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Currently, infections produced by the gram-positive bacteria S. aureus represent a significant healthcare burden throughout the world. This is attributed to the ability of this bacterium to develop antibiotic resistance and efficiently evade human immune response. Therefore, research effort of many scientific laboratories worldwide is directed toward characterization of the genetic organization and molecular mechanisms responsible for S. aureus pathogenesis. This report is aimed to describe the growing body of evidence related to our understanding of the genetic organization and molecular interactions of the S. aureus leukocidins with the human cells that play an important role in bacterial pathogenesis, and represent a significant healthcare burden. Understanding of the genetic organization linked with additional mechanisms responsible for the realization of toxic potential, can help us to develop a better personalized approach for therapy against S. aureus infections. Thus, improved understanding of the molecular interactions between S. aureus leucocidins, and cell surface receptors may lead to the development of the alternative anti-microbial agents, that either independently or in combination with the current antibiotic treatment regimens will be used as an effective treatment strategy in clinic.

The microbiota can play important roles in the development of human immunity and the establishment of immune homeostasis. Lifestyle factors including diet, hygiene, and exposure to viruses or bacteria, and medical interventions with antibiotics or anti‐ulcer medications, regulate phylogenetic variability and the quality of cross talk between innate and adaptive immune cells via mucosal and skin epithelia. More recently, microbiota and their composition have been linked to protective effects for health. Imbalance, however, has been linked to immune‐related diseases such as allergy and cancer, characterized by impaired, or exaggerated immune tolerance, respectively. In this AllergoOncology position paper, we focus on the increasing evidence defining the microbiota composition as a key determinant of immunity and immune tolerance, linked to the risk for the development of allergic and malignant diseases. We discuss novel insights into the role of microbiota in disease and patient responses to treatments in cancer and in allergy. These may highlight opportunities to improve patient outcomes with medical interventions supported through a restored microbiome.

Chronic rhinosinusitis (CRS) is thought to be a multifactorial disease that includes a direct involvement of bacteria that trigger inflammation and contribute to CRS pathogenesis. Staphylococcus aureus infection and persistence is associated with chronic rhinosinusitis (CRS), and it may be particularly relevant in the form with nasal polyps (CRSwNP). The large array of exotoxins deployed by S. aureus is instrumental for the bacterium to warrant its infection and dissemination in different human body districts. Here, we analyze the common Th2 environment in CRSwNP and prospect a possible dynamic role played by S. aureus leukocidins in promoting this chronic inflammation, considering leukocidin ED (LukED) as a strong prototype candidate worth of therapeutic investigation. CCR5 is an essential target for LukED to exert its cytotoxicity towards T cells, macrophages and dendritic cells. Therefore, CCR5 blockade might be an interesting therapeutic option for CRS and, more specifically, persistent and relapsing CRSwNP. In this perspective, the arsenal of CCR5 antagonists being developed to inhibit HIV-1 entry (CCR5 being the major HIV-1 co-receptor) could be easily repurposed for CRS therapeutic investigation. Finally, direct targeting of LukED by neutralizing antibodies could represent an important additional solution to S. aureus infection.

Learning from the lengthy fight against HIV-1, influenza, and Ebola virus infection, broadly neutralizing antibodies (bnAbs), directed at conserved regions of surface proteins crucial to virus entry (Env, hemagglutinin, and GP, respectively), are an essential resource for passive as well as active immunization. Rare in their emergence and antigen recognition mode, bnAbs are active toward a large set of different viral strains. Isolation, characterization and production of bnAbs lead to their possible use in passive immunotherapy and form the basis for an educated effort in the development of vaccines for universal coverage. SARS-CoV-2-specific antibodies targeting the spike receptor binding domain (RBD) may lead to antibody dependent enhancement (ADE) of infection, possibly hampering the field of vaccine development. This perspective points to the identification of conserved regions in the spike of SARS-CoV-2, SARS-CoV, and MERS-CoV through investigation, dissection and recombinant production of isolated moieties. These spike moieties should be capable of independent folding and allow the detection as well as the elicitation of bnAbs, thus setting the basis for an effective passive immunotherapy and the development of a universal vaccine against human epidemic coronaviruses (HCoVs). SARS, MERS and, most of all, COVID-19 demonstrate that humanity is the target of HCoV, preparedness for future hits is thus no longer an option.

Omalizumab is an anti-IgE humanized monoclonal antibody approved for the treatment of severe asthma and chronic spontaneous urticaria. Omalizumab binds free serum IgE and antagonizes its interaction with FceRI, which is considered the main pharmacodynamic mechanism responsible for the clinical response to the treatment. The reduction of IgE serum concentration down-regulates the cellular expression of FceRI on basophils. However, the biological events occurring on basophils during the therapy with omalizumab are multiple and complex. Here we review the current evidence regarding the specific biological effects of omalizumab on basophils in patients with asthma and chronic spontaneous urticaria. In addition to the modulation of IgE receptors, omalizumab may affect basophils homeostasis, intra-cellular signaling, cellular responsiveness/activation and cytokine release. These effects may be partially responsible for the clinical success of omalizumab and potentially provide useful biological markers for future assessment of the clinical response to the treatment. However, further investigation is required to better elucidate the role of basophils during the treatment with omalizumab.

