Characterisation of local immune responses induced by a novel nano-particle based carrier-adjuvant in sheep.

Most adjuvants require danger signals to promote immune responses against vaccine antigens. Our previous studies have characterised a powerful nano-particulate antigen delivery system, which by itself does not induce inflammation, and which further appears to induce substantial immune responses in mice and sheep without the requirement for added stimulators of toll like receptors or other pathogen recognition receptors. In the present study we dissect the nature of the early induction phase of the immune response stimulated by such a vaccine comprising 40 nm polystyrene nano-particles conjugated to the antigen. We analyse the kinetics of export from an individual draining lymph node from the sheep, of antibodies and cytokines as well as antigen responsive CD4 and CD8 T cells. Our results indicate that simple inert nano-bead based antigen delivery into the draining area of the lymph node is highly efficient at priming combined humoral and T cell antigen specific immunity without the need for added 'danger signals'. Furthermore this nano-bead adjuvant is a potent agent capable of promoting cross-priming for CD8 T cell induction in sheep. Interestingly, using nano-beads, similarly to what has been observed with natural pathogen based lymph node stimulation, a phase of CD4 T cell priming and export preceded CD8 T cell induction, suggesting the engagement of natural priming processes and kinetics.

Transgenic mice expressing human FcgammaRIIa have enhanced sensitivity to induced autoimmune arthritis as well as elevated Th17 cells.

The major human Fc receptor, huFcgammaRIIa, is implicated in the development of autoimmune arthritis in humans but until recently has not been studied in mouse models. We evaluated potential roles of FcgammaRIIa by using transgenic mice expressing the receptor. We examined two models of induced autoimmune arthritis pristane-induced arthritis (PIA) and collagen-induced arthritis (CIA) as well as the anti-collagen-II antibody-induced arthritis (CAIA) model. In the induced arthritis models PIA and CIA, the transgenic mice developed a more severe arthritis than the other arthritis-prone SJL or DBA1 mice. Interestingly, anti-collagen-II antibodies were elevated in PIA in the susceptible mice. In the CIA model, the highly susceptible transgenic mouse had IgG subclass levels equivalent to the unaffected and disease resistant C57BL/6 mouse strain implying that the FcgammaRIIa lowers the threshold of IgG dependent leukocyte activation. This is consistent with the greatly enhanced sensitivity of the FcgammaRIIa transgenic mice to CAIA which clearly indicates a role for the receptor at least at the inflammatory effector cell level. Other roles for huFcgammaRIIa or other gene products in the development of autoimmunity cannot be ruled out however, especially as the mice exhibited elevated Th1 or Th17 CD4 T cells in the draining lymph nodes.

Inhibition of destructive autoimmune arthritis in FcgammaRIIa transgenic mice by small chemical entities.

The interaction of immune complexes with the human Fc receptor, FcgammaRIIa, initiates the release of inflammatory mediators and is implicated in the pathogenesis of human autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, so this FcR is a potential target for therapy. We have used the three-dimensional structure of an FcgammaRIIa dimer to design small molecule inhibitors, modeled on a distinct groove and pocket created by receptor dimerization, adjacent to the ligand-binding sites. These small chemical entities (SCEs) blocked immune complex-induced platelet activation and aggregation and tumor necrosis factor secretion from macrophages in a human cell line and transgenic mouse macrophages. The SCE appeared specific for FcgammaRIIa, as they inhibited only immune complex-induced responses and had no effect on responses to stimuli unrelated to FcR, for example platelet stimulation with arachidonic acid. In vivo testing of the SCE in FcgammaRIIa transgenic mice showed that they inhibited the development and stopped the progression of collagen-induced arthritis (CIA). The SCEs were more potent than methotrexate and anti-CD3 in sustained suppression of CIA. Thus, in vitro and in vivo activity of these SCE FcgammaRIIa receptor antagonists demonstrated their potential as anti-inflammatory agents for autoimmune diseases involving immune complexes.

Type 1 and 2 immunity following vaccination is influenced by nanoparticle size: formulation of a model vaccine for respiratory syncytial virus.

