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Josef Thalhamer

Publications and source records attributed to Josef Thalhamer.

At least 19 recordsLinked to original sources
PubMed2006

Gene gun immunization with clinically relevant allergens aggravates allergen induced pathology and is contraindicated for allergen immunotherapy.

Gene gun immunization has been associated with the induction of a heterologous type of immune response characterized by a T(H)1-like immune reaction on the cellular level, i.e. generation of IFN-gamma secreting CD8(+) T-cells, yet a T(H)2 biased serology as indicated by high IgG1:IgG2a ratios and induction of IgE. Nevertheless, gene gun immunization using the model molecule beta-galactosidase has been argued to prevent IgE induction and to promote T(H)1 cells with respect to allergy DNA immunization. In our current study, we evaluated the potential of gene gun immunization to prevent type I allergic reactions comparing beta-galactosidase with two clinically relevant allergens, and further investigated the effect of gene gun immunization on relevant lung parameters. BALB/c mice were immunized with plasmids encoding the birch pollen allergen Bet v 1, the grass pollen allergen Phl p 5, or the model molecule beta-galactosidase, either by gene gun or intradermal injection followed by sensitization and intranasal provocation with the respective allergen. IgG1 and IgG2a antibody titers were determined by ELISA. IgE levels were evaluated in a rat basophil release assay. The severity of eosinophilia was determined in bronchoalveolar lavages, and the overall infiltrate was analyzed by histology on lung paraffin sections. Gene gun immunization induced a T(H)2-biased immune reaction, which did not prevent from production of IgE after subsequent sensitization. This T(H)2 effect was influenced by the nature of the antigen, with a more pronounced T(H)2-bias for the allergens Bet v 1 and Phl p 5 compared to beta-galactosidase. Gene gun immunization with all three antigens promoted eosinophil influx into the lung and did not alleviate lung pathology after intranasal provocation. In contrast to needle injection of plasmid DNA, which triggers a clearly T(H)1-biased and allergy-preventing immune response, gene gun application fails to induce anti-allergic reactions with all tested antigens and is therefore contraindicated for allergen-specific immunotherapy.

Allergens↗
PubMed2006

Epidermal inoculation of Leishmania-antigen by gold bombardment results in a chronic form of leishmaniasis.

Experimental leishmaniasis represents a suitable model to analyze Th1-type associated immunity. In C57BL/6 mice healing of leishmaniasis correlates with activation of Th1 cells. Recently, it could be demonstrated that dermal dendritic cells rather than epidermal Langerhans cells are responsible for the activation of Th1 cells after infection, indicating a necessary reconsideration of the role of Langerhans cells. In our current work, epidermal application of Leishmania-antigen prior to infection resulted in an atypical course of disease that is characterized by an impaired Leishmania-specific Th1 response. Consequently, these mice cannot manage an efficient elimination of the parasites at the site of infection. These data point to the activation of immunomodulatory effects by epidermal incorporation of antigen.

Animals↗
PubMed2006

Immunization with a low-dose replicon DNA vaccine encoding Phl p 5 effectively prevents allergic sensitization.

BACKGROUND: Replicase-based DNA vaccines stimulate T(H)1-biased immune responses at ultralow doses and induce self-removal of transfected cells through apoptosis. Both aspects are important requirements for efficient and safe DNA-based immunotherapy of type I allergies. OBJECTIVE: A Sindbis virus replicon-based DNA vaccine encoding the major timothy grass pollen allergen Phl p 5 was evaluated for its antiallergic potential compared with a conventional DNA vaccine in a BALB/c mouse model of allergy. METHODS: Mice were intradermally prevaccinated with plasmid DNA, followed by sensitization and intranasal allergen provocation with recombinant Phl p 5. In vitro proliferation and cytokine secretion was measured in splenocyte cultures. Distribution of IgG1, IgG2a, and IgE antibody subclasses was determined by means of ELISA. IgE-mediated degranulation was measured with the basophil release assay. Bronchoalveolar lavage fluid was analyzed for eosinophils, IL-4, IL-5, IL-13, and IFN-gamma. Mucus production, inflammatory infiltrates, and epithelial damage were determined in lung sections. RESULTS: Both vaccines induced T(H)1-biased immune responses, resulting in suppression of functional IgE, reduction of eosinophilia in bronchoalveolar lavage fluid, and alleviation of lung pathology. However, immunization with the replicon DNA vaccine elicited these effects at a 100-fold lower dose compared with the conventional DNA vaccine. CONCLUSIONS: The increased immunogenicity of replicon-based DNA vaccines allows for application of extremely low doses, thereby eliminating the concerns associated with conventional DNA vaccines, which have to be administered at milligram amounts to induce immune reactions in human subjects. CLINICAL IMPLICATIONS: Their high safety profile makes replicon-based DNA vaccines promising candidates for treatment of type I allergies in the clinic.

