Tuning antiviral CD8 T-cell response via proline-altered peptide ligand vaccination

Viral escape from CD8+ cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8+ T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape

In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation Capacity

Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients

Cloning, expression, purification, crystallization and preliminary X-ray analysis of the pilus-associated sortase C from Streptococcus pneumoniae

Crystallization conditions and preliminary X-ray diffraction analysis of the S. pneumoniae-derived pilus-associated protein sortase C are reported

Crystallization and preliminary X-ray diffraction analysis of a protease inhibitor from the haemolymph of the Indian tasar silkworm Antheraea mylitta

A protein with inhibitory activity against fungal proteases was purified from the haemolymph of the Indian tasar silkworm Antheraea mylitta and was crystallized using the hanging-drop vapour-diffusion method. Polyethylene glycol 3350 was used as a precipitant. Crystals belonged to space group P6<SUB>3</SUB>22, with unit-cell parameters a = b = 60.6, c = 85.1 Å. X-ray diffraction data were collected and processed to a maximum resolution of 2.1 Å

Crystal Structure of the Dog Lipocalin Allergen Can f 2: Implications for Cross-reactivity to the Cat Allergen Fel d 4

Allergic diseases are a major health problem, affecting up to 30% of the population in affluent countries. Domestic pets such as cats, dogs and horses are among the most common agents of IgE-mediated allergies. Conventional diagnostics and allergen-specific immunotherapy (SIT) still rely on crude allergen extracts, even though several commercial extracts have demonstrated poor quality with regard to the included allergen content. Particularly in dog allergy, SIT may show a low clinical effectiveness upon treatment with allergenic extracts. As an alternative, recombinant DNA technologies allow for the construction of recombinant (r) allergens for improved allergy diagnostics and induction of a long-lasting protection by SIT. However, this therapy is still limited by unwanted side-effects such as IgE-mediated acute reactions, or late phase reactions (LPR). To combat these limitations, allergens have been modified for safer use in SIT, i.e. hypoallergens, or several allergenic molecules have been combined in one construct for improved diagnostics and treatment of allergic patients. The aim of this thesis was to demonstrate how development in molecular allergology may increase the understanding of the allergenicity and cross-reactivity of dog allergens. We also set out to demonstrate how this knowledge can be used to engineer safer and more efficacious allergenic constructs for improved diagnostics and therapy. In paper I, we described the crystal structure and the structural characteristics of the dog lipocalin allergen Can f 2. The recombinant protein was shown to mimick the IgEbinding characteristics of the natural allergen. Furthermore, we demonstrated an IgEmediated cross-reactivity between Can f 2 and the cat lipocalin allergen Fel d 4, and suggested a potential conserved epitope as a cross-reactive site. In paper II, the primary structures for Fel d 4 and the major horse allergen Equ c 1 were used to identify a novel dog lipocalin allergen, Can f 6. The recombinant allergen elicited a positive IgE-reaction in ~40% of dog-sensitized subjects, and most individuals also had a positive reaction to the homologous allergens Fel d 4 and Equ c 1. The biological activity of rCan f 6 and the homologous allergens was demonstrated by basophil activation test (BAT). Finally, we identified IgE-mediated cross-reactivity between these three allergens, which may account for cross-species sensitization between dog, cat and horse. In paper III, the method phage display was for the first time used to construct hypoallergenic mutants of the major cat allergen, Fel d 1. The four vaccine candidates were produced as folded proteins in E. coli and showed a lower IgE-binding than the wild type protein in ELISA. By T cell proliferation and BAT assays, we demonstrated that two of the four candidates had a significantly lower T cell activation capacity. Moreover all mutants had a lower allergenic activity than rFel d 1, which could indicate a safer profile for SIT of allergic patients. The effectiveness of the mutants was demonstrated in a mouse model for cat allergy, by the induction of blocking IgG antibodies. In paper IV, a different vaccine concept was described, where four dog lipocalin allergens were assembled in one molecule. The corresponding fusion protein comprised the biochemical and immunological properties of the original allergens, investigated by CD-spectra and ELISA. Importantly, the linked construct bound more IgE than the individual allergens, demonstrating the usefulness for diagnostics. Finally, the linked molecule induced comparable IgG levels to all included allergens compared with an equimolar mix, while showing a lower T cell reactivity which could implicate a lower risk of LPR. To summarize, this thesis demonstrates how molecular approaches may be used to improve our understanding of allergenicity, to characterize new allergens and define their relationship with other allergens. This detailed knowledge was used to formulate modified allergen constructs for improved diagnosis and vaccination of allergic patients