A ball resonator, positioned on the tip of an optical fiber, has been developed as a biosensor for the prototypic detection of thrombin. The device was fabricated with a fast and repeatable CO2 laser splicing method, followed by gold-sputtering and functionalization for the measurement of various protein concentrations. The ball resonator acts as a weak interferometer with a return loss below − 50 dB, and it is interrogated with an optical backscatter reflectometer measuring the reflection spectrum. We report here a sample presenting high sensitivity (1273.74 nm/RIU, RIU = refractive index units), which allows protein detection in the range 0.4–100 pM, with a limit of detection of 1.56 pM in logarithmic response.

Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both antitumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE-mediated immune responses against cancer, to derive novel insights into more effective treatments. Coincident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state-of-the-art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intratumoral innate allergy effector cells and mediators, IgE-mediated tumour antigen cross-presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the crosstalk between allergic response and cancer is paving the way for new avenues of treatment.

An aptasensor based on etched tilted fiber Bragg grating (eTFBG) is developed on a single-mode optical fiber targeting biomolecule detection. TFBGs were chemically etched using hydrofluoric acid (HF) to partially remove the fiber cladding. The sensor response was coarsely interrogated, resulting on a sensitivity increase from 1.25 nm/RIU (refractive index unit) at the beginning of the process, up to 23.38 nm/RIU at the end of the etching, for a RI range from 1.3418 to 1.4419 RIU. The proposed aptasensor showed improved RI sensitivity as compared to the unetched TFBG, without requiring metal depositions on the fiber surface or polarization control during the measurements. The proposed sensor was tested for the detection of thrombin-aptamer interactions based on silane-coupling surface chemistry, with thrombin concentrations ranging from 2.5 to 40 nM. Functionalized eTFBGs provided a competitive platform for biochemical interaction measurements, showing sensitivity values ranging from 2.3 to 3.3 p.m./nM for the particular case of thrombin detection.&nbsp

Efforts to improve existing anti-HIV-1 therapies or develop preventatives have identified CCR5 as an important target and CCL5 as an ideal scaffold to sculpt potent HIV-1 entry inhibitors. We created novel human CCL5 variants that exhibit exceptional anti-HIV-1 features using recombinant lactobacilli (exploited for live microbicide development) as a screening platform. Protein design, expression and anti-HIV-1 activity flowed in iterative cycles, with a stepwise integration of successful mutations and refinement of an initial CCL5 mutant battery towards the generation of two ultimate CCL5 derivatives, a CCR5 agonist and a CCR5 antagonist with similar anti-HIV-1 potency. The CCR5 antagonist was tested in human macrophages and against primary R5 HIV-1 strains, exhibiting cross-clade low picomolar IC50 activity. Moreover, its successful combination with several HIV-1 inhibitors provided the ground for conceiving therapeutic and preventative anti-HIV-1 cocktails. Beyond HIV-1 infection, these CCL5 derivatives may now be tested against several inflammation-related pathologies where the CCL5:CCR5 axis plays a relevant role.

CCR5 and its interaction with chemokine ligands have been crucial for understanding and tackling HIV-1 entry into target cells. However, over time, CCR5 has witnessed an impressive transition from being considered rather unimportant in physiology and pathology to becoming central in a growing number of pathophysiological conditions. It now turns out that the massive efforts devoted to combat HIV-1 entry by interfering with CCR5, and the subsequent production of chemokine ligand variants, small chemical compounds, and other molecular entities and strategies, may set the therapeutic standards for a wealth of different pathologies. Expressed on various cell types, CCR5 plays a vital role in the inflammatory response by directing cells to sites of inflammation. Aside HIV-1, CCR5 has been implicated in other infectious diseases and non-infectious diseases such as cancer, atherosclerosis, and inflammatory bowel disease. Individuals carrying the CCR5Δ32 mutation live a normal life and are warranted a natural barrier to HIV-1 infection. Therefore, CCR5 antagonism and gene-edited knockout of the receptor gained growing interest for the therapeutic role that CCR5 blockade may play in the attenuation of the severity or progression of numerous diseases.

While desired for the cure of allergy, regulatory immune cell subsets and non-classical Th2-biased inflammatory mediators in the tumour microenvironment can contribute to immune suppression and escape of tumours from immunological detection and clearance. A key aim in the cancer field is therefore to design interventions that can break immunological tolerance and halt cancer progression, whereas on the contrary allergen immunotherapy exactly aims to induce tolerance. In this position paper, we review insights on immune tolerance derived from allergy and from cancer inflammation, focusing on what is known about the roles of key immune cells and mediators. We propose that research in the field of AllergoOncology that aims to delineate these immunological mechanisms with juxtaposed clinical consequences in allergy and cancer may point to novel avenues for therapeutic interventions that stand to benefit both disciplines. This article is protected by copyright. All rights reserved.

