MHCII3D-Robust Structure Based Prediction of MHC II Binding Peptides.

Knowledge of MHC II binding peptides is highly desired in immunological research, particularly in the context of cancer, autoimmune diseases, or allergies. The most successful prediction methods are based on machine learning methods trained on sequences of experimentally characterized binding peptides. Here, we describe a complementary approach called MHCII3D, which is based on structural scaffolds of MHC II-peptide complexes and statistical scoring functions (SSFs). The MHC II alleles reported in the Immuno Polymorphism Database are processed in a dedicated 3D-modeling pipeline providing a set of scaffold complexes for each distinct allotype sequence. Antigen protein sequences are threaded through the scaffolds and evaluated by optimized SSFs. We compared the predictive power of MHCII3D with different sequence-based machine learning methods. The Pearson correlation to experimentally determine IC50 values for MHC II Automated Server Benchmarks data sets from IEDB (Immune Epitope Database) is 0.42, which is in the competitor methods range. We show that MHCII3D is quite robust in leaving one molecule out tests and is therefore not prone to overfitting. Finally, we provide evidence that MHCII3D can complement the current sequence-based methods and help to identify problematic entries in IEDB. Scaffolds and MHCII3D executables can be freely downloaded from our web pages.

In silico Design of Phl p 6 Variants With Altered Fold-Stability Significantly Impacts Antigen Processing, Immunogenicity and Immune Polarization.

Introduction: Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. Methods: MAESTRO software was used for in silico prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in E. coli, and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. Results: In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from -5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing in vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. In vivo, stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21. Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

A hybrid of two major Blomia tropicalis allergens as an allergy vaccine candidate.

INTRODUCTION: Allergen-specific immunotherapy (AIT) represents a curative approach for treating allergies. In the tropical and subtropical regions of the world, Blomia tropicalis (Blo t 5 and Blo t 21) is the likely dominant source of indoor allergens. AIM: To generate a hypoallergenic Blo t 5/Blo t 21 hybrid molecule that can treat allergies caused by B tropicalis. METHODS: Using in silico design of B tropicalis hybrid proteins, we chose two hybrid proteins for heterologous expression. Wild-type Blo t 5/Blo t 21 hybrid molecule and a hypoallergenic version, termed BTH1 and BTH2, respectively, were purified by ion exchange and size exclusion chromatography and characterized by physicochemical, as well as in vitro and in vivo immunological, experiments. RESULTS: BTH1, BTH2 and the parental allergens were purified to homogeneity and characterized in detail. BTH2 displayed the lowest IgE reactivity that induced basophil degranulation using sera from allergic rhinitis and asthmatic patients. BTH2 essentially presented the same endolysosomal degradation pattern as the shortened rBlo t 5 and showed a higher resistance towards degradation than the full-length Blo t 5. In vivo immunization of mice with BTH2 led to the production of IgG antibodies that competed with human IgE for allergen binding. Stimulation of splenocytes from BTH2-immunized mice produced higher levels of IL-10 and decreased secretion of IL-4 and IL-5. In addition, BTH2 stimulated T-cell proliferation in PBMCs isolated from allergic patients, with secretion of higher levels of IL-10 and lower levels of IL-5 and IL-13, when compared to parental allergens. CONCLUSIONS AND CLINICAL RELEVANCE: BTH2 is a promising hybrid vaccine candidate for immunotherapy of Blomia allergy. However, further pre-clinical studies addressing its efficacy and safety are needed.

The Cell Wall PAC (Proline-Rich, Arabinogalactan Proteins, Conserved Cysteines) Domain-Proteins Are Conserved in the Green Lineage.

