Immunobiology of the Tomatine adjuvant.

Soluble or sub-unit protein vaccines alone are incapable of generating antigen-specific cellular immune responses. This failure can be attributed to the manner in which the immune system processes antigen; endogenous antigens are cycled through the MHC class I pathway to stimulate CD8+ restricted responses and exogenous antigens are processed through the MHC class II pathway to generate humoral immunity. Traditionally sub-unit vaccines have been formulated with adjuvants to enhance immunogenicity, however in the last decade a number of adjuvants have been developed that effectively stimulate the generation of both humoral and cellular immune responses, although the manner in which they exert their effects has not been investigated. Here we describe Tomatine, a glycoalkaloid based adjuvant, capable of stimulating potent antigen-specific humoral and cellular immune responses that contribute to protection against malaria, Francisella tularensis and regression of experimental tumors. Using in vivo models we investigated the manner in which cellular immune responses were generated by Tomatine. We established that Tomatine did not require either lymph node or splenic macrophages to generate cytotoxic T lymphocytes (CTL) and delivered soluble protein into a pathway not dependant on the machinery of the classical MHC class I pathway. We also observed that at the molecular level Tomatine required both CD80 and CD86 costimulation to engender antigen-specific cellular immunity.

The apoptotic and necrotic effects of tomatine adjuvant.

Tomatine adjuvant, consisting of tomatine, n-octyl-beta-d-glucopyranoside (OGP), phosphatidylethanolamine and cholesterol is unique in that when combined with soluble protein antigen it elicits a cytotoxic T lymphocyte (CTL) response in immunized animals. The mechanisms underlying this property are unknown. In an attempt to understand how tomatine activates cellular immunity, we examined its potential to induce apoptosis. Thus in the present study, cell death of EL4 thymoma cells induced by whole adjuvant and the surface-active components in the formulation was examined. Cytotoxicity was monitored using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and lactate dehydrogenase release assays, apoptosis and necrosis were quantified by flow cytometry using Annexin V and propidium iodide staining, and morphology was examined by Hoechst 33342 staining. Flow cytometric analysis demonstrated the appearance of the sub-G1 phase in cells treated with these agents and Annexin V/PI staining showed that all three agents induced both apoptosis and necrosis in EL4 cells in a concentration-dependent manner. Tomatine was effective at much lower concentrations than OGP, suggesting that the majority of the effect of whole adjuvant could be attributed to this component. Microscopic examination of EL4 cells after treatment with these agents revealed morphological features of apoptosis, including chromatin condensation and DNA fragmentation. Pretreatment with zVAD-fmk did not block cell death induced by these agents, showing that tomatine adjuvant-induced EL4 cell apoptosis is caspase-independent.

Cell death induced by vaccine adjuvants containing surfactants.

Many vaccine adjuvants contain surface-active agents, but the immunological roles played by these components have been essentially ignored. The objective of this study was to examine possible apoptotic and necrotic effects of the surface-active agents, Pluronic L121 and Tween 80, which are components of L121-adjuvant (a formulation we synthesized with the aim of representing several commercially produced adjuvants), on EL4 lymphoma cells. Cell viability and cytolytic effects were analyzed using the MTT and LDH release assays, and the distribution of cells in different stages of the cell cycle after treatment with these agents was analyzed by propidium iodide (PI) staining and flow cytometry. L121-adjuvant was shown to induce cell cycle arrest and inhibit cell proliferation. Treatment of EL4 cells with surface-active agents resulted in a concentration-dependent increase in the apoptotic/necrotic cell populations. Fluorescence microscopy using Hoechst 33342 staining demonstrated chromosome condensation and DNA fragmentation in cells treated with surfactants or adjuvant. The apoptotic and necrotic effects of vaccine adjuvant containing surface-active agents were confirmed by Annexin V/propidium iodide staining and flow cytometric analysis. Pretreatment of EL4 cells with zVAD-fmk, a broad range caspase inhibitor, partially prevented apoptosis induced by Pluronic L121, but did not prevent the cell death induced by Tween 80 or L121-adjuvant. These findings suggested that Tween 80 and L121-adjuvant induced apoptosis in EL4 cells via a "non-classical" caspase-independent pathway. Results presented in this study suggest mechanisms of elicitation of CD8(+), class I-restricted CTL response by soluble antigens mediated by the vaccine adjuvant containing surface-active agents.

Isolation of human leukocyte antigen (HLA)-associated peptide(s) in the absence of HLA-restricted specific cytolytic T lymphocytes.

