Biomedical subjects

Josef Thalhamer

Publications and source records attributed to Josef Thalhamer.

27 records · Page 2Linked to original sources

PubMed2003

Prevention of allergen-specific IgE production and suppression of an established Th2-type response by immunization with DNA encoding hypoallergenic allergen derivatives of Bet v 1, the major birch-pollen allergen.

In atopic patients, programming towards a preferential Th2 immunity leads to IgE antibody production and cellular Th2 immunity against otherwise harmless antigens. We report the development of prophylactic and therapeutic DNA vaccines for the major birch-pollen allergen, Bet v 1. We constructed three DNA vaccines, coding for the complete cDNA, coding for two hypoallergenic fragments or coding for a hypoallergenic Bet v 1 mutant. The protective effect was studied in mice pretreated by intradermal DNA injections, then sensitized with Bet v 1 protein. Mice pretreated with any of the three Bet v 1-specific DNA vaccines were protected against allergic sensitization to Bet v 1. Protection was characterized by a lack of Bet v 1-specific IgE production, a lack of basophil activation and an enhanced IFN-gamma expression. DNA vaccines with wild-type Bet v 1 induced strong Bet v 1-specific antibody responses whereas DNA vaccines with hypoallergenic Bet v 1 derivatives induced no (fragments) or only transient (mutant) Bet v 1-specific antibody responses. A therapeutic approach with the fragment-DNA vaccine reduced IgE production and stimulated a sustained Th1 cytokine milieu. Our results demonstrate that DNA vaccines with hypoallergenic forms of the allergen specifically protect against sensitization and suppress established Th2-type responses. This concept may be applied for the development of safe and specific DNA vaccines for the prophylaxis and therapy of allergic diseases.

Allergens↗

PubMed2003

TH1-promoting DNA immunization against allergens modulates the ratio of IgG1/IgG2a but does not affect the anaphylactic potential of IgG1 antibodies: no evidence for the synthesis of nonanaphylactic IgG1.

BACKGROUND: In mouse, IgG1 has been reported to make up 2 functionally distinct phenotypes that also differ in their induction requirements. One of these phenotypes lacks anaphylactic activity. OBJECTIVE: We hypothesized that nonanaphylactic IgG1 could modulate allergic reactions and investigated whether such antibodies are induced by DNA immunization. METHODS: Mice were immunized with allergen-encoding plasmid DNA or with recombinant allergens and alum. Sera were analyzed for IgG subclasses by ELISA for anaphylactic IgE by rat basophil degranulation, and after heat inactivation of IgE for anaphylactic IgG by passive cutaneous anaphylaxis assay. IFN-gamma and IL-5 from in-vitro restimulated spleen cells were quantitated by ELISA. RESULTS: After protein immunization, mice produced IgG1 and IgE, whereas DNA immunization elicited IgG1 and IgG2a but no IgE. However, all sera were positive for non-IgE-mediated passive cutaneous anaphylaxis. In the presence of anaphylactic IgG1, the additional occurrence of nonanaphylactic IgG1 cannot be strictly ruled out. To circumvent this problem, we immunized IL-4 receptor-deficient mice against Bet v 1a, because anaphylactic but not nonanaphylactic IgG1 has been reported to depend on IL-4. These animals produced only low amounts of IgG1, but sera were again positive for non-IgE-mediated anaphylactic activity. CONCLUSIONS: Our results revealed no evidence for the production of nonanaphylactic IgG1. Furthermore, our data indicate that the development of non-IgE-mediated anaphylaxis does not require IL-4 receptor signaling.

Allergens↗

PubMed2003

A DNA vaccine encoding the outer surface protein C from Borrelia burgdorferi is able to induce protective immune responses.

