Allergens displayed on virus-like particles are highly immunogenic but fail to activate human mast cells.

BACKGROUND: The goal of allergen-specific immunotherapy is the induction of protective immune responses in the absence of anaphylactic reactions. We have previously shown that Fel d 1, the major cat allergen, displayed in a repetitive fashion on virus-like particles (VLPs) may fulfill these criteria. Specifically, Fel d 1 on VLPs induced strongly increased protective IgG responses compared to free allergen in mice while anaphylactic reactions were essentially abolished. Here we extend these findings to human mast cells and offer a mechanistic explanation for the reduced anaphylactic activity. METHODS: We differentiated human mast cells in vitro from blood-derived stem cell progenitors and sensitized the cells with a monoclonal Fel d 1-specific IgE. We compared the capability of Fel d 1 to induce mast cell activation in its free form versus displayed on VLPs and we performed allergen binding studies by surface plasmon resonance as well as flow cytometry. RESULTS: We show that free Fel d 1 induces degranulation of IgE-sensitized mast cells whereas Fel d 1 displayed on VLPs fails to induce mast cell activation. We demonstrate that this inability to activate mast cells is based on a biophysical as well as a biochemical mechanism. Firstly, Fel d 1 on VLPs showed a strongly impaired ability to bind to surface-bound IgE. Secondly, despite residual binding, repetitively displayed allergen on VLPs failed to cause mast cell activation. CONCLUSION: These findings indicate that repetitively displaying allergens on VLPs increases their immunogenicity while reducing their potential to cause anaphylactic reactions by essentially eliminating IgE-mediated activation of mast cells.

Allergen-specific immunotherapy: is it vaccination against toxins after all?

IgE-mediated allergies, in particular allergic rhinoconjunctivitis and asthma, have reached epidemic proportions, affecting about one-third of the population in developed countries. The most effective treatment for allergies is specific immunotherapy (SIT), which involves the injection of increasing doses of an allergen extract to allergic individuals. The current form of SIT was first introduced in 1911 and recently celebrated its 100th birthday for the treatment of hay fever. The concept of this therapy at the time was straightforward, as it was believed that pollen contained toxins against which the patient could be vaccinated. However, the understanding became blurred with the discovery that IgE antibodies were the effector molecules of the allergic response. Subsequent research focused on the idea that SIT should induce tolerance keeping the IgE antibodies at bay. In this review, we will discuss the various hypotheses for the mechanism of SIT and we will put forward the concept that allergens may be viewed as 'protoxins' which need to be activated by IgE antibodies. Within this framework, protoxin-neutralizing antibodies are the key effector molecules while a shift to Th1 or Treg cells mainly contributes to the efficacy of SIT by helping B cells to produce neutralizing IgG antibodies.

IgG-mediated down-regulation of IgE bound to mast cells: a potential novel mechanism of allergen-specific desensitization.

BACKGROUND: Allergen-specific IgGs are known to inhibit IgE-mediated mast cell degranulation by two mechanisms, allergen-neutralization and engagement of the inhibitory FcγRIIB recruiting the phosphatase SHIP-1. Here we unravel an additional mechanism of IgG-mediated mast cell desensitization in mice: down-regulation of allergen-specific IgE. METHODS: Mast cells were loaded in vitro and in vivo with monoclonal IgE antibodies specific for Fel d1 and exposed to immune complexes consisting of Fel d1-specific IgG antibodies recognizing different epitopes. Down regulation of IgE was followed by flow cytometry. RESULTS: Mast cells loaded with 2 different IgE antibodies efficiently internalized the IgE antibodies if exposed to recombinant Feld d1. In contrast, no down-regulation occurred if mast cells were loaded with IgE antibodies exhibiting a single specificity before stimulation with recombinant Fel d1 [corrected]. Interestingly, however, IgEs of a single specificity were rapidly down-regulated in vitro and in vivo in the presence of Fel d1-specific monoclonal IgGs recognizing another epitope on Fel d1. Despite FceRI-internalization, little calcium flux or mast cell degranulation occurred. FcγRIIB played a dual role in the process since it enhanced IgE internalization and prevented cellular activation as documented by the inhibited calcium flux and mast cell degranulation. Similar observations were made in the presence of low concentrations of IgEs recognizing several epitopes on Fel d1. CONCLUSION: We demonstrate here that Fel d1-specific IgG antibodies interact with FcγRIIB which (i) promotes IgE internalization; and (ii) inhibits mast cell activation. These results broaden our understanding of allergen-specific desensitization and may provide a mechanism for long-term desensitization of mast cells by selective removal of long-lived IgE antibodies on mast cells.