Animals↗
PubMed2006

Generation of hypoallergenic DNA vaccines by forced ubiquitination: preventive and therapeutic effects in a mouse model of allergy.

BACKGROUND: Hypoallergenic immunotherapy of type I allergies aims at inducing T-cell immunity while avoiding cross-linking of pre-existing IgE. DNA-based immunotherapy depends on the recruitment of antigen-specific T(H)1 cells and therefore has to provide the whole repertoire of T-cell epitopes. Ubiquitination offers a general approach for the production of hypoallergenic DNA vaccines. OBJECTIVE: A DNA-based vaccine encoding the major birch pollen allergen Bet v 1 stably linked to ubiquitin was evaluated for its antiallergic potential in a BALB/c mouse model of allergy. METHODS: Plasmid DNA was applied to mice before (preventive) or after (therapeutic) sensitization with recombinant Bet v 1. In the preventive setting, mice were exposed to aerosolized allergen in addition. Cytokine production was monitored via ELISPOT and Luminex. IgG(1), IgG(2a), and IgE subclass antibody titers were determined by ELISA. In vitro antigen-specific cross-linking of IgE was measured in a degranulation assay. Bronchoalveolar lavages were analyzed for leukocyte subsets as well as for IFN-gamma and IL-5, and paraffin sections of lungs were examined for mucus production and endothelial damage. RESULTS: Prevaccination with ubiquitinated Bet v 1-stimulated T(H)1-biased immune responses with concomitant suppression of functional IgE, reduction of eosinophil counts in bronchoalveolar lavages, and alleviation of lung pathology, and could also suppress an ongoing IgE response in a therapeutic setting. CONCLUSION: The data clearly demonstrate that hypoallergenic DNA vaccines encoding ubiquitin fusion constructs induce effective antiallergic immune responses. CLINICAL IMPLICATIONS: Ubiquitination of allergen gene vaccines eliminates the risk of IgE cross-linking, thereby meeting the safety requirements for clinical applications.

Allergens↗
PubMed2006

Is genetic vaccination against allergy possible?

Genetic immunization has proven a powerful method to induce antiallergic immune responses. The underlying functional principle has been described to be based on the recruitment of allergen-specific Th1 cells, CD8+ cells and the establishment of a Th1 cytokine milieu, which prevent the development of a Th2-biased response in a protective setup and can balance an ongoing Th2-type response in a therapeutic situation. Genetic immunization with plasmid DNA offers innovative solutions to the major problems associated with protein immunization, such as crosslinking of pre-existing immunoglobulin E on mast cells/basophils or induction of de novo synthesis of immunoglobulin E by the protein immunization itself. It easily enables the routine production of hypoallergenic vaccines, which do not translate native allergens, thus avoiding potential anaphylactic side effects. DNA vaccines can also be applied as mixtures of single vaccines, making them interesting candidates for treatment based on component-resolved diagnosis, followed by an individualized therapy with the relevant allergens. In addition to the description of up-to-date allergen gene vaccine approaches, this review gives an overview of animal studies dealing with the following topics: danger signals as the inherent adjuvant properties, methods to optimize the vaccine immunogenicity, modulation of the immune response, nonparenteral applications and low-dose vaccination strategies.

Animals↗
PubMed2006

Bicistronic expression plasmid encoding allergen and anti-IgE single chain variable fragment antibody as a novel DNA vaccine for allergy therapy and prevention.

