Epitope Structure Research Articles

Specificity of Anti-Citrullinated Protein Antibodies to Citrullinated α-Enolase Peptides as a Function of Epitope Structure and Composition.

Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1–2% of the world population. In addition to the first discovered serologic markers for RA, the rheumatoid factors (RFs), anti-citrullinated protein antibodies (ACPAs) are even more specific for the disease compared to RFs and are found in 70–80% of RA patient sera. RA etiopathogenesis still needs to be elucidated, as different factors are proposed to be involved, such as Epstein–Barr virus infection. Hence, understanding the interaction between ACPAs and their citrullinated peptide targets is relevant for a better knowledge of RA pathophysiology and for diagnostic purposes. In this study, a cohort of RA sera, healthy control sera and multiple sclerosis sera were screened for reactivity to a variety of citrullinated peptides originating from α-enolase, pro-filaggrin, proteoglycan and Epstein–Barr nuclear antigen-2 by enzyme-linked immunosorbent assay. ACPA reactivity to citrullinated α-enolase peptides was found to depend on peptide length and peptide conformation, favouring cyclic (disulfide bond) conformations for long peptides and linear peptides for truncated ones. Additional investigations about the optimal peptide conformation for ACPA detection, employing pro-filaggrin and EBNA-2 peptides, confirmed these findings, indicating a positive effect of cyclization of longer peptides of approximately 20 amino acids. Moreover, screening of the citrullinated peptides confirmed that ACPAs can be divided into two groups based on their reactivity. Approximately 90% of RA sera recognize several peptide targets, being defined as cross-reactive or overlapping reactivities, and whose reactivity to the citrullinated peptide is considered primarily to be backbone-dependent. In contrast, approximately 10% recognize a single target and are defined as nonoverlapping, primarily depending on the specific amino acid side-chains in the epitope for a stable interaction. Collectively, this study contributed to characterize epitope composition and structure for optimal ACPA reactivity and to obtain further knowledge about the cross-reactive nature of ACPAs.

A conserved epitope III on hepatitis C virus E2 protein has alternate conformations facilitating cell binding or virus neutralization

Epitope III, a highly conserved amino acid motif of 524APTYSW529 on the hepatitis C virus (HCV) E2 glycoprotein, resides in the critical loop that binds to the host receptor CD81, thus making it one of the most important antibody targets for blocking HCV infections. Here, we have determined the X-ray crystal structure of epitope III at a 2.0-Å resolution when it was captured by a site-specific neutralizing antibody, monoclonal antibody 1H8 (mAb1H8). The snapshot of this complex revealed that epitope III has a relatively rigid structure when confined in the binding grooves of mAb1H8, which confers the residue specificity at both ends of the epitope. Such a high shape complementarity is reminiscent of the "lock and key" mode of action, which is reinforced by the incompatibility of an antibody binding with an epitope bearing specific mutations. By subtly positioning the side chains on the three residues of Tyr527, Ser528, and Trp529 while preserving the spatial rigidity of the rest, epitope III in this cocrystal complex adopts a unique conformation that is different from previously described E2 structures. With further analyses of molecular docking and phage display-based peptide interactions, we recognized that it is the arrangements of two separate sets of residues within epitope III that create these discrete conformations for the epitope to interact selectively with either mAb1H8 or CD81. These observations thus raise the possibility that local epitope III conformational dynamics, in conjunction with sequence variations, may act as a regulatory mechanism to coordinate "mAb1H8-like" antibody-mediated immune defenses with CD81-initiated HCV infections.

