Bioengineering of Virus-like Particles for the Prevention or Treatment of Allergic Diseases.

Recent findings on the mechanism of allergen-specific immunotherapy (AIT) have revisited the role of immunoglobulin G (IgG) as the development of specific blocking IgG antibodies appeared critical for the successful suppression of T-helper 2 (Th2)-biased allergic responses. Consequently, any form of molecular AIT-promoting potent allergen-specific neutralizing antibodies would be preferred to conventional administration of allergen extracts. The potent immunogenicity of virus-like particles (VLPs) could be harnessed for that purpose. The particle size (20-200 nm) optimizes uptake by antigen-presenting cells as well as lymphatic trafficking. Moreover, the display of antigens in repetitive arrays promotes potent B cell activation for the development of sustained antibody responses. The presentation of self-antigens on the particle surface was even capable to break B cell tolerance. In this review, we describe the immunomodulatory properties of the 3 VLP-based strategies designed so far for the treatment of allergic disease: VLP packaged with CpG motifs as well as chimeric particles displaying pro-Th2/Th2 cytokines or allergens (full-length or B cell epitopes).

Molecular approaches to allergen-specific immunotherapy: Are we so far from clinical implementation?

Conventional allergen-specific immunotherapy (AIT), based on administrations of allergen extracts, represents up to now the unique protocol for the desensitization of allergic patients. Whereas the effectiveness of AIT was evidenced for the treatment of allergic rhinitis and allergic asthma, such strategy remains experimental for food allergies up to now. However, important issues are commonly associated with AIT as the quality of natural allergen extracts, the long duration and adverse side-effects which negatively affect successful desensitization together with the patient compliance. The rapid progression of molecular allergology made possible the quest of safer, shorter and more effective immunotherapeutic approaches. The aim of this review was to provide an update on these different innovative recombinant derivatives including their efficacy but also their limitations. Despite promising preclinical and early clinical studies, the absence of convincing data in large phase III trials precludes so far the translation of these immunotherapeutic candidates into the clinic.

Bioconversion of chitosan into chito-oligosaccharides (CHOS) using family 46 chitosanase from Bacillus subtilis (BsCsn46A).

BsCsn46A, a GH family 46 chitosanase from Bacillus subtilis had been previously shown to have potential for bioconversion of chitosan to chito-oligosaccharides (CHOS). However, so far, in-depth analysis of both the mode of action of this enzyme and the composition of its products were lacking. In this study, we have employed size exclusion chromatography, 1H NMR, and mass spectrometry to reveal that BsCsn46A can rapidly cleave chitosans with a wide-variety of acetylation degrees, using a non-processive endo-mode of action. The composition of the product mixtures can be tailored by varying the degree of acetylation of the chitosan and the reaction time. Detailed analysis of product profiles revealed differences compared to other chitosanases. Importantly, BsCsn46A seems to be one of the fastest chitosanases described so far. The detailed analysis of preferred endo-binding modes using H218O showed that a hexameric substrate has three productive binding modes occurring with similar frequencies.

OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system.

The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing. Proteins destined for secretion contain an N-terminal signal peptide that is cleaved by secretion machinery in the plasma membrane. The resulting protein is released in an active mature form. In this study, Bacillus subtilis chitosanase (Csn) was used as a model protein to compare the effect of two signal peptides on the secretion of heterologous recombinant protein. The results showed that the E. coli secretion machinery could recognize both native bacillus and E. coli signal peptides. However, only the native bacillus signal peptide could generate the same N-terminal sequence as in the wild type bacteria. When the recombinant Csn constructs contained the E. coli OmpA signal peptide, the secreted enzymes were heterogeneous, comprising a mixed population of secreted enzymes with different N-terminal sequences. Nevertheless, the E. coli OmpA signal peptide was found to be more efficient for high expression and secretion of bacillus Csn. These findings may be used to help engineer other recombinant proteins for secretory production in E. coli.

Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system.

BACKGROUND: Heterologous production of hydrolytic enzymes is important for green and white biotechnology since these enzymes serve as efficient biocatalysts for the conversion of a wide variety of raw materials into value-added products. Lactic acid bacteria are interesting cell factories for the expression of hydrolytic enzymes as many of them are generally recognized as safe and require only a simple cultivation process. We are studying a potentially food-grade expression system for secretion of hydrolytic enzymes into the culture medium, since this enables easy harvesting and purification, while allowing direct use of the enzymes in food applications. RESULTS: We studied overexpression of a chitosanase (CsnA) and a ß-mannanase (ManB), from Bacillus licheniformis and Bacillus subtilis, respectively, in Lactobacillus plantarum, using the pSIP system for inducible expression. The enzymes were over-expressed in three forms: without a signal peptide, with their natural signal peptide and with the well-known OmpA signal peptide from Escherichia coli. The total production levels and secretion efficiencies of CsnA and ManB were highest when using the native signal peptides, and both were reduced considerably when using the OmpA signal. At 20 h after induction with 12.5 ng/mL of inducing peptide in MRS media containing 20 g/L glucose, the yields and secretion efficiencies of the proteins with their native signal peptides were 50 kU/L and 84% for ManB, and 79 kU/L and 56% for CsnA, respectively. In addition, to avoid using antibiotics, the erythromycin resistance gene was replaced on the expression plasmid with the alanine racemase (alr) gene, which led to comparable levels of protein production and secretion efficiency in a suitable, alr-deficient L. plantarum host. CONCLUSIONS: ManB and CsnA were efficiently produced and secreted in L. plantarum using pSIP-based expression vectors containing either an erythromycin resistance or the alr gene as selection marker.

Production of recombinant Bacillus subtilis chitosanase, suitable for biosynthesis of chitosan-oligosaccharides.

Chitosanases are enzymes that catalyse the hydrolysis of the ß-1,4 glycosidic bond of chitosan. One of the most promising applications of this enzyme is for the bioconversion of chitosan into value-added chitosan-oligosaccharides (COS). GH46 chitosanase (Csn) from Bacillus subtilis 168 was expressed in Escherichia coli by fusing the gene encoding mature Csn to the E. coli OmpA signal peptide sequence. The recombinant enzyme was secreted into the culture supernatant. The recombinant Csn showed high specific activity and stability over a wide range of pH. The enzyme was >100 times more thermostable in the presence of the substrate, with a half-life time of activity (τ(1/2)) of approximately 20 h at 50 °C and pH 5.5. Efficient bioconversion of chitosan into different mixtures of COS, using crude culture supernatant containing secreted enzyme was demonstrated.

Expression and characterization of Bacillus licheniformis chitinase (ChiA), suitable for bioconversion of chitin waste.

Chitinase (EC 3.2.1.14) is an enzyme with multiple industrial applications. These include bioconversion of chitin waste, a highly resistant and abundant biopolymer from crustacean food industry, into glucosamine and chito-oligosaccharide value-added products. This paper reports on the expression of endochitinase (ChiA) from Bacillus licheniformis strain DSM8785 in E. coli and characterization of the recombinant enzyme. Recombinant ChiA could efficiently convert colloidal chitin to N-acetyl glucosamine and chitobiose at pH 4.0, 6.0 and 9.0 at 50 degrees C and retained its activity up to 3days under these conditions, suggesting that this enzyme is suitable for bioconversion of chitin waste.