BACKGROUND/METHODS: In this study, immunobead purification, dot-blot, immunocytochemical staining, and SDS-PAGE techniques in combination with high-performance liquid chromatography were used to isolate human leukocyte antigen (HLA) class I antigens and associated peptides from a bladder tumour cell line (Fen) before and after gene transfection. RESULTS: The results showed that: (1) Transfection of the class I negative Fen cell line with normal beta-microglobulin (beta(2)-m) gene resulted in the restoration of missing class I antigens. (2) The intact class I antigens could be isolated from lysate of the beta(2)-m gene transfected cells using Sepharose CNBr-W6/32 beads. (3) Dissociation of class I antigens from beads and analysis by the SDS-PAGE showed the presence of both free heavy and light chains of class I antigens. (4) More than 22 class I-associated peptides with a molecular weight of 700-3,000 daltons could be isolated from W6/32-loaded beads but only from lysate of HLA-positive Fen cell line. The data also showed that 1 x 10(6) of positive Fen cells contained about 200 microg total protein of which about 0.10 microg was class I and about 2 ng was class I-associated peptides. CONCLUSIONS: These findings demonstrated that the gene transfection approach could be used to restore missing class I antigens on an otherwise class I negative bladder tumour cell line. The results also showed the feasibility of using above techniques for isolation of HLA-associated peptides. These approaches may provide a realistic possibility for identification of putative tumour-specific peptide(s) from tumour specimens with the long-term aim to use such peptide(s) for immunotherapy in cancer patients.

The ultrastructure of tomatine adjuvant.

The tomatine adjuvant, consisting of tomatine, n-octyl-beta-D-glucopyranoside, phosphatidylethanolamine, cholesterol, and ovalbumin, has recently been shown to potentiate the immunogenicity of protein antigen and elicit cytotoxic T-lymphocyte responses in immunized animals. The physicochemical properties of tomatine adjuvant have not been characterized. The aim of this study was to examine the microstructure of this complex formulation, as directly related to its physicochemical properties. To elucidate the micromorphology of this system, the tomatine adjuvant was separated by isopycnic ultracentrifugation, followed by freeze fracturing and examination by transmission and scanning electron microscopy. The adjuvant mixture was shown to be composed of several micro- and nano-structures. The major fraction obtained from isopycnic separation was shown to consist of flaky needle-like microcrystals, approximately 80-160 nm in width and 2-4 microm in length. The tomatine crystals alone in 0.9% NaCl, on the other hand, were shown to be elongated hollow tubular crystals of hundreds of nanometers up to a few microns in length, along which n-octyl-beta-glucopyranoside was speculated to serve as a seeding microtemplate for gel crystallization of protein complexes. Indented marks within the gel phase were observed in the freeze fractured replicas of the adjuvant, suggesting that protein complexes may have been crystallized or precipitated within the gels. Several other forms of micro- and nano-structures were also observed, showing multiple-dispersion features with gel characteristics. The presence of gel crystalline and multiple-dispersed phases is postulated to contribute to the sustained immunopotentiation effect of tomatine adjuvant.

Immunization with a DNA vaccine expressing a truncated form of varicella zoster virus glycoprotein E.

The gE glycoprotein of varicella zoster virus (VZV) is involved with cell entry and it is the most abundant glycoprotein produced in VZV-infected cells. It is also the first glycoprotein to be recognized by the immune system and induces neutralizing antibodies and cellular immunity. We have shown previously that immunization with a DNA vaccine encoding full length gE induces high antibody titres in BALB/c mice. In this study, we engineered a truncated form of gE to facilitate secretion of the glycoprotein, which is thought to increase the quantity of antigen available for B cells to mount an immune response. This hypothesis was tested by using inverse PCR mutagenesis (IPCRM) to engineer a mutated form of gE that was secreted from the cell. This construct was then evaluated as a potential DNA vaccine. Following immunization studies, the magnitude of the immune response induced with the mutant form of gE was found to be similar to that induced by membrane bound protein. This finding suggests that, in the case of VZV, a DNA vaccine expressing a secreted protein has no advantage over one expressing a membrane bound protein. However, mice immunized with the truncated form of gE (gED) displayed responses favouring IgG1 (Th2) in comparison with mice immunized with the full length gE construct, which generated an IgG2a (Th1) response. This observation indicates that immunization with a truncated form of a gene may induce immune modulation, a phenomenon that should be taken into account for the design of vaccines.

Potentiation by a novel alkaloid glycoside adjuvant of a protective cytotoxic T cell immune response specific for a preerythrocytic malaria vaccine candidate antigen.