The Impact of Specific Factor VIII Domains on Uptake By Dendritic Cells

Introduction: The role of dendritic cells in the presentation of foreign antigens to induce an adaptive immune response is critical for protection from potentially harmful pathogens. However in the treatment of hemophilia A, the recognition, processing, and presentation of factor VIII (fVIII) by dendritic cells that results in anti-fVIII inhibitors is a devastating complication of modern hemophilia A care. FVIII structure consists of A1, A2, B, A3, C1, and C2 domains. While prior studies have described a role for the C1 and C2 domains in mediating fVIII uptake by dendritic cells, the contribution of the remaining fVIII domains has not been fully explored. In this study we analyzed the full spectrum of fVIII domains on uptake by dendritic cells as a means to better understand the pathogenic innate immune response to fVIII. Methods: Bone marrow cells were harvested from the femurs and tibias of 8-10 week old E16 hemophilia A knockout mice, stimulated with GM-CSF, and cultured at 37°C for 6-7 days to induce bone marrow derived dendritic cells (BMDCs). BMDCs were incubated with dylight650-conjugated recombinant full length fVIII (DyL650-rfVIII) in combination with a panel of 17 previously characterized anti-fVIII monoclonal antibodies (MAbs) from our repertoire of anti-human murine derived MAbs representing B cell epitopes from all fVIII domains. Internalization of 10 nM fVIII by CD11c+ BMDCs in the presence of molar excess of individual anti-fVIII MAbs (80 nM) following a 30 minute incubation at 37°C was compared to uptake of fVIII alone. FVIII uptake by BMDCs at 4°C was used as a negative control. FVIII internalization was analyzed by flow cytometry and imaged using an Imagestream X Mark II flow cytometer. A supraphysiologic dose of fVIII was utilized in these experiments for optimal visualization and measurement of fVIII internalization by flow cytometry, though similar trends in uptake pattern at more physiologic concentrations of fVIII have also been observed in previous studies performed. Results: The panel of 17 anti-fVIII MAbs tested included 1 anti-A1, 2 anti-A2, 2 anti-B, 2 anti-A3, 3 anti-C1, and 7 anti-C2 domain MAbs. In this model, the presence of anti-C1 domain and anti-C2 domain MAbs reduced fVIII uptake by BMDCs as shown in prior studies. Anti-C1 domain MAb B136, a group B MAb that binds a critical structural epitope within the 2077-2084 region of the C1 domain, reduced fVIII uptake by 56%. Additionally classical group A anti-C2 domain MAb 3E6 reduced fVIII uptake by 35%. Both MAbs B136 and 3E6 bind to epitopes that are important for fVIII binding to von Willebrand factor and phospholipid surfaces suggesting that these epitopes may play a role in fVIII recognition by dendritic cells, particularly in instances when fVIII is unbound by either ligand in plasma. Interestingly anti-A1 domain MAb 2-116 and anti-A3 domain MAb I92 that binds residues 1818-1916 located at the C terminal end of the A3 domain significantly increased fVIII uptake by BMDCs two-fold (113% and 95% respectively) compared to fVIII alone. These findings indicate that epitopes within the A1 and A3 domains may play a role in limiting fVIII recognition by dendritic cells. All of the anti-A2 and anti-B domain MAbs, 2 of the 3 anti-C1 MAbs, and 5 of the 7 anti-C2 MAbs tested did not alter fVIII uptake. The figure below depicts a bar graph summarizing fVIII internalization by BMDCs with or without the anti-fVIII MAbs tested (figure 1A) and a representative Imagestream image of a dendritic cell (Ch01), CD11c+ surface staining of the dendritic cell (Ch07), DyL650-rfVIII alone without an anti-fVIII MAb (Ch11), and a merged image of CD11c+ dendritic cell and DyL650-rfVIII (Ch07/Ch11) (figure 1B). Conclusions: The C1 and C2 domains of fVIII primarily mediate fVIII uptake by dendritic cells. However the A1 and A3 domains may play a protective role in antigen uptake and ultimately antigen presentation by dendritic cells. Further investigation into the innate immune response to fVIII in patients with hemophilia A and differences between patients who do and do not develop inhibitors would provide insight into key aspects of the immune response that regulates fVIII tolerance. Download : Download high-res image (136KB) Download : Download full-size image Figure 1 . Disclosures Batsuli: Genentech, Octapharma: Membership on an entity's Board of Directors or advisory committees.

An epitope-specific chemically defined nanoparticle vaccine for respiratory syncytial virus

Respiratory syncytial virus (RSV) can cause severe respiratory disease in humans, particularly in infants and the elderly. However, attempts to develop a safe and effective vaccine have so far been unsuccessful. Atomic-level structures of epitopes targeted by RSV-neutralizing antibodies are now known, including that bound by Motavizumab and its clinically used progenitor Palivizumab. We developed a chemically defined approach to RSV vaccine design, that allows control of both immunogenicity and safety features of the vaccine. Structure-guided antigen design and a synthetic nanoparticle delivery platform led to a vaccine candidate that elicits high titers of palivizumab-like, epitope-specific neutralizing antibodies. The vaccine protects preclinical animal models from RSV infection and lung pathology typical of vaccine-derived disease enhancement. The results suggest that the development of a safe and effective synthetic epitope-specific RSV vaccine may be feasible by combining this conformationally stabilized peptide and synthetic nanoparticle delivery system.

