Distribution Of Epitopes Research Articles

Protective Antimicrobial Effect of the Potential Vaccine Created on the Basis of the Structure of the IgA1 Protease from Neisseria meningitidis

Background/Objectives: IgA1 protease is one of the virulence factors of Neisseria meningitidis, Haemophilus influenzae and other pathogens causing bacterial meningitis. The aim of this research is to create recombinant proteins based on fragments of the mature IgA1 protease A28–P1004 from N. meningitidis serogroup B strain H44/76. These proteins are potential components of an antimeningococcal vaccine for protection against infections caused by pathogenic strains of N. meningitidis and other bacteria producing serine-type IgA1 proteases. Methods: To obtain promising antigens for creating a vaccine, we designed and obtained several recombinant proteins. These proteins consisted of single or directly connected fragments selected from various regions of the IgA1 protease A28–P1004. The choice of these fragments was based on our calculated data on the distribution of linear and conformational B-cell epitopes and MHC-II T-cell epitopes in the structure of IgA1 protease, taking into account the physicochemical properties of potential compounds and the results of a comparative analysis of the spatial structures of the original IgA1 protease and potential recombinant proteins. We studied the immunogenic and protective effects of the obtained proteins on the BALB/c mice against meningococci of serogroups A, B and C. Results: Proteins MA28–P1004-LEH6, MW140–K833-LEH6, MW329–P1004-LEH6, M(W140–H328)-(W412–D604)-(Y866–P1004)-LEH6 and M(W140–Q299)-(Y866–P1004)-LEH6 have shown the following antibody titers, 103/titer: 11 ± 1, 6 ± 2, 6 ± 1, 9 ± 1 and 22 ± 3, respectively. Also, the last two proteins have shown the best average degree of protection from N. meningitidis serogroups A, B and C, %: 62 ± 6, 63 ± 5, 67 ± 4 respectively for M(W140–H328)-(W412–D604)-(Y866–P1004)-LEH6 and 70 ± 5, 66 ± 6, 83 ± 3 respectively for M(W140–Q299)-(Y866–P1004)-LEH6. Conclusions: We selected two recombinant proteins consisting of two (M(W140–Q299)-(Y866–P1004)-LEH6) or three (M(W140–H328)-(W412–D604)-(Y866–P1004)-LEH6) linked fragments of IgA1 protease A28–P1004 as candidate active component for an antimeningococcal vaccine.

Prolonged Omicron-specific B cell maturation alleviates immune imprinting induced by SARS-CoV-2 inactivated vaccine

ABSTRACT SARS-CoV-2 ancestral strain-induced immune imprinting poses great challenges to updating vaccines for new variants. Studies showed that repeated Omicron exposures could override immune imprinting induced by inactivated vaccines but not mRNA vaccines, a disparity yet to be understood. Here, we analyzed the immune imprinting alleviation in inactivated vaccine (CoronaVac) cohorts after a long-term period following breakthrough infections (BTI). We observed in CoronaVac-vaccinated individuals who experienced BA.5/BF.7 BTI, the proportion of Omicron-specific memory B cells (MBCs) substantially increased after an extended period post-Omicron BTI, with their antibodies displaying enhanced somatic hypermutation and neutralizing potency. Consequently, the neutralizing antibody epitope distribution encoded by MBCs post-BA.5/BF.7 BTI after prolonged maturation closely mirrors that in BA.5/BF.7-infected unvaccinated individuals. Together, these results indicate the activation and expansion of Omicron-specific naïve B cells generated by first-time Omicron exposure helped to alleviate CoronaVac-induced immune imprinting, and the absence of this process should have caused the persistent immune imprinting seen in mRNA vaccine recipients.

Systematic analysis of SARS-CoV-2 Omicron subvariants' impact on B and T cell epitopes.

