E. coli. OBP  was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in  Caenorhabditis elegans to mammals [E. coli [O-GlcNAcylation is a highly dynamic and reversible PTM in the serine/threonine residues of nuclear, cytosolic, and mitochondrial proteins ,2. Only  mammals . Unlike [E. coli .Although more than 4000 proteins (including many disease-related proteins) have been found to be O-GlcNAcylated, the function of O-GlcNAcylation in most proteins has not been clarified ,18. It iE. coli and the chemical synthesis of modified peptide. The chemical synthesis of O-GlcNAc peptide/protein, selectively adding GlcNAc to specific sites of target peptides/proteins using solid phase or chemoenzymatic synthesis, is widely used to generate a specific antigen and desired antibody by immunizing animals [At present, the synthesis methods of O-GlcNAc-modified peptide/protein mainly include OGT co-expression modified protein in  animals , such as animals , histone animals , Tau -tagged strategy was developed to increase the O-GlcNAc level of a protein through the co-expression of OGT and tagged protein. This strategy successfully improved the O-GlcNAc level of Tau, H2B, and c-Myc proteins, which was far more efficient than control proteins without P1 and could conveniently and efficiently produce milligram-scale quantities of O-GlcNAc protein with a high O-GlcNAc level. Compared with Tau, O-GlcNAc of P1Tau had obvious interference with P1Tau aggregation in vitro. This strategy provides convenience for further preparation of a large number of O-GlcNAc-modified proteins and functional research.E. coli  or C-terminal (TauP1) of Tau, respectively. The protein expression level of P1Tau (or TauP1) and O-GlcNAc level of P1Tau (or TauP1) were detected in the lysis of ference. E,F.The O-GlcNAc Tau protein was purified under different induction temperatures. It was found that P1Tau and TauP1 were all expressed well under 16 °C, 25 °C, or 37 °C . Then, tm/z 126.06, 138.06, 144.07, 168.07, 186.08, and 204.09) generated upon HCD were characteristic of N-acetylglucosamine (GlcNAc). GlcNAc oxonium ion provided evidence of an O-GlcNAc-modified peptide. Based on the mass difference of 203.08 Da in the HCD/ETD spectra, we identified nineteen O-GlcNAcylated sites on Tau, seven sites on P1Tau, and eight sites on TauP1. The locations of these O-GlcNAc sites in the Tau, P1Tau, and TauP1 proteins are shown in Tau, P1Tau, and TauP1 were purified using Ni-NTA, and the O-GlcNAc sites were detected using HCD/ETD. The array of low-mass oxocarbenium ions  without OGT co-expression, Tau, and P1Tau protein with OGT co-expression were purified using SP cation exchange chromatography for aggregation experiment A. AnotheThe absorbance at 550 nm represents the amount of Tau fibrils to monitor the kinetic effect on the formation of Tau protein amyloid fibers induced by Congo red. Tau (no OGT) was used as a positive control and HEPES buffer as a negative control. The empirical Hill equation was fitted to the kinetic data in The heparin induced Tau protein aggregation, which was monitored through fluorescence spectroscopy using Thioflavin T (ThT) . Tau proOverall, these results confirmed that increasing the O-GlcNAc level of P1Tau significantly slowed the formation of amyloid fibrils during Tau protein aggregation.E. coli were detected using anti-His antibody and RL2 antibody, respectively  and the O-GlcNAcylation of tagged Myc in the lysis of ectively A. Comparectively B. Similaectively C. Comparectively D. P1 shoE. coli could produce O-GlcNAc recombinant proteins using the dual-plasmid system [In this paper, the OBP-tagged strategy was used to successfully improve the O-GlcNAc level of Tau, H2B, and c-Myc proteins. It has been reported that the co-expression of OGT with its target substrates in d system ,31,32. HE. coli. Therefore, the effectiveness of this tag-bound nanobody in producing O-GlcNAc-modified protein is worthy of further study and verification in E. coli. Previous studies have implied that the asparagines in the N-terminal superhelical tetratricopeptide repeat (TPR) lumen proximal to the catalytic domain of OGT play a critical role in the recognition of most substrates [Although P1 tag is the substrate of OGT, the O-GlcNAc modification of P1 depends on the fusion proteins. The Ser on P1 in P1p53 was O-GlcNAcylation, but not in P1Tau. These data indicated that the function of the P1 tag is to promote the binding of OGT and the substrate protein, rather than compete for O-GlcNAc sites of the substrate. The OBP-tagged strategy with a high affinity of OGT is a successful approach. We will attempt to find more peptides with higher affinity for OGT to further optimize this OBP-tagged strategy. Ramirez et al. reported that OGT fused with tag-bound nanobody could enhance the O-GlcNAc level of the target protein in HEK293T cells , but thibstrates ,41,42. Hbstrates . The amiThe results of Tau and tagged Tau O-GlcNAc sites showed that this strategy not only improved the O-GlcNAc modification level, but also reduced the diversity and microheterogeneity of O-GlcNAc sites, which may be more similar to the intracellular modification status in the microenvironment. S400 is an important O-GlcNAcylated site in the Tau protein that plays a major role in regulating Tau aggregation because the S400A mutation in recombinant O-GlcNAcylated Tau eliminates the inhibition of wild-type O-GlcNAcylation on Tau aggregation in vitro ,35. The Many O-GlcNAc-modified proteins have been identified with multiple O-GlcNAc sites, some of which have important functions ,37. MutaIn conclusion, we report an in vitro method for enhancing protein O-GlcNAcylation using the OBP-tagged strategy, which provides highly modified and homogeneous samples for studying the biological functions of protein O-GlcNAcylation.2 was inserted between P1 and Tau. Using the Tau gene as the template, a pair of primers, P1Tau-Fa/Tau-R (P1Tau-Fa: CTC GTG CGG GCG GCG GCG GCA GCG GCG GCG GCG GCA GCG CTG AGC CCC GCC AGG AG and Tau-R: GGT GGT GGT GGT GCT CGA GCT GCA GCA AAC CCT GCT TGG CCA G), were designed for amplification. Then, using the above PCR product as a template, P1Tau-Fb/Tau-R (P1Tau-Fb: AAG GAG ATA TAC CAT GGT CCA TAT GAA AAA GGT GCC GGT CTC TCG TGC GGG CGG CGG CG and Tau-R: GGT GGT GGT GGT GCT CGA GCT GCA GCA AAC CCT GCT TGG CCA G) were designed for the second amplification. This second PCR product was inserted into the pET-4CDS vector between Nde1 and Pst1. A pair of PCR primers, OGT-F/R (OGT-F: GCG GAT GGG ATC CGA ATT CGT CGA CAT GAA AAT CGA AGA AGG TAA AC and OGT-R: TGC CCA CGG AAG ACG CCA TGT CGA CGA TAT CGC GGC CGC CCA TGG), were designed to amplify the OGT gene. The PCR product was inserted into pET-4CDS at the Sal1 site (restriction enzymes were purchased from Takara). The pET-4CDS was given by Dr. Yanting Su. Amplification of the genes was carried out using high-fidelity polymerase enzyme 2×Phanta Flash Master Mix (Vazyme). 