Anton Shmelev - John Innes Centre
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Anton Shmelev
John Innes Centre
Molecular Microbiology
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Papers by Anton Shmelev
Structural basis for recognition of two HLA-A2-restricted SARS-CoV-2 spike epitopes by public and private T cell receptors
Research Square (Research Square)
, Jul 31, 2021
T cells play a vital role in combatting SARS-CoV-2 and in forming long-term memory responses. Whe...
more
T cells play a vital role in combatting SARS-CoV-2 and in forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T cell receptors (TCRs) bound to their peptide-MHC targets is lacking. We determined structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures revealed the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of both TCRs to recognize natural variants of YLQ and RLQ but not homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.
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T cells can effectively respond to SARS-CoV-2 even without antibodies, as seen in agammaglobulinemia patients recovering from COVID-19.
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Transgenic HA-1-Specific CD8+ T-Lymphocytes Selectively Target Leukemic Cells
Cancers
A significant share of allogeneic hematopoietic stem cell transplantations (allo-HSCT) results in...
more
A significant share of allogeneic hematopoietic stem cell transplantations (allo-HSCT) results in the relapse of malignant disease. The T cell immune response to minor histocompatibility antigens (MiHAs) promotes a favorable graft-versus-leukemia response. The immunogenic MiHA HA-1 is a promising target for leukemia immunotherapy, as it is predominantly expressed in hematopoietic tissues and presented by the common HLA A*02:01 allele. Adoptive transfer of HA-1-specific modified CD8+ T cells could complement allo-HSCT from HA-1- donors to HA-1+ recipients. Using bioinformatic analysis and a reporter T cell line, we discovered 13 T cell receptors (TCRs) specific for HA-1. Their affinities were measured by the response of the TCR-transduced reporter cell lines to HA-1+ cells. The studied TCRs showed no cross-reactivity to the panel of donor peripheral mononuclear blood cells with 28 common HLA alleles. CD8+ T cells after endogenous TCR knock out and introduction of transgenic HA-1-spec...
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Proposed using adoptive transfer of HA-1 specific T cells as a targeted approach to eliminate residual leukemia while preserving healthy tissues.
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Structural basis for recognition of two HLA-A2-restricted SARS-CoV-2 spike epitopes by public and private T cell receptors
T cells play a vital role in combatting SARS-CoV-2 and in forming long-term memory responses. Whe...
more
T cells play a vital role in combatting SARS-CoV-2 and in forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T cell receptors (TCRs) bound to their peptide–MHC targets is lacking. We determined structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures revealed the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of both TCRs to recognize natural variants of YLQ and RLQ but not homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.
Suntio T, Shmelev A, Lund M, Makarow M. The sorting determinant guiding Hsp150 to the COPI-independent transport pathway in yeast. J Cell Sci. 1999 22:3889-98
Journal of Cell Science
, 1999
The sorting determinant guiding Hsp150 to the COPI-independent transport pathway in yeast
Journal of Cell Science
, 1999
The COPI coatomer is thought to be required in yeast directly for retrograde transport from the G...
more
The COPI coatomer is thought to be required in yeast directly for retrograde transport from the Golgi to the endoplasmic reticulum (ER), and directly or indirectly for ER-to-Golgi transport. Unexpectedly, the secretory glycoproteins Hsp150 and invertase have been found not to require COPI for ER exit. The features according to which cargo proteins are selected for the COPI-independent pathway are not known. The ER form of Hsp150 has three distinct domains: an N-terminal fragment of 54 amino acids (subunit I) is followed by 11 repeats of a 19 amino acid peptide plus a unique C-terminal fragment of 114 amino acids (subunit II). By fusing heterologous proteins to different Hsp150 domains and expressing them in sec21-1 and sec21-3 mutants with temperature-sensitive mutations in the γ-COPI subunit, we show here that the repeats of subunit II function as sorting determinants for COPI-independent ER exit. The C-terminal fragment of Hsp150 could be replaced by E. coli β-lactamase or rat ner...