Epitopes are specific structures in antigens that are recognized by the immune system. They are widely used in the context of immunology-related applications, such as vaccine development, drug design, and diagnosis / treatment / prevention of disease. The SARS-CoV-2 virus has represented the main point of interest within the viral and genomic surveillance community in the last four years. Its ability to mutate and acquire new characteristics while it reorganizes into new variants has been analyzed from many perspectives. Understanding how epitopes are impacted by mutations that accumulate on the protein level cannot be underrated. With a focus on Omicron-named SARS-CoV-2 lineages, including the last WHO-designated Variants of Interest, we propose a workflow for data retrieval, integration, and analysis pipeline for conducting a database-wide study on the impact of lineages' characterizing mutations on all T cell and B cell linear epitopes collected in the Immune Epitope Database (IEDB) for SARS-CoV-2. Our workflow allows us to showcase novel qualitative and quantitative results on 1) coverage of viral proteins by deposited epitopes; 2) distribution of epitopes that are mutated across Omicron variants; 3) distribution of Omicron characterizing mutations across epitopes. Results are discussed based on the type of epitope, the response frequency of the assays, and the sample size. Our proposed workflow can be reproduced at any point in time, given updated variant characterizations and epitopes from IEDB, thereby guaranteeing to observe a quantitative landscape of mutations' impact on demand. A big data-driven analysis such as the one provided here can inform the next genomic surveillance policies in combatting SARS-CoV-2 and future epidemic viruses.

SUB-immunogold-SEM reveals nanoscale distribution of submembranous epitopes

Electron microscopy paired with immunogold labeling is the most precise tool for protein localization. However, these methods are either cumbersome, resulting in small sample numbers and restricted quantification, or limited to identifying protein epitopes external to the membrane. Here, we introduce SUB-immunogold-SEM, a scanning electron microscopy technique that detects intracellular protein epitopes proximal to the membrane. We identify four critical sample preparation factors contributing to the method’s sensitivity. We validate its efficacy through precise localization and high-powered quantification of cytoskeletal and transmembrane protein distribution. We evaluate the capabilities of SUB-immunogold-SEM on cells with highly differentiated apical surfaces: (i) auditory hair cells, revealing the presence of nanoscale MYO15A-L rings at the tip of stereocilia; and (ii) respiratory multiciliate cells, mapping the distribution of the SARS-CoV-2 receptor ACE2 along the motile cilia. SUB-immunogold-SEM extends the application of SEM-based nanoscale protein localization to the detection of intracellular epitopes on the exposed surfaces of any cell.

Comparative Label-Free Proteomics Study on Celiac Disease-Active Epitopes in Common Wheat, Spelt, Durum Wheat, Emmer, and Einkorn.

Wheat species with various ploidy levels may be different regarding their immunoreactive potential in celiac disease (CD), but a comprehensive comparison of peptide sequences with known epitopes is missing. Thus, we used an untargeted liquid chromatography tandem mass spectrometry method to analyze the content of peptides with CD-active epitope in the five wheat species common wheat, spelt, durum wheat, emmer, and einkorn. In total, 494 peptides with CD-active epitope were identified. Considering the average of the eight cultivars of each species, spelt contained the highest number of different peptides with CD-active epitope (193 ± 12, mean ± SD). Einkorn showed the smallest variability of peptides (63 ± 4) but higher amounts of certain peptides compared to the other species. The wheat species differ in the presence and distribution of CD-active epitopes; hence, the entirety of peptides with CD-active epitope is crucial for the assessment of their immunoreactive potential.

SARS-Cov-2 antigen exposure history shapes post-infection antibody epitope and immune pressure distribution

Abstract The COVID-19 pandemic has led to a complex immunological landscape shaped by various vaccination regimens and SARS-CoV-2 variant infections. The effect of diverse exposure history on antibody response and virus evolution is a critical, unsolved issue. Using high-throughput single-cell V(D)J sequencing, high-throughput deep mutational scanning, and neutralization assays, we characterized epitope distributions and immune pressure of more than 6000 Receptor Binding Domain (RBD)-antibodies from various SARS-CoV-2 exposure histories, including ancestral strain vaccinated or unvaccinated individuals with single or dual infections with different variants. We found that single variant breakthrough infections post-vaccination induce a high proportion of non-neutralizing antibodies due to immune imprinting. In contrast, dual breakthrough infections exhibit fewer non-neutralizing antibodies, akin to those in unvaccinated individuals. Furthermore, the initial infection variant influences the antibody repertoire to second infections, indicating that variant infections also induce immune imprinting. Antibody profiles shaped by diverse exposure histories exert distinct pressures on RBD, promoting the virus to evolve at convergent immune pressure sites. This is evidenced by frequent RBD mutations at positions 420, 455, 456, and 475 across different variants. These findings highlight the potential of diversifying the antibody pressure across populations to reduce the pace of viral evolution.