2×MultiF Seamless Assembly Mix  was used to link genes with vectors. The final constructs were verified through DNA sequence analysis.The OGT target proteins used in this study were human Tau441, H2B, and c-Myc. Taking the P1Tau fusion gene as an example, we constructed a fusion gene of P1 and Tau. The flexible and non-helical linker (GSSSS)E. coli BL21 (DE3) prepared in our laboratory. The bacterial strains were grown in LB containing kanamycin (50 μg/mL) at 37 °C until OD 600 reached 0.6~0.8. Then, the culture was induced with 1 mM IPTG and further incubated at 25 °C for 12 h. The bacterial solution after induction was centrifuged at 3500 rpm for 25 min, and the supernatant was discarded.The above plasmids were transformed into The remaining sediment was resuspended in binding buffer  and broken ultrasonically. After centrifugation , the supernatant was purified as recombinant protein using a Ni-NTA affinity column (TransGen Biotech). The target proteins adsorbed on the Ni-NTA affinity column were eluted with an elution buffer , and the protein sample was added to 5× loading buffer, boiled at 100 °C for 10 min, and separated in SDS-PAGE for LC-MS/MS analysis.2HPO4·12H2O, 20 mM NaH2PO4·2H2O, 1 mM EDTA, 0.5% β-Mercaptoethanol, 1 mM PMSF, pH 6.8) and were then broken by sonication on ice. The supernatants were filtered, loaded onto a cation exchanger HiTrap SP FF , and washed with SP buffer. A linear gradient of salt (0–400 mM NaCl) in the same buffer as used to elute the Tau protein. BCA Protein Assay (Yeasen) and Coomassie brilliant blue staining of 10% SDS-PAGE were used to estimate protein concentration and purity, respectively. The purified proteins were dialyzed three times with 10 mM HEPES buffer , concentrated with a 10K Amicon centrifugal filter (Millipore) for kinetic analysis, and transferred to −80 °C for long-term storage.The above bacteria were harvested and resuspended in SP buffer  is the absorbance at 550 nm in the long time limit, t50 is the elapsed time at which A is equal to one-half of A(∞), and n is a cooperativity parameter.Congo red, widely employed as an inducer for Tau aggregation in vitro, can react specifically with β-sheet-rich amyloid fibrils, and the bound form displays a characteristic red shift in its maximum absorbance from 490 to 550 nm ,48. A frF is the fluorescence intensity, k the rate constant for the growth of fibrils, and tm is the time to 50% of maximal fluorescence. F0 describes the initial baseline during the lag time. A + ct describes the final baseline after the growth phase has ended. The lag time is determined to be tm − 2/k.The aggregation of Tau protein normally occurs through the nucleation-dependent fibril polymerization in three phases: activation and nucleation (Lag phase), elongation (growth phase), and steady phase  . A smallThe target proteins were fractionated with SDS-PAGE  and visualized by staining with Coomassie blue. For Western blot analysis, the proteins were transferred to PVDF membrane , and blocked with 5% non-fat milk  for 1 h at room temperature or overnight at 4 °C, after separating on SDS-PAGE. Target proteins Tau, H2B, and Myc were identified by using anti-His antibody conjugated to HRP (1:10000) . Likewise, the OGT was detected by using mouse anti-Flag (1:1000)  as the primary antibody and goat anti-mouse antibody conjugated to HRP  as the secondary antibody. The O-GlcNAcylation of the target was identified by using anti-O-GlcNAc antibody RL2 , CTD110.6  and anti-H2B S112GlcNAc (Abcam). Super ECL Detection Reagent  was used for developing and capturing proteins. Blots were visualized using an automatic chemiluminescence image analysis system (Tanon 5200). The expression of proteins was analyzed using ImageJ and Graphpad prism 7.m/z 204.09) and a series of HexNAc fragments , which are significant to the diagnosis of O-GlcNAcylated peptides. The samples were analyzed using Fourier transform ion cyclotron resonance . The FT-ICR/Orbitrap mass spectrometer was equipped with an online nano-electrospray ionization (ESI) source. The database search results were imported into Peaks Studio 10.6. Search parameters included a precursor tolerance of 10 ppm and a fragment tolerance of 0.02 Da. Enzyme specificity was set to trypsin, and up to two missed cleavage sites were allowed. Carbamidomethylation was set as a fixed modification. For the main GlcNAc search, HexNAc (S/T) was set as a variable modification. GlcNAc was considered positively localized to serine or threonine residues with FDR less than 1%. Putative O-GlcNAcylated peptides were manually confirmed by checking for the presence of the expected HexNAc neutral loss from the precursor ion and/or fragmented HexNAc oxonium ions. SDS-PAGE-separated O-GlcNAcylated proteins Tau, P1Tau, and TauP1 were reduced using 10 mM dithiothreitol and alkylated in 55 mM iodoacetamide  prior to in-gel proteolytic digestion with trypsin  for 12 h. The digested peptides were extracted from the gel pieces by sequentially adding 0.1% formic acid in 50% acetonitrile, 0.1% formic acid in 80% acetonitrile, and 100% acetonitrile. The extraction products were combined and lyophilized for the following step. The MS approach employed in this study for sequencing O-GlcNAcylated peptides and assigning the modification sites is a combination of HCD and ETD fragmentation. When analyzing O-GlcNAcylated peptides, the unique advantage of HCD fragmentation is the generation of distinct HexNAc oxonium ions (


