Liposomes containing lipid A: a potent nontoxic adjuvant for a human malaria sporozoite vaccine

Liposomes containing lipid A have been developed as adjuvants for inducing humoral immunity to synthetic antigens containing repeat sequence epitopes from the circumsporozoite protein of Plasmodium falciparum. Preclinical studies demonstrated that liposomes containing lipid A and encapsulated antigen could overcome immunosuppression observed with antigen alone. When liposomes containing lipid A were adsorbed with aluminum hydroxide (alum), further stimulation of humoral immunity against encapsulated antigen was observed in animals. In the presence of huge doses of liposomal lipid A pyrogenicity was not observed and adjuvant activity was enhanced. A phase I human clinical trial has been initiated utilizing a vaccine containing a synthetic recombinant antigen and monophosphoryl lipid A in liposomes and nonliposomal alum as a further adjuvant. Preliminary results confirm that the vaccine lacks significant acute toxicity in humans and causes very strong specific humoral immunity against the appropriate epitopes of the target antigen.     

Use of Liposomes as an Immunopotentiating Delivery System: in Perspective of Vaccine Development

Liposomes have been widely used to deliver antigens to the antigen-presenting cells (APCs) and also to modify their immunological behaviour in model animals. We recently demonstrated the potential of yeast lipid liposomes to undergo membrane-membrane fusion with cytoplasmic membrane of the target cells. Interestingly, studies in the present report revealed that antigen encapsulated in yeast lipid liposomes could be successfully delivered simultaneously into the cytosolic as well as endosomal processing pathways of APCs, leading to the generation of both CD4+ T helper and CD8+ cytotoxic T cells. In contrast, encapsulation of same antigen in egg phosphatidyl-choline (PC) liposomes, just like its free form, has inefficient access to the cytosolic pathway of major histocompatibility complex (MHC) I dependent antigen presentation and failed to generate antigen specific CD8+ cytotoxic T-cell response. However, both egg PC as well as yeast lipid liposomes have elicited strong antigen specific antibody responses in immunized animals. These results imply usage of liposome encapsulated antigen as potential candidate vaccine capable of eliciting both cell mediated as well as humoral immune responses.     

Antigen Presenting Cells in Human Decidual Tissue: III. Role of Accessory Cells in the Activation of Suppressor Cells

ABSTRACT: Human decidual antigen presenting cells (DAPCs) exposed to fetal cells in vitro induce generation of suppressor T cells among a population of peripheral blood lymphocytes. Human lymphocyte antigen (HLA)-class II positive antigen presenting cells were isolated from early normal human decidual tissue and from peripheral blood (PAPCs) by adhering Ficoll-Pa-que separated cell suspensions to fibronectin. In contrast to PAPCs, DAPCs pulsed with fetal antigens induced a radio-sensitive, Leu 1,2-positive T suppressor cell population. A nylon wool adherent B cell population is required during the in vitro induction of the suppressor cells. These suppressor cells impair primary mitogen and mixed lymphocyte culture (MLC) responses, generation of anti-trinitrophe-nyl (TNP) cytotoxic T lymphocytes, and antibody response of autologous and allogeneic lymphocytes. Only intact viable embryonic cells can effectively confer upon DAPCs the ability to induce T suppressor cells. The T suppressor cell induction by DAPCs primed with fetal antigens is restricted by the major histocompatibility complex. Our results show that the HLA-DR molecules are the most prominent restriction elements.     

Serum-treated antigen-antibody complexes inhibit the production of C2 and factor B by mononuclear phagocytes

Since inactivated virus preparations are poor inducers of influenza-specific cytotoxic T cells (Tc), studies were undertaken utilizing artificial vesicles (liposomes) as a means of delivering viral and H-2 antigens in a multivalent form and oriented with respect to a lipid bilayer. Liposomes prepared from extracted mouse cell lipids efficiently incorporated influenza-viral proteins and were not toxic in culture. Using polybrene to promote greater contact of liposomes with cells, liposomes prepared from whole virus could effectively stimulate memory Tc from spleens of intranasally infected mice in vitro. H-2 was not required in the liposomes to obtain stimulation, and its presence did not improve responses, which were always lower than in parallel stimulations using virally infected syngeneic cells. Liposomes prepared from purified influenza virion subunits (haemagglutinin, neuraminidase, matrix protein) were only slightly stimulatory in vitro, and were unable to prime mice for significant Tc memory.     

