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.

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.

Enzymatically releasable polyethylene glycol - host defense peptide conjugates with improved activity and biocompatibility.

Host defense peptides (HDPs) have been the subject of great interest for the treatment of multidrug-resistant bacterial infections due to their multimodal activity and low induction of resistance. However, aggregation, toxicity, and short biological half-life have limited their applicability for clinical treatment. Many methods have been explored to alleviate these issues, such as polymer (e.g., polyethylene glycol (PEG)) conjugation, but these are often accompanied by reductions in the activity of the HDP. Here, we detail the design of a novel PEG-HDP conjugate incorporating an enzymatic cleavage sequence targeting matrix metalloproteinases (MMPs) that accumulate at sites of inflammation and infection. Addition of the cleavage sequence onto either the N- or the C-terminal region of the parent peptide (peptide 73, a derivative of the HDP aurein 2.2) was explored to determine the location for optimal antimicrobial activity following MMP cleavage; furthermore, the susceptibility of the peptide to MMP cleavage after conjugation to 2 kDa or 5 kDa PEG was examined. The top candidate, L73, utilized an N-terminal cleavage site that was subsequently conjugated to a 2 kDa PEG polymer. Both L73 and the conjugate exhibited no antimicrobial activity in vitro until cleaved by purified MMP, which liberated a peptide fragment with 16- or 63-fold improved activity, respectively, corresponding to a minimum inhibitory concentration (MIC) of 8 µg/mL, comparable to that of peptide 73 (4 µg/mL). Furthermore, PEG conjugation improved the blood compatibility and reduced the aggregation tendency of the HDP in vitro, indicating enhanced biocompatibility. When administered as a single subcutaneous dose (~3.6 mg, or a peptide concentration of 142 mg/kg) in a mouse abscess model of high-density methicillin-resistant Staphylococcus aureus (MRSA) infection, the conjugate displayed strong activity, reducing abscess size and bacterial load by 73.3% and 58-fold, respectively. This activity was completely lost when the cleavage site was rendered resistant to MMPs by the substitution of two d-amino acids, supporting the hypothesis that antimicrobial activity was dependent on cleavage by MMPs, which were shown here to increasingly accumulate at the abscess site up to 18 h post infection. Finally, the conjugate displayed biocompatibility in vivo, with no identifiable toxicity or aggregation.

Effect of structural stability on endolysosomal degradation and T-cell reactivity of major shrimp allergen tropomyosin.

BACKGROUND: Tropomyosins are highly conserved proteins, an attribute that forms the molecular basis for their IgE antibody cross-reactivity. Despite sequence similarities, their allergenicity varies greatly between ingested and inhaled invertebrate sources. In this study, we investigated the relationship between the structural stability of different tropomyosins, their endolysosomal degradation patterns, and T-cell reactivity. METHODS: We investigated the differences between four tropomyosins-the major shrimp allergen Pen m 1 and the minor allergens Der p 10 (dust mite), Bla g 7 (cockroach), and Ani s 3 (fish parasite)-in terms of IgE binding, structural stability, endolysosomal degradation and subsequent peptide generation, and T-cell cross-reactivity in a BALB/c murine model. RESULTS: Tropomyosins displayed different melting temperatures, which did not correlate with amino acid sequence similarities. Endolysosomal degradation experiments demonstrated differential proteolytic digestion, as a function of thermal stability, generating different peptide repertoires. Pen m 1 (Tm 42°C) and Der p 10 (Tm 44°C) elicited similar patterns of endolysosomal degradation, but not Bla g 7 (Tm 63°C) or Ani s 3 (Tm 33°C). Pen m 1-specific T-cell clones, with specificity for regions highly conserved in all four tropomyosins, proliferated weakly to Der p 10, but did not proliferate to Bla g 7 and Ani s 3, indicating lack of T-cell epitope cross-reactivity. CONCLUSIONS: Tropomyosin T-cell cross-reactivity, unlike IgE cross-reactivity, is dependent on structural stability rather than amino acid sequence similarity. These findings contribute to our understanding of cross-sensitization among different invertebrates and design of suitable T-cell peptide-based immunotherapies for shrimp and related allergies.