A relapse of the malignant disease frequently occurs after allogeneic hematopoietic stem cell transplantation. Immune recognition of minor histocompatibility antigens, the polymorphic peptides that differ between donor and recipient, often triggers a beneficial graft-versus-leukemia response. The transgenic donor-derived cytotoxic T cells, which recognize patient-specific minor histocompatibility antigens presented by hematopoietic cells, allow precise elimination of malignant recipient cells while sparing both donor and non-hematopoietic patient cells. We generated the MiHA-specific T cells by gene editing to knock out the endogenous T cell receptor, followed by lentiviral transduction of HA-1-specific T cell receptors. Modified T cells demonstrated cytotoxicity against leukemia cells from HA-1+ donors with acute myeloid leukemia, acute T-cell, and B-cell lymphoblastic leukemia. Transgenic T cells showed no cytotoxicity against donor cells lacking HA-1 surface presentation. The proposed therapeutic approach could be used after allogeneic hematopoietic stem cell transplantation to prevent and treat leukemia relapse.+ 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-specific TCR were able to lyse hematopoietic cells from HA-1+ patients with acute myeloid, T-, and B-cell lymphocytic leukemia (n = 15). No cytotoxic effect was observed on cells from HA-1- or HLA-A*02-negative donors (n = 10). The results support the use of HA-1 as a target for post-transplant T cell therapy.A significant share of allogeneic hematopoietic stem cell transplantations  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 Allogeneic hematopoietic stem cell transplantation  is widely used for the treatment of acute myeloid leukemia (AML) and lymphoblastic leukemia (ALL) ,2. The tDonor and recipient cells could be distinguished by their cell surface presentation of minor histocompatibility antigens (MiHAs), the peptides derived from proteins with polymorphic amino acids and presented by HLA molecules ,23. The + T cells could be modified by lentiviral transduction to express a high-avidity transgenic HA-1-specific T cell receptor (TCR) [The minor histocompatibility antigen HA-1 is a promising target for several reasons. First, it is presented by the HLA allele (HLA-A*02:01) common in the Caucasian population . Second,or (TCR) ,35.In this study, we report the development of HA-1-specific T cell immunotherapy. Using the Jurkat J76 reporter T cell line , we have+ T cells; a selected HA-1-specific TCR was then introduced by lentiviral transduction. Such HA-1-specific CD8+ T cells showed in vitro cytotoxicity against blood cells from HA-1–positive leukemia patients, but not against cells from HA-1 or HLA-A*02-negative patients. The described pipeline for the selection of the HA-1-specific TCRs and the production of the MiHA-specific CD8+ cells could be applied to a wide variety of different MiHAs presented by the other HLA alleles.Endogenous TCR was knocked out by CRISPR/Cas in primary CD8The study was approved by the Research Ethics Committee of the National Research Center for Hematology . All donors and patients gave informed consent before enrollment. Blood samples were collected during the patients’ hospitalization. Peripheral mononuclear blood cells (PBMC) were isolated from a whole blood sample by Ficoll gradient centrifugation . PBMC were stored frozen in FBS  in the presence of 7% DMSO at −80 °C.DNA from the blood samples was extracted using QIAamp DNA Blood Mini kit and QIAcube system  according to the manufacturer’s protocol. HLA typing was performed either by next-generation sequencing (NGS)  or by fl+ T cells from HA-1 donors (rs1801284 G/G) were isolated from PBMC using a naive T cell immunomagnetic isolation kit  according to the manufacturer’s protocol. Cells were cultured in RPMI medium  supplemented with 10% FBS, 50 U/mL IL-2 , 0.5 U/mL IL-7 and 0.8 U/mL IL-15 , and 100 U/mL penicillin/streptomycin . For the isolation of dendritic cells (DCs), monocytes from the same donor were purified using anti-CD14 immunomagnetic beads  and cultured in RPMI medium supplemented with 10%FBS, 100 U/mL penicillin/streptomycin, 800 U/mL GM-CSF , 100 U/mL IL-4 , 10 ng/mL lipopolysaccharide , and 100 U/mL IFN-γ . After 3 days, DCs were detached with a cell scraper and irradiated for 50 min with a total dose of 50 Gy using Biobeam GM 8000 . Irradiated DCs were pulsed with synthetic HA-1 peptide  at a final concentration of 5000 ng/mL (4.88 nmol/mL).Naive CD8+ T cells were co-cultured with the irradiated DCs of the same donor  for 10 days as described previously [Naive CD8eviously .5 to 1 × 106 per well in RPMI media supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, 30 ng/mL IL-21 . The ratio of plated CD8+ naive T cells to DC cells was 2:1 or 4:1. Both cytokines IL-7 and IL-15  were added at concentrations of 100 U/mL each on days 3, 5, and 7. After co-culture, the T cell cultures were screened for antigen specificity by restimulation followed by CD137 or direct tetramer staining as described in the + T cell activity assays and flow cytometry analysis).Briefly, cells were seeded in 48-well suspension plates at a density of 2 × 10+ or CD137+ cells of T cell expansions were selected using anti-PE immunomagnetic beads , and RNA was purified using RNEasy mini columns  followed by cDNA library synthesis as described previously [p < 0.05) and strong (10-fold or greater) enrichment. α- and β-chains enriched in CD137+ or tetramer+ fractions from the same expansion well were used for cloning into lentiviral modules as described below.Tetramereviously . Brieflyeviously . After reviously ,45. The For transgenic TCR reactivity assays, the J76 reporter cell line was used . This ce5 of the obtained TCR transgenic J76 cells were incubated overnight with 1 × 106 K562-HLA-A*02 cells that were pulsed with HA-1 peptide to a final concentration of 4,88 nmol/mL. Activation of J76 cells was determined by GFP expression using flow cytometry (4 ng/mL (24.4 nmol/mL). We co-cultured 1.25 × 105 of J76 cells with K562-A*02:01 cells in a 1:2 ratio with an indicated amount of peptide in a 96-well plate in duplicate. After overnight stimulation, the percentage of GFP-expressing DID-positive J76 cells was analyzed using a MACS Quant flow cytometer . EC50 values for the plotted TCR titration curves were estimated using GraphPad Prizm 9 software .J76 cells harboring a transgenic TCR were prepared as described  was added and as a negative control, mock (PBS) transduced effectors were added. After overnight cocultivation at 37 °C and 5% CO2, cells were