Antigenic Structure Research Articles

PHYLOGENETIC CHARACTERISTICS AND ANALYSIS OF THE ANTIGENIC EPITOPES OF RUSSIAN ROTAVIRUSES IN A COMPARATIVE ASPECT WITH VACCINE STRAINS

Abstract Accumulation of mutations in the amino acid sequence of immunologically significant regions of the outer capsid proteins in locally circulating rotaviruses may reduce the effectiveness of vaccine-generated protection. The aim of the work was to comparatively analyze Russian rotaviruses and strains of the Indian pentavalent vaccine approved for use in the Russian Federation in 2020. Methods. There were used 38 rotavirus-positive samples derived from children with acute intestinal infection identified in 2022-2023. cDNA fragments of the VP7 gene 877 bp long were sequenced by two strands using Nanofor 05 device. Phylogenetic analysis was performed using BEAST software package. The final sample included 161 VP7 gene sequences of RVA isolates from three Russian cities (Nizhny Novgorod, Moscow, Novosibirsk), other countries, and vaccine strains. Results. Based on the results of phylogenetic analysis, Russian rotaviruses were found to belong to 13 lineages and/or sublineages (G1-I-A, G1-II-C, G2-IVa-1, G2-IV-3, G3-I, G3-3-а, G3-3-е, G4-I-с, G6-I, G8-IV, G9-III-d, G9-VI-е, G12-III). Vaccine strains (D, WI79-9, A41CB052A, DS-1, SC2-9, P, WI78-8, ST3, BrB-9, WI79-4, AU-32, 116E) were grouped separately in each case (G1-III, G1-II-A, G2-I, G2-II, G3-3-d, G4-I-а, G6-IV, G9-I, G9-II). Comparative analysis in the regions of antigenic epitopes targeted by neutralizing antibodies showed 3 to 6 amino acid differences between Russian and homotypic vaccine strains. The highest number was observed in isolates of sublineages G1-I-A, G2-IVa-1 and lineage G3-I. In the regions of T-cell epitopes, 1 to 4 substitutions were found. The greatest number of differences had rotaviruses of the G3-I lineage and the G4-I-c sublineage. Conclusions. For the G3P[8] variant of the G3-I lineage, which is widespread in Russia, 6 substitutions in neutralizing epitopes and 4 substitutions in T-cell epitopes were found in comparison with homotypic vaccine strains. The study results are important for understanding a potential impact of vaccines on the antigenic structure of the rotavirus population in Russia.

Neutralising and Non-neutralising Antibodies to SARS-CoV-2: Role during Infection and in the Evolution of Antigenic structure

Relevance. COVID-19, caused by the SARS-CoV-2 virus, remains a global public health threat despite the end of the pandemic. In the four years since the onset of the pandemic, the SARS-CoV-2 genome has undergone significant changes, particularly in the gene encoding the spike (S) protein. These changes resulted from the accumulation of immune responses in the human population, allowing the virus to evade the immune response. A significant proportion of the population was infected early in the pandemic or vaccinated with vaccines based on earlier variants of the virus. The emergence of new mutant variants raises concerns about the potential for severe COVID-19 in previously infected or vaccinated individuals.Аim. To examine the specifics of antibody formation, as well as the spectrum and functional activity of these antibodies in patients with COVID-19.Conclusions. Antibodies produced in response to infection or vaccination show diversity in spectrum and functional activity. Changes in the viral genome may reduce antibody effectiveness, highlighting the importance of monitoring new SARS-CoV-2 variants and developing adapted vaccines. These data will be key in shaping COVID-19 vaccination and treatment strategies in a changing epidemiological situation.

Monoclonal antibody development and antigenic epitope identification of chicken pro-IL-1β.

