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Background: Rubella is an infection caused by rubella virus (RV) and is generally regarded as a mild childhood disease. The disease continues to be of public health importance mainly because when the infection is acquired during early pregnancy, it often results in fetal abnormalities, which are classified as congenital rubella syndrome (CRS). An accurate diagnosis of rubella is thus of pivotal importance for proper treatment. Objective: The aim of the study was to produce a recombinant multiepitope protein (rMERUB) for the diagnosis of rubella, based on conserved immunodominant epitopes of glycoprotein E1 and E2. Methods: A synthetic gene was designed and cloned into vector pET21a with a 6xHis tag at the C-terminal for affinity purification and overexpressed in Escherichia coli cells. Biophysical analysis of rMERUB was performed by circular dichroism. Biological activity was assessed using an in-house ELISA assay. Results: Expression in Escherichia coli showed a ~22 kDa protein that was purified and used to per-form structural assays and an IgG ELISA. Structural analyses reveal that rMERUB has a β leaf pattern that promotes the exposure of epitopes, thus allowing antibody recognition. Evaluation of 33 samples (22=positive; 11=negative) was performed using in-house ELISA and this was compared with a com-mercial kit. The sensitivity was 100% (95% CI: 85-100) and specificity 90.91% (95% CI: 62-99). Excellent agreement (Kappa index = 0.9) was obtained between ELISA assays. Conclusions: The careful choice of epitopes and the high epitope density, coupled with simple-step purification, pinpoints rMERUB as a promising alternative for rubella diagnosis, with potential for the development of a diagnostic kit.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. With the expectation of improved survival, tremendous efforts and resources have been invested in the discovery of specific biomarkers for early detection of the disease. Several investigators have reported the presence of cancer-associated autoantibodies in the plasma or serum of lung cancer patients. Previously, we used a monoclonal antibody (mAb) proteomics technology platform for the discovery of novel lung cancer-associated proteins. OBJECTIVE: The identification of specific protein epitopes associated with various cancers is a promising method in biomarker discovery. Here, in a preliminary study, we aimed to detect autoantibody-leucine-rich alpha-2-glycoprotein 1 (LRG1) immunocomplexes using epitope-specific monoclonal antibodies (mAbs). METHODS: We performed sandwich ELISA assays using the LRG1 epitope-specific capture mAbs, Bsi0352 and Bsi0392, and an IgG-specific polyclonal antibody coupled to a reporter system as the detection reagent. We tested the plasma of lung cancer patients and apparently healthy controls. RESULTS: Depending on the epitope specificity of the capture mAb, we were either unable to distinguish the control from LC-groups or showed a higher level of LRG1 and IgG autoantibody containing immunocomplexes in the plasma of non-small cell lung cancer and small cell lung cancer subgroups of lung cancer patients than in the plasma of control subjects. CONCLUSIONS: Our findings underline the importance of protein epitope-specific antibody targeted approaches in biomarker research, as this may increase the accuracy of previously described tests, which will need further validation in large clinical cohorts.

Graphical abstract, Introduction The SARS-CoV-2 pandemic has endangered global health, the world economy, and societal values. Despite intensive measures taken around the world, morbidity and mortality remain high as many countries face new waves of infection and the spread of new variants. Worryingly, more and more variants are now being identified, such as 501Y.V1 (B.1.1.7) in the UK, 501Y.V2 (B.1.351) in South Africa, 501Y.V3 in Manaus, Brazil, and B.1.617/B.1.618 in India, which could lead to a severe epidemic rebound. Moreover, some variants have a stronger immune escape ability. To control the new SARS-CoV-2 variant, we may need to develop and redesign new vaccines repeatedly. So it is important to investigate how our immune system combats and responds to SARS-CoV-2 infection to develop safe and effective medical interventions. Objectives In this study, we performed a longitudinal and proteome-wide analysis of antibodies in the COVID-19 patients to revealed some immune processes of COVID-19 patients against SARS-CoV-2 and found some dominant epitopes of a potential vaccine. Methods Microarray assay, Antibody depletion assays, Neutralization assay. Results We profiled a B-cell linear epitope landscape of SARS-CoV-2 and identified the epitopes specifically recognized by either IgM, IgG, or IgA. We found that epitopes more frequently recognized by IgM are enriched in non-structural proteins. We further identified epitopes with different immune responses in severe and mild patients. Moreover, we identified 12 dominant epitopes eliciting antibodies in most COVID-19 patients and identified five key amino acids of epitopes. Furthermore, we found epitope S-82 and S-15 are perfect immunogenic peptides and should be considered in vaccine design. Conclusion This data provide useful information and rich resources for improving our understanding of viral infection and developing a novel vaccine/neutralizing antibodies for the treatment of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic respiratory infectious disease COVID-19. However, clinical manifestations and outcomes differ significantly among COVID-19 patients, ranging from asymptomatic to extremely severe, and it remains unclear what drives these disparities. Here, we studied 159 sequentially enrolled hospitalized patients with COVID-19-associated pneumonia from Brescia, Italy using the VirScan phage-display method to characterize circulating antibodies binding to 96,179 viral peptides encoded by 1,276 strains of human viruses. SARS-CoV-2 infection was associated with a marked increase in immune antibody repertoires against many known pathogenic and non-pathogenic human viruses. This antiviral antibody response was linked to longitudinal trajectories of disease severity and was further confirmed in additional 125 COVID-19 patients from the same geographical region in Northern Italy. By applying a machine-learning-based strategy, a viral exposure signature predictive of COVID-19-related disease severity linked to patient survival was developed and validated. These results provide a basis for understanding the role of memory B-cell repertoire to viral epitopes in COVID-19-related symptoms and suggest that a unique anti-viral antibody repertoire signature may be useful to define COVID-19 clinical severity.