Thermal and Acidic Treatments of Gluten Epitopes Affect Their Recognition by HLA-DQ2 in silico

Celiac disease (CD) is a prevalent disorder with autoimmune features. Dietary exposure of wheat gluten (including gliadins and glutenins) to the small intestine activates the gluten-reactive CD4+ T cells and controls the disease development. While the human leukocyte antigen (HLA) is the single most important genetic factor of this polygenic disorder, HLA-DQ2 recognition of gluten is the major biological step among patients with CD. Gluten epitopes are often rich in Pro and share similar primary sequences. Here, we simulated the solution structures changes of a variety of gluten epitopes under different pH and temperatures, to mimic the fermentation and baking/cooking processes. Based on the crystal structure of HLA-DQ2, binding of differently processed gluten epitopes to DQ2 was studied in silico. This study revealed that heating and pH change during the fermentation process impact the solution structure of gluten epitope. However, binding of differently treated gluten epitope peptide (GEP) to HLA-DQ2 mainly depended on its primary amino acid sequence, especially acidic amino acid residues that play a pivotal role in their recognition by HLA-DQ2.

Phage Display Screening of Bovine Antibodies to Foot-and-Mouth Disease Virus and Their Application in a Competitive ELISA for Serodiagnosis.

For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals.

Synthesis of Glycodendrimers with Antiviral and Antibacterial Activity.

Glycodendrimers are an important class of synthetic macromolecules that can be used to mimic many structural and functional features of cell-surface glycoconjugates. Their carbohydrate moieties perform key important functions in bacterial and viral infections, often regulated by carbohydrate-protein interactions. Several studies have shown that the molecular structure, valency and spatial organisation of carbohydrate epitopes in glycoconjugates are key factors in the specificity and avidity of carbohydrate-protein interactions. Choosing the right glycodendrimers almost always helps to interfere with such interactions and blocks bacterial or viral adhesion and entry into host cells as an effective strategy to inhibit bacterial or viral infections. Herein, the state of the art in the design and synthesis of glycodendrimers employed for the development of anti-adhesion therapy against bacterial and viral infections is described.

Extensin, an underestimated key component of cell wall defence?

Extensins are plant cell wall hydroxyproline-rich glycoproteins known to be involved in cell wall reinforcement in higher plants, and in defence against pathogen attacks. The ability of extensins to form intra- and intermolecular cross-links is directly related to their role in cell wall reinforcement. Formation of such cross-links requires appropriate glycosylation and structural conformation of the glycoprotein. Although the role of cell wall components in plant defence has drawn increasing interest over recent years, relatively little focus has been dedicated to extensins. Nevertheless, new insights were recently provided regarding the structure and the role of extensins and their glycosylation in plant-microbe interactions, stimulating an interesting debate from fellow cell wall community experts. We have previously revealed a distinct distribution of extensin epitopes in Arabidopsis thaliana wild-type roots and in mutants impaired in extensin arabinosylation, in response to elicitation with flagellin 22. That study was recently debated in a Commentary by Tan and Mort (Tan L, Mort A. 2020. Extensins at the front line of plant defence. A commentary on: 'Extensin arabinosylation is involved in root response to elicitors and limits oomycete colonization'. Annals of Botany 125: vii-viii) and several points regarding our results were discussed. As a response, we herein clarify the points raised by Tan and Mort, and update the possible epitope structure recognized by the anti-extensin monoclonal antibodies. We also provide additional data showing differential distribution of LM1 extensin epitopes in roots between a mutant defective in PEROXIDASES 33 and 34 and the wild type, similarly to previous observations from the rra2 mutant defective in extensin arabinosylation. We propose these two peroxidases as potential candidates to specifically catalyse the cross-linking of extensins within the cell wall. Extensins play a major role within the cell wall to ensure root protection. The cross-linking of extensins, which requires correct glycosylation and specific peroxidases, is most likely to result in modulation of cell wall architecture that allows enhanced protection of root cells against invading pathogens. Study of the relationship between extensin glycosylation and their cross-linking is a very promising approach to further understand how the cell wall influences root immunity.