Pro-IL-1β is an important inflammatory factor and is also a biomarker for detecting early pro-inflammatory immune responses. However, commercially available antibodies against chicken inflammatory factors are lacking, which prohibits an in-depth exploration of the mechanism of chicken inflammation. This study cloned and expressed chicken pro-IL-1β, and developed a hybridoma cell line 1E12 capable of stably secreting chicken pro-IL-1β monoclonal antibody (mAb). The secreted mAb 1E12 can recognize exogenous or endogenous chicken pro-IL-1β by Western blot and IFA techniques, and for the first time, a novel antigen epitope 13SSLSEETFY21 of chicken pro-IL-1β recognized by this mAb was identified. This study not only provides an important tool for the detection and research of chicken pro-IL-1β, but also has significant implications for understanding the antigenic structure and biochemical characteristics of chicken pro-IL-1β.

Expression and characterization of canine distemper virus hemagglutinin protein in suspension mammalian cells.

Hundreds of millions of the domestic dogs worldwide are routinely inoculated with the modified live vaccines for canine distemper virus (CDV) every year. However, the corresponding serological diagnostic and detections are always lacking, thus, there is an urgent demand to establish its unique diagnostic technologies to produce high-quality antigenic biomolecules. In the present study, the ectodomain (et) of CDV hemagglutinin (H) protein was firstly expressed in a soluble and secreted forms by an Expi293F transient transfection system based on its antigenic secondary structure analysis. The yields of CDV H(et) protein was 2.6 g/L with purity over 95 % in supernatant of Expi293F cells. The CDV H(et) protein elicited comparative antibodies levels to the CDV virions in rabbit by ELISA and neutralization test. The purified polyclonal antibodies of immunized with CDV H(et) protein recognized both wild-type and vaccine CDV strains. More importantly, the purified polyclonal antibodies of CDV H(et) protein revealed significantly higher viral neutralizing activity than those from CDV-3 virions, which highlighted that the critical role of CDV H protein to elicit viral specific and protective neutralizing antibodies. Taken together, the CDV H(et) protein produced in mammalian expression systems was high-quality and good immunogenicity, and would be with great potential to serve as a serological diagnostic antigen or a novel CDV subunit vaccine in future.

In-silico Studies of Some Novel Pyrazoline Derivatives Focusing on Melanoma, Antimicrobial and Anti-inflammatory Activity

Microbial infections often produce pain and chronic inflammation, leading cause of cancer. The compound possessing anti-cancer potency with wide spectrum anti-microbial and anti-inflammatory activity is not common. Study focuses on designing a series of novel pyrazoline derivatives active towards anti-neoplastic, anti-microbial and anti-inflammatory response. The pyrazoline derivatives were investigated against the structure of vanin-1: defining the link between metabolic disease, oxidative stress and inflammation and Crystal structure of antigen 43 from uropathogenic Escherichia coli UTI89. Furthermore, the antitumor activity of pyrazoline compounds was evaluated against the crystal structure of human melanoma-associated antigen B1 (MAGEB1). The reported data highlight the impact of chemical structure variations on biological activity. Specific structural modifications consistently altered activity across all tests. Introducing p-nitro and p-hydroxy groups into the aryl moiety of the pyrazoline analogs resulted in compounds with significant anti-inflammatory, antimicrobial, and anticancer properties. The enhanced activities are attributed to the presence of 4-NO₂, 4-OH, and 4-Cl groups in the phenyl ring at the 5-position of the pyrazoline ring. In certain cases, these compounds demonstrated activities equal to or exceeding those of standard drugs.

An appreciation of the seminal contributions of John Brooksby and Fred Brown on foot and mouth disease.

