Rezultaty
In humans, TLR 7 and TLR 8 have been identified as receptors for synthetic compounds belonging to the imidazoquinoline family, which have potent adjuvant activity. We have demonstrated that the presence of TLR 7/8 ligands during myeloid dendritic cell differentiation impairs the function of the differentiated dendritic cells.
This result is important in the context of development of vaccines, which contain compounds activating TLR 7/8. These data extend the 'state of the art' in terms of knowledge of the immunological consequences of TLR 7/8 activation.
Suppressor T cells are a promising tool for therapies of immunologic disorders like allergies or autoimmune diseases. We could identify IL-2 and particularly plasmid DNA vaccination using pIL-2 to strongly activate suppressor function in CD25+ Foxp3 regulatory T cells (Treg).
Following gene-gun vaccination with IL-2 Treg were able to suppress antigen specific T cell proliferation in vivo following vaccination with an antigen-coding plasmid. The suppression depends on TGF-b and IL-10. and can be transferred by regulatory T cells.
This strategy to activate Treg suppressor activity might be helpful for the treatment or prevention of allergies or autoimmune disease.
A new ELISA protocol for detection of Ovalbumin-specific IgE antibody in serum has been developed.
The wells of a polistyrene plate were coated overnight with anti-IgE Ab. Following blocking, various dilutions of the serum were added to the plate.
After 1h incubation with biotinylated Ovalbumin followed by 1h incubation with streptavidin bound to alkaline posphatase, plates were developed using specific substrate.
The absorbance at 405 nm was determined by an ELISA reader.
A mouse model of allergy to Ovalbumin for studying the inflammatory immune response has been established in collaboration with Dr. Sarah Howie (University of Edinburgh).
The protocol we have followed implies 2 intra-peritoneal administrations of alum-precipitated-Ovalbumin at weekly intervals, followed by 3 intra-tracheal (i.t.) injections of soluble Ovalbumin at 4 day intervals and sacrifice of mouse with collection of blood sample/ organs/ BALF at different time points after the last i.t. administration of antigen.
We have generated a DNA vaccine which delivers the antigen directly into the MHC class II compartment. This is achieved by a fusion of the antigen (ovalbumin) with the leader sequence of the invariant chain (Ii80).
This results in effective antigen presentation by transfected cells in vivo and in vitro. Furthermore such plasmids induce strong Th1 differentiation without a detectable B cell /antibiody response.
At the same time the antigens is retained within transfected cells, this prevents activation of the humoral arm of the immune system (B cells or antibody-laden mast cells) and may provide a safer DNA vaccine.
The plasmid construct might be useful to redirect pathogenic Th2 responses without triggering allergic reactions or to selectively increase CD4 T cell responses.
Plasmid pCMVDerp1 is an in-house developed construct containing the Der P1 gene, encoding the dust mite allergen of Dermatophagoides pteridissinus.
In this plasmid, DerP1 is cloned as fusion to a tPA leader sequence, in order to allow Derp1 secretion by transfected cells. The plasmid is able to express the encoded antigen in vitro transiently transfected cells. Furthermore, the plasmid is capable of inducing allergen-specific cellular and humoral responses in non-allergic mice.
The construct is particularly designed for DNA immunization experiments aimed at evaluating the immunogenicity of DNA-based allergy vaccine in murine models of naïve and allergic mice.
The plasmid is suitable for cell transfection assays aimed at studying allergen expression and presentation by antigen presenting cells and cell activation pathways upon DNA uptake. The application can be disseminated as further R&D.
Plasmid pCMVIC80Derp1 is an in-house developed construct containing the Der P1 gene, to be used for cell transfection and in vivo immunization experiments.
In this plasmid, DerP1 is cloned as fusion to the leader sequence of the murine MHC class II invariant chain, in order to target Derp1 expression into MHC class II pathway.
The plasmid is able to express the encoded antigen in vitro transfected cells. This construct is particularly suitable for immunogenicity studies in naïve and allergic murine models.
The plasmid is also suitable for studies aimed at evaluating antigen processing and presentation and APCs activation upon DNA uptake. The application can be disseminated as further R&D.
To test the efficacy of experimental DNA allergen vaccines a reliable model of the response to a 'real' lung allergen in an intact immune system was needed.
The model not only had to be immunologically robust and produce signs of disease it also had to involve the minimal amount of handling and stress.
To this end we used the natural, house dust mite derived, human allergen Der p1 from the faeces of Dermatophagoides pteronyssimus, a major allergen in non-seasonal allergic asthma.
Two sensitising and two challenge doses of allergen were necessary to induce allergic airway disease characterised by lung inflammation, eosinophilia and goblet cell differentiation.
