SARS-CoV-2 Epitopes Are Recognized by a Public and Diverse Repertoire of Human T Cell Receptors.

Understanding the hallmarks of the immune response to SARS-CoV-2 is critical for fighting the COVID-19 pandemic. We assessed antibody and T cell reactivity in convalescent COVID-19 patients and healthy donors sampled both prior to and during the pandemic. Healthy donors examined during the pandemic exhibited increased numbers of SARS-CoV-2-specific T cells, but no humoral response. Their probable exposure to the virus resulted in either asymptomatic infection without antibody secretion or activation of preexisting immunity. In convalescent patients, we observed a public and diverse T cell response to SARS-CoV-2 epitopes, revealing T cell receptor (TCR) motifs with germline-encoded features. Bulk CD4+ and CD8+ T cell responses to the spike protein were mediated by groups of homologous TCRs, some of them shared across multiple donors. Overall, our results demonstrate that the T cell response to SARS-CoV-2, including the identified set of TCRs, can serve as a useful biomarker for surveying antiviral immunity.

Evaluating performance of existing computational models in predicting CD8+ T cell pathogenic epitopes and cancer neoantigens.

T cell recognition of a cognate peptide-major histocompatibility complex (pMHC) presented on the surface of infected or malignant cells is of the utmost importance for mediating robust and long-term immune responses. Accurate predictions of cognate pMHC targets for T cell receptors would greatly facilitate identification of vaccine targets for both pathogenic diseases and personalized cancer immunotherapies. Predicting immunogenic peptides therefore has been at the center of intensive research for the past decades but has proven challenging. Although numerous models have been proposed, performance of these models has not been systematically evaluated and their success rate in predicting epitopes in the context of human pathology has not been measured and compared. In this study, we evaluated the performance of several publicly available models, in identifying immunogenic CD8+ T cell targets in the context of pathogens and cancers. We found that for predicting immunogenic peptides from an emerging virus such as severe acute respiratory syndrome coronavirus 2, none of the models perform substantially better than random or offer considerable improvement beyond HLA ligand prediction. We also observed suboptimal performance for predicting cancer neoantigens. Through investigation of potential factors associated with ill performance of models, we highlight several data- and model-associated issues. In particular, we observed that cross-HLA variation in the distribution of immunogenic and non-immunogenic peptides in the training data of the models seems to substantially confound the predictions. We additionally compared key parameters associated with immunogenicity between pathogenic peptides and cancer neoantigens and observed evidence for differences in the thresholds of binding affinity and stability, which suggested the need to modulate different features in identifying immunogenic pathogen versus cancer peptides. Overall, we demonstrate that accurate and reliable predictions of immunogenic CD8+ T cell targets remain unsolved; thus, we hope our work will guide users and model developers regarding potential pitfalls and unsettled questions in existing immunogenicity predictors.

MHC-II alleles shape the CDR3 repertoires of conventional and regulatory naïve CD4+ T cells.

T cell maturation and activation depend upon T cell receptor (TCR) interactions with a wide variety of antigenic peptides displayed in a given major histocompatibility complex (MHC) context. Complementarity-determining region 3 (CDR3) is the most variable part of the TCRα and -ß chains, which govern interactions with peptide-MHC complexes. However, it remains unclear how the CDR3 landscape is shaped by individual MHC context during thymic selection of naïve T cells. We established two mouse strains carrying distinct allelic variants of H2-A and analyzed thymic and peripheral production and TCR repertoires of naïve conventional CD4+ T (Tconv) and naïve regulatory CD4+ T (Treg) cells. Compared with tuberculosis-resistant C57BL/6 (H2-Ab) mice, the tuberculosis-susceptible H2-Aj mice had fewer CD4+ T cells of both subsets in the thymus. In the periphery, this deficiency was only apparent for Tconv and was compensated for by peripheral reconstitution for Treg We show that H2-Aj favors selection of a narrower and more convergent repertoire with more hydrophobic and strongly interacting amino acid residues in the middle of CDR3α and CDR3ß, suggesting more stringent selection against a narrower peptide-MHC-II context. H2-Aj and H2-Ab mice have prominent reciprocal differences in CDR3α and CDR3ß features, probably reflecting distinct modes of TCR fitting to MHC-II variants. These data reveal the mechanics and extent of how MHC-II shapes the naïve CD4+ T cell CDR3 landscape, which essentially defines adaptive response to infections and self-antigens.

Comprehensive analysis of structural and sequencing data reveals almost unconstrained chain pairing in TCRαß complex.