Previous studies compared uptake by dendritic cells (DC) of 20, 40, 100, 200, 500, 1000, and 2000 nm beads in vivo. When beads were used as antigen carriers, bead size influenced antibody responses and induction of IFN-gamma-producing CD4 and CD8 T cells. Beads of 40-50 nm were taken up preferentially by DC and induced particularly strong immunity. Herein, we examine immunity induced by minute differences in nanobead size, specifically within a narrow viral-sized range (20, 40, 49, 67, 93, 101, and 123 nm), to see if bead carrier size influenced the induction of type 1 or type 2 cells as demonstrated by the production of IFN-gamma or IL-4. In vivo uptake by DC was assessed for selected sizes in this range. Responses to whole ovalbumin (OVA) or the OVA-derived CD8 T cell peptide epitope (SIINFEKL) were tested. After one immunization with beads-OVA, IFN-gamma responses to both OVA and SIINFEKL were significantly better with 40 and 49 nm beads than other sizes, while, in contrast, IL-4 responses to OVA were higher after immunization with OVA conjugated to larger beads (93, 101, and 123 nm). Thus IFN-gamma induction from CD8 T cells was limited to 40-49 nm beads, while CD4 T cell activation and IL-4 were induced by 93-123 nm beads-OVA. After two immunizations, there were comparable high levels of IFN-gamma produced with 40 and 49 beads and IL-4 reactivity was still higher for larger beads (93, 101, 123 nm). Production of IgG1 was seen across the full range of bead sizes, increasing after two immunizations. Since protection against respiratory syncytial virus (RSV) depends on strong IFN responses, while IL-4 responses are reported to cause asthma-like symptoms, immunization with RSV antigens on the 49 nm carrier beads could provide the basis for a suitable vaccine. When the 49 nm beads were conjugated to RSV proteins G88 (surface) or M2.1 (internal capsid), one immunization with G88 induced high levels of IFN-gamma and low levels of IL-4. IL-4 increased with two immunizations. Beads-M2.1 induced only moderate levels of IFN-gamma and low titer antibody after two immunizations. Mice vaccinated once with G88-conjugated 49 nm beads and challenged intranasally with RSV strain A2 subtype showed reduced viral titers and recovered from weight loss more rapidly than mice immunized with M2.1-conjugated 49 nm beads or naive control mice. These results show that precise selection of nanobead size for vaccination can influence the type 1/type 2 cytokine balance after one immunization, and this will be useful in the development of effective vaccines against common human pathogens such as RSV.

FcgammaRII and multi-system autoimmune disease.

The FcR are a crucial link in the immune response between humoral and cellular immunity and cell-based effector systems, mediating a wide variety of physiological and biochemical responses. The FcR for IgG (FcgammaR) and in particular the most widely expressed of these, FcgammaRII, are important in regulating adaptive immunity. Disruption of their function is a key factor in the development of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), which are characterized by chronic, multi-organ inflammation. Studies of the FcgammaRII include structure/function relationships, investigation of the associations between FcR polymorphisms and human disease and animal studies using knockout or transgenic mouse models. These investigations showed that the various forms of FcgammaRII interact with immune complexes to either initiate or inhibit inflammation. In conjunction with environmental antigens and genotype, the FcgammaRII activating and inhibitory receptors determine the nature and magnitude of response to antigens. In this review, the structure and function of the FcgammaRIIs and their role in immune complex-mediated auto-immunity are discussed.

Pathogen recognition and development of particulate vaccines: does size matter?