TGFß1 mimetic peptide modulates immune response to grass pollen allergens in mice.

BACKGROUND: Transforming growth factor ß1 (TGFß1) is a cytokine that exerts immunosuppressive functions, as reflected by its ability to induce regulatory T (Treg) cell differentiation and inhibit Th1 and Th2 responses. Hence, peptides that mimic the active core domain of TGFß1 may be promising candidates for modulation of the allergic response. This study aimed to investigate a synthetic TGFß1 mimetic peptide (TGFß1-mim) for its ability to modulate the immune response during allergic sensitization to grass pollen allergens. METHODS: The in vitro action of TGFß1-mim was evaluated in human lung epithelial cells, Jurkat cells, and rat basophilic leukemia cells. The in vivo action was evaluated in a murine model of Phl p 5 allergic sensitization. Additionally, the Th2 modulatory response was evaluated in IL-4 reporter mice. RESULTS: In vitro, TGFß1-mim downregulated TNF-α production, IL-8 gene expression, and cytokine secretion, upregulated IL-10 secretion, and inhibited Phl p 5-induced basophil degranulation. During Phl p 5 sensitization in mice, TGFß1-mim downregulated IL-2, IL-4, IL-5, IL-13, and IFN-γ, upregulated IL-10, and induced Treg cell production. Furthermore, mice treated with TGFß1-mim had lower levels of IgE, IgG1, IgG2a and higher levels of IgA antibodies than control mice. In a reporter mouse, the mimetic inhibited Th2 polarization. CONCLUSION: The TGFß1-mim efficiently modulated various important events that exacerbate the allergic microenvironment, including the production of main cytokines that promote Th1 and Th2 differentiation, and the induction of allergen-specific regulatory T cells, highlighting its potential use in therapeutic approaches to modulate the immune response toward environmental allergens.

Moonlighting matrix metalloproteinase substrates: Enhancement of proinflammatory functions of extracellular tyrosyl-tRNA synthetase upon cleavage.

Tyrosyl-tRNA synthetase ligates tyrosine to its cognate tRNA in the cytoplasm, but it can also be secreted through a noncanonical pathway. We found that extracellular tyrosyl-tRNA synthetase (YRS) exhibited proinflammatory activities. In addition to acting as a monocyte/macrophage chemoattractant, YRS initiated signaling through Toll-like receptor 2 (TLR2) resulting in NF-κB activation and release of tumor necrosis factor α (TNFα) and multiple chemokines, including MIP-1α/ß, CXCL8 (IL8), and CXCL1 (KC) from THP1 monocyte and peripheral blood mononuclear cell-derived macrophages. Furthermore, YRS up-regulated matrix metalloproteinase (MMP) activity in a TNFα-dependent manner in M0 macrophages. Because MMPs process a variety of intracellular proteins that also exhibit extracellular moonlighting functions, we profiled 10 MMPs for YRS cleavage and identified 55 cleavage sites by amino-terminal oriented mass spectrometry of substrates (ATOMS) positional proteomics and Edman degradation. Stable proteoforms resulted from cleavages near the start of the YRS C-terminal EMAPII domain. All of the MMPs tested cleaved at ADS386↓387LYV and VSG405↓406LVQ, generating 43- and 45-kDa fragments. The highest catalytic efficiency for YRS was demonstrated by MMP7, which is highly expressed by monocytes and macrophages, and by neutrophil-specific MMP8. MMP-cleaved YRS enhanced TLR2 signaling, increased TNFα secretion from macrophages, and amplified monocyte/macrophage chemotaxis compared with unprocessed YRS. The cleavage of YRS by MMP8, but not MMP7, was inhibited by tyrosine, a substrate of the YRS aminoacylation reaction. Overall, the proinflammatory activity of YRS is enhanced by MMP cleavage, which we suggest forms a feed-forward mechanism to promote inflammation.