Antigen recognition by T-cells is guided by the T-cell receptor (TCR) heterodimer formed by α and ß chains. A huge diversity of TCR sequences should be maintained by the immune system in order to be able to mount an effective response towards foreign pathogens, so, due to cooperative binding of α and ß chains to the pathogen, any constraints on chain pairing can have a profound effect on immune repertoire structure, diversity and antigen specificity. By integrating available structural data and paired chain sequencing results we were able to show that there are almost no constraints on pairing in TCRαß complexes, allowing naive T-cell repertoire to reach the highest possible diversity. Additional analysis reveals that the specific choice of contacting amino acids can still have a profound effect on complex conformation. Moreover, antigen-driven selection can distort the uniform landscape of chain pairing, while small, yet significant, differences in the pairing can be attributed to various specialized T-cell subsets such as MAIT and iNKT T-cells, as well as other TCR sets specific to certain antigens.

Biological Evaluation of a New Brassinosteroid: Antiproliferative Effects and Targeting Estrogen Receptor α Pathways.

Brassinosteroids (BS), a class of plant-specific steroid hormones, are considered as new potential anticancer agents for the treatment of tumors of different origin, including hormone-dependent cancers. Effects of a synthetic brassinosteroid BS4 ((22R,23R,24R)-22,23-dihydroxy-24-methyl-B-homo-7-oxa-5α-cholest-2-en-6-one ((3aS,7aR,7bS,9aS,10R,12aS,12bS)-10-[(2S,3R,4R,5R)-3,4-dihydroxy-5,6-dimethylheptan-2-yl]-7a,9a-dimethyl-1,3a,4,7,7a,7b,8,9,9a,10,11,12,12a,12b-tetradecahydro-3H-benzo[c]indeno[5,4-e]oxepin-3-one)) on hormone-dependent breast cancer cells and normal epithelial cells and its impact on the estrogen receptor signaling were evaluated. Cytotoxicity was assessed by MTT-test; expression of estrogen receptor α and survivin was measured by immunoblotting. Transactivation analysis of luciferase reporter gene was performed for ERα and AP-1 factors after the brassinosteroid treatment. Dock6 and Autodock Vina were used for molecular docking. BS4 revealed a significant antiproliferative effect towards the hormone-dependent breast cancer cells and was not active against normal epithelial cells. BS4 action on MCF-7 breast cancer cells was found to be complex: a decrease in ERα expression as well as in its transcription activity was accompanied by inhibition of ERα-related signaling pathways (AP-1 complex and survivin). BS4 binding mode to ERα ligand-binding domain was analyzed by molecular docking. The obtained results show that antiproliferative and antiestrogenic properties of the brassinosteroid BS4, as well as its ability to inhibit the anti-apoptotic protein survivin may be of interest for further development of anticancer agents.

Computer-aided design of aptamers for cytochrome p450.

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with Kd values in the range of 10(-6)-10(-7) M.

Large-scale template-based structural modeling of T-cell receptors with known antigen specificity reveals complementarity features.

Introduction: T-cell receptor (TCR) recognition of foreign peptides presented by the major histocompatibility complex (MHC) initiates the adaptive immune response against pathogens. While a large number of TCR sequences specific to different antigenic peptides are known to date, the structural data describing the conformation and contacting residues for TCR-peptide-MHC complexes is relatively limited. In the present study we aim to extend and analyze the set of available structures by performing highly accurate template-based modeling of these complexes using TCR sequences with known specificity. Methods: Identification of CDR3 sequences and their further clustering, based on available spatial structures, V- and J-genes of corresponding T-cell receptors, and epitopes, was performed using the VDJdb database. Modeling of the selected CDR3 loops was conducted using a stepwise introduction of single amino acid substitutions to the template PDB structures, followed by optimization of the TCR-peptide-MHC contacting interface using the Rosetta package applications. Statistical analysis and recursive feature elimination procedures were carried out on computed energy values and properties of contacting amino acid residues between CDR3 loops and peptides, using R. Results: Using the set of 29 complex templates (including a template with SARS-CoV-2 antigen) and 732 specificity records, we built a database of 1585 model structures carrying substitutions in either TCRα or TCRß chains with some models representing the result of different mutation pathways for the same final structure. This database allowed us to analyze features of amino acid contacts in TCR - peptide interfaces that govern antigen recognition preferences and interpret these interactions in terms of physicochemical properties of interacting residues. Conclusion: Our results provide a methodology for creating high-quality TCR-peptide-MHC models for antigens of interest that can be utilized to predict TCR specificity.

HLA variants have different preferences to present proteins with specific molecular functions which are complemented in frequent haplotypes.