Changes were seen in local immune responses in draining lymph nodes - high secretion of cytokines associated with allergy [IL-5], and/or Th2 responses [IL-10 and IL-13].
Antigen reactive cells were present throughout but effector responses were only induced by lung challenge and were transient. IgE antibody induction required lung challenge and was transient.
IgG1 antibodies were found regardless of challenge, were boosted by challenge and continued increasing after challenge.
Th1 cytokines or antibodies were not detected at any timepoint. The model was set such that it could be modulated to monitor both increased and decreased inflammation.
Unmethylated olygodesoxinucleotides, Imidazoquinolines-derived compounds (Resiquimod and Imiquimod) and modified adenines redirect in vitro the allergen-specific Th2 response by activating APC via TL9, TLR7/8 and TLR7 triggering respectively.
These signals induce the overproduction of endogeneous pro-inflammatory cytokines (namely IL-12 and IL-18). These factors, together with antigen, are able to switch in vitro the profile of polarized effector Th2 cells from allergic patients into IFN-gamma producing T cells. This represents a very easy assay to evaluate the plasticity of Th2 response of a given subject.
It is hypothesized that these compounds can be usefully associated to traditional specific immunotherapy or DNA vaccines for novel strategies of treatment of allergic disorders or Th2-oriented diseases. The in vitro assay as well the new formulations of vaccines can be disseminated as further R&D.
An ELISPOT test for detection and enumeration of Ovalbumin-specific IgE-secreting plasma cells in several organs during an inflammatory response in mouse has been developed.
The wells of a polistyrene plate are coated ON with anti-IgE Ab. Following blocking, various dilutions of the cell suspension prepared from pooled organs were culture on the plate.
After 1 h incubation with biotinylated Ovalbumin and additional 1h with streptavidin bound to alkaline posphatase, plate are developed with specific substrate. Spots were counted using a dissecting microscope.
The partner provided data on functional activity of CD4+CD25+ T cells purified from post-natal human thymuses, the so-called Natural Treg Cells.
Their suppressive activity was contact-dependent and appeared to be related to their ability to down-regulate the IL-2 receptor (IL-2R) - chain expression in target T cells via the combined action of TGF-1 and CTLA-4 expressed on their membrane.
Moreover, both CD4+ Treg cells completely suppressed the proliferation of Th1 clones, but exhibited significantly lower suppressive activity on the proliferation of Th2 clones.
Another mechanism concerns Mesenchymal stem cells (MSC), which are progenitor cells with immunoregulatory properties. The anti-proliferative effect of MSC on T cells and NK cells requires the presence of IFN-gamma produced by activated T cells and NK cells. IFN-gamma acts by enhancing indoleamine 2,3-diooxygenase which induces kinurenines showing anti-proliferative and pro-apoptotic activities.
These findings allow defining some molecules as therapeutical targets to be associated with DNA vaccines in vitro or in vivo models.
Plasmid pCMVOva is an in-house developed construct containing the ovalbumin gene, to be used for cell transfection and in vivo immunization experiments.
In this plasmid, the ovalbumin gene is cloned as fusion to the tPA leader sequence, in order to allow Derp1 secretion by transfected cells. The plasmid is able to express the encoded antigen in vitro-transfected cells.
Furthermore, the plasmid is capable of inducing allergen-specific cellular and humoral responses in non-allergic mice. The construct is particularly designed for DNA immunization experiments aimed at evaluating the immunogenicity of DNA-based allergy vaccine in murine models of naïve and allergic mice.
The plasmid is suitable for cell transfection assays aimed at studying allergen expression and presentation by antigen presenting cells and cell activation pathways upon DNA uptake. The application can be disseminated as further R&D.
A mouse model of allergy to Ovalbumin has been established as described (see result 38144). By use of the Elispot technique (see result 38145), we could detect high numbers of Ovalbumin-specific IgE-secreting plasma cells in bone marrow of allergic mice, as soon as d4-d7 after the last i.t. administration of antigen.
The early presence of antigen-specific IgE- secreting plasma cells in the bone marrow, i.e. a primary lymphoid organ, is a new finding that could be important for the design of new strategy of vaccination against allergic diseases and could be a factor influencing the efficiency of vaccines.
Therapeutic DNA vaccination was used to modulate clinical signs in the model of allergic airway disease induced by the human allergen house dust mite derived Der p1 by use of gene gun delivery after sensitisation and before challenge with allergen.
It was reasoned this would most closely mimic therapeutic application i.e. patients with allergies could be vaccinated to prevent symptoms rather than aiming vaccination at the whole population to prevent allergy. Various vaccine preparations were supplied by partner 4 at Chiron.