John Brooksby was an outstanding Scottish veterinary virologist who worked at the Pirbright Institute (Pirbright) for 40 years, including 16 as the institute's director. He devised quantitative methods for measuring neutralising antibodies and perfected a complement fixation test for the diagnosis, typing and strain differentiation of foot and mouth disease (FMD), especially when combined with neutralisation. He identified four of the seven types of FMD virus (FMDV) and many subtypes. Consequently, the institute was designated the World Reference Laboratory for FMD. As director, Brooksby also oversaw advances in the pathogenesis, epidemiology and aerobiology of FMD and other diseases. His advice on the prevention and control of FMD was widely sought by international organisations and individual countries. Fred Brown was an eminent English biochemist and molecular virologist. He joined the Biochemistry Department at Pirbright in 1955, became head of the department in 1964, and in 1980 became deputy director of the institute. Advances under his leadership included the use of aziridines as inactivating agents for vaccine production, purification of FMDV suitable for biochemical analyses, demonstration of the infectivity of isolated RNA, analysis of the genomic and antigenic structure of FMDV, solving of the atomic structure of FMDV and demonstration of the potential for synthetic peptide vaccines to protect animals against virus challenge.

Mechanistic insights into structure-based design of a Lyme disease subunit vaccine.

The quality of protective immunity plays a critical role in modulating vaccine efficacy, with native antigens often not able to trigger sufficiently strong immune responses for pathogen killing. This warrants creation of structure-based vaccine design, leveraging high-resolution antigen structures for mutagenesis to improve protein stability and efficient immunization strategies. Here, we investigated the mechanisms underlying structure-based vaccine design using CspZ-YA, a vaccine antigen from Borrelia burgdorferi, the bacteria causing Lyme disease (LD), the most common vector-borne disease in the Northern Hemisphere. Compared to wild-type CspZ-YA, we found CspZ-YAI183Y and CspZ-YAC187S required lower immunization frequency to protect mice from LD-associated manifestations and bacterial colonization. We observed indistinguishable human and mouse antigenicity between wild-type and mutant CspZ-YA proteins after native infection or active immunization. This supports our newly generated, high-resolution structures of CspZ-YAI183Y and CspZ-YAC187S, showing no altered surface epitopes after mutagenesis. However, CspZ-YAI183Y and CspZ-YAC187S favored the interactions between helices H and I, consistent with their elevated thermostability. Such findings are further strengthened by increasing ability of protective CspZ-YA monoclonal antibodies in binding to CspZ-YA at a physiological temperature (37°C). Overall, this study demonstrated enhanced intramolecular interactions improved long-term stability of antigens while maintaining protective epitopes, providing a mechanism for structure-based vaccine design. These findings can ultimately be extended to other vaccine antigens against newly emerging pathogens for the improvement of protective immunity.

Hydrolysates of mare's milk proteins. Immunochemical and physico-chemical characteristics

In recent years, the attention of specialists has been attracted by a significant increase in allergic diseases, especially among children. Enzymatic hydrolysis with the participation of peptidases is considered the most effective measure to reduce the allergenicity of milk proteins. The study of the features of protein proteolysis by various enzymes, aimed at establishing optimal process parameters, is the subject of research in the production of hydrolysates with specified properties: a certain peptide and amino acid profile, reduced antigenic properties. At the same time, for a correct preclinical assessment of the safety and potential effectiveness of specialized food products, it is not enough only to determine the molecular weight distribution of peptide fractions in the composition. A necessary additional and informative indicator of reducing the potential allergenicity of the hydrolysate is the degree of preservation of the antigenic properties of the initial protein (residual antigenicity), determined by immunochemical methods. The preparation and subsequent comprehensive assessment of enzymatic hydrolysates of mare's milk proteins is of considerable interest, since, according to the results of clinical studies, it was suggested that upon allergy to cow's milk proteins mare's milk can be considered hypoallergenic. The purpose of this work was to obtain enzymatic hydrolysates of mare's milk proteins using domestic enzymes and their further physico-chemical and immunochemical characteristics. The object of the study was mare's milk protein obtained from skimmed mare's milk powder as a protein substrate. Two samples of enzymes from various manufacturers of the Russian Federation were used in the work. The results of the conducted studies show that the peptide profiles of hydrolysates vary greatly depending on the enzyme used, however, the multiplicity of antigenicity reduction practically does not depend on the nature of the enzyme, and does not depend on an increase in the enzyme/substrate ratio. The peptide profile and the multiplicity of reducing the antigenicity of hydrolysates of mare's milk proteins are influenced by ultrafiltration treatment of the obtained hydrolysates, which allows reducing the content of antigenic structures in the final product by several orders of magnitude.

