Conformational Epitopes Research Articles

Allergenicity of Egg White Powder Compared to Baked Egg White Powder

There are several new products with baked egg powder marketed for possible prevention of food allergies. Previous studies have shown that standard egg white powder is equivalent to raw egg whites in allergenicity. It is known that heating destroys many conformational epitopes and reduces allergenicity in eggs, thus our aim was to compare the binding of serum IgE from egg-allergic patients to different formulations of egg white powders to evaluate the possible allergenicity of these baked egg products.

Potent transmission-blocking monoclonal antibodies from naturally exposed individuals target a conserved epitope on Plasmodium falciparum Pfs230

Pfs230 is essential for Plasmodium falciparum transmission to mosquitoes and is the protein targeted by the most advanced malaria-transmission-blocking vaccine candidate. Prior understanding of functional epitopes on Pfs230 is based on two monoclonal antibodies (mAbs) with moderate transmission-reducing activity (TRA), elicited from subunit immunization. Here, we screened the B cell repertoire of two naturally exposed individuals possessing serum TRA and identified five potent mAbs from sixteen Pfs230 domain-1-specific mAbs. Structures of three potent and three low-activity antibodies bound to Pfs230 domain 1 revealed four distinct epitopes. Highly potent mAbs from natural infection recognized a common conformational epitope that is highly conserved across P.falciparum field isolates, while antibodies with negligible TRA derived from natural infection or immunization recognized three distinct sites. Our study provides molecular blueprints describing P.falciparum TRA, informed by contrasting potent and non-functional epitopes elicited by natural exposure and vaccination.

New, Structural Database of Allergenic Proteins (SDAP) Provides 3D models with Incorporated Bioinformatics Tools

The 3D structures of allergenic proteins are needed to determine conformational epitopes contributing to allergen cross-reactivity. SDAP now includes new models and bioinformatics tools for predicting allergenic propensity and epitopes.

Epitope Mapping of an Anti-EpCAM Monoclonal Antibody (EpMab-37) Using the Alanine Scanning Method.

The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, and plays critical roles in cell adhesion, proliferation, and tumorigenesis. EpCAM has been considered as a promising target for tumor diagnosis and therapy. Anti-EpCAM monoclonal antibodies (mAbs) have been developed for EpCAM-overexpressed tumors, and several clinical trials have demonstrated promising outcomes. We previously established an anti-EpCAM mAb, EpMab-37 (mouse IgG1, kappa), using the Cell-Based Immunization and Screening method. EpMab-37 was revealed to recognize the conformational epitope of EpCAM. In this study, we determined the critical epitope of EpMab-37 by flow cytometry using the 1 × alanine scanning (1 × Ala-scan) and the 2 × alanine scanning (2 × Ala-scan) method. We first performed flow cytometry by 1 × Ala-scan using one alanine (or glycine)-substituted EpCAM mutants, which were expressed on Chinese hamster ovary-K1 cells, and found that the EpMab-37 did not recognize the R163A mutant of EpCAM. We next performed flow cytometry by 2 × Ala-scan using two alanine (or glycine) residues-substituted EpCAM mutants, and confirmed that EpMab-37 did not recognize R163A-including mutants of EpCAM. The results indicated that the critical binding epitope of EpMab-37 includes Arg163 of EpCAM.

Effect of Glycan Shift on Antibodies against Hepatitis C Virus E2 412-425 Epitope Elicited by Chimeric sHBsAg-Based Virus-Like Particles.