The main advantage of using the gene gun is that 80-100 fold less DNA is used compared to subcutaneous injection, thus minimising vaccine reactions. The results showed that disease was prevented by any bacterial plasmid given between sensitisation and challenge.
This induced switching of subsequent immunity to a conventional Th2 [characterised by IgG1 antibody] rather than an allergic Th2 [characterised by lung eosinophilia and serum IgE antibody]. This effect may have been due to non-specific effects of bacterial DNA products [eg cpG motifs] on circulating APCs.
However, this indicates that use of such motifs may represent a possible therapeutic approach to prevent development of allergic symptoms and may obviate the need to produce allergen specific therapeutic vaccines.
Plasmid pCMVDerp1 cyt is an in-house developed construct containing the Der P1 gene, to be used for cell transfection and DNA immunization purposes.
In this plasmid, DerP1 is cloned directly downstream from the CMV promoter, in order to avoid Derp1 secretion and retain Derp1 expression in the cytoplasmic compartment of transfected cells.
The plasmid is able to express the encoded antigen in vitro transiently transfected cells. This construct is particularly suitable for in vitro transfection studies aimed at evaluating Derp1 processing and presentation and to investigate cell activation pathways upon DNA uptake.
The plasmid is also suitable for DNA immunization purposes, to study plasmid immunogenicity in the murine models of naïve and allergic mice. The application can be disseminated as further R&D.
Successful vaccination and development of new effective vaccination strategies and vaccines depends on direct monitoring of the in vivo effects of vaccination.
We have developed tools to directly monitor T cell activation and differentiation in vivo and to monitor the endogenous CD4 T cell response to a vaccine. Naïve T cells can be monitored by adoptive transfer of small numbers of TCR transgenic T cells into congenic recipients.
The total pool of endogenous memory T cell response in a non-manipulated animals can be visualized by ex vivo antigenic restimulation followed by analysis of CD40 ligand (CD154) expression versus functional markers such as cytokines.
These technologies will be helpful to directly assess the effect of a particular vaccine on the antigen-specific T helper cell response and provide a rational for development of vaccination strategies and testing.
The formulation of the pCMVDerP1 and pCMVIC80DerP1 plasmids with cationic particles is aimed at increasing the immunogenicity of the corresponding naked plasmid DNA, to be used in murine models of naïve and allergic mice. The formulations are capable of inducing an allergen-specific cellular response in non-allergic mice.
Plasmid pCMVHuIC80Derp1 is an in-house developed construct containing the Der P1 gene, to be used for cell transfection experiments. In this plasmid, DerP1 is cloned as fusion to the leader sequence of the human MHC class II invariant chain, in order to target Derp1 expression into MHC class II pathway.
The plasmid is able to express the encoded antigen in vitro transiently transfected cells. This construct is particularly suitable for in vitro studies aimed at analysing the antigen processing and presentation by human APCs, and to evaluate cell activation upon DNA uptake. The application can be disseminated as further R&D.
Testing of two antisera produced by Partner 4 (Chiron, Siena) allowed us to validate the specificity of these antisera in human cells.
This result can be used in the overall development of new reagents for the detection of DerP1 protein expression.
CXCL10 (which is the ligand of the variant CXCR3-A) up-regulates the production of IFN-gamma and down-regulates the production of type 2 cytokines, whereas CXCL4 (the ligand of the new isophorm called CXCR3-B) down-regulates the production of IFN-gamma and up-regulates the production of IL-4, IL-5 and IL-13 in antigen or allergen-specific T cells.
CXCL10 and CXCL4 induce two different signalling pathways inside T cells. CXCL10 up-regulates the expression of T-bet and down-regulates GATA-3, whereas CXCL4 inhibites T-bet transcription and associates with increased GATA-3 expression. Finally, CXCL4 is able to induce the direct activation of IL-5 and IL-13 transcription.
These findings demonstrate that CXCL10 and CXCL4 exert opposite effects on the production of human Th1 and Th2 cytokines through their interaction with CXCR3-A or CXCR3-B and the subsequent activation of distinct signal transduction pathways, thus providing evidence for the existence of a novel internal regulatory model of Th1- and Th2-cell polarization.
CXCL10 can be a fruitful candidate as adjuvants of DNA vaccines for allergy.
The expression of the Der P1 protein was validated after nucleofection of the plasmid produced by Partner 4 (Chiron) in cells of the myeloid lineage (K562). Immunoblotting, FACS analysis and confocal microscopy were all positive for expression of DerP1
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