Cell wall epitope distribution in the functional compartments of galls induced by Palaeomystella oligophaga (Lepidoptera) in Macairea radula (Melastomataceae)

Context The parasitic interaction between the galling insect Palaeomystella oligophaga (Lepidoptera) and the host plant tissues of Macairea radula (Melastomataceae) leads to the formation of globoid galls. These galls have storage and typical nutritive tissues (outer and inner compartments, respectively), whose functions may be related to differential cell wall component distributions and to the stage of development of the gall inducer. Aims We evaluated whether the cell wall composition of the gall compartments (storage and nutritive tissues) changes according to the developmental stage of the gall inducer P. oligophaga. Methods The galls occupied by the gall inducer in different stages of development (i.e. larval and pupal stages, as well as empty galls) were collected and submitted to histological examination, immunocytochemical analyses using monoclonal antibodies for pectins, hemicelluloses, and glycoproteins, and histochemical analyses using phloroglucinol for lignins. Key results The histological results showed that nutritive tissues have smaller cells compared with storage ones when the galling insect is in the larval stage. Immunocytochemical analyses also showed the occurrence of both methyl-esterified and unesterified pectins, as well hemicelluloses in the nutritive tissue during the larval stage. The dynamic of some cell wall components changes when the gall inducer is in the pupal stage, or the galls are empty. Conclusions The changes in the composition of the cell walls, especially regarding homogalacturonans and xyloglucans, support the high metabolism and resource availability in cell walls of nutritive tissue for the gall inducer. Implications The cell wall composition may indicate important metabolic steps during gall formation and galling insect diet.

InflANNet: a neural network predictor for Influenza A CTL and HTL epitopes to aid robust vaccine design

BackgroundAn efficient and reliable data-driven method is essential to aid robust vaccine design, particularly in the case of an epidemic like Influenza A. Although various prediction tools are existing, most of them focus on the MHC-peptide binding affinity predictions. A tool which can incorporate more features other than binding affinity which characterizes the T-cell epitopes as vaccine candidates would be of much value in this scenario. The objective of this study is to develop two separate neural network models for the predictions of CTLs (cytotoxic T lymphocyte) and HTLs (helper T lymphocyte) with the manually curated datasets as a part of this study from the raw viral sequences of Influenza A.ResultsThe epitope datasets curated from the raw sequences of the broadly protective Neuraminidase protein were utilized for building and training the models for CTLs and HTLs. Each set consisted of nearly a balanced mix of vaccine candidates and non-vaccine candidates for both CTLs and HTLs. These were fed to neural networks as they are proven to be powerful for the predictions when compared with the other machine/deep learning algorithms. A set of epitopes experimentally proved were chosen to validate the model which was also tested through mutational analysis and cross-reactivity. The prepared dataset gave some valuable insights into the epitope distribution statistics and their conservancy in various outbreaks. An idea about the most probable range of peptide-MHC binding affinities was also obtained. Both the models performed well giving high accuracies when validated. These epitopes were checked for cross-reactivity with other antigens upon which it proved to be highly conservative and ideal for vaccine formulation.ConclusionsThe combination of various features and the resulting model efficiencies in turn proved that the collected features are valuable in the easy identification of the vaccine candidates. This suggests that our proposed models have more potential for conserved epitope prediction compared to other existing models trained on similar data and features. The possibility of refining the model with more set threshold values based on more parameters is an added feature that makes it more user driven. Furthermore, the uniqueness of the model due to exclusive set of Neuraminidase epitopes paves a robust way for rapid vaccine design.Graphical abstract

Epitope Binning of Monoclonal and Polyclonal Antibodies by Biolayer Interferometry.

Understanding the epitopes of antibodies elicited by infection and vaccination is often useful in immunogen design. In this chapter, we describe biolayer interferometry (BLI)-based methods to evaluate such epitopes and permit simultaneous analysis of antibodies from several sources, including monoclonal antibodies (mAbs) and polyclonal serum antibodies (pAbs). Using previously characterized antibodies with known epitopes as controls, the distribution of epitopes for the influenza hemagglutinin (HA) is shown for isolated human mAbs and pooled serum from HA-immunized mice. This method is versatile, high-throughput, and can be adapted to several antigens.

