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.     

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).     

TLR7 imidazoquinoline ligand 3M-019 is a potent adjuvant for pure protein prototype vaccines

Cancer vaccines, while theoretically attractive, present difficult challenges that must be overcome to be effective. Cancer vaccines are often poorly immunogenic and may require augmentation of immunogenicity through the use of adjuvants and/or immune response modifiers. Toll-like receptor (TLR) ligands are a relatively new class of immune response modifiers that may have great potential in inducing and augmenting both cellular and humoral immunity to vaccines. TLR7 ligands produce strong cellular responses and specific IgG2a and IgG2b antibody responses to protein immunogens. This study shows that a new TLR7 ligand, 3M-019, in combination with liposomes produces very strong immune responses to a pure protein prototype vaccine in mice. Female C57BL/6 mice were immunized subcutaneously with ovalbumin (OVA, 0.1 mg/dose) weekly 4x. Some groups were immunized to OVA plus 3M-019 or to OVA plus 3M-019 encapsulated in liposomes. Both antibody and cellular immune responses against OVA were measured after either two or four immunizations. Anti-OVA IgG antibody responses were significantly increased after two immunizations and were substantially higher after four immunizations in mice immunized with OVA combined with 3M-019. Encapsulation in liposomes further augmented antibody responses. IgM responses, on the other hand, were lowered by 3M-019. OVA-specific IgG2a levels were increased 625-fold by 3M-019 in liposomes compared to OVA alone, while anti-OVA IgG2b levels were over 3,000 times higher. In both cases encapsulation of 3M-019 in liposomes was stronger than either liposomes alone or 3M-019 without liposomes. Cellular immune responses were likewise increased by 3M-019 but further enhanced when it was encapsulated in liposomes. The lack of toxicity also indicates that this combination may by safe, effective method to boost immune response to cancer vaccines.     

Interleukin-2/liposomes potentiate immune responses to a soluble protein cancer vaccine in mice

A critical element in improving the potency of cancer vaccines, especially pure protein or peptide antigens, is to develop procedures that can strongly but safely increase their ability to induce immune responses. Here, we describe that encapsulation of a pure protein antigen and interleukin-2 (IL-2) together into liposomes significantly improves immune responses and tumor protection. Groups of C57Bl/6 mice were immunized weekly ×4 with –0.1 mg of ovalbumin (OVA) injected subcutaneously in PBS or encapsulated in liposomes with or without human recombinant IL-2. Control groups included mice immunized to irradiated E.G7-OVA cells (that express ovalbumin), or to PBS. Sera were collected and pooled by immunization group at baseline and at weeks 2 and 4 to measure antibody responses to OVA by ELISA. Splenocytes obtained at week 4 were tested for anti-OVA cellular responses by ELISPOT. Mice were then challenged to a lethal dose of E.G7-OVA cells to measure tumor-protective immunity. IL-2 liposomes caused no detectable toxicity. Antibody, CD8⁺ T cell, and tumor-protective immune responses were markedly enhanced in mice immunized to OVA + IL-2 in liposomes compared to mice immunized to OVA, either alone or encapsulated into liposomes without IL-2. These results indicate that IL-2 liposomes enhance antibody, cellular, and tumor-protective immune responses to immunization with a soluble protein. This may provide a simple, safe, and effective way to enhance the immunogenicity of vaccines that consist of pure protein antigens. Supported by grant CA096804 (DJ)     

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).     

Surface-linked liposomal antigen induces ige-selective unresponsiveness regardless of the lipid components of liposomes

We have previously reported that antigen coupled with liposomes induced antigen-specific and IgE-selective unresponsiveness in mice. This antigen preparation was investigated for application in a novel vaccine protocol to induce minimal IgE synthesis. In this study, ovalbumin (OVA)-liposome conjugates were made using liposomes of four different lipid components, including unsaturated carrier lipid and three different saturated carrier lipids, after which the induction of anti-OVA antibody production was investigated in mice. All of the OVA-liposome conjugates induced IgE-selective unresponsiveness. The membrane fluidity of liposomes, as measured by detecting changes in the fluorescence polarization of a 1,6-diphenyl-1,3,5-hexatriene (DPH) probe located in the bilayers, was significantly higher in liposomes consisting of unsaturated carrier lipids than those of the other liposomes consisting of saturated carrier lipids. The highest titer of anti-OVA IgG was observed in mice immunized with OVA-liposomes made using liposomes consisting of unsaturated carrier lipids. In addition, among these OVA-liposomes, the one possessing the longest carbon chain induced the lowest IgG antibody production. These results suggest that the membrane fluidity of liposomes might affect the adjuvant effect of liposomes but not the induction of IgE-selective unresponsiveness in immunizations with surface-linked liposomal antigens.     