Vaccine-induced human monoclonal antibodies to PfRH5 show broadly neutralizing activity against P. falciparum clinical isolates

Vaccines to the Plasmodium falciparum reticulocyte binding-like protein homologue 5 (PfRH5) target the blood-stage of the parasite life cycle. PfRH5 has the potential to trigger the production of strain-transcendent antibodies and has proven its efficacy both in pre-clinical and early clinical studies. Vaccine-induced monoclonal antibodies (mAbs) to PfRH5 showed promising outcomes against cultured P. falciparum laboratory strains from distinct geographic areas. Here, we assessed the functional impact of vaccine-induced anti-PfRH5 mAbs on more genetically diverse P. falciparum clinical isolates. We used mAbs previously isolated from single B cells of UK adult PfRH5 vaccinees and used ex-vivo growth inhibition activity (GIA) assays to assess their efficacy against P. falciparum clinical isolates. Next-generation sequencing (NGS) was used to assess the breadth of genetic diversity in P. falciparum clinical isolates and to infer the genotype/phenotype relationship involved in antibody susceptibility. We showed a dose-dependent inhibition of clinical isolates with three main GIA groups: high, medium and low. Except for one isolate, our data show no significant differences in the mAb GIA profile between P. falciparum clinical isolates and the 3D7 reference strain, which harbors the vaccine allele. We also observed an additive relationship for mAb combinations, whereby the combination of GIA-low and GIA-medium antibodies resulted in increased GIA, having important implications for the contribution of specific clones within polyclonal IgG responses. While our NGS analysis showed the occurrence of novel mutations in the pfrh5 gene, these mutations were predicted to have little or no functional impact on the antigen structure or recognition by known mAbs. Our present findings complement earlier reports on the strain transcendent potential of anti-PfRH5 mAbs and constitute, to our knowledge, the first report on the susceptibility of P. falciparum clinical isolates from natural infections to vaccine-induced human mAbs to PfRH5.

Discovery, recognized antigenic structures, and evolution of cross-serotype broadly neutralizing antibodies from porcine B-cell repertoires against foot-and-mouth disease virus.

It is a great challenge to isolate the broadly neutralizing antibodies (bnAbs) against foot-and-mouth disease virus (FMDV) due to its existence as seven distinct serotypes without cross-protection. Here, by vaccination of pig with FMDV serotypes O and A whole virus antigens, we obtained 10 bnAbs against serotypes O, A and/or Asia1 by dissecting 216 common clonotypes of two serotypes O and A specific porcine B-cell receptor (BCR) gene repertoires containing total 12720 B cell clones, indicating the induction of cross-serotype bnAbs after sequential vaccination with serotypes O and A antigens. The majority of porcine bnAbs (9/10) were derived from terminally differentiated B cells of different clonal lineages, which convergently targeted the conserved "RGDL" motif on structural protein VP1 of FMDV by mimicking receptor recognition to inhibit viral attachment to cells. Cryo-EM complex structures revealed that the other bnAb pOA-2 specifically targets a novel inter-pentamer antigen structure surrounding the viral three-fold axis, with a highly conserved determinant at residue 68 on VP2. This unique binding pattern enabled cross-serotype neutralization by destabilizing the viral particle. The evolutionary analysis of pOA-2 demonstrated its origin from an intermediate B-cell, emphasizing the crucial role of somatic hypermutations (SHMs) in balancing the breadth and potency of neutralization. However, excessive SHMs may deviate from the trajectory of broad neutralization. This study provides a strategy to uncover bnAbs against highly mutable pathogens and the cross-serotype antigenic structures to explore broadly protective FMDV vaccine.