Human leukocyte antigen (HLA) genes are the most polymorphic loci in the human genome and code for proteins that play a key role in guiding adaptive immune responses by presenting foreign and self peptides (ligands) to T cells. Each person carries up to 6 HLA class I variants (maternal and paternal copies of HLA-A, HLA-B and HLA-C genes) and also multiple HLA class II variants, which cumulatively define the landscape of peptides presented to T cells. Each HLA variant has its own repertoire of presented peptides with a certain sequence motif which is mainly defined by peptide anchor residues (typically the second and the last positions for HLA class I ligands) forming key interactions with the peptide-binding groove of HLA. In this study, we aimed to characterize HLA binding preferences in terms of molecular functions of presented proteins. To focus on the ligand presentation bias introduced specifically by HLA-peptide interaction we performed large-scale in silico predictions of binding of all peptides from human proteome for a wide range of HLA variants and established which functions are characteristic for proteins that are more or less preferentially presented by different HLA variants using statistical calculations and gene ontology (GO) analysis. We demonstrated marked distinctions between HLA variants in molecular functions of preferentially presented proteins (e.g. some HLA variants preferentially present membrane and receptor proteins, while others - ribosomal and DNA-binding proteins) and reduced presentation of extracellular matrix and collagen proteins by the majority of HLA variants. To explain these observations we demonstrated that HLA preferentially presents proteins enriched in amino acids which are required as anchor residues for the particular HLA variant. Our observations can be extrapolated to explain the protective effect of certain HLA alleles in infectious diseases, and we hypothesize that they can also explain susceptibility to certain autoimmune diseases and cancers. We demonstrate that these differences lead to differential presentation of HIV, influenza virus, SARS-CoV-1 and SARS-CoV-2 proteins by various HLA alleles. Taking into consideration that HLA alleles are inherited in haplotypes, we hypothesized that haplotypes composed of a combination of HLA variants with different presentation preferences should be more advantageous as they allow presenting a larger repertoire of peptides and avoiding holes in immunopeptidome. Indeed, we demonstrated that HLA-A/HLA-B and HLA-A/HLA-C haplotypes which have a high frequency in the human population are comprised of HLA variants that are more distinct in terms of functions of preferentially presented proteins than the control pairs.

The impact of long-distance mutations on the Ω-loop conformation in TEM type ß-lactamases.

ß-lactamases are hydrolytic enzymes primarily responsible for occurrence and abundance of bacteria resistant to ß-lactam antibiotics. TEM type ß-lactamases are formed by the parent enzyme TEM-1 and more than two hundred of its mutants. Positions for the known amino acid substitutions cover ∼30% of TEM type enzyme's sequence. These substitutions are divided into the key mutations that lead to changes in catalytic properties of ß-lactamases, and the secondary ones, which role is poorly understood. In this study, Residue Interaction Networks were constructed from molecular dynamic trajectories of ß-lactamase TEM-1 and its variants with two key substitutions, G238S and E240K, and their combinations with secondary ones (M182T and Q39K). Particular attention was paid to a detailed analysis of the interactions that affect conformation and mobility of the Ω-loop, representing a part of the ß-lactamase active site. It was shown that key mutations weakened the stability of contact inside the Ω-loop thus increasing its mobility. Combination of three amino acid substitutions, including the 182 residue, leads to the release of R65 promoting its new contacts with N175 and D176. As a result, Ω-loop is fixed on the protein globule. The second distal mutation Q39K prevents changes in spatial position of R65, which lead to the weakening of the effect of M182T substitution and the recovery of the Ω-loop mobility. Thus, the distal secondary mutations are directed for recovering the mobility of enzyme disturbed by the key mutations responsible for expansion of substrate specificity. AbbreviationsESBLextended spectrum beta-lactamasesIRinhibitor resistant beta-lactamasesMDmolecular dynamicsRINresidue interaction networksRMSDroot mean square deviationRMSFroot mean square fluctuations.Communicated by Ramaswamy H. Sarma.

The mechanism of a one-substrate transketolase reaction.

Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.

Memory of Divisional History Directs the Continuous Process of Primitive Hematopoietic Lineage Commitment.

Hematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.

Mutual influence of secondary and key drug-resistance mutations on catalytic properties and thermal stability of TEM-type ß-lactamases.