transferred to a round-bottomed 96-well plate, pelleted by centrifugation at 300× g for 5 min, and stained using the CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit  according to the manufacturer’s protocol. Plates were analyzed on a MACS Quant flow cytometer. The percentage of caspase-3/7 and 7AAD-positive events in the DID-negative fraction was measured to determine the cytotoxic effect.For the caspase-killing assay, 2.5 × 10+ T cells was followed by the amount of IFN-γ produced in the media, measured by the IFN-γ ELISA assay  according to the manufacturer’s protocol. Cells in the functional assay were stimulated with serial dilutions of HA-1 peptide as described above.The functional activity of TCR-modified CD8+ T clones were screened with irradiated B lymphoblastoid cells (B-LCL) from the same donor. The B-LCL cells  and either used directly for Golden Gate assembly of the final lentiviral constructs or subcloned into the pJet1.2 plasmid . Unwanted BpiI sites were removed from the variable chains by PCR mutagenesis.For rapid cloning of the desired TCR, we developed a modular Golden Gate assembly system B. Human BpiI restriction endonuclease  and T4 ligase . The Golden Gate assembly was performed by 9 to 30 cycles of 15 min restriction at 37 °C followed by 10 min ligation at 16 °C. The reaction was terminated by 5 min at 55 °C. The reaction mix was then transformed into NEB stable competent cells , and transformants were screened by PCR. Plasmid constructs were isolated using the Gene Jet Miniprep kit  and sequenced using the SeqStudio Genetic Analyzer .The final plasmid was constructed from modules containing two variable and two constant chain regions and the lentiviral backbone vector by the Golden Gate assembly reaction  using Bp+ T cells were activated using T cell anti-CD3/2/28 activation/expansion kit  according to the manufacturer’s protocol, and further cultivated for 72h in the RPMI media supplemented with 10% FBS, 500 U of IL-2, 100 U of IL-15, and 100 U/mL penicillin/streptomycin. The non-treated 24-well culture plates  were coated with 30 µg/mL retronectin  and blocked with 2% BSA solution  at 37 °C and 5% CO2 overnight to be used for lentiviral transduction. Activated CD8+ T cells (1 × 106/well) were seeded into retronectin-coated plates, and thawed lentiviral supernatant (lentivirus manufacturing) was added to achieve MOI~6. Plates were then centrifuged at 1000× g during 45 min at 32 °C and cultured at 37 °C and 5% CO2 for 72h. Cells expressing transgenic murine TCR were purified by staining with anti-mouse TCR β antibody, followed by the anti-APC immunomagnetic separation on beads , and further expanded for a total of 4–5 weeks with fresh medium changed every week.The purified primary CD86 activated T cells were electroporated with ribonucleoprotein (RNP) complexes (see Ribonucleoprotein complexes) using the Neon transfection system . We added 20 µL of RNP complexes to T cells in 90 µL of electroporation buffer T supplemented with 11 µM electroporation enhancer . Electroporation pulse parameters were 1600 V, 10 ms, 3 pulses. If further lentiviral transduction was required, T cells were transferred to retronectin-coated plates with virus-containing medium immediately after electroporation and were then proceeded to spinfection as described previously. Alternatively, T cells were immediately transferred into 1.5 mL of pre-warmed medium in a 24-well plate and incubated for 72 h at 37 °C and 5% CO2 for further flow cytometric analysis to determine TCR knockout efficacy. For the gRNA efficacy screening, Jurkat E6-1 cells (ATCC TIB-152™) were electroporated as described above.A total of 2.5 × 10+ T cells of five healthy HLA-A02:01+ HA-1 donors. We complemented them with expansions of T cells from three HLA-A02:01 donors (p < 0.05) enriched in the antigen-specific fraction, indicating they belonged to the HA-1–specific TCRs  currently dominate, with more than 600 active clinical trials underway . CompareAnother area where TCR-T has some advantages over CAR-T is in the treatment of solid tumors . The facThe efficiency of antigen presentation by HLA is the key factor to consider in the design of an efficient TCR-T therapy. In addition, the immunogenicity of the peptide and the differences in the expression pattern of the antigen source in healthy and tumor cells are two other factors influencing the effect of TCR-T therapy. These limitations drastically limit the choice of antigens: the majority of TCR-T therapies use the NY-ESO-1 and WT-1 targets against solid and hematological malignancies, respectively . The higThe use of MiHAs as immunotherapy targets may be more advantageous in the context of allo-HSCT therapy than targeting the TAA and tumor neoantigens. MiHAs are more immunogenic than TAA because they are completely foreign to the MiHA-negative immune system of the donor . In addiCurrently, only a few clinical trials are focused on the use of MiHA-specific transgenic T cells for the treatment of relapsed and refractory hematologic diseases, especially AML . Taking The pipeline of the antigen-specific expansion and TCR discovery outlined in our work  could be+ T cells developing an immune response to the peptides derived from the foreign sequences of the genetic construct [+ responses directed against murinized TCR, there is evidence that such responses impair the efficacy of CAR-T therapy [The efficient expression and exposure of the transgenic TCR could be improved not only by the choice of the V-gene, but also by the use of the murine TCR constant chains ,59. Inevonstruct . While l therapy ,90. TherThe generated transgenic TCR-T cell products pose a potential safety risk because transgenic and endogenous TCR chains could form heterodimers of unknown reactivity . To circThe persistence of modified T cells is essential for therapeutic efficacy . CAR-T cAdvanced methods of gene engineering and novel bioinformatic tools for peptide immunogenicity prediction have great potential to make TCR T therapy a highly efficient and specific method of choice when other approaches fail, particularly for the therapy of relapsed and refractory AML. MiHAs are particularly promising targets for this purpose because, unlike other classes of antigens, they allow efficient discrimination between donor and recipient cells.