Assessment of clinical efficacy of CYT003-QbG10 in patients with allergic rhinoconjunctivitis: a phase IIb study.

BACKGROUND: Allergic symptoms are generally caused by exposure to substances to which people have become sensitized. Associated with this is an 'unbalanced' Th1/Th2 immune response with T cell responses skewed towards the production of Th2 cytokines, IL-4, 5, and 13 and high levels of IgE antibodies. Current immune modulating therapies require the use of allergens, carrying the risk to induce potentially severe allergic reactions. OBJECTIVE: Goal of the present study was to assess the safety and efficacy of an allergen-free immune modulator in patients suffering from perennial allergy. METHODS: In order to be protected from immediate degradation upon injection, a toll-like receptor 9 (TLR9) agonist was packaged into virus-like particles. These nanoparticles loaded with TLR9 ligands (CYT003-QbG10) were injected six times, at weekly intervals, into patients with house dust mite allergy in an attempt to ameliorate allergic symptoms by modifying the immune response towards allergens. Two different doses were compared against placebo in this double-blind, randomized phase IIb study (n=299). Public trial registry: http://clinicaltrials.gov (NCT00800332). RESULTS: The treatment was safe and generally well tolerated. Rhinoconjunctivitis symptoms were significantly lower in patients treated with high dose of CYT003-QbG10 as compared with placebo (scores 0.31 vs. 0.52, P=0.04) based on a standardized average combined symptom and medication score. Furthermore, patients in the high dose group reported a significantly better quality of life score post-treatment than patients on placebo (scores 0.71 vs. 1.21, P=0.02). The conjunctival provocation test revealed a median 10-fold increase in allergen tolerance in the high dose group while in the placebo group it remained unchanged. CONCLUSION AND CLINICAL RELEVANCE: Treatment with high-dose CYT003-QbG10 improved disease symptoms and reduced medication use in allergic individuals thus providing first evidence for a new potential immunotherapeutic.

Use of A-type CpG oligodeoxynucleotides as an adjuvant in allergen-specific immunotherapy in humans: a phase I/IIa clinical trial.

BACKGROUND: B-type CpG oligodeoxynucleotides (ODN) is currently used in clinical trials because of its prolonged half-life, which is due to its phosphorothioate backbone. A-type CpG ODN is a stronger inducer of IFN but has an unstable phosphodiester backbone that has so far prohibited its clinical use. However, upon association with virus-like particles (VLP) consisting of the bacteriophage Qbeta coat protein, A-type CpG ODN can be stabilized and can become an efficient adjuvant in mice. Therefore, the phase I/IIa study presented represents the first test of A-type CpGs in humans. OBJECTIVE: To test the safety, tolerability and clinical efficacy of QbG10 as an adjuvant for subcutaneous immunotherapy with a house dust mite (HDM) allergen extract in allergic patients. METHODS: A single centre, open-label phase I/IIa study evaluated the safety, tolerability and clinical efficacy of QbG10 as an adjuvant to immunotherapy with a subcutaneous HMD allergen extract in 20 patients suffering from HDM allergy. Twenty-one patients were enrolled between March and July 2005. Individual immunotherapy lasted 10 weeks. Clinical end-points included questionnaires, conjunctival provocation, skin prick tests and the measurement of allergen-specific IgG and IgE. RESULTS: QbG10 was well tolerated. Almost complete tolerance to the allergen was observed in conjunctival provocation testing after treatment with QbG10, and symptoms of rhinitis and allergic asthma were significantly reduced. Within 10 weeks of therapy, patients were nearly symptom-free and this amelioration lasted for at least 38 weeks post-treatment. Following injections of QbG10 and HDM allergen extract, allergen-specific IgG increased, while there was a transient increase in allergen-specific IgE titres. Skin reactivity to HDM was reduced. CONCLUSION: The subcutaneous application of HDM allergen, together with A-type CpG ODN packaged into VLP, was safe. All patients achieved practically complete alleviation of allergy symptoms after 10 weeks of immunotherapy. This promising clinical outcome calls for larger placebo-controlled phase II studies.