Matrix metalloproteinases inactivate the proinflammatory functions of secreted moonlighting tryptophanyl-tRNA synthetase.

Tryptophanyl-tRNA synthetase (WRS) is a cytosolic aminoacyl-tRNA synthetase essential for protein synthesis. WRS is also one of a growing number of intracellular proteins that are attributed distinct noncanonical "moonlighting" functions in the extracellular milieu. Moonlighting aminoacyl-tRNA synthetases regulate processes such as inflammation, but how these multifunctional enzymes are themselves regulated remains unclear. Here, we demonstrate that WRS is secreted from human macrophages, fibroblasts, and endothelial cells in response to the proinflammatory cytokine interferon γ (IFNγ). WRS signaled primarily through Toll-like receptor 2 (TLR2) in macrophages, leading to phosphorylation of the p65 subunit of NF-κB with associated loss of NF-κB inhibitor α (IκB-α) protein. This signaling initiated secretion of tumor necrosis factor α (TNFα) and CXCL8 (IL8) from macrophages. We also demonstrated that WRS is a potent monocyte chemoattractant. Of note, WRS increased matrix metalloproteinase (MMP) activity in the conditioned medium of macrophages in a TNFα-dependent manner. Using purified recombinant proteins and LC-MS/MS to identify proteolytic cleavage sites, we demonstrated that multiple MMPs, but primarily macrophage MMP7 and neutrophil MMP8, cleave secreted WRS at several sites. Loss of the WHEP domain following cleavage at Met48 generated a WRS proteoform that also results from alternative splicing, designated Δ1-47 WRS. The MMP-cleaved WRS lacked TLR signaling and proinflammatory activities. Thus, our results suggest that moonlighting WRS promotes IFNγ proinflammatory activities, and these responses can be dampened by MMPs.

Neutrophil elastase-cleaved corticosteroid-binding globulin is absent in human plasma.

Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.

Granzyme B is elevated in autoimmune blistering diseases and cleaves key anchoring proteins of the dermal-epidermal junction.

In healthy skin, epidermis and dermis are anchored together at the dermal-epidermal junction (DEJ), a specialized basement membrane pivotal for skin integrity and function. However, increased inflammation in the DEJ is associated with the disruption and separation of this junction and sub-epidermal blistering. Granzyme B (GzmB) is a serine protease secreted by immune cells. Dysregulated inflammation may lead to increased GzmB accumulation and proteolysis in the extracellular milieu. Although elevated GzmB is observed at the level of the DEJ in inflammatory and blistering skin conditions, the present study is the first to explore GzmB in the context of DEJ degradation in autoimmune sub-epidermal blistering. In the present study, GzmB induced separation of the DEJ in healthy human skin. Subsequently, α6/ß4 integrin, collagen VII, and collagen XVII were identified as extracellular substrates for GzmB through western blot, and specific cleavage sites were identified by mass spectrometry. In human bullous pemphigoid, dermatitis herpetiformis, and epidermolysis bullosa acquisita, GzmB was elevated at the DEJ when compared to healthy samples, while α6/ß4 integrin, collagen VII, and collagen XVII were reduced or absent in the area of blistering. In summary, our results suggest that regardless of the initial causation of sub-epidermal blistering, GzmB activity is a common final pathway that could be amenable to a single targeted treatment approach.

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.

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.

Phosphocholine-Specific Antibodies Improve T-Dependent Antibody Responses against OVA Encapsulated into Phosphatidylcholine-Containing Liposomes.