Two of the most important mechanisms of hepatitis C virus (HCV) immune evasion are the high variability of the amino acid sequence and epitope shielding via heavy glycosylation of the envelope (E) proteins. Previously, we showed that chimeric sHBsAg (hepatitis B virus [HBV] small surface antigen)-based virus-like particles (VLPs) carrying highly conserved epitope I from the HCV E2 glycoprotein (sHBsAg_412-425) elicit broadly neutralizing antibodies (bnAbs). However, many reports have identified escape mutations for such bnAbs that shift the N-glycosylation site from N417 to N415. This shift effectively masks the recognition of epitope I by antibodies raised against the wild-type glycoprotein. To investigate if glycan-shift-mediated immune evasion could be overcome by targeted vaccination strategies, we designed sHBsAg-based VLPs carrying epitope I with an N417S change (sHBsAg_N417S). Studies in BALB/c mice revealed that both sHBsAg_412-425 and sHBsAg_N417S VLPs were immunogenic, eliciting antibodies that recognized peptides encompassing epitope I regardless of the N417S change. However, we observed substantial differences in E1E2 glycoprotein binding and cell culture-derived HCV (HCVcc) neutralization between the sera elicited by sHBsAg_412-425 and those elicited by sHBsAg_N417S VLPs. Our results suggest a complex interplay among antibodies targeting epitope I, the E1E2 glycosylation status, and the epitope or global E1E2 conformation. Additionally, we observed striking similarities in the E1E2 glycoprotein binding patterns and HCVcc neutralization between sHBsAg_412-425 sera and AP33, suggesting that the immunization of mice with sHBsAg_412-425 VLPs can elicit AP33-like antibodies. This study emphasizes the role of antibodies against epitope I and represents an initial effort toward designing an antigen that elicits an immune response against epitope I with a glycan shift change. IMPORTANCE Epitope I, located within amino acids 412 to 423 of the HCV E2 glycoprotein, is an important target for an epitope-based HCV vaccine. One interesting feature of epitope I is the N417 glycosylation site, where a single change to S417 or T417 can shift the glycosylation site to position N415. This shift can effectively prevent the binding of broadly neutralizing antibodies targeting epitope I. Aiming to overcome glycan-shift-mediated immune evasion, we constructed sHBsAg_N417S VLPs carrying E2 epitope I, with N417S, and compared them with VLPs carrying wild-type epitope I. We show that antibodies elicited by the sHBsAg-based VLPs presenting two variants of the 412-425 epitope targeted two distinct glycan variants of the HCV E1E2 heterodimer. Our study suggests that due to the conformational flexibility of the E2 glycoprotein and epitope I, future vaccine antigens should elicit antibodies targeting more than one conformation and glycosylation variant of the 412-423 epitope.

Structural basis for a conserved neutralization epitope on the receptor-binding domain of SARS-CoV-2

Antibody-mediated immunity plays a crucial role in protection against SARS-CoV-2 infection. We isolated a panel of neutralizing anti-receptor-binding domain (RBD) antibodies elicited upon natural infection and vaccination and showed that they recognize an immunogenic patch on the internal surface of the core RBD, which faces inwards and is hidden in the “down” state. These antibodies broadly neutralize wild type (Wuhan-Hu-1) SARS-CoV-2, Beta and Delta variants and some are effective against other sarbecoviruses. We observed a continuum of partially overlapping antibody epitopes from lower to upper part of the inner face of the RBD and some antibodies extend towards the receptor-binding motif. The majority of antibodies are substantially compromised by three mutational hotspots (S371L/F, S373P and S375F) in the lower part of the Omicron BA.1, BA.2 and BA.4/5 RBD. By contrast, antibody IY-2A induces a partial unfolding of this variable region and interacts with a conserved conformational epitope to tolerate all antigenic variations and neutralize diverse sarbecoviruses as well. This finding establishes that antibody recognition is not limited to the normal surface structures on the RBD. In conclusion, the delineation of functionally and structurally conserved RBD epitopes highlights potential vaccine and therapeutic candidates for COVID-19.

Immunotherapy-induced neutralizing antibodies disrupt allergen binding and sustain allergen tolerance in peanut allergy.