Inhibition of antibody binding to viable cells by liposomes containing purified rat spleen glycoprotein

The capacity of liposomes with inserted RT-1 histocompatibility antigen to bind anti-RT-1 antibodies varies depending on their lipid composition and mode of preparation. The binding capacity of liposomes prepared by dialysis is different from that of liposomes prepared by gel filtration or of small membrane protein micelles. A new assay for analysis of membrane antigens has been developed. Liposomes are used to compete for antibody binding with in vitro cultured cells while binding of antibody to the same adherent liposomes is simultaneously assayed.     

CD1 Expression in Human Atherosclerosis : A Potential Mechanism for T Cell Activation by Foam Cells

Atherosclerotic plaques are chronic inflammatory lesions composed of dysfunctional endothelium, smooth muscle cells, lipid-laden macrophages, and T lymphocytes. This study analyzed atherosclerotic tissue specimens for expression of CD1 molecules, a family of cell surface proteins that present lipid antigens to T cells, and examined the possibility that CD1+ lipid-laden macrophages might present antigen to T cells. Immunohistochemical studies using a panel of specific monoclonal antibodies demonstrated expression of each of the four previously characterized human CD1 proteins (CD1a, -b, -c, and -d) in atherosclerotic plaques. Expression of CD1 was not observed in normal arterial specimens and appeared to be restricted to the CD68+ lipid-laden foam cells of atherosclerotic lesions. CD1 molecules colocalized in areas of the arterial wall that also contained abundant T lymphocytes, suggesting potential interactions between CD1+ cells and plaque-infiltrating lymphocytes in situ. Using CD1-expressing foam cells derived from macrophages in vitro, we demonstrated the ability of such cells to present lipid antigens to CD1 restricted T cells. Given the abundant T cells, CD1+ macrophages, and lipid accumulation in atherosclerotic plaques, we propose a potential role for lipid antigen presentation by CD1 proteins in the generation of the inflammatory component of these lesions.     

Adjuvant effects of liposomes containing lipid A: enhancement of liposomal antigen presentation and recruitment of macrophages.

Liposomes containing lipid A induced potent humoral immune responses in mice against an encapsulated malaria antigen (R32NS1) containing NANP epitopes. The immune response was not enhanced by lipid A alone or by empty liposomes containing lipid A. Experiments to investigate the adjuvant mechanisms of liposomes and lipid A revealed that liposome-encapsulated R32NS1 was actively presented by bone marrow-derived macrophages to NANP-specific cloned T cells. The degree of presentation was related to the amount of liposomal antigen added per macrophage in the culture medium. At high cell densities, poor presentation occurred when liposomes lacked lipid A but excellent presentation occurred when the liposomes contained lipid A. Liposomes containing lipid A and encapsulated antigen also activated gamma interferon-treated macrophages to produce nitric oxide. Macrophage activation and antigen presentation occurred with liposomes that could not be detected by the Limulus amebocyte lysis assay. Intraperitoneal injection of liposomal lipid A caused a marked increase in the recruitment of immature (peroxidase-positive) macrophages to the peritoneum. On the basis of these experiments, we propose that the mechanism of the adjuvant action of liposomal lipid A is partly due to increased antigen presentation by macrophages and partly due to recruitment of an increased number of macrophages serving as antigen-presenting cells.     

Expression of immune system-associated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer's disease

HLA-DR is a class II major histocompatibility complex antigen which in the periphery confers antigen presenting capability. We have previously shown that this marker is profusely expressed in cortex of elderly and Alzheimer's disease (AD) patients, as is the receptor for the lymphokine interleukin-2. We now report presence of additional immune-related antigens in AD, and distributional differences from normal elderly controls. In gray matter, HLA-DR immunoreactivity is normally sparse, except in AD where it co-localizes with virtually all neuritic plaques. HLA-DR positive T cells can be demonstrated in Alzheimer's disease brain tissue, as can instances of apposition between putative brain microglia and T cells. In addition, cells with the morphologic characteristics of astrocytes label for natural killer cell antigen (Leu-11), and apparent lymphocytes bearing T helper and T cytotoxic/suppressor cell antigens are observed. These and other data suggest that the glial proliferation and scavenger activity characteristic of Alzheimer's disease may occur in an immune context and may play an important role in the pathogenesis of the disorder.     

