Target Epitopes Research Articles

Endogenous CD4 T cells that recognize ALK and the NPM1::ALK fusion protein can be expanded from human peripheral blood.

Anaplastic lymphoma kinase (ALK)-fusion proteins resulting from chromosomal rearrangements are promising targets for cancer immunotherapy. While ALK-specific CD8+ T cells and epitopes presented on MHC class I have been identified in patients with ALK-positive malignancies, little is known about ALK-specific CD4+ T cells. We screened peripheral blood of ten ALK-positive anaplastic large cell lymphoma (ALK+ALCL) patients in remission and six healthy donors for CD4+ T-cell responses to the whole ALK-fusion protein, nucleophosmin (NPM1)::ALK. ALK-specific CD4+ T cells were detected in 15 individuals after stimulation with autologous dendritic cells pulsed with long-overlapping ALK peptide pools. CD4+ T-cell epitopes were predominantly located within three specific regions (p102-188, p257-356, p593-680) in the ALK portion of the fusion protein. We detected CD4+ T cells in one patient that recognized the NPM1::ALK fusion neoepitope and identified a corresponding T-cell receptor (TCR) by TCR single-cell sequencing. The NPM1::ALK fusion-specific TCR was HLA-DR13 restricted and conferred antigen specificity when expressed in a TCR- reporter cell line (58--). Together, our data provide evidence of ALK-specific CD4+ T cells in human peripheral blood, describe target epitopes in patients and support the consideration of CD4+ T cells in the development of ALK-specific immunotherapies.

DECODE enables high-throughput mapping of antibody epitopes at single amino acid resolution.

Antibodies are extensively used in biomedical research, clinical fields, and disease treatment. However, to enhance the reproducibility and reliability of antibody-based experiments, it is crucial to have a detailed understanding of the antibody's target specificity and epitope. In this study, we developed a high-throughput and precise epitope analysis method, DECODE (Decoding Epitope Composition by Optimized-mRNA-display, Data analysis, and Expression sequencing). This method allowed identifying patterns of epitopes recognized by monoclonal or polyclonal antibodies at single amino acid resolution and predicted cross-reactivity against the entire protein database. By applying the obtained epitope information, it has become possible to develop a new 3D immunostaining method that increases the penetration of antibodies deep into tissues. Furthermore, to demonstrate the applicability of DECODE to more complex blood antibodies, we performed epitope analysis using serum antibodies from mice with experimental autoimmune encephalomyelitis (EAE). As a result, we were able to successfully identify an epitope that matched the sequence of the peptide inducing the disease model without relying on existing antigen information. These results demonstrate that DECODE can provide high-quality epitope information, improve the reproducibility of antibody-dependent experiments, diagnostics and therapeutics, and contribute to discover pathogenic epitopes from antibodies in the blood.

Immunoinformatics design of a novel multiepitope vaccine candidate against non-typhoidal salmonellosis caused by Salmonella Kentucky using outer membrane proteins A, C, and F.

The global public health risk posed by Salmonella Kentucky (S. Kentucky) is rising, particularly due to the dissemination of antimicrobial resistance genes in human and animal populations. This serovar, widespread in Africa, has emerged as a notable cause of non-typhoidal gastroenteritis in humans. In this study, we used a bioinformatics approach to develop a peptide-based vaccine targeting epitopes from the outer membrane proteins A, C, and F of S. Kentucky. Additionally, we employed flagellin protein (fliC) from Salmonella Typhimurium (S. Typhimurium) as an adjuvant to enhance the vaccine's effectiveness. Through this approach, we identified 14 CD8+ and 7 CD4+ T-cell epitopes, which are predicted to be restricted by various MHC class I and MHC class II alleles. The predicted epitopes are expected to achieve a population coverage of 94.91% when used in vaccine formulations. Furthermore, we identified seven highly immunogenic linear B-cell epitopes and three conformational B-cell epitopes. These T-cell and B-cell epitopes were then linked using appropriate linkers to create a multi-epitope vaccine (MEV). To boost the immunogenicity of the peptide construct, fliC from S. Typhimurium was included at the N-terminal. The resulting MEV construct demonstrated high structural quality and favorable physicochemical properties. Molecular docking studies with Toll-like receptors 1, 2, 4, and 5, followed by molecular dynamic simulations, suggested that the vaccine-receptor complexes are energetically feasible, stable, and robust. Immune simulation results showed that the MEV elicited significant responses, including IgG, IgM, CD8+ T-cells, CD4+ T-cells, and various cytokines (IFN-γ, TGF-β, IL-2, IL-10, and IL-12), along with a noticeable reduction in antigen levels. Despite these promising in-silico findings, further validation through preclinical and clinical trials is required to confirm the vaccine's efficacy and safety.

