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.     

A vaccine based on biodegradable microspheres induces protective immunity against scuticociliatosis without producing side effects in turbot

The histiophagous scuticociliate parasite Philasterides dicentrarchi is an emergent pathogen in aquaculture and causes significant economic losses on turbot (Scophthalmus maximus) farms. In this study, the surface antigens (Ag) of the parasite were encapsulated and covalently linked to a polymeric microparticle formulation composed of two biodegradable polymers (chitosan and Gantrez). The antigenicity of the formulation and the protection provided were compared in mice and turbot. This formulation induced a higher antibody (Ab) response in mice at doses of 5mg of microspheres (MS) conjugated with approximately 230 μg of Ag (MS-Ag(c)). However, Ab levels were significantly lower than in mice vaccinated with the same concentration of Ag in complete Freund's adjuvant (FCA). In turbot, the MS-Ag(c) formulation induced a higher level of Abs than that induced by the same vaccine emulsified in FCA. The challenge experiments performed with P. dicentrarchi and vaccinated turbot also showed a clear correlation between Ab levels and survival levels. Growth was significantly affected in fish vaccinated with FCA, but not in fish vaccinated with MS. The high adjuvant capacity of MS, together with its biodegradability and low toxicity to fish, makes this new vaccine an economical, effective and safe alternative to oil-based adjuvants for the immunoprophylaxis of scuticociliatosis in turbot.     

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.     

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.     

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.     

Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying

Disaccharides are well-known reagents to protect biostructures like proteins and phospholipid-based liposomes during freezing and drying. We have investigated the ability of the two disaccharides trehalose and sucrose to stabilize a novel, non-phospholipid-based liposomal adjuvant composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) upon freeze-drying. The liposomes were freeze-dried using a human dose concentration containing 2.5 mg/ml DDA and 0.5 mg/ml TDB with varying concentrations of the two sugars. The influence on particle size upon rehydration was investigated using photon correlation spectroscopy (PCS) and the gel to fluid phase transition was examined by differential scanning calorimetry (DSC). Data revealed that concentrations above 211 mM trehalose protected and preserved DDA/TDB during freeze-drying, and the liposomes were readily rehydrated. Sucrose was less efficient as a stabilizer and had to be used in concentrations above 396 mM in order to obtain the same effect. Immunization of mice with the tuberculosis vaccine candidate Ag85B-ESAT-6 in combination with the trehalose stabilized adjuvant showed that freeze-dried DDA/TDB liposomes retained their ability to stimulate both a strong cell-mediated immune response and an antibody response. These findings show that trehalose at isotonic concentrations protects cationic DDA/TDB-liposomes during freeze-drying. Since this is not the case for liposomes based on DDA solely, we suggest that the protection is facilitated via direct interaction with the headgroup of TDB and a kosmotropic effect, whereas direct interaction with DDA plays a minor role.     

Liposomal muramyl dipeptide therapy of experimental M5076 liver metastases in mice

Summary The effectiveness ofN-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) or of liposomes containing a lipophilic MDP derivative, MDP-glyceroyldipalmitate MDP-GDP in inhibiting the growth of M5076 reticulum cell sarcoma liver metastases in C57BL/6 mice has been determined. MDP (100 µg) or liposomal MDP-GDP (2.5 µmol containing 1 µg) were equally effective in inhibiting liver metastatic growth when given as a single treatment 3 days before tumor cell injection. Therapeutic treatment, initiated 3 days after tumor cell injection and continued for a period of 2 weeks, failed to inhibit metastatic growth. Activation of thioglycollate-elicited peritoneal macrophages or Kupffer cells in vitro with MDP or liposomal MDP-GDP resulted in the expression of tumoricidal activity against M5076 tumor cells. Adoptive cellular therapy with four injections of 2 × 10⁶ macrophages was ineffective: activation of the macrophages with either MDP or liposomal MDP-GDP prior to injection was effective in inhibiting liver metastatic growth. Incorporation of the macrophage toxin dichlorodimethylene diphosphonate within liposomes containing MDP-GDP abolished the ability of such liposomes to induce macrophage or Kupffer cell tumoricidal activity in vitro as well as the antitumor activity when administered 3 days before tumor cell challenge.     

Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying

Disaccharides are well-known reagents to protect biostructures like proteins and phospholipid-based liposomes during freezing and drying. We have investigated the ability of the two disaccharides trehalose and sucrose to stabilize a novel, non-phospholipid-based liposomal adjuvant composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6′-dibehenate (TDB) upon freeze-drying. The liposomes were freeze-dried using a human dose concentration containing 2.5 mg/ml DDA and 0.5 mg/ml TDB with varying concentrations of the two sugars. The influence on particle size upon rehydration was investigated using photon correlation spectroscopy (PCS) and the gel to fluid phase transition was examined by differential scanning calorimetry (DSC). Data revealed that concentrations above 211 mM trehalose protected and preserved DDA/TDB during freeze-drying, and the liposomes were readily rehydrated. Sucrose was less efficient as a stabilizer and had to be used in concentrations above 396 mM in order to obtain the same effect. Immunization of mice with the tuberculosis vaccine candidate Ag85B-ESAT-6 in combination with the trehalose stabilized adjuvant showed that freeze-dried DDA/TDB liposomes retained their ability to stimulate both a strong cell-mediated immune response and an antibody response. These findings show that trehalose at isotonic concentrations protects cationic DDA/TDB-liposomes during freeze-drying. Since this is not the case for liposomes based on DDA solely, we suggest that the protection is facilitated via direct interaction with the headgroup of TDB and a kosmotropic effect, whereas direct interaction with DDA plays a minor role.     

Effective immunization against cutaneous leishmaniasis with defined membrane antigens reconstituted into liposomes

The abundant Leishmania promastigote surface Ag gp63 and Leishmania promastigote lipophosphoglycan were reconstituted into liposomes and used as a vaccine against the agent of New World cutaneous leishmaniasis, Leishmania mexicana. The Ag were inoculated s.c., i.p., and i.v. into CBA/ca and BALB/c mice. Even at low Ag dosages, 8 to 10 micrograms/mouse, the Ag induced appreciable levels of protection. In CBA/ca mice complete protection was obtained by s.c. inoculation of antigen-containing liposomes. Protection could be transferred with T cells to naive mice. Interestingly, the Ag-containing liposomes did not cause the disease exacerbation observed in previous vaccine studies with crude parasite extracts.     

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.     

Induction of cytotoxic T lymphocytes in vivo with protein antigen entrapped in membranous vehicles.

Intravenous administration of APC such as splenocytes loaded with a soluble protein Ag has been shown to prime for an Ag-specific CTL response. It is thought that the APC directly presents loaded Ag in a MHC-restricted manner. However, it is demonstrated in this study that allogeneic splenocytes, MHC-free RBC, and even synthetic lipid vesicles (liposomes) after loading with OVA can elicit an OVA-specific and MHC-restricted CTL response. Biodistribution studies of these Ag-associated vehicles showed that the liver, spleen, and lung were the major organs responsible to scavenge these carriers, suggesting that the monocyte-macrophage system was involved in the Ag presentation for CTL. Depletion of macrophages by a specific macrophage killer, Cl2MDP, containing liposomes, abolished the CTL induction by immunization with OVA Ag carried by these vehicles except the induction by syngeneic splenocytes. Thus, the syngeneic splenocytes present Ag directly to the T cells, but other membranous vehicles carry the Ag to the host APC including macrophages, which then present it to the T cells. These results indicate that formulation of an Ag in membranous/colloidal vehicles may be a way to prime for a CTL response.     

Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6'-dibehenate)-a novel adjuvant inducing both strong CMI and antibody responses

Incorporation of the glycolipid trehalose 6,6'-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4 degrees C and 25 degrees C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B-ESAT-6 in DDA-TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-gamma cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-gamma was identified as CD4 T cells.     

Recombinant trehalose-6-phosphate phosphatase of Brugia malayi cross-reacts with human Wuchereria bancrofti immune sera and engenders a robust protective outcome in mice

Trehalose-6-phosphate phosphatase of Brugia malayi (Bm-TPP) represents an attractive vaccine candidate because it is present in all the major life stages of parasite, but is absent in mammals. We have previously cloned, purified and biochemically characterized Bm-TPP. In the present study, we investigated the cross-reactivity of recombinant Bm-TPP (r-Bm-TPP) with the sera of human bancroftian patients belonging to different disease categories. In silico study using bioinformatics tool demonstrated that Bm-TPP is highly immunogenic in nature. BALB/c mice administered with r-Bm-TPP alone or in combination with Freund's complete adjuvant (FCA) generated a strong IgG response. Further investigations on the antibody isotypes showed generation of a mixed T helper cell response which was marginally biased towards Th1 phenotype. r-Bm-TPP with or without adjuvant lead to significantly increased accumulation of CD4+ and CD8+ T cells in the spleen of infected mice and increased the activation of peritoneal macrophages. Additionally, r-Bm-TPP enhanced the production of both proinflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and mice immunized with r-Bm-TPP alone or in combination with FCA showed 54.5% and 67% protection respectively against B. malayi infective larvae challenge. Taken together, our findings suggest that Bm-TPP is protective in nature and might be a potential candidate for development of vaccine against lymphatic filarial infections.     