SARS-CoV-2 Epitopes are Recognized by a Public and Diverse Repertoire of Human T-Cell Receptors
SSRN Electronic Journal
, 2020
Understanding the determinants of adaptive immune response to SARS-CoV-2 is critical for fighting...
more
Understanding the determinants of adaptive immune response to SARS-CoV-2 is critical for fighting the ongoing COVID-19 pandemic. Here we assayed both antibody and T-cell reactivity to SARS-CoV-2 antigens in COVID-19 convalescent patients and healthy donors sampled before and during the pandemic. Our results show that while anti-SARS-CoV-2 antibodies can distinguish convalescent patients from healthy donors, the magnitude of T-cell response was more pronounced in healthy donors sampled during COVID-19 pandemic than in donors sampled before the outbreak. This hints at the possibility that some individuals have encountered the virus but were protected by T-cell cross-reactivity observed. A public and diverse T-cell response was observed for two A*02-restricted SARS-CoV-2 epitopes, revealing a set of T-cell receptor motifs displaying germline-encoded features. Bulk CD4+ and CD8+ T-cell response to SARS-CoV-2 glycoprotein S is characterized by multiple groups of homologous T-cell receptor sequences some of which are shared across multiple donors, indicating the existence of immunodominant epitopes. Overall, our findings indicate that T cells form an efficient response to SARS-CoV-2 and alongside the antibodies can serve as a useful biomarker for surveying SARS-CoV-2 exposure and immunity. We hope that data, including the set of specific Tcell receptors identified in this study can serve as a basis for future developments of SARS-CoV-2 vaccinations and monitoring.
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Healthy donors exhibited S-protein specific T-cells similar to convalescent patients, indicating potential pre-exposure or cross-reactive immune responses.
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Transgenic lymphocytes targeting minor histocompatibility antigens for post-transplant immunotherapy
Cytotherapy
, 2020
Background & Aim The relapse of acute leukemia occurs in one third of the patients after HLA-...
more
Background & Aim The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Methods, Results & Conclusion Naive CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis. To prevent off-target effects caused by the mispairing of endogenous and recombinant TCR chains, the endogenous TCR knockout was made using gRNA/Cas9 ribonucleoprotein complexes. Transgenic alpha and beta-chain constant domains were modified to be resistant to Cas9 cleavage and to form more stable TCR. Antigen-specificity was measured by binding to MHC-tetramers loaded with the MiHA peptide and by TCR-mediated activation of the Jurkat 76 triple parameter reporter cell line upon antigen stimulation. Most of the generated cell lines were specific to the desired antigen, and none were activated in response to irrelevant peptides presented in the same HLA. After analysis of cross-reactivity to allogeneic HLA on the panel of Epstein-Barr virus-transformed lymphoblastoid cell lines, the best receptors would be selected for further therapeutic application. Adoptive transfer of MiHA-specific genetically modified T cells holds great promise for the treatment of leukemia relapse after allo-HSCT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156.
Development of T-Cell Therapy Targeting Hematopoietic Minor Histocompatibility Antigen HA-1
Blood
, 2019
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only curative the...
more
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only curative therapy for hematological malignancies yet in nearly one-third of patients, it is followed by a relapse of the disease contributing to high mortality. In fully HLA-matched allo-HSCT graft versus leukemia reaction is driven by the recognition of the minor histocompatibility antigens (MiHAs) - endogenous polymorphic peptides presented by MHC. Particularly, HA-1 MiHA is a promising target for immunotherapy. HA-1 is presented by frequent among Caucasians HLA allele - A*02:01. The single nucleotide variation in ARGHAP45 gene which generates the MiHA has the optimal allelic distribution, thus immunogenic mismatch occurs in 30% of allo-HSCT. Also, ARGHAP45 is overexpressed in certain types of leukemia. Here we aim to develop HA-1-specific T-cells for post-transplant relapse therapy. To obtain the sequences of HA-1-specific T-cell receptors (TCRs), naive CD8+ T-cells from 3 HLA-A*02:01 positive and...