Genetic characterization of diagnostic epitopes of cardiac troponin I in African rhinoceros.

African rhinoceros undergo chemical immobilization and prolonged transport during translocations for conservation purposes and, hence, experience several pathophysiologic changes, including skeletal muscle injury. Potential concurrent myocardial injury has not been investigated due to a lack of validated immunoassays. We aimed to use inferred cardiac troponin I (cTnI) amino acid sequences of southern white (Ceratotherium simum simum) and southern-central black (Diceros bicornis minor) rhinoceros to assess the potential usefulness of several commercial cTnI immunoassays for detecting cTnI in African rhinoceros. We extracted RNA from the myocardium of deceased rhinoceros (2 white, 1 black rhinoceros) followed by primer design, cDNA synthesis via RT-PCR, and Sanger sequencing. The inferred cTnI amino acid sequences were obtained from the mRNA transcript sequences. The homology of epitope binding sites recognized by capture and detection antibodies in 6 human immunoassays was visually evaluated using aligned inferred rhinoceros cTnI amino acid sequences. Percentage identity between white and black rhinoceros cDNA nucleotide sequences was 99%; inferred amino acid sequences were identical. There were 5 amino acid differences between humans and rhinoceros in the epitope binding sites of immunoassay antibodies; 5 assays contained antibodies against epitopes that were not conserved. For one assay, the single capture antibody targeted a short heterologous epitope (residue 87-91), and cross-reactivity with rhinoceros cTnI was deemed unlikely. For the other 5 assays, complete antibody-epitope homology, or the inclusion of multiple detection or capture antibodies, or targeting of long epitopes, indicated that these assays could be suitable for further investigation of cTnI measurement in African rhinoceros.

A comparison of tau aggregation and seeding competency of anti‐tau antibodies under clinical trials and their target epitopes itself

A comparison of tau aggregation and seeding competency of anti‐tau antibodies under clinical trials and their target epitopes itself

Evolving antibody response to SARS-CoV-2 antigenic shift from XBB to JN.1

The continuous evolution of SARS-CoV-2, particularly the emergence of the BA.2.86/JN.1 lineage replacing XBB, necessitates re-evaluation of vaccine compositions1, 2–3. Here, we provide a comprehensive analysis of the humoral immune response to XBB and JN.1 human exposure. We demonstrate the antigenic distinctiveness of XBB and JN.1 lineages in SARS-CoV-2-naive individuals and show that infection with JN.1 elicits superior plasma neutralization against its subvariants. We highlight the strong immune evasion and receptor-binding capability of KP.3, supporting its foreseeable prevalence. Extensive analysis of the B cell receptor repertoire, in which we isolate approximately 2,000 receptor-binding-domain-specific antibodies, with targeting epitopes characterized by deep mutational scanning, underscores the superiority of JN.1-elicited memory B cells4,5. Class 1 IGHV3-53/3-66-derived neutralizing antibodies (NAbs) are important contributors to the wild-type reactivity of NAbs against JN.1. However, KP.2 and KP.3 evade a substantial subset of these antibodies, even those induced by JN.1, supporting a need for booster updates. JN.1-induced Omicron-specific antibodies also demonstrate high potency across Omicron. Escape hotspots for these NAbs have already been mutated, resulting in a higher immune barrier to escape and indicating probable recovery of escaped NAbs. In addition, the prevalence of IGHV3-53/3-66-derived antibodies and their ability to compete with all Omicron-specific NAbs suggests that they have an inhibitory effect on the activation of Omicron-specific naive B cells, potentially explaining the heavy immune imprinting in mRNA-vaccinated individuals6, 7–8. These findings delineate the evolving antibody response to the antigenic shift of Omicron from XBB to JN.1 and highlight the importance of developing the JN.1 lineage, especially KP.2- and KP.3-based vaccine boosters.