Several approaches have been applied in order to alleviate the difficulties allergic patients are suffering from. Among them DNA vaccination and anti-IgE antibody have shown promising results. Herewith, a combination of both strategies is proposed to minimize IgE production while inducing high levels of blocking IgG and strong Th1 immune responses. A bicistronic expression plasmid including an internal ribosomal entry site (IRES) can express both, allergen and a single chain variable fragment (scFv) antibody against human IgE within antigen presenting cells (APCs) including B cells. Presentation of allergen derived peptides via MHC I and MHC II stimulates specific Th1 responses resulting in high levels of IFN-gamma and IgG. Anti-IgE scFv antibody binds to newly synthesized IgE molecules within B cell cytoplasm and also to free serum IgE, thereby inhibiting attachment of IgE to its receptors on basophils and mast cells. Also, IgE-anti-IgE complex functions as blocking antibody and neutralizes allergens entering the body. Additionally, anti-IgE scFv antibody binds to membrane bound IgE (mIgE) on B cells and interferes with IgE expression. Using assays, such as enzyme linked immunosorbent assay (ELISA), IgG and IgE production in response to this expression system can be evaluated. Also, rat basophil leukemia cell assay (using RBL-2H3 cells) can show the amount of functional IgE in sera as basophil mediator release is regarded as an indicator of the allergic hypersensitive reactions. The proposed approach may result in high levels of blocking IgG and low levels of IgE secretion from B cells. Additionally, it can inhibit activity of IgE in degranulation of basophils and mast cells.

Animals↗
PubMed2006

Genetic vaccination approaches against malaria based on the circumsporozoite protein.

Malaria is the world's major parasitic disease, for which effective control measures are urgently needed. Despite considerable efforts, no successful vaccine against malaria has been developed so far. The method of DNA-based immunization offers the possibility to induce both antibody- and cell-mediated immune responses to a variety of antigens. The flexibility of the DNA vaccine technology permits the combination of several antigens from different developmental stages of the parasite's complicated life cycle. This review covers the development of DNA-based immunization against malaria from initial experiments in small animals to recently conducted clinical studies. Focusing on one of the best characterized malaria vaccine candidate antigens, the circumsporozoite protein, an overview of strategies to enhance vaccine efficacy is provided. Advanced application methods such as the gene gun technology or the needle-less jet injection device are described. As DNA vaccination represents a relatively new methodology, safety concerns associated with planned clinical applications are discussed. In summary, this novel type of vaccine has to be considered as a promising tool for future malaria vaccination strategies.

Animals↗
PubMed2006

Replicase-based DNA vaccines for allergy treatment.

Replicase-based vaccines were introduced to overcome some of the deficiencies of conventional DNA- and RNA-based vaccines, including poor efficiency and low stability. At ultra-low doses, these alphavirus-derived vectors elicit cellular as well as humoral immune responses. Additionally, replicase-based vectors induce "self-removal" of the vaccine via apoptosis of transfected cells. This chapter describes the construction of a replicon-based DNA vaccine vector from commercially available plasmids. We present protocols for monitoring cellular immune responses following replicase-based immunization including measurement of allergen-specific proliferation of splenocytes, ELISPOT, a FACS-based cytokine secretion assay providing information about T-helper subsets, and a cytokine fluorescent bead immunoassay.

Animals↗
PubMed2006

DNA vaccines for allergy treatment.

The ability of DNA vaccines to stimulate Th1 type reactions has rendered them a promising tool for immunotherapy of type I allergy. In this chapter, we describe strategies for up-to-date anti-allergic DNA-based immunization. This includes codon optimization of allergen genes, CpG-enrichment of plasmid vectors for enhanced Th1-bias, and the creation of hypoallergenic DNA vaccines either by gene fragmentation or by forced ubiquitination, both reducing the risk of side effects. Also, detailed protocols for plasmid DNA purification, intradermal immunization, and subcutaneous allergen sensitization are provided. Read-out systems presented in this chapter are focused on humoral immune responses and comprise measurement of mediator release from basophils induced by functional IgE and an ELIS A protocol based on chemiluminescence technology.

Allergens↗
PubMed2006

Modified recombinant allergens for safer immunotherapy.