The Future Perspective of Gut Microbiota and Autoimmune Thyroid Disease

The human digestive tract contains a large number of gut microbiota. Where the composition of the bacteria is influenced by various factors, ranging from geographical differences, to various other factors. The gut microbiota plays an important role not only in the stability of digestive function, but also in immune function, hormones, and metabolic homeostasis. Recent research results indicate that there is an autoimmune process that occurs after bacterial infection, where the mechanism involves molecular mimicry, epitope distribution, bystander activation, and cryptic antigens. The condition of dysbiosis has been associated with various gastrointestinal disorders and systemic disorders, one of which is in the group of autoimmune diseases. Increased intestinal permeability causes the flow of toxins, antigens, and bacterial metabolites from the intestine into the blood vessels. Where this condition is a hypothesis of the initiation process of autoimmune thyroid disease. A balance between protective reactions and tolerance is required to maintain intestinal homeostasis. Changes in this balance can cause auto-aggressive disturbances triggered by differences in the composition of the microbiota. The deiodinase enzyme is an important factor in thyroid hormone metabolism. Several microbes were identified as regulators of certain enzymes involved in iodothyronine metabolism. Autoimmune thyroid disease and gut microbiota have a potential relationship which, if further investigated, could be the future of autoimmune thyroid disease therapy.

Antibody feedback contributes to facilitating the development of Omicron-reactive memory B cells in SARS-CoV-2 mRNA vaccinees.

In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)-specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.

Physical and chemical evolution of sugar beet pulp during aqueous ammonia pretreatment and its impacts on the recalcitrance of cellulose

Recognition and deconstruction of biomass recalcitrance are essential steps in fuel ethanol production. Aqueous ammonia (AA) is an efficient pretreatment to improve the enzymatic digestibility of sugar beet pulp (SBP), but the precise mechanism is still ambiguous. Multidimensional methods, e.g., physical properties, wet chemistry compositions, and glycome profiling were integrated to elucidate the impacts of AA pretreatment on recalcitrance of SBP semi-quantitatively. Additionally, the variations in enzyme adsorption and cell wall morphology during the enzymatic hydrolysis were systematically observed. The results confirmed that the constitution, association, and distribution of glycan epitopes within cell walls significantly correlated to recalcitrance. Specifically, the arabinogalactan and rhamnogalacturonan epitopes negatively correlated to the recalcitrance. And xylans only bound to cellulose contributed to recalcitrance. Dislocation and fragmentation of cell walls through sequential removal of several glycan epitopes could facilitate enzymatic digestibility, in terms of enzyme adsorption, reducing sugar yields, and enzymatic saccharification behaviors. Whereas, cell corners seemed to be highly resistant to enzymatic digestion due to some gathered epitopes. The maximum reducing sugar yields ranging from 437.7 to 441.48 mg/g were obtained from the pretreated SBP (3, 5, and 7 h), which nearly increased by 87% compared to the control. Selecting a proper pretreatment to precisely strip out the vital glycan epitopes might be an economically feasible strategy in fuel ethanol production.

Epitope-Evaluator: An interactive web application to study predicted T-cell epitopes.

Multiple immunoinformatic tools have been developed to predict T-cell epitopes from protein amino acid sequences for different major histocompatibility complex (MHC) alleles. These prediction tools output hundreds of potential peptide candidates which require further processing; however, these tools are either not graphical or not friendly for non-programming users. We present Epitope-Evaluator, a web tool developed in the Shiny/R framework to interactively analyze predicted T-cell epitopes. Epitope-Evaluator contains six tools providing the distribution of epitopes across a selected set of MHC alleles, the promiscuity and conservation of epitopes, and their density and location within antigens. Epitope-Evaluator requires as input the fasta file of protein sequences and the output prediction file coming out from any predictor. By choosing different cutoffs and parameters, users can produce several interactive plots and tables that can be downloaded as JPG and text files, respectively. Using Epitope-Evaluator, we found the HLA-B*40, HLA-B*27:05 and HLA-B*07:02 recognized fewer epitopes from the SARS-CoV-2 proteome than other MHC Class I alleles. We also identified shared epitopes between Delta, Omicron, and Wuhan Spike variants as well as variant-specific epitopes. In summary, Epitope-Evaluator removes the programming barrier and provides intuitive tools, allowing a straightforward interpretation and graphical representations that facilitate the selection of candidate epitopes for experimental evaluation. The web server Epitope-Evaluator is available at https://fuxmanlab.shinyapps.io/Epitope-Evaluator/

Predicting Epitope Candidates for SARS-CoV-2.