Measures of Population Immunity Can Predict the Dominant Clade of Influenza A (H3N2) in the 2017-2018 Season and Reveal Age-Associated Differences in Susceptibility and Antibody-Binding Specificity.

For antigenically variable pathogens such as influenza, strain fitness is partly determined by the relative availability of hosts susceptible to infection with that strain compared with others. Antibodies to the hemagglutinin (HA) and neuraminidase (NA) confer substantial protection against influenza infection. We asked if a cross-sectional antibody-derived estimate of population susceptibility to different clades of influenza A (H3N2) could predict the success of clades in the following season. We collected sera from 483 healthy individuals aged 1 to 90 years in the summer of 2017 and analyzed neutralizing responses to the HA and NA of representative strains using focus reduction neutralization tests (FNRT) and enzyme-linked lectin assays (ELLA). We estimated relative population-average and age-specific susceptibilities to circulating viral clades and compared those estimates to changes in clade frequencies in the following 2017-2018 season. The clade to which neutralizing antibody titers were lowest, indicating greater population susceptibility, dominated the next season. Titer correlations between viral strains varied by age, suggesting age-associated differences in epitope targeting driven by shared past exposures. Yet substantial unexplained variation remains within age groups. This study indicates how representative measures of population immunity might improve evolutionary forecasts and inform selective pressures on influenza.

Cardiac myosin-binding protein-C: assessing fragmentation patterns to differentiate between forms of myocardial injury

Abstract Background Cardiac myosin-binding protein-C (cMyC) is a novel biomarker of myocardial injury. We hypothesize that fragmentation of cMyC differs according to aetiology of myocardial injury. Assessing the fragmentation pattern of the protein may aid diagnosis of these conditions. Purpose This work represents an examination of cMyC fragmentation in human serum, and studies its utility in differentiating between forms of myocardial injury. Methods Patients who were admitted to a hospital during October-November 2023 with acute myocardial injury were recruited. Acute myocardial injury was defined as a dynamic troponin rise above the 99th centile (Abbott Alinity: cTnI>34ng/L male, cTnI >16ng/L female). Following collection, samples were left at room temperature for 30 minutes and then centrifuged. Serum was separated for cMyC measurement, frozen in liquid nitrogen and stored at -80 °C. Recruited patients were followed up during their inpatient journey. Their final diagnosis was defined using the 4th universal definition of MI by two cardiologists, with a third cardiologist acting as adjudicator, if required. The serum samples were analysed using two separate electrochemiluminescence sandwich ELISA assays detecting different portions of cMyC. One assay formulated for total concentration of cMyC using high affinity mouse N-terminal monoclonal antibodies (mAb) targeting epitopes in close proximity (Limit of Detection (LoD) = 9.0ng/L). The other assay uses a N-terminal and a C-terminal mouse mAb which straddle the major proteolytic cleavage site of cMyC. This assay detects the intact (fuller-length) cMyC (LoD= 14.2ng/L). Both assays perform with good sensitivity, low variability and are capable of detecting cMyC at concentrations well below the 99th centile. Results 11 patient samples were collected with final diagnoses of; type 1 MI (STEMI n=3, NSTEMI n=4), type 4 MI (alcohol septal ablation n=1), acute myocardial injury (myocarditis n=2) and type 2 MI (chest sepsis with known coronary disease n=2). Figure one details the percentage of intact cMyC relative to total cMyC measured in serum for each patient, and is split into their adjudicated diagnosis. The bars display the median and interquartile range (IQR). Our findings suggest that the fragmentation pattern of cMyC could help differentiate between forms of myocardial injury. The ratio of intact: total cMyC was higher in the type 2 MI than any other condition. The results did not reach statistical significance (p = 0.06) due to the small sample size. Conclusions The fragmentation pattern of cMyC is dependent on the type of myocardial injury and could aid in the differentiation between type 2 MI and other forms of myocardial injury.Figure One