Molecular cloning and recombinant production of allergens offered new perspectives for the increasing problem of allergies. A variety of preparations are being developed aiming to increase safety and improve efficacy of specific immunotherapy. Recombinant-based approaches are mostly focused on genetic modification of allergens to produce molecules with reduced allergenic activity and conserved antigenicity, i.e. hypoallergens. Studies dealing with genetic modifications of allergen genes reported the production of site-directed mutants, deletion mutants, allergen fragments and oligomers, and allergen chimeras. An alternative to genetic engineering is the chemical modification of pure recombinant allergens. It has been shown that allergens modified with immunostimulatory DNA sequences (allergen-ISS conjugates), which masks IgE epitopes and adds a desirable Th1-inducing character to the allergen molecule. Other chemical modifications include oligomerization by aldehydes (allergoids) and maleylation, which seems to target allergens to particular antigen presenting cells. Several of these modified allergen preparations have been already evaluated for their safety in clinical provocation studies. So far, clinical trials showed the efficacy and safety of immunotherapy with an Amb a 1-ISS conjugate for ragweed pollen-allergic patients. In addition, a preparation consisting of hypoallergenic fragments of Bet v 1 was evaluated for immunotherapy of birch pollen-allergic patients. In parallel, several animal studies have now demonstrated the potential of genetic immunization for allergy treatment in the future.

Allergens↗
PubMed2005

C3d binding to the circumsporozoite protein carboxy-terminus deviates immunity against malaria.

The immunogenicity of recombinant protein or anti-viral DNA vaccines can be significantly improved by the addition of tandem copies of the complement fragment C3d. We sought to determine if the efficacy of a circumsporozoite protein (CSP)-based DNA vaccine delivered to mouse skin by gene gun was improved by using this strategy. Instead, we found that C3d suppressed the protective immunity against Plasmodium berghei malaria infection and deviated immunity, most notably by suppressing the induction of antibodies specific for the CSP C-terminal flanking sequence and by suppressing the induction of CSP-specific IL-4-producing spleen cells. We further showed that C3d bound to the C-terminal flanking sequence of the CSP, which may explain the immune deviation observed in CS/C3d chimeric antigen. We have thus identified C3d-mediated epitope masking and shifting of both the humoral and cellular immune responses as a potential novel escape mechanism, which plasmodia may use to divert the induction of protective immunity.

Animals↗
PubMed2005

A hybrid molecule resembling the epitope spectrum of grass pollen for allergy vaccination.

BACKGROUND: Allergy vaccines based on natural allergen extracts contain greatly varying amounts of individual allergens with different immunogenicity. OBJECTIVE: To develop a novel type of allergy vaccine for complex allergen sources that combines defined amounts of the major allergens in the form of single hybrid molecules. METHODS: A hybrid molecule was engineered by PCR-based mending and expression of the cDNAs coding for the 4 major grass pollen allergens and compared with its single components by circular dichroism analysis, T-cell proliferation, ELISA competition, and histamine release assays. Immune responses to the hybrid molecule were studied in BALB/c mice and rat basophil leukemia assays. RESULTS: The hybrid contained most of the B-cell epitopes of grass pollen and could be used to diagnose allergy in 98% (n = 652) of patients allergic to grass pollen. Immunization of mice and rabbits with the hybrid induced stronger and earlier IgG antibody responses than equimolar mixtures of the components, which can be explained by the induction of stronger T-cell responses by the hybrid versus the individual components. IgG antibodies induced by vaccination with the hybrid blocked immediate allergic reactions, as demonstrated by rat basophil degranulation assays in a murine model of grass pollen allergy. CONCLUSION: We demonstrate for grass pollen allergy that recombinant hybrid molecules covering the spectrum of the disease-eliciting epitopes of complex allergen sources can be engineered.

Allergens↗
PubMed2005

Design of protective and therapeutic DNA vaccines for the treatment of allergic diseases.