IgE is a highly elusive antibody class, yet a tremendously powerful elicitor of immune reactions. Despite huge efforts spent on the characterization and understanding of the IgE system many questions remain either unanswered or only marginally addressed. One above all relates to the role of IgE. A common doubt is based on whether IgE mode of action should only be relegated to anti-parasite immunity and allergic manifestations. In search for a hidden role of IgE, reports from several laboratories are described herein in which a natural IgE link to cancer or the experimental redirection of IgE against cancer have been investigated. Epidemiological and investigational studies are trying to elucidate a possible direct intervention of endogenous IgE against cancer, raising thus far no definitive evidence. Conversely, experimental approaches implementing several strategies and engineered IgE formats built up a series of convincing results indicating that cancer might be tackled by the effector functions of this immunoglobulin class. Because of its peculiar immune features, IgE may present a superior anti-tumor performance as compared to IgG. However, extreme care should be taken on how IgE-based anti-tumor approaches should be devised. Overall, IgE appears as a promising resource, likely destined to enrich the anti-cancer arsenal.

The IgE-mediated immune system activation can be redirected to combat tumors. Mouse and human IgE have been shown to provide a potent adjuvant effect in antitumor vaccination, with a crucial role played by FcεRI. This effect results from T cell-mediated adaptive immune response. Modified vaccinia virus Ankara (MVA) has been used to infect IgE-loaded tumor cells. These results led to a shift toward a highly safe protocol employing membrane IgE (mIgE), thus eliminating any possible anaphylactogenicity caused by circulating IgE. Evidence that human mIgE and a truncated version lacking IgE Fabs (tmIgE) bind and activate FcεRI has been fundamental and forms the core of this report. Human tmIgE has been engineered into a recombinant MVA (rMVA-tmIgE), and the expression of tmIgE and its transport to the surface of rMVA-tmIgE–infected cells has been detected by Western blot and cytofluorimetry, respectively. FcεRI activation by tmIgE has been confirmed by the release of β-hexosaminidase in a cell-to-cell contact assay using human FcεRI-transfected RBL-SX38 cells. The rMVA-tmIgE antitumor vaccination strategy has been investigated in FcεRIα−/− human FcεRIα+ mice, with results indicating a level of protection comparable to that obtained using soluble human IgE tumor cell loading. The rMVA-tmIgE vector represents a device that suits safe IgE-based antitumor vaccines, harboring the possibility to couple tmIgE with other gene insertions that might enhance the antitumor effect, thus bringing the field closer to the clinics.

SummaryCCR5, the major HIV-1 coreceptor, is a primary target for HIV-1 entry inhibition strategies. CCL5/RANTES, a natural CCR5 ligand, is one of the most potent HIV-1 entry inhibitors and, therefore, an ideal candidate to derive HIV-1 blockers. Peptides spanning the RANTES N-loop/β1-strand region act as specific CCR5 antagonists, with their hydrophobic N- and C termini playing a crucial role in virus blockade. Here, hydrophobic surfaces were enhanced by tryptophan substitution of aromatic residues, highlighting position 27 as a critical hot spot for HIV-1 blockade. In a further molecular evolution step, C-terminal engraftment of RANTES 40′ loop produced a peptide with the highest solubility and anti-HIV-1 activity. These modified peptides represent leads for the development of effective HIV-1 inhibitors and microbicides.

The chemokine receptor CCR5 is utilized as a critical coreceptor by most primary HIV-1 strains. While the lack of structural information on CCR5 has hampered the rational design of specific inhibitors, mimetics of the chemokines that naturally bind CCR5 can be molecularly engineered. We used a structure-guided approach to design peptide mimetics of the N-loop and β1-strand regions of regulated on activation normal T-cell-expressed and secreted (RANTES)/CCL5, which contain the primary molecular determinants of HIV-1 blockade. Rational modifications were sequentially introduced into the N-loop/β1-strand sequence, leading to the generation of mimetics with potent activity against a broad spectrum of CCR5-specific HIV-1 isolates (IC50 range: 104–640 nM) but lacking activity against CXCR4-specific HIV-1 isolates. Functional enhancement was initially achieved with the stabilization of the N loop in the β-extended conformation adopted in full-length RANTES, as confirmed by nuclear magnetic resonance (NMR) analysis. However, the most dramatic increase in antiviral potency resulted from the engraftment of an in silico-optimized linker segment designed using de novo structure-prediction algorithms to stabilize the C-terminal α-helix and experimentally validated by NMR. Our mimetics exerted CCR5-antagonistic effects, demonstrating that the antiviral and proinflammatory functions of RANTES can be uncoupled. RANTES peptide mimetics provide new leads for the development of safe and effective HIV-1 entry inhibitors.—Lusso, P., Vangelista, L., Cimbro, R., Secchi, M., Sironi, F., Longhi, R., Faiella, M., Maglio, O., Pavone, V. Molecular engineering of RANTES peptide mimetics with potent anti-HIV-1 activity.