Rapid Systematic Screening of Bispecific Antibody Surrogate Geometries for T-Cell Engagement Using DNA Nanotechnology.

Bispecific antibodies (bsAbs) are emerging immune-therapeutics, and many formats exist that differ considerably in structure. However, little systematic data exist about how the spatial organization of their components influences activity, requiring innovative approaches combining empirical and quantitative frameworks. This study presents a modular DNA nanotechnology platform to generate numerous bsAbs with surrogate geometries that span the structural features of the BiTE, IgG-like, and IgG-conjugate platforms to screen for T-cell engagement. Results highlight interesting structure-activity relationships regarding bsAb potency and selectivity and raise questions regarding the molecular phenomena underlying activity. To elucidate some effects, the platform was paired with a simple mathematical model. This work is thus one of the first to systematically investigate and reveal the importance of the spatial organization of bsAb components on activity and equally provides an accessible and convenient tool for rapidly mapping out such trends for other combinations of target epitopes.

Direct Mapping of Polyclonal Epitopes in Serum by HDX-MS.

Elucidating the interactions that drive antigen recognition is central to understanding antibody-mediated protection and is vital for the rational design of immunogens. Often, structural knowledge of epitopes targeted by antibodies is derived from isolated studies of monoclonal antibodies, for which numerous structural techniques exist. In contrast, there are very few approaches capable of mapping the full scope of antigen surfaces targeted by polyclonal sera through the course of a natural antibody response. Here, we develop an approach using immobilized antigen coupled to hydrogen/deuterium exchange with mass spectrometry (HDX-MS) to probe epitope targeting in the context of the fully native serum environment. Using the well-characterized Staphylococcal enterotoxin B (SEB) as a model system, we show that complex combinations of epitopes can be detected and subtle differences across different antisera can be discerned. This work reveals new insight into how neutralizing antibodies and antisera target SEB and, more importantly, establishes a novel method for directly mapping the epitope landscape of polyclonal sera.

CD4 downregulation precedes Env expression and protects HIV-1-infected cells from ADCC mediated by non-neutralizing antibodies.

Antibody-dependent cellular cytotoxicity (ADCC) represents an effective immune response for clearing virally infected cells, making ADCC-mediating antibodies promising therapeutic candidates for HIV-1 cure strategies. Broadly neutralizing antibodies (bNAbs) target epitopes present on the native "closed" envelope glycoprotein (Env), while non-neutralizing antibodies (nnAbs) recognize epitopes exposed upon Env-CD4 interaction. Here, we provide evidence that env mRNA is predominantly expressed by productively infected cells that have already downmodulated cell-surface CD4. This indicates that CD4 downmodulation by HIV-1 precedes Env expression, making productively infected cells resistant to ADCC mediated by nnAbs but sensitive to those mediated by bnAbs. These findings offer critical insights for the development of immunotherapy-based strategies aimed at targeting and eliminating productively infected cells in people living with HIV.