Generation and epitope mapping of novel neutralizing monoclonal antibodies against glycoprotein E2 of CSFV

Classical swine fever virus (CSFV) is a highly contagious and economically important pathogen threatening pig industry worldwide, the envelope glycoprotein E2 of CSFV is the dominant antigen inducing strong antiviral neutralizing immunity. In this study, 7 monoclonal antibodies (mAbs) with neutralizing potency were generated using E2 protein of CSFV Shimen strain (SM) expressed by eukaryotic cells. Their reactivity with 116 CSFV strains in cell cultures and E2 proteins of 10 subgenotypes in western blots showed different CSFV spectrums they recognized. Of them, three (HCL-001, HCL-005 and HCL-010) reacted with all CSFV subgenotypes, while HCL-014 and HCL-002 reacted with most CSFV strains, except for some variants in genotype 2.3. In contrast, mAb HCL-009 reacted only with a few subgenotype 1.1 strains including SM, field strains and some vaccine strains. Interestingly, mAb HCL-018 reacted only with SM and field subgenotype 1.1 strains, not with any vaccine strains. Further epitope mapping using chimeric and site-directed mutated E2 proteins showed that HCL-001, HCL-005 and HCL-010 recognized a conservative epitope motif 143SPT145,L147, and HCL-002 recognized a conformational epitope with key aa motifs of 95GDD97,157RX(D/E)K(R)XFXXR164. HCL-014 recognized a new conservative epitope with key aa motifs of 41D,58XNVVXRR64. HCL-009 and HCL-018 recognized the epitope with key aa motifs of 36D,40ND41,45KXI47 and 69LHXGXLLT76, respectively. Taken together, present study has provided not only new insights into the antigenic structure of E2 protein, but also key reagents for antigenic characterization of CSFV strains and development of antibody assay for evaluation of the vaccination efficacy.

Pertussis is still not a completely controlled infection

The lecture focuses on pertussis, an infection managed by specific preventive measures, which continues to threaten both child and maternal health, as well as overall healthcare system. Despite high vaccination rates, pertussis remains an “under-controlled” infection and a significant contributor to childhood morbidity and mortality. Moreover, in many countries, the incidence of pertussis is clearly rising among both children and adults, with a notable proportion of cases occurring in vaccinated individuals. This increase may be associated with changes in the antigenic structure of the pathogen, the limited duration of post-vaccination immunity, reduced vaccination coverage, and the use of more sensitive laboratory diagnostic methods. The lecture addresses modern aspects of the etiopathogenesis, epidemiology, clinical presentation, diagnosis, treatment, and prevention of pertussis. Given the rising incidence of this infection in Russia and several other countries in 2023, the information presented will be especially valuable for practitioners, particularly family doctors.

MVSF-AB: Accurate antibody-antigen binding affinity prediction via multi-view sequence feature learning.

Predicting the binding affinity between antigens and antibodies accurately is crucial for assessing therapeutic antibody effectiveness and enhancing antibody engineering and vaccine design. Traditional machine learning methods have been widely used for this purpose, relying on interfacial amino acids' structural information. Nevertheless, due to technological limitations and high costs of acquiring structural data, the structures of most antigens and antibodies are unknown, and sequence-based methods have gained attention. Existing sequence-based approaches designed for protein-protein affinity prediction exhibit a significant drop in performance when applied directly to antibody-antigen affinity prediction due to imbalanced training data and lacking design in the model framework specifically for antibody-antigen, hindering the learning of key features of antibodies and antigens. Therefore, we propose MVSF-AB, a Multi-View Sequence Feature learning for accurate Antibody-antigen Binding affinity prediction. MVSF-AB designs a multi-view method that fuses semantic features and residue features to fully utilize the sequence information of antibody-antigen and predicts the binding affinity. Experimental results demonstrate that MVSF-AB outperforms existing approaches in predicting unobserved natural antibody-antigen affinity and maintains its effectiveness when faced with mutant strains of antibodies. Datasets we used and source code are available on our public GitHub repository https://github.com/TAI-Medical-Lab/MVSF-AB.