Structural assessment of HLA-A2-restricted SARS-CoV-2 spike epitopes recognized by public and private T-cell receptors
Nature Communications
, 2022
T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Wherea...
more
T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T-cell receptors (TCRs) bound to their peptide–MHC targets is lacking. Here we determine the structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures reveal the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of the TCRs to recognize natural variants of RLQ but not YLQ. Neither TCR recognizes homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit p...
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Study limitations include potential unnatural pairing of TCRs, affecting affinity characteristics, which might question TCR specificity confidence.
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Folding and Selective Exit of Reporter Proteins from the Yeast Endoplasmic Reticulum
The present study analyses the traffic of Hsp150 fusion proteins through the endoplasmic reticulu...
more
The present study analyses the traffic of Hsp150 fusion proteins through the endoplasmic reticulum (ER) of yeast cells, from their post-translational translocation and folding to their exit from the ER via a selective COPI-independent pathway. The reporter proteins used in the present work are: Hsp150p, an O-glycosylated natural secretory protein of Saccharomyces cerevisiae, as well as fusion proteins consisting of a fragment of Hsp150 that facilitates in the yeast ER proper folding of heterologous proteins fused to it. It is thought that newly synthesized polypeptides are kept in an unfolded form by cytosolic chaperones to facilitate the post-translational translocation across the ER membrane. However, beta-lactamase, fused to the Hsp150 fragment, folds in the cytosol into bioactive conformation. Irreversible binding of benzylpenicillin locked beta-lactamase into a globular conformation, and prevented the translocation of the fusion protein. This indicates that under normal conditi...
[C-terminal domain of saccharomyces cerevisiae protein ChI4 binds to centromere DNA fragment of yeast chromosome III]
Tsitologiia
, 1999
We have expressed in Escherichia coli a recombinant protein consisting of N-terminal peptide omeg...
more
We have expressed in Escherichia coli a recombinant protein consisting of N-terminal peptide omega 10s10 (11 aa) fused with part (aa 135-458) of yeast protein Chl4 involved in the chromosome segregation in Saccharomyces cerevisiae. Mice were immunized with the antigen purified from inclusion bodies, and a polyclonal serum against yeast protein Chl4 was raised. MW of the detected yeast protein Chl4 was approximately 54 kDa, corresponding to the full length ORF translation. C-terminal portion of Chl4 (aa 376-458), containing Helin-Turn-Helix (HTH) motif of DNA-binding, was fused in frame after E. coli maltose binding protein MalE. The soluble fusion was affinity purified using an alternative procedure on the preswollen amylose column. This protein and a 32P labelled 620 bp fragment of yeast CEN3 DNA were used in the DNA-mobility shift assay in polyacrylamide and agarose gels. The binding was detected in the presence and absence of Zn2+ ions. The data obtained could support participati...
[Multiple drug resistance in yeasts and its effect on stability and karyotype of transformants]
Molekuliarnaia biologiia
Inhibition of Translocation of beta -Lactamase into the Yeast Endoplasmic Reticulum by Covalently Bound Benzylpenicillin
Journal of Biological Chemistry
, 2001
We found recently that beta-lactamase folds in the yeast cytosol to a native-like, catalytically ...
more
We found recently that beta-lactamase folds in the yeast cytosol to a native-like, catalytically active, and trypsin-resistant conformation, and is thereafter translocated into the ER and secreted to the medium. Previously, it was thought that pre-folded proteins cannot be translocated. Here we have studied in living yeast cells whether beta-lactamase, a tight globule in authentic form, must be unfolded for ER translocation. A beta-lactamase mutant (E166A) binds irreversibly benzylpenicillin via Ser(70) in the active site. We fused E166A to the C terminus of a yeast-derived polypeptide having a post-translational signal peptide. In the presence of benzylpenicillin, the E166A fusion protein was not translocated into the endoplasmic reticulum, whereas translocation of the unmutated variant was not affected. The benzylpenicillin-bound protein adhered to the endoplasmic reticulum membrane, where it prevented translocation of BiP, carboxypeptidase Y, and secretory proteins. Although the 321-amino acid-long N-terminal fusion partner adopts no regular secondary structure and should have no constraints for pore penetration, the benzylpenicillin-bound protein remained fully exposed to the cytosol, maintaining its signal peptide. Our data suggest that the beta-lactamase portion must unfold for translocation, that the unfolding machinery is cytosolic, and that unfolding of the remote C-terminal beta-lactamase is required for initiation of pore penetration.