7337 High Parathyroid Hormone (PTH) Due To Immunoassay Interference

Abstract Disclosure: V.A. Mendpara: None. A.J. McShane: None. L.Z. Khan: None. Background: PTH levels are not always accurate. This case illustrates immunoassay interference in a female with markedly abnormal PTH values. Understanding immunoassay interference in diverse hormonal assessments can prevent diagnostic inaccuracies. Clinical Case: A 63-year-old female with osteopenia underwent serum parathyroid hormone (PTH) testing as part of a thorough bone evaluation. The initial PTH level revealed an abnormal level of 4008 pg/mL (normal range: 15 to 65 pg/mL). The follow-up PTH level with the same assay continued to result in elevated values of >5000 pg/mL. The medication list was negative for biotin use, which is a well-known interfering substance in the PTH assay. Clinical evaluation was negative for hyperparathyroidism symptoms, and thyroid/parathyroid ultrasound was normal, as were serum levels of phosphate, vitamin D, and calcium. No PTH values had been checked for patients in the past after an extensive review of prior records. The initial PTH testing used the Roche Elecsys PTH STAT assay. To investigate a potential immunoassay interference, the following studies were performed: First, the sample was diluted with polyethylene glycol (PEG) to precipitate large molecular weight proteins; second, the sample was treated with a commercial heterophilic blocking reagent (Scantibodies Laboratory); and lastly, the sample was assayed utilizing a different PTH vendor (Siemens Atellica, chemiluminescent immunoassay). The PEG and heterophilic blocking treatments resulted in significantly lower PTH concentrations of 895.6 and 40.7 pg/mL, respectively. We do not anticipate a change in PTH levels in samples supplemented with blocking reagents or PEG without interference. The result from Siemens’ assay was markedly lower at 43.4 pg/mL. Both the Siemens and Roche assays employ mouse antibodies; however, the antibodies are proprietary between the vendors. This results in potentially different anti-mouse antibody epitope targets. Due to a history of advanced ovarian cancer, the patient was taking Oregovomab, a murine monoclonal antibody, as therapy for her advanced disease. It is unclear if this medication resulted in assay interference, but it is possible. Discussion: In summary, the discrepancy in PTH assay results highlights the need to consider immunoassay interference, particularly in patients with a history of cancer on immunotherapy agents. Our case highlights the importance of the clinician’s review of medications when assessing patients. While prior PTH assays were not conducted before the patient started Oregovomab, the potential development of interfering antibodies warrants consideration. Clinicians should be vigilant for atypical results and collaborate closely with laboratory professionals, as detecting interfering substances can result in misdiagnosis. Presentation: 6/2/2024

High-affinity antibodies specific to the core region of the tau protein exhibit diagnostic and therapeutic potential for Alzheimer’s disease

BackgroundRecent advances in blood-based biomarker discovery are paving the way for simpler, more accessible diagnostic tools that can detect early signs of Alzheimer’s disease (AD). Recent successes in the development of amyloid-targeting immunotherapy approaches mark an important advancement in providing new options for the treatment of AD. We have developed a set of high-affinity monoclonal antibodies (mAbs) to tau protein that have the potential as tools for diagnosis and treatment of AD.MethodsSheep were immunised with either full-length tau (1-441) or truncated paired helical filament (PHF)-core tau (297–391). A stringent bio-panning and epitope selection strategy, with a particular focus directed to epitopes within the disease-relevant PHF-core tau, was used to identify single-chain antibodies (scAbs). These scAbs were ranked by affinity for each epitope class, with leads converted to high-affinity mAbs. These antibodies and their potential utility were assessed by their performance in tau immunoassays, as well as their ability to prevent tau aggregation and propagation. Further characterisation of these antibodies was performed by immunohistochemical staining of brain sections and immuno-gold electronmicroscopy of isolated PHFs.ResultsOur work resulted in a set of high-affinity antibodies reacting with multiple epitopes spanning the entire tau protein molecule. The tau antibodies directed against the core tau unit of the PHF inhibited pathological aggregation and seeding using several biochemical and cell assay systems. Through staining of brain sections and PHFs, the panel of antibodies revealed which tau epitopes were available, truncated, or occluded. In addition, highly sensitive immunoassays were developed with the ability to distinguish between and quantify various tau fragments.ConclusionThis article introduces an alternative immunodiagnostic approach based on the concept of a “tauosome” – the diverse set of tau fragments present within biological fluids. The development of an antibody panel that can distinguish a range of different tau fragments provides the basis for a novel approach to potential diagnosis and monitoring of disease progression. Our results further support the notion that tau immunotherapy targeting the PHF-core needs to combine appropriate selection of both the target epitope and antibody affinity to optimise therapeutic potential.