ObjectivesThe human leukocyte antigen-shared epitope (HLA-SE) alleles and smoking are the most prominent genetic and environmental risk factors for rheumatoid arthritis (RA). However, at which pre-arthritis stage (asymptomatic/symptomatic) they exert their effect is unknown. We aimed to determine whether HLA-SE and smoking are involved in the onset of autoantibody positivity, symptoms (clinically suspect arthralgia (CSA)) and/or progression to clinical arthritis.MethodsWe performed meta-analyses on results from the literature on associations of HLA-SE and smoking with anti-citrullinated protein antibodies (ACPAs) in the asymptomatic population. Next, we studied associations of HLA-SE and smoking with autoantibody positivity at CSA onset and with progression to clinical inflammatory arthritis (IA) during follow-up. Associations in ACPA-positive patients with CSA were validated in meta-analyses with other arthralgia cohorts. Analyses were repeated for rheumatoid factor (RF), anti-carbamylated protein antibodies (anti-CarP) and anti-acetylated protein antibodies (AAPA).ResultsMeta-analyses showed that HLA-SE is not associated with ACPA positivity in the asymptomatic population (OR 1.06 (95% CI:0.69 to 1.64)), whereas smoking was associated (OR 1.37 (95% CI: 1.15 to 1.63)). At CSA onset, both HLA-SE and smoking associated with ACPA positivity (OR 2.08 (95% CI: 1.24 to 3.49), OR 2.41 (95% CI: 1.31 to 4.43)). During follow-up, HLA-SE associated with IA development (HR 1.86 (95% CI: 1.23 to 2.82)), in contrast to smoking. This was confirmed in meta-analyses in ACPA-positive arthralgia (HR 1.52 (95% CI: 1.08 to 2.15)). HLA-SE and smoking were not associated with RF, anti-CarP or AAPA-positivity at CSA onset. Longitudinally, AAPA associated with IA development independent from ACPA and RF (HR 1.79 (95% CI: 1.02 to 3.16)), anti-CarP did not.ConclusionsHLA-SE and smoking act at different stages: smoking confers risk for ACPA and symptom development, whereas HLA-SE mediates symptom and IA development. These data enhance the understanding of the timing of the key risk factors in the development of RA.

Background: Administration of potent anti-receptor-binding domain (RBD) monoclonal antibodies has been shown to curtail viral shedding and reduce hospitalization in patients with SARS-CoV-2 infection. However, the structure-function analysis of potent human anti-RBD monoclonal antibodies and its links to the formulation of antibody cocktails remains largely elusive. Methods: Previously, we isolated a panel of neutralizing anti-RBD monoclonal antibodies from convalescent patients and showed their neutralization efficacy in vitro. Here, we elucidate the mechanism of action of antibodies and dissect antibodies at the epitope level, which leads to a formation of a potent antibody cocktail. Results: We found that representative antibodies which target non-overlapping epitopes are effective against wild type virus and recently emerging variants of concern, whilst being encoded by antibody genes with few somatic mutations. Neutralization is associated with the inhibition of binding of viral RBD to ACE2 and possibly of the subsequent fusion process. Structural analysis of representative antibodies, by cryo-electron microscopy and crystallography, reveals that they have some unique aspects that are of potential value while sharing some features in common with previously reported neutralizing monoclonal antibodies. For instance, one has a common VH 3-53 public variable region yet is unusually resilient to mutation at residue 501 of the RBD. We evaluate the in vivo efficacy of an antibody cocktail consisting of two potent non-competing anti-RBD antibodies in a Syrian hamster model. We demonstrate that the cocktail prevents weight loss, reduces lung viral load and attenuates pulmonary inflammation in hamsters in both prophylactic and therapeutic settings. Although neutralization of one of these antibodies is abrogated by the mutations of variant B.1.351, it is also possible to produce a bi-valent cocktail of antibodies both of which are resilient to variants B.1.1.7, B.1.351 and B.1.617.2. Conclusions: These findings support the up-to-date and rational design of an anti-RBD antibody cocktail as a therapeutic candidate against COVID-19.

During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2–α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1–α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1–α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1–α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins., During apoptosis, Bak and Bax are activated by BH3-only proteins binding to a specific hydrophobic groove. Here, the authors show that antibodies can also activate Bak and mitochondrial Bax by binding to the α1-α2 loop, thus identifying a potential clinical target.

INTRODUCTION The HLA (Human Leukocyte Antigen) system plays a pivotal role both in transplantation and immunology. While classical HLA genotypes matching is made at the allelic level, recent progresses were developed to explore antibody-antigen recognition by studying epitopes. Donor to recipient matching at the epitopic level is becoming a trending topic in the transplantation research field because anti-HLA antibodies are epitope-specific rather than allele-specific. Indeed, different HLA alleles often share common epitopes. METHODS We present the HLA-Epi tool (hla.univ-nantes.fr) to study an HLA genotype at the epitope level. Using the international HLA epitope registry (Epregistry.com.br) as a reference, we developed HLA-Epi to easily determine epitopic and allelic compatibility levels between several HLA genotypes. RESULTS The epitope database covers the most common HLA alleles (N=2,976 HLA alleles), representing more than 99% of the total observed frequency of HLA alleles. The freely accessible web tool HLA-Epi calculates an epitopic mismatch load between different sets of potential recipient-donor pairs at different resolution levels. We have characterized the epitopic mismatches distribution in a cohort of more than 10,000 kidney transplanted pairs from European ancestry, which showed low number of epitopic mismatches: 56.9 incompatibilities on average. CONCLUSIONS HLA-Epi allows the exploration of epitope pairing matching to better understand epitopes contribution to immune responses regulation, particularly during transplantation. This free and ready-to-use bioinformatics tool not only addresses limitations of other related tools, but also offers a cost-efficient and reproducible strategy to analyze HLA epitopes as an alternative to HLA allele compatibility. In the future, this could improve sensitization prevention for allograft allocation decisions and reduce the risk of alloreactivity. This article is protected by copyright. All rights reserved.

Delta’s spike Understanding the molecular mechanisms of the increased transmissibility and immune evasion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is critical to guiding current and future intervention strategies. Zhang et al . determined cryo–electron microscopy structures of the full-length spike protein trimers of the Delta, Kappa, and Gamma variants of SARS-CoV-2 and studied their function and antigenic properties. The Delta spike protein fused membranes more efficiently at low levels of the cellular receptor ACE2, and its pseudotyped viruses infected target cells substantially more rapidly than all other variants tested, possibly at least partly accounting for its heightened transmissibility. Mutations of each variant rearranged the antigenic surface of the N-terminal domain of the spike protein but only caused local changes in the receptor-binding domain, consistent with greater resistance to neutralizing antibodies. These findings elucidate the molecular events that have led these viruses to adapt in human communities and to evade host immunity. —VV

Identification of antibodies targeting diverse functional epitopes on an antigen is highly crucial for discovering effective therapeutic candidates. Employing a traditional stepwise antibody "screening funnel" as well as prioritizing affinity-based selections over epitope-based selections, result in lead antibody panels lacking epitope diversity. In the present study, we employed an array-based surface plasmon resonance (SPR) platform to perform high-throughput epitope binning analysis on a large number of monoclonal antibodies (mAbs) generated in the early drug discovery process. The mAb panel contained clones from different antibody generation techniques and diverse transgenic mouse strains. The epitope binning results were analyzed in unique ways using various visualizations in the form of dendrograms and network plots, which assisted in determining diversity and redundancy in the mAb sample set. The binning data were further integrated with affinity information to evaluate the performance of seven different transgenic mouse strains. The combination of epitope binning results with binding kinetics and sequence analysis provided an effective and efficient way of selecting high affinity antibodies representing a diverse set of sequence families and epitopes.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

A prophylactic vaccine would be a powerful tool in the fight against HIV. Passive immunization of animals with broadly neutralizing antibodies (bnAbs) affords protection against viral challenge, and recent data from the Antibody Mediated Prevention clinical trials support the concept of bnAbs providing protection against HIV in humans, albeit only at broad and potent neutralizing antibody titers. Moreover, it is now clear that a successful vaccine will also need to induce bnAbs against multiple neutralizing epitopes on the HIV envelope (Env) glycoprotein. Here, we review recent clinical trials evaluating bnAb-based vaccines, and discuss key issues in the development of an HIV vaccine capable of targeting multiple Env neutralizing epitopes.

