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Human epidermal growth factor receptor-2 (HER2) is overexpressed in various solid tumor types, acting as an established therapeutic target. Over the last three decades, the fast-paced development of diverse HER2-targeted agents, notably marked by the introduction of the antibody-drug conjugate (ADC), yielding substantial improvements in survival rates. However, resistance to anti-HER2 treatments continues to pose formidable challenges. The complex structure and dynamic dimerization properties of HER2 create significant hurdles in the development of novel targeted therapeutics. In this review, we synthesize the latest insights into the structural intricacies of HER2 and present an unprecedented overview of the epitope characteristics of HER2-targeted antibodies and their derivatives. Furthermore, we delve into the correlation between anti-HER2 antibody binding epitopes and their respective functions, with a particular focus on their efficacy against resistant tumors. In addition, we highlight the potential of emerging anti-HER2 agents that target specific sites or non-overlapping epitopes, poised to transform the therapeutic landscape for HER2-positive tumors in the foreseeable future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Coagulation factor replacement therapy for the X-linked bleeding disorder Haemophilia, characterized by a deficiency of coagulation protein factor VIII (FVIII), is severely complicated by antibody (inhibitors) formation. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with a tremendous increase in morbidity as well as treatment costs. The ultimate goal of inhibitor control is antibody elimination. Immune tolerance induction (ITI) is the only clinically established approach for developing antigen-specific tolerance to FVIII. This work aims to establish a novel cost-effective strategy to produce FVIII molecules in fusion with cholera toxin B (CTB) subunit at the N terminus using the Bacillus subtilis expression system for oral tolerance, as the current clinical immune tolerance protocols are expensive. Regions of B-Domain Deleted (BDD)-FVIII that have potential epitopes were identified by employing Bepipred linear epitope prediction; 2 or more epitopes in each domain were combined and cDNA encoding these regions were fused with CTB and cloned in the Bacillus subtilis expression vector pHT43 and expression analysis was carried out. The expressed CTB-fused FVIII epitope domains showed strong binding affinity towards the CTB-receptor GM1 ganglioside. To conclude, Bacillus subtilis expressing FVIII molecules might be a promising candidate for exploring for the induction of oral immune tolerance., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

To visualize the cellular and subcellular localization of neuromodulatory G-protein-coupled receptors in Drosophila , we implement a molecular strategy recently used to add epitope tags to ionotropic receptors at their endogenous loci. Leveraging evolutionary conservation to identify sites more likely to permit insertion of a tag, we generated constitutive and conditional tagged alleles for Drosophila 5-HT1A , 5-HT2A , 5-HT2B , Oct β 1R , Oct β 2R , two isoforms of OAMB , and mGluR The conditional alleles allow for the restricted expression of tagged receptor in specific cell types, an option not available for any previous reagents to label these proteins. We show expression patterns for these receptors in female brains and that 5-HT1A and 5-HT2B localize to the mushroom bodies (MBs) and central complex, respectively, as predicted by their roles in sleep. By contrast, the unexpected enrichment of Octβ1R in the central complex and of 5-HT1A and 5-HT2A to nerve terminals in lobular columnar cells in the visual system suggest new hypotheses about their functions at these sites. Using an additional tagged allele of the serotonin transporter, a marker of serotonergic tracts, we demonstrate diverse spatial relationships between postsynaptic 5-HT receptors and presynaptic 5-HT neurons, consistent with the importance of both synaptic and volume transmission. Finally, we use the conditional allele of 5-HT1A to show that it localizes to distinct sites within the MBs as both a postsynaptic receptor in Kenyon cells and a presynaptic autoreceptor., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Background: Trastuzumab and pertuzumab combination has been approved for the treatment of patients with HER2-positive metastatic breast cancer. However, trastuzumab and pertuzumab combination did not show improvement in overall survival in patients with HER2-positive metastatic gastric cancer., Methods: We developed a new HER2-targeted monoclonal antibody, HLX22, targeting HER2 subdomain IV as trastuzumab but with non-overlapping epitopes. We examined the antitumor effects of this novel HER2-antibody in gastric cell lines and cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models., Results: HLX22 in combination with HLX02 (trastuzumab biosimilar) induced enhancement of HER2/HER2 homodimers and HER2/EGFR heterodimers internalization, which ultimately led to the reduction in signal transductions involving STAT3, P70 S6, and AKT; gene expressions of FGF-FGFR-PI3K-MTOR, EGF-EGFR-RAS, TGF-β-SMAD, PLCG and cell cycle progression related pathways that favor tumor development, proliferation, progression, migration and survival in gastric cancer cell line NCI-N87 were also reduced. These differing but complementary actions contributed to the synergistic antitumor efficacy of the HLX22 and HLX02 combination in gastric cancer cell lines, CDX and PDX. In addition, HLX22 in combination with HLX02 demonstrated stronger antitumor efficacy than HLX02 and HLX11 (a potential pertuzumab biosimilar) combination treatment both in vitro and in vivo., Conclusions: These results suggested that the application of non-competing antibodies HLX22 and HLX02 targeting HER2 subdomain IV together may be of substantial benefit to gastric cancer patients who currently respond suboptimal to trastuzumab therapy., (© 2024. The Author(s).)

Currently, SARS-CoV-2 has evolved into various variants, including the numerous highly mutated Omicron sub-lineages, significantly increasing immune evasion ability. The development raises concerns about the possibly diminished effectiveness of available vaccines and antibody-based therapeutics. Here, we describe those representative categories of broadly neutralizing antibodies (bnAbs) that retain prominent effectiveness against emerging variants including Omicron sub-lineages. The molecular characteristics, epitope conservation, and resistance mechanisms of these antibodies are further detailed, aiming to offer suggestion or direction for the development of therapeutic antibodies, and facilitate the design of vaccines with broad-spectrum potential.

Objective: Autoantibodies against MDA5 (melanoma differentiation-associated protein 5) serve as a biomarker for DM (dermatomyositis) and indicate a risk factor for interstitial lung disease (ILD). MDA5 is a protein responsible for sensing RNA virus infection and activating signalling pathways against it. However, little is known about the antigen epitopes on MDA5 autoantibodies. We aimed to determine the interaction of the MDA5 autoantibody-antigen epitope., Methods: Cell-based assays (CBAs), immunoprecipitation-immunoblot assays, and various immunoblotting techniques were used in the study., Results: We demonstrated that DM patient autoantibodies recognize MDA5 epitopes in a native conformation-dependent manner. Furthermore, we identified the central helicase domain （3Hel） formed by Hel1, Hel2i, Hel2, and pincer as the major epitopes. As proof of principle, the purified 3Hel efficiently absorbed MDA5 autoantibodies from patient sera through immunoprecipitation-immunoblot assay., Conclusion: Our study uncovered the nature of the antigen epitopes on MDA5 and can provide guidance for diagnosis and a targeted therapeutic approach development., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Acquired Immunodeficiency Syndrome is caused by the Human Immunodeficiency Virus (HIV), and a significant number of fatalities occur annually. There is a dire need to develop an effective vaccine against HIV-1. Understanding the structural proteins of viruses helps in designing a vaccine based on immunogenic peptides. In the current experiment, we identified gp120 epitopes using bioinformatic epitope prediction tools, molecular docking, and MD simulations. The Gb-1 peptide was considered an adjuvant. Consecutive sequences of GTG, GSG, GGTGG, and GGGGS linkers were used to bind the B cell, Cytotoxic T Lymphocytes (CTL), and Helper T Lymphocytes (HTL) epitopes. The final vaccine construct consisted of 315 amino acids and is expected to be a recombinant protein of approximately 35.49 kDa. Based on docking experiments, molecular dynamics simulations, and tertiary structure validation, the analysis of the modeled protein indicates that it possesses a stable structure and can interact with Toll-like receptors. The analysis demonstrates that the proposed vaccine can provoke an immunological response by activating T and B cells, as well as stimulating the release of IgA and IgG antibodies. This vaccine shows potential for HIV-1 prophylaxis. The in-silico design suggests that multiple-epitope constructs can be used as potentially effective immunogens for HIV-1 vaccine development.