Production and characterization of a panel of anti-mouse placenta-specific protein 1 (plac1) monoclonal antibodies

Placenta-Specific Protein 1 (PLAC1) is essential for normal placental and embryonic development. It is widely expressed in various types of cancer cells. We produced a panel of anti-mouse plac1 monoclonal antibodies (mAbs) with different applications. Two recombinant proteins were produced containing either the extracellular domain (ED) plus tetanus toxin P2, P30, pan-DR epitope (PADRE), and KDEL3 (main plac1) or ED plus KDEL3 (control plac1). Recombinant proteins were used for immunization and screening. Positive clones were selected by ELISA and flow cytometry. Purified mAbs were tested by ELISA, WB, flow cytometry, immunohistochemistry (IHC), and immunofluorescent (IF). A combination of bioinformatics tools was used to predict the target epitope(s) of the mAbs. Eight anti-mouse plac1 mAbs (all IgG1/κ1) were generated, all reacting with high affinity in ELISA. Seven clones recognized plac1 in both reduced and non-reduced Western blots, while one only recognized the non-reduced form. Cross-inhibition ELISA revealed that all mAbs recognized overlapping epitopes with a shared motif except for 5C9. Four clones reacted with the native antigen in flow cytometry, but none were functional in IF or IHC staining. The produced multifunctional mAbs can be used to investigate different aspects of PLAC1 biology in reproduction and cancer.

Sensitive Electrochemical and Thermal Detection of Human Noroviruses Using Molecularly Imprinted Polymer Nanoparticles Generated against a Viral Target.

Norovirus (NoV) is the predominant cause of foodborne illness globally; current detection methods are typically expensive, have inadequate sensitivities, and utilize biological receptors with poor stability. Therefore, accurate, cost-effective, and highly stable detection methods are needed to screen for NoV in foods. We developed molecularly imprinted polymer nanoparticles (nanoMIPs) to detect NoV using a small target epitope (12 amino acids) with a solid-phase synthesis approach. The performance of three batches of nanoMIPs with varying monomer compositions (nanoMIP-1, -2, and -3) were compared both experimentally and computationally. Surface plasmon resonance examined nanoMIP binding affinity to norovirus virus-like particles (NoV-LPs), whereby nanoMIP-1 had the lowest KD value of 0.512 μM. This is significant, as traditional targets for generation of norovirus ligands previously reported were generated against drastically larger norovirus capsid segments that have limitations in ease of production. Further, an electrochemical sensor was developed by covalently attaching the nanoMIPs to glassy carbon electrodes. In agreement with our predictions from density functional theory simulations, electrochemical impedance spectroscopy showed a sensitive response toward NoV-LPs for nanoMIP batches tested; however, nanoMIP-1 was optimal, with an excellent detection limit of 3.4 pg/mL (1.9 × 105 particles/mL). Due to its exceptional performance, nanoMIP-1 was immobilized to screen-printed electrodes and utilized within a thermal sensor, where it exhibited a low detection limit of 6.5 pg/mL (3.7 × 105 particles/mL). Crucially, we demonstrated that nanoMIP-1 could detect NoV in real food samples (romaine lettuce) by using electrochemical and thermal sensors. Consequently, the study highlights the exceptional potential of nanoMIPs to replace traditional biological materials (e.g., antibodies) as sensitive, versatile, and highly stable receptors within NoV sensors.