Epitopes are short amino acid sequences that define the antigen signature to which an antibody or T cell receptor binds. In light of the current pandemic, epitope analysis and prediction are paramount to improving serological testing and developing vaccines. In this paper, known epitope sequences from SARS-CoV, SARS-CoV-2, and other Coronaviridae were leveraged to identify additional antigen regions in 62K SARS-CoV-2 genomes. Additionally, we present epitope distribution across SARS-CoV-2 genomes, locate the most commonly found epitopes, and discuss where epitopes are located on proteins and how epitopes can be grouped into classes. The mutation density of different protein regions is presented using a big data approach. It was observed that there are 112 B cell and 279 T cell conserved epitopes between SARS-CoV-2 and SARS-CoV, with more diverse sequences found in Nucleoprotein and Spike glycoprotein.

Specific Components Associated With the Endothelial Glycocalyx Are Lost From Brain Capillaries in Cerebral Malaria.

Cerebral malaria (CM) is a rare, but severe and frequently fatal outcome of infection with Plasmodium falciparum. Pathogenetic mechanisms include endothelial activation and sequestration of parasitized erythrocytes in the cerebral microvessels. Increased concentrations of glycosaminoglycans in urine and plasma of malaria patients have been described, suggesting involvement of endothelial glycocalyx. We used lectin histochemistry on postmortem samples to compare the distribution of multiple sugar epitopes on cerebral capillaries in children who died from CM and from nonmalarial comas. N-acetyl glucosamine residues detected by tomato lectin are generally reduced in children with CM compared to controls. We used the vascular expression of intercellular adhesion molecule 1 and mannose residues on brain capillaries of CM as evidence of local vascular inflammation, and both were expressed more highly in CM patients than controls. Sialic acid residues were found to be significantly reduced in patients with CM. By contrast, the levels of other sugar epitopes regularly detected on the cerebral vasculature were unchanged, and this suggests specific remodeling of cerebral microvessels in CM patients. Our findings support and expand upon earlier reports of disruptions of the endothelial glycocalyx in children with severe malaria.

Evaluation of Spike Protein Epitopes by Assessing the Dynamics of Humoral Immune Responses in Moderate COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike protein (S) of SARS-CoV-2 is a major target for diagnosis and vaccine development because of its essential role in viral infection and host immunity. Currently, time-dependent responses of humoral immune system against various S protein epitopes are poorly understood. In this study, enzyme-linked immunosorbent assay (ELISA), peptide microarray, and antibody binding epitope mapping (AbMap) techniques were used to systematically analyze the dynamic changes of humoral immune responses against the S protein in a small cohort of moderate COVID-19 patients who were hospitalized for approximately two months after symptom onset. Recombinant truncated S proteins, target S peptides, and random peptides were used as antigens in the analyses. The assays demonstrated the dynamic IgM- and IgG recognition and reactivity against various S protein epitopes with patient-dependent patterns. Comprehensive analysis of epitope distribution along the spike gene sequence and spatial structure of the homotrimer S protein demonstrated that most IgM- and IgG-reactive peptides were clustered into similar genomic regions and were located at accessible domains. Seven S peptides were generally recognized by IgG antibodies derived from serum samples of all COVID-19 patients. The dynamic immune recognition signals from these seven S peptides were comparable to those of the entire S protein or truncated S1 protein. This suggested that the humoral immune system recognized few conserved S protein epitopes in most COVID-19 patients during the entire duration of humoral immune response after symptom onset. Furthermore, in this cohort, individual patients demonstrated stable immune recognition to certain S protein epitopes throughout their hospitalization period. Therefore, the dynamic characteristics of humoral immune responses to S protein have provided valuable information for accurate diagnosis and immunotherapy of COVID-19 patients.