Peptide antibodies have become one of the most important classes of reagents in molecular biology and clinical diagnostics. For this reason, methods for their production and characterization continue to be developed, including basic peptide synthesis protocols, peptide-conjugate production and characterization, conformationally restricted peptides, immunization procedures, etc. Detailed mapping of peptide antibody epitopes has yielded important information on antibody-antigen interaction in general and specifically in relation to antibody cross-reactivity and theories of molecular mimicry. This information is essential for detailed understanding of paratope-epitope dynamics, design of antibodies for research, design of peptide-based vaccines, development of therapeutic peptide antibodies, and de novo design of antibodies with predetermined specificity., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unpredictable manifestations of coronavirus disease (COVID-19) and variable clinical course with some patients being asymptomatic whereas others experiencing severe respiratory distress, or even death. We aimed to evaluate the immunoglobulin G (IgG) response towards linear peptides on a peptide array containing sequences from SARS-CoV-2, Middle East respiratory syndrome-related coronavirus (MERS) and common-cold coronaviruses 229E, OC43, NL63 and HKU1 antigens, in order to identify immunological indicators of disease outcome in SARS-CoV-2 infected patients., Methods: We included in the study 79 subjects, comprising 19 pediatric and 30 adult SARS-CoV-2 infected patients with increasing disease severity, from mild to critical illness, and 30 uninfected subjects who were vaccinated with one dose of SARS-CoV-2 spike mRNA BNT162b2 vaccine. Serum samples were analyzed by a peptide microarray containing 5828 overlapping 15-mer synthetic peptides corresponding to the full SARS-CoV-2 proteome and selected linear epitopes of spike (S), envelope (E) and membrane (M) glycoproteins as well as nucleoprotein (N) of MERS, SARS and coronaviruses 229E, OC43, NL63 and HKU1 (isolates 1, 2 and 5)., Results: All patients exhibited high IgG reactivity against the central region and C-terminus peptides of both SARS-CoV-2 N and S proteins. Setting the threshold value for serum reactivity above 25,000 units, 100% and 81% of patients with severe disease, 36% and 29% of subjects with mild symptoms, and 8% and 17% of children younger than 8-years reacted against N and S proteins, respectively. Overall, the total number of peptides in the SARS-CoV-2 proteome targeted by serum samples was much higher in children compared to adults. Notably, we revealed a differential antibody response to SARS-CoV-2 peptides of M protein between adults, mainly reacting against the C-terminus epitopes, and children, who were highly responsive to the N-terminus of M protein. In addition, IgG signals against NS7B, NS8 and ORF10 peptides were found elevated mainly among adults with mild (63%) symptoms. Antibodies towards S and N proteins of other coronaviruses (MERS, 229E, OC43, NL63 and HKU1) were detected in all groups without a significant correlation with SARS-CoV-2 antibody levels., Conclusions: Overall, our results showed that antibodies elicited by specific linear epitopes of SARS-CoV-2 proteome are age dependent and related to COVID-19 clinical severity. Cross-reaction of antibodies to epitopes of other human coronaviruses was evident in all patients with distinct profiles between children and adult patients. Several SARS-CoV-2 peptides identified in this study are of particular interest for the development of vaccines and diagnostic tests to predict the clinical outcome of SARS-CoV-2 infection., (© 2023. The Author(s).)

Cancer-related epitopes can engage the immune system against tumor cells, thus exploring epitopes derived from non-coding regions is emerging as a fascinating field in cancer immunotherapies. Here, we described a database, IEAtlas (http://bio-bigdata.hrbmu.edu.cn/IEAtlas), which aims to provide and visualize the comprehensive atlas of human leukocyte antigen (HLA)-presented immunogenic epitopes derived from non-coding regions. IEAtlas reanalyzed publicly available mass spectrometry-based HLA immunopeptidome datasets against our integrated benchmarked non-canonical open reading frame information. The current IEAtlas identified 245 870 non-canonical epitopes binding to HLA-I/II allotypes across 15 cancer types and 30 non-cancerous tissues, greatly expanding the cancer immunopeptidome. IEAtlas further evaluates the immunogenicity via several commonly used immunogenic features, including HLA binding affinity, stability and T-cell receptor recognition. In addition, IEAtlas provides the biochemical properties of epitopes as well as the clinical relevance of corresponding genes across major cancer types and normal tissues. Several flexible tools were also developed to aid retrieval and to analyze the epitopes derived from non-coding regions. Overall, IEAtlas will serve as a valuable resource for investigating the immunogenic capacity of non-canonical epitopes and the potential as therapeutic cancer vaccines., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Structural characterization of infection- and vaccination-elicited antibodies in complex with antigen provides insight into the evolutionary arms race between the host and the pathogen and informs rational vaccine immunogen design. We isolated a germ line-encoded monoclonal antibody (mAb) from plasmablasts activated upon mRNA vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and determined its structure in complex with the spike glycoprotein by electron cryomicroscopy (cryo-EM). We show that the mAb engages a previously uncharacterized neutralizing epitope on the spike N-terminal domain (NTD). The high-resolution structure reveals details of the intermolecular interactions and shows that the mAb inserts its heavy complementarity-determining region 3 (HCDR3) loop into a hydrophobic NTD cavity previously shown to bind a heme metabolite, biliverdin. We demonstrate direct competition with biliverdin and that, because of the conserved nature of the epitope, the mAb maintains binding to viral variants B.1.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta), and B.1.1.529 (omicron). Our study describes a novel conserved epitope on the NTD that is readily targeted by vaccine-induced antibody responses. IMPORTANCE We report the first structure of a vaccine-induced antibody to SARS-CoV-2 spike isolated from plasmablasts 7 days after vaccination. The genetic sequence of the antibody PVI.V6-14 suggests that it is completely unmutated, meaning that this type of B cell did not undergo somatic hypermutation or affinity maturation; this cell was likely already present in the donor and was activated by the vaccine. This is, to our knowledge, also the first structure of an unmutated antibody in complex with its cognate antigen. PVI.V6-14 binds a novel, conserved epitope on the N-terminal domain (NTD) and neutralizes the original viral strain. PVI.V6-14 also binds the newly emerged variants B.1.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta), and B.1.1.529 (omicron). Given that this antibody was likely already present in the donor prior to vaccination, we believe that this antibody class could potentially "keep up" with the new variants, should they continue to emerge, by undergoing somatic hypermutation and affinity maturation.