The polymorphism analysis and therapy vaccine target epitopes screening of HPV-35 E6 E7 among the threaten α-9 HPV in Sichuan area

High-risk human papilloma virus (HR-HPV) persistent infection is closely associated with the development of cervical cancer and squamous intraepithelial lesion (SIL).The α-9 HPVs, which is predominantly composed of HR-HPV types, account for 75% of HR-HPV infection in Sichuan. The oncoproteins E6 and E7 of HPV play a crucial role in tumor initiation and progression. Notably, HPV-35 is the only HR-HPV type within the α-9 genus that is not included in the nine-valent HPV prophylactic vaccine. Cervical cell samples obtained from Sichuan were collected for HPV detection and genotyping. Among the 406 HPV-positive samples, 31 HPV-35 were detected, 24 HPV-35 E6 and 26 E7 were successfully amplified and sequenced, five nucleotide mutations in E6 and three in E7 were detected, T232C, T434G of E6 (W78R, I145R) and C67T, G84T of E7 (H23Y, L28F) were non-synonymy mutation. PAML 4.8 server was used to detect positive selection sites of HPV-35 E6, E7, and E6 is W78R. Phyre2 were used to predict and analyze protein structures, W78R made influences on protein structure. IEDB were used to screen epitopes vaccine target for HPV-35 affection therapy, and 5 HPV-35 E6 and 3 HPV-35 E7 most potential epitopes were obtained, the most potential peptides for therapy vaccine design were 79-91YRYSVYGETLEKQ, 45-60FACYDLCIVREGQPY, 124-135RFHNIGGRWTGR of E6; 3-19GEITTLQDYVLDLEPEA, 38-47TIDGPAGQAK, 70-88VQSTHIDIRKLEDLLMGTF of E7 and W78R mainly decreased the epitopes affinity.Conclusions Amino acid substitution in the positive selection sites of HPV-35 E6 and E7 genes have been found to influence protein structure and to decrease the overall affinity of antigen epitopes. This observation aligns with the evolutionary significance of positive selection site, which may confer advantages to the virus by making infected cells more challenging for the immune system to detect, thereby enhancing HPV’s adaptability to the host environment. The polymorphism analysis of HPV-35 E6, E7 contributes to the enrichment of α-9 HPV data in Sichuan China, which is instrumental in improving the effectiveness of clinical detection. Furthermore, these findings provide a relevant theoretical foundation for the prevention and treatment of HPV-related diseases.

Exploring the nuclear proteins, viral capsid protein, and early antigen protein using immunoinformatic and molecular modeling approaches to design a vaccine candidate against Epstein Barr virus

The available Epstein Barr virus vaccine has tirelessly harnessed the gp350 glycoprotein as its target epitope, but the result has not been preventive. Right here, we designed a global multi-epitope vaccine for EBV; with special attention to making sure all strains and preventive antigens are covered. Using a robust computational vaccine design approach, our proposed vaccine is armed with 6–16 mers linear B-cell epitopes, 4–9 mer CTL epitopes, and 8–15 mer HTL epitopes which are verified to induce interleukin 4, 10 & IFN-gamma. We employed deep computational mining coupled with expert intelligence in designing the vaccine, using human Beta defensin-3—which has been reported to induce the same TLRs as EBV—as the adjuvant. The tendency of the vaccine to cause autoimmune disorder is quenched by the assurance that the construct contains no EBNA-1 homolog. The protein vaccine construct exhibited excellent physicochemical attributes such as Aliphatic index 59.55 and GRAVY − 0.710; and a ProsaWeb Z score of − 3.04. Further computational analysis revealed the vaccine docked favorably with EBV indicted TLR 1, 2, 4 & 9 with satisfactory interaction patterns. With global coverage of 85.75% and the stable molecular dynamics result obtained for the best two interactions, we are optimistic that our nontoxic, non-allergenic multi-epitope vaccine will help to ameliorate the EBV-associated diseases—which include various malignancies, tumors, and cancers—preventively.