Irradiated sporozoite vaccine induces cytotoxic T lymphocytes that recognize malaria antigens on the surface of infected hepatocytes

The observation that protective immunity induced by immunization with radiation attenuated Plasmodium berghei and Plasmodium yoelii sporozoites is dependent on CD8+ T lymphocytes in some strains of mice led us to speculate that immunization with sporozoites induces cytotoxic T lymphocytes (CTL) that recognize malaria antigens on the surface of malaria-infected hepatocytes. In this report we summarize a series of experiments that confirm this hypothesis. We first showed that when immune mice are challenged with live sporozoites they develop malaria-specific, CD8+ T cell-dependent infiltrates in their livers. Next we demonstrated that spleen cells from immune mice eliminate malaria infected hepatocytes from in vitro culture in an antigen specific and genetically restricted manner, indicating that these immune cells recognize malaria antigens on the surface of infected hepatocytes. Finally we defined a CTL epitope of the P. yoelii CS protein, and demonstrated that CTL against this 16-amino-acid peptide (PYCTL1) eliminate infected hepatocytes from culture in an antigenic specific, and MHC restricted manner, indicating that this 16-amino-acid peptide from the CS protein is present on the surface of the infected hepatocytes. We are currently working on constructing vaccines that induce protective CTL against PYCTL1, and identifying additional pre-erythrocytic stage targets of CTL mediated protective immunity.     

Stimulation of antigen-specific murine T cell clones in vitro with antigen-pulsed adherent cells fixed to a carrier

Antigen pulsed antigen presenting cells (APCs) were fixed to a carrier (culture flask or microtiter plate) to stimulate antigen-specific T cell clones in vitro. The resulting stimulation was comparable with that in which free antigen together with irradiated APCs were used and the antigen concentration was less critical in the case of toxic antigens. It was shown that the carriers coated with pulsed APCs could be stored for 12 weeks without loss of stimulatory capacities, provided that the APCs were fixed. In addition it was demonstrated that coated carriers could be used thrice to stimulate T cells in vitro without affecting the stimulating properties. The observed T cell proliferation was both antigen specific and MHC restricted. The main advantages of this novel method were the standardization of antigen stimulation of T cells achieved in vitro and the availability of a 100% pure T cell population immediately after stimulation, both features contributing to more reproducible experiments with T cell clones or lines in vivo and in vitro.     

CD1 proteins: targets of T cell recognition in innate and adaptive immunity

The CD1 family consists of antigen presenting molecules encoded by genes located outside of the major histocompatibility complex. CD1 proteins are conserved among mammalian species and are expressed on the surface of cells involved in antigen presentation. The CD1 system has been shown to be involved in activation of cell-mediated responses, and T cells specific for either CD1 molecules or antigens presented by CD1 have been isolated. Structural and biochemical analyses demonstrate that antigens presented by CD1 are nonpeptide lipid or glycolipid structures, including examples found in the cell walls of pathogenic mycobacteria. The hydrophobic part of these antigens most likely binds in the CD1 ligand-binding groove, whereas the polar headgroup of these antigens appears to make direct contact with the T cell receptor and determines specific recognition. Presentation of antigens by CD1 molecules requires uptake and intracellular processing by antigen presenting cells and can be achieved for both exogenous and endogenous antigens. T cells recognizing CD1 restricted antigens have a broad range of functional activities that suggest that the CD1 system is involved in both innate and adaptive immune responses against microbial infections.     

Unraveling the Mystery of γδ T Cell Recognizing Lipid A

Traditionally, the materials which are regarded as antigens recognized by γδ T lymphocytes are protein and carbohydrate, not nucleic acid or lipid. Recently, it has been demonstrated that γδ T cells can recognize lipid A and directly induce immune responses that involve CD1 （cluster of differentiation type 1） family and Toll like receptors （TLRs）. This is a review about the interacting-mechanism, immunological effect and clinical application of them.     

Liposome potentiation of humoral immune response to lipopolysaccharide and O-polysaccharide antigens of Brucella abortus.

Liposomes were evaluated for their effectiveness as vaccine carriers in the potentiation of the mouse humoral response to the lipopolysaccharide (LPS) and O-polysaccharide (OPS) antigens of Brucella abortus. LPS and OPS were extracted from a pathogenic strain of B. abortus and were encapsulated within multilamellar vesicles. Groups of mice, immunized with liposome-encapsulated and free LPS or OPS, were bled weekly and the specific IgM and IgG levels in the sera were determined by an indirect fluorogenic enzyme-linked immunosorbent assay. Humoral response to these antigens were found to be dose-dependent. Mice immunized with LPS and OPS encapsulated within liposomes were found to have significantly higher IgG levels than mice immunized with free LPS and OPS. In addition, the antibody levels in mice that were immunized with liposome-encapsulated LPS and OPS were more sustained and remained at elevated levels--even after 5 weeks post immunization. As expected, OPS was found to be less immunogenic than LPS, but multiple injections of OPS encapsulated within liposomes greatly improved the immunogenicity. These results indicate that the humoral response to LPS and OPS of B. abortus can be enhanced when these antigens are encapsulated within liposomes.     