In IgE-mediated food allergies, exposure to the allergen activates systemic allergic responses. Oral immunotherapy (OIT) treats food allergies through incremental increases in oral allergen exposure. However, OIT only induces sustained clinical tolerance and decreased basophil sensitivity in a subset of individuals despite increases in circulating allergen-specific IgG in all treated individuals. Therefore, we examined the allergen-specific antibodies from 2 OIT cohorts of patients with sustained and transient responses. Here, we compared antibodies from individuals with sustained or transient responses and discovered specific tolerance-associated conformational epitopes of the immunodominant allergen Ara h 2 recognized by neutralizing antibodies. First, we identified what we believe to be previously unknown conformational, intrahelical epitopes using x-ray crystallography with recombinant antibodies. We then identified epitopes only recognized in sustained tolerance. Finally, antibodies recognizing tolerance-associated epitopes effectively neutralized allergen to suppress IgE-mediated effector cell activation. Our results demonstrate the molecular basis of antibody-mediated protection in IgE-mediated food allergy, by defining how these antibodies disrupt IgE-allergen interactions to prevent allergic reactions. Our approach to studying the structural and functional basis for neutralizing antibodies demonstrates the clinical relevance of specific antibody clones in antibody-mediated tolerance. We anticipate that our findings will form the foundation for treatments of peanut allergy using neutralizing antibodies and hypoallergens.

Reducing the Immunogenicity of Pulchellin A-Chain, Ribosome-Inactivating Protein Type 2, by Computational Protein Engineering for Potential New Immunotoxins

Pulchellin is a plant biotoxin categorized as a type 2 ribosome-inactivating protein (RIPs) which potentially kills cells at very low concentrations. Biotoxins serve as targeting immunotoxins (IT), consisting of antibodies conjugated to toxins. ITs have two independent protein components, a human antibody and a toxin with a bacterial or plant source; therefore, they pose unique setbacks in immunogenicity. To overcome this issue, the engineering of epitopes is one of the beneficial methods to elicit an immunological response. Here, we predicted the tertiary structure of the pulchellin A-chain (PAC) using five common powerful servers and adopted the best model after refining. Then, predicted structure using four distinct computational approaches identified conformational B-cell epitopes. This approach identified some amino acids as a potential for lowering immunogenicity by point mutation. All mutations were then applied to generate a model of pulchellin containing all mutations (so-called PAM). Mutants’ immunogenicity was assessed and compared to the wild type as well as other mutant characteristics, including stability and compactness, were computationally examined in addition to immunogenicity. The findings revealed a reduction in immunogenicity in all mutants and significantly in N146V and R149A. Furthermore, all mutants demonstrated remarkable stability and validity in Molecular Dynamic (MD) simulations. During docking and simulations, the most homologous toxin to pulchellin, Abrin-A was applied as a control. In addition, the toxin candidate containing all mutations (PAM) disclosed a high level of stability, making it a potential model for experimental deployment. In conclusion, by eliminating B-cell epitopes, our computational approach provides a potential less immunogenic IT based on PAC.

Prediction of Conformational and Linear B-Cell Epitopes on Envelop Protein of Zika Virus Using Immunoinformatics Approach.

The current spread of Zika virus infection in India has become a public health issue due to the virus's possible link to birth abnormalities and neurological disorders. There is a need for enhanced vaccines or drugs as a result of its epidemic outbreak and the lack of potential medication. B-cell mediated adaptive immunity is capable of developing pathogen-specific memory that confers immunological protection. Therefore, in this study, the envelope protein of the Zika virus was retrieved from the NCBI protein database. The ABCpred and BepiPred software were used to discover linear B-cell epitopes on envelope protein. Conformational B-cell epitopes on envelope protein were identified using SEPPA 3.0 and Ellipro tools. Predicted B-cell epitopes were evaluated for allergenicity, toxicity, and antigenicity. Two consensus linear B-cell epitopes, envelope165-180 (AKVEITPNSPRAEATL) and envelope224-238 (PWHAGADTGTPHWNN) were identified using ABCpred and BepiPredtools. SEPPA 3.0 and Elliprotools predicted consensus conformational envelope98-110 (DRGWGNGCGLFGK) and envelope248-251 (AHAK) epitopes and one residue (75PRO) within envelope protein as a component of B-cell epitopes. These predicted linear and conformational B-cell epitopes will help in designing peptide vaccines that will activate the humoral response. However, in-vitro and in-vivo laboratory experimental confirmations are still needed to prove the application's feasibility.