The DNA vaccine revolution has opened a vast scope of novel approaches for protective and therapeutic treatments of type I allergy. This review gives an overview on the current status of allergy DNA vaccines and presents advances in the design of vaccine constructs. An immense number of concurring studies have proven the stimulation of Th1 cells and the induction of a balanced Th1/Th2 cytokine milieu as the fundamental mechanisms underlying the anti-allergic effects of DNA vaccines. Basic vaccine formulations thus can be optimized by improving the cellular immunogenicity via co-administration of cytokines, co-expression or co-application of immunostimulatory DNA sequences or adapting the codon usage. The latter is a frequent and major reason for impaired vaccine expression (e.g. translation of plant allergen genes in mammal cells). Because of unwanted side effects during conventional specific immunotherapy with allergen extracts, safety is increasingly demanded for both, protein and DNA vaccines for allergy treatment. We discuss the creation of hypoallergenic DNA vaccines based on deliberate allergen gene fragmentation, the use of mutations and the routine production of hypoallergenic DNA vaccines by forced ubiquitination. Furthermore, allergen-expressing DNA replicon vaccines are introduced, which enable a drastic reduction of the vaccine dose without loss of anti-allergic efficacy. Finally, the development of DNA multi vaccines and fusion vaccines for protective and therapeutic applications against certain groups of allergens is addressed.

Allergens↗
PubMed2004

Generation of an allergy vaccine by disruption of the three-dimensional structure of the cross-reactive calcium-binding allergen, Phl p 7.

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients' IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients' IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen's three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.

Allergens↗
PubMed2004

DNA vaccines for allergy treatment.

In the past 10 years, a great number of studies have demonstrated that injection of plasmid DNA coding for certain genes results in the induction of humoral and cellular immune responses against the respective gene product. This vaccination approach covers a broad range of possible applications, including the induction of protective immunity against viral, bacterial, and parasitic infections, and it opens new perspectives for treatment of cancer. Surprisingly, DNA immunization also turned out as a promising novel type of immunotherapy against allergy. In this paper, we describe the construction of DNA vaccines for application in allergy models. Beyond, we offer a palette of recently developed modulations to optimize DNA vaccines for allergy treatment by increasing their immunogenicity and minimizing their anaphylactic potential.

Allergens↗
PubMed2003

Mucosal co-application of lactic acid bacteria and allergen induces counter-regulatory immune responses in a murine model of birch pollen allergy.

Recent epidemiological studies and clinical trials suggest a possible role of certain lactic acid bacteria (LAB) strains in the prevention of allergic diseases. In this study, we aimed at evaluating the immunomodulatory potential of two LAB strains, Lactococcus lactis and Lactobacillus plantarum, for prophylaxis and therapy of allergic immune responses. Both LAB strains-induced high levels of IL-12 and IFN-gamma in naive murine spleen cell cultures. Intranasal co-application with recombinant Bet v 1, the major birch pollen allergen, prior or after allergic sensitization, led to increased levels of allergen-specific IgG2a antibodies and in vitro IFN-gamma production, indicating a shift towards Th1 responses. Successful immunomodulation by the mucosal pre-treatment was further demonstrated by suppression of allergen-induced basophil degranulation. We conclude that these LAB strains in combination with an allergen could be promising candidates for mucosal vaccination against type I allergy.

Adjuvants, Immunologic↗
PubMed2003

Induction of murine ras oncogene peptide-specific T cell responses by immunization with plasmid DNA-based minigene vectors.

In a BALB/c mouse model, we have previously identified a ras oncogene peptide that contained both CD8(+) and CD4(+) T cell epitopes in a nested configuration. In this study, we developed several plasmid DNA minigene vectors encoding these determinants and examined whether they could induce antigen (Ag)-specific CD8(+) cytotoxic and CD4(+) lymphoproliferative responses. Furthermore, we compared two different immunization procedures, epidermal gene gun inoculation and intradermal (i.d.) injection of saline plasmid DNA, along with several approaches addressing different aspects of immune modulation. We demonstrated that each DNA plasmid induced the relevant Ag-specific cellular immune response. Gene gun inoculation was superior to that of needle injection for induction of the CTL response. Moreover, DNA plasmids containing both ras epitopes induced the highest CTL response, as compared with vector preparations containing only the CD8(+) epitope. These results suggested that a DNA plasmid expressing nested mutant ras epitope-specific CD4(+) and CD8(+) T cell epitopes can be processed in vivo to induce both subset-specific T cell responses, and that the addition of the helper epitope quantitatively improved the development of the CTL response, which may have implications for DNA-based anti-tumor immunotherapies.

Adjuvants, Immunologic↗