The use of particulate carriers holds great promise for the development of effective and affordable recombinant vaccines. Rational development requires a detailed understanding of particle up-take and processing mechanisms to target cellular pathways capable of stimulating the required immune responses safely. These mechanisms are in turn based on how the host has evolved to recognize and process pathogens. Pathogens, as well as particulate vaccines, come in a wide range of sizes and biochemical compositions. Some of these also provide 'danger signals' so that antigen 'senting cells (APC), usually dendritic cells (DC), acquire specific stimulatory activity. Herein, we provide an overview of the types of particles currently under investigation for the formulation of vaccines, discuss cellular uptake mechanisms (endocytosis, macropinocytosis, phagocytosis, clathrin-dependent and/or caveloae-mediated) for pathogens and particles of different sizes, as well as antigen possessing and presentation by APC in general, and DC in particular. Since particle size and composition can influence the immune response, inducing humoral and/or cellular immunity, activating CD8 T cells and/or CD4 T cells of T helper 1 and/or T helper 2 type, particle characteristics have a major impact on vaccine efficacy. Recently developed methods for the formulation of particulate vaccines are presented in this issue of Methods, showcasing a range of "cutting edge" particulate vaccines that employ particles ranging from nano to micro-sized. This special issue of Methods further addresses practical issues of production, affordability, reproducibility and stability of formulation, and also includes a discussion of the economic and regulatory challenges encountered in developing vaccines for veterinary use and for common Third World infectious diseases.

Methods for nano-particle based vaccine formulation and evaluation of their immunogenicity.

Nano- and microparticles have long been used for the delivery of drugs and are currently being evaluated as vaccine delivery systems. Particulates can elicit potent immune responses, either by direct immuno-stimulation of antigen presenting cells (APC) or/and by delivering antigen to specific cellular compartments and promoting antigen uptake by appropriate stimulatory cell types. Herein, we describe a detailed method for the preparation of a novel nanoparticle-based antigen delivery system which induces strong cellular and humoral immune responses in mice and sheep. This simple system is based on the use of 40 nanometer (nm) inert solid carrier beads to which antigen is covalently coupled before injection. Covalent conjugation of antigen to the nanobeads, assessment of conjugation efficiency, characterisation and measurement of in vivo immunogenicity by cytokine ELISPOT (to measure antigen-specific T-cell responses) and ELISA (to measure antibody titers), are described. Emphasis is placed on providing trouble-shooting advice to enable the reproducible production of soluble nano-size formulations that do not suffer from common problems such as aggregation, as well as understanding the causes and thus avoiding a range of prevalent technical problems that occur when using immune response detection assays, such as the cytokine ELISPOT assay and ELISA.

Systemic immune responses in sheep, induced by a novel nano-bead adjuvant.

Although a number of adjuvants are currently approved for use in veterinary species, only alum has been widely used in humans. While it induces strong antibody responses, cell mediated responses are often low and inflammatory reactions at the site of injection are common. We investigated the immunological properties of a novel nano-bead adjuvant in a sheep large-animal model. In contrast to alum, antigen covalently coupled to nano-beads induced substantial cell mediated responses along with moderate humoral responses. No adverse reactions were seen at the site of immunisation in the sheep. Thus, nano-bead adjuvants in veterinary species may be useful for the induction of immunity to viral pathogens, where a cell mediated response is required. These findings also highlight the potential usefulness of nano-bead vaccines for intracellular pathogens in humans.

Development of spontaneous multisystem autoimmune disease and hypersensitivity to antibody-induced inflammation in Fcgamma receptor IIa-transgenic mice.

OBJECTIVE: The major human Fc receptor, FcgammaRIIa, is the most widespread activating FcR. Our aim was to determine the role of FcgammaRIIa in a transgenic mouse model of immune complex-mediated autoimmunity and to characterize the development of spontaneous autoimmune disease. METHODS: Arthritis was induced in normal and FcgammaRIIa-transgenic mice by immunization with type II collagen (CII) or by transfer of arthritogenic anti-CII antibodies. Also, mice that spontaneously developed autoimmune disease were assessed by clinical scoring of affected limbs, histology and serology, and measurement of autoantibody titers and cytokine production. RESULTS: FcgammaRIIa-transgenic mice developed collagen-induced arthritis (CIA) more rapidly than did archetypal CIA-sensitive DBA/1 (H-2q) mice, while nontransgenic C57BL/6 (H-2b) mice did not develop CIA when similarly immunized. Passive transfer of a single dose of anti-CII antibody induced a more rapid, severe arthritis in FcgammaRIIa-transgenic mice than in nontransgenic animals. In addition, most immune complex-induced production of tumor necrosis factor alpha by activated macrophages occurred via FcgammaRIIa, not the endogenous mouse FcR. A spontaneous, multisystem autoimmune disease developed in aging (>20 weeks) transgenic mice (n = 25), with a 32% incidence of arthritis, and by 45 weeks, all mice had developed glomerulonephritis and pneumonitis, and most had antihistone antibodies. Elevated IgG2a levels were seen in mice with CIA and in those with spontaneous disease. CONCLUSION: The presence of enhanced passive and induced autoimmunity, as well as the emergence of spontaneous autoimmune disease at 20-45 weeks of age, suggest that FcgammaRIIa is a very important factor in the pathogenesis of autoimmune inflammation and a possible target for therapeutic intervention.