IgE epitopes of Ara h 9, Jug r 3, and Pru p 3 in peanut-allergic individuals from Spain and the US.

Non-specific lipid transfer proteins (LTPs) are well studied allergens that can lead to severe reactions, but often cause oral allergy syndrome in the Mediterranean area and other European countries. However, studies focused on LTP reactivity in allergic individuals from the United States are lacking because they are not considered major allergens. The goal of this study is to determine if differences in immunoglobulin (Ig) E binding patterns to the peanut allergen Ara h 9 and two homologous LTPs (walnut Jug r 3 and peach Pru p 3) between the US and Spain contribute to differences observed in allergic reactivity. Synthetic overlapping 15-amino acid-long peptides offset by five amino acids from Ara h 9, Jug r 3, and Pru p 3 were synthesized, and the intact proteins were attached to microarray slides. Sera from 55 peanut-allergic individuals from the US were tested for IgE binding to the linear peptides and IgE binding to intact proteins using immunofluorescence. For comparison, sera from 17 peanut-allergic individuals from Spain were also tested. Similar IgE binding profiles for Ara h 9, Jug r 3, and Pru p 3 were identified between the US and Spain, with slight differences. Certain regions of the proteins, specifically helices 1 and 2 and the C-terminal coil, were recognized by the majority of the sera more often than other regions of the proteins. While serum IgE from peanut-allergic individuals in the US binds to peptides of Ara h 9 and its homologs, only IgE from the Spanish subjects bound to the intact LTPs. This study identifies Ara h 9, Jug r 3, and Pru p 3 linear epitopes that were previously unidentified using sera from peanut-allergic individuals from the US and Spain. Certain regions of the LTPs are recognized more often in US subjects, indicating that they represent conserved and possible cross-reactive regions. The location of the epitopes in 3D structure models of the LTPs may predict the location of potential conformational epitopes bound by a majority of the Spanish patient sera. These findings are potentially important for development of peptide or protein-targeting diagnostic and therapeutic tools for food allergy.

The Nuclear Dense Fine Speckled (DFS) Immunofluorescence Pattern: Not All Roads Lead to DFS70/LEDGFp75.

The monospecific dense fine speckled (DFS) immunofluorescence assay (IFA) pattern is considered a potential marker to aid in exclusion of antinuclear antibody (ANA)-associated rheumatic diseases (AARD). This pattern is typically produced by autoantibodies against transcription co-activator DFS70/LEDGFp75, which are frequently found in healthy individuals and patients with miscellaneous inflammatory conditions. In AARD patients, these antibodies usually co-exist with disease-associated ANAs. Previous studies reported the occurrence of monospecific autoantibodies that generate a DFS-like or pseudo-DFS IFA pattern but do not react with DFS70/LEDGFp75. We characterized this pattern using confocal microscopy and immunoblotting. The target antigen associated with this pattern partially co-localized with DFS70/LEDGFp75 and its interacting partners H3K36me2, an active chromatin marker, and MLL, a transcription factor, in HEp-2 cells, suggesting a role in transcription. Immunoblotting did not reveal a common protein band immunoreactive with antibodies producing the pseudo-DFS pattern, suggesting they may recognize diverse proteins or conformational epitopes. Given the subjectivity of the HEp-2 IFA test, the awareness of pseudo-DFS autoantibodies reinforces recommendations for confirmatory testing when reporting patient antibodies producing a putative DFS pattern in a clinical setting. Future studies should focus on defining the potential diagnostic utility of the pseudo-DFS pattern and its associated antigen(s).

Component-Resolved Diagnosis Based on a Recombinant Variant of Mus m 1 Lipocalin Allergen.

