Primary in vitro immunogenicity of liposomal model membranes in mouse spleen cell cultures.

Neither 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) nor fluoresceinthiocarbamylphosphatidylethanolamine (F1-PE) induces hapten-specific plaque-forming cells (PFC) when incubated with suspensions of spleen cells from unimmunized C57BL/6J mice. However, PFC are produced after incorporation of these synthetic lipid antigens into liposomal model membranes. The in vitro response is characterized by the following: a) it is time and dose dependent; b) the frequency of IgM PFC exceeds IgG PFC; c) both nonadherent and adherent cells are required (2-mercaptoethanol can replace the requirement for adherent cells in some experiments); d) depletion of thymus-derived cells by treatment with anti-theta antiserum plus complement does not diminish the response; e) spleen cells from nude BALB/c mice also produce PFC. Thus, the essential features of the in vivo immunogenicity of DNP-Cap-PE and F1-PE sensitized liposomes, which have been previously described, can be replicated in an in vitro cell culture system.     

Effect of liposomal model membrane composition on immunogenicity

We have examined the effect of composition on the immunogenicity in mice of liposomal model membranes sensitized with dinitrophenyl-epsilon-aminocaproyl-phosphatidylethanolamine (DNP-Cap-PE) derivatives. Neither cholesterol content nor incorporation of exogenous charged amphiphile (dicetylphosphate, stearylamine) exerted a significant influence on the in vivo anti-DNP response as measured by the appearance of direct plaque-forming cells in the spleen. Similarly, the nature of the fatty acids (saturated vs unsaturated) present in DNP-Cap-PE had no effect. In contrast, the nonpolar region of the basic phospholipids comprising the liposomal bilayers played an important role as revealed by a comparative study of model membranes prepared with beef sphingomyelin (SM), egg phosphatidylcholine (PC), and synthetic distearoyl-, dimyristoyl-, dilauroyl-, and dioleoyl-phosphatidylcholines (DSPC, DMPC, DLPC, DOPC). Thus, liposomes with a large content of phospholipids possessing a high transition temperature (e.g., beef SM, DSPC) were more immunogenic than those containing phospholipids of low transition temperature (e.g., egg PC, DOPC). This correlation held for both unsonicated and sonicated liposomes. These findings may have a bearing on the phenomenon of membrane-localized antigen expression.     

The inhibitory effect of anti-allergic agent suplatast tosilate (IPD-1151T) on methacholine- and allergen-induced bronchoconstriction in sensitized mice. asakazu@med.showa-u.dc.jp

The influence of an anti-allergic agent, suplatast tosilate (IPD-1151T; (+/-)-[2-[4-(3-ethoxy-2-hydroxypropoxy)phenyl-carbamoyl]-ethyl] dimethylsulfonium p-toluenesulfonate) on allergic bronchoconstriction induced by allergen and methacholine (MCh) were examined in mice. BALB/c mice were sensitized by intraperitoneal injection of dinitrophenylated-keyhole limpet hemocyanin (DNP-KLH) mixed with A1(OH)3 (DNP-KLH). IPD-1151T was administered orally once a day for either 5 or 14 days in doses of 10, 30 or 100 mg/kg. Bronchoconstriction was measured 24h after the final drug administration. IPD-1151T inhibited both antigen- and MCh-mediated bronchoconstriction in actively sensitized mice. The inhibition induced was closely related to the dose and frequency of oral administration of the agent. We also examined the effect of IPD-1151T on IgE production in response to DNP-KLH immunization. IPD-1151T inhibited dose-dependently both total and specific IgE concentrations in serum prepared from mice 15 days after immunization. These results strongly indicate that IPD-1151T inhibits IgE production in vivo and results in attenuating effect on bronchoconstriction.     