Introduction: Molecular studies of hen’s egg allergens help define allergic phenotypes, with IgE to sequential (linear) epitopes on the ovomucoid (OVM) protein associated with a persistent disease. Epitope profiles of other egg allergens are largely unknown. The objective of this study was to construct an epitope library spanning across 7 allergens and further evaluate sequential epitope-specific (ses-)IgE and ses-IgG4 among baked-egg reactive or tolerant children. Methods: A Bead-Based Epitope Assay was used to identify informative IgE epitopes from 15-mer overlapping peptides covering the entire OVM and ovalbumin (OVA) proteins in 38 egg allergic children. An amalgamation of 12 B-cell epitope prediction tools was developed using experimentally identified epitopes. This ensemble was used to predict epitopes from ovotransferrin, lysozyme, serum albumin, vitellogenin-II fragment, and vitellogenin-1 precursor. Ses-IgE and ses-IgG4 repertoires of 135 egg allergic children (82 reactive to baked-egg, the remaining 52 tolerant), 46 atopic controls, and 11 healthy subjects were compared. Results: 183 peptides from OVM and OVA were screened and used to create an aggregate algorithm, improving predictions of 12 individual tools. A final library of 65 sequential epitopes from 7 proteins was constructed. Egg allergic children had higher ses-IgE and lower ses-IgG4 to predominantly OVM epitopes than both atopic and healthy controls. Baked-egg reactive children had similar ses-IgG4 but greater ses-IgE than tolerant group. A combination of OVA-sIgE with ses-IgEs to OVM-023 and OVA-028 was the best predictor of reactive phenotype. Conclusion: We have created a comprehensive epitope library and showed that ses-IgE is a potential biomarker of baked-egg reactivity.

Tuberculosis (TB) is a highly contagious disease that mostly affects the lungs and is caused by a bacterial pathogen, Mycobacterium tuberculosis. The associated mortality rate of TB is much higher compared to any other disease and the situation is more worrisome by the rapid emergence of drug resistant strains. Bacillus Calmette–Guerin (BCG) is the only licensed attenuated vaccine available for use in humans however, many countries have stopped its use as it fails to confer protective immunity. Therefore, urgent efforts are required to identify new and safe vaccine candidates that are not only provide high immune protection but also have broad spectrum applicability. Considering this, herein, I performed an extensive computational vaccine analysis to investigate 200 complete sequenced genomes of M. tuberculosis to identify core vaccine candidates that harbor safe, antigenic, non-toxic, and non-allergic epitopes. To overcome literature reported limitations of epitope-based vaccines, I carried out additional analysis by designing a multi-epitopes vaccine to achieve maximum protective immunity as well as to make experimental follow up studies easy by selecting a vaccine that can be easily analyzed because of its favorable physiochemical profile. Based on these analyses, I identified two potential vaccine proteins that fulfill all required vaccine properties. These two vaccine proteins are diacylglycerol acyltransferase and ESAT-6-like protein. Epitopes: DSGGYNANS from diacylglycerol acyltransferase and AGVQYSRAD, ADEEQQQAL, and VSRADEEQQ from ESAT-6-like protein were found to cover all necessary parameters and thus used in a multi-epitope vaccine construct. The designed vaccine is depicting a high binding affinity for different immune receptors and shows stable dynamics and rigorous van der Waals and electrostatic binding energies. The vaccine also simulates profound primary, secondary, tertiary immunoglobulin production as well as high interleukins and interferons count. In summary, the designed vaccine is ideal to be evaluated experimentally to decipher its real biological efficacy in controlling drug resistant infections of M. tuberculosis.

Epstein-Barr virus (EBV) is associated with a range of epithelial and B cell malignancies as well as autoimmune disorders, for which there are still no specific treatments or effective vaccines. Here, we isolate EBV gH/gL-specific antibodies from an EBV-infected individual. One antibody, 1D8, efficiently neutralizes EBV infection of two major target cell types, B cells and epithelial cells. In humanized mice, 1D8 provides protection against a high-dose EBV challenge by substantially reducing viral loads and associated tumor burden. Crystal structure analysis reveals that 1D8 binds to a key vulnerable interface between the D-I/D-II domains of the viral gH/gL protein, especially the D-II of the gH, thereby interfering with the gH/gL-mediated membrane fusion and binding to target cells. Overall, we identify a potent and protective neutralizing antibody capable of reducing the EBV load. The novel vulnerable site represents an attractive target that is potentially important for antibody and vaccine intervention against EBV infection., Currently, there are no treatments or vaccines against Epstein-Barr virus (EBV) infection. Here, Zhu et al. isolate gH/gL-specific antibodies from infected donors and identify 1D8, that substantially reduces infection levels in both, B- and epithelial cells, and reduces tumor burdens in EBV-challanged humanized mice due to interferance with the gH/gL-mediated membrane fusion and binding.

Antibody-Framework-to-Antigen Distance (AFAD) – the distance between the body of an antibody and a protein antigen – is an important parameter governing antibody recognition. Here, we quantify AFAD for ~2,000 non-redundant antibody-protein-antigen complexes in the Protein Data Bank. AFADs showed a gaussian distribution with mean of 16.3 Å and standard deviation (σ) of 2.4 Å. Notably, antibody-antigen complexes with extended AFADs (>3σ) were exclusively human immunodeficiency virus-type 1 (HIV-1)-neutralizing antibodies. High correlation (R2 = 0.8110) was observed between AFADs and glycan coverage, as assessed by molecular dynamics simulations of the HIV-1-envelope trimer. Especially long AFADs were observed for antibodies targeting the glycosylated trimer apex, and we tested the impact of introducing an apex-glycan hole (N160K); the cryo-EM structure of the glycan hole-targeting HIV-1-neutralizing antibody 2909 in complex with an N160K-envelope trimer revealed a substantially shorter AFAD. Overall, extended AFADs exclusively recognized densely glycosylated surfaces, with the introduction of a glycan hole enabling closer recognition., Here, the authors analyse the distance between the body of an antibody and a protein antigen denoted as the Antibody-Framework-to-Antigen Distance (AFAD) for about 2000 non-redundant antibody-protein antigen complexes in the Protein Data Bank. They observe that antibodies with exceptionally long AFADs were all broad HIV-1-neutralizing antibodies that targeted densely glycosylated regions on the HIV-1-envelope trimer. The connection between long AFAD and dense glycan was further validated by the cryo-EM structure of antibody 2909 recognizing a glycan hole and by glycan shielding analyses based on molecular dynamics simulations.

Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance., Krebs and colleagues identify multiple mAbs that recognize either the RBD or the NTD of SARS-CoV-2 spike protein that have potent cross-neutralizing activities against variants of concern. Combinatorial mAb cocktails have complementary effects on viral neutralization and Fc effector functions and can protect against SARS-CoV-2 escape mutants.

Sarcoplasmic calcium-binding protein is a stable allergen in Scylla paramamosain and named Scy p 4. To explore the importance of linear epitopes in the immunoglobulin G (IgG)/immunoglobulin E (IgE)-binding capacity of Scy p 4, chemical denaturants were used to destroy the structure. Scy p 4 was reduced with dithiothreitol and subsequently alkylated with iodoacetamide (IAA). Furthermore, the structural analysis indicated that IAA-Scy p 4 was an unstructured protein. The inhibition enzyme-linked immunosorbent assay showed that IAA-Scy p 4 could inhibit the binding of Scy p 4 to sensitize serum, with inhibition rates reached 55%. Moreover, the linear mimotopes of Scy p 4 were predicted in silico. Three linear epitopes were verified by serological tests and named L-Scy p 4-1 (AA76-91), L-Scy p 4-2 (AA111-125), and L-Scy p 4-3 (AA137-146). Overall, these data provide an understanding of the relationship between the structure and allergenicity about Scy p 4, and the identified linear epitopes can be used for diagnosis and food processing of shellfish allergy.

Food allergy has become a major global public health concern. In the past decades, enzymatic crosslinking technique has been employed to mitigate the immunoreactivity of food allergens. It is an emerging non-thermal technique that can serve as a great alternative to conventional food processing approaches in developing hypoallergenic food products, owing to their benefits of high specificity and selectivity. Enzymatic crosslinking via tyrosinase (TYR), laccase (LAC), peroxidase (PO), and transglutaminase (TG) modifies the structural and biochemical properties of food allergens that subsequently cause denaturation and masking of the antigenic epitopes. LAC, TYR, and PO catalyze the oxidation of tyrosine side chains to initiate protein crosslinking, while TG initiates isopeptide bonding between lysine and glutamine residues. Enzymatic treatment produces a high molecular weight crosslinked polymer with reduced immunoreactivity and IgE-binding potential. Crosslinked allergens further inhibit mast cell degranulation due to the lower immunostimulatory potential that assists in the equilibration of T-helper (Th)1/Th2 immunobalance. This review provides an updated overview of the studies carried out in the last decade on the potential application of enzymatic crosslinking for mitigating food allergenicity that can be of importance in the context of developing hypoallergenic/non-allergenic food products.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS‑CoV‑2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS‑CoV‑2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS‑CoV‑2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS‑CoV‑2 virus and the Spike protein receptor-binding domain at high titers. Supplementary information The online version contains supplementary material available at 10.1134/S0006297921100096.

Classical swine fever virus (CSFV) is a member of the genus Pestivirus, which causes serious economic losses. The re-emergence of the disease in Japan in 2018 has increased awareness of CSFV. In this study, Balb/c mice were immunized with plant-derived E2 protein, and four monoclonal antibodies (mAbs) 4B11, 7B3, 11A5 and 6F3 were generated. Two of these mAbs, 4B11 and 7B3, effectively blocked CSFV infection of PK-15 cells. Both mAbs recognized a novel linear epitope, 256CLIGNTTVKVHASDER271. The neutralizing ability of anti-CSFV serum decreased 63%, when pre-incubated with the linear peptide at 200 μg/mL. Structural analysis showed that this linear epitope is present at the border of Domain C and Domain D on the surface of the E2 protein. Alignment of amino acid sequences showed that the epitope was conserved in different subgroups of CSFV but not in other members of the Pestivirus genus. Consistently with the analysis above, this epitope distinguished antibodies against CSFV from those against bovine viral diarrhea virus (BVDV). Our study provides an ideal candidate peptide for new vaccine design and differential diagnosis of CSFV. These findings will contribute to the control and eradication of classical swine fever.

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes β-coronavirus lineage B (β-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional “down” conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD “up”. Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-β-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against β-CoV-B and newly emerging SARS-CoV-2 variants of concern. Supplementary Information The online version contains supplementary material available at 10.1007/s13238-021-00871-6.

Background The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important. Methods In this study, we analyzed the SARS‐CoV‐2 polyclonal antibody response in a large population of clinically well‐characterized patients after mild and severe COVID‐19 using a panel of microarrayed structurally folded and unfolded SARS‐CoV‐2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor‐binding domain (RBD) of the virus. Results S‐ and RBD‐specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1, and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin‐converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD‐specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus‐neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants. Conclusion These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS‐CoV‐2–neutralizing antibodies conferring sterilizing immunity., IgG response in convalescent COVID‐19 patients is directed to folded but not to unfolded RBD or RBD peptides. IgGs to folded RBD are required for virus neutralization. Twenty percent of convalescent COVID‐19 patients selectively lack RBD‐specific IgG. Only immunization with folded, but not with unfolded RBD, induces antibodies with virus‐neutralizing activity.

A substantial body of work has been carried out describing the structural features of the complex between single-domain antibodies (VHHs) and antigens, and the preeminence for epitopes located at concave surfaces of the antigen. However, the thermodynamic basis of binding is far less clear. Here, we have analysed the energetic profiles of five VHHs binding to the catalytic cleft or to a noncleft epitope of hen egg lysozyme. Various binding energetic profiles with distinctive enthalpic/entropic contributions and structural distribution of critical residues were found in the five antibodies analysed. Collectively, we suggest that from an energetic point of view the binding mechanism is influenced by the shape of the epitope. This information may be beneficial for the design of tailored epitopes for VHHs and their practical use.