Towards the accurate modelling of antibody-antigen complexes from sequence using machine learning and information-driven docking.

Antibody-antigen complex modelling is an important step in computational workflows for therapeutic antibody design. While experimentally determined structures of both antibody and the cognate antigen are often not available, recent advances in machine learning-driven protein modelling have enabled accurate prediction of both antibody and antigen structures. Here, we analyse the ability of protein-protein docking tools to use machine learning generated input structures for information-driven docking. In an information-driven scenario, we find that HADDOCK can generate accurate models of antibody-antigen complexes using an ensemble of antibody structures generated by machine learning tools and AlphaFold2 predicted antigen structures. Targeted docking using knowledge of the complementary determining regions on the antibody and some information about the targeted epitope allows the generation of high-quality models of the complex with reduced sampling, resulting in a computationally cheap protocol that outperforms the ZDOCK baseline. The source code of HADDOCK3 is freely available at github.com/haddocking/haddock3. The code to generate and analyse the data is available at github.com/haddocking/ai-antibodies. The full runs, including docking models from all modules of a workflow have been deposited in our lab collection (data.sbgrid.org/labs/32/1139) at the SBGRID data repository.

The Autonomous Fusion Activity of Human Cytomegalovirus Glycoprotein B Is Regulated by Its Carboxy-Terminal Domain.

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection.

Guiding Diffusion Models for Antibody Sequence and Structure Co-design with Developability Properties

Recent advances in deep generative methods have allowed antibody sequence and structure co-design. This study addresses the challenge of tailoring the highly variable complementarity-determining regions (CDRs) in antibodies to fulfill developability requirements. We introduce a guidance approach that integrates property information into the antibody design process using diffusion probabilistic models. This approach allows us to simultaneously design CDRs conditioned on antigen structures while considering critical properties like solubility and folding stability. Our property-guided diffusion model offers versatility by accommodating diverse property constraints, presenting a promising avenue for computational antibody design in therapeutic applications. Published by the American Physical Society 2024

EpiScan: accurate high-throughput mapping of antibody-specific epitopes using sequence information

The identification of antibody-specific epitopes on virus proteins is crucial for vaccine development and drug design. Nonetheless, traditional wet-lab approaches for the identification of epitopes are both costly and labor-intensive, underscoring the need for the development of efficient and cost-effective computational tools. Here, EpiScan, an attention-based deep learning framework for predicting antibody-specific epitopes, is presented. EpiScan adopts a multi-input and single-output strategy by designing independent blocks for different parts of antibodies, including variable heavy chain (VH), variable light chain (VL), complementary determining regions (CDRs), and framework regions (FRs). The block predictions are weighted and integrated for the prediction of potential epitopes. Using multiple experimental data samples, we show that EpiScan, which only uses antibody sequence information, can accurately map epitopes on specific antigen structures. The antibody-specific epitopes on the receptor binding domain (RBD) of SARS coronavirus 2 (SARS-CoV-2) were located by EpiScan, and the potentially valuable vaccine epitope was identified. EpiScan can expedite the epitope mapping process for high-throughput antibody sequencing data, supporting vaccine design and drug development. Availability: For the convenience of related wet-experimental researchers, the source code and web server of EpiScan are publicly available at https://github.com/gzBiomedical/EpiScan.

The Role of Metal Nanoparticles in the Pathogenesis of Stone Formation.