The outer surface protein C (OspC) of Borrelia burgdorferi, the spirochete that causes Lyme disease, is a promising candidate for a vaccine against borreliosis. BALB/c and C3H/HeJ mice were immunized either with recombinant OspC protein or with plasmid DNA encoding OspC fused to the human tissue plasminogen activator leader sequence (pCMV-TPA/ZS7). The influence of the route of administering the DNA and the use of oligodeoxynucleotides containing CpG-motifs on the development of the immune response was investigated. In both mouse strains, protein as well as gene-gun immunization induced Th2 type responses, whereas needle injection of plasmid DNA resulted in Th1 type antibody production. Co-injection of CpG-motifs did not significantly modify the response type in any immunization group, as indicated by only marginal changes of antibody subclass distribution. The protection rate after challenge with 10(4) B. burgdorferi organisms per mouse was between 80% and 100% for all groups. These results demonstrate, for the first time, that a DNA vaccine encoding OspC of B. burgdorferi is suitable for inducing protection against Lyme borreliosis.

Animals↗

PubMed2003

DNA vaccines for non-infectious diseases: new treatments for tumour and allergy.

The last decade of DNA vaccine research was characterised by a pioneer spirit and enormous enthusiasm, with a large number of publications demonstrating the usefulness of this approach. Unfortunately, DNA vaccines have not necessarily met the high clinical expectations and a number of complications need to be overcome. In the case of cancer and allergy, the requirements for achieving the objectives are very different. Vaccines against allergies need to suppress or alter an unwanted immune response, while a cancer DNA vaccine has to overcome tolerance and/or immune suppression and initiate a powerful immune response. This review addresses currently used general optimisation strategies for DNA vaccines such as modification of immunisation regimens, improving the delivery systems and using molecular adjuvants. In addition, cancer-specific approaches, such as the stimulation of innate and adaptive immunity with replicase-based DNA vaccines, and targeting non-tumour-associated antigens (TAAs) are discussed. Specifically for the optimisation of DNA vaccination against allergies, procedures such as allergen gene recoding, T helper (Th)1 modulation, and the creation of safe DNA vaccines by gene fragmentation, ubiquitination or using artificial hypoallergens are being analysed. These strategies, individually or in combination, hold the potential of making DNA vaccines useful for application in the clinic.

Adjuvants, Immunologic↗

PubMed2003

Strategies for the development of safe and effective DNA vaccines for allergy treatment.

During the past ten 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. The features of DNA vaccines enable a broad range of applications, including the induction of protective immunity against viral, bacterial, and parasitic infections, and open up new perspectives for the treatment of cancer. Furthermore, based on their Th1-promoting properties, DNA vaccines also turned out to balance Th2-mediated immune reactions, a quality which renders them a promising alternative for immunotherapy against allergy. Their unique immunological properties offer new possibilities for the development of vaccines, which do not cause anaphylactic side effects, a major drawback of specific immunotherapy (SIT). In this review, we present approaches to avoid the translation of native allergenic determinants, thus preventing release of allergy mediators stimulated by crosslinking of pre-existing or vaccine-induced IgE antibodies on mast cells. Three approaches are described, which fulfill these requirements: (i) cutting the allergen gene into overlapping fragments, which lack any antigenic determinant of the native allergen, but display the original repertoire of T cell epitopes, (ii) using hypoallergenic derivatives or (iii) fusing the allergen with ubiquitin, thus fragmenting the antigen and destroying its native structure. The presented experiments demonstrate that DNA vaccines are suitable to balance an allergic response in a protective as well as a therapeutic experimental design, thus demonstrating their potential for allergy treatment. In addition to conventional plasmid DNA vaccines, aspects and perspectives of replicon-based DNA vaccines will be discussed.

Allergens↗

PubMed2002

Quantification of histamine in blood plasma and cell culture supernatants: a validated one-step gas chromatography-mass spectrometry method.