Pattern recognition by B cells: the role of antigen repetitiveness versus Toll-like receptors.

Viruses induce excellent antibody responses due to several intrinsic features. Their repetitive, organised structure is optimal for the activation of the B cell receptor (BCR), leading to an increased humoral response and a decreased dependence on T cell help. Viruses also trigger Toll-like receptors (TLRs), which in addition to increasing overall Ig levels, drive the switch to the IgG2a isotype. This isotype is more efficient in viral and bacterial clearance and will activate complement, which in turn lowers the threshold of BCR activation. Exploiting these characteristics in vaccine design may help us to create vaccines which are as safe as a recombinant vaccine yet still as effective as a virus in inducing B cell responses.

Bioprocess applications of a Sindbis virus-based temperature-inducible expression system.

The production and study of toxic proteins requires inducible expression systems with low basal level expression and high inducibility. Here, we describe bioprocess applications of the pCytTS temperature-regulatable Sindbis virus replicon-based expression system. We used green fluorescent protein as a marker protein to optimize the selection of stable transfected clones with increased expression levels. Using the optimized protocol, clones were constructed that produced the growth-inhibiting, anti-viral protein interferon beta (beta-IFN). Selected clones were analyzed for temperature-dependent beta-IFN production in adherent and suspension cultures in serum free medium. Specific expression levels were around 1.0 x 10(5) IU/10(6) cells/day (0.5 microg/10(6) cells/day) in suspension cultures and over 1.5 x 10(6) IU/mL/day (7.5 microg/mL/day) in hollow fiber reactors using adherent cells. Hexahistidine-tagged beta-IFN purified from T-flask cultures was highly glycosylated and showed high specific activity. beta-IFN mRNA amplified by the viral replicase for 10 days did not show an accumulation of mutations. These data suggest the applicability of the pCytTS-inducible expression system for the production of high-quality glycoproteins in different reactors.

A high-throughput alphavirus-based expression cloning system for mammalian cells.

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.

Normal pathogen-specific immune responses mounted by CTLA-4-deficient T cells: a paradigm reconsidered.

CTLA-4 is a critical negative regulator of T cell responses and CTLA-4-deficient (CTLA-4(-/-)) mice die of a lymphproliferative disease. Nevertheless, RAG-2-deficient mice reconstituted with a mixture of CTLA-4(-/-) and normal (CTLA-4(+/+)) bone marrow survive in the absence of any signs of disease, although 50% of their T cells do not express CTLA-4. Using such mixed chimeras, we analyzed the role of CTLA-4 in specific T cell responses to lymphocytic choriomeningitis virus, Leishmania major and mouse mammary tumor virus, which cause acute, chronic and persistent infections, respectively. The populations of antigen-specific CTLA-4(-/-)CD4(+) and CTLA-4(-/-)CD8(+) T cells became activated, expanded and contracted indistinguishably from CTLA-4(+/+)CD4(+) and CTLA-4(+/+)CD8(+) T cells after infection with all three pathogens. Thus, CTLA-4 is not involved in the down-regulation of specific T cell responses and peripheral deletion in a T cell-autonomous fashion.

New applications of alphavirus-based expression vectors.

Alphaviruses are positive stranded RNA viruses that replicate to extremely high titers. Sindbis and Semliki Forest viral vectors are widely used tools for high-level production of recombinant proteins. Recent studies have broadened their scope to vaccine production, gene therapy, and analysis of cell function. Here we discuss the development of non-cytopathic and inducible expression vectors which can be applied to bioprocess development strategies. Furthermore, a Sindbis-based expression cloning system has been developed that allows for the rapid identification of genes encoding proteins with a selected functional activity.

The antigen dose determines T helper subset development by regulation of CD40 ligand.