Top Down Computational Approach: A Vaccine Development Step to Find Novel Superantigenic HLA Binding Epitopes from Dengue Virus Proteome.

Dengue virus (DENV) is a major mosquito vector based human pathogenic flavivirus which is causing major threat worldwide, yet the availability of therapeutic treatment and several vaccines, still called for advance treatment and vaccine development. The present top down computational approach is a vaccine development step to find novel super antigenic HLA binding epitopes from DENV proteome. The approach used sequence based screening to find complete conserve and high population coverage, common epitopes among all DENV serotype. Propred and Immune Epitope Data Base were used for sequence based screening with recommended parameters. Among top 29 identified epitopes, five structural protein epitopes viz. 33LQGRGPLKL41, 249VVVLGSQEG257, 172LVGIVTLYL180, 146MKILIGVVI154, 72YIIVGVEPG80 and one nonstructural protein epitope 18LKNDIPMTG26 were showed high conserve nature and high population coverage from complete DENV proteome. Further structure based study involving docking and molecular dynamic simulation to confirm stable behavior of HLA allele–peptide complex to give potent cell mediated immune response. Docking of epitope 72YIIVGVEPG80–DRB1 0401 allele and epitope 33LQGRGPLKL41–B*5101 allele complexes showed the best binding energy of − 7.71 and − 7.20 kcal/mol, respectively and stable binding pattern over the time window during molecular dynamic simulation. This computational approach resulted novel epitopes which can be used in the design and development of short epitope based vaccines as well as diagnosis tools for dengue infection.

Construction and Expression of Eukaryotic and Prokaryotic Recombinant Vectors of Pepck and Gp63 Dominant Epitopes of Leishmania infantum

To construct eukaryotic and prokaryotic recombinant vectors containing Pepck- Gp63 and to achieve protein expression by selecting the dominant epitope genes of Pepck and Gp63 of Leishmania infantum. The secondary structure and HLA epitopes of phosphoenolpyruvate carboxylase (PEPCK) were predicted by in silico analysis, and the dominant epitopes were picked out. According to the analysis results of glycoprotein of 63×10 3(GP63) epitopes identified by the same method in our laboratory, the dominant epitope genes of Pepck and Gp63 were used to construct pET32a- Pepck- Gp63 and pVAX1- Pepck- Gp63 by overlapping PCR and enzyme reaction. Then, for protein expression, the prokaryotic vectors were transfected into E.coil while the eukaryotic vectors were transfected into NIH3T3 cells by liposome transfection. There were multiple dominant epitopes in Pepckand there were Pepck-Gp63 sequences in the polyclonal site of expression vector. The expression of Pepck-Gp63 in E.coil appeared in inclusion form and led to 74 kDa band in SDS-PAGE. The immunofluorescence results of NIH3T3 cells transfected by pVAX1- Pepck-Gp63 were positive. The recombinant prokaryotic expression plasmids pET32a- Pepck-Gp63 and eukaryotic expression plasmids pVAX1- P epck -Gp63 were successfully constructed, and it was shown that the recombinant plasmids were able to express the corresponding target proteins in E. coli and NIH3T3 cells, respectively, providing a preliminary experimental basis for the subsequent study of immunization strategies.

SARS-CoV-2-specific CD8+ T cell responses in convalescent COVID-19 individuals.

Characterization of the T cell response in individuals who recover from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 coronavirus disease 2019 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis and from humoral and inflammatory responses. There were 132 SARS-CoV-2-specific CD8+ T cell responses detected across 6 different HLAs, corresponding to 52 unique epitope reactivities. CD8+ T cell responses were detected in almost all convalescent individuals and were directed against several structural and nonstructural target epitopes from the entire SARS-CoV-2 proteome. A unique phenotype for SARS-CoV-2-specific T cells was observed that was distinct from other common virus-specific T cells detected in the same cross-sectional sample and characterized by early differentiation kinetics. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem cell and transitional memory states (subsets), which may be key to developing durable protection.