Ascaris suum: Immunosuppression in mice during acute infection

Mice inoculated by stomach intubation with 10,000 embryonated Ascaris suum eggs, 4, 11, or 21 days before an intraperitoneal (ip) immunization with 2 × 10⁸ sheep erythrocytes (SRBC) had reduced numbers of direct (IgM) splenic hemolytic plaques measured at 4 days after immunization and only a marginal reduction in indirect plaques (IgG) measured at 9 days after immunization. Lower dosages of Ascaris eggs or simultaneous inoculation of Ascaris eggs and SRBC did not suppress antibody responses to SRBC. No reduction in a secondary antibody response to SRBC injected 4 days after Ascaris inoculation was observed. IgM and IgG hemagglutinin titers, as distinguished by 2-mercaptoethanol sensitivity, were suppressed in mice injected ip with 10⁸ SRBC 10 days following inoculation of 10,000 Ascaris eggs, but titers in both Ig classes were similar in infected and control mice injected with 2 × 10⁹ SRBC. At Day 20, antibody titers following ip injection of 1.0 or 100 μg of ovalbumin in alum were reduced in mice infected with 10,000 Ascaris eggs 4 days before antigen injection.Contact hypersensitivity to oxazalone was not altered in mice sensitized at 5 or 14 days after inoculation of 10,000 Ascaris eggs. The delayed hypersensitivity response, measured by footpad swelling, to an optimum intravenous sensitizing dosage of SRBC was inhibited in mice sensitized 10 days after Ascaris infection, but not inhibited in mice sensitized at 21 or 32 days after infection. In contrast, the delayed hypersensitivity response to subcutaneous sensitization with SRBC 10 days after Ascaris infection was not altered.     

Cationization of protein antigens. II. Alteration of regulatory properties

Immunoregulatory effects of cationized bovine serum albumin (cBSA) and native bovine serum albumin (nBSA) have been investigated. Intravenous administration of nBSA to BDF1 mice substantially suppressed the antibody response to subsequent immunization with either nBSA or cBSA, whereas pretreatment with cBSA by the same route significantly enhanced the responses to both antigens. The functional properties of BSA-specific T and B cells from mice immunized with cBSA or nBSA were examined in reconstitution experiments in which splenic T populations together with B cells were transferred into irradiated syngeneic recipients. Transfer of splenic T cells from mice primed with nBSA caused profound suppression of the response to subsequent immunization with nBSA or cBSA, whereas transfer of either B or T cells from cBSA treated mice produced an enhanced response to both antigens. C57BL/6 mice, which are considered to be low responders to BSA, produced a significant antibody response to BSA when immunized with cBSA. In contrast, immunization with nBSA did not produce measureable amounts of antibody in mice of this strain. Our data clearly demonstrate that cationized BSA exhibits unique immunogenic properties due to alterations in the self-regulation of the immune response.     

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.     

Antibody mediated suppression of secondary IgM response in nude mice against vesicular stomatitis virus

Nude mice immunized with either of the two serotypes of vesicular stomatitis virus (VSV), VSV Indiana (VSV-Ind) or VSV-New Jersey (VSV-NJ), showed an early T cell independent immunoglobulin (Ig) M antibody response comparable with normal euthymic mice. Unlike euthymic mice, however, nude mice reinjected with the homologous serotype were unable to mount a second measurable serum neutralizing (SN) antibody response; a second injection with the heterologous serotype induced a normal primary type of SN antibody response. The serotype-specific refractoriness to a second challenge recovered at about 10 wk after primary infection. When antibody responses were assayed by enzyme-linked immunoabsorbent assay (ELISA), suppressive effects by previous immunization could be observed even after challenge with the heterologous serotypes; this finding probably reflects the known existence of common nonneutralizing determinants shared between the two serotypes. A weak 2-mercaptoethanol (2-ME)-resistant anti-VSV IgG SN antibody response was noticed during the primary response in nude mice and was also found in ELISA; after second infections, this 2-ME-resistant response did not develop. Serum transfer studies in nude and +/+ mice confirmed that the serotype-specific transitory refractoriness of a second response in nude mice was mediated through the anti-VSV-specific IgM antibodies.     

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)     

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.     

Immunosuppressive properties of a peptic fragment of BSA.