The development of effective microbicides for the prevention of HIV-1 sexual transmission represents a primary goal for the control of AIDS epidemics worldwide. A promising strategy is the use of bacteria belonging to the vaginal microbiota as live microbicides for the topical production of HIV-1 inhibitors. We have engineered a human vaginal isolate of Lactobacillus jensenii to secrete the anti-HIV-1 chemokine RANTES, as well as C1C5 RANTES, a mutated analogue that acts as a CCR5 antagonist and therefore is devoid of proinflammatory activity. Full-length wild-type RANTES and C1C5 RANTES secreted by L. jensenii were purified to homogeneity and shown to adopt a correctly folded conformation. Both RANTES variants were shown to inhibit HIV-1 infection in CD4 + T cells and macrophages, displaying strong activity against HIV-1 isolates of different genetic subtypes. This work provides proof of principle for the use of L. jensenii -produced C1C5 RANTES to block HIV-1 infection of CD4 + T cells and macrophages, setting the basis for the development of a live anti-HIV-1 microbicide targeting CCR5 in an antagonistic manner.

Development of effective topical microbicides for the prevention of HIV-1 sexual transmission represents a primary goal for the control of the AIDS pandemic. The viral co-receptor CCR5, used by the vast majority of primary HIV-1 isolates, is considered a primary target molecule. RANTES and its derivatives are the most suitable protein-based compounds to fight HIV-1 via CCR5 targeting. Yet, receptor activation should be avoided to prevent pro-inflammatory effects and possibly provide anti-inflammatory properties. C1C5 RANTES is a chemokine mutant that exhibits high anti-HIV-1 potency coupled with CCR5 antagonism. However, the need for the formation of an N-terminal intramolecular disulphide bridge between non-natural cysteine residues at position 1 and 5 represents a challenge for the correct folding of this protein in recombinant expression systems, a crucial step towards its development as a microbicide against HIV-1. We report here a rare case of superior folding in a prokaryote as compared to an eukaryotic expression system. Production of C1C5 RANTES was highly impaired in CHO cells, with a dramatic yield reduction compared to that of wild type RANTES and secretion of the molecule as disulphide-linked dimer. Conversely, a human vaginal isolate of Lactobacillus jensenii engineered to secrete C1C5 RANTES provided efficient delivery of the monomeric protein. This and other reports on successful secretion of complex proteins indicate that lactic acid bacteria are an excellent system for the expression of therapeutic proteins, which can be used as a platform for the engineering of conceptually novel RANTES mutants with potent anti-HIV-1 activity.

The G protein-coupled receptor CCR5 is a human chemokine receptor involved in the activation and migration of leukocytes. CCR5 is also the major HIV-1 coreceptor that, together with human CD4 and the viral glycoprotein gp120, promotes virus entry into host cells. Thus inhibition of the CCR5-gp120 interaction presents a promising route to prevent HIV infections. Atomic structural details of the interaction between CCR5 and its cognate chemokines or gp120 are presently unknown due to the general difficulties of membrane protein structure determination. Here, we report the high-yield expression of human CCR5 in baculovirus-infected Sf9 insect cells. Highly purified (>90%) CCR5 is obtained in detergent-solubilized form at yields of about 1 mg/l cell culture. The conformational integrity of recombinant CCR5 after purification is shown by immunoprecipitation with the conformation-dependent monoclonal antibody 2D7, CD and NMR spectroscopy. The detergent micelles contain CCR5 in monomeric and dimeric forms, which can be separated by size exclusion chromatography and characterized individually. Further functional characterization by isothermal titration calorimetry indicates that the recombinant receptor interacts with its cognate chemokine RANTES. This interaction is strongly suppressed when sulfation of CCR5 is inhibited in the insect cells.

Interaction of secretory IgE with FcεRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcεRIα to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcεRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cε2-Cε3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igα/Igβ BCR accessory proteins), and both εBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcεRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca2+ responses in the basophil cell line, while membrane IgE-FcεRI complexes were detected by immunoprecipitation. FcεRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcεRI in several cellular entities suggests a possible membrane IgE-FcεRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.