Human heavy-chain ferritin (HFn) and hepatitis B virus core (HBc) are both nanoparticle proteins presenting a well-oriented architecture with constant size and shape, which can be engineered to carry epitopes on the surface of the nanoparticle protein cage, enabling vaccine design. This study aims to investigate the immunogenicity differences between engineered HFn and chimeric HBc bearing the same epitope. As a proof of concept, the model epitope Epstein-Barr nuclear antigen 1 (EBNA1) is inserted at the N-terminus of the HFn and HBc subunit to produce two vaccine candidates named EBNA1-HFn (E1F1) and EBNA1-HBc (E1H1), respectively. From

The COVID-19 pandemic highlights the need for platform technologies enabling rapid development of vaccines for emerging viral diseases. The current vaccines target the SARS-CoV-2 spike (S) protein and thus far have shown tremendous efficacy. However, the need for cold-chain distribution, a prime-boost administration schedule, and the emergence of variants of concern (VOCs) call for diligence in novel SARS-CoV-2 vaccine approaches. We studied 13 peptide epitopes from SARS-CoV-2 and identified three neutralizing epitopes that are highly conserved among the VOCs. Monovalent and trivalent COVID-19 vaccine candidates were formulated by chemical conjugation of the peptide epitopes to cowpea mosaic virus (CPMV) nanoparticles and virus-like particles (VLPs) derived from bacteriophage Qβ. Efficacy of this approach was validated first using soluble vaccine candidates as solo or trivalent mixtures and subcutaneous prime-boost injection. The high thermal stability of our vaccine candidates allowed for formulation into single-dose injectable slow-release polymer implants, manufactured by melt extrusion, as well as microneedle (MN) patches, obtained through casting into micromolds, for prime-boost self-administration. Immunization of mice yielded high titers of antibodies against the target epitope and S protein, and data confirms that antibodies block receptor binding and neutralize SARS-CoV and SARS-CoV-2 against infection of human cells. We present a nanotechnology vaccine platform that is stable outside the cold-chain and can be formulated into delivery devices enabling single administration or self-administration. CPMV or Qβ VLPs could be stockpiled, and epitopes exchanged to target new mutants or emergent diseases as the need arises.

The repertoire of antibodies (Abs) produced upon vaccination against a particular antigenic site is rarely studied due to the complexity of the immunogens. We received such an opportunity when one rhesus macaque was immunized six times at 0, 4, 10, 16, 32, and 143 weeks with C4-447 peptide containing the 8-mer epitope for human monoclonal Ab (mAb) 447-52D specific to the V3 region of gp120 HIV-1. Strong anti-V3 antibody responses reached 50% binding titer in serum of 10−5 at week 10 that declined to 10−3 by week 70. After an additional boost of C4-447 peptide at week 143, titers rebounded to 10−5 at week 146, or 2.7 years after the first immunization. Using the blood sample at week 146, we produced 41 V3-specific recombinant mAbs by single B cell isolation and cloning. Sequence analysis revealed 21B cell lineages, single and clonally related, based on immunoglobulin gene usage and CDR3s. The broad repertoire of Abs directed to a small antigenic site shows the targeting potency of a vaccine-elicited immune response in rhesus macaques.

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection., Neutralizing nanobodies (Nb) are of considerable interest as therapeutic agents for COVID-19 treatment. Here, the authors functionally and structurally characterize Nbs that bind with high affinity to the receptor binding domain of the SARS-CoV-2 spike protein and show that an engineered homotrimeric Nb prevents disease progression in a Syrian hamster model of COVID-19 when administered intranasally.

The emergence of numerous variants of SARS-CoV-2, the causative agent of COVID-19, has presented new challenges to the global efforts to control the COVID-19 pandemic. Here, we obtain two cross-neutralizing antibodies (7D6 and 6D6) that target Sarbecoviruses’ receptor-binding domain (RBD) with sub-picomolar affinities and potently neutralize authentic SARS-CoV-2. Crystal structures show that both antibodies bind a cryptic site different from that recognized by existing antibodies and highly conserved across Sarbecovirus isolates. Binding of these two antibodies to the RBD clashes with the adjacent N-terminal domain and disrupts the viral spike. Both antibodies confer good resistance to mutations in the currently circulating SARS-CoV-2 variants. Thus, our results have direct relevance to public health as options for passive antibody therapeutics and even active prophylactics. They can also inform the design of pan-sarbecovirus vaccines., Antibodies (Abs) targeting highly conserved epitopes are important tools against emerging virus variants. Here, the authors characterize Abs that recognize a cryptic epitope in the receptor-binding domain of SARS-CoV-2 spike that is well conserved and show that these Abs can neutralize several variants of concerns.

Background The efficacy assessment of human anti-IgE monoclonal antibodies (mAbs) in animal models before clinical trials is hampered due to the lack of cross-reactivity of anti-IgE mAbs between species. Objective We developed CRE-DR (an anti-dog IgE monoclonal antibody), an anti-IgE mouse mAb that recognizes canine and human IgE, and then examined its IgE specificity and cross-reactivity between three animal and human species. Methods After mouse immunization with a synthetic peptide derived from canine IgE (282 NTNDWIEGETYYC294 ), we generated a hybridoma producing CRE-DR. The CRE-DR purified from the ascites of hybridoma-inoculated mice was used for ELISA and Western blot analysis to examine reactivity to dog, human, and rodent IgEs as well as recombinant bovine serum albumin (BSA)-conjugated to canine, human, and rodent IgE amino acid peptides corresponding to the immunizing sequence. We then performed enzyme-linked immunosorbent assays (ELISAs) for dog IgE using sera from dogs with atopic dermatitis (AD) after inhibition with canine IgE and IgG. The amino acid sequence recognized by CRE-DR was identified by ELISA using synthetic peptides. Results CRE-DR is a monoclonal mouse IgG1κ specific for dog IgE, and the ELISA values in atopic dog sera were inhibited by dog IgE, but not dog IgG. The binding of CRE-DR to human IgE was relatively maintained, but not to rodent IgEs, which results were confirmed with the BSA-conjugated IgE peptides of the various species. The CRE-DR reactivity was supported by the comparison of amino acid sequence of CRE-DR epitope, DWIEGETYYC, in dog IgE; one, two, and three amino acids were substituted in the human, rat, and mouse IgE epitopes, respectively. Conclusions and clinical relevance CRE-DR is a mAb cross-reactive to dog and human IgEs, which can allow the use of a dog model of allergy to test the efficacy of a CRE-DR-derived anti-IgE therapeutic mAb before human clinical trials.

De novo design methods hold the promise of reducing the time and cost of antibody discovery, while enabling the facile and precise targeting of predetermined epitopes. Here we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterisation showed that all designs are highly stable, and bind their intended targets with affinities in the nanomolar range without any in vitro affinity maturation. We further discuss how a high-resolution input antigen structure is not required, as our method yields similar predictions when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides a starting point for the rapid generation of lead antibodies binding to pre-selected epitopes.summaryA combinatorial method can rapidly design nanobodies for predetermined epitopes, which bind with KDs in the nanomolar range.