Immunodominant conformational and linear IgE epitopes lie in a single segment of Ara h 2

Contribution of conformational epitopes to the IgE reactivity of peanut allergens Ara h 2 and Ara h 6 is at least as important as that of the linear epitopes. However, little is known about these conformational IgE-binding epitopes. We investigated the distribution of conformational epitopes on chimeric 2S-albumins. Recombinant chimeras were generated by exchanging structural segments between Ara h 2 and Ara h 6. Well-refolded chimeras, as verified by circular dichroism analysis, were then used to determine the epitope specificity of mAbs by performing competitive inhibition of IgG binding. Furthermore, we delineated the contribution of each segment to the overall IgE reactivity of both 2S-albumins by measuring the chimeras' IgE-binding capacity with sera from 21 patients allergic to peanut. We finally assessed chimeras' capacity to trigger mast cell degranulation. Configuration of the conformational epitopes was preserved in the chimeras. Mouse IgG mAbs, raised against natural Ara h 6, and polyclonal human IgE antibodies recognized different conformational epitopes distributed all along Ara h 6. In contrast, we identified human IgG mAbs specific to different Ara h 2 linear or conformational epitopes located in all segments except the C-terminal one. The major conformational IgE-binding epitope of Ara h 2 was located in a segment located between residues 33 and 81 that also contains the major linear hydroxyproline-containing epitope. Accordingly, this segment is critical for the capacity of Ara h 2 to induce mast cell degranulation. Chimeric 2S-albumins provide new insights on the conformational IgE-binding epitopes of Ara h 2 and Ara h 6. Proximity of the immunodominant linear and conformational IgE-binding epitopes probably contributes to the high allergenicpotency of Ara h 2.

Recombinantly Expressed Chimeric Fibers Demonstrate Discrete Type-Specific Neutralizing Epitopes in the Fowl Aviadenovirus E (FAdV-E) Fiber, Promoting the Optimization of FAdV Fiber Subunit Vaccines towards Cross-Protection in vivo.

ABSTRACTVaccines against inclusion body hepatitis in chickens are complicated by the involvement of antigenically diverse fowl adenovirus types. Though immunization with fiber protein confers robust protection, type specificity of fiber antibodies is an obstacle for the desired broad coverage. In this study, we utilized information on multiple linear epitopes predicted in the Fowl Aviadenovirus E (FAdV-E) fiber head (knob) to develop chimeric fibers with an exchange between two serotypes’ sequences, each containing proposed epitopes. Two consecutive segments pertaining to amino acid positions 1 to 441 and 442 to 525/523 in the fibers of FAdV-8a and -8b, types of Fowl Aviadenovirus E that cause inclusion body hepatitis, were swapped reciprocally to result in novel chimeras, crecFib-8a/8b and crecFib-8b/8a. crecFib was indistinguishable from monospecific recombinant fibers in its eactivity with different FAdV antisera in Western blotting. However, contrary to the results for monospecific fibers, crecFib induced cross-neutralizing antibodies against both serotypes in chickens. This demonstrates three nonidentical epitopes in the FAdV-E fiber, the conserved epitope detected in Western blotting and at least two epitopes participating in neutralization, being type specific and located opposite residue position 441-442. Furthermore, we supply conformational evidence for a site in the fiber knob with accessibility critical for neutralization. With such an extended neutralization spectrum compared to those of individual fibers, crecFib was anticipated to fulfill and even extend the mechanistic basis of fiber-mediated protection toward bivalent coverage. Accordingly, crecFib, administered as a single-antigen component, protected chickens simultaneously against challenge with FAdV-8a or -8b, demonstrated by up-to-complete resistance to clinical disease, prevention of target organ-related changes, and significant reduction of viral load.IMPORTANCE The control of inclusion body hepatitis, a disease of economic importance for chicken production worldwide, is complicated by an etiology involving multiple divergent fowl adenovirus types. The fiber protein is principally efficacious in inducing neutralizing and protective antibodies in vaccinated chickens; however, it faces limitations due to its intrinsic type specificity for neutralization. In this study, based on an in silico-guided prediction of multiple epitopes in the fowl adenovirus fiber head’s loops, we designed chimeric proteins, swapping N- and C-distal fiber portions, each containing putative epitopes, between divergent types FAdV-8a and -8b. In in vitro and in vivo studies, the chimeric fiber displayed extended properties compared to those of individual monotype-specific fibers, allowing the number, distribution, functionality, and conformational bearings of epitopes of the fowl adenovirus fiber to be characterized in more detail. Importantly, the chimeric fiber induced cross-neutralizing antibodies and protective responses in chickens against infections by both serotypes, promoting the advancement of broadly protective subunit vaccination strategies against FAdV.

