Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive Bacillus subtilis 1012 for the potential application as vaccines for immunotherapy of atopic allergy

<p>Abstract</p> <p>Background</p> <p>Genetic fusion of the major birch pollen allergen (Bet v1) to bacterial surface-(S)-layer proteins resulted in recombinant proteins exhibiting reduced allergenicity as well as immunomodulatory capacity. Thus, S-layer/allergen fusion proteins were considered as suitable carriers for new immunotherapeutical vaccines for treatment of Type I hypersensitivity. Up to now, endotoxin contamination of the fusion protein which occurred after isolation from the gram-negative expression host <it>E. coli </it>had to be removed by an expensive and time consuming procedure. In the present study, in order to achieve expression of pyrogen-free, recombinant S-layer/allergen fusion protein and to study the secretion of a protein capable to self-assemble, the S-layer/allergen fusion protein rSbpA/Bet v1 was produced in the gram-positive organism <it>Bacillus subtilis </it>1012.</p> <p>Results</p> <p>The chimaeric gene encoding the S-layer protein SbpA of <it>Lysinibacillus sphaericus </it>CCM 2177 as well as Bet v1 was cloned and expressed in <it>B. subtilis </it>1012. For that purpose, the <it>E. coli-B. subtilis </it>shuttle vectors pHT01 for expression in the <it>B. subtilis </it>cytoplasm and pHT43 for secretion of the recombinant fusion protein into the culture medium were used. As shown by western blot analysis, immediately after induction of expression, <it>B. subtilis </it>1012 was able to secret rSbpA/Bet v1 mediated by the signal peptide amyQ of <it>Bacillus amyloliquefaciens</it>. Electron microscopical investigation of the culture medium revealed that the secreted fusion protein was able to form self-assembly products in suspension but did not recrystallize on the surface of the <it>B. subtilis </it>cells. The specific binding mechanism between the N-terminus of the S-layer protein and a secondary cell wall polymer (SCWP), located in the peptidoglycan-containing sacculi of <it>Ly. sphaericus </it>CCM 2177, could be used for isolation and purification of the secreted fusion protein from the culture medium. Immune reactivity of rSbpA/Bet v1 could be demonstrated in immunoblotting experiments with Bet v1 specific IgE containing serum samples from patients suffering birch pollen allergy.</p> <p>Conclusions</p> <p>The impact of this study can be seen in the usage of a gram-positive organism for the production of pyrogen-free self-assembling recombinant S-layer/allergen fusion protein with great relevance for the development of vaccines for immunotherapy of atopic allergy.</p

The effect of birch pollen immunotherapy on apple and rmal d 1 challenges in adults with apple allergy

Background: A proportion of patients allergic to birch pollen are also allergic to pit fruit. The objective of this study was to investigate the effect of immunotherapy with birch pollen on birch-pollen-related apple allergy. Method: Patients with birch pollen immunotherapy underwent a skin-prick test with birch pollen, apple and rMal d 1, global assessments and nasal challenges with birch pollen, open food challenge with apple and a double-blind, placebo-controlled test with rMal d 1 at the start of and during the immunotherapy. Measurements of specific IgE in response to Bet v 1 and rMal d 1 and IgG4 in response to Bet v 1 and rMal d 1 took place. Results: Six of eight patients demonstrated an improvement of nasal challenge test results and all patients improved on global assessment during the immunotherapy. The median oral dose of apple required to elicit a reaction increased but was not statistically significant. The patients showed a decrease in skin-prick test values in response to birch pollen (1.05 to 0.36), apple (0.78 to 0.25) and rMal d 1 (0.51 to 0.10) with p-values of 0.04, 0.03 and 0.06, respectively and a decrease of specific IgE in response to Bet v 1 (10.66 kU/L to 5.19 kU/L) and rMal d 1 (0.99 to 0.61 kU/L) with p-values of 0.01 and 0.05, respectively. Only the median specific IgG4 value to Bet v 1 increased from 0.05 to 1.85 mg/L (p-value of 0.02) and not to IgG4 rMal d 1 (0.07 to 0.08 kU/L). Conclusion: The beneficial effects of immunotherapy for birch pollen were accompanied by a limited effect on apple allergy