Hepatitis B Immune Globulin (HBIG) is a human plasma-derived IgG concentrate that contains a high titer of neutralizing antibodies (anti-HBs) to the hepatitis B virus (HBV) surface antigen (HBsAg). HBIG is known to be highly effective in treating HBV infections, however, a more systematic characterization of the antibody binding sites on HBsAg and their correlation with emerging "escape" mutations in HBsAg was lacking. By using anti-HBs antibodies from HBIG lots to screen random peptide phage display libraries, we identified five clusters of peptides that corresponded to five distinct anti-HBs binding sites on the HBsAg. Three sites, Site II (C121-C124), Site III (M133-P135), and Site IV (T140-G145), were mapped within the "a" determinant, while the two other sites, Site I (Q101-M103) and Site V (I152-S154), were outside the "a" determinant. We then tested in binding assays HBsAg peptides containing clinically relevant mutations previously reported within these sites, such as Y134S, P142S and G145R, and observed a significant reduction in anti-HBs binding activity to the mutated sites, suggesting a mechanism the virus may use to avoid HBIG-mediated neutralization. The current HBIG treatment could be improved by supplementing it with site-specific neutralizing monoclonal antibodies that target these mutations for control of HBV infections. This article is protected by copyright. All rights reserved.

Avidity is defined as the binding strength of immunoglobulin G (IgG) toward its target epitope. Avidity is directly related to affinity, as both processes are determined by the best fit of IgG to epitopes. We confirm and extend data on incomplete avidity maturation of IgG toward severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) nucleoprotein (NP), spike protein‐1 (S1), and its receptor‐binding domain (RBD) in coronavirus disease 2019 (COVID‐19) patients. In SARS‐CoV‐2‐infected individuals, an initial rise in avidity maturation was ending abruptly, leading to IgG of persistently low or intermediate avidity. Incomplete avidity maturation might facilitate secondary SARS‐CoV‐2 infections and thus prevent the establishment of herd immunity. Incomplete avidity maturation after infection with SARS‐CoV‐2 (with only 11.8% of cases showing finally IgG of high avidity, that is, an avidity index > 0.6) was contrasted by regular and rapid establishment of high avidity in SARS‐CoV‐2 naïve individuals after two vaccination steps with the BioNTech messenger RNA (mRNA) Vaccine (78% of cases with high avidity). One vaccination step was not sufficient for induction of complete avidity maturation in vaccinated SARS‐CoV‐2 naïve individuals, as it induced high avidity only in 2.9% of cases within 3 weeks. However, one vaccination step was sufficient to induce high avidity in individuals with previous SARS‐CoV‐2 infection., Highlights After severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, immunoglobulin G (IgG) toward viral nucleoprotein (NP), receptor‐binding domain (RBD), and spike protein‐1 (S1) shows immature avidity.The breakpoint of avidity maturation correlates with that of IgG production.Patients with more severe clinical symptoms show increased avidity maturation.Two vaccination steps lead to complete avidity maturation.Therefore, vaccination seems to induce protective immunity toward SARS‐CoV‐2 and coronavirus disease 2019 (COVID‐19).Preceding SARS‐CoV‐2 infections enhance avidity maturation after subsequent vaccination.

The protective efficacy of neutralizing antibodies (nAbs) elicited during natural infection with SARS-CoV-2 and by vaccination based on its spike protein has been compromised with emergence of the recent SARS-CoV-2 variants. Residues E484 and K417 in the receptor-binding site (RBS) are both mutated in lineages first described in South Africa (B.1.351) and Brazil (B.1.1.28.1). The nAbs isolated from SARS-CoV-2 patients are preferentially encoded by certain heavy-chain germline genes and the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2) can each bind the RBS in two different binding modes. However, their binding and neutralization are abrogated by either the E484K or K417N mutation, whereas nAbs to the cross-reactive CR3022 and S309 sites are largely unaffected. This structural and functional analysis illustrates why mutations at E484 and K417 adversely affect major classes of nAbs to SARS-CoV-2 with consequences for next-generation COVID-19 vaccines.

Antigen-specific immunotherapies (ASI) require successful loading and presentation of antigen peptides into the major histocompatibility complex (MHC) binding cleft. One route of ASI design is to mutate native antigens for either stronger or weaker binding interaction to MHC. Exploring all possible mutations is costly both experimentally and computationally. To reduce experimental and computational expense, here we investigate the minimal amount of prior data required to accurately predict the relative binding affinity of point mutations for peptide-MHC class II (pMHCII) binding. Using data from different residue subsets, we interpolate pMHCII mutant binding affinities by Gaussian process (GP) regression of residue volume and hydrophobicity. We apply GP regression to an experimental data set from the Immune Epitope Database, and theoretical data sets from NetMHCIIpan and Free Energy Perturbation calculations. We find that GP regression can predict binding affinities of nine neutral residues from a six-residue subset with an average R2 coefficient of determination value of 0.62 ± 0.04 (±95% CI), average error of 0.09 ± 0.01 kcal/mol (±95% CI), and with an receiver operating characteristic (ROC) AUC value of 0.92 for binary classification of enhanced or diminished binding affinity. Similarly, metrics increase to an R2 value of 0.69 ± 0.04, average error of 0.07 ± 0.01 kcal/mol, and an ROC AUC value of 0.94 for predicting seven neutral residues from an eight-residue subset. Our work finds that prediction is most accurate for neutral residues at anchor residue sites without register shift. This work holds relevance to predicting pMHCII binding and accelerating ASI design.

Antibodies recognize their target with high affinity and specificity. This is important for pathogen neutralization, which plays a crucial role in defense against disease. Antibodies are powerful tools in the development of new therapeutics, such as vaccines, to fight diseases such as viral infections and even cancer. The development of monoclonal and specific antibodies is time-consuming and expensive, but it can be greatly simplified with structural and allosteric information. Nuclear magnetic resonance (NMR) is a powerful technique to study protein structure and dynamics, and it has proven to be efficient to analyze large protein complexes, despite the overall size limitation. Here, we discuss NMR approaches efficiently used to conformational epitope mapping.

Until recently, glycan epitopes have not been documented by the WHO/IUIS Allergen Nomenclature Sub-Committee. This was in part due to scarce or incomplete information on these oligosaccharides, but also due to the widely held opinion that IgE to these epitopes had little or no relevance to allergic symptoms. Most IgE-binding glycans recognized up to 2008 were considered to be “classical” cross-reactive carbohydrate determinants (CCD) that occur in insects, some helminths and throughout the plant kingdom. Since 2008, the prevailing opinion on lack of clinical relevance of IgE-binding glycans has been subject to a reevaluation. This was because IgE specific for the mammalian disaccharide galactose-alpha-1,3-galactose (alpha-gal) was identified as a cause of delayed anaphylaxis to mammalian meat in the United States, an observation that has been confirmed by allergists in many parts of the world. Several experimental studies have shown that oligosaccharides with one or more terminal alpha-gal epitopes can be attached as a hapten to many different mammalian proteins or lipids. The classical CCDs also behave like haptens since they can be expressed on proteins from multiple species. This is the explanation for extensive in vitro cross-reactivity related to CCDs. Because of these developments, the Allergen Nomenclature Sub-Committee recently decided to include glycans as potentially allergenic epitopes in an adjunct section of its website (www.allergen.org). In this article, the features of the main glycan groups known to be involved in IgE recognition are revisited, and their characteristic structural, functional, and clinical features are discussed.