Background: The IgE-mediated activation of effector cells and antigen-presenting cells through the high-affinity receptor for IgE (FceRI) represents a key pathomechanism in type I allergy and many forms of asthma. Objective: We sought to establish an in vitro molecular model for the interaction of human FceRI, IgE, and the corresponding allergen and to identify monoclonal anti-human IgE antibodies with a therapeutic profile different from previously established anti-IgE antibodies. Methods: Human FceRI α chain, a human monoclonal allergen-specific IgE antibody (chimeric Bip 1), and the corresponding allergen, the major birch pollen allergen Bet v 1, were produced as recombinant proteins and analyzed by means of circular dichroism and native overlays, respectively. Using this molecular model, as well as negative stain immunoelectron microscopic analysis, and in vitro cultivated human basophils, we characterized mouse anti-human IgE antibodies. Results: We established a molecular model for the interaction of human IgE with FceRI. Using this molecular model, we identified a nonanaphylactic anti-human IgE antibody fragment (Fab12), which blocked the IgE-FceRI interaction and reacted with effector cell‐bound IgE. Conclusion: Fab12 represents a candidate molecule for therapy of atopy and asthma because it can be used for the depletion of circulating IgE antibodies, as well as for the depletion of IgEbearing cells. (J Allergy Clin Immunol 2001;108:409-16.)

Membrane-bound immunoglobulins have, in addition to the transmembrane and cytoplasmic portions, an extracellular membrane-proximal domain (EMPD), absent in the secretory forms. EMPDs of immunoglobulin isotypes α, γ, and e contain cysteines whose role has so far not been elucidated. Using a genetic strategy, we investigated the ability of these cysteines to form disulfide bridges. Shortened versions of human membrane immunoglobulins, depleted of cysteines known to form intermolecular disulfide bonds, were constructed and expressed on the surface of a B-cell line. The resulting membrane proteins contain a single chain fragment of variable regions (scFv) linked to the dimerizing domain from the immunoglobulin heavy chains (CH3 for α and γ or CH4 for e isotypes), followed by the corresponding EMPD and the transmembrane and cytoplasmic domains. The two functional membrane versions of the e chain, containing the short and long EMPD, were analyzed. Our results show that the single cysteine within α1L and γ1 EMPD...

We report the characterization of a domain of the major timothy grass pollen allergen, Phl p 5A, which contains the binding site for a human monoclonal IgE antibody. The human monoclonal IgE antibody fragment (Fab) was obtained from an IgE combinatorial library constructed from lymphocytes of a grass pollen-allergic patient. An 11.2-kD N-terminal fragment representing approximately one third of the complete Phl p 5A allergen could be identified to contain the binding site for the IgE Fab. The rPhl p 5A fragment revealed an extremely high allergenic activity in skin test experiments which in some cases equaled that of the complete Phl p 5A allergen. The rPhl p 5A domain thus represents an allergen fragment containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation.

BACKGROUND—Almost 4% of the population suffer from food allergy which is an adverse reaction to food with an underlying immunological mechanism. AIMS—To characterise one of the most frequent IgE defined food allergens, fish parvalbumin. METHODS—Tissue and subcellular distribution of carp parvalbumin was analysed by immunogold electron microscopy and cell fractionation. Parvalbumin was purified to homogeneity, analysed by mass spectrometry and circular dichroism (CD) spectroscopy, and its allergenic activity was analysed by IgE binding and basophil histamine release tests. RESULTS—The isoelectric point (pI) 4.7 form of carp parvalbumin, a three EF-hand calcium-binding protein, was purified to homogeneity. CD analysis revealed a remarkable stability and refolding capacity of calcium-bound parvalbumin. This may explain why parvalbumin, despite cooking and exposure to the gastrointestinal tract, can sensitise patients. Purified parvalbumin reacted with IgE of more than 95% of individuals allergic to fish, induced dose-dependent basophil histamine release and contained, on average, 83% of the IgE epitopes present in other fish species. Calcium depletion reduced the IgE binding capacity of parvalbumin which, according to CD analysis, may be due to conformation-dependent IgE recognition. CONCLUSIONS—Purified carp parvalbumin represents an important cross reactive food allergen. It can be used for in vitro and in vivo diagnosis of fish-induced food allergy. Our finding that the apo-form of parvalbumin had a greatly reduced IgE binding capacity indicates that this form may be a candidate for safe immunotherapy of fish-related food allergy. Keywords: food allergy; parvalbumin; circular dichroism; epitopes; antibodies; immunochemistry

Atopic allergy is a genetically determined immunodisorder that affects almost 20% of the population worldwide. Immediate symptoms of type I allergy are caused by the release of biologic mediators from effector cells induced by IgE-allergen complexes that cross-link the high-affinity receptor for IgE (FceRI). Chronic disease manifestations result from allergen-specific T-cell activation, a process that is enhanced when allergens are presented via FceRI-bound IgE. We report the baculovirus expression, as soluble recombinant proteins, of the minimal units required for human IgE and FceRI interaction: Ce3 represents the third constant domain of the IgE heavy chain, and α2 is the membrane-proximal Ig-like module from FceRIα. Native overlay experiments showed binding of human FceRIα to recombinant Ce3 and of natural or recombinant human IgE to recombinant α2. Moreover, recombinant Ce3 inhibited binding of natural IgE antibodies to α2, and preincubation of human IgE with α2 inhibited anti-IgE‐triggered histamine release from human basophils. Isolated Ce3 and α2 can now be used for the molecular and structural analysis of the IgE-FceRI interaction, as well as for diagnostic and therapeutic applications.