Recombinant L-HN Fusion Antigen Derived from the L and HN Domains of Botulinum Neurotoxin B Stimulates a Protective Antibody Response Against Active Neurotoxin.

Botulinum neurotoxin (BoNT) is a neurotoxin produced by Clostridium botulinum in an anaerobic environment. BoNT is the most toxic protein among bacteria, animals, plants, and chemical substances reported to date. BoNTs are 150kDa proteins composed of three major functional domains: catalytic (L domain, 50kDa), translocation (HN domain, 50kDa), and receptor-binding (Hc domain, 50kDa) domains. Most studies have focused on the use of the Hc domain as an antigen because it is capable of generating robust protective immunity and contains some functional neutralizing epitopes. In the present study, we produced and characterized a recombinant L-HN fusion fragment of the parent BoNT/B (BL-HN) composed of L and HN domains with a deletion in the Hc domain (BHc). When the BL-HN protein was expressed in E. coli, it retained its stable structure and antigenicity. As a vaccine antigen, the recombinant BL-HN protein was found to induce sufficient protection against native BoNT/B in a mouse model. The BL-HN subunit vaccine could also induce a strong humoral immune response and generate sufficient neutralizing antibodies in immunized mice. Therefore, BL-HN may retain the native neurotoxin structure and critical epitopes responsible for inducing serum neutralizing antibodies. Studies of the dose-dependent immunoprotective effects further confirmed that the BL-HN antigen could provide potent protective immunity. This finding suggests that BL-HN can play an important role in immune protection against BoNT/B. Therefore, the BL-HN fusion fragment provides an excellent platform for the design of recombinant botulinum vaccines and neutralizing antibodies.

Human Proteinase 3, an important autoantigen of c-ANCA associated vasculitis, shares cross-reactive epitopes with serine protease allergens from mites: an in silico analysis

Background: In autoimmune vasculitis, autoantibodies to Human Proteinase 3 (PR3), a human serine protease, seems to have a role on the inception of c-ANCA associated vasculitis. The origin of this autoreactive response remains unclear. However, for several autoreactive responses, molecular mimicry between environmental antigens and human proteins is key to trigger autoantibodies and finally autoimmunity manifestations. Considering that PR3 is a serine protease and house dust mite (HDM) group 3 allergens share this biochemical activity, the aim of this study was to identify cross-reactive epitopes between serine proteases from human and mites using an in silico approach. Methods: Multi alignment among amino acid sequences of PR3 and HDM group 3 allergens was performed to explore identity and structural homology. ElliProandBepiPred in silicotoolswereusedtopredictBand T cell epitopes. Consurf tool was used to conduct identification of conserved regions in serine proteases family. Results: PR3 and HDM group 3 allergens shared moderate identity and structural homology (rootmeansquaredeviation<1). One B cell cross reactive epitope among serine proteases was identified (29I, 30V, 31G, 32G, 34E, 36K, 37A, 38L, 39A and 54C) and two T cell epitopes. Conclusions: PR3 have structural homology and share cross reactive epitopes with HDM group 3 allergens.

Exploration of structure-activity relationship between IgG1 and IgE binding ability and spatial conformation in ovomucoid with pulsed electric field treatment

Ovomucin (OVM) is one of the most main allergens in egg white, which limits the application of egg in the field of food processing. In this study, the effect of pulsed electric fields (PEF) on the IgG1 and IgE binding capability and the composition changes of OVM was studied by indirect competitive ELISA, SDS-PAGE, Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) spectroscopy, contact angle meter, fluorescence spectrophotometer and scanning electron microscopes (SEM). The results suggested that PEF treatment couldsignificantly reduce the IgG1 and IgE binding capacity of OVM, especially under the condition of 30 kV/cm. PEF treatment could cause partial unfolding of the protein, which was attributed to the destruction of the epitope structure that binds to IgG1 and IgE in OVM. Results indicated that PEF treatment significantly changed the primary structure, secondary structure and tertiary structure of the protein. Specifically, the secondary structure of the protein was partially unwound, and hydrophobic amino acids were exposed after PEF treatment. In addition, we observed that the microstructure of the OVM also changed significantly after PEF treatment.