Identification of a Novel Neutralizing Epitope on the N-Terminal Domain of the Human Coronavirus 229E Spike Protein.

The receptor binding domain (RBD) of the coronavirus spike protein (S) has been verified to be the main target for potent neutralizing antibodies (nAbs) in most coronaviruses, and the N-terminal domain (NTD) of some betacoronaviruses has also been indicated to induce nAbs. For alphacoronavirus HCoV-229E, its RBD has been shown to have neutralizing epitopes, and these epitopes could change over time. However, whether neutralizing epitopes exist on the NTD and whether these epitopes change like those of the RBD are still unknown. Here, we verified that neutralizing epitopes exist on the NTD of HCoV-229E. Furthermore, we characterized an NTD targeting nAb 5H10, which could neutralize both pseudotyped and authentic HCoV-229E VR740 in vitro. Epitope mapping indicated that 5H10 targeted motif E1 (147-167 aa) and identified F159 as critical for 5H10 binding. More importantly, our results revealed that motif E1 was highly conserved among clinical isolates except for F159. Further data proved that mutations at position 159 gradually appeared over time and could completely abolish the neutralizing ability of 5H10, supporting the notion that position 159 may be under selective pressure during the human epidemic. In addition, we also found that contemporary clinical serum has a stronger binding capacity for the NTD of contemporary strains than historic strains, proving that the epitope on the NTD could change over time. In summary, these findings define a novel neutralizing epitope on the NTD of HCoV-229E S and provide a theoretical basis for the design of vaccines against HCoV-229E or related coronaviruses. IMPORTANCE Characterization of the neutralizing epitope of the spike (S) protein, the major invasion protein of coronaviruses, can help us better understand the evolutionary characteristics of these viruses and promote vaccine development. To date, the neutralizing epitope distribution of alphacoronaviruses is not well known. Here, we identified a neutralizing antibody that targeted the N-terminal domain (NTD) of the alphacoronavirus HCoV-229E S protein. Epitope mapping revealed a novel epitope that was not previously discovered in HCoV-229E. Further studies identified an important residue, F159. Mutations that gradually appeared over time at this site abolished the neutralizing ability of 5H10, indicating that selective pressure occurred at this position in the spread of HCoV-229E. Furthermore, we found that the epitopes within the NTD also changed over time. Taken together, our findings defined a novel neutralizing epitope and highlighted the role of the NTD in the future prevention and control of HCoV-229E or related coronaviruses.

Mapping Pectic-Polysaccharide Epitopes in Cell Walls of Forage Chicory (Cichorium intybus) Leaves.

The cell walls of forage chicory (Cichorium intybus) leaves are known to contain high proportions of pectic polysaccharides. However, little is known about the distribution of pectic polysaacharides among walls of different cell types/tissues and within walls. In this study, immunolabelling with four monoclonal antibodies was used to map the distribution of pectic polysaccharides in the cell walls of the laminae and midribs of these leaves. The antibodies JIM5 and JIM7 are specific for partially methyl-esterified homogalacturonans; LM5 and LM6 are specific for (1→4)-β-galactan and (1→5)-α-arabinan side chains, respectively, of rhamnogalacturonan I. All four antibodies labelled the walls of the epidermal cells with different intensities. JIM5 and JIM7, but not LM5 or LM6, labelled the middle lamella, tricellular junctions, and the corners of intercellular spaces of ground, xylem and phloem parenchyma. LM5, but not LM6, strongly labelled the walls of the few sclerenchyma fibres in the phloem of the midrib and lamina vascular bundles. The LM5 epitope was absent from some phloem parenchyma cells. LM6, but not LM5, strongly labelled the walls of the stomatal guard cells. The differential distribution of pectic epitopes among walls of different cell types and within walls may reflect the deposition and modification of these polysaccharides which are involved in cell wall properties and cell development.