Effect of Liposomal Formulations and Immunostimulating Peptidoglycan Monomer (PGM) on the Immune Reaction to Ovalbumin in Mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-l-Ala-d-isoGln-mesoDpm(εNH₂)-d-Ala-d-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).     

Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

Enhancement of carrier-specific helper T cell function by the synthetic adjuvant, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP).

The adjuvant effect of a synthetic peptidoglycan, muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine, MDP), was studied by using the anti-Tnp PFC and hemagglutinin responses of BALB/c mice to hapten-carrier conjugates. Administration of Tnp-OVA and MDP in saline to mice, followed 2 weeks later by a boost of Tnp-OVA in saline, led to significantly higher IgM and IgG anti-Tnp PFC and total anti-Tnp-hemagglutinin responses than those obtained in mice not treated with MDP in the initial immunization. A similar adjuvant effect by MDP on anti-hapten PFC responses was seen if mice were primed with KLH together with MDP and challenged with Tnp-KLH 2 weeks later. This apparent effect on carrier priming for helper function was confirmed and quantitated by double adoptive transfer experiments with graded numbers of spleen cells from KLH +/- MDP-primed mice and a fixed number of hapten-primed spleen cells from syngeneic Tnp-OVA immunized animals. These data suggest that at least one mode of action of the synthetic adjuvant MDP is via the enhanced stimulation of the helper T cell function.     

Effect of Macrophage Elimination Using Liposome-Encapsulated Dichloromethylene Diphosphonate on Tissue Distribution of Liposomes

Abstract

Effect of macrophage elimination using liposomal dichloromethylene diphosphonate (C1₂MDP)1 on tissue distribution of different types of liposomes was examined in mice. Intravenously administration into mice with CI2MDP encapsulated in liposomes composed of phosphatidylcholine, cholesterol and phosphatidylserine exhibits a temporary blockade of liver and spleen function for liposome uptake. At a low dose of 90 (ig/mouse, the liposome uptake by the liver was significantly decreased. Such decrease was accompanied by an increase in liposome accumulation in either spleen or blood depending on liposome composition and size. Direct correlation between the administration dose of liposomal CI2MDP and the liposome circulation time in blood was also obtained even for liposomes with an average diameter of more than 500 nm. These results indicate that temporary elimination of macrophages of the liver and spleen using liposomal CI2MDP may prove to be useful to enhance the drug delivery efficiency of liposomes.     

流行性感冒病毒血凝素亚单位疫苗在小鼠体内免疫效果观察

流感病毒表面抗原——血凝素(HA)亚单位,在人工合成的胞壁酰二肽(MDP)佐剂配合下,注射小白鼠所产生的免疫效果与常用的Al(OH)_3及福氏佐剂相似。含MDP佐剂的流感病毒HA亚单位疫苗腹腔注射小白鼠,能产生与福尔马林灭活的流感疫苗相似的免疫反应,而皮下注射,前者的免疫效果比后者明显为佳。     

Effect of liposomal formulations and immunostimulating peptidoglycan monomer (PGM) on the immune reaction to ovalbumin in mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDpm(epsilonNH2)-D-Ala-D-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).     

In vitro activation of immune cells by a mycolic acid-containing glycolipid, trehalose-2,3,6'-trimycolate, derived from Gordona aurantiaca

A mycolic acid-containing glycolipid, trehalose-2,3,6'-trimycolate (GaGM), derived from Gordona aurantiaca, an acid-fast bacteria closely related taxonomically to Mycobacterium, was investigated for its immune adjuvant activity in vitro. The liposomes containing GaGM showed strong mitogenic effects on murine spleen cells at the doses used (25-100 micrograms/ml), but not on T-cell-depleted spleen cells or macrophage-depleted spleen cells. These results suggest that the mitogenic property of liposomes containing GaGM differs from that of such as lipopolysaccharide, a B-cell mitogen and that its mitogenic effects depend on the presence of macrophages. In addition, liposomes containing GaGM augmented the mixed lymphocyte reaction (MLR) and in vitro induction of cytotoxic T-lymphocytes (CTLs) against allogeneic tumor cells. These results suggest that liposomes containing GaGM have immune adjuvant properties in vitro and the adjuvant activity may be related to such cytokines as interleukin-1 and -2.