A novel one-step ethylchloroformate (ECF) derivatization of histamine in biological liquid matrices that allows the sensitive quantification by gas chromatography and mass spectroscopic detection (GC-MS) from small volumes of blood plasma or cell culture supernatants within 15 min is described. After addition of ECF/chloroform directly to the crude sample, histamine has been found to be quantitatively derivatized within seconds. Following centrifugation, the organic phase is transferred to a fresh vial, dried by addition of anhydrous sodium sulfate, and subjected to GC-MS analysis. The reliability of the results is verified by use of two different ion pairs for detection. The method is validated according to DIN 38402. Linearity is given from 0.0054 to 13 microg/ml and the limit of detection is 2 ng/ml (10 pg absolute, at a signal to noise ratio of 3:1). The limit of quantification, as calculated at a confidence level of 95%, is 15.6 ng/ml. Practical application is exemplified by the determination of the histamine content in blood plasma of birch pollen-sensitized mice and in the culture supernatant of rat basophil leukemia cells after Ca(2+) ionophore-mediated degranulation.

Allergens↗

PubMed2002

Gene gun bombardment with gold particles displays a particular Th2-promoting signal that over-rules the Th1-inducing effect of immunostimulatory CpG motifs in DNA vaccines.

The mode of administering a DNA vaccine can influence the type of immune response induced by the vaccine. For instance, application of a DNA vaccine by gene gun typically induces a Th2-type reaction, whereas needle inoculation triggers a Th1 response. It has been proposed that the approximately 100-fold difference in the amount of DNA administered by these two methods is the critical factor determining whether a Th1 or a Th2 response is made. To test this hypothesis, BALB/c mice were immunized with two plasmid DNA constructs encoding different proteins (OspC/ZS7 of Borrelia burgdorferi and Bet v 1a, the major birch pollen allergen). Both vaccines were applied by needle and/or by gene gun immunization at the same and at different sites of injection. An analysis of the IgG subclass distribution and measurement of IFN-gamma after antigen-specific lymphoproliferation does not support the widely accepted view that Th2-type immunity induced by gene gun application is solely due to the low amount of injected plasmid DNA thus falling below the critical concentration of CpG motifs necessary for Th1-induction. Furthermore, the data also indicate a strong and even systemic adjuvant effect of the gene gun shot itself.

Adjuvants, Immunologic↗

PubMed2002

Genetic engineering of allergens: future therapeutic products.

Genetic engineering of allergens for specific immunotherapy should aim at the production of modified molecules with reduced IgE-binding epitopes (hypoallergens), while preserving structural motifs necessary for T cell recognition (T cell epitopes) and for induction of IgG antibodies reactive with the natural allergen (blocking antibodies). Common approaches for engineering of hypoallergens usually require knowledge of T and B cell epitopes and involve changing specific base pairs (mutated gene), introduction of a new piece of DNA into the existing DNA molecule (chimeric or hybrid gene), and deletions (truncated gene or fragments). DNA family shuffling has the advantage that it does not require a priori knowledge of structural and functional properties for efficient generation of hypoallergens. The combination of the hypoallergen concept with the Th1-inducing genetic immunization approach might be an attractive alternative for protein-based immunotherapy.

Allergens↗

PubMed2002

Molecular and immunological characterization of profilin from mugwort pollen.

In late summer in Europe, pollen of mugwort is one of the major sources of atopic allergens. No information about the complete molecular structure of any mugwort allergen has been published so far. Here we report the isolation and characterization of mugwort pollen cDNA clones coding for two isoforms of the panallergen profilin. Thirty-six percent of the mugwort-allergic patients tested displayed IgE antibodies against natural and recombinant profilin, and no significant differences were observed in the IgE-binding properties of the isoforms. One profilin isoform was purified to homogeneity and detailed structural analysis indicated that the protein exists in solution as dimers and tetramers stabilized by sulfydryl and/or ionic interactions. Profilin monomers were detectable only after exposure of multimers to harsh denaturing conditions. Dimers and tetramers did not significantly differ in their ability to bind serum IgE from mugwort pollen-allergic patients. However, oligomeric forms might have a higher allergenic potential than monomers because larger molecules would have additional epitopes for IgE-mediated histamine release. Profilin isolated from mugwort pollen also formed multimers. Thus, oligomerization is not an artifact resulting from the recombinant production of the allergen. Inhibition experiments showed extensive IgE cross-reactivity of recombinant mugwort profilin and profilin from various pollen and food extracts.

Allergens↗