+ T cells with HA-1-specific transgenic TCRs after CRISPR/Cas knockout of an endogenous TCR. The resulting T cells showed clear cytotoxic activity against PBMC of patients with various hematological malignancies, including AML, B-, and T-cell ALL. The described pipeline could be applied to the development of novel therapies targeting other minor histocompatibility antigens (MiHAs), which represent a promising class of antigens for the treatment of hematological malignancies.We reported the experimental pipeline for the development of MiHA HA-1 specific TCR-T therapy. We described the repertoire of HA-1-specific TCRs, identified a number of functional/TCR chain combinations, and then selected several TCR receptors with sufficient affinity and no alloreactivity. The ability of the cloned TCRs to effectively recognize endogenously processed HA-1 antigens on the surface of cells derived from healthy and leukemia patient PBMCs was demonstrated. We proposed a method for rapid TCR cloning and demonstrated its use for subsequent modification of CD8


Actinobacillus succinogenes was recognized as one of the most promising SA producers; however, lack of genetic background and the scarcity of genetic manipulation tools hinder the improvement in A. succinogenes by metabolic engineering. Here, for the first time, we successfully developed a series of A. succinogenes base editors (BEs) mediated by the fusion of Cas9 nickase and deaminase, including CBE, ABE, Td-GABE, and Td-CBE. Among these, ABE and Td-CBE based on a fusion of Cas9 nickase and TadA-8e variant (Escherichia coli TadA) can efficiently convert A to G and C to T, respectively, with editing efficiencies of up to 100%. We also investigated the multiplex base editing of ABE and Td-CBE, and the results showed that the editing efficiency of ABE reached 100% for six sites and 10% editing efficiency of Td-CBE for two sites. In addition, cytosine base editors were applied to inactivate hypothetical sugar and SA transporters of A. succinogenes. We found that the inactivation of Asuc_0914 encoding sucrose-specific IIBC subunit enhanced SA production, while the inactivation of hypothetical SA transporters Asuc_0715 and Asuc_0716 significantly reduced SA production. Therefore, the tools have great application potential in the metabolic engineering of A. succinogenes.The production of bio-succinic acid (SA) from renewable feedstocks is a promising and sustainable approach to mitigating the high carbon emissions associated with the current energy crisis. The online version contains supplementary material available at 10.1186/s13068-023-02443-8. A. succinogenes is a strain that naturally accumulates high concentrations of SA and is recognized as one of the most promising SA producers [A. succinogenes to be behind those of other strains, such as Escherichia coli, Corynebacterium glutamicum, and Mannheimia succiniciproducens [A. succinogenes was dissatisfactory, and the screening process was time-consuming and complicated. Nevertheless, the “scar” or screening marker usually remained in cells, preventing further engineered strains from being achieved.Succinic acid (SA) is a crucial platform C4 chemical, since it has great commercial value and a wide range of application in chemical, food, pharmaceutical, and other fields of industry –3. It isroducers , 7–11. Iroducens , 7, 12. roducens –16, mutacas9 and cas12a, respectively [C. glutamicum, Bacillus amyloliquefaciens, Staphylococcus aureus, cyanobacteria, and Bacillus subtilis [Clustered regularly interspersed short palindromic repeats (CRISPR) and associated proteins (Cas) genome editing system was successfully employed in various plants, animals, and microorganisms –20. The ectively , 22. In ectively , 23. Depectively , 25. Howectively , 27. Recectively –32. It wsubtilis , 33–36.A. succinogenes were developed for the first time. Four types of BEs for converting targeted C to T, A to G, and G to T in A. succinogenes were designed by engineering the fusion of Cas9 nickase and deaminase , dCpf1(D917A), and nCas9(D10A) , which encodes a β-galactosidase in A. succinogenes, was selected as the editing target. The mutant will not show blue phenotype on the TSB plates containing X-gal by disrupting lacZ [In our previous study, the inogenes . To cons0A) Fig. a. The pl element –39. To fene Asuc_98, whichlacZ gene -UGI and rAPOBEC1-dCpf1(D917A)-UGI had no base-editing ability in A. succinogenes. Only one white colony was obtained by transforming rAPOBEC1-nCas9(D10A)-UGI and culturing for the first and second passages. One white colony and nine random blue colonies from the first and second passages were picked and sequenced. The sequencing results showed that only two white colonies had base mutation at Cs positions of -2, -1, 1, and 6 upstream of CGG PAM, which indicated that rAPOBEC1-nCas9(D10A)-UGI had the potential for base editing in A. succinogenes , but no C-to-G or C-to-A conversion was observed, indicating that rAPOBEC1-nCas9(D10A)-udg could not convert C-to-G or C-to-A in A. succinogenes.In addition, glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a UDG, cause C-to-A transversions in an cells . To consA. succinogenes, the efficiency was unsatisfactory. The number of cells expressing base editors and/or functional editor proteins produced by each cell is a major bottleneck of editing efficiency [A. succinogenes, the linker length between fusion proteins and UGI copy number were also increased, yielding dCpf1(D917A-E1006A)max, and dCpf1(D917A)max max was detected after multiple subcultures on TSB plates containing 100 μg/mL X-gal. As shown in Fig. Although rAPOBEC1-nCas9(D10A)-UGI could convert C to T successfully in ficiency . In addificiency . To imprmax Fig. c. After lacZ gene in A. succinogenes max, other two sgRNAs were designed and employed for modifying 0Amax, otAnc689, optimization, and ancestral reconstruction containing 36 amino acid substitutions relative to rAPOBEC1 replaced rAPOBEC1 of nCas9(D10A)max to construct Anc689-nCas9(D10A)max to improve expected editing efficiency. The results showed that the conversion of C to T at Cs positions of 6, 7, and 8 were achieved and that the editing efficiency was 20%–30%  amino terminus of dCpf1(D917A-E1006A), dCpf1(D917A), and nCas9(D10A) to generate the ABEs , and A-to-G conversion at position 6 (50%) and position 8 (12%) .Although CBEs were successfully developed in the above works, the editing efficiency was dissatisfactory. Recent studies have shown that the TadA8e variant (with an N46L mutation) eliminates adenine deaminase activity, resulting in a TadA8e-derived C-to-G base editing (Td-CGBE) and further fusion with UGI resulting in C-to-T base editing (Td-CBE) that can be edited efficiently and precisely . The mutA. succinogenes, several targets were tested. It was found that Td-CBE and Td-CBEmax showed efficient C-to-T editing at positions 6–8, similar to a previous study [A. succinogenes -ABE and Td-CBEs showed highly efficient base editing in A. succinogenes, we selected them for the subsequent experiment.Simultaneous action of multiple sites can save a large amount of time and processes, providing great convenience for metabolic engineering. However, this was not feasible in the past in A. succinogenes, nCas9(D10A)-ABE and Td-CBEs were constructed containing two sgRNAs. Asuc_1034 and Asuc_1575 were target genes of nCas9(D10A)-ABE and Td-CBEs, respectively. After being transformed to A. succinogenes, ten colonies were randomly picked for sequencing. The results showed that simultaneous A-to-G base editing at two sites targeting Asuc_1034 was achieved with efficiency of 100% by nCas9(D10A)-ABE, suggesting that multiple base editing by nCas9(D10A)-ABE was feasible -ABE, resulting in multiple loci editing plasmids. The results showed that the A-to-G editing efficiency by nCas9(D10A)-ABE were 100% for each site, indicating that nCas9(D10A)-ABE exhibited efficient A-to-G conversion at multiple loci in A. succinogenes. However, simultaneous C-to-T base editing at two sites of Asuc_1575 was achieved with efficiency of 10% by Td-CBE and Td-CBEmax. The editing efficiency of the first site was 100%, and the editing efficiency of the second site was only 10%. These results demonstrated that nCas9(D10A)-ABE, Td-CBE, and Td-CBEmax can perform multiple base editing, but nCas9(D10A)-ABE is more efficient.To confirm the possibility of multiple targets being edited simultaneously in A. succinogenes, we used them for metabolic engineering with a view of enhancing the production of SA. Transporters of substrates and products were crucial for the production of SA in cells. The SucE1 gene of C. glutamicum and yjjPB gene of E. coli were identified as succinate exporters that were required for succinate export [Pichia kudriavzevii were found to effectively import succinate. Simultaneous inactivation of both PkJEN transporters enhanced extracellular accumulation of SA in the late stage of fermentation [ptsG, which encodes glucose-specific permease of the phosphotransferase system–protein EIICBglc, shifted the fermentative metabolism strongly toward succinate in fermentation of glucose by E. coli [mgtA and mgtBC genes were found to be magnesium transporters in E. coli and Salmonella enterica serovar Typhimurium [A. succinogenes. By BLAST, Asuc_0023 and Asuc_0715-0716 were found to be homologous with succinate exporter, and Asuc_0750 and Asuc_2056 were found to be homologous with succinate importer in A. succinogenes. Asuc_0914 was homologous with ptsG, and Asuc_1034 was homologous with a magnesium transporter. A stop codon was expectedly created in A. succinogenes using nCas9(D10A)-CBEmax or Td-CBE to target the above genes  the lack of expressing plasmids, (2) no promoters to express gRNA element, and (3) weak DNA repair ability. To develop a CRISPR/Cas genome editing system, the one and only plasmid pLGZ922 was chosen as backbone, which has been demonstrated to express foreign proteins [frd (fumarate reductase) promoter was used to drive gRNA element. However, no positive colony was observed due to its weak DNA repair ability. Recently, although we expressed Cas9 from Streptococcus pyogenes and Cpf1 from Francisella tularensis and developed a CRISPR interference (CRISPRi)-mediated gene repression system based on dCpf1 in A. succinogenes [A. succinogenes. Unlike CRISPRi, however, we found that the dFnCpf1 was incompatible with rAPOBEC1/TadA8e, while nCas9(D10A) with TadA8e exhibit excellent performance for base editing in A. succinogenes. Thus, we successfully achieved precise genome editing at a single-nucleotide resolution with Td-CBE and further multiplex genome editing with ABE and Td-CBE, which not only provided a new genome editing tools, but also laid the foundation for the development of CRISPR/Cas genome editing system in A. succinogenes. Base editor perform genome editing without DSB, it is not fully applicable to introduce STOP codon in the open-reading frames of A. succinogenes due to the PAM dependence of current Cas9-guided base editor. Therefore, an efficient base-editing toolbox of debottlenecking PAM is further required for A. succinogenes. Different Cas proteins such as Cpf1 that requires a T-rich PAM sequence for target-DNA recognition were employed to develop base editor by fusing deaminase with UGI, yielding C-to-T conversion in human cells [A. succinogenes. Currently, highly efficient PAM-less base-editing toolbox for B. subtilis based on a variant of Cas9 nickase (nSpRY) was developed, yielding PAM-less adenine and/or cytosine base editors [uccinate , 52. Howproteins . The endinogenes , the exian cells , 54. How editors . TherefoA. succinogenes produces SA as part of a mixed acid fermentation, along with the production of acetate and formate as by-products, leading to difficult separation and purification of SA and increases the production costs. It is desirable to genetically engineer A. succinogenes to produce SA as the sole fermentation end product. Carbon flux distribution to succinate and alternative products was determined for understanding of the metabolic pathways by gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy, and found that formate and pyruvate dehydrogenases were contributing to NADH production, and oxaloacetate and malate were being decarboxylated to pyruvate [E. coli, C. glutamicum, and P. kudriavzevii, were identified [A. succinogenes. This study focused on the sugar and SA transporters and found that several hypothetical transporters were required for SA synthesis in A. succinogenes, providing the alternative promising metabolic engineering strategies for SA production.pyruvate , 57. Furpyruvate , suggestentified –48. HoweA. succinogenes for the first time. Here, two base editors, ABE and Td-CBE, were engineered with Cas9 and TadA mutant, which can efficiently convert A to G and C to T, respectively, with editing efficiency of up to 100%. Further, multiplex genome editing was achieved by these two BEs. We applied the BEs to inactivation of sugars and SA transporters to study their effects on SA production in A. succinogenes, and we found that Asuc_0914, Asuc_0715, and Asuc_0716 are significantly associated with SA production. This study established an efficient and fast genetic manipulation tool, which will provide critical insights into the development of a CRISPR/Cas genome editing system and SA production through metabolic engineering of A. succinogenes.In summary, we have developed a series of BEs by fusion of Cas nuclease and cytidine/adenine deaminase in E. coli JM109 was used as the host for the plasmid clone and cultured with LB medium at 37 °C or on LB agar plate with ampicillin (100 μg/mL) when necessary. A. succinogenes CGMCC1593 was anaerobically cultured with TSB medium or TSB agar plate with ampicillin (100 μg/mL) or X-gal when necessary. The fermentation medium was described in Reference [https://chopchop.cbu.cib.no/), and the protospacer sequences are listed in Additional file The strains and plasmids used in this study are listed in Additional file The plasmids pLGZ-nCas9(D10A), pLGZ-dCpf1(D917A), and pLGZ-dCpf1(d917A-E1006A), reported in our previous study, harbor Cas protein and sgRNA/crRNA. They were used as the backbone to construct base editors . Condon-To construct a cytosine base editor based on nCas9D10A) or dCpf1, rAPOBEC1 was fused with the amino terminus of dCpf1(D917A-E1006A), dCpf1(D917A), and nCas9(D10A). Here, the plasmids pLGZ-nCas9(D10A), pLGZ-dCpf1(D917A), and pLGZ-dCpf1(D917A-E1006A), respectively, were used as the template to generate linearized vectors for constructing different CBEs. The linearized vectors and rAPOBEC1 fragment were amplified using the relevant primers. The PCR product of rAPOBEC1 fragment was assembled into linearized vectors using ClonExpress II One Step Cloning Kit  to yield rAPOBEC1-nCas9(D10A), rAPOBEC1-dCpf1(D917A), and rAPOBEC1-dCpf1(D917A-E1006A), respectively , pLGZ-dCpf1(D917A), and pLGZ-dCpf1(D917A-E1006A) were used as the templates to generate linearized vectors for constructing different ABEs. The TadA8e fragment was amplified and assembled into the linearized vector to construct TadA8e-nCas9(D10A), TadA8e-dCpf1(D917A), and TadA8e-dCpf1(D917A-E1006A), respectively. Td-CGBE was created by PCR to introduce an N46L mutation. Thereafter, one copy of UGI and two copies of UGI were assembled into Td-CGBE to generate Td-CBE and Td-CBEmax, respectively.To cure the plasmid, the temperature-sensitive replicon Rep was amplified from the plasmid pKCcas9dO and assembled into the plasmid of BE to yielding Rep-nCas9(D10A)-ABE, Rep-Td-CBE, and Rep-Td-CBEmax.A. succinogenes, the method for the transformation refers to references [For ferences , 37.After transformation, we randomly transferred one colony to TSB liquid medium containing 100 μg/mL ampicillin for cultivation for 24 h and then spread it on TSB agar plate overnight. Once the colonies had developed, the colony PCR was performed by randomly picking ten colonies, and the PCR products were sent for sequencing. The mutation was determined based on the sequencing resultsA. succinogenes grows slowly at 30 °C, which leads to a prolonged experiment period. Indeed, the other plasmid curing procedure was performed by subculture of mutants in TSB medium overnight and plating on TSBG agar without antibiotics. Likewise, the target mutants that did not grow in the presence of antibiotics and that grew in the absence of antibiotics were the target strains.To curie plasmids, a temperature-sensitive replication protein, Rep from the plasmid pKCcas9dO, was introduced into the plasmid of BEs. After the mutant was obtained at 30 °C, the mutant was cultured at 37 °C for 24 h to eliminate the plasmid. The target mutants that did not grow in the presence of antibiotics and that grew in the absence of antibiotics were the target strains. However, Asuc_1034. These sgRNAs were driven by the frd promoter and assembled into the nCas9(D10A)-ABE plasmid. Similarly, two sgRNAs were assembled into Td-CBE and Td-CBEmax to target Asuc_1575.To construct multiplex-site ABE, one, two, three, four, five, and six sgRNAs were designed and assembled by ClonExpress II One Step Cloning Kit  by targeting different sites on A. succinogenes was cultivated anaerobically in 25 mL of TSB medium at 37 °C for 12–16 h, and a 10% v/v inoculum was added into shake flasks containing 50 g/L glucose fermentation medium with the pH regulator MgCO3 powder for 48 h. For a 3 L fermenter, the temperature and agitation were 38 °C and 200 rpm, respectively. MgCO3 was used as pH regulator in the early stage of fermentation, and 3 M Na2CO3 was maintained at pH 6.5 in the late stage of fermentation. The initial glucose concentration was 70 g/L, and we supplemented glucose to maintain it between 15 and 30 g/L when the glucose fell to below 15 g/L.A. succinogenes was monitored by spectrophotometry at 660 nm (OD660). Glucose and organic acids in the fermentation broth were analyzed by HPLC. The special determination methods refer to Reference [The optical density of eference .Additional file 1: Table S1. Strains and plasmids used in this study. Table S2. The primer sequences used in this study. Table S3. The protospacer sequences used in this study. Figure S1. The sequencing results of Anc689-nCas9(D10A) targeting LacZ gene in A. succinogenes. Figure S2. The sequencing results of STOP codon introduction targeting genes of transporters in A. succinogenes. Sequences S1. The nCas9(D10A)-CBEmax sequences in this study. Sequences S2. The nCas9(D10A)-ABE sequences in this study. Sequences S3. The Td-GABE sequences in this study. Sequences S4. The Td-CBE sequences in this study.


Maternal vitamin D intake and BMI during pregnancy in relation to child’s growth and weight status from birth to 8 years: a large national cohort study. BMJ Open 2021;11:e048980. doi: 10.1136/bmjopen-2021-048980Amberntsson A, Papadopoulou E, Winkvist A, The authors want to alert the readers on the updates done for tables 2-3 in the published version.