Superoxide-producing NADPH oxidase, gp91 phox /NOX2, in phagocytes plays a critical role in the host defenses against pathogens. Moreover, gp91 phox /NOX2 contributes to the oxidative stress in endothelial cells. Therefore, investigating the level of gp91 phox /NOX2 with immunoblotting is important for estimating the amount of superoxide produced. Here, we showed that the epitopes in human gp91 phox /NOX2 recognized by monoclonal antibodies (mAbs) CL-5 and 48 were in amino acids 132-147 and 136-144, respectively. Although the epitopes were close to the N-glycosylation sites, N-glycan maturation did not affect mAbs recognition. When Pro-136 was substituted with Arg, the corresponding mouse residue, human gp91 phox /NOX2 was not recognized by mAbs CL-5 and 48; however, the substitution did not affect gp91 phox /NOX2-based oxidase activity. This finding explains why these mAbs specifically recognize the human but not mouse gp91 phox /NOX2. Hence, these mAbs are useful for investigating the level of gp91 phox /NOX2 without amino acid substitutions in the epitopes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Enzyme-linked immunosorbent assay (ELISA) is a plate-based immunological assay designed to detect and quantify peptides, proteins, antibodies, and hormones. Fluorescence polarization (FP) is a solution-phase technique that can be used to determine equilibrium dissociation constant of ligand for the protein of interest. Here we describe the protocols for different ELISAs and for Fluorescence Polarization, and how they can be used to determine relative or absolute binding of macrocyclic peptides to the target proteins. In ELISA, the target protein is used as the antigen, and the binding of antigen is quantified using cyclic peptides and enzyme-linked antibodies. In Fluorescence Polarization assays, a cyclic ligand is fluorescent dye-labeled and titrated with serial concentrations of the non-labeled target protein to determine the equilibrium dissociation constant (K D ) of ligand for protein. Detailed descriptions of sample preparation and the ELISA and FP experiments are provided in this chapter., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Interleukin-1 (IL-1) family cytokines are potent mediators of inflammation, acting to coordinate local and systemic immune responses to a wide range of stimuli. Aberrant signaling by IL-1 family cytokine members, however, is linked to myriad inflammatory syndromes, autoimmune conditions and cancers. As such, blocking the inflammatory signals inherent to IL-1 family signaling is an established and expanding therapeutic strategy. While several FDA-approved IL-1 inhibitors exist, including an Fc fusion protein, a neutralizing antibody, and an antagonist cytokine, none specifically targets the co-receptor IL-1 receptor accessory protein (IL-1RAcP). Most IL-1 family cytokines form productive signaling complexes by binding first to their cognate receptors - IL-1RI for IL-1α and IL-1β; ST2 for IL-33; and IL-36R for IL-36α, IL-36β and IL-36γ - after which they recruit the shared secondary receptor IL-1RAcP to form a ternary cytokine/receptor/co-receptor complex. Recently, IL-1RAcP was identified as a biomarker for both AML and CML. IL-1RAcP has also been implicated in tumor progression in solid tumors and an anti-IL1RAP antibody (nadunolimab, CAN04) is in phase II clinical studies in pancreatic cancer and non-small cell lung cancer (NCT03267316). As IL-1RAcP is common to all of the abovementioned IL-1 family cytokines, targeting this co-receptor raises the possibility of selective signaling inhibition for different IL-1 family cytokines. Indeed, previous studies of IL-1β and IL-33 signaling complexes have revealed that these cytokines employ distinct mechanisms of IL-1RAcP recruitment even though their overall cytokine/receptor/co-receptor complexes are structurally similar. Here, using functional, biophysical, and structural analyses, we show that antibodies specific for IL-1RAcP can differentially block signaling by IL-1 family cytokines depending on the distinct IL-1RAcP epitopes that they engage. Our results indicate that targeting a shared cytokine receptor is a viable therapeutic strategy for selective cytokine signaling inhibition., Competing Interests: DL, GF, and GB are employees of Cantargia AB (Medicon Village, Lund, Sweden). KS is an employee of Innovagen AB (Lund, Sweden). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received partial funding from Cantargia AB. The funder had the following involvement in the study: data collection and analysis, decision to publish. Cantargia AB is the owner of the intellectual property rights for CAN03 and CAN04 for use in the treatment and diagnosis of neoplastic disorders., (Copyright © 2021 Fields, Kihn, Birkedal, Klontz, Sjöström, Günther, Beadenkopf, Forsberg, Liberg, Snyder, Deredge and Sundberg.)

Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Bivalent V H Hs have been shown to display better functional affinity compared with their monovalent counterparts. Bivalency can be achieved either by inserting a hinge region between both V H Hs units or by using modules that lead to dimerization. In this report, a small self-associating peptide originating from the tetramerization domain of p53 was developed as a tool for devicing nanobody dimerization. This E3 peptide was evaluated for the dimerization of an anti-eGFP nanobody (nano-eGFP-E3) whose activity was compared to a bivalent anti-eGFP constructed in tandem using GS rich linker. The benefit of bivalency in terms of avidity and specificity was assessed in different in vitro and in cellulo assays. In ELISA and SPR, the dimeric and tandem formats were nearly equivalent in terms of gain of avidity compared to the monovalent counterpart. However, in cellulo, the nano-eGFP-E3 construct showed its superiority over the tandem format in terms of specificity with a highest and better ratio signal-to-noise. All together, the E3 peptide provides a universal suitable tool for the construction of dimeric biomolecules, in particular antibody fragments with improved functional affinity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objectives: There is a need for precision medicine and an unspoken promise of an optimal approach for identification of the right patients for value-based medicine based on big data. However, there may be a misconception that measurement of proteins is more valuable than measurement of fewer selected biomarkers. In population-based research, variation may be somewhat eliminated by quantity. However, this fascination of numbers may limit the attention to and understanding of the single. This review highlights that protein measurements (with collagens as examples) may mean different things depending on the targeted epitope - formation or degradation of tissues, and even signaling potential of proteins., Design and Methods: PubMed was searched for collagen, neo-epitope, biomarkers., Results: Ample examples of assays with specific epitopes, either pathological such as HbA1c, or domain specific such as pro-peptides, which total protein arrays would not have identified were evident., Conclusions: We suggest that big data may be considered as the funnel of data points, in which most important parameters will be selected. If the technical precision is low or the biological accuracy is limited, and we include suboptimal quality of biomarkers, disguised as big data, we may not be able to fulfill the promise of helping patients searching for the optimal treatment. Alternatively, if the technical precision of the total protein quantification is high, but we miss the functional domains with the most considerable biological meaning, we miss the most important and valuable information of a given protein. This review highlights that measurements of the same protein in different ways may provide completely different meanings. We need to understand the pathological importance of each epitope quantified to maximize protein measurements., (Copyright © 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Very recently, the genome of the modern durum wheat cv. Svevo was fully sequenced, and its assembly is publicly available. So, we exploited the opportunity to carry out an in-depth study for the systematic characterization of the γ-gliadin gene family in the cv. Svevo by combining a bioinformatic approach with transcript and protein analysis. We found that the γ-gliadin family consists of nine genes that include seven functional genes and two pseudogenes. Three genes, Gli-γ1a , Gli-γ3a and Gli-γ4a , and the pseudogene Gli-γ2a* mapped on the A genome, whereas the remaining four genes, Gli-γ1b , Gli-γ2b , Gli-γ3b and Gli-γ5b , and the pseudogene Gli-γ4b* mapped on the B genome. The functional γ-gliadins presented all six domains and eight-cysteine residues typical of γ-gliadins. The Gli-γ1b also presented an additional cysteine that could possibly have a role in the formation of the gluten network through binding to HMW glutenins. The γ-gliadins from the A and B genome differed in their celiac disease (CD) epitope content and composition, with the γ-gliadins from the B genome showing the highest frequency of CD epitopes. In all the cases, almost all the CD epitopes clustered in the central region of the γ-gliadin proteins. Transcript analysis during seed development revealed that all the functional γ-gliadin genes were expressed with a similar pattern, although significant differences in the transcript levels were observed among individual genes that were sometimes more than 60-fold. A progressive accumulation of the γ-gliadin fraction was observed in the ripening seeds that reached 34% of the total gliadin fraction at harvest maturity. We believe that the insights generated in the present study could aid further studies on gliadin protein functions and future breeding programs aimed at the selection of new healthier durum wheat genotypes.

Focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) are common forms of idiopathic nephrotic syndrome. The causes of these diseases are incompletely understood, but the response of patients to immunosuppressive therapies suggests that their pathogenesis is at least in part immune mediated. Preclinical and clinical research indicates that activation of the classical pathway of complement contributes to glomerular injury in FSGS. Glomerular IgM deposits are also prominent in some patients, raising the possibility that IgM is a trigger of classical pathway activation. In the present study, we examined the pattern of complement activation in the glomeruli and plasma of patients with nephrotic syndrome. We also tested whether patients with FSGS and MCD have elevated levels of natural IgM reactive with epitopes on glomerular endothelial cells and cardiolipin. We found evidence of classical pathway activation in patients with idiopathic nephrotic syndrome compared with healthy control subjects. We also detected higher levels of self-reactive IgM to both targets. Based on these results, IgM and classical pathway activation may contribute to disease pathogenesis in some patients with FSGS and MCD. NEW & NOTEWORTHY IgM is detected in biopsies from some patients with nephrotic syndrome, although this has been attributed to passive trapping of the protein. We found, however, that IgM colocalizes with complement activation fragments in some glomeruli. We also found that affected patients had higher levels of IgM reactive to glomerular endothelial cell epitopes. Thus, IgM activates the complement system in the glomeruli of some patients with nephrotic syndrome and may contribute to injury.

Although not a standard-of-care yet, adoptive immunotherapeutic approaches have gradually earned a place within the list of antiviral therapies for some of fatal and hard-to-treat viral diseases. To maintain robust antiviral immunity and to effectively target the viral particles and virally-infected cells, immune cells capable of recognizing the viral antigens are required. While conventional vaccination can induce these cells in vivo; another option is to prime and generate antigen-specific immune cells ex vivo. This approach has been successfully trialed for virulent opportunistic viral infections after bone marrow transplantation. Amid the crisis of SARS-CoV2 pandemic, which has been followed by the success of certain early-authorized vaccines; some institutions and companies have explored the effects of viral-specific adoptive cell transfers (ACTs) in trials, as alternative treatments. Aimed at outlining a perspective on antigen-specific adoptive immunotherapy for viral infections, this review article specifically provides an appraisal of ACT-based studies/trials on SARS-CoV2 infection., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Overexpression of nonmutated proteins involved in oncogenesis is a mechanism by which such proteins become immunogenic. We questioned whether overexpressed colorectal cancer associated proteins found at higher incidence and associated with poor prognosis could be effective vaccine antigens. We explored whether vaccines targeting these proteins could inhibit the development of intestinal tumors in the azoxymethane (AOM)-induced colon model and APC Min mice., Methods: Humoral immunity was evaluated by ELISA. Web-based algorithms identified putative Class II binding epitopes of the antigens. Peptide and protein specific T-cells were identified from human peripheral blood mononuclear cells using IFN-gamma ELISPOT. Peptides highly homologous between mouse and man were formulated into vaccines and tested for immunogenicity in mice and in vivo tumor challenge. Mice treated with AOM and APC Min transgenic mice were vaccinated and monitored for tumors., Results: Serum IgG for CDC25B, COX2, RCAS1, and FASCIN1 was significantly elevated in colorectal cancer patient sera compared to volunteers (CDC25B p=0.002, COX-2 p=0.001, FASCIN1 and RCAS1 p<0.0001). Epitopes predicted to bind to human class II MHC were identified for each protein and T-cells specific for both the peptides and corresponding recombinant protein were generated from human lymphocytes validating these proteins as human antigens. Some peptides were highly homologous between mouse and humans and after immunization, mice developed both peptide and protein specific IFN-γ-secreting cell responses to CDC25B, COX2 and RCAS1, but not FASCIN1. FVB/nJ mice immunized with CDC25B or COX2 peptides showed significant inhibition of growth of the syngeneic MC38 tumor compared to control (p<0.0001). RCAS1 peptide vaccination showed no anti-tumor effect. In the prophylactic setting, after immunization with CDC25B or COX2 peptides mice treated with AOM developed significantly fewer tumors as compared to controls (p<0.0002) with 50% of mice remaining tumor free in each antigen group. APC Min mice immunized with CDC25B or COX2 peptides developed fewer small bowel tumors as compared to controls (p=0.01 and p=0.02 respectively)., Conclusions: Immunization with CDC25B and COX2 epitopes consistently suppressed tumor development in each model evaluated. These data lay the foundation for the development of multi-antigen vaccines for the treatment and prevention of colorectal cancer., Competing Interests: MD is a stockholder in EpiThany and receives grant support from Celgene, EMD Serono, Pfizer, Janssen, and Precigen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Corulli, Cecil, Gad, Koehnlein, Coveler, Childs, Lubet and Disis.)

Aims: Brazil is nowadays one of the epicentres of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and new therapies are needed to face it. In the context of specific immune response against the virus, a correlation between Major Histocompatibility Complex Class I (MHC-I) and the severity of the disease in patients with COVID-19 has been suggested. Aiming at better understanding the biology of the infection and the immune response against the virus in the Brazilian population, we analysed SARS-CoV-2 protein S peptides in order to identify epitopes able to elicit an immune response mediated by the most frequent MHC-I alleles using in silico methods., Methods: Our analyses consisted in searching for the most frequent Human Leukocyte Antigen (HLA)-A, HLA-B and HLA-C alleles in the Brazilian population, excluding the genetic isolates; then, we performed: molecular modelling for unsolved structures, MHC-I binding affinity and antigenicity prediction, peptide docking and molecular dynamics of the best fitted MHC-I/protein S complexes., Results: We identified 24 immunogenic epitopes in the SARS-CoV-2 protein S that could interact with 17 different MHC-I alleles (namely, HLA-A*01:01; HLA-A*02:01; HLA-A*11:01; HLA-A*24:02; HLA-A*68:01; HLA-A*23:01; HLA-A*26:01; HLA-A*30:02; HLA-A*31:01; HLA-B*07:02; HLA-B*51:01; HLA-B*35:01; HLA-B*44:02; HLA-B*35:03; HLA-C*05:01; HLA-C*07:01 and HLA-C*15:02) in the Brazilian population., Conclusions: Being aware of the intrinsic limitations of in silico analysis (mainly the differences between the real and the Protein Data Bank (PDB) structure; and accuracy of the methods for simulate proteasome cleavage), we identified 24 epitopes able to interact with 17 MHC-I more frequent alleles in the Brazilian population that could be useful for the development of strategic methods for vaccines against SARS-CoV-2., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