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.     

Structural characterization of archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) formulations prepared by different protocols and their efficacy upon intranasal immunization of mice

Intranasal administration of ovalbumin (OVA) formulated in an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system prepared by the addition of CaCl2 to small unilamellar archaeosomes (liposomes made from archaeal polar lipids) containing encapsulated OVA, was recently shown to elicit strong and sustained OVA-specific mucosal and systemic immune responses. In this study, we show that the centrifugation/washing and antigen quantization steps required in the standard protocol for obtaining OVA/AMVAD model vaccine formulations can be eliminated by using simpler protocols such as admixing OVA with preformed empty archaeosomes, or by changing the starting ratio (w/w) of archaeal lipid to antigen at the archaeosome preparation stage, prior to the addition of CaCl2 to convert to the AMVAD structures. Irrespective of the vaccine preparation protocol, the AMVAD particle typically comprised of larger spherical structures that had aggregated like a bunch of grapes, and it contained aqueous compartment(s). The anti-OVA IgA antibody responses in vaginal wash, nasal wash, serum, and bile samples, and the anti-OVA IgG antibody responses in sera, in mice intranasally immunized with the OVA/AMVAD formulations prepared by the simplified or the standard protocols, were comparable.     

Intratracheal and intranasal immunization with ovalbumin conjugated withBacillus firmus as a carrier in mice

Inactivated Bacillus firmus (BF), G+ nonpathogenic bacterium of the external environment, was coupled to ovalbumin (OVA) and used in immunization experiments as antigen carrier. Balb/c mice were immunized thrice intra-tracheally and intra-nasally with conjugates of OVA and BF. Surprisingly, administration of OVA-BF conjugates inhibited anti-OVA IgG response in both sera and mucosal secretions if compared to an exposure to OVA alone. The suppression of antigen-specific antibody production was accompanied by promotion of TH1 phenotype.     

Induction of differential T-cell epitope by plain- and liposome-coupled antigen

The T-cell receptors of CD4(+) T lymphocytes recognize immunogenic peptide sequences bound within the groove of MHC class II molecules, and the peptides that bind to these molecules are known to share common structural motifs. For example, OVA(323-339), an I-A(d)-binding peptide, involves a motif of the I-A(d) peptide-binding groove. In the present study, OVA peptides of up to 26-mer were sequentially synthesized and screened, and two additional I-A(d) binding OVA peptides, OVA(20-43) and OVA(264-286), were found to stimulate CD4(+) T cells of OVA-immune BALB/c mice. OVA(20-43) involved structural motifs of the I-A(d) peptide-binding groove, while OVA(264-286) did not. The ability of these three I-A(d) binding OVA peptides to induce antigen-specific cytokine production was compared among CD4(+) T cells of mice immunized either with alum-adsorbed OVA (OVA-alum) or OVA chemically coupled to the surface of liposome (OVA-liposome). CD4(+) T cells of mice immunized with OVA-alum produced more cytokines when stimulated with OVA(264-286) than with OVA(323-339), while CD4(+) T cells of mice immunized with OVA-liposome conjugates produced more cytokines when stimulated with OVA(323-339) than with OVA(264-286). OVA(20-43) induced production of comparable levels of cytokines in mice immunized either with OVA-alum or OVA-liposome. Confocal laser scanning microscopic analysis demonstrated that chemically coupled OVA and liposomes were colocalized in APCs until OVA received processing. Three-dimensional structural analysis demonstrated that both OVA(264-286) and OVA(323-339) were present on the surface of OVA, but OVA(20-43) was not. These results suggested that the chemical coupling of OVA to liposome affected antigen processing in APCs and thus resulted in the induction of differential T-cell epitopes as compared with those induced by plain OVA.     