Exposure to the Mus m 1 aeroallergen is a significant risk factor for laboratory animal allergy. This allergen, primarily expressed in mouse urine where it is characterized by a marked and dynamic polymorphism, is also present in epithelium and dander. Considering the relevance of sequence/structure assessment in protein antigenic reactivity, we compared the sequence of the variant Mus m 1.0102 to other members of the Mus m 1 allergen, and used Discotope 2.0 to predict conformational epitopes based on its 3D-structure. Conventional diagnosis of mouse allergy is based on serum IgE testing, using an epithelial extract as the antigen source. Given the heterogeneous and variable composition of extracts, we developed an indirect ELISA assay based on the recombinant component Mus m 1.0102. The assay performed with adequate precision and reasonable diagnostic accuracy (AUC = 0.87) compared to a routine clinical diagnostic test that exploits the native allergen. Recombinant Mus m 1.0102 turned out to be a valuable tool to study the fine epitope mapping of specific IgE reactivity to the major allergen responsible for mouse allergy. We believe that advancing in its functional characterization will lead to the standardization of murine lipocalins and to the development of allergen-specific immunotherapy.

Critical review of conformational B-cell epitope prediction methods.

Accurate in silico prediction of conformational B-cell epitopes would lead to major improvements in disease diagnostics, drug design and vaccine development. A variety of computational methods, mainly based on machine learning approaches, have been developed in the last decades to tackle this challenging problem. Here, we rigorously benchmarked nine state-of-the-art conformational B-cell epitope prediction webservers, including generic and antibody-specific methods, on a dataset of over 250 antibody-antigen structures. The results of our assessment and statistical analyses show that all the methods achieve very low performances, and some do not perform better than randomly generated patches of surface residues. In addition, we also found that commonly used consensus strategies that combine the results from multiple webservers are at best only marginally better than random. Finally, we applied all the predictors to the SARS-CoV-2 spike protein as an independent case study, and showed that they perform poorly in general, which largely recapitulates our benchmarking conclusions. We hope that these results will lead to greater caution when using these tools until the biases and issues that limit current methods have been addressed, promote the use of state-of-the-art evaluation methodologies in future publications and suggest new strategies to improve the performance of conformational B-cell epitope prediction methods.

Crystal Structure Analysis of Sarcoplasmic-Calcium-Binding Protein: An Allergen in Scylla paramamosain.

The structure of allergenic proteins provides important information about the binding of allergens to antibodies. In this study, the crystal structure of Scy p 4 with a resolution of 1.60 Å was obtained by X-ray diffraction. Epitope mapping of Scy p 4 revealed that linear epitopes are located on the surface of Scy p 4. Also, conformational epitopes are mostly located in the structural conservative region. Further structural comparison, surface electrostatic potential, and hydrogen bond force analysis showed that mutation of Asp70 and Asp18/20/70 would lead to calcium-binding capacity being lost and destruction of allergenicity. Furthermore, a comparative analysis of structure showed that sarcoplasmic-calcium-binding protein (SCP) had high sequence, secondary, and spatial structural identity in crustaceans, which may be an important factor leading to cross-reactivity among crustaceans. The structure of Scy p 4 provides a template for epitope evaluation and localization of SCPs, which will help to reveal cross-reactivity among species.

OMIP-088: Twenty-target imaging mass cytometry panel for major cell populations in mouse formalin fixed paraffin embedded liver.