The protection of current influenza vaccines is limited due to the viral antigenic shifts and antigenic drifts. The universal influenza vaccine is a new hotspot in vaccine research that aims to overcome these problems. Polydopamine (PDA), a versatile biomaterial, has the advantages of an excellent biocompatibility, controllable particle size, and distinctive drug loading approach in drug delivery systems. To enhance the immunogenicities and delivery efficiencies of H9N2 avian influenza virus (AIV) epitope peptide vaccines, PDA nanoparticles conjugated with the BPP-V and BP-IV epitope peptides were used to prepare the nano BPP-V and BP-IV epitope peptide vaccines, respectively. The characteristics of the newly developed epitope peptide vaccines were then evaluated, revealing particle sizes ranging from approximately 240 to 290 nm (PDI<0.3), indicating that the synthesized nanoparticles were stable. Simultaneously, the immunoprotective effects of nano BPP-V and BP-IV epitope peptide vaccines were assessed. The nano BPP-V and BP-IV epitope vaccines, especially nano BP-IV epitope vaccine, quickly induced anti-hemagglutinin (HA) antibody production and a sustained immune response, significantly promoted humoral and cellular immune responses, reduced viral lung damage and provided effective protection against AIV viral infection. Together, these results reveal that PDA, as a delivery carrier, can improve the immunogenicities and delivery efficiencies of H9N2 AIV nano epitope vaccines, thereby providing a theoretical basis for the design and development of PDA as a carrier of new universal influenza vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Wang, Zhou, Shi, Shen, Chen, Zhang, Liao and Wang.)

Allergen-specific immunotherapy (AIT) is the only means of altering the natural immunological course of allergic diseases and achieving long-term remission. Pharmacological measures are able to suppress the immune response and/or ameliorate the symptoms but there is a risk of relapse soon after these measures are withdrawn. Current AIT approaches depend on the administration of intact allergens, often comprising crude extracts of the allergen. We propose that the challenges arising from current approaches, including the risk of serious side-effects, burdensome duration of treatment, poor compliance and high cost, are overcome by application of peptides based on CD4 + T cell epitopes rather than whole allergens. Here we describe evolving approaches, summarize clinical trials involving peptide AIT in allergic rhinitis and asthma, discuss the putative mechanisms involved in their action, address gaps in evidence and propose future directions for research and clinical development., (© 2021 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

A major goal of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine efforts is to elicit antibody responses that confer protection. Mapping the epitope targets of the SARS-CoV-2 antibody response is critical for vaccine design, diagnostics, and development of therapeutics. Here, we develop a pan-coronavirus phage display library to map antibody binding sites at high resolution within the complete viral proteomes of all known human-infecting coronaviruses in patients with mild or moderate/severe coronavirus disease 2019 (COVID-19). We find that the majority of immune responses to SARS-CoV-2 are targeted to the spike protein, nucleocapsid, and ORF1ab and include sites of mutation in current variants of concern. Some epitopes are identified in the majority of samples, while others are rare, and we find variation in the number of epitopes targeted between individuals. We find low levels of SARS-CoV-2 cross-reactivity in individuals with no exposure to the virus and significant cross-reactivity with endemic human coronaviruses (CoVs) in convalescent sera from patients with COVID-19., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Natural killer cell engagers gained enormous interest in recent years due to their potent anti-tumor activity and favorable safety profile. Simultaneously, chicken-derived antibodies entered clinical studies paving the way for avian-derived therapeutics. In this study, we describe the affinity maturation of a common light chain (cLC)-based, chicken-derived antibody targeting EGFR, followed by utilization of the same light chain for the isolation of CD16a- and PD-L1-specific monoclonal antibodies. The resulting binders target their respective antigen with single-digit nanomolar affinity while blocking the ligand binding of all three respective receptors. Following library-based humanization, bispecific and trispecific variants in a standard 1 + 1 or a 2 + 1 common light chain format were generated, simultaneously targeting EGFR, CD16a, and PD-L1. The trispecific antibody mediated an elevated antibody-dependent cellular cytotoxicity (ADCC) in comparison to the EGFR×CD16a bispecific variant by effectively bridging EGFR/PD-L1 double-positive cancer cells with CD16a-positive effector cells. These findings represent, to our knowledge, the first detailed report on the generation of a trispecific 2 + 1 antibodies exhibiting a common light chain and illustrate synergistic effects of trispecific antigen binding. Overall, this generic procedure paves the way for the engineering of tri- and oligospecific therapeutic antibodies derived from avian immunizations., Competing Interests: JG and BH were employed by the company Ferring Pharmaceuticals. JB, SC, and DF are employed by TU Darmstadt in frame of a collaboration project with Ferring Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bogen, Carrara, Fiebig, Grzeschik, Hock and Kolmar.)

Background: The host's immune system develops in equilibrium with both cellular self-antigens and non-self-antigens derived from microorganisms which enter the body during lifetime. In addition, during the years, a tumor may arise presenting to the immune system an additional pool of non-self-antigens, namely tumor antigens (tumor-associated antigens, TAAs; tumor-specific antigens, TSAs)., Methods: In the present study, we looked for homology between published TAAs and non-self-viral-derived epitopes. Bioinformatics analyses and ex vivo immunological validations have been performed., Results: Surprisingly, several of such homologies have been found. Moreover, structural similarities between paired TAAs and viral peptides as well as comparable patterns of contact with HLA and T cell receptor (TCR) α and β chains have been observed. Therefore, the two classes of non-self-antigens (viral antigens and tumor antigens) may converge, eliciting cross-reacting CD8 + T cell responses which possibly drive the fate of cancer development and progression., Conclusions: An established antiviral T cell memory may turn out to be an anticancer T cell memory, able to control the growth of a cancer developed during the lifetime if the expressed TAA is similar to the viral epitope. This may ultimately represent a relevant selective advantage for patients with cancer and may lead to a novel preventive anticancer vaccine strategy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In 2016 Delong et al. discovered a new type of neoepitope formed by the fusion of two unrelated peptide fragments. Remarkably these neoepitopes, called hybrid insulin peptides, or HIPs, are recognized by pathogenic CD4 + T cells in the NOD mouse and human pancreatic islet-infiltrating T cells in people with type 1 diabetes. Current data implicates CD4 + T-cell responses to HIPs in the immune pathogenesis of human T1D. Because of their role in the immune pathogenesis of human T1D it is important to identify new HIPs that are recognized by CD4 + T cells in people at risk of, or with, T1D. A detailed knowledge of T1D-associated HIPs will allow HIPs to be used in assays to monitor changes in T cell mediated beta-cell autoimmunity. They will also provide new targets for antigen-specific therapies for T1D. However, because HIPs are formed by the fusion of two unrelated peptides there are an enormous number of potential HIPs which makes it technically challenging to identify them. Here we review the discovery of HIPs, how they form and discuss approaches to identifying new HIPs relevant to the immune pathogenesis of human type 1 diabetes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mannering, Rubin, Wang and Bhattacharjee.)