In-depth immunogenicity studies of tixagevimab-cilgavimab (T-C) are lacking, including following breakthrough coronavirus disease 2019 (COVID-19) in vaccinated patients with hematologic malignancy (HM) receiving T-C as pre-exposure prophylaxis.We performed a prospective, observational cohort study and detailed immunological analyses of 93 patients with HM who received T-C from May 2022, with and without breakthrough infection, during a follow-up period of 6 months and dominant Omicron BA.5 variant.+ (but not CD8+) T cells post infection, comparable to previously infected healthy controls.In 93 patients who received T-C, there was an increase in Omicron BA.4/5 receptor-binding domain (RBD) immunoglobulin G (IgG) antibody titers that persisted for 6 months and was equivalent to 3-dose-vaccinated uninfected healthy controls at 1 month postinjection. Omicron BA.4/5 neutralizing antibody was lower in patients receiving B-cell–depleting therapy within 12 months despite receipt of T-C. COVID-19 vaccination during T-C treatment did not incrementally improve RBD or neutralizing antibody levels. In 16 patients with predominantly mild breakthrough infection, no change in serum neutralization of Omicron BA.4/5 postinfection was detected. Activation-induced marker assay revealed an increase in CD4Our study provides proof-of-principle for a pre-exposure prophylaxis strategy and highlights the importance of humoral and cellular immunity post–breakthrough COVID-19 in vaccinated patients with HM. + T cells postinfection.In 93 vaccinated patients with hematologic malignancy receiving tixagevimab-cilgavimab, humoral and cellular immunity was assessed. In 16 patients with mild breakthrough infection, there was no change in serum neutralization of Omicron BA.4/5, but there was an increase in CD4 Multiple strategies including vaccination and early antiviral therapies are now in place to reduce the risk of severe coronavirus disease 2019 (COVID-19) in patients with hematologic malignancy (HM). Despite these measures, patients with HM remain at high risk of morbidity and mortality from breakthrough (BT) infection . It is aClinical effectiveness studies of T-C have reported a modest BT infection rate 3%–15%) in patients with HM, depending on the prevailing variant, underlying disease, follow-up period, and COVID-19 prevalence %–15% in . ImmunogTo guide future monoclonal antibody preexposure prophylaxis studies, we report the dynamics of humoral and cellular immune responses in a cohort of vaccinated patients with HM receiving T-C as pre-exposure prophylaxis during Omicron BA.5 and the evolution of immune responses with BT infection in comparison to a healthy control population.An observational, prospective, single-center cohort study was performed from 1 May 2022 of adult patients ≥18 years of age with HM eligible to receive T-C 150/150 mg). The primary outcome was the development of BT COVID-19 infection after T-C with planned follow-up of 6 months post injection. Breakthrough COVID-19 was defined as compatible symptoms and positive detection by polymerase chain reaction or rapid antigen testing for SARS-CoV-2, occurring any time after T-C. Sequencing of SARS-CoV-2 variants of concern was not routinely reported for individual patients, but is monitored by a national real-time genomic surveillance program 0/150 mg.. SeveritPatients were recruited from the outpatient setting. Exclusion criteria included febrile illness in the preceding week, recipient of intravenous immunoglobulin (±30 days), and severe thrombocytopenia or other coagulopathy that precluded safe intramuscular injection of T-C.Data including demographics, HM treatment, COVID-19 infection, and vaccination history were collected and recorded into an electronic database . Blood was collected from each participant for sera, plasma, granulocytes, and peripheral blood mononuclear cells (PBMCs) at a baseline visit prior to T-C , then days 30 (V1), 90 (V3), and 180 (V6) after injection (±2 weeks). Patients were followed closely with monthly phone calls for BT COVID-19, attributable side effects, and/or further COVID-19 vaccination. Patients were able to additionally consent to blood collection 2–4 weeks post–COVID-19 vaccination or infection.2), a prospectively collected, ethics-approved (HREC/75984/Austin-2021) blood sample biobank of COVID-19–vaccinated individuals. Samples from 2 control groups were utilized: (1) 15 healthy participants with no history of COVID-19 after 3 COVID-19 vaccine doses (cohort 1); and (2) 10 healthy participants after mild BT COVID-19 during Omicron BA.1 and 2-dose vaccinated (cohort 2).Healthy control participants were included from the Victorian Critical Vaccinees Collection  were assessed by immunoglobulin G (IgG) enzyme-linked immunosorbent assay as previously described , 19. AllThe GenScript SARS-CoV-2 surrogate virus neutralization test (sVNT)  was performed with ancestral and BA.4/5 Omicron RBD constructs (GenScript number L00847-A) as described previously , 20–22. 6 PBMCs/well. For the activation-induced marker (AIM) assay, cells were stimulated in complete RPMI with 10 mg/mL SARS-CoV-2 spike peptide pool  or dimethyl sulfoxide (DMSO) , as a negative control, and cultured at 37°C/5% CO2 for 24 hours. Cells were washed and stained  and Wilcoxon signed-rank test (paired) used for comparisons between 2 groups. Given the HM patients acted as their own controls between time points, over a relatively short period of time (6 months), these analyses were not adjusted for. Key clinically relevant questions were analyzed by subgroup analysis including the effect of vaccination post T-C, receipt of B-cell–depleting therapy within the last 12 months, and BT infection. McNemar test was used to compare proportions of positive versus negative Omicron BA.4/5 sVNT at each time point in patients with HM who received T-C.The primary analysis focused on comparisons between HM patients, with Mann-Whitney Univariable and multivariable linear mean regression or quantile median regression were used to compare HM cases and healthy controls at baseline (V0) as appropriate. All outcome variables including IgG titers, neutralization, and T-cell frequency were formally tested for skew, with normally distributed outcome variables being compared using mean regression and significantly skewed outcomes analyzed with median regression. All outcomes were adjusted for age, sex, and time from last vaccine dose until blood sample (cohort 1) or age, sex, and time from BT infection until blood sample (cohort 2).P < .05 was considered significant. Figures were prepared using GraphPad Prism version 9 software. All statistical analyses were performed using IBM SPSS (version 28.0), GraphPad Prism (version 9), or Stata (version 17) software.For all analyses, Approval (HREC/85611/PMCC) was granted from the Human Research Ethics Committee of the Peter MacCallum Cancer Centre, and patients enrolled provided written informed consent.A) who received T-C at a dose of 150 mg/150 mg from 1 May to August 2022. Clinical characteristics of BT infection in this cohort have been described elsewhere  or chimeric antigen receptor T-cell [CAR-T] therapy) or other therapies and in patients with aggressive or indolent lymphoma, who are common recipients of B-cell–depleting therapies.In patients who had received any B-cell–depleting therapies in the last 12 months (n = 40), median V0 IgG titers against ancestral RBD and Omicron BA.4/5 RBD E were sireceived B and undase type C and 1D In summary, a significant rise in the ancestral and Omicron BA.4/5 RBD IgG was observed after T-C in patients with HM, achieving levels comparable to 3-dose-vaccinated uninfected healthy controls, with no additional serological benefit seen in those receiving vaccination in the 6-month follow-up period and negative influence in those who had received recent B-cell–depleting therapy.A), sVNT on sera against ancestral and Omicron BA.4/5 strains was performed for all time points. In contrast to the IgG titers, at V1 (A), there was improvement in percentage neutralization against ancestral (P < .001) but not Omicron BA.4/5 (P = .80). The findings remained consistent when patients with prior COVID-19 were excluded from the analysis (A). The proportion of patients with HM achieving a positive sVNT ≥30% and ≥50% against ancestral and Omicron BA.4/5 strains was analyzed (B) increased at V1 (P = .052) but then declined significantly at V3 . In comparison to healthy controls, ancestral sVNT (B) was equivalent at V0 on adjusted median regression analysis