Kamin et al. beschreiben den Fall einer anaphylaktischen Reaktion als Nebenwirkung auf einen gelatinehaltigen FSME-Impfstoff. Anaphylaktische Reaktionen nach Impfungen sind sehr selten [1], aber potenziell lebensbedrohlich bis zum tödlichen Verlauf im anaphylaktischen Schock. Eine entsprechende Diagnose hat u. a. für die Frage von Folgeimpfungen mit dem gleichen Impfstoff Relevanz. Der Fall wurde noch im gleichen Jahr, 1996, dem pharmazeutischen Hersteller des Impfstoffes zur Kenntnis gebracht. Nach Markteinführung von Medikamenten sind Berichte zu Verdachtsfällen von Nebenwirkungen das wesentliche Instrument, das Profil des Produktes weiter zu beobachten und gegebenenfalls optimieren zu können.

The Mycobacterium bovis BCG vaccine for commercial use is classically produced as surface pellicles by culture on synthetic medium. Under these conditions, reproducibility of the cultures and quality assessment are hampered by slow growth of the bacilli, the formation of bacterial aggregates and a high proportion of dead bacilli after processing and final formulation of the vaccine. Here, we established dispersed cultures of M. bovis BCG in synthetic media in small-scale bioreactors. These cultures allow recording and adjusting of culture parameters and give rise to single bacilli with a high degree of live bacteria. In the murine model, bioreactor-grown M. bovis BCG exhibited slightly stronger replication and persistence than the vaccine produced under the classical conditions. The protective efficacy against challenge with M. tuberculosis was identical for both vaccine preparations.

The heat-stable and soluble glycoprotein gp200 (molecular weight 200 kDa) is part of the cell wall of S. cerevisiae. Recently, an association was shown between IgA and IgG against gp200 and inflammation in Crohn's disease. Gp200 is able to induce a proliferation of human lymphocytes in vitro, together with a natural killer cell associated cytotoxicity. Specific IgE against Saccharomyces cerevisiae (baker's or brewer's yeast) may be detected in approximately 73%, against Candida albicans in 68% of those patients suffering from severe atopic dermatitis. The aim of this study was to elucidate the possible role of an anti-gp200 immune response for the pathogenesis of atopic dermatitis by immunoblot analysis.Anti-gp200 IgE was found in 55% of healthy individuals, in 67% of individuals with atopic predisposition without eczema, in 63% of the patients with mild atopic dermatitis, and in 86% of patients with severe atopic dermatitis, respectively. On the contrary, anti-gp200 IgG could be shown in 55% of healthy individuals, in 89% of individuals with atopic predisposition but without eczema, in 100% of patients with mild atopic dermatitis, and in 79% with severe atopic dermatitis, respectively. No immunoreactivity was found when an extract of Arxula adeninivorans was used as antigen. These results underline the specificity of the immunoblot results with gp200 from Saccharomyces cerevisiae. It can be concluded that occurrence of specific IgE against Saccharomyces cerevisiae cannot be explained by a cross reactivity, e.g., against Candida albicans allergens. Further investigations with the recombinant gp200 will give information on the role of this glycoprotein both in atopic dermatitis and Morbus Crohn.

The chitinase gene FB7-1 of Nicotiana tabacum cv. samsun line 5 was expressed in the two Saccharomyces cerevisiae strains, INVSC2 and H4, under the control of the GAL1 promoter from S. cerevisiae and a multicopy plasmid vector. Both yeast strains express the plant gene as enzymatic active proteins. In transformants of the strain INVSC2, 94% of the total plant chitinase is contained inside the cells, probably within the vacuole which has been confirmed by subcellular fractionation as well as immunohistochemical experiments. This retention inside the cells is due to the C-terminally located 7 amino acids long vacuolar targeting peptide of the prochitinase. When this sequence was removed, chitinase was transported into the culture medium. Pulse-chase experiments revealed that during translation in transformants of both yeast strains one chitinase polypeptide can be immunoadsorbed with specific antibodies. In the case of INVSC2-transformants, newly formed chitinase is modified in a 60 min chase to slightly increase its molecular mass, whereas in H4-transformants the molecular mass constantly remained 32 kDa. By Western blot analysis two chitinase corresponding polypeptides of 32 and 37 kDa were accumulated in the culture medium of both transformants carrying the chitinase gene without the vacuolar targeting sequence. The larger one was very likely O-glycosylated. Whereas, both polypepitdes were also detected in cell extracts of the H4-transformant, only the smaller one was found in the INVSC2-transformant. The plant chitinase passed through the endoplasmic reticulum on its way to the vacuole. The N-terminal signal peptide responsible for the uptake into the endoplasmic reticulum is cleaved correctly. However, cleavage of the vacuolar targeting peptide located at the C-terminus, to give the mature chitinase is obviously influenced by the genetic background of the host strain. In INVSC2-transformants chitinase accumulates in its mature form whereas both the polypeptides of H4-transformants retain their vacuolar targeting peptide. Our results demonstrate that in the case of plant class I chitinase, the plant sorting signal is recognized in yeast cells but post-translational modifications are influenced by the host strain.