Liposomes containing phosphatidylcholine have been widely used as adjuvants. Recently, we demonstrated that B-1 cells produce dipalmitoyl-phosphatidylcholine (DPPC)-specific IgM upon immunization of BALB/c mice with DPPC-liposomes encapsulating ovalbumin (OVA). Although this preparation enhanced the OVA-specific humoral response, the contribution of anti-DPPC antibodies to this effect was unclear. Here, we demonstrate that these antibodies are secreted by B-1 cells independently of the presence of OVA in the formulation. We also confirm that these antibodies are specific for phosphocholine. The anti-OVA humoral response was partially restored in B-1 cells-deficient BALB/xid mice by immunization with the liposomes opsonized with the serum total immunoglobulin (Ig) fraction containing anti-phosphocholine antibodies, generated in wild-type animals. This result could be related to the increased phagocytosis by peritoneal macrophages of the particles opsonized with the serum total Ig or IgM fractions, both containing anti-phosphocholine antibodies. In conclusion, in the present work, it has been demonstrated that phosphocholine-specific antibodies improve T-dependent antibody responses against OVA carried by DPPC-liposomes.

Potential of nanoparticles for allergen-specific immunotherapy - use of silica nanoparticles as vaccination platform.

INTRODUCTION: Allergen-specific immunotherapy is the only curative approach for the treatment of allergies. There is an urgent need for improved therapies, which increase both, efficacy and patient compliance. Novel routes of immunization and the use of more advanced vaccine platforms have gained heightened interest in this field. Areas covered: The current status of allergen-specific immunotherapy is summarized and novel routes of immunization and their challenges in the clinics are critically discussed. The use of nanoparticles as novel delivery system for allergy vaccines is comprehensively reviewed. Specifically, the advantages of silica nanoparticles as vaccine carriers and adjuvants are summarized. Expert opinion: Future allergen-specific immunotherapy will combine engineered hypoallergenic vaccines with novel routes of administration, such as the skin. Due to their biodegradability, and the easiness to introduce surface modifications, silica nanoparticles are promising candidates for tailor-made vaccines. By covalently linking allergens and polysaccharides to silica nanoparticles, a versatile vaccination platform can be designed to specifically target antigen-presenting cells, render the formulation hypoallergenic, and introduce immunomodulatory functions. Combining potent skin vaccination methods, such as fractional laser ablation, with nanoparticle-based vaccines addresses all the requirements for safe and efficient therapy of allergic diseases.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity.

Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen.

BACKGROUND: The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties. OBJECTIVE: We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1. METHODS: By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model. RESULTS: Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment. CONCLUSIONS: Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.

Bio-analytical method based on MALDI-MS analysis for the quantification of CIGB-300 anti-tumor peptide in human plasma.

A fully validated bio-analytical method based on Matrix-Assisted-Laser-Desorption/Ionization-Time of Flight Mass Spectrometry was developed for quantitation in human plasma of the anti-tumor peptide CIGB-300. An analog of this peptide acetylated at the N-terminal, was used as internal standard for absolute quantitation. Acid treatment allowed efficient precipitation of plasma proteins as well as high recovery (approximately 80%) of the intact peptide. No other chromatographic step was required for sample processing before MALDI-MS analysis. Spectra were acquired in linear positive ion mode to ensure maximum sensitivity. The lower limit of quantitation was established at 0.5 µg/mL, which is equivalent to 160 fmol peptide. The calibration curve was linear from 0.5 to 7.5 µg/mL, with R(2)>0.98, and permitted quantitation of highly concentrated samples evaluated by dilution integrity testing. All parameters assessed for five validation batches met the FDA guidelines for industry. The method was successfully applied to analysis of clinical samples obtained in a phase I clinical trial following intravenous administration of CIGB-300 at a dose of 1.6 mg/kg body weight. With the exception of Cmax and AUC, pharmacokinetic parameters were similar for ELISA and MALDI-MS methods.

Role of B-1 cells in the immune response against an antigen encapsulated into phosphatidylcholine-containing liposomes.