Production of folate by bacteria isolated from oat bran
by
Matti Korhola
and
Anton Shmelev
International Journal of Food Microbiology
, 2010
Twenty bacteria isolated from three commercial oat bran products were tested for their folate pro...
more
Twenty bacteria isolated from three commercial oat bran products were tested for their folate production capability. The bacteria as well as some reference organisms were grown until early stationary phase on a rich medium (YPD), and the amount of total folate in the separated cell mass and the culture medium (supernatant) was determined by microbiological assay. Folate vitamer distribution was determined for eight bacteria including one isolated from rye flakes. For seven bacteria the effect of temperature and pH on folate production was studied in more detail. Relatively large amount of folate was both produced in the cell biomass (up to 20.8 μg/g) and released to the culture medium (up to 0.38 μg/g) by studied bacteria. The best producers were characterized as Bacillus subtilis ON4, Chryseobacterium sp. NR7, Janthinobacterium sp. RB4, Pantoea agglomerans ON2, and Pseudomonas sp ON8. The level of folate released in culture medium was the highest for B. subtilis ON5, Chryseobacterium sp. NR7, Curtobacterium sp. ON7, Enterococcus durans ON9, Janthinobacterium sp. RB4, Paenibacillus sp. ON10, Propionibacterium sp. RB9, and Staphylococcus kloosii RB7. Marked differences in the distribution of folate vitamers among the bacterial strains were revealed by the HPLC analysis. The main vitamers were tetrahydrofolate, 5,10-methenyltetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate. Increase in the folate content during bacterial growth was accompanied by proportional increase in the 5-methyltetrahydrofolate content and decrease of 5-formyltetrahydrofolate. 10-Formylfolic acid dominated in the culture media of four bacteria, and Janthinobacterium sp. RB4 was also found to excrete 5-methyltetrahydrofolate. Intracellular folate content was higher when the bacteria were grown at 28°C than at 18°C or 37°C and also higher at pH 7 than at pH 5.5.
Production of folate by bacteria isolated from oat bran
by
Anton Shmelev
and
Matti Korhola
International Journal of Food Microbiology
, 2010
Twenty bacteria isolated from three commercial oat bran products were tested for their folate pro...
more
Twenty bacteria isolated from three commercial oat bran products were tested for their folate production capability. The bacteria as well as some reference organisms were grown until early stationary phase on a rich medium (YPD), and the amount of total folate in the separated cell mass and the culture medium (supernatant) was determined by microbiological assay. Folate vitamer distribution was determined for eight bacteria including one isolated from rye flakes. For seven bacteria the effect of temperature and pH on folate production was studied in more detail. Relatively large amount of folate was both produced in the cell biomass (up to 20.8 μg/g) and released to the culture medium (up to 0.38 μg/g) by studied bacteria. The best producers were characterized as Bacillus subtilis ON4, Chryseobacterium sp. NR7, Janthinobacterium sp. RB4, Pantoea agglomerans ON2, and Pseudomonas sp ON8. The level of folate released in culture medium was the highest for B. subtilis ON5, Chryseobacterium sp. NR7, Curtobacterium sp. ON7, Enterococcus durans ON9, Janthinobacterium sp. RB4, Paenibacillus sp. ON10, Propionibacterium sp. RB9, and Staphylococcus kloosii RB7. Marked differences in the distribution of folate vitamers among the bacterial strains were revealed by the HPLC analysis. The main vitamers were tetrahydrofolate, 5,10-methenyltetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate. Increase in the folate content during bacterial growth was accompanied by proportional increase in the 5-methyltetrahydrofolate content and decrease of 5-formyltetrahydrofolate. 10-Formylfolic acid dominated in the culture media of four bacteria, and Janthinobacterium sp. RB4 was also found to excrete 5-methyltetrahydrofolate. Intracellular folate content was higher when the bacteria were grown at 28°C than at 18°C or 37°C and also higher at pH 7 than at pH 5.5.
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