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind-pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF-hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen-specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7-homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium-bound and apo-rPhl p 7 indicated that differences in IgE recognition may be due to calcium-induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation-dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.

The Bühlmann CAST 2000 enzyme-linked immunosorbent assay is a potentially useful assay for measuring sulfidoleukotrienes released in vitro by allergen-challenged basophils. However, we observed that the positive-control reagent yielded positive signals in cell-free systems. These false-positive results depended on using a mouse anti-FcεRI monoclonal antibody and were prevented by degranulation-inducing reagents other than mouse monoclonal antibodies.

The vertebrate striated muscle protein titin is thought to play a critical role in myofibril assembly and passive tension. The recently determined complete primary structure of titin revealed a modular architecture that opens the way to a structural characterisation and the understanding of essential properties of this molecule through dissection into units that are structurally and/or functionally relevant. To understand the assembly process of titin, and ultimately the molecular basis of its elastic behaviour, we studied the thermodynamic properties of module pairs, the smallest structural unit that includes a module-module interface. Thus, selected module pairs and their component single modules from the I-band part of the titin molecule were expressed in Escherichia coli and their heat-induced and denaturant-induced unfolding was investigated with a combination of techniques (circular dichroism, fluorescence spectroscopy and nuclear magnetic resonance). The stabilities of single modules and pairs were determined from denaturation experiments. The module interface was also modelled on the basis of the sequence alignment of all approximately 40 immunoglobulin like modules from the I-band and the known structure of one of them. Our results show that all modules and module pairs examined are independently folded in solution. When covalently linked, although weakly interacting, they still behave as autonomous co-operative units upon unfolding. These observations lead us to suggest that folding of titin in vitro is a hierarchical event and that weak interactions between its adjacent modules must only partly account for its presumed elastic function. J Mol Biol

Tetanus neurotoxin (TeNT) blocks neuroexocytosis via a zinc-endopeptidase activity highly specific for vescicle-associated membrane protein(VAMP)/synaptobrevin. TeNT is the prototype of clostridial neurotoxins, a new family of metalloproteinases. They consist of three domains and the proteolytic activity is displayed by the 50-kDa light chain (L chain). The L chain was isolated here in the native state from bacterial filtrates of Clostridium tetani and its structure was studied via circular dichroism (CD) and fluorescence spectroscopy. The secondary structure content (27% alpha-helix and 43% beta-sheet), estimated by far-ultraviolet CD measurements, was in reasonable agreement with that obtained by standard predictive methods (25% alpha-helix and 49% beta-sheet). Moreover, the hypothetical zinc-binding motif, encompassing residues His-Glu-Leu-Ile-His, was correctly predicted to be in alpha-helical conformation, as also expected on the basis of the geometrical requirements for a correct coordination of the zinc ion. Both near-ultraviolet CD and fluorescence data strongly suggest that the single Trp43 residue is buried and constrained in a hydrophobic environment, likely distant from the zinc ion located in the active-site cleft. The contribution of the bound zinc ion to the overall conformation of TeNT L chain was investigated by different and complementary techniques, including spectroscopic (far- and near-ultraviolet CD, fluorescence, second derivative absorption spectroscopy) as well as proteolytic probes. The results indicate that the zinc ion plays little, if any, role in determining the structural properties of the L chain molecule. Similarly, the metal-free apo-enzyme and the holo-protein share common stability features evaluated in respect to different physico-chemical parameters (pH, temperature and urea concentration). These results parallel those obtained on thermolysin, a zinc-dependent neutral endoprotease from Bacillus thermoproteolyticus, where both conformational and stability properties are unchanged upon zinc removal.

Background: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. Methods: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. Results: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. Conclusions: in silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders. Copyright (C) 2011 S. Karger AG, Basel Ist Sci San Raffaele, IT-20132 Milan, Italy Univ Parma, Dept Expt Med, I-43100 Parma, Italy Univ Parma, Dept Gen & Inorgan Chem Analyt Chem & Phys Chem, I-43100 Parma, Italy CNR, Inst Chem & Mol Recognit, I-20133 Milan, Italy Universidade Federal de São Paulo, Dept Biophys, São Paulo, Brazil Universidade Federal de São Paulo, Dept Biophys, São Paulo, Brazil Web of Science