Highly mutable ß-lactamases are responsible for the ability of Gram-negative bacteria to resist ß-lactam antibiotics. Using site-directed mutagenesis technique, we have produced in vitro a number of recombinant analogs of naturally occurring TEM-type ß-lactamases, bearing the secondary substitution Q39K and key mutations related to the extended-spectrum (E104K, R164S) and inhibitor-resistant (M69V) ß-lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of ß-lactamases. However, in combination with the key mutations, this substitution resulted in decreased KM values towards hydrolysis of a chromogenic substrate, CENTA. The ability of enzymes to restore catalytic activity after exposure to elevated temperature has been examined. All double and triple mutants of ß-lactamase TEM-1 bearing the Q39K substitution showed lower thermal stability compared with the enzyme with Q39 intact. A sharp decrease in the stability was observed when Q39K was combined with E104K and M69V. The key R164S substitution demonstrated unusual ability to resist thermal inactivation. Computer analysis of the structure and molecular dynamics of ß-lactamase TEM-1 revealed a network of hydrogen bonds from the residues Q39 and K32, related to the N-terminal α-helix, towards the residues R244 and G236, located in the vicinity of the enzyme's catalytic site. Replacement of Q39 by lysine in combination with the key drug resistance mutations may be responsible for loss of protein thermal stability and elevated mobility of its secondary structure elements. This effect on the activity of ß-lactamases can be used as a new potential target for inhibiting the enzyme.

Impact of titanium dioxide nanoparticles on purification and contamination of nematic liquid crystals.

We have investigated the impact of titanium dioxide nanoparticles on the ionic contamination of liquid crystals. Nematic liquid crystals with high and low initial ionic contamination have been examined. It has been shown that titanium dioxide nanoparticles reduced the ion density of liquid crystals with high initial ionic contamination from 134.5 × 1012 cm-3 to 63.2 × 1012 cm-3. In the case of liquid crystals with low initial ionic contamination, the nanoparticles led to an insignificant increase of ion density from 19.8 × 1012 cm-3 to 25.7 × 1012 cm-3.

Stability of the PHF10 subunit of PBAF signature module is regulated by phosphorylation: role of ß-TrCP.

The PBAF chromatin-remodeling complexes are multi-protein machines, regulating expression of genes involved in proliferation and differentiation. PHF10 is a subunit of the PBAF essential for its association with chromatin. Mammalian PHF10 is expressed as four ubiquitous isoforms, which are alternatively incorporated in the complex and differ by their influence on transcription of target genes. PHF10 have different domain structure and two of them (PHF10-S isoforms) lack C-terminal PHD domains, which enables their phosphorylation by CK-1. Here we have found that PBAF subunits have low turnover rate, except for PHF10 which has much lower half-life, and is degraded by ß-TrCP. The ß-TrCP knockdown stabilizes PBAF core subunits - BRG1 and BAF155 and specific subunits - PHF10, BAF200, BAF180 and BRD7. PHF10 isoforms contain two non-canonical ß-TrCP degrons and are degraded by ß-TrCP in a phospho-dependent manner. But phosphorylation of PHF10-S degrons by CK-1, contrary to previously described degrons, prevents their degradation. Targeted molecular docking demonstrated that phosphorylated forms of PHF10 bind to ß-TrCP with much lower affinity than non-phosphorylated ones, contrary to previously described degrons. This unorthodox mechanism proposes that phosphorylation of ß-TrCP degrons by CK-1 could not only degrade a set of proteins, but also stabilize a different set of targets.

Chlamydial Type III Secretion System Needle Protein Induces Protective Immunity against Chlamydia muridarum Intravaginal Infection.

Chlamydia trachomatis imposes serious health problems and causes infertility. Because of asymptomatic onset, it often escapes antibiotic treatment. Therefore, vaccines offer a better option for the prevention of unwanted inflammatory sequelae. The existence of serologically distinct serovars of C. trachomatis suggests that a vaccine will need to provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of structural and effector proteins which is an essential virulence factor. In this study, we expressed the T3SS needle protein of Chlamydia muridarum, TC_0037, an ortholog of C. trachomatis CdsF, in a replication-defective adenoviral vector (AdTC_0037) and evaluated its protective efficacy in an intravaginal Chlamydia muridarum model. For better immune responses, we employed a heterologous prime-boost immunization protocol in which mice were intranasally primed with AdTC_0037 and subcutaneously boosted with recombinant TC_0037 and Toll-like receptor 4 agonist monophosphoryl lipid A mixed in a squalene nanoscale emulsion. We found that immunization with TC_0037 antigen induced specific humoral and T cell responses, decreased Chlamydia loads in the genital tract, and abrogated pathology of upper genital organs. Together, our results suggest that TC_0037, a highly conserved chlamydial T3SS protein, is a good candidate for inclusion in a Chlamydia vaccine.