Proteasome-catalyzed peptide splicing (PCPS) of cancer-driving antigens could generate attractive neoepitopes to be targeted by T cell receptor (TCR)-based adoptive T cell therapy. Based on a spliced peptide prediction algorithm, TCRs were generated against putative KRAS G12V - and RAC2 P29L -derived neo-splicetopes with high HLA-A*02:01 binding affinity. TCRs generated in mice with a diverse human TCR repertoire specifically recognized the respective target peptides with high efficacy. However, we failed to detect any neo-splicetope-specific T cell response when testing the in vivo neo-splicetope generation and obtained no experimental evidence that the putative KRAS G12V - and RAC2 P29L -derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2 P29L -derived neo-splicetopes was generated by in vitro PCPS. The experiments pose severe questions on the notion that available algorithms or the in vitro PCPS reaction reliably simulate in vivo splicing and argue against the general applicability of an algorithm-driven 'reverse immunology' pipeline for the identification of cancer-specific neo-splicetopes., Competing Interests: GW, CB, LI, GP, VS, AG, HH, TB, PK No competing interests declared, (© 2021, Willimsky et al.)

The mechanisms underlying type 1 diabetes (T1D) pathogenesis remain largely unknown. While autoantibodies to pancreatic beta-cell antigens are often the first biological response and thereby a useful biomarker for identifying individuals in early stages of T1D, their role in T1D pathogenesis is not well understood. Recognition of these antigenic targets by autoreactive T-cells plays a pathological role in T1D development. Recently, several beta-cell neoantigens have been described, indicating that both neoantigens and known T1D antigens escape central or peripheral tolerance. Several questions regarding the mechanisms by which tolerance is broken in T1D remain unanswered. Further delineating the timing and nature of antigenic responses could allow their use as biomarkers to improve staging, as targets for therapeutic intervention, and lead to a better understanding of the mechanisms leading to loss of tolerance. Multiple factors that contribute to cellular stress may result in the generation of beta-cell derived neoepitopes and contribute to autoimmunity. Understanding the cellular mechanisms that induce beta-cells to produce neoantigens has direct implications on development of therapies to intercept T1D disease progression. In this perspective, we will discuss evidence for the role of neoantigens in the pathogenesis of T1D, including antigenic responses and cellular mechanisms. We will additionally discuss the pathways leading to neoepitope formation and the cross talk between the immune system and the beta-cells in this regard. Ultimately, delineating the timing of neoepitope generation in T1D pathogenesis will determine their role as biomarkers as well as therapeutic targets., Competing Interests: JLD is an employee of Janssen Research and Development, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rodriguez-Calvo, Johnson, Overbergh and Dunne.)

Our previous studies have shown that inoculation of the oral cavity of "humanized" B6.DR1/4 mice with the periodontal pathogen Porphyromonas gingivalis results in an increase in the percentage of circulating Th17 cells, loss of bone and an exacerbation of experimental autoimmune arthritis. The aim of this study was to assess the role played by the human HLA-DRβ molecule containing the shared epitope supplied as a transgene to I-A˚ (murine class II null) C57BL/6 (B6) mice in driving these findings. We compared various immune response parameters as well as alveolar and peri-articular bone loss between humanized B6.DR1 (or B6.DR4) mice and their WT (B6) counterparts. We found that the presence of the shared epitope in the context of inoculation with P. gingivalis enhanced the percentage of Th17 cells generated, dramatically enhanced bone loss and importantly allowed for the generation of CCP2⁺ ACPAs that are not found in C57BL/6 or DBA/1 arthritic mouse serum. Due to the exceedingly complex nature of environmental factors impacting on genetic elements, it has been difficult to unravel mechanisms that drive autoimmune arthritis in susceptible individuals. The findings in this study may provide one small piece of this puzzle that can help us to better understand part of this complexity., Competing Interests: The authors have declared that no competing interests exist.

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types., Competing Interests: ES, JR, AH, PG, ZT, MW, XL, AS, RG, TT, TB, LG, PS No competing interests declared, CL Cara Lord is affiliated with GlaxoSmithKline. The author has no financial interests to declare., (© 2021, Swanson et al.)

Naturally occurring antibodies are abundant in human plasma, but their importance in the defence against bacterial pathogens is unclear. We studied the role of the most abundant of such antibodies, the antibody against terminal galactose-α-1,3-galactose (anti-αGal), in the protection against pneumococcal infections (Streptococcus pneumonia). All known pneumococcal capsular polysaccharides lack terminal galactose-α-1,3-galactose, yet highly purified human anti-αGal antibody of the IgG class reacted with 48 of 91 pneumococcal serotypes. Anti-αGal was found to contain multiple antibody subsets that possess distinct specificities beyond their general reactivity with terminal galactose-α-1,3-galactose. These subsets in concert targeted a wide range of microbial polysaccharides. We found that anti-αGal constituted up to 40% of the total antibody reactivity to pneumococci in normal human plasma, that anti-αGal drives phagocytosis of pneumococci by human neutrophils and that the anti-αGal level was twofold lower in patients prone to pneumococcal infections compared with controls. Moreover, during a 48-year period in Denmark, the 48 anti-αGal-reactive serotypes caused fewer invasive pneumococcal infections (n = 10 927) than the 43 non-reactive serotypes (n = 18 107), supporting protection on the population level. Our findings explain the broad-spectrum pathogen reactivity of anti-αGal and support that these naturally occurring polyreactive antibodies contribute significantly to human protective immunity., (© 2020 John Wiley & Sons Ltd.)