The process of stone formation in the human body remains incompletely understood, which requires clinical and laboratory studies and the formulation of a new endogenous, nanotechnological concept of the mechanism of origin and formation of crystallization centers. Previously, the mechanism of sialolithiasis was considered a congenital disease associated with the pathology of the ducts in the structure of the glands themselves. To date, such morphological changes of congenital nature can be considered from the position of the intrauterine formation of endogenous bacterial infections complicated by the migration of antigenic structures initiating the formation of crystallization centers. The present work is devoted to the study of the morphology and composition of stones obtained as a result of surgical interventions for sialolithiasis. Presumably, nanoparticles of metals and other chemical compounds can be structural components of crystallization centers or incorporated into the conditions of chronic endogenous inflammation and the composition of antigenic structures, in complexes with protein and bacterial components. X-ray microtomography, X-ray fluorescence analysis, scanning transmission electron microscopy and microanalysis, mass spectrometry, and Raman spectroscopy were used to study the pathogenesis of stone formation. Immunoglobulins (Igs) of classes A and G, as well as nanoparticles of metals Pb, Fe, Cr, and Mo, were found in the internal structure of the stones. The complex of antigenic structures was an ovoid calcified layered matrix of polyvid microbial biofilms, with the inclusion of metal nanoparticles and chemical elements, as well as immunoglobulins. The obtained results of clinical and laboratory studies allow us to broaden the view on the pathogenesis of stone formation and suggest that the occurrence of the calcification of antigenic structures may be associated with the formation of IgG4-associated disease.

Dissecting the Enterococcal Polysaccharide Antigen (EPA) structure to explore innate immune evasion and phage specificity

Streptococci, Lactococci and Enterococci all produce L-rhamnose-containing cell wall polysaccharides which define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The L-rhamnose containing Enterococcal Polysaccharide Antigen (EPA), produced by the opportunistic pathogen Enterococcus faecalis, plays a critical role in normal growth, division, biofilm formation, antimicrobial resistance, phage susceptibility, and innate immune evasion. Despite the critical role of this polymer in E. faecalis physiology and host-pathogen interactions, little information is available on its structure and biosynthesis. Here, using an NMR approach, we elucidate the structure of EPA and propose a model for biosynthesis. We report the structure of the EPA-peptidoglycan linkage unit and reveal an unprecedented complexity of the EPA rhamnose backbone and decoration subunits. Finally, we explore the impact of several EPA structural modifications on innate immune evasion and recognition by bacteriophages. This work represents a first step towards the functional characterisation of EPA and the rational design of therapeutic strategies against a group of important pathogens.

A comparison of antibody-antigen complex sequence-to-structure prediction methods and their systematic biases.

The ability to accurately predict antibody-antigen complex structures from their sequences could greatly advance our understanding of the immune system and would aid in the development of novel antibody therapeutics. There have been considerable recent advancements in predicting protein-protein interactions (PPIs) fueled by progress in machine learning (ML). To understand the current state of the field, we compare six representative methods for predicting antibody-antigen complexes from sequence, including two deep learning approaches trained to predict PPIs in general (AlphaFold-Multimer and RoseTTAFold), two composite methods that initially predict antibody and antigen structures separately and dock them (using antibody-mode ClusPro), local refinement in Rosetta (SnugDock) of globally docked poses from ClusPro, and a pipeline combining homology modeling with rigid-body docking informed by ML-based epitope and paratope prediction (AbAdapt). We find that AlphaFold-Multimer outperformed other methods, although the absolute performance leaves considerable room for improvement. AlphaFold-Multimer models of lower quality display significant structural biases at the level of tertiary motifs (TERMs) toward having fewer structural matches in non-antibody-containing structures from the Protein Data Bank (PDB). Specifically, better models exhibit more common PDB-like TERMs at the antibody-antigen interface than worse ones. Importantly, the clear relationship between performance and the commonness of interfacial TERMs suggests that the scarcity of interfacial geometry data in the structural database may currently limit the application of ML to the prediction of antibody-antigen interactions.