Antigen delivery via two molecules on the CD8- dendritic cell subset induces humoral immunity in the absence of conventional "danger".

Targeting antigen to dendritic cells (DC) in vivo might be an effective method of modulating immune responses. Given the functional specializations among DC subsets, we investigated how targeting different receptors on different DC subsets may influence antibody (Ab) production. We show here that targeting FIRE (F4/80-like receptor) or CIRE (C-type lectin receptor), two molecules expressed on the surface of immature CD8- DC in the mouse, increases Ab production 100-1000-fold over a non-targeted control. This response was equivalent to that achieved with CpG adjuvant. In contrast, targeting CD205, which is primarily expressed on CD8+ DC, did not elicit an Ab response unless an adjuvant was added. Strong Ab responses in FcRgamma-/- mice, and with the use of F(ab')2 fragments, confirmed that FIRE and CIRE targeting was due to specific rather than FcR or complement binding. Our findings may reflect differences in the ability of CD8+ and CD8- DC subsets to stimulate immune responses in vivo. Although the consensus view is that Ag presentation on DC in their steady state leads to tolerance, the Ab enhancement from FIRE and CIRE targeting in the apparent absence of any "danger" or inflammatory signal would suggest that targeting certain DC molecules can supplant the need for external adjuvants for eliciting immune responses.

Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors.

Infection can protect against subsequent disease by induction of both humoral and cellular immunity, but inert protein-based vaccines are not as effective. In this study, we present a new vaccine design, with Ag covalently conjugated to solid core nano-beads of narrowly defined size (0.04-0.05 microm) that localize to dendritic cells (DEC205(+) CD40(+), CD86(+)) in draining lymph nodes, inducing high levels of IFN-gamma production (CD8 T cells: precursor frequencies 1/5000 to 1/1000) and high Ab titers in mice. Conjugation of Ag to these nano-beads induced responses that were significantly higher (2- to 10-fold) than those elicited by other bead sizes, and higher than a range of currently used adjuvants (alum, QuilA, monophosphoryl lipid A). Responses were comparable to CFA/IFA immunization for Abs and ex vivo peptide-pulsed dendritic cell immunization for CD8 T cells. A single dose of Ag-conjugated beads protected mice from tumors in two different model challenges and caused rapid clearance of established tumors in mice. Thus, a range of Ags conjugated to nano-beads was effective as immunogens in both therapeutic and prophylactic scenarios.

Past, present and future drug treatment for rheumatoid arthritis and systemic lupus erythematosus.

Historically, treatment of complex autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus has aimed to relieve symptoms, and in severe cases, use broad-spectrum immunosuppressive treatments in attempts to induce permanent remission. Recent research into the causes of chronic autoimmune inflammatory activation have not only explored the mechanism of action of known therapies, but also provided a number of new targets for therapy, by identifying the cells, cytokines and signalling pathways activated during autoimmune antibody mediated processes. This review briefly outlines progress in the understanding of the autoimmune nature of rheumatoid diseases and the expansion of treatment options, from broad to specific immunotherapies for these closely related diseases.

The role of FcgammaRIIa as an inflammatory mediator in rheumatoid arthritis and systemic lupus erythematosus.