SARS-CoV-2 is the coronavirus that originated in Wuhan in December 2019 and has spread globally. Studies have shown that smokers are less likely to be diagnosed with or be hospitalized for COVID-19 but, once hospitalized, have higher odds for an adverse outcome. We have previously presented the potential interaction between SARS-CoV-2 Spike glycoprotein and nicotinic acetylcholine receptors (nAChRs), due to a "toxin-like" epitope on the Spike glycoprotein, with homology to a sequence of a snake venom toxin. This epitope coincides with the well-described cryptic epitope for the human anti-SARS-CoV antibody CR3022. In this study, we present the molecular complexes of both SARS-CoV and SARS-CoV-2 Spike glycoproteins, at their open or closed conformations, with the model of the human α7 nAChR. We found that all studied protein complexes' interface involves a large part of the "toxin-like" sequences of SARS-CoV and SARS-CoV-2 Spike glycoproteins and toxin binding site of human α7 nAChR. Our findings provide further support to the hypothesis about the protective role of nicotine and other cholinergic agonists. The potential therapeutic role of CR3022 and other similar monoclonal antibodies with increased affinity for SARS-CoV-2 Spike glycoprotein against the clinical effects originating from the dysregulated cholinergic pathway should be further explored., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tuberculosis remains a serious global health problem. BCG is the only prophylactic TB vaccine and it shows variable protective efficacy. Chimeric protein subunit vaccines hold great potential as stand-alone vaccines or heterologous BCG prime boosters. We have designed a protein chimera, PP31, by combining Mtb ESAT-6 family antigen Rv1198 and MoCo biosynthesis family antigen Rv3111. Further, PP31 was extended by addition of latency antigen Rv1813c to yield PP43. Immunization of BALB/c mice with PP31 or PP43 with FIA adjuvant elicited strong humoral immune response. Restimulation of splenocytes of the immunized mice lead to significant proliferation of lymphocytes, secretion of cytokines IFN-γ, TNF, IL-2 of the Th1 class, IL-17A of the Th17 class, and IL-6. PP31 and PP43 also induced intracellular cytokine expression (IFN-γ, TNF, and IL-2) from both CD4 + -CD44 high and CD8 + -CD44 high T-cells. Antigen-specific IFN-γ + /IL-2 + double positive CD4 + T-cells were significantly higher in case of PP43 than PP31-immunized mice and control group. PP43 showed protection equivalent to heat-inactivated BCG in response to challenge of the immunized mice with Mtb H37Ra. Based on its immunogenicity and protective efficacy, PP43 appears to be a potential candidate for further development as a subunit vaccine against TB., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) is a global health problem, being prevalent in the developing countries. A rapid, reliable and cost effective diagnostic method would help in controlling TB in the endemic populations. Development of suitable fusion molecules detecting multiple antibodies produced against Mycobacterium tuberculosis antigens would enhance sensitivity of serodiagnostic assays. In this study, EspC, CFP7 and PPE57 antigens of M. tuberculosis were selected for constructing fusion molecules after prediction of B-cell epitopes using in silico tools. Fusion proteins EspC-CFP7, HspX-EspC-CFP7 and HspX-EspC-CFP7-PPE57 were expressed in E.coli (BL21). The serodiagnostic potential of the individual antigens and their fusions was analyzed by screening 230 plasma samples of pulmonary TB patients. The single antigens HspX, EspC, CFP7, PPE57 showed sensitivities of 30%, 31%, 22% and 35%, respectively. The fusion protein EspC-CFP7 showed sensitivity of 43%. Linking of HspX antigen to the N-terminus of EspC-CFP7 fusion molecule increased sensitivity to 58%, while joining PPE57 antigen to the C-terminus of HspX-EspC-CFP7 increased sensitivity to 69%. The fusion protein HspX-EspC-CFP7-PPE57 seems to be a promising molecule for use in the development of fusions with higher sensitivity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that is potentially pathogenic in immunosuppressed individuals and pregnant females during primary infection. The HCMV envelope glycoprotein B (gB) facilitates viral entry into all cell types and induces a potent immune response. AD-2 epitope is a highly conserved linear neutralizing epitope of gB and a critical target for antibodies; however, only 50% of sero-positive individuals make IgG antibodies to this site and IgA responses have not been fully investigated. This study aimed to compare IgG and IgA responses against gB and the AD-2 epitope in naturally exposed individuals and those receiving a recombinant gB/MF59 adjuvant vaccine. Thus, vaccination of sero-positive individuals improved pre-existing gB-specific IgA and IgG levels and induced de novo gB-specific IgA and IgG responses in sero-negative recipients. Pre-existing AD-2 IgG and IgA responses were boosted with vaccination, but de novo AD-2 responses were not detected. Naturally exposed individuals had dominant IgG responses towards gB and AD-2 compared with weaker and variable IgA responses, although a significant IgA binding response to AD-2 was observed within human breastmilk samples. All antibodies binding AD-2 contained kappa light chains, whereas balanced kappa/lambda light chain usage was found for those binding to gB. V region-matched AD-2-specific recombinant IgG and IgA bound both to gB and to AD-2 and neutralized HCMV infection in vitro. Overall, these results indicate that although human IgG responses dominate, IgA class antibodies against AD-2 are a significant component of human milk, which may function to protect neonates from HCMV., (© 2020 The Authors. Immunology published by John Wiley & Sons Ltd.)

The influenza virus neuraminidase (NA) is becoming a focus for novel vaccine designs. However, the epitopes of human anti-NA antibodies have been poorly defined. Using a panel of 10 anti-N2 monoclonal antibodies (MAbs) that bind the H3N2 virus A/Switzerland/9715293/2013, we generated five escape mutant viruses. These viruses contained mutations K199E/T, E258K, A272D, and S331N. We found that mutations at K199 and E258 had the largest impact on MAb binding, NA inhibition and neutralization activity. In addition, a natural isolate from the 2017-2018 season was found to contain the E258K mutation and was resistant to numerous antibodies tested. The mutation S331N, was identified in virus passaged in the presence of antibody; however, it had little impact on MAb activity and greatly decreased viral fitness. This information aids in identifying novel human MAb epitopes on the N2 and helps with the detection of antigenically drifted NAs. IMPORTANCE The influenza virus neuraminidase is an emerging target for universal influenza virus vaccines. However, in contrast to influenza virus hemagglutinin, we know little about antibody epitopes and antigenic sites on the neuraminidase. Characterizing and defining these sites is aiding vaccine development and helping to understand antigenic drift of NA., (Copyright © 2021 Kirkpatrick Roubidoux et al.)

Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.

Antibodies have been attracting attention as therapeutic tools owing to their high affinity and specificity. To develop potent antibodies, affinity maturation, epitope regulation, and using target antigens in native form are pivotal requirements. Here we describe a method to conduct epitope-directed affinity maturation of antibodies using engineered mammalian cells. This method utilizes protein chimeras that transduce cell death signaling in response to antibody binding. As the competition of antibody binding inhibits the cell death signaling, only affinity-matured antibodies retaining the same epitope as an original one can be selected using cell survival as readout.

Advances in several key technologies, including MHC peptidomics, have helped fuel our understanding of basic immune regulatory mechanisms and the identification of T cell receptor targets for the development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. However, the current multistep analytical process is challenging, and improvements in throughput and reproducibility would enable rapid characterization of multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines such as MC38, can be enriched in a semiautomated fashion on reusable, dry-storage, customized antibody cartridges. Using this method, a researcher, with very little hands-on time and in a single day, can perform up to 96 simultaneous enrichments at a similar level of quality as a manual workflow. TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification), a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation. This unique combination of robust semiautomated MHC-I peptide isolation and high-throughput multiplexed targeted quantitation allows for both the routine analysis of >4000 unique MHC-I peptides from 250 million cells using nontargeted methods, as well as quantitative sensitivity down to the low amol/μl level using TOMAHAQ targeted MS., Competing Interests: Conflict of interest We are employed by Genentech, a member of the Roche group. The authors have no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Peptide-major histocompatibility complex class II (pMHCII) multimers have emerged as a convenient and powerful tool for characterization of CD4 T cell immune responses in a large variety of human diseases. Peptide-MHCII multimers can rapidly identify peptide antigens recognized by CD4 T cells via high-throughput peptide screening procedures. The specificity and phenotype of antigen-specific CD4 T cells can be effectively visualized by pMHCII multimers from unmanipulated immune cell populations. Functional attributes of antigen-specific CD4 T cells can also be defined with the multimer technology in combination with immune functional assays such as intracellular cytokine staining (ICS).