Plant cell wall proteins play major roles during plant development and in response to environmental cues. A bioinformatic search for functional domains has allowed identifying the PAC domain (Proline-rich, Arabinogalactan proteins, conserved Cysteines) in several proteins (PDPs) identified in cell wall proteomes. This domain is assumed to interact with pectic polysaccharides and O-glycans and to contribute to non-covalent molecular scaffolds facilitating the remodeling of polysaccharidic networks during rapid cell expansion. In this work, the characteristics of the PAC domain are described in detail, including six conserved Cys residues, their spacing, and the predicted secondary structures. Modeling has been performed based on the crystal structure of a Plantago lanceolata PAC domain. The presence of ß-sheets is assumed to ensure the correct folding of the PAC domain as a ß-barrel with loop regions. We show that PDPs are present in early divergent organisms from the green lineage and in all land plants. PAC domains are associated with other types of domains: Histidine-rich, extensin, Proline-rich, or yet uncharacterized. The earliest divergent organisms having PDPs are Bryophytes. Like the complexity of the cell walls, the number and complexity of PDPs steadily increase during the evolution of the green lineage. The association of PAC domains with other domains suggests a neo-functionalization and different types of interactions with cell wall polymers.

Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins.

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70-90% coverage of the allergenic epitopes from mugwort pollen-allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3-specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

Rational Design, Structure-Activity Relationship, and Immunogenicity of Hypoallergenic Pru p 3 Variants.

SCOPE: Allergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy. METHODS AND RESULTS: Pru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild-type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long-term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE-binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross-inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate-based adjuvant formulation in a mouse model. CONCLUSIONS: An in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy.

Synergistic cross-talk of hedgehog and interleukin-6 signaling drives growth of basal cell carcinoma.

Persistent activation of hedgehog (HH)/GLI signaling accounts for the development of basal cell carcinoma (BCC), a very frequent nonmelanoma skin cancer with rising incidence. Targeting HH/GLI signaling by approved pathway inhibitors can provide significant therapeutic benefit to BCC patients. However, limited response rates, development of drug resistance, and severe side effects of HH pathway inhibitors call for improved treatment strategies such as rational combination therapies simultaneously inhibiting HH/GLI and cooperative signals promoting the oncogenic activity of HH/GLI. In this study, we identified the interleukin-6 (IL6) pathway as a novel synergistic signal promoting oncogenic HH/GLI via STAT3 activation. Mechanistically, we provide evidence that signal integration of IL6 and HH/GLI occurs at the level of cis-regulatory sequences by co-binding of GLI and STAT3 to common HH-IL6 target gene promoters. Genetic inactivation of Il6 signaling in a mouse model of BCC significantly reduced in vivo tumor growth by interfering with HH/GLI-driven BCC proliferation. Our genetic and pharmacologic data suggest that combinatorial HH-IL6 pathway blockade is a promising approach to efficiently arrest cancer growth in BCC patients.

Influence of protein fold stability on immunogenicity and its implications for vaccine design.

INTRODUCTION: In modern vaccinology and immunotherapy, recombinant proteins more and more replace whole organisms to induce protective or curative immune responses. Structural stability of proteins is of crucial importance for efficient presentation of antigenic peptides on MHC, which plays a decisive role for triggering strong immune reactions. Areas covered: In this review, we discuss structural stability as a key factor for modulating the potency of recombinant vaccines and its importance for antigen proteolysis, presentation, and stimulation of B and T cells. Moreover, the impact of fold stability on downstream events determining the differentiation of T cells into effector cells is reviewed. We summarize studies investigating the impact of protein fold stability on the outcome of the immune response and provide an overview on computational methods to estimate the effects of point mutations on protein stability. Expert commentary: Based on this information, the rational design of up-to-date vaccines is discussed. A model for predicting immunogenicity of proteins based on their conformational stability at different pH values is proposed.

Tackling Bet v 1 and associated food allergies with a single hybrid protein.

BACKGROUND: Allergy vaccines should be easily applicable, safe, and efficacious. For Bet v 1-mediated birch pollen and associated food allergies, a single wild-type allergen does not provide a complete solution. OBJECTIVE: We aimed to combine immunologically relevant epitopes of Bet v 1 and the 2 clinically most important related food allergens from apple and hazelnut to a single hybrid protein, termed MBC4. METHODS: After identification of T cell epitope-containing parts on each of the 3 parental allergens, the hybrid molecule was designed to cover relevant epitopes and evaluated in silico. Thereby a mutation was introduced into the hybrid sequence, which should alter the secondary structure without compromising the immunogenic properties of the molecule. RESULTS: MBC4 and the parental allergens were purified to homogeneity. Analyses of secondary structure elements revealed substantial changes rendering the hybrid de facto nonreactive with patients' serum IgE. Nevertheless, the protein was monomeric in solution. MBC4 was able to activate T-cell lines from donors with birch pollen allergy and from mice immunized with the parental allergens. Moreover, on immunization of mice and rabbits, MBC4 induced cross-reactive IgG antibodies, which were able to block the binding of human serum IgE. CONCLUSION: Directed epitope rearrangements combined with a knowledge-based structural modification resulted in a protein unable to bind IgE from allergic patients. Still, properties to activate specific T cells or induce blocking antibodies were conserved. This suggests that MBC4 is a suitable vaccine candidate for the simultaneous treatment of Bet v 1 and associated food allergies.

MAESTROweb: a web server for structure-based protein stability prediction.

UNLABELLED: The prediction of change in stability upon point mutations in proteins has many applications in protein analysis and engineering. We recently adjoined a new structure-based method called MAESTRO, which is distributed as command line program. We now provide access to the most important features of MAESTRO by an easy to use web service. MAESTROweb allows the prediction of change in stability for user-defined mutations, provides a scan functionality for the most (de)stabilizing n-point mutations for a maximum of n = 5, creates mutation sensitivity profiles and evaluates potential disulfide bonds. MAESTROweb operates on monomers, multimers and biological assemblies as defined by PDB. AVAILABILITY AND IMPLEMENTATION: MAESTROweb is freely available for non-commercial use at https://biwww.che.sbg.ac.at/maestro/web CONTACT: peter.lackner@sbg.ac.at.

MAESTRO--multi agent stability prediction upon point mutations.

BACKGROUND: Point mutations can have a strong impact on protein stability. A change in stability may subsequently lead to dysfunction and finally cause diseases. Moreover, protein engineering approaches aim to deliberately modify protein properties, where stability is a major constraint. In order to support basic research and protein design tasks, several computational tools for predicting the change in stability upon mutations have been developed. Comparative studies have shown the usefulness but also limitations of such programs. RESULTS: We aim to contribute a novel method for predicting changes in stability upon point mutation in proteins called MAESTRO. MAESTRO is structure based and distinguishes itself from similar approaches in the following points: (i) MAESTRO implements a multi-agent machine learning system. (ii) It also provides predicted free energy change (Δ ΔG) values and a corresponding prediction confidence estimation. (iii) It provides high throughput scanning for multi-point mutations where sites and types of mutation can be comprehensively controlled. (iv) Finally, the software provides a specific mode for the prediction of stabilizing disulfide bonds. The predictive power of MAESTRO for single point mutations and stabilizing disulfide bonds is comparable to similar methods. CONCLUSIONS: MAESTRO is a versatile tool in the field of stability change prediction upon point mutations. Executables for the Linux and Windows operating systems are freely available to non-commercial users from http://biwww.che.sbg.ac.at/MAESTRO.

D-Light on promoters: a client-server system for the analysis and visualization of cis-regulatory elements.

BACKGROUND: The binding of transcription factors to DNA plays an essential role in the regulation of gene expression. Numerous experiments elucidated binding sequences which subsequently have been used to derive statistical models for predicting potential transcription factor binding sites (TFBS). The rapidly increasing number of genome sequence data requires sophisticated computational approaches to manage and query experimental and predicted TFBS data in the context of other epigenetic factors and across different organisms. RESULTS: We have developed D-Light, a novel client-server software package to store and query large amounts of TFBS data for any number of genomes. Users can add small-scale data to the server database and query them in a large scale, genome-wide promoter context. The client is implemented in Java and provides simple graphical user interfaces and data visualization. Here we also performed a statistical analysis showing what a user can expect for certain parameter settings and we illustrate the usage of D-Light with the help of a microarray data set. CONCLUSIONS: D-Light is an easy to use software tool to integrate, store and query annotation data for promoters. A public D-Light server, the client and server software for local installation and the source code under GNU GPL license are available at http://biwww.che.sbg.ac.at/dlight.