Activation of the antigen-IgE-FcɛRI axis results in a potent inflammatory state. The redirection of this IgE-mediated activation of the immune system from allergic reactions toward tumors is the main theme of the new AllergoOncology field. Our particular approach has been to employ IgE as an adjuvant in anti-tumor vaccination. IgE-coated tumor cells can protect against tumor challenge, an observation that supports the involvement of IgE in anti-tumor immunity. The adjuvant effect of IgE was shown to result from eosinophil-dependent priming of the T-cell-mediated adaptive immune response. Moreover, the role of FcɛRI in IgE anti-tumor adjuvanticity has been recently demonstrated. The interaction of tumor cell-bound IgE with receptors triggers the release of mediators with following recruitment of effector cells, cell killing and tumor antigen cross-priming. Starting from these evidences, several improvements toward a simple and universal use of IgE in anti-tumor cellular vaccines have been accomplished. In view of narrowing the gap between experimental models and therapeutic applications, the field is now shifting toward a humanized systems, employing human IgE and human FcɛRIα transgenic mice.

Working with C57BL/6 mouse tumor models, we had previously demonstrated that vaccination with IgE-coated tumor cells can protect against tumor challenge, an observation that supports the involvement of IgE in antitumor immunity. The adjuvant effect of IgE was shown to result from eosinophil-dependent priming of the T cell-mediated adaptive immune response. The protective effect is likely to be mediated by the interaction of tumor cell-bound IgE with receptors, which then trigger the release of mediators, recruitment of effector cells, cell killing and tumor Ag cross-priming. It was therefore of utmost importance to demonstrate the strict dependence of the protective effect on IgE receptor activation. First, the protective effect of IgE was confirmed in a BALB/c tumor model, in which IgE-loaded modified VV Ankara-infected tumor cells proved to be an effective cellular vaccine. However, the protective effect was lost in Fc(epsilon)RIalpha(-/-) (but not in CD23(-/-)) knockout mice, showing the IgE-Fc(epsilo)nRI interaction to be essential. Moreover, human IgE (not effective in BALB/c mice) had a protective effect in the humanized knockin mouse (Fc(epsilon)RIalpha(-/-) hFc(epsilon)RIalpha(+)). This finding suggests that the adjuvant effect of IgE could be exploited for human therapeutics.

Working with C57BL/6 mouse tumor models, we had previously demonstrated that vaccination with IgE-coated tumor cells can protect against tumor challenge, an observation that supports the involvement of IgE in antitumor immunity. The adjuvant effect of IgE was shown to result from eosinophil-dependent priming of the T cell-mediated adaptive immune response. The protective effect is likely to be mediated by the interaction of tumor cell-bound IgE with receptors, which then trigger the release of mediators, recruitment of effector cells, cell killing and tumor Ag cross-priming. It was therefore of utmost importance to demonstrate the strict dependence of the protective effect on IgE receptor activation. First, the protective effect of IgE was confirmed in a BALB/c tumor model, in which IgE-loaded modified VV Ankara-infected tumor cells proved to be an effective cellular vaccine. However, the protective effect was lost in FcεRIα−/− (but not in CD23−/−) knockout mice, showing the IgE-FcεRI interaction to be essential. Moreover, human IgE (not effective in BALB/c mice) had a protective effect in the humanized knockin mouse (FcεRIα−/− hFcεRIα+). This finding suggests that the adjuvant effect of IgE could be exploited for human therapeutics.

Background: The interaction of immune complexes consisting of allergens and allergen–specific IgE with the high–affinity Fcε receptor represents the key event in the induction of symptoms in type I allergic individuals. Immediate–type symptoms result from the release of biological mediators due to allergen–induced cross–linking of FcεRI receptors on mast cells and basophils, whereas FcεRI–mediated presentation of allergen–IgE complexes may contribute to late–phase symptoms through enhanced T cell activation. The interaction of allergens/allergen–specific IgE/FcεRI represents, therefore, an important target for therapeutic intervention strategies in type I allergy. Methods and Results: A molecular model of the allergen–IgE–FcεRI interaction was established. It consists of recombinant purified Bet v 1, the major birch pollen allergen, a chimeric Bet v 1 specific monoclonal IgE antibody, and the baculovirus–expressed purified human alpha chain of FcεRI. The chimeric Bet v 1–specific IgE antibody consists of the light chain and the heavy chain variable region of a mouse monoclonal Bet v 1 specific antibody, Bip 1, and the constant region of human IgE. The interaction of rBet v 1, chimeric Bip 1, and human alpha chain was investigated by overlay experiments. Nitrocellulose–immobilized recombinant alpha chains was incubated with chimeric Bip 1 and, for control purposes, with mouse–derived Bip 1. Bound chimeric Bip 1 was detected with 125I–labeled rBet v 1. The specific interaction of rBetv 1, chimeric Bip 1, and recombinant human alpha chain is demonstrated. We thus establish a molecular model of the allergen/IgE/alpha chain interaction. The usefulness of the described in vitro system is exemplified by the identification of a mouse monoclonal antihuman IgE antibody which blocked the IgE–alpha chain interaction. Conclusions: The module system consisting of rBet v 1, chimeric Bip 1, and recombinant alpha chain may be used for the identification of competitors of the allergic effector reaction by means of high throughput screening of compounds or by combinatorial chemistry.