Induction of antigen-specific immunity by pH-sensitive carbonate apatite as a potent vaccine carrier

The ability of carbonate apatite (CO₃Ap) to enhance antigen-specific immunity was examined in vitro and in vivo to investigate its utility as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) containing CO₃Ap more effectively than free OVA. Interestingly, mice immunized with OVA-containing CO₃Ap produced OVA-specific antibodies more effectively than mice immunized with free OVA. Furthermore, immunization of C57BL/6 mice with OVA-containing CO₃Ap induced the proliferation and antigen-specific production of IFN-γ by splenocytes more strongly than immunization with free OVA. Moreover, no significant differences were detected in the induction of delayed-type hypersensitivity responses, an immune reaction involving an antigen-specific, cell-mediated immune response between OVA-containing CO₃Ap and OVA-containing alumina salt (Alum), suggesting that CO₃Ap induced cell-mediated immune response to the same degree as Alum, which is commonly used for clinical applications. This study is the first to demonstrate the induction of antigen-specific immune responses in vivo by CO₃Ap.     

Protective effect of antiganglioside antibodies against experimental Trypanosoma brucei infection in mice

Liposome-associated ganglioside antigens (ganglioside GM1 or bovine brain gangliosides) were prepared to facilitate the potential protective efficacy for Trypanosoma brucei. Mice were immunized with liposome-associated ganglioside GM1 or bovine brain gangliosides intraperitoneally (i.p.). After immunization, significantly higher antigen-specific IgG and IgM antibodies were detected in sera than in the nonimmunized control group. When sera from immunized mice were analyzed for isotype distribution, antigen-specific IgG1, IgG2a, and IgG3 antibody responses were also noted. After immunization, mice were challenged i.p. with 1 x 10(2) cells of T. brucei. Sixty percentage of liposome-associated ganglioside GM1-immunized mice survived the infection, and all the mice immunized with bovine brain gangliosides-containing liposomes survived. However, all control mice died within 7 days after infection. These data demonstrate that liposomes containing ganglioside antigens have the potential usefulness for the induction of a protective immune response against T. brucei infection and suggest the possibility of developing vaccines that may ultimately be used for the prevention of trypanosomiasis.     

Effect of respiratory syncytial virus infection on the uptake of and immune response to other inhaled antigens

Groups of BALB/c mice were sham infected or inoculated intranasally (IN) with live RSV. From Day 4 to 8 after infection, the animals were exposed IN to ovalbumin (OVA) with or without alum adjuvant. At different intervals, levels of OVA concentration in serum, IgG-anti-OVA antibody activity in serum, and IgA-anti-OVA antibody activity in bronchial washings were determined, employing the ELISA technique. IgE-anti-OVA antibody titers in serum and bronchial washings were assessed by PCA. OVA concentrations in serum were significantly higher in RSV-infected animals compared to uninfected controls. The use of alum adjuvant also increased OVA uptake in uninfected animals but to a lesser extent than RSV infection. RSV-infected animals developed significantly higher OVA-specific antibody titers of IgG isotype in serum and IgA isotype in bronchial washings than the uninfected controls, while alum enhanced the immune response less markedly but still significantly in uninfected mice. An IgE antibody response to OVA in serum was demonstrable in 50% of RSV-infected mice immunized IN with OVA and alum, while all uninfected animals and RSV-infected animals immunized with OVA alone (without adjuvant) failed to develop a detectable IgE response. These findings suggest that infections with viral agents such as RSV may function as adjuvants for other antigens inhaled during acute respiratory infection. These observations may explain the alterations in the immune response to other antigens in patients with acute viral-induced bronchopulmonary diseases.     

Purified protein derivative (PPD) as an immunogen carrier elicits high antigen specificity to haptens.