The immunogenic properties of a peptic fragment of BSA were investigated. BSA was subjected to limited proteolysis by pepsin and the resulting fragments were separated on DEAE cellulose. The fragment under consideration, Fraction Ia (m.w. 8000 to 10,000), did not precipitate with anti-BSA serum but did inhibi, the binding of specific antibody to labeled BSA, indicating the presence of determinants found on the native antigen. BDF1 mice immunized with Fraction Ia in A1 (OH)3 gel or in complete Freund's adjuvant produced no significant antibody response as measured by passive cutaneous anaphylited a (PCA) or by a modified Farr assay. The fragment elicited a PCA reaction in mouse skin sensitized with anti-BSA serum. Treatment of mice with single doses of Fraction Ia at various time intervals before immunization with BSA resulted in significant suppression of the formation of anti-BSA antibody. The conditions of suppresion of the IgE response by the peptic fragment were studied in greater detail. Evidence is presented that such suppression can be attributed to the presence of specific T suppressor cells in our system.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

C57BL/6 and AKR mice were treated with hamster erythrocytes (HRBC) in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and the development of delayed hypersensitivity and antibody production were examined. 1) Delayed hypersensitivity against HRBC antigen, as determined by the peritoneal macrophage disappearance test, was detected in mice sensitized with HRBC in CFA but not in those sensitized with HRBC in IFA. 2) Antibody production against HRBC or hapten TNP after a booster injection of HRBC or trinitrophenylated HRBC (TNP-HRBC) in saline was enhanced by pretreatment with HRBC in CFA or IFA. 3) Delayed hypersensitivity was not detectable after a booster sensitization with HRBC in CFA in mice which had been pretreated with HRBC in IFA 2 weeks earlier. In the mice treated with both HRBC in IFA (day ?21) and in CFA (day ?7), however, an enhanced antibody production against HRBC or TNP was detected after an intravenous injection with HRBC or TNP-HRBC in saline (day 0). These results suggest that sensitized effector lymphocytes in delayed hypersensitivity and helper cells in antibody production may be derived from the same pool of unprimed T cells. The pool of unprimed T cells with a capacity to differentiate into either type of primed T cells may be exhausted after pretreatment with the antigen in IFA, and the primed helper T cells may not be able to differentiate into sensitized lymphocytes even after sensitization with the antigen in CFA, which favors development of delayed hypersensitivity in normal controls.     

Universal vaccine carrier. Liposomes that provide T-dependent help to weak antigens

The design of a thymus-dependent synthetic vaccine that will provide a universal T cell epitope for B cell epitopes is described in this study. Simultaneous incorporation into liposomes of both a peptide recognized by Th lymphocytes and a lipophilic hapten and the IgG antibody responses to this hapten were assessed in outbred mice. DNP-aminocaproyl phosphatidylethanolamine (DNP-CapPE) is a well characterized T-independent hapten Ag. HA2 peptide derived from the hemagglutinin protein of influenza virus contains amino acid sequences recognized by Th and T cytotoxic lymphocytes. In addition, HA2 contains a sequence of hydrophobic amino acids near the carboxyl terminus, allowing its incorporation into liposomes. Results of immunization show that (i), when DNP-CapPE is carried by liposomes without the HA2 peptide, an IgM antibody response is induced, (ii) liposomes carrying both HA2 and DNP-CapPE elicit an IgG antibody response to DNP in a dose-dependent fashion for both HA2 and DNP, (iii) the liposomes must be processed intracellularly in order to elicit a response, (iv) the system leads to a memory response for DNP, and (v) all of the IgG subclasses are elicited. These data suggest that liposomes containing the HA2 peptide exhibit a T-dependent carrier effect for a T-independent Ag. The significance of these findings is discussed in conjunction with the characteristics of the liposome model used.     

Production of soluble suppressor factor by spleen cells from mice immunized with type III pneumococcal polysaccharide

Supernatant fluid (SF) derived from spleen cell cultures, obtained from mice 16 hr after immunization with 0.5 microgram of Type III pneumococcal polysaccharide (SSS-III), suppressed the antibody response when SF was given (i.v.) 3 hr before immunization with SSS-III. Such suppression was antigen specific and could be reproduced by SF derived from cultures of T cells from mice immunized with SSS-III (0.5 microgram) or by SF derived from cultures of spleen cells from mice primed with a subimmunogenic dose of SSS-III (0.005 microgram). Adsorption of SF with SSS-III covalently bound to a Sepharose 4B column did not alter the ability of SF to suppress the SSS-III-specific antibody response. However, adsorption of SF with Ig+ (B) cells from mice immunized with 0.5 microgram SSS-III completely removed the suppressive activity. Significant (p less than 0.05) suppression of the antibody response was observed only when SF was administered (i.v.) 24 hr before to 24 hr after immunization with 0.5 microgram of SSS-III. These results suggest that suppressor T cells generated in response to SSS-III function by releasing a soluble factor(s) that binds to determinants on B cells rather than antigen; this soluble factor(s) acts directly on antigen-stimulated B cells or inhibits the induction of amplifier T cells.     