We have recently demonstrated that the novel glycoalkaloid tomatine, derived from leaves of the wild tomato Lycopersicon pimpinellifolium, can act as a powerful adjuvant for the elicitation of antigen-specific CD8+ T cell responses. Here, we have extended our previous investigation with the model antigen ovalbumin to an established malaria infection system in mice and evaluated the cellular immune response to a major preerythrocytic stage malaria vaccine candidate antigen when administered with tomatine. The defined MHC H-2kd class I-binding 9-mer peptide (amino acids 252-260) from Plasmodium berghei circumsporozoite (CS) protein was prepared with tomatine to form a molecular aggregate formulation and this used to immunise BALB/c (H-2kd) mice. Antigen-specific IFN-gamma secretion and cytotoxic T lymphocyte activity in vitro were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunised control mice. Moreover, when challenged with P. berghei sporozoites, mice immunised with the CS 9-mer-tomatine preparation had a significantly delayed onset of erythrocytic infection compared to controls. The data presented validate the use of tomatine to potentiate a cellular immune response to antigenic stimulus by testing in an important biologically relevant system. Specifically, the processing of the P. berghei CS 9-mer as an exogenous antigen and its presentation via MHC class I molecules to CD8+ T cells led to an immune response that is an in vitro correlate of protection against preerythrocytic malaria. This was confirmed by the protective capacity of the 9-mer-tomatine combination upon in vivo immunisation. These findings merit further work to optimise the use of tomatine as an adjuvant in malaria vaccine development.

Autoantibodies and overlap syndromes in autoimmune rheumatic disease.

Many patients diagnosed with autoimmune rheumatic disease cannot be categorised easily into one of the established clinical entities such as systemic lupus erythematosus, dermatomyositis, or systemic sclerosis. The term "overlap syndrome" has been increasingly used to identify such patients and is useful in terms of clarifying prognosis and facilitating disease management. This article reviews overlap syndrome in autoimmune rheumatic disease, with particular emphasis on the associated serological markers.

Helicobacter pylori vaccine strategies--triggering a gut reaction.

Helicobacter pylori causes peptic ulcer disease and some forms of gastric cancer; it is one of the most common chronic bacterial infections of humans. Although several prototype protein-based vaccines have shown promising results, they have not cleared infection and/or prevented reinfection. Nucleic acid vaccination offers a useful alternative to protein immunization, especially now that two complete H. pylori genome sequences are available, and facilitates the selection of antigenic targets.

Localized monocyte chemotactic protein-1 production correlates with T cell infiltration of synovium in patients with psoriatic arthritis.

OBJECTIVE: To examine normal and psoriatic skin and synovial tissue from patients with psoriatic arthritis (PsA) for evidence of monocyte chemotactic protein-1 (MCP-1) mediated T cell chemotaxis. METHODS: Peripheral blood (PB), synovial fluid (SF), normal and psoriatic skin, and synovial biopsies were obtained from patients with PsA (n = 19) and compared to samples from normal (n = 5) and disease (n = 5) controls (NC, DC). Immune cell populations in PB and SF samples were assessed by immunofluorescent labeling and flow cytometry, levels of soluble MCP-1 were determined by quantitative ELISA, and immunohistochemistry was used to detect T cell subsets and macrophages and MCP-1 protein in frozen skin and synovial tissue sections. RESULTS: CD8+ but not CD4+ T cells were elevated in SF compared to PB, and the majority of these cells expressed CD45RO. Plasma MCP-1 levels in PsA were elevated relative to NC. MCP-1 levels were significantly higher than paired plasma samples in patients with recent onset (< 6 mo) synovitis (n = 10). A positive correlation was observed between synovial T cell numbers and MCP-1 levels in SF. MCP-1 protein was present in all tissues examined, but most intense expression was observed in synovium. CONCLUSION: Elevated concentrations of MCP-1 concomitant with memory T cell infiltration in PsA SF suggests that MCP-1 mediated chemotaxis is involved in the recruitment of T lymphocytes into the synovial compartment of patients with PsA.

Immunization with a DNA expression vector encoding the varicella zoster virus glycoprotein E (gE) gene via intramuscular and subcutaneous routes.

In this study we constructed a plasmid containing the gene encoding varicella-zoster virus transmembrane glycoprotein gE (VZV gE) and evaluated its utility for DNA immunization in mice. Our initial work demonstrates that intramuscular and subcutaneous injection of VZV gE DNA, without the use of costimulatory molecules or other adjuvant materials, results in the generation of antigen-specific antibodies of primarily the IgG2a subclass, indicating that this vaccine can stimulate Th1 type immunity. This is the first report of a prototype DNA vaccine for varicella-zoster virus.