The effectiveness of the carrier protein in eliciting antigen-specific antibodies was investigated. The effect of the carrier protein was independent of the conjugation chemistry involved. Keyhole limpet hemocyanin (KLH), purified protein derivative (PPD), and ovalbumin (OVA) were used as carrier proteins in the immunization of mice. Three antigens were studied: LY170881 (a small drug molecule), 4-[1'-cyanobenz(f)isoindolyl]butyric acid (CBI-butyric acid), and a seven residue peptide GPGRGPG (KLE1). The serum antibody response to the antigen or antigen:BSA conjugate was superior in the case where the PPD:antigen conjugates were used as the immunogen when compared to KLH and OVA. The specificity of the antibodies to the respective antigens vs cross-reactivity with the carrier protein was investigated. PPD-coupled antigen immunized mice generated a higher percentage of antigen-specific hybridomas compared to the other carrier proteins. These findings confirmed PPD as the best carrier molecule for the production of both polyclonal and monoclonal antibodies.     

Cytotoxic T-cells specific for natural IgE peptides downregulate IgE production

Immunoglobulin E (IgE) plays a central role in IgE-mediated immediate type hypersensitivity. Since production of IgE depends on Th2, efforts to block IgE production and control allergic reactions include tolerization of Th2 or deviating development of Th2. We hypothesized that cytotoxic T lymphocytes targeting natural IgE peptides/MHC I complexes can eliminate IgE-producing cells and inhibit centrally IgE production. CTL to self-IgE peptides were elicited in mice immunized with nonameric p109-117, p113-121, and p103-141 (CHepsilon2 domain), which encompass both peptides with an OVA helper peptide (OVAp restricted for H-2d/b) in liposomes and presented by dendritic cells (DC). CTL from BALB/c lysed IgE peptide-pulsed P815 target as well as IgE-producing 26.82 hybridomas (H-2d). Natural tolerance to self-IgE peptides was tested in IgE sufficient (IgE +/+) as well as IgE-deficient (IgE -/-) 129/SvEv mice (H-2b). Comparable magnitude of CTL responses was observed in both strains immunized with p109-117 or p103-141 concomitantly with CD4 T-cell costimulation. CTL from 129/SvEv lysed not only IgE peptide-pulsed EL-4 but also IgE-producing B4 hybridomas (H-2b). This observation strongly suggests a correspondence of epitope of immunogenic peptide to that of physiologically processed IgE peptides presented on IgE-producing cells. Moreover, CTL were generated in 129/SvEv, immunized with the recombinant antigenized antibody in liposomes encompassing p107-123, p109-117, and p113-121 expressed in CDR3 of VH62/human gamma1. Polyclonal IgE production was inhibited by coincubation with MHC I-restricted CTL in vitro. Furthermore, antigen-specific IgE responses were inhibited in mice, immunized with p109-117 and p103-141 while IgG responses were not suppressed. Since IgE peptide sequences of CHepsilon2 are ubiquitous to all murine IgE heavy chain, peptides made as such can serve as a universal IgE vaccine to prevent allergy for a myriad of allergens in rodents. This observation suggests that similar human IgE peptides should be identified and employed to downregulate human IgE production.     

Induction of immunity against experimental tuberculosis with mycobacterial mannophosphoinositides encapsulated in liposomes containing lipid A

Abstract Mycobacterial mannophosphoinositides (PIMs) encapsulated in liposomes made of egg phosphatidylcholine (EPC) and cholesterol (CH) (2:1.5 molar ratio) were able to induce humoral and delayed type hypersensitivity (DTH, foot-pad swelling reaction) responses in mice without the help of any carrier protein. Animals immunized with this glycophospholipid antigen demonstrated enhanced percent survival on intravenous challenge with virulent M. tuberculosis . On fractionation of PIMs, pentamannophosphoinositide (PIM₅) was found to induce higher antibody and DTH reaction and proved to be more immunoprotective than other fractions. Inclusion of lipid A as immunomodulator in liposomes containing PIMs or PIM₅ resulted in a significantly increased immune response. Further, mice immunized with PIMs or PIM₅ in lipid A-containing liposomes exhibited decreased mortality on challenge with M. tuberculosis H₃₇Rv, which was comparable to BCG vaccinated animals.     