The purpose of this 20-target imaging mass cytometry (IMC) panel is to identify the main cell types in formalin fixed paraffin embedded (FFPE) mouse liver tissue with the Hyperion™ mass cytometer from Standard BioTools (formerly Fluidigm). The antibody panel includes markers to identify hepatocytes (E-cadherin, HNF4α (hepatocyte nuclear factor 4 alpha), Arginase-1), liver sinusoidal endothelial cells (LSECs; CD206), Kupffer cells (F4/80, CD206), neutrophils (Ly6G, CD11b), bone marrow derived myeloid cells (BMDMs; CD11b), cholangiocytes (E-cadherin high), endothelial cells (CD31, α-SMA), plasmacytoid dendritic cells (CD317), B cells (CD19), T cells (CD3e, CD4, CD8a), NK cells (CD161) as well markers of cell activation (CD44, CD74), proliferation (Ki-67) and to aid in cell segmentation (Pan-Actin, E-cadherin, histone H3). The panel has been tested in other mouse tissues, namely the spleen, colon and lung, and therefore is likely to work across various mouse FFPE samples of interest. It has not been tested using human samples, frozen samples or in suspension mass cytometry because FFPE treatment profoundly changes epitope conformation. In summary, this panel is a powerful tool for pre-clinical research to determine cellular abundance and spatial distribution within mouse tissues and serves as a scaffold, to which more targets can be added for project specific requirements.

Mapping Polyclonal Antibody Responses to Infection Using Next-Generation Phage Display.

Peptide phage display has historically been used to epitope map monoclonal antibodies. More recently, by coupling this method with next-generation sequencing (so-called next-generation phage display, NGPD) to mass screen peptide binding events, the methodology has been successfully applied to map polyclonal antibody responses to infection. This leads to the identification of panels of mimotopes that represent the pathogen's epitopes. One potential advantage of using such an approach is that the mimotopes can represent not just linear epitopes but also conformational epitopes or those produced from post-translational modifications of proteins or from other non-protein macromolecules. The mapping of such complex immunological recognition of a pathogen can inform novel serological assay development and vaccine design. Here, we provide detailed methods for the application of NGPD to identify panels of mimotopes that are recognized specifically by antibodies from individuals with a particular infection.

Immunobiology and Host Response to HEV.

Hepatitis E virus (HEV) usually causes acute self-limiting hepatitis but sometimes leads to chronic infection in immunocompromised persons. HEV is not directly cytopathic. Immunologically mediated events after HEV infection are believed to play important roles in the pathogenesis and clearance of infection. The anti-HEV antibody responses have been largely clarified since the determination of major antigenic determinant of HEV, which is located in the C-terminal portion of ORF2. This major antigenic determinant also forms the conformational neutralization epitopes. Robust anti-HEV immunoglobulin M (IgM) and IgG responses usually develop 3-4weeks after infection in experimentally infected nonhuman primates. In humans, potent specific IgM and IgG responses occur in the very early phase of the disease and are critical in eliminating the virus, in concert with the innate and adaptive T-cell immune responses. Testing anti-HEV IgM is valuable in the diagnosis of acute hepatitis E. The long-term persistence and protection of anti-HEV IgG provide the basis for estimating the prevalence of HEV infection and for the development of a hepatitis E vaccine. Although human HEV has four genotypes, all the viral strains are considered to belong to a single serotype. It is becoming increasingly clear that the innate and adaptive T-cell immune responses play critical roles in the clearance of the virus. Potent and multispecific CD4+ and CD8+ T cell responses to the ORF2 protein occur in patients with acute hepatitis E, and weaker HEV-specific CD4+ and CD8+ T cell responses appear to be associated with chronic hepatitis E in immunocompromised individuals.

Identification of Novel Nontoxic Mutants of Diphtheria Toxin Unable to ADP-ribosylate EF2 Using Molecular Dynamics Simulations and Free Energy Calculations