Epidemiological studies have suggested inverse associations between allergic diseases and malignancies. As a proof of concept for the capability of immunoglobulin E (IgE) to destruct tumor cells, several experimental strategies have evolved to specifically target this antibody class towards relevant tumor antigens. It could be demonstrated that IgE antibodies specific to overexpressed tumor antigens have been superior to any other immunoglobulin class with respect to antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) reactions. In an alternative approach, IgE nonspecifically attached to tumor cells proved to be a powerful adjuvant establishing tumor-specific immune memory. Active Th2 immunity could also be achieved by applying an oral immunization regimen using mimotopes, i.e. epitope mimics of tumor antigens. The induced IgE antibodies could be cross-linked by live tumor cells leading to tumoricidic mediator release. Thus, IgE antibodies may not only act in natural tumor surveillance, but could possibly also be exploited for tumor control in active and passive immunotherapy settings. Thereby, eosinophils, mast cells and macrophages can be armed with the cytophilic IgE and become potent anti-tumor effectors, able to trace viable tumor cells in the tissues. It is strongly suggested that the evolving new field AllergoOncology will give new insights into the role of IgE-mediated allergy in malignancies, possibly opening new avenues for tumor therapy.

The discovery that the CC chemokines RANTES, MIP-1alpha and MIP-1beta act as potent natural inhibitors of HIV-1, the causative agent of AIDS, and the subsequent identification of CCR5 as a major virus coreceptor have triggered a wealth of basic and applied research approaches aimed at developing safe and effective viral entry inhibitors. Some of these efforts have focused on RANTES engineering with the goal of enhancing the antiviral activity of the native molecule while reducing or abrogating its inflammatory properties. The wavefront generated a decade ago is still on its course, with a flow of promising leads constantly emerging and being evaluated in preclinical studies. Here, we present an overview of this rapidly evolving field, highlighting the most important features of RANTES molecular architecture and structure-function relationships.

HIV initiates its infectious cycle by docking to CD4 and a chemokine receptor, most commonly CCR5. RANTES, a natural CCR5 ligand, is a potent inhibitor of HIV-1. Despite the lack of structural information on the RANTES-CCR5 complex, determinants of HIV blockade were previously identified within the RANTES N-loop and beta1-strand regions. A prototype N-loop/beta1-strand peptide, named R11-29, contains two terminal hydrophobic stretches separated by a central hydrophilic region. Here, the role of the terminal hydrophobic clusters was investigated by means of amino acid substitutions or deletions. Most hydrophobic residues in these clusters were shown to be fundamental for the anti-HIV activity. However, increasing the hydrophobicity of the two clusters using non-natural amino acids did not significantly improve the potency of the peptides. These results may provide instrumental knowledge for the rational design of RANTES-derivative molecules with increased anti-HIV activity.

We previously identified the major structural determinants of CCR5 binding and HIV blockade in RANTES, describing linear RANTES-derived peptides with biological activity in the low micromolar range (Nardese et al., 2001). To deepen our understanding of RANTES structure-function relations and obtain more potent antiviral peptido-mimetics, we have extensively mutagenized the prototypic peptide, R11-29. This presents two clusters of hydrophobic residues at its termini, (corresponding to RANTES N-loop and b1-strand) connected by a positivelycharged linker. Single or multiple alanine substitutions within the Nor C-terminal hydrophobic clusters resulted in a dramatic loss of antiviral activity, whereas deletion of selected residues within the hydrophilic linker had no major functional consequences. Based on RANTES 3D structure, we designed a series of modified peptides, resulting in a progressive increase in specific antiviral activity. These peptides also displayed anti-inflammatory properties blocking RANTES-elicited lymphocyte chemotaxis. Through serial passages in culture in the presence of increasing concentrations of the most effective antiviral peptides, we have derived variants of the R5 HIV-1 isolate BaL resistant to the peptide inhibitory activity. Complete sequencing of the envelope genes from such variants is currently underway. Our results provide new insights into the structure of the receptor-binding region of RANTES and identify new antiviral peptides that may be instrumental in the development of effective HIV-1 entry inhibitors. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Interaction of secretory IgE with FcepsilonRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcepsilonRIalpha to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcepsilonRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cepsilon2-Cepsilon3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igalpha/Igbeta BCR accessory proteins), and both epsilonBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcepsilonRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca(2+) responses in the basophil cell line, while membrane IgE-FcepsilonRI complexes were detected by immunoprecipitation. FcepsilonRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcepsilonRI in several cellular entities suggests a possible membrane IgE-FcepsilonRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.