MAbTope is a docking-based method for the determination of epitopes. It has been used to successfully determine the epitopes of antibodies with known 3D structures. However, during the antibody discovery process, this structural information is rarely available. Although we already have evidence that homology models of antibodies could be used instead of their 3D structure, the choice of the template, the methodology for homology modeling and the resulting performance still have to be clarified. Here, we show that MAbTope has the same performance when working with homology models of the antibodies as compared to crystallographic structures. Moreover, we show that even low-quality models can be used. We applied MAbTope to determine the epitope of dupilumab, an anti- interleukin 4 receptor alpha subunit therapeutic antibody of unknown 3D structure, that we validated experimentally. Finally, we show how the MAbTope-determined epitopes for a series of antibodies targeting the same protein can be used to predict competitions, and demonstrate the accuracy with an experimentally validated example.3D: three-dimensionalRMSD: root mean square deviationCDR: complementary-determining regionCPU: central processing unitsVH: heavy chain variable regionVL: light chain variable regionscFv: single-chain variable fragmentsVHH: single-chain antibody variable regionIL4Rα: Interleukin 4 receptor alpha chainSPR: surface plasmon resonancePDB: protein data bankHEK293: Human embryonic kidney 293 cellsEDTA: Ethylenediaminetetraacetic acidFBS: Fetal bovine serumANOVA: Analysis of varianceEGFR: Epidermal growth factor receptorPE: PhycoerythrinAPC: AllophycocyaninFSC: forward scatterSSC: side scatterWT: wild typeKeywords: MAbTope, Epitope Mapping, Molecular docking, Antibody modeling, Antibody-antigen docking.

T-cell engaging biologics is a class of novel and promising immune-oncology compounds that leverage the immune system to eradicate cancer. Here, we compared and contrasted a bispecific diabody-Fc format, which displays a relatively short antigen-binding arm distance, with our bispecific IgG platform. By generating diverse panels of antigen-expressing cells where B cell maturation antigen is either tethered to the cell membrane or located to the juxtamembrane region and masked by elongated structural spacer units, we presented a systematic approach to investigate the role of antigen epitope location and molecular formats in immunological synapse formation and cytotoxicity. We demonstrated that diabody-Fc is more potent for antigen epitopes located in the membrane distal region, while bispecific IgG is more efficient for membrane-proximal epitopes. Additionally, we explored other parameters, including receptor density, antigen-binding affinity, and kinetics. Our results show that molecular format and antigen epitope location, which jointly determine the intermembrane distance between target cells and T cells, allow decoupling of cytotoxicity and cytokine release, while antigen-binding affinities appear to be positively correlated with both readouts. Our work offers new insight that could potentially lead to a wider therapeutic window for T-cell engaging biologics in general.

Sensitive and reproducible pharmacokinetic (PK) assays and immunogenicity assessment are required as part of the complex and lengthy development process for biotherapeutic proteins. Ligand binding assays (LBAs) are included in a range of approaches applied to understand the nature and properties of the drug as well as the induction of anti-drug antibodies (ADA) against the therapeutic, which can cause adverse events and loss of efficacy. Currently, most biotherapeutics are monoclonal human or humanized antibodies. Anti-idiotypic antibodies, targeting the idiotopic determinants of individual antibody drugs are recognized as perfect reagents for such LBAs. Here we describe the typical setups for these assays and how different types of anti-biotherapeutic antibodies can be used to establish selective and sensitive assays.

Legionella pneumophila , an intracellular bacterium, may cause life-threatening pneumonia in immunocompromised individuals. Mononuclear cells and antibodies have been reported to be associated with the host defense response against L. pneumophila. This study is to determine whether Legionella peptidoglycan-associated lipoprotein (PAL)-specific CD8 + T cells are directly associated with protection against L. pneumophila , with a focus on potential epitopes. Synthetic peptides derived from PAL of L. pneumophila were obtained and tested through in vitro and in vivo cytotoxic T lymphocyte (CTL) assays for immunogenicity. PAL DNA vaccines or a peptide epitope with or without CpG-oligodeoxynucleotides (ODN) was evaluated for protection against L. pneumophila infection in animal models. When mice were immunized with DNA vaccines expressing the PAL of L. pneumophila , they were significantly protected against a lethal challenge with L. pneumophila through induction of antigen-specific CD8 + CTLs. Of the 13 PAL peptides tested, PAL 92-100 (EYLKTHPGA) was the most immunogenic and induced the strongest CTL responses. When mice were immunized with the PAL 92-100 peptide plus CpG-ODN, they were protected against the lethal challenge, while control mice died within 3-6 days after the challenge. Consistent with lung tissue histological data, bacterial counts in the lungs of immunized mice were significantly lower than those in control mice. Also, the amino acid sequence of PAL 92-100 peptides is conserved among various Legionella species. To our knowledge, this study is the first to demonstrate that PAL 92-100 -specific CD8 + T cells play a central role in the host defense response against L. pneumophila ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Kim, Sin and Kim.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused COVID-19 disease in China. So far, no vaccine has licensed to protect against infection with COVID-19, therefore an effective COVID-19 vaccine needed. The aim of this study was to predict antigenic peptides of SARS-CoV-2 for designing the COVID-19 vaccine using immunoinformatic analysis. In this study, T and B-cell epitopes of S protein were predicted and screened based on the antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. The epitopes were joined by the appropriate linker. LT-IIc as an adjuvant was attached to the end of the structure. The secondary and 3D structure of the vaccine was predicted. The refinement process was performed to improve the quality of the 3D model structure; the validation process is performed using the Ramachandran plot and ProSA z-score. The proposed vaccine's binding affinity to the HLA-A11:01 and HLA-DRB1&#95;01:01 molecule was evaluated by molecular docking. Using molecular dynamics, the stability of vaccine-HLA complexes was also evaluated. Finally, in silico gene cloning was performed in the pET30a (+) vector. The findings suggest that the current vaccine may be a promising vaccine to prevent SARS-CoV-2 infection., Competing Interests: Declaration of competing interest The authors have declared no competing interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a K D of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH-FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHβ subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing., Competing Interests: The authors declare no competing interest.

Yellow fever virus (YFV) is a mosquito-borne member of the genus flavivirus, including other important human-pathogenic viruses, such as dengue, Japanese encephalitis, and Zika. Herein, we report identifying 129 YFV Class II epitopes in donors vaccinated with the live attenuated YFV vaccine (YFV-17D). A total of 1156 peptides predicted to bind 17 different common HLA-DRB1 allelic variants were tested using IFNγ ELISPOT assays in vitro re-stimulated peripheral blood mononuclear cells from twenty-six vaccinees. Overall, we detected responses against 215 YFV epitopes. We found that the capsid and envelope proteins, as well as the non-structural (NS) proteins NS3 and NS5, were the most targeted proteins by CD4 + T cells from YF-VAX vaccinated donors. In addition, we designed and validated by flow cytometry a CD4 + mega pool (MP) composed of structural and non-structural epitopes in an independent cohort of vaccinated donors. Overall, this study provides a comprehensive prediction and validation of YFV epitopes in a cohort of YF-17D vaccinated individuals. With the design of a CD4 epitope MP, we further provide a useful tool to detect ex vivo responses of YFV-specific CD4 T cells in small sample volumes.