Despite their essential role in host protection, immunoglobulins are also involved in autoimmune processes where antibodies recognize the host's own tissue, triggering inflammatory responses that result in extensive tissue damage. A complex interaction of genetic predisposition, together with environment factors, is thought to trigger immune dysfunction. Although recent studies have dissected the essential role of Fc receptors in autoimmune antibody mediated processes, the uniquely human FcgammaRIIa has not been studied in detail. This Fc receptor is of particular interest, as it is the most abundantly expressed Fc receptor in humans and is implicated in immune complex disease. Investigation of its role has been hampered to date due to lack of suitable animal models. This review examines the evidence for the direct role of this receptor in diseases such as systemic lupus erythematosus and rheumatoid arthritis.

Unique monoclonal antibodies define expression of Fc gamma RI on macrophages and mast cell lines and demonstrate heterogeneity among subcutaneous and other dendritic cells.

The mouse Fc gamma RI is one of the most fundamentally important FcRs. It participates in different stages of immunity, being a low affinity receptor for T-independent IgG3 and yet a high affinity receptor for IgG2a, the product of a Th1 immune response. However, analysis of this receptor has been difficult due largely to the failure to generate specific Abs to this FcR. We have made use of the polymorphic differences between BALB/c and NOD/Lt mice to generate mAb specific for the Fc gamma RI of BALB/c and the majority of in-bred mouse strains. Three different mAb were obtained that detected Fc gamma RI encoded by the more common Fcgr1(a) and Fcgr1(b) alleles, and although they identified different epitopes, none inhibited the binding of IgG to Fc gamma RI. When bound to Fc gamma RI, these mAb induced calcium mobilization upon cross-linking. Several novel observations were made of the cellular distribution of Fc gamma RI. Resting and IFN-gamma-induced macrophages expressed Fc gamma RI as well as mast cell lines. Both bone marrow-derived and freshly isolated dendritic cells from spleen and lymph nodes expressed Fc gamma RI. A class of DC, uniquely found in s.c. lymph nodes, expressed the highest level of Fc gamma RI and also high levels of MHC class II, DEC205, CD40, and CD86, with a low level of CD8 alpha, corresponding to the phenotype for Langerhans-derived DC, which are highly active in Ag processing. Thus, in addition to any role in effector functions, Fc gamma RI on APC may act as a link between innate and adaptive immunities by binding and mediating the uptake of T-independent immune complexes for presentation, thereby assisting in the development of T-dependent immune responses.

Remission and pancreas isograft survival in recent onset diabetic NOD mice after treatment with low-dose anti-CD3 monoclonal antibodies.

Diabetes in NOD mice is an autoimmune disease similar to Type I diabetes in humans. Prior to hypoglycemia, changes in the islet infiltrate led to autoreactive T cell activation and destruction of the insulin-producing beta cells. If T cell activation can be inhibited before beta cell destruction is complete, islet cell rescue and regeneration can occur. Female NOD mice > 100 days old with blood glucose levels > 20 mM/l for less than 7 days were selected as 'recent onset' mice. Untreated, all of these animals would die of diabetes in < 40 days. Mice treated with anti-CD4 (GK1.5) achieved 14.3% permanent remission, while those treated with anti-CD8 (53.6.7) showed 33.3% permanent remission. Mice treated with anti-CD3 (145-2C1) also achieved 33.3% permanent remission, but 14% of these died of first dose syndrome. In mice treated with a low dose of anti-CD3 (10 microg KT3), which did not induce first dose syndrome, 50% remained in remission for > 100 days. This dose of mAb reduced insulitis but did not deplete splenic CD3 cells. When mice in remission were challenged with a vascularized pancreas isograft at 50 days, 9/22 remained normal and 13/22 had recurrent disease in both transplanted and native pancreas. Of the long-surviving isografts 7/9 were in KT3 treated recipients. Histology showed activated T cell infiltration in the native and transplanted pancreases of mice with transient remission. Benign insulitis with macrophages, B cells, CD4 > CD8 T cells and low levels of IL-2R, IL-2, IFN-gamma and IL-4 was seen in islets from the native pancreas and in long surviving pancreas isografts in mice that remained in remission. Thus, using low dose KT3, it was possible to halt the development of diabetes in 50% of animals treated soon after diagnosis, despite significant islet cell destruction at this stage. Of the KT3 treated mice in permanent remission, 70% had re-established tolerance to autoantigen and did not destroy vascularized pancreas isografts.