Hydrothermal synthesis in the systems LaCl 3 (CeCl 3 )-Al(OH) 3 (Ga 2 O 3 )-GeO 2 yielded crystals of composition La 3 AlGe 5 O 16 , La 3 GaGe 5 O 16 and Ce 3 GaGe 5 O 16 , respectively. The syntheses were carried out at temperatures of 1023 K-1073 K under pressures of 1000 bar. The space group of the three substances was determined as P1 with the pseudosymmetry P2 1 /c. All compounds are isostructural. The lattice parameters are a = 4.794(4) A, b = 8.102(8) A, c = 15.722(8) A, α = 90.42(3)°, β = 94.46(2)°, γ = 89.91(2)° for La 3 AlGe 5 O 16 , a = 4.827(4) A, b = 8.090(4) A, c = 15.707(6) A, α = 90.76(5), β = 94.01(7)°, γ = 90.01(7)° for La 3 GaGe 5 O 16 and a = 4.768(7) A, b = 8.088(5) A, c = 15.600(2) A, α = 90.55(4)°, β = 94.46(4)°, γ = 89.63(4)° for Ce 3 AlGe 5 O 16 . There are two formula units in the unit cell.

According to Heelan et al. patients suffering from Crohn's disease (CD) produce antibodies against a cell wall associated glycoprotein antigen gp200 of the yeast Saccharomyces cerevisiae, while healthy people do not. Here the authors show, that antibodies against this glycoprotein gp200 can also be detected in the sera of healthy humans. The intensity of the antibody titer which is measured by immunoblot experiments is independent from the state of health. The Saccharomyces cerevisiae specific gp200 is a highly glycosylated protein localized not only in the cell wall but also accumulated in the culture medium. Some of the tested sera from CD patients, as well as from healthy adults, also reacted with a 120-kDa glycoprotein which is to be found in preparations containing secreted proteins. Because the binding of antibodies is greatly reduced by periodate treatment of gp200 and by the 120-kDa polypeptide, it is very likely that their carbohydrate moieties are the antigenic determinants against which the specific human antibodies are directed. The human humoral immune response applies only to Saccharomyces cerevisiae antigens, because no analogous immune responses could be detected against antigens derived from the yeast Arxula adeninivorans.

A high molecular weight glycoprotein antigen was isolated by size exclusion chromatography on Sepharose 4B from an extract of the yeast Saccharomyces cerevisiae. The glycoprotein antigen Sc 500 was shown to be identical to the antigen termed gp200 previously isolated (Heelan et al., 1991). The MW of Sc 500 was determined to be about 500 kDa by size exclusion chromatography on Superose 6 and 460 kDa +/- 20k Da by size-exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). Sc 500 contained 90% mannose and traces of N-acetylglucosamine. The amino acid composition revealed that serine and threonine were the most abundant amino acids of the protein part. By alkaline borohydride treatment some, but not all bonds between protein and carbohydrate were broken. This indicates that the main type of linkage between protein and carbohydrate is O-glycosidic and that a minor type is of N-glycosidic nature. Methylation analysis revealed that the mannose residues were connected by 1-->2 and 1 --> 3 linkages with 1 --> 2, 1 --> 6 linked branch points.Purified Sc 500 was subjected to a series of chemical and enzymatic modifications followed by studies of antibody binding activity. Treatments with both periodate and alkaline sodium borohydride reduced the human serum IgA, IgG and monoclonal IgM antibody binding activity of Sc 500 whereas trypsin and pronase did not affect its ability to bind these antibodies. The mannosidase Man alpha 1 --> 2,3,6Man reduced the IgM binding to Sc 500 while the other enzymes included in this experiment (Mana 1 --> 2Man, Man beta 1 --> 4GlcNAc and PNGase Fl had no effect on the antibody binding. Copyright (C) 1996 Elsevier Science Ltd

Recombinant outer membrane proteins (Oprs) of Pseudomonas aeruginosa were expressed in Escherichia coli as glutathione S-transferase (GST)-linked fusion proteins. GST-linked Oprs F and I (GST-OprF190-350 [GST linked to OprF spanning amino acids 190 to 350] and GST-OprI21-83, respectively) and recombinant hybrid Oprs (GST-OprF190-342-OprI21-83 and GST-OprI21-83-OprF190-350) were isolated and tested for their efficacy as vaccines in immunodeficient mice. GST-OprF-OprI protected the mice against a 975-fold 50% lethal dose of P. aeruginosa. Expression of GST-unfused OprF-OprI failed in E. coli, although this hybrid protein has been expressed without a fusion part in Saccharomyces cerevisiae and used for immunizing rabbits. The immune rabbit sera protected severe combined deficient (SCID) mice against a 1,000-fold 50% lethal dose of P. aeruginosa. Evidence is provided to show that the most C-terminal part of OprF (i.e., amino acids 332 to 350) carries an important protective epitope. Opr-based hybrid proteins may have implications for a clinical vaccine against P. aeruginosa.