Immuno-informatics approach for B-cell and T-cell epitope based peptide vaccine design against novel COVID-19 virus

COVID-19 has brought the world to a standstill with a wave of destruction in country after country with tremendous loss of lives and livelihood in advanced to developing nations. Whole world is staring at the prospect of repeated lockdowns with another wave of COVID-19 predicted to hit the world in September of 2020. The second wave is assumed to be even more destructive with severe impact across much of the world. The only way to defeat this pandemic is to quickly develop a safe and effective vaccine against this raging menace and initiate a global vaccination drive. Our study is an attempt to deploy various computational methods to identify B-cell and T-cell epitopes from the spike surface glycoprotein of SARS-COV-2 which have the novel potential for vaccine development against COVID-19. For this we have taken 8 unique strains with one each from India, China, France, USA, Italy, Australia, Iran and Pakistan. The strain data was extracted from NCBI Database. By analyzing the immune parameters like surface accessibility, antigenicity, variability, conservancy, flexibility, hydrophilicity, allergenicity and toxicity of the conserved sequences of spike glycoprotein using various databases and bioinformatics tools, we identified two potential novel linear (SGTNGTKRFDN and ASVYAWNRK) and one structural B-cell epitope as well as two T-cell epitopes (RLFRKSNLK and IPTNFTISV) which can be used as epitope-based peptide vaccines. Docking simulation assay revealed that above T-cell epitopes have minimum free binding energy and showed strong hydrogen bond interaction which strengthened its potential as being a T-cell epitope for the epitope-based novel vaccine against SARS-CoV-2. This study allows us to claim that B-cell and T-cell epitopes mentioned above provide potential pathways for developing an exploratory vaccine against spike surface glycoprotein of SARS-CoV-2 with high confidence for the identified strains. We will need to confirm our findings with biological assays.

Development of a structural epitope mimic: an idiotypic approach to HCV vaccine design

HCV vaccine development is stymied by the high genetic diversity of the virus and the variability of the envelope glycoproteins. One strategy to overcome this is to identify conserved, functionally important regions—such as the epitopes of broadly neutralizing antibodies (bNAbs)—and use these as a basis for structure-based vaccine design. Here, we report an anti-idiotype approach that has generated an antibody that mimics a highly conserved neutralizing epitope on HCV E2. Crucially, a mutagenesis screen was used to identify the antibody, designated B2.1 A, whose binding characteristics to the bNAb AP33 closely resemble those of the original antigen. Protein crystallography confirmed that B2.1 A is a structural mimic of the AP33 epitope. When used as an immunogen B2.1 A induced antibodies that recognized the same epitope and E2 residues as AP33 and most importantly protected against HCV challenge in a mouse model.

Effects of thermal processing on the allergenicity, structure, and critical epitope amino acids of crab tropomyosin.

Food processing can change the structure and immunoreactivity of purified allergens, but the effect of food processing on the immunoreactivity of the processed and purified allergen is still poorly understood. In this study, tropomyosin (TM) was obtained from Scylla paramamosain and purified after different treatments. A basophil activation test was employed to detect the allergenicity of allergens. The protein structure was detected by mass spectrometry, circular dichroism spectroscopy and surface hydrophobicity. Critical amino acids were identified by Dot blot. Heating obviously affects the biochemical characteristics of TM. The allergenicity of TM was decreased in high temperature-pressure-processed crabs, due to alteration in the protein structure (e.g. denaturation). Seven critical amino acids, namely, R21, E103, E104, E115, A116, E122 and E156, related to the maintenance of the IgE-binding activity of TM were identified. This research of thermal processing helps to accurately reduce or eliminate the immunoreactivity of crabs by food processing.

N-terminal domain mutations of the spike protein are structurally implicated in epitope recognition in emerging SARS-CoV-2 strains