The in vivo elimination of CD4+ T cells prevents the induction but not the expression of carrier-induced epitopic suppression.

Injection of mice with an immunogenic dose of carrier (keyhole limpet hemocyanin (KLH)) followed by immunization with hapten-carrier conjugate (TNP-KLH) selectively suppresses anti-hapten antibody response. In this study, the cellular basis of this epitopic suppression and also of the suppression induced by a high dose of carrier were analyzed by in vivo depletion of CD4+ or CD8+ T cell subsets by using mAb. The mAb treatments were performed either at the time of carrier priming or at the time of hapten-carrier immunization. The elimination of CD8+ T cells has not modified the anti-carrier antibody response, whether this treatment was performed at the time of KLH-priming or during TNP-KLH immunization. Moreover, the in vivo treatment with the anti-CD8 mAb did not modify the carrier-induced epitopic suppression induced either by a low immunogenic dose of KLH or by a high dose of this Ag. The elimination of CD4+ T cells at the time of KLH immunization has prevented the induction of a memory response to KLH, clearly establishing that CD4+ T cells are essential in memory B cell development to T-dependent Ag. Moreover, this treatment has totally abrogated the epitopic suppression induced either by low or high dosages of KLH. In contrast, the in vivo elimination of CD4+ T cells after carrier immunization did not abolish the secondary anti-carrier antibody response and did not prevent the expression of epitopic suppression. These data indicate that primed CD4+ T cells are required neither for memory B cell expression nor for the expression of suppression. Finally, once induced, the suppression can be evidenced after in vivo depletion of both primed CD4+ and CD8+ T cells. These data support the view that epitopic suppression is induced through the expansion of carrier-specific B cells and resulted from intramolecular antigenic competition between hapten and carrier epitopes.     

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.     

Determinant selection in murine experimental autoimmune myasthenia gravis. Effect of the bm12 mutation on T cell recognition of acetylcholine receptor epitopes.

C57BL/6 (B6) mice respond to immunization with acetylcholine receptor (AChR) from Torpedo californica as measured by T cell proliferation, antibody production, and the development of muscle weakness resembling human myasthenia gravis. The congenic strain B6.C-H-2bm12 (bm12), which differs from B6 by three amino acid substitutions in the beta-chain of the MHC class II molecule I-A, develops a T cell proliferative response but does not produce antibody or develop muscle weakness. By examining the fine specificity of the B6 and bm12 T cell responses to AChR by using T cell clones and synthetic AChR peptides, we found key differences between the two strains in T cell epitope recognition. B6 T cells responded predominantly to the peptide representing alpha-subunit residues 146-162; this response was cross-reactive at the clonal level to peptide 111-126. Based on the sequence homology between these peptides and the T cell response to a set of truncated peptides, the major B6 T cell epitope was determined to be residues 148-152. The cross-reactivity of peptides 146-162 and 111-126 could also be demonstrated in vivo. Immunization of B6 mice with either peptide primed for T cell responses to both peptides. In contrast, immunization of bm12 mice with peptide 111-126 primed for an anti-peptide response, which did not cross-react with 146-162. Peptide-reactive T cells were not elicited after immunization of bm12 mice with 146-162. These results define a major T cell fine specificity in experimental autoimmune myasthenia gravis-susceptible B6 mice to be directed at alpha-subunit residues 148-152. T cells from disease-resistant bm12 mice fail to recognize this epitope but do recognize other portions of AChR. We postulate that alpha-148-152 is a disease-related epitope in murine experimental autoimmune myasthenia gravis. In this informative strain combination, MHC class II-associated determinant selection, rather than Ag responsiveness per se, may play a major role in determining disease susceptibility.     