Engineering antigen‐specific NK cell lines against the melanoma‐associated antigen tyrosinase via TCR gene transfer

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T‐lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL‐TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off‐target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen‐specific TCR complex on NK cell lines NK‐92 and YTS, demonstrated by using a TCR against the HLA‐A2‐restricted tyrosinase‐derived melanoma epitope, Tyr_368‐377. Most importantly, the introduction of a TCR complex to NK cell lines enables MHC‐restricted, antigen‐specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of “mispairing” in TCR gene therapy.     

B淋巴细胞对兔免疫球蛋白和兔抗鼠IgM抗体的提呈作用

本文采用兔免疫蛋白特异的鼠T细胞株为靶细胞,以T细胞的增殖和IL-2的产生活性为反应指标,探讨了小鼠B淋巴细胞兔免疫球蛋白和兔抗鼠IgM等抗原的提呈作用。实验结果显示,B细胞对兔抗鼠IgM抗体有很强的提呈作用,提呈兔抗鼠IgM抗体所需的抗原浓度较低,比兔免疫球蛋白低1000倍以上,当用巨噬细胞作为抗原提呈细胞时,则无上述差异。故提示可用兔抗鼠IgM抗体模拟抗原特异性B细胞的高效抗原提呈作用。此研究结果为进一步探讨抗原特异性B细胞抗原提呈作用的特点,以及T—B细胞间相互作用的途径和机制,提供了有用的实验模型。     

Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones.

Experimental studies have suggested that antigen-specific T lymphocytes are important mediators of resistance to infection with the pathogenic fungus Histoplasma capsulation. To gain a better understanding of the role of T lymphocytes, we developed murine T-cell lines and clones that recognized Histoplasma antigens. These T cells were of the helper/inducer phenotype (Thy-1.2+ Lyt-1+ L3T4+ Lyt-2-) and exerted multiple immunological functions. T-cell lines and 12 clones proliferated vigorously in response to histoplasmin; the T-cell lines and 6 clones also were reactive with heterologous fungal antigens prepared from either Blastomyces dermatitidis or Coccidioides immitis. Recognition of antigen by T cells was H-2 restricted; in the absence of antigen, four clones demonstrated alloreactivity. All T-cell clones conferred local delayed-type hypersensitivity responses when injected with antigen into footpads of mice. Ten of 12 T-cell clones released interleukin-2 after stimulation with antigen, and all clones tested secreted interferon. Moreover, culture supernatants from antigen-stimulated clones armed peritoneal macrophages to inhibit intracellular growth of H. capsulatum yeast cells. All clones assayed exerted nonspecific help. Thus, development of T-cell clones should facilitate analysis of the regulatory properties of Histoplasma-specific T cells.     

Effect of liposomes on lymphocytes: induction of proliferation of B lymphocytes and potentiation of the cytotoxic response of T lymphocytes to alloantigens

Liposomes of certain lipid composition prepared by the detergent removal method (Brunner, J. et al., Biochim. Biophys. Acta 1976. 455: 322) induced the proliferation of spleen cells from different mouse strains. Spleen cell populations enriched in B lymphocytes and those obtained from nude mice were induced to proliferate, whereas spleen cell fractions enriched in T lymphocytes and thymocytes were not. The mitogenic effect of liposomes resembled that of lipopolysaccharide (LPS) and it depended upon their lipid composition. Liposomes prepared from dimyristoyl lecithin (DML), 2:1 dimyristoyl lecithin:cholesterol (DML:C), 2:1 dioleoyl lecithin: cholesterol (DOL:C), and 2:1 egg yolk lecithin:cholesterol (EYL:C) were mitogenic, whereas liposomes prepared from egg yolk lecithin (EYL) alone were not mitogenic for spleen cells. The mitogenic effects of these liposome preparations were in the decreasing order DML greater than DOL:C greater than or equal to EYL:C greater than DML:C greater than EYL. The results suggest a correlation between the membrane fluidity of liposomes and their mitogenic effect. Although no proliferative response was induced on T lymphocytes, two of these liposomes, DML and EYL:C, had the ability to potentiate the cytotoxic response of T lymphocytes to alloantigens in mixed leukocyte culture. In responder-stimulator combinations which differed for the H-2K, H-2D or the entire H-2 region, these liposomes potentiated the cytotoxic response significantly. The results suggest that liposomes have an ability to modulate T lymphocyte response.     