Universality of Symmetry and Mixed-symmetry Collective Nuclear States

The global correlation in the observed variation with mass number of the $E2$ and summed $M1$ transition strengths is examined for rare earth nuclei. It is shown that a theory of correlated $S$ and $D$ fermion pairs with a simple pairing plus quadrupole interaction leads naturally to this universality. Thus a unified and quantitative description emerges for low-lying quadrupole and dipole strengths.Comment: In press, Phys. Rev. Lett. 199

O(12) limit and complete classification of symmetry schemes in proton-neutron interacting boson model

It is shown that the proton-neutron interacting boson model (pnIBM) admits new symmetry limits with O(12) algebra which break F-spin but preserves the quantum number M_F. The generators of O(12) are derived and the quantum number `v' of O(12) for a given boson number N is determined by identifying the corresponding quasi-spin algebra. The O(12) algebra generates two symmetry schemes and for both of them, complete classification of the basis states and typical spectra are given. With the O(12) algebra identified, complete classification of pnIBM symmetry limits with good M_F is established.Comment: 22 pages, 1 figur

Prevention of Birch Pollen-Related Food Allergy by Mucosal Treatment with Multi-Allergen-Chimers in Mice

Among birch pollen allergic patients up to 70% develop allergic reactions to Bet v 1-homologue food allergens such as Api g 1 (celery) or Dau c 1 (carrot), termed as birch pollen-related food allergy. In most cases, specific immunotherapy with birch pollen extracts does not reduce allergic symptoms to the homologue food allergens. We therefore genetically engineered a multi-allergen chimer and tested if mucosal treatment with this construct could represent a novel approach for prevention of birch pollen-related food allergy.BALB/c mice were poly-sensitized with a mixture of Bet v 1, Api g 1 and Dau c 1 followed by a sublingual challenge with carrot, celery and birch pollen extracts. For prevention of allergy sensitization an allergen chimer composed of immunodominant T cell epitopes of Api g 1 and Dau c 1 linked to the whole Bet v 1 allergen, was intranasally applied prior to sensitization.Intranasal pretreatment with the allergen chimer led to significantly decreased antigen-specific IgE-dependent β-hexosaminidase release, but enhanced allergen-specific IgG2a and IgA antibodies. Accordingly, IL-4 levels in spleen cell cultures and IL-5 levels in restimulated spleen and cervical lymph node cell cultures were markedly reduced, while IFN-γ levels were increased. Immunomodulation was associated with increased IL-10, TGF-β and Foxp3 mRNA levels in NALT and Foxp3 in oral mucosal tissues. Treatment with anti-TGF-β, anti-IL10R or anti-CD25 antibodies abrogated the suppression of allergic responses induced by the chimer.Our results indicate that mucosal application of the allergen chimer led to decreased Th2 immune responses against Bet v 1 and its homologue food allergens Api g 1 and Dau c 1 by regulatory and Th1-biased immune responses. These data suggest that mucosal treatment with a multi-allergen vaccine could be a promising treatment strategy to prevent birch pollen-related food allergy

Nuclear Scissors Mode with Pairing

The coupled dynamics of the scissors mode and the isovector giant quadrupole resonance are studied using a generalized Wigner function moments method taking into account pair correlations. Equations of motion for angular momentum, quadrupole moment and other relevant collective variables are derived on the basis of the time dependent Hartree-Fock-Bogoliubov equations. Analytical expressions for energy centroids and transitions probabilities are found for the harmonic oscillator model with the quadrupole-quadrupole residual interaction and monopole pairing force. Deformation dependences of energies and $B(M1)$ values are correctly reproduced. The inclusion of pair correlations leads to a drastic improvement in the description of qualitative and quantitative characteristics of the scissors mode.Comment: 36 pages, 5 figures, the results of calculation by another method and the section concerning currents are adde

Temperature Dependence of Damping and Frequency Shifts of the Scissors Mode of a trapped Bose-Einstein Condensate

We have studied the properties of the scissors mode of a trapped Bose-Einstein condensate of $^{87}$Rb atoms at finite temperature. We measured a significant shift in the frequency of the mode below the hydrodynamic limit and a strong dependence of the damping rate as the temperature increased. We compared our damping rate results to recent theoretical calculations for other observed collective modes finding a fair agreement. From the frequency measurements we deduce the moment of inertia of the gas and show that it is quenched below the transition point, because of the superfluid nature of the condensed gas.Comment: 5 pages, 4 figure