During the past two years, the world has been ravaged by a global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Acquired mutations in the SARS-CoV-2 genome affecting virus infectivity and/or immunogenicity have led to a number of novel strains with higher transmissibility compared to the original Wuhan strain. Mutations in the receptor binding domain (RBD) of the SARS-CoV-2 spike protein have been extensively studied in this context. However, mutations and deletions within the N-terminal domain (NTD) located adjacent to the RBD are less studied. Many of these are found within certain β sheet-linking loops, which are surprisingly long in SARS-CoV-2 in comparison to SARS-CoV and other related β coronaviruses. Here, we perform a structural and epidemiological study of novel strains carrying mutations and deletions within these loops. We identify short and long-distance interactions that stabilize the NTD loops and form a critical epitope that is essential for the recognition by a wide variety of neutralizing antibodies from convalescent plasma. Among the different mutations/deletions found in these loops, Ala 67 and Asp 80 mutations as well as His 69/Val 70 and Tyr 144 deletions have been identified in different fast-spreading strains. Similarly, deletions in amino acids 241–243 and 246–252 have been found to affect the network of NTD loops in strains with high transmissibility. Our structural findings provide insight regarding the role of these mutations/deletions in altering the epitope structure and thus affecting the immunoreactivity of the NTD region of spike protein.

Current Biomarkers for Precision Medicine in Breast Cancer.

Breast cancer has become the prototypical solid tumor where targets have been identified within the tumor allowing for personalized approach for systemic therapy. Biomarkers are beginning to play an important role in preparing the way for precision treatment. Mandatory biomarkers for every newly diagnosed case of breast cancer are estrogen receptors and progesterone receptors in selecting patients for endocrine treatment and HER2 for identifying patients likely to benefit from antiHER2 therapy. Although methodological problems exist in the determination of Ki67, because of its clearly established clinical value, wide availability, and low costs relative to the available multianalyte signatures, Ki67 may be used for determining prognosis, especially if values are low or high. Also, the androgen receptor (AR) pathway is emerging as a potential therapeutic target in breast cancer. AR-targeted treatments for breast cancer are in development and have shown promising preliminary results. While, most established biomarkers in breast cancer require tissue samples, serum tumor markers are easily accessible and require a less invasive procedure. Among them, tissue polypeptide-specific antigen (TPS), a specific epitope structure of a peptide in serum associated with human cytokeratin 18, is linked to the proliferative activity of tumors. TPS may be a valuable and independent prognostic biomarker for breast cancer.In order to accelerate progress towards precision treatment for women with breast cancer, we need additional predictive biomarker, especially for enhancing the positive predictive value for endocrine and antiHER2 therapies, as well as biomarkers for predicting response to specific forms of chemotherapy.

In Ukrainian

The review material belongs to the scientific and practical issues related to the interdisciplinary direction on the border of nanotechnology, surface chemistry and physics, biology and medicine and is based on the use of natural components in the composition of iron-containing bioactive nanocomposites and magnetic fluids in creating effective vector systems for antitumor therapy with minimized side effects on the human body and improved compatibility with other drugs. Such natural components, which have unique properties, significant and not yet realized potential opportunities for practical use, include, in particular, lectins. The aim of the work is to select and analyze the results of works on the extraction of lectins, the study of their properties and application in biology and medicine. Lectins are a group of substances of protein nature (proteins and glycoproteins) of non-immune origin, which have the ability to reversely and selectively bind carbohydrates and carbohydrate determinants of biopolymers without changes in covalent structure and recognize them with extremely high specificity. Due to this property, they are an ideal tool for reading of code in the structure of specific sugar epitopes on the surface of all cells. Lectins are substances of primary synthesis and are present in all kingdoms, types and classes of living organisms. They mediate cellular communication at the molecular level and are involved in many physiological and pathophysiological processes. Pathogenic bacteria and viruses use lectins to attach to the host tissue, which is one of the prerequisites for the development of infection. Blocking of specific pathogen adhesion with lectin inhibitors is the basis of anti-adhesive therapy, an alternative method of treatment of infections caused by multidrug-resistant bacterial strains. Numerous lectins show antitumor activity and are being studied as potential antitumor drugs. To date, they have found practical application in a number of specialized medical fields, such as histology (detection of carbohydrate structures on the surface of cells and tissues), diagnosis of immunodeficiency and chromosomal abnormalities, transplantology (separation of blood cells and lymphoid cells with different antigenic properties). The prospect of use of lectins in the purification of blood from viruses, pathologically altered glycoproteins, in the targeted delivery of drugs to normal or pathologically altered cells and tissues of the body or to infectious agents is considered very significant. The combination of properties of lectins and magnetically sensitive iron-containing nanocomposites in the composition of magnetic fluids for use in oncology is considered relevant and promising.