The use of clonal mRNA as an antigenic format for the detection of antigen-specific T lymphocytes in IFN-gamma ELISPOT assays

Most assay systems for the quantification of antigen-specific T-cell responses in infectious, malignant and autoimmune disease depend on the peptide antigen format and are therefore restricted to known epitopes and their presenting HLA molecules. Here we tested in ELISPOT assays the application of in vitro-transcribed clonal mRNA as an alternative antigen format covering all potential epitopes of a given antigen. As model antigens, we chose pp65 of human cytomegalovirus (HCMV) and human tyrosinase (hTyr). Antigen-presenting cells (APC) were K562 cells stably transfected with single HLA class I alleles and autologous dendritic cells (DC). As effectors, we applied in vitro-generated anti-tyrosinase T-cell populations as well as ex vivo-CD8(+) lymphocytes from HCMV-seropositive donors. APC electroporated with clonal mRNA were efficient inducers of spot formation by antigen-experienced CD8(+) T cells. They were equivalent to peptide-loaded targets. mRNA electroporation did not induce non-specific spot formation. While the use of autologous mRNA-electroporated DC can uncover the complete individual T-cell response towards an antigen, mRNA-electroporated K562 cells stably transfected with single HLA class I alleles help to detect CD8(+) T-cell responses restricted by single HLA class I molecules.     

Impaired functional capacities of liver dendritic cells from murine hepatitis B virus (HBV) carriers: relevance to low HBV-specific immune responses

The chronic hepatitis B virus (HBV) carrier exhibits ongoing replication of HBV and expresses abundant amounts of HBV-related antigens in the liver. However, HBV-specific immune responses are either absent or narrowly focused in these subjects. With the postulation that impaired functional abilities of liver dendritic cells (DCs) might be responsible for this, we assessed the functions of liver DCs in HBV transgenic mice (HBV-TM), an animal model of the HBV carrier state. Liver DCs were isolated from normal C57BL/6 mice and HBV-TM without the use of cytokines or growth factors. Lymphoproliferative assays were conducted to evaluate the ability of liver DCs to induce the proliferation of allogenic T lymphocytes and hepatitis B surface antigen (HBsAg)-enriched T lymphocytes. Liver DCs were stimulated with viral and bacterial products to assess their cytokine-producing capacities. In comparison to liver DCs from normal C57BL/6 mice, liver DCs from HBV-TM exhibited significantly decreased T cell proliferation-inducing capacities in allogenic mixed leucocyte reaction (P <0.05) and HBsAg-enriched T lymphocytes proliferation assays (P <0.05). Liver DCs from HBV-TM produced significantly lower levels of interleukin-12p70, tumour necrosis factor-alpha, interferon-gamma, and interleukin-6 (P <0.05) compared to liver DCs from normal C57BL/6 mice. This study provides evidence that liver DCs from HBV-TM had impaired ability to induce both innate and adaptive immune responses. This might account for a weak and almost undetectable HBV-specific immune response in chronic HBV carriers. This inspires hope that up-regulation of the functions of liver DCs in situ may have therapeutic implications in chronic HBV carriers.     

Specific suppression of antibody responses by soluble protein-specific, class II-restricted cytolytic T lymphocyte clones

Antigenic stimulation with exogenous soluble proteins induces cytotoxic T lymphocytes (CTL) recognizing antigenic peptides presented on class II major histocompatibility complex (MHC) molecules. Such CTL have been shown to lyse preferentially B cells expressing immunoglobulin receptors reactive with the relevant antigens, presumably because such B cells can efficiently trap and present the antigen. Therefore, possible involvement of soluble protein antigen-specific CTL in specific suppression of antibody responses has been hypothesised. In this report, keyhole limpet hemocyanin and ovalbumin-specific, class II-restricted CD4+ CTL clones established from lymph nodes of immunized mice were examined for their suppressive activities on antibody production. When these CTL clones were added to in vitro secondary cultures, genetically restricted, carrier-specific suppression of anti-2,4,6-trinitrophenyl antibody production was observed. These data therefore demonstrate that CTL directed toward soluble antigens are capable of mediating specific suppression of antibody responses. Furthermore, the antibody response of MHC-heterozygous F1 lymphocytes was almost completely suppressed by a CTL clone restricted to one parental class II MHC antigen, indicating that the mechanism of suppression by these CTL is distinct from that by classical suppressor T cells.