with the same antigens. Both the protective immunity and the inhibition of its induction could be adoptively transferred with specific Lyt-2- T cells. Here, we present evidence showing that protection and disease promotion resulting from i.v. or s.c. immunization, respectively, are mediated by functionally distinct subsets of T cells. In a series of titration experiments, it was found that freshly isolated T cells derived from prophylactically i.v. immunized BALB/c mice were either protective (greater than 10(7) cells/recipient) or ineffective (less than 10(7) cells/recipient). No exacerbation of disease was observed at any dose. Conversely, T cells from mice immunized s.c. either accelerated disease development and inhibited protective immunization (greater than 10(7) cells/recipient) or had no effect (less than 10(7) cells/recipient). No protection was observed at any dose tested. In mixed transfer experiments, increasing numbers of T cells from s.c. immunized donors progressively inhibited the protective effect of T cells from i.v. immunized donors. Supernatant of T cell cultures from protectively immunized donors contained substantial macrophage-activating factor whereas such activity was not detectable in the supernatant of T cell culture from s.c. immunized donors. Analysis by flow cytometry showed that the spleen and lymph nodes of normal, i.v., or s.c. immunized BALB/c mice contained similar ratios of L3T4+ cells and Lyt-2+ cells.     

SIR2-deficient Leishmania infantum induces a defined IFN-gamma/IL-10 pattern that correlates with protection

The ability to manipulate the Leishmania genome to create genetically modified parasites by introducing or eliminating genes is considered a powerful alternative for developing a new generation vaccine against leishmaniasis. Previously, we showed that the deletion of one allele of the Leishmania infantum silent information regulatory 2 (LiSIR2) locus was sufficient to dramatically affect amastigote axenic proliferation. Furthermore, LiSIR2 single knockout (LiSIR2(+/-)) amastigotes were unable to replicate in vitro inside macrophages. Because this L. infantum mutant persisted in BALB/c mice for up to 6 wk but failed to establish an infection, we tested its ability to provide protection toward a virulent L. infantum challenge. Strikingly, vaccination with a single i.p. injection of LiSIR2(+/-) single knockout elicits complete protection. Thus, vaccinated BALB/c mice showed a reversal of T cell anergy with specific anti-Leishmania cytotoxic activity and high levels of NO production. Moreover, vaccinated mice simultaneously generated specific anti-Leishmania IgG Ab subclasses suggestive of both type 1 and type 2 responses. A strong correlation was found between the elimination of the parasites and an increased Leishmania-specific IFN-gamma/IL-10 ratio. Therefore, we propose that the polarization to a high IFN-gamma/low IL-10 ratio after challenge is a clear indicator of vaccine success. Furthermore these mutants, which presented attenuated virulence, represent a good model to understand the correlatives of protection in visceral leishmaniasis.     

Prophylactic immunization against experimental leishmaniasis. V. Mechanism of the anti-protective blocking effect induced by subcutaneous immunization against Leishmania major infection

The responsiveness of BALB/c mice to protective i.v. immunization with 150,000-rad irradiated or heat-killed Leishmania major promastigotes can be totally suppressed by prior subcutaneous (s.c.) injection of the same "vaccine." Induction of this effect is leishmania specific for although prevention of protection against L. major infection can be obtained with either homologous or Leishmania donovani promastigotes, it does not follow s.c. administration of an immunogenic Trypanosoma cruzi epimastigote preparation. Multiple s.c. injections of irradiated L. major promastigotes do not inhibit the subsequent antibody response of any major isotype to i.v. immunization, but rather induce some priming. The same s.c. injections induced delayed-type hypersensitivity (DTH) reactivity that could be transferred locally or systemically, although it was weaker than in mice with cured infections. Parallel cell-mediated immunity (CMI) responses were also reflected in vitro in specific lymphocyte transformation assays. Despite this evidence of a DTH/helper type of T cell response, transfer of 5 X 10(7) viable T cell-enriched spleen cells from 4 X s.c. immunized donors to normal recipients completely abrogated the protective response to i.v. immunization. Conversely, T cell-depleted (anti-Thy-1.2 + C treated) cells were without effect. The inhibitory T cells were defined by monoclonal antibody pretreatment as possessing an Lyt-1+2-,L3T4+ phenotype. T cells from s.c. immunized donors were also shown, by mixed transfer experiments, to counteract completely the protective effect of T cells from i.v. immunized donors in 550-rad irradiated recipients. They were as potent as suppressor T cells from donors with progressive disease both in this capacity and in abrogating the prophylactic effect of sublethal irradiation itself. The similarities and differences between suppressor and immune effector T cells induced by s.c. or i.v. immunization and those arising in response to leishmanial infection itself are discussed.     

Dissociation between vasodilation and Leishmania infection-enhancing effects of sand fly saliva and maxadilan.

In this study, the ability of maxadilan and Lutzomyia longipalpis salivary gland lysate to enhance the infection of CBA mice by Leishmania major and of BALB/c mice by L. braziliensis was tested. No difference was observed between sizes of lesion in CBA mice infected with L. major and treated or not with salivary gland lysate or maxadilan, although they were injected in concentrations that induced cutaneous vasodilation. Although parasites were more frequently observed in foot pads and spleens of animals treated with maxadilan than in the animals treated with salivary gland lysate or saline, the differences were small and not statistically significant. The lesions in BALB/c mice infected with L. braziliensis and treated with maxadilan were slightly larger than in animals that received Leishmania alone. Such differences disappeared 14 weeks after infection, and were statistically significant only in one of two experiments.     

The role of liposome charge on immune response generated in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63)

Liposomes as a lipid-based system have been shown to be an effective adjuvant formulation. In this study, the role of liposome charge in induction of a Th1 type of immune response and protection against leishmaniasis in BALB/c mice was studied. Liposomes containing rgp63 were prepared by Dehydration-Rehydration Vesicle (DRV) method. Neutral liposomes consisted of dipalmitoylphosphatidylcholine and cholesterol. Positively and negatively charged liposomes were prepared by adding dimethyldioctadecylammonium bromide (DDAB) or dicetyl phosphate (DCP) to the neutral liposome formulation, respectively. Female BALB/c mice were immunized subcutaneously with negatively, positively charged or neutral liposomes encapsulated with rgp63, rgp63 in soluble form or PBS, three times in 3 week intervals. The extent of protection and type of immune response generated were studied in different groups of mice. The group of mice immunized with rgp63 encapsulated in neutral liposomes showed a significantly (P < 0.01) smaller footpad swelling upon challenge with Leishmania major compared with positively or negatively charged liposomes. The mice immunized with neutral liposomes also showed a significantly (P < 0.01) the lowest splenic parasite burden, the highest IgG2a/IgG1 ratio and IFN-γ production and the lowest IL-4 level compared to the other groups. The results indicated that a Th1 type of immune response was induced in mice immunized with neutral liposomes more efficiently than positively charged liposomes and conversely negatively charged liposomes induced a Th2 type of immune response.