Development of a first-in-class antibody and a specific assay for α-1,6-fucosylated prostate-specific antigen

Prostate-specific antigen (PSA) levels are widely used to screen for prostate cancer, yet the test has poor sensitivity, specificity and predictive value, which leads to overdiagnosis and overtreatment. Alterations in the glycosylation status of PSA, including fucosylation, may offer scope for an improved biomarker. We sought to generate a monoclonal antibody (mAb) targeting α-1,6-fucosylated PSA (fuc-PSA) and to develop a tissue-based immunological assay for fuc-PSA detection. Immunogens representing fuc-PSA were used for immunisation and resultant mAbs were extensively characterised. The mAbs reacted specifically with fuc-PSA-specific glycopeptide, but not with aglycosylated PSA or glycan without the PSA peptide. Reactivity was confirmed using high-throughput surface plasmon resonance spectroscopy. X-ray crystallography investigations showed that the mAbs bound to an α-helical form of the peptide, whereas the native PSA epitope is linear. Protein unfolding was required for detection of fuc-PSA in patient samples. Peptide inhibition of fuc-PSA mAbs was observed with positive screening reagents, and target epitope specificity was observed in formalin-fixed, paraffin-embedded tissue samples. This research introduces a well-characterised, first-in-class antibody targeting fuc-PSA and presents the first crystal structure of an antibody demonstrating glycosylation-specific binding to a peptide.

Identification of Two Linear Epitopes on MGF_110-13L Protein of African Swine Fever Virus with Monoclonal Antibodies.

African swine fever caused by African swine fever virus (ASFV) is an acute, highly contagious swine disease with high mortality. To facilitate effective vaccine development and find more serodiagnostic targets, fully exploring the ASFV antigenic proteins is urgently needed. In this study, the MGF_110-13L was identified as an immunodominant antigen among the seven transmembrane proteins. The main outer-membrane domain of MGF_110-13L was expressed and purified. Two monoclonal antibodies (mAbs; 8C3, and 10E4) against MGF_110-13L were generated. The epitopes of two mAbs were preliminary mapped with the peptide fusion proteins after probing with mAbs by enzyme-linked immunosorbent assay (ELISA) and Western blot. And the two target epitopes were fine-mapped using further truncated peptide fusion protein strategy. Finally, the core sequences of mAbs 8C3 and 10E4 were identified as 48WDCQDGICKNKITESRFIDS67, and 122GDHQQLSIKQ131, respectively. The peptides of epitopes were synthesized and probed with ASFV antibody positive pig sera by a dot blot assay, and the results showed that epitope 10E4 was an antigenic epitope. The epitope 10E4 peptide was further evaluated as a potential antigen for detecting ASFV antibodies. To our knowledge, this is the first report of antigenic epitope information on the antigenic MGF_110-13L protein of ASFV.

Effect of food processing on the immunoreactivity of gliadin

SummaryWheat is the most important crop in the world mainly because of the feature of its storage protein, gluten, such as stretching and extending, that can hold the produced gas. However, some people have immunoreactivity against wheat gluten, which is generally called gluten disorder. Coeliac disease (CD) and non‐coeliac gluten sensitivity are types of gluten disorders. Several food processing techniques have been used to reduce gliadin immunoreactivity such as using thermal processing, fermentation, high‐pressure processing and pulsed light with different wheat products such as wheat flour, flatbread and gluten suspension. CD is the most known gluten disorder; therefore, this review will focus on the reduction effect of food processing on it. Gluten consists of gliadins and glutenin; the allergic effect of gliadins is well known, and several epitopes were identified, which are usually used to follow gluten immunoreactivity. Till now, consuming a gluten‐free diet is the only way for people who are suffering from gluten disorder. Up to now, all the attempts to reduce the immunoreactivity of gliadin (IRG) by food processing have not fully succeeded. The main techniques used to follow the target epitopes are enzyme‐linked immunosorbent assay (ELISA), western blot, LC–MS/MS, etc. The fermentation process and adding enzymes are the most promising food processing in terms of reducing IRG. Worth mentioning that using different techniques gave different gliadin reductions; beyond that, using different ELISA kits gave different reductions. Combining different techniques is recommended to monitor the IRG reduction by using the most effective method till now.