Human ferritin heavy chain, an example of a protein nanoparticle, has recently been used as a vaccine delivery platform. Human ferritin has advantages of uniform architecture, robust thermal and chemical stabilities, and good biocompatibility and biodegradation. There is however a lack of understanding about the relationship between insertion sites in ferritin (N-terminus and C-terminus) and the corresponding humoral and cell-mediated immune responses. To bridge this gap, we utilized an Epstein-Barr Nuclear Antigen 1 (EBNA1) epitope as a model to produce engineered ferritin-based vaccines E1F1 (N-terminus insertion) and F1E1 (C-terminus insertion) for the prevention of Epstein-Barr virus (EBV) infections. X-ray crystallography confirmed the relative positions of the N-terminus insertion and C-terminus insertion. For N-terminus insertion, the epitopes were located on the exterior surface of ferritin, while for C-terminus insertion, the epitopes were inside the ferritin cage. Based on the results of antigen-specific antibody titers from in-vivo tests, we found that there was no obvious difference on humoral immune responses between N-terminus and C-terminus insertion. We also evaluated splenocyte proliferation and memory lymphocyte T cell differentiation. Both results suggested C-terminus insertion produced a stronger proliferative response and cell-mediated immune response than N-terminus insertion. C-terminus insertion of EBNA1 epitope was also processed more efficiently by dendritic cells (DCs) than N-terminus insertion. This provides new insight into the relationship between the insertion site and immunogenicity of ferritin nanoparticle vaccines.

A comprehensive analysis of the humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential in understanding COVID-19 pathogenesis and developing antibody-based diagnostics and therapy. In this work, we performed a longitudinal analysis of antibody responses to SARS-CoV-2 proteins in 104 serum samples from 49 critical COVID-19 patients using a peptide-based SARS-CoV-2 proteome microarray. Our data show that the binding epitopes of IgM and IgG antibodies differ across SARS-CoV-2 proteins and even within the same protein. Moreover, most IgM and IgG epitopes are located within nonstructural proteins (nsps), which are critical in inactivating the host’s innate immune response and enabling SARS-CoV-2 replication, transcription, and polyprotein processing. IgM antibodies are associated with a good prognosis and target nsp3 and nsp5 proteases, whereas IgG antibodies are associated with high mortality and target structural proteins (Nucleocapsid, Spike, ORF3a). The epitopes targeted by antibodies in patients with a high mortality rate were further validated using an independent serum cohort (n = 56) and using global correlation mapping analysis with the clinical variables that are associated with COVID-19 severity. Our data provide fundamental insight into humoral immunity during SARS-CoV-2 infection. SARS-CoV-2 immunogenic epitopes identified in this work could also help direct antibody-based COVID-19 treatment and triage patients.

Immunoglobulin G (IgG) autoantibodies reactive to fetal brain proteins in mothers of children with ASD have been described by several groups. To understand their pathologic significance, we developed a mouse model of maternal autoantibody related ASD (MAR-ASD) utilizing the peptide epitopes from human autoantibody reactivity patterns. Male and female offspring prenatally exposed to the salient maternal autoantibodies displayed robust deficits in social interactions and increased repetitive self-grooming behaviors as juveniles and adults. In the present study, neuroanatomical differences in adult MAR-ASD and control offspring were assessed via high-resolution ex vivo magnetic resonance imaging (MRI) at 6 months of age. Of interest, MAR-ASD mice displayed significantly larger total brain volume and of the 159 regions examined, 31 were found to differ significantly in absolute volume (mm3) at an FDR of

Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR- analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody

A combined chemo‐enzymatic synthesis/NMR‐based methodology is presented to identify, in unambiguous manner, the distinctive binding epitope within repeating sugar oligomers when binding to protein receptors. The concept is based on the incorporation of 13C‐labels at specific monosaccharide units, selected within a repeating glycan oligomeric structure. No new chemical tags are added, and thus the chemical entity remains the same, while the presence of the 13C‐labeled monosaccharide breaks the NMR chemical shift degeneracy that occurs in the non‐labeled compound and allows the unique identification of the different components of the oligomer. The approach is demonstrated by a proof‐of‐concept study dealing with the interaction of a polylactosamine hexasaccharide with five different galectins that display distinct preferences for these entities., Repeating carbohydrate patterns are commonly found in Nature in extended oligosaccharides and polysaccharides. When recognized by protein receptors, dissecting the contribution of each repeating unit to the binding event is not easy. Here, the introduction of 13C‐labels at specific positions of a tri‐LacNAc repeating hexasaccharide has been used in STD‐HSQC NMR experiments to reveal the preferred binding epitopes for a panel of human galectins.

The SARS-CoV-2 B.1.617 lineage was identified in October 2020 in India1–5. Since then, it has become dominant in some regions of India and in the UK, and has spread to many other countries6. The lineage includes three main subtypes (B1.617.1, B.1.617.2 and B.1.617.3), which contain diverse mutations in the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein that may increase the immune evasion potential of these variants. B.1.617.2—also termed the Delta variant—is believed to spread faster than other variants. Here we isolated an infectious strain of the Delta variant from an individual with COVID-19 who had returned to France from India. We examined the sensitivity of this strain to monoclonal antibodies and to antibodies present in sera from individuals who had recovered from COVID-19 (hereafter referred to as convalescent individuals) or who had received a COVID-19 vaccine, and then compared this strain with other strains of SARS-CoV-2. The Delta variant was resistant to neutralization by some anti-NTD and anti-RBD monoclonal antibodies, including bamlanivimab, and these antibodies showed impaired binding to the spike protein. Sera collected from convalescent individuals up to 12 months after the onset of symptoms were fourfold less potent against the Delta variant relative to the Alpha variant (B.1.1.7). Sera from individuals who had received one dose of the Pfizer or the AstraZeneca vaccine had a barely discernible inhibitory effect on the Delta variant. Administration of two doses of the vaccine generated a neutralizing response in 95% of individuals, with titres three- to fivefold lower against the Delta variant than against the Alpha variant. Thus, the spread of the Delta variant is associated with an escape from antibodies that target non-RBD and RBD epitopes of the spike protein. The SARS-CoV-2 Delta variant partially evades neutralization by several monoclonal antibodies and by sera from individuals who have had COVID-19, but two doses of anti-COVID-19 vaccines still generate a strong neutralizing response.