Abrogation of oral tolerance by feeding encapsulated antigen

Results from previous studies have indicated that suppression of immune responses cannot be abrogated once oral tolerance has been established. Using a new methodology, OVA was encapsulated and fed to tolerized BDF1 mice. Results from these studies indicated that although IgG2a titers remained low, total IgG and IgG1 antibody titers were no longer suppressed compared to controls. Furthermore, in vitro splenocyte proliferation was not significantly suppressed in tolerized mice fed encapsulated OVA. To determine whether oral tolerance could be abrogated in other strains of mice, BALB/c mice were tolerized and fed encapsulated OVA. Results from these studies indicated that IgG2a as well as IgG1 and total anti-OVA antibody titers were no longer suppressed. Although splenocyte proliferation did remain significantly suppressed in these mice, IFN-gamma and IL-4 levels were no longer decreased. To the best of our knowledge, this is the first time that abrogation of an established oral tolerance has been demonstrated.     

Cholesterol inclusion in liposomes affects induction of antigen-specific IgG and IgE antibody production in mice by a surface-linked liposomal antigen

In the previous study, we investigated the induction of ovalbumin (OVA)-specific antibody production in mice by OVA-liposome conjugates made using four different lipid components, including unsaturated carrier lipid and three different saturated carrier lipids. All of the OVA-liposome conjugates tested induced IgE-selective unresponsiveness. The highest titer of anti-OVA IgG was observed in mice immunized with OVA-liposomes made using liposomes with the highest membrane fluidity, suggesting that the membrane fluidity of liposomes affects their adjuvant effect. In this study, liposomes with five different cholesterol inclusions, ranging from 0% to 43% of the total lipid, were made, and the induction of OVA-specific antibody production by OVA-liposome conjugates was compared among these liposome preparations. In contrast to the results in the previous study, liposomes that contained no cholesterol and possessed the lowest membrane fluidity demonstrated the highest adjuvant effect for the induction of IgG antibody production. In addition, when the liposomes with four different lipid compositions were used, OVA-liposome conjugates made using liposomes that did not contain cholesterol induced significantly higher levels of anti-OVA IgG antibody production than did those made using liposomes that contained cholesterol and, further, induced significant production of anti-OVA IgE. These results suggest that cholesterol inclusion in liposomes affects both adjuvanticity for IgG production and regulatory effects on IgE synthesis by the surface-coupled antigen of liposomes.     

Liposome-encapsulated antigens engender lysosomal processing for class II MHC presentation and cytosolic processing for class I presentation

Liposome-encapsulated protein Ag were used to dissect the roles of various subcellular compartments in Ag processing for class I and class II MHC-restricted presentation. Macrophages exhibited efficient processing of Ag encapsulated in acid-resistant dioleoylphosphatidylcholine/dioleoylphosphatidylserine liposomes, which sequester their contents from potential endosomal processing events and release them only after delivery to lysosomes. Lysosomal processing was demonstrated for all four Ag studied (OVA, murine hemoglobin, bovine ribonuclease A, and hen egg lysozyme), establishing the recycling of immunogenic peptides from lysosomes after Ag processing. These acid-resistant liposomes did not engender class I processing. Ag encapsulated within acid-sensitive dioleoylphosphatidylethanolamine/palmitoylhomocysteine liposomes were also processed via the class II pathway. Of the four Ag encapsulated in liposomes, one, OVA, was tested for ability to stimulate a class I-specific response. OVA in acid-resistant liposomes did not engender a class I-specific response. In contrast, OVA encapsulated in acid-sensitive liposomes was presented by class I molecules, albeit less efficiently than it was presented by class II molecules. We interpret this to be the result of the release of a minor portion of the encapsulated Ag into the cytosol.     

Comparative effect on humoral response of four different proteins covalently linked on BSA-containing liposomes

The humoral response to encapsulated BSA appears to be a classical TD antigen response with a high ratio of IgG to IgM, whereas that to covalently-linked antigen is more complex, characterized by an enhanced synthesis of IgM, leading to an equal production of IgM and IgG. In a recent paper, we observed that surface-linked Con A on BSA-containing liposomes changed the isotype distribution to encapsulated BSA so as to mimic the response to surface-linked antigen. In the present study, we compared the immune response to BSA in BALB/c mice immunized with the antigen encapsulated into liposomes coated with one of four different proteins: Con A, Myo, MSA, or PWM. The humoral response was analyzed by measurements of antibody production (total Ig, IgM, and IgG isotypes) on serum samples obtained by cardiac puncture. It can be concluded from our results that any surface-linked protein may affect the interaction between liposome-associated antigen and immunocompetent cells.