Delivery systems for molecular vaccination.

Vaccination is one of the medical success stories of the 20th century, however, there are many diseases for which no prophylactic regimes are available. A major hindrance that has prevented the development of effective mass immunization programs is the inability to induce an appropriate, protective, immune response. For example, for vaccines against intracellular pathogens there is a requirement for cell-mediated immunity as characterized by cytolytic T-lymphocyte activity. However, such a response can be extremely difficult to elicit, especially those employing recombinant, soluble protein subunits. This deficiency is due to the inability of these antigens to access the machinery of the appropriate antigen-processing pathway. Following an improved understanding of the mechanisms underlying such processing, as well as the realization that delivery systems can affect, quantitatively and qualitatively, the resulting immune response, the last decade has witnessed an intense research effort in this field. In this article we will review the major developments in the area of antigen delivery as related to vaccination.

Nucleic acid immunization: concepts and techniques associated with third generation vaccines.

A radical change in vaccine methodology arrived nine years ago with the advent of nucleic acid immunization. Aspects such as plasmid design, gene selection, the use of immunostimulatory complexes and clinical trials are discussed in this review. Furthermore, concepts and protocols involved in the construction, evaluation and immunization of a DNA vaccine have been examined as new strategies to enhance this technology continues to grow.

Novel aggregate structure adjuvants modulate lymphocyte proliferation and Th1 and Th2 cytokine profiles in ovalbumin immunized mice.

Cytokines are important mediators of effector lymphoid cell function during an immune response. The principal cytokine producers are the T helper (Th) cells and macrophages. Vaccine strategies need to take into account the balance of Th (Th1/Th2) cytokines they induce. Adjuvants are compounds that, when combined with an antigen, potentiate an immune response in an immunized species. The use of adjuvants has been shown to activate differentially Th1 and Th2 subsets. In this study we describe the immunopotentiating properties of three novel molecular aggregate formulations based on tomatine (RAM1), a glycosylamide lipid (RAM2) and a fifth generation dendrimeric polymer (RAM3) respectively. These formulations were evaluated for their ability to augment Th1 or Th2 cytokine responses when administered with a soluble protein antigen. Of the three formulations, RAM1 was found to induce predominantly Th1 cytokines; the levels of which were substantially higher than those induced by reference control adjuvants. It was also found that at a late post-vaccinated period, RAM1 can stimulate Th2 responses. In contrast, RAM2 and RAM3 induced cytokine profiles typically associated with Th2 responses.

Anti-tumour activity against B16-F10 melanoma with a GM-CSF secreting allogeneic tumour cell vaccine.

Genetic modification of tumour cells with the GM-CSF encoding gene renders these cells more potent, as autologous tumour cell vaccine, than their wild-type counterparts. However, autologous vaccines are impractical for wide-scale clinical use and we have therefore investigated the efficacy of the GM-CSF genetic modification approach with an allogeneic whole cell tumour vaccine. In this report, we show that the allogeneic K1735-M2 (H-2k) melanoma cell vaccine induces a specific protective anti-tumour response against the syngeneic B16-F10 (H-2b) melanoma tumour in C57BL/6J mice. In vitro T cell work demonstrated that vaccination of animals with the allogeneic cell vaccine generated cytotoxic T cells specific for the autologous tumour. In vivo T cell subset depletion experiments also illustrated that this anti-tumour effect was mediated by both CD4+ve and CD8+ve T cells, suggesting that the allogeneic vaccine may operate through the 'cross-priming' phenomenon whereby tumour antigens are processed and presented to T cells by the host's own antigen presenting cells (APC). Thus, we transduced K1735-M2 cells with a GM-CSF expressing retroviral vector and showed anti-tumour activity of the GM-CSF secreting K1735-M2 cells as a therapeutic vaccine against the syngeneic B16-F10 tumour. Our data imply that GM-CSF genetically modified allogeneic whole cell tumour vaccines could be successful in the clinic. In addition, more potent combination gene therapy strategies could be tested using this therapeutic allogeneic vaccine model.

Generation of antigen specific CD8+ cytotoxic T cells following immunization with soluble protein formulated with novel glycoside adjuvants.