Rapid changes in the regulatory potential of autologous anti-idiotopic T cells during an antigen-driven primary response

The antibody response of C57BL/6 strain mice to Streptococcus pneumoniae R36a (Pn) is dominated by the T15 idiotype, but the responding cells appear to be idiotypically heterogeneous, in that individual antibody plaque-forming cells (PFC) may express some but not all idiotopes (Id) of the T15 complex. The presence of these distinct Id on the PFC was detected by a plaque-inhibition assay with three different monoclonal anti-Id antibodies, designated AB1-2, MaId5-4, and B36-82. A periodic change in the expression of AB1-2 and MaId5-4 Id was observed during primary (IgM) antibody response to Pn in the spleen. Those two Id were poorly expressed in the log phase of the response between day 2 and day 4 after immunization (few PFC in the spleen bore the Id), but they became detectable on the majority of PFC at the peak of the response, day 5 to day 7. The proportion of the Id-(AB1-2 or MaId5-4) positive PFC declined, again at day 10 after immunization. In contrast, the B36-82 Id was expressed on greater than or equal to 80% PFC throughout the entire primary response. The possibility that the apparent changes in the Pn-reactive cell populations are regulated by autologous anti-Id T cells was tested in vitro. Normal, unimmunized B cells were cultured with Pn, either alone or in the presence of syngeneic T cells isolated from the spleen of mice at the appropriate intervals after immunization: day 2 (T2), day 5 (T5), and days 10 to 14 (T10 to T14); T cells from unimmunized donors (T0) served as a control. The specific response after 4 days in culture was determined in regard to the total PFC as well as the proportion of PFC expressing the Id. Pn-stimulated B cells, alone or with the control T0 cells, produced moderate, variable levels of AB1-2+ and MaId5-4+ PFC. The expression of these two Id in the assay cultures was suppressed by addition of either T2 cells or T10-14 cells, but it was enhanced if T5 cells were added. However, these various T cell populations did not differ in their effect on the total PFC response. Also, the proportion of PFC bearing the third Id, B36-82 was high, and it was not consistently influenced by the added T cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipid vesicle-cell interactions. III. Introduction of a new antigenic determinant into erythrocyte membranes

The introduction of a new antigenic determinant, 2,4-dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-Cap-PE), into the surface membranes of intact human erythrocytes is described. Fresh cells were incubated in the presence of liposomes composed of 10% DNP-Cap-PE, 5% stearylamine, 20% lysolecithin, and 65% lecithin. Such liposome-treated erythrocytes are shown to be susceptible to immune lysis by anti-DNP serum in the presence of complement. Uptake of DNP-Cap-PE by erythrocyte membranes is also demonstrated by immunofluorescence using indirect staining with rabbit anti-DNP serum followed by fluroescein-conjugated goat anti-rabbit IgG and by electron microscopy using ferritin-conjugated antibody. Antigen uptake did not occur at low temperatures or from vesicles lacking lysolecithin and stearylamine. Fluorescence microscopy shows that the antigen-antibody complexes are free to diffuse over the cell surface, eventually coalescing into a single area on the cell membrane. Electron microscopy suggests that a substantial proportion of the lipid antigen is incorporated by fusion of vesicles with the cell membrane. There are indications that vesicle treatment causes a small proportion of cells to invaginate.     

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.     

Activation of T cells by glutaraldehyde-fixed erythrocyte antigens: radiosensitivity of cells mediating delayed-type hypersensitivity reactions in the mouse.

Mice immunized with glutaraldehyde-fixed sheep red blood cells (G-SRBC) show delayed-type hypersensitivity (DTH) reactions to G-SRBC or SRBC. The specificity of the DTH reaction of mice sensitized with glutaraldehyde-fixed antigens is similar to that found after sensitization with unfixed antigens. The dose-response curve for sensitization by glutaraldehyde-fixed SRBC was very different from the curve for normal SRBC. At low doses, both antigens were effective in sensitizing to show DTH but neither induced an antibody response. However, at high antigen doses, only the glutaraldehyde-fixed antigen was efficient in sensitizing to show DTH and it failed to raise an antibody titer. Spleen cells of mice sensitized with fixed RBC can transfer DTH locally but if the donor cells are irradiated (500 R), the transfer is abrogated. In contrast, the transfer of DTH by spleen cells of mice immunized with unfixed antigen is not affected by 500 R. The transfer of DTH by spleen cells of mice immunized with fixed antigen can be blocked by “in vitro desensitization” while the transfer of DTH by spleen cells from mice primed with normal antigen is resistant to “in vitro desensitization.” These results suggest that immunization of mice with different physical states of the same antigen can result in the activation of antigen-specific T cells which exhibit markedly different properties.