David Walgraffe

A hypoallergenic variant of Der p 1 as a candidate for mite allergy vaccines.

BACKGROUND Recombinant hypoallergens that display reduced allergenicity but retain T-cell reactivity represent promising candidates to improve the safety and efficacy of allergen-specific vaccines or immunotherapy. OBJECTIVE The current study reports the immunologic characterization of a hypoallergenic variant of the major mite allergen Der p 1. METHODS The recombinant proform of Der p 1 (ProDer p 1) was expressed in Escherichia coli (ProDer p 1 coli), purified and characterized at the level of its secondary structure, and IgE and T-cell reactivities. Moreover, the prophylactic potential of ProDer p 1 coli vaccinations was evaluated in a murine Der p 1 sensitization model. RESULTS After purification and refolding, ProDer p 1 coli remained aggregated with a higher beta-sheet content and altered Der p 1 conformational epitopes compared with the correctly folded monomeric ProDer p 1 produced in Chinese hamster ovary cells. Both ProDer p 1 forms were able to retain the Der p 1-specific T-cell reactivity but direct ELISA, competitive inhibition, and rat basophil leukemia assays clearly showed that ProDer p 1 coli displays a very weak IgE reactivity. Mice vaccinations with aggregated ProDer p 1 adjuvanted with alum induced a T(H)1-biased immune response that prevented the subsequent allergic response after Der p 1 sensitization and airway challenge with aerosolized mite extracts. Furthermore, ProDer p 1 coli treatment inhibited the development of airway eosinophilia and airway hyperresponsiveness to inhaled methacholine. CONCLUSION Aggregated forms of Der p 1 could represent hypoallergens suitable for the prevention of mite allergy.

Characterization of a TFIIH homologue from Trypanosoma brucei.

Trypanosomes are protozoans showing unique transcription characteristics. We describe in Trypanosoma brucei a complex homologous to TFIIH, a multisubunit transcription factor involved in the control of transcription by RNA Pol I and RNA Pol II, but also in DNA repair and cell cycle control. Bioinformatics analyses allowed the detection of five genes encoding four putative core TFIIH subunits (TbXPD, TbXPB, Tbp44, Tbp52), including a novel XPB variant, TbXPBz. In all cases sequences known to be important for TFIIH functions were conserved. We performed a molecular analysis of this core complex, focusing on the two subunits endowed with a known enzymatic (helicase) activity, XPD and XPB. The involvement of these T. brucei proteins in a bona fide TFIIH core complex was supported by (i) colocalization by immunofluorescence in the nucleus, (ii) direct physical interaction of TbXPD and its interacting regulatory subunit Tbp44 as determined by double‐hybrid assay and tandem affinity purification of the core TFIIH, (iii) involvement of the core proteins in a high molecular weight complex and (iv) occurrence of transcription, cell cycle and DNA repair phenotypes upon either RNAi knock‐down or overexpression of essential subunits.

Identification and characterization of two trypanosome TFIIS proteins exhibiting particular domain architectures and differential nuclear localizations.

Nuclear transcription of Trypanosoma brucei displays unusual features. Most protein‐coding genes are organized in large directional gene clusters, which are transcribed polycistronically by RNA polymerase II (pol II) with subsequent processing to generate mature mRNA. Here, we describe the identification and characterization of two trypanosome homologues of transcription elongation factor TFIIS (TbTFIIS1 and TbTFIIS2‐1). TFIIS has been shown to aid transcription elongation by relieving arrested pol II. Our phylogenetic analysis demonstrated the existence of four independent TFIIS expansions across eukaryotes. While TbTFIIS1 contains only the canonical domains II and III, the N‐terminus of TbTFIIS2‐1 contains a PWWP domain and a domain I. TbTFIIS1 and TbTFIIS2‐1 are expressed in procyclic and bloodstream form cells and localize to the nucleus in similar, but distinct, punctate patterns throughout the cell cycle. Neither TFIIS protein was enriched in the major pol II sites of spliced‐leader RNA transcription. Single RNA interference (RNAi)‐mediated knock‐down and knockout showed that neither protein is essential. Double knock‐down, however, impaired growth. Repetitive failure to generate a double knockout of TbTFIIS1 and TbTFIIS2‐1 strongly suggests synthetical lethality and thus an essential function shared by the two proteins in trypanosome growth.

Development of recombinant stable house dust mite allergen Der p 3 molecules for component-resolved diagnosis and specific immunotherapy

The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the diagnosis of allergy and the safety and efficacy of immunotherapy.