Although the amount of antigen and the strength of T cell stimulation have been suggested to regulate Th1 vs. Th2 polarization, it remains unclear how the antigen dose and the strength of signal is detected by the T cell and translated into differential cytokine production. Using co-cultures of dendritic cells (DC) and ovalbumin (OVA)-specific CD4+ T cells obtained from RAG-2)(-/-) DO11.10 mice, we show here that high-dose antigen induced Th1 development by up-regulation of CD40 ligand (CD40L), whereas low-dose antigen stimulation failed to induce CD40L and promoted Th2 development. CD40-CD40L interaction was essential for IL-12 production by DC. In the absence, de novo IL-4 production by T cells and autocrine Th2 development was induced. Furthermore, our results demonstrate that LFA-1/ ICAM interaction promotes Th1 differentiation by lowering the antigen dose required for CD40L up-regulation. Thus, we propose that (1) peptide-MHC density and (2) accessory molecules such as LFA-1 determine T helper polarization by regulation of CD40L.

Inducible costimulator protein (ICOS) controls T helper cell subset polarization after virus and parasite infection.

It has been shown that certain pathogens can trigger efficient T cell responses in the absence of CD28, a key costimulatory receptor expressed on resting T cells. Inducible costimulator protein (ICOS) is an inducible costimulator structurally and functionally related to CD28. Here, we show that in the absence of CD28 both T helper cell type 1 (Th1) and Th2 responses were impaired but not abrogated after infection with lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), and the nematode Nippostrongylus brasiliensis. Inhibition of ICOS in CD28-deficient mice further reduced Th1/Th2 polarization. Blocking of ICOS alone had a limited but significant capacity to downregulate Th subset development. In contrast, cytotoxic T lymphocyte (CTL) responses, which are regulated to a minor and major extent by CD28 after LCMV and VSV infection, respectively, remained unaffected by blocking ICOS. Together, our results demonstrate that ICOS regulates both CD28-dependent and CD28-independent CD4(+) subset (Th1 and Th2) responses but not CTL responses in vivo.

A temperature-regulated replicon-based DNA expression system.

We present a temperature-regulated, alphavirus replicon-based DNA expression system. The system is regulated by a viral temperature-sensitive RNA-dependent RNA replicase, creating a temperature-dependent RNA amplification loop. Because of this positive feedback, the system exhibits both low background and high inducibility. We observed 700-fold induction in transiently transfected cells, and over 104-fold induction in stably transfected cells. The high stringency of inducibility allowed the generation of stable cell lines expressing a highly toxic protein upon temperature shift. These data suggest that the present expression system could simplify bioprocess engineering strategies, especially in situations where the cloned protein has detrimental effects on host cell metabolism.

Antiviral B cell memory in the absence of mature follicular dendritic cell networks and classical germinal centers in TNFR1-/- mice.

TNFR1-/- mice have been shown to lack networks of mature follicular dendritic cells (FDCs) and they do not form germinal centers. With nonreplicating Ags, IgG titers were inefficiently induced and not maintained. In this study, the neutralizing Ab response and the establishment of B cell memory in TNFR1-/- mice after infection with vesicular stomatitis virus (VSV) were analyzed histologically and functionally. Immunization with VSV-derived protein Ags without adjuvant induced only IgM but no IgG Abs in TNFR1-/- mice, whereas VSV glycoprotein emulsified in CFA or IFA induced IgM and IgG responses that were short-lived and of moderate titer. However, infection with live VSV induced excellent neutralizing IgM and IgG responses in TNFR1-/- mice, and adoptively transferable B cell memory was generated and persisted for more than 300 days. In contrast, IgG levels and Ab-forming cells in the bone marrow declined within 300 days by 90-95% compared with controls. These findings suggest that 1) increased Ag dose and time of Ag availability can substitute for FDC-stored Ab-complexed Ag in the induction of efficient IgG responses in TNFR1-/- mice devoid of classical germinal centers; 2) the induction and maintenance of adoptively transferable B cell memory can occur in the absence of Ag bound to mature FDCs; and 3) the long-term maintenance of elevated IgG titers is largely dependent on FDC-associated persisting Ag. However, about 5-10% of the Ab production remained in the absence of detectable persisting Ag in TNFR1-/- mice, probably either due to immature FDCs being partially functional and/or due to long-lived plasma cells.