Substitution of His-35 with arginine in staphylococcal alpha-toxin leads to loss of its membrane perturbating properties in vitro. In this study, we report that the inactive mutant toxin is also devoid of toxic properties when injected intravenously into rabbits. Whereas a single application of native toxin at a dose of 20 micrograms/kg proved uniformly lethal within 3 hours (n = 3), all animals receiving 20, 100 or 200 micrograms/kg mutant toxin (n = 3 for each group) remained in unaltered, healthy states over a 15 day period of observation. Furthermore, all animals receiving inactive toxin developed antibody titers on day 10-15. No dose dependency was noted in the tested range. Thus, the site-directed alpha-toxin mutant (His-35:Arg) is an excellent vaccine candidate.

We tested the ability of the recombinant outer membrane proteins of Pseudomonas aeruginosa to serve as a protective vaccine against this Gram-negative pathogen in the presence of two main pathophysiological events leading to P. aeruginosa sepsis: (i) systemic infection during immunosuppression; and (ii) bacterial translocation. A hybrid vaccine was cloned which combined the protective epitopes of outer membrane protein F (OprF) and outer membrane protein I (OprI). This vaccine proved to be highly protective against an intraperitoneal challenge with P. aeruginosa in immunosuppressed mice. Oral immunization of mice with recombinant OprI expressing Salmonella dublin, induced s-IgA antibodies in the gut mucosa against OprI. These provided protection against translocation of P. aeruginosa in an immunosuppressed mouse model. To test whether OprI is effective in man, recombinant OprI was purified and used for the immunization of human volunteers. Immunization was tolerated well, and no side effects were observed. Antibody titers against OprI were measured in 90% of the volunteers after immunization.

Murine monoclonal antibodies (mAbs) were selected against a cell wall glycoprotein of Saccharomyces cerevisiae. One of the mAbs (92-276/018) specifically identified S. cerevisiae and the sibling species S. paradoxus, S. pastorianus and S. bayanus in immunofluorescence studies and immunoblot analyses, while no other yeast genera except Saccharomyces were recognized. Further analysis indicated that the mAb 92-276/018 reacts with an epitope in the carbohydrate chain of the cell wall glycoproteins.

Monoclonal antibodies (MAbs) were raised against the gag protein of human immunodeficiency virus type 1 (HIV-1). The reactivity of the selected Mabs with the matrix protein p17 of HIV-1 were investigated in several tests, i.e. ELISA, immunostaining of Western blots, and alkaline phosphatase anti-alkaline phosphatase immunocytochemistry (APAAP). All three Mabs reacted exclusively with HIV-1 and showed specific binding to the virus surface in pre-embedding immuno-electron-microscopy and useful as diagnostic agents. In an "in vitro" cultivation experiment the MAbs showed antiviral activity in concentrations in the range of 25-100 micrograms/ml. No binding region could be defined using overlapping peptides consisting of the p17 protein sequence of HIV-1 in an epitope mapping system and therefore we concluded, that the MAbs recognize a conformational epitope.

Purified whole virus preparations of HIV-1 were produced from supernatants of infected cells and concentrated 5000-fold. After inactivation with formaldehyde, the concentrates were combined with one of three different adjuvants, and used to immunize three groups of three chimpanzees each. The chimpanzees were monitored for HIV-specific humoral and cellular immune responses by ELISA, immunoblot, virus neutralization, delayed-type hypersensitivity, lymphocyte proliferation and antibody-dependent cell-mediated cytotoxicity. Weak and inconsistent responses were observed in animals that received HIV-1 formulated with alum as adjuvant, whereas HIV-1 formulated with incomplete Freund's adjuvant or an experimental adjuvant (BWZL) induced good humoral and cellular immune responses to the virus. The three animals that received HIV-1 with the BWZL adjuvant generated overall the best immune responses; therefore, 2 weeks after the sixth immunization these animals were challenged with infectious HIV-1. Despite the presence of good humoral and cell-mediated immunity, all three immunized animals and a control animal became infected within 4 weeks, as evidenced by repeated isolation of HIV-1 from peripheral blood mononuclear cells and anamnestic antibody responses. The new experimental adjuvant has to be further investigated in other vaccine trials and different animal models.

The secretion of Escherichia coli beta-lactamase was studied in a Saccharomyces cerevisiae ts1 mutant strain. The signal sequence of pre-beta-lactamase was recognized by the yeast cell and the precursor protein was processed to an enzymatically active mature beta-lactamase. In contrast to conventional strains, the mutant strain was able to secrete bla-gene-encoded beta-lactamase into the culture broth. These results indicate the potential usefulness of ts1 mutant strains for biotechnological purposes.

The e antigen of the hepatitis B virus (HBeAg) was expressed in S. cerevisiae. Yeast-derived HBeAg exhibits high HBe antigenicity while lacking any HBc antigenicity. The production yield of HBeAg expressed in yeast is dependent on the host strains and the nature of the leader sequences used in the plasmid constructions. The recombinant antigen is not secreted into the medium, independent from the leader sequences which are used. A simple extraction procedure was developed, enabling the isolation of HBeAg from the cells without killing them. Recombinant HBeAg derived from yeast can replace plasma-derived antigen in ELISA for determining antibodies to HBeAg.