Presentation of peptide on MHC class I molecules is essential to elicit cytolytic T cell (CTL) activity. Such peptides are a result of the cytosolic, or class I, antigen processing pathway. Due to the segregation of the class I and the exogenous processing pathway, soluble protein cannot enter the class I pathway and is thus incapable of inducing CTL. However careful formulation with adjuvants can overcome this obstacle. In this study we evaluated the capacity of two novel amphiphilic adjuvants, better termed delivery vehicles, to elicit CTL activity in a C57Bl/6 murine model with ovalbumin (OVA) as an antigen. Incomplete Freund's adjuvant and aluminium hydroxide (Alhydrogel) were used as reference adjuvants. In addition the oil-in-water emulsion Provax was used throughout as a positive control adjuvant. Both amphiphile preparations were capable of eliciting potent CTL activity after administration of one immunizing dose of ovalbumin. CTL were CD8+ restricted as assessed by in vitro depletion of CD8+ and CD4+ T cells. CTL activity was also MHC-restricted as well as specific for the H-2Kb OVA motif SIINFEKL.

Evaluation of novel aggregate structures as adjuvants: composition, toxicity studies and humoral responses.

Adjuvants are compounds that, when combined with an antigen, potentiate an immune response in an immunized species. There are numerous pathogens for which there are no protective vaccines and since alum is the only adjuvant licensed for use in humans, there is a clear need for more effective adjuvant preparations. In this study we describe the immunopotentiating properties of three novel molecular aggregate formulations based on tomatine (RAM1), a glycosylamide lipid (RAM2) and a fifth generation dendrimeric polymer (RAM3) respectively. These formulations were evaluated for their ability to augment antigen-specific antibody responses when administered with a soluble protein antigen. All three adjuvants were shown to be nontoxic to mice and elicited antigen-specific antibody responses. Of the three formulations, RAM1 was found to induce the highest titers of antibody; these were substantially higher than those induced by reference control adjuvants. RAM1 elicited antibodies of the IgG1 and IgG2a subclasses indicating, indirectly, that this adjuvant can stimulate Th2 and Th1 type immunity.

Integrin signalling defects in T-lymphocytes in systemic lupus erythematosus.

OBJECTIVE: To establish the relationship between T cell responses to integrin coreceptor stimulation and B cell hyperreactivity as measured by pathologic autoantibody production. METHODS: Peripheral blood mononuclear cells from 42 patients with SLE according to the American Rheumatism Association criteria were examined for their ability to adhere to plate-immobilised fibronectin. Co-stimulation assays were performed on the same cells using anti-CD3 antibody alone or co-immobilised with an anti-beta1-integrin antibody. Proliferative responses were measured by 3[H]thymidine pulsing on day 3 and activation was determined using a commercial protein kinase C assay, the protocol being established by our group in association with Promega. Beta-integrin expression was established by FACS analysis. RESULTS: An impaired PKC response to integrin-mediated activation was found in T-lymphocytes from 6/21 (29%) SLE patients, which correlated significantly with an absence of anti-dsDNA antibody in patient sera, irrespective of prednisolone treatment. Integrin co-stimulation of TcR/CD3-induced proliferation and T cell adhesion to fibronectin were also impaired among 5/21 (24%) and 6/15 (40%) patients studied, respectively. CONCLUSION: We hypothesise that the integrity of beta1-integrin signalling pathways may influence pathological antibody production in SLE by affecting T-lymphocyte activation and interactions between T- and B-lymphocytes.

Pathogenic mechanisms in psoriatic arthritis.

Psoriatic arthritis is an inflammatory arthritis which can develop in some psoriasis patients. The absence of a serological test means that diagnosis must be based on consideration of clinical indices. This article summarizes the inflammatory mediators and immunological events which characterize psoriatic arthritis and examines evidence for involvement of an infectious agent in its aetiology.

Comparison of four strategies for tumour vaccination in the B16-F10 melanoma model.

We have compared four cell-based tumour vaccine strategies in prevention experiments using the B16-F10 melanoma model. Two of these are thought to favour the direct antigen presentation pathway (B16-F10 expressing B7.1 and hybrids made between B16-F10 cells and macrophages) and the other two strategies are thought to act by an indirect pathway of presentation (allogeneic tumour cells and autologous tumour cells combined with a powerful adjuvant (Provax-IDEC Pharmaceuticals)). Only the two latter vaccines promoted antitumour activity, whereas the vaccines consisting of B7.1-expressing tumour cells or the hybrid vaccine failed to provide any antitumour activity. Recently human trials have commenced using transfection of the B7.1 molecule, as well as employing the hybrid technology to make tumour-B cell hybrids or tumour and dendritic cell hybrids. Our results suggest that these approaches could be disappointing in the clinics if not optimised.