Introduction: The Diphtheria vaccine is currently manufactured by chemical inactivation of Diphtheria toxin (DT) following large-scale cultivation of the pathogenic Corynebacterium diphtheriae. The development of non-toxic variants of DT in non-pathogenic recombinant hosts improves the process safety aspects considerably and is therefore desirable. The cytotoxicity of DT was exerted through ADPribosylation of elongation factor 2 (EF2) by the catalytic (C) domain of DT using NAD as the ADP donor. Aim: We, therefore, aim to design DT variants unable to perform ADP-ribosylation of EF2. Methods: We used molecular dynamics simulations (MDS) for docking of the C-domain of DT onto EF2 to identify interacting amino acid fluctuations using the root mean square fluctuation (RMSF). Amino acids of the C domain possessing low fluctuations and hence low flexibility were then specified and used for the mutant design. The C domain and the mutants were docked with NAD and ADENYLYL-3'-5'- PHOSPHO-URIDINE3'-MONOPHOSPHATE (APU) as a C domain inhibitor. G52E showing negligible NAD binding was also included in our study. Free binding energies of the complexes were calculated and used to select the desired mutants. Moreover, contact maps of the C domain and the selected mutants were compared to elucidate their structural differences. The SCRATCH tool was used to estimate their solubility upon recombinant expression in E. coli. ElliPro was further used to determine their B-cell epitopes. Results: Our results indicated that amino acids Y20, V80, V81, V83, and Y149 within the C domain showed low flexibility. The constructed mutants, including Y20E and Y80E were able to bind APU, whereas Y149E was not able to bind it. Both Y20E and Y149E were unable to bind NAD. Thus, these mutants did not have ADP-ribosylation activity and were nontoxic. These were structurally different from the C domain. However, their solubility in E. coli and their conformational B-cell epitopes were similar to those of the C domain. Conclusion: Therefore, Y20E and Y149E are applicable for the vaccine design with DT using a much safer process compared to the commercial process.

Recombinant Protein Mimicking the Antigenic Structure of the Viral Surface Envelope Protein Reinforces Induction of an Antigen-Specific and Virus-Neutralizing Immune Response Against Dengue Virus.

Dengue virus (DENV), belonging to the family Flaviviridae, is the causative agent of dengue and comprises four serotypes. A second heterologous DENV infection is a critical risk factor for severe dengue, and no effective vaccine is available to prevent infection by all four DENV serotypes. Recombinant DENV vaccines are primarily based on the envelope proteins, prM and E. The E protein and its envelope domain III (EDIII) have been investigated as candidate antigens (Ags) for recombinant subunit vaccines. However, most EDIII-based Ags are monomers that do not display the cognate antigenic structure of E protein, which is essential for induction of virus-neutralizing immunity. Here, we developed recombinant DENV-2 envelope domain (r2ED) protein as an Ag that mimics the quaternary structure of E protein on the DENV surface. We confirmed that r2ED retained the conformational epitope displayed at the E-dimer interface, which reportedly exhibits broad virus-neutralizing capacity, without displaying the fusion loop epitope that causes antibody (Ab)-dependent enhancement. Furthermore, compared with EDIII alone, r2ED elicited stronger Ag-specific and cross-reactive neutralizing Ab and T cell-mediated immune responses in mice. This Ag-specific immunity was maintained at an elevated level 6months after the last immunization, suggesting sustained Ag-specific immune memory. Taken together, these observations suggest that r2ED could be used to develop an improved subunit vaccine capable of inducing a broadly cross-reactive and long-lasting immune response against DENV infection.

Covalent polyphenol modification of a reactive cysteine in the major apple allergen Mal d 1

Naturally occurring polyphenols can modify the molecular properties of food allergens. For the major apple allergen Mal d 1 it has been postulated that chemical reactions with polyphenols cause permanent changes in the tertiary structure, causing a loss of conformational IgE epitopes and reducing allergenicity. In our study, we investigated the effect that reactions with oxidized polyphenols have on the structure of Mal d 1 by mass spectrometry and NMR spectroscopy. We showed that a surface-exposed cysteine residue in this allergen spontaneously reacts with oxidized polyphenols under formation of a defined covalent adduct. Chemical modification of Mal d 1 did not destabilize or perturb the three-dimensional fold, nor did it interfere with ligand binding to its internal pocket. A structural model of the chemically modified apple allergen is presented, which reveals that the bound polyphenol partially covers a conformational IgE epitope on the protein surface.