B-1 lymphocytes comprise a unique subset of B cells that differ phenotypically, ontogenetically and functionally from conventional B-2 cells. A frequent specificity of the antibody repertoire of peritoneal B-1 cells is phosphatidylcholine. Liposomes containing phosphatidylcholine have been studied as adjuvants and their interaction with dendritic cells and macrophages has been demonstrated. However, the role of B-1 cells in the adjuvanticity of liposomes composed of phosphatidylcholine has not been explored. In the present work, we studied the contribution of B-1 cells to the humoral response against ovalbumin (OVA) encapsulated into dipalmitoylphosphatidylcholine (DPPC) and cholesterol-containing liposomes. BALB/X-linked immunodeficient (xid) mice, which are deficient in B-1 cells, showed quantitative and qualitative differences in the anti-OVA antibody response compared with wild-type animals after immunization with these liposomes. The OVA-specific immune response was significantly increased in the BALB/xid mice when reconstituted with B-1 cells from naive BALB/c mice. Our results indicate the internalization of DPPC-containing liposomes by these cells and their migration from the peritoneal cavity to the spleen. Phosphatidylcholine significantly contributed to the immunogenicity of liposomes, as DPPC-containing liposomes more effectively stimulated the anti-OVA response compared with vesicles composed of dipalmitoylphosphatidylglycerol. In conclusion, we present evidence for a cognate interaction between B-1 cells and phosphatidylcholine liposomes, modulating the immune response to encapsulated antigens. This provides a novel targeting approach to assess the role of B-1 cells in humoral immunity.

New approaches to transcutaneous immunotherapy: targeting dendritic cells with novel allergen conjugates.

PURPOSE OF REVIEW: This review summarizes recent preclinical and human studies evaluating allergen-specific immunotherapy via the transcutaneous route, and provides a rationale for the application of modified allergens with reduced allergenicity. Furthermore, it covers approaches to generate hypoallergenic conjugates for specific dendritic cell targeting. RECENT FINDINGS: Efficacy and safety of specific immunotherapy by application of allergens to the skin have been demonstrated in both animal models as well as clinical trials. However, localized adverse events have been reported, and delivery of antigens via barrier-disrupted skin has been linked to the induction of unwanted T helper 2-biased immune responses and allergic sensitization. Coupling of carbohydrates to allergens has been shown to induce formation of nanoparticles, which can specifically target dendritic cells and potentiate immune responses, and by masking B-cell epitopes, can render the molecules hypoallergenic. SUMMARY: Due to its abundance of immunocompetent cells, the skin represents an attractive target tissue for novel and enhanced immunotherapeutic approaches. However, in order to avoid adverse events and therapy-induced sensitizations, transcutaneous immunotherapy requires the use of formulations with reduced allergenic potential. Combining novel hypoallergenic conjugates with painless transcutaneous immunization techniques may provide an efficient and patient-friendly alternative to the standard specific immunotherapy practices.

Isoelectric point optimization using peptide descriptors and support vector machines.

IPG (Immobilized pH Gradient) based separations are frequently used as the first step in shotgun proteomics methods; it yields an increase in both the dynamic range and resolution of peptide separation prior to the LC-MS analysis. Experimental isoelectric point (pI) values can improve peptide identifications in conjunction with MS/MS information. Thus, accurate estimation of the pI value based on the amino acid sequence becomes critical to perform these kinds of experiments. Nowadays, pI is commonly predicted using the charge-state model [1], and/or the cofactor algorithm [2]. However, none of these methods is capable of calculating the pI value for basic peptides accurately. In this manuscript, we present an new approach that can significant improve the pI estimation, by using Support Vector Machines (SVM) [3], an experimental amino acid descriptor taken from the AAIndex database [4] and the isoelectric point predicted by the charge-state model. Our results have shown a strong correlation (R(2)=0.98) between the predicted and observed values, with a standard deviation of 0.32 pH units across the complete pH range.