Objective Analysis of the quantity and quality of epitope-specific CD8+ T-cell responses is crucial for understanding the mechanism of HIV/simian immunodeficiency virus (SIV) replication control. We have previously shown that acute-phase passive infusion of neutralizing antibodies (NAbs) results in augmented broad T-cell responses and robust SIVmac239 control in rhesus macaques. Analyzing long-term dynamics of CD8+ T-cell responses in these SIV controllers provides important insights into designing lasting anti-HIV immunity. Design We analyzed dynamics and metabolic/functional profiles of SIV-specific CD8+ T-cell responses in rhesus macaques that controlled SIVmac239 replication following acute-phase passive NAb infusion. Methods SIV epitope-specific CD8+ T-cell responses in peripheral blood at multiple chronic-phase time points were investigated in four passive NAb-infused SIV controllers. In particular, expression patterns of Eomesodermin (Eomes), phosphorylated AMP kinase (pAMPK), CD28 and programmed death-1 (PD-1) were examined. Results In the NAb-infused SIV controllers, a single epitope-specific CD8+ T-cell response detected from acute infection and maintaining low levels up to year 1 showed a surge thereafter, up to year 2 post-challenge. Retention of an effector-skewed and unexhausted Eomes-high/pAMPK-low/CD28-negative/PD-1-low subpopulation in these epitope-specific CD8+ T cells implicated their front-line commitment in residual viral replication control. Conclusions In long-term SIV control following acute-phase passive NAb infusion, a single-epitope, high-quality CTL response was dominantly induced in the chronic phase. These results likely describe one favorable pattern of immunodominant epitope-specific CD8+ T-cell preservation and suggest the importance of incorporating metabolic marker signatures for understanding NAb/T-cell synergism-based HIV/SIV control.

The Cap of porcine circovirus type 2 (PCV2) can be assembled into virus like particles (VLPs) in vitro that have multiple loops located on the particle surface. This would make it a good vehicle for displaying exogenous proteins or epitopes. We derived two epitopes, epitope B (EpB, S37HIQLIYNL45) and epitope 7 (Ep7, Q196WGRL200) from Gp5 of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). We replaced the core region of Loop CD (L75PPGGGSN82) and the carboxyl terminus (K222DPPL226) of PCV2 Cap, respectively, to construct a bi-epitope chimeric PCV2 Cap. Its immunogenicity and protective effects were evaluated as one PRRSV subunit vaccine. The chimeric PCV2 Cap was soluble, efficiently expressed in an Escherichia coli expression system, and could be self-assembled into chimeric virus like particles (cVLPs) with a diameter of 12-15 nm. Western blotting confirmed that the cVLPs could be specifically recognized by anti-PCV2, anti-EpB and anti-Ep7 antibodies. The cVLPs vaccine could alleviate the clinical symptoms and reduce the viral loads after HP-PRRSV challenge in 100-120 days old pigs. These data suggest that the cVLPs vaccine could provide pigs with partial protection against homologous PRRSV strains, and it provides a new design for additional PRRSV subunit vaccines.

Systemic lupus erythematosus is a complex immunological disease where both environmental factors and genetic predisposition lead to the dysregulation of important immune mechanisms. Eventually, the combination of these factors leads to the production of self-reactive antibodies that can target any organ or tissue of the human body. Autoantibodies can form immune complexes responsible for both the organ damage and the most severe complications. Involvement of the central nervous system defines a subcategory of the disease, generally known with the denomination of neuropsychiatric systemic lupus erythematosus. Neuropsychiatric symptoms can range from relatively mild manifestations, such as headache, to more severe complications, such as psychosis. The evaluation of the presence of the autoantibodies in the serum of these patients is the most helpful diagnostic tool for the assessment of the disease. The scientific progresses achieved in the last decades helped researchers and physicians to discover some of autoepitopes targeted by the autoantibodies, although the majority of them have not been identified yet. Additionally, the central nervous system is full of epitopes that cannot be found elsewhere in the human body, for this reason, autoantibodies that selectively target these epitopes might be used for the differential diagnosis between patients with and without the neuropsychiatric symptoms. In this review, the most relevant data is reported with regard to mechanisms implicated in the production of autoantibodies and the most important autoantibodies found among patients with systemic lupus erythematosus with and without the neuropsychiatric manifestations.

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq (1) ) to the spike ectodomain (S-ECD (2) ) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro , we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å (2) ). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning (3,4) our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

A newly-emergent beta-coronavirus, SARS-CoV-2, rapidly has become a pandemic since 2020. It is a serious respiratory disease and caused more than 100 million of deaths in the world. WHO named it COVIA-19 and there is no effective targeted drug for it. The main treatment strategies include chemical medicine, traditional Chinese medicine (TCM) and biologics. Due to SARS-CoV-2 uses the spike proteins (S proteins) on its envelope to infect human cells, monoclonal antibodies that neutralize the S protein have become one of the hot research areas in the current research and treatment of SARS-CoV-2. In this study, we reviewed the antibodies that have been reported to have neutralizing activity against the SARS-CoV-2 infection. According to their different binding epitope regions in RBD or NTD, they are classified, and the mechanism of the representative antibodies in each category is discussed in depth, which provides potential foundation for future antibody and vaccine therapy and the development of antibody cocktails against SARS-CoV-2 mutants.

Influenza viruses are a major public health problem. Vaccines are the best available countermeasure to induce effective immunity against infection with seasonal influenza viruses; however, the breadth of antibody responses in infection versus vaccination is quite different. Here, we show that nasal infection controls two sequential processes to induce neutralizing IgG antibodies recognizing the hemagglutinin (HA) of heterotypic strains. The first is viral replication in the lung, which facilitates exposure of shared epitopes that are otherwise hidden from the immune system. The second process is the germinal center (GC) response, in particular, IL-4 derived from follicular helper T cells has an essential role in the expansion of rare GC-B cells recognizing the shared epitopes. Therefore, the combination of exposure of the shared epitopes and efficient proliferation of GC-B cells is critical for generating broadly-protective antibodies. These observations provide insight into mechanisms promoting broad protection from virus infection., The reasons why influenza infection promotes a broader antibody response compared with vaccines are not fully understood. Here the authors show that unmasking of haemagglutinin epitopes and IL-4 signals in the germinal centre contribute to broader antibody responses after infection.

Maltose binding protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The monoclonal antibody B48 binds MBP tightly and has no cross‐reactivity to other proteins in an Escherichia coli lysate. This high level of specificity suggested that MBP contains an epitope that could prove useful as a purification and visualization tag for proteins expressed in E. coli. To discover the MBP epitope, a co‐crystal structure was determined for MBP bound to its antibody and four amino acids of MBP were identified as critical for the binding interaction. Fusions of various fragments of MBP to the glutathione S‐transferase protein were engineered in order to identify the smallest fragment still recognized by the α‐MBP antibody. Stabilization of the epitope via mutational engineering resulted in a minimized 14 amino‐acid tag.