Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Art v 1 IgE epitopes of patients and humanized mice are conformational.

BACKGROUND: Worldwide, pollen of the weed mugwort (Artemisiavulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and the corresponding HLA have been made. OBJECTIVE: We sought to characterize IgE epitopes of Art v 1-sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. METHODS: Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. RESULTS: Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients' IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. CONCLUSIONS: Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy.

Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CLpro substrate degradome.

The main viral protease (3CLpro) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CLpro by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CLpro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CLpro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike in healthy lungs. The 3CLpro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.

Component-Resolved Diagnosis of American Cockroach (Periplaneta americana) Allergy in Patients From Different Geographical Areas.

Background: Manifestation of respiratory allergy to American cockroach (Periplaneta americana) is prominent in the subtropical and tropical areas. However, co-existing perennial indoor inhalant allergies frequently compromise clinical diagnosis of cockroach allergy, and the analysis of sensitization pattern is limited by the lack of Periplaneta allergens widely available for component-resolved diagnostics (CRD). Objective: To evaluate a collection of previously described recombinant Periplaneta allergens for CRD in cockroach allergy. Methods: A panel of nine recombinant Periplaneta allergens (Per a 1-5, 7-10) was generated, purified, and subjected to physicochemical characterization by applying circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), amino acid (AA) analysis, and mass spectrometry (MS). Patients (n = 117) from India, Korea, Venezuela, and Iran, reporting perennial respiratory indoor allergies with IgE sensitization to cockroach (P. americana and/or Blattella germanica), were included. The sensitization profile was monitored by the experimental ImmunoCAP testing. Results: ImmunoCAP testing confirmed IgE sensitization to Periplaneta and/or Blattella extract in 98 of 117 patients (r = 0.95). Five out of 117 patients were sensitized to only one of the two cockroach species. Within the whole study group, the prevalence of sensitization to individual allergens varied from 4% (Per a 2) to 50% (Per a 9), with the highest IgE values to Per a 9. Patients from four countries displayed different sensitization profiles at which Per a 3 and Per a 9 were identified as major allergens in India and Korea. Periplaneta-derived lipocalin and myosin light chain were characterized as new minor allergens, designated as Per a 4 and Per a 8. Periplaneta extract showed higher diagnostic sensitivity than all individual components combined, suggesting the existence of allergens yet to be discovered. Conclusion: Utilization of a panel of purified Periplaneta allergens revealed highly heterogeneous sensitization patterns and allowed the classification of lipocalin and myosin light chain from Periplaneta as new minor allergens.

Similar Allergenicity to Different Artemisia Species Is a Consequence of Highly Cross-Reactive Art v 1-Like Molecules.

Background and objectives: Pollens of weeds are relevant elicitors of type I allergies. While many Artemisia species occur worldwide, allergy research so far has only focused on Artemisia vulgaris. We aimed to characterize other prevalent Artemisia species regarding their allergen profiles. Materials and Methods: Aqueous extracts of pollen from seven Artemisia species were characterized by gel electrophoresis and ELISA using sera from mugwort pollen-allergic patients (n = 11). The cDNA sequences of defensin-proline-linked proteins (DPLPs) were obtained, and purified proteins were tested in a competition ELISA, in rat basophil mediator release assays, and for activation of Jurkat T cells transduced with an Art v 1-specific TCR. IgE cross-reactivity to other allergens was evaluated using ImmunoCAP and ISAC. Results: The protein patterns of Artemisia spp. pollen extracts were similar in gel electrophoresis, with a major band at 24 kDa corresponding to DPLPs, like the previously identified Art v 1. Natural Art v 1 potently inhibited IgE binding to immobilized pollen extracts. Six novel Art v 1 homologs with high sequence identity and equivalent IgE reactivity were identified and termed Art ab 1, Art an 1, Art c 1, Art f 1, Art l 1, and Art t 1. All proteins triggered mediator release and cross-reacted at the T cell level. The Artemisia extracts contained additional IgE cross-reactive molecules from the nonspecific lipid transfer protein, pectate lyase, profilin, and polcalcin family. Conclusions: Our findings demonstrate that DPLPs in various Artemisia species have high allergenic potential. Therefore, related Artemisia species need to be considered to be allergen elicitors, especially due to the consideration of potential geographic expansion due to climatic changes.

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.

Author Correction: Conjugation of wildtype and hypoallergenic mugwort allergen Art v 1 to flagellin induces IL-10-DC and suppresses allergen-specific TH2-responses in vivo.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Conjugation of wildtype and hypoallergenic mugwort allergen Art v 1 to flagellin induces IL-10-DC and suppresses allergen-specific TH2-responses in vivo.

Allergies to weed pollen including members of the Compositae family, such as mugwort, ragweed, and feverfew are spreading worldwide. To efficiently treat these newly arising allergies, allergen specific immunotherapy needs to be improved. Therefore, we generated novel vaccine candidates consisting of the TLR5-ligand Flagellin A from Listeria and the major mugwort allergen Art v 1 including either the wild type Art v 1 sequence (rFlaA:Artv1) or a hypoallergenic variant (rFlaA:Artv1hyp) with reduced IgE-binding capacity. Immune modulating capacity of these constructs and respective controls was evaluated in vitro and in vivo. Incorporation of hypoallergenic Art v 1 derivative did not interfere with the resulting fusion proteins' immune stimulatory capacity. Both rFlaA:Artv1 and rFlaA:Artv1hyp induced a prominent, mTOR-dependent, IL-10 secretion from murine dendritic cells, and suppressed allergen-specific TH2-cytokine secretion in vitro and in vivo. Both conjugates retained the capacity to induce rFlaA-specific antibody responses while efficiently inducing production of Art v 1-specific IgG1 and IgG2a antibodies in mice. Interestingly, only the suppression of TH2-cytokine secretion by rFlaA:Artv1 (but not rFlaA:Artv1hyp) was paralleled by a strong secretion of IFN-γ. In summary, we provided evidence that incorporating hypoallergens into flagellin:allergen fusion proteins is a suitable strategy to further improve these promising vaccine candidates.

Synergistic effects of dendritic cell targeting and laser-microporation on enhancing epicutaneous skin vaccination efficacy.

Due to its unique immunological properties, the skin is an attractive target tissue for allergen-specific immunotherapy. In our current work, we combined a dendritic cell targeting approach with epicutaneous immunization using an ablative fractional laser to generate defined micropores in the upper layers of the skin. By coupling the major birch pollen allergen Bet v 1 to mannan from S. cerevisiae via mild periodate oxidation we generated hypoallergenic Bet-mannan neoglycoconjugates, which efficiently targeted CD14+ dendritic cells and Langerhans cells in human skin explants. Mannan conjugation resulted in sustained release from the skin and retention in secondary lymphoid organs, whereas unconjugated antigen showed fast renal clearance. In a mouse model, Bet-mannan neoglycoconjugates applied via laser-microporated skin synergistically elicited potent humoral and cellular immune responses, superior to intradermal injection. The induced antibody responses displayed IgE-blocking capacity, highlighting the therapeutic potential of the approach. Moreover, application via micropores, but not by intradermal injection, resulted in a mixed TH1/TH17-biased immune response. Our data clearly show that applying mannan-neoglycoconjugates to an organ rich in dendritic cells using laser-microporation is superior to intradermal injection. Due to their low IgE binding capacity and biodegradability, mannan neoglycoconjugates therefore represent an attractive formulation for allergen-specific epicutaneous immunotherapy.

Proteomic profiling of the weed feverfew, a neglected pollen allergen source.

Feverfew (Parthenium hysterophorus), an invasive weed from the Asteraceae family, has been reported as allergen source. Despite its relevance, knowledge of allergens is restricted to a partial sequence of a hydroxyproline-rich glycoprotein. We aimed to obtain the entire sequence for recombinant production and characterize feverfew pollen using proteomics and immunological assays. Par h 1, a defensin-proline fusion allergen was obtained by cDNA cloning and recombinantly produced in E. coli. Using two complementary proteomic strategies, a total of 258 proteins were identified in feverfew pollen among those 47 proteins belonging to allergenic families. Feverfew sensitized patients' sera from India revealed IgE reactivity with a pectate lyase, PR-1 protein and thioredoxin in immonoblot. In ELISA, recombinant Par h 1 was recognized by 60 and 40% of Austrian and Indian sera, respectively. Inhibition assays demonstrated the presence of IgE cross-reactive Par h 1, pectate lyase, lipid-transfer protein, profilin and polcalcin in feverfew pollen. This study reveals significant data on the allergenic composition of feverfew pollen and makes recombinant Par h 1 available for cross-reactivity studies. Feverfew might become a global player in weed pollen allergy and inclusion of standardized extracts in routine allergy diagnosis is suggested in exposed populations.

Pollen Allergens for Molecular Diagnosis.

Pollen allergens are one of the main causes of type I allergies affecting up to 30% of the population in industrialized countries. Climatic changes affect the duration and intensity of pollen seasons and may together with pollution contribute to increased incidences of respiratory allergy and asthma. Allergenic grasses, trees, and weeds often present similar habitats and flowering periods compromising clinical anamnesis. Molecule-based approaches enable distinction between genuine sensitization and clinically mostly irrelevant IgE cross-reactivity due to, e. g., panallergens or carbohydrate determinants. In addition, sensitivity as well as specificity can be improved and lead to identification of the primary sensitizing source which is particularly beneficial regarding polysensitized patients. This review gives an overview on relevant pollen allergens and their usefulness in daily practice. Appropriate allergy diagnosis is directly influencing decisions for therapeutic interventions, and thus, reliable biomarkers are pivotal when considering allergen immunotherapy in the context of precision medicine.

Induction of leukocyte infiltration at metastatic site mediates the protective effect of NGcGM3-based vaccine.

While the NGcGM3/VSSP vaccine, a preparation consisting in very small sized proteoliposomes (VSSP) obtained by the incorporation of the NGcGM3 ganglioside into the outer membrane protein (OMP) complex of Neisseria meningitides, is currently studied in late stage clinical trials in breast cancer and melanoma patients, mechanisms involved in the vaccine's antitumor effect are insufficiently understood. Here we have addressed the role of adaptive and innate immune cells in mediating the protective effect of the vaccine. To this aim we selected the 3LL-D122 Lewis lung spontaneous metastasis model. Unexpectedly, inoculation of the vaccine in tumor bearing C57BL/6 mice, either by subcutaneous (sc) or intraperitoneal (ip) routes, induced similar anti-metastatic effect. Regardless the T-independent nature of NGcGM3 ganglioside as antigen, the antimetastatic effect of NGcGM3/VSSP is dependent on CD4(+) T cells. In a further step we found that the vaccine was able to promote the increase, maturation, and cytokine secretion of conventional DCs and the maturation of Bone Marrow-derived plasmacytoid DCs. In line with this result the in vivo IFNα serum level in ip vaccinated mice increased as soon as 2h after treatment. On the other hand the infiltration of NK1.1(+)CD3(-) and NK1.1(+)CD3(+) cells in lungs of vaccinated mice was significantly increased, compared with the presence of these cells in control animal lungs. In the same way NGcGM3/VSSP mobilized acquired immunity effector cells into the lungs of vaccinated tumor bearing mice. Finally and not less noteworthy, leukocyte infiltration in lungs of tumor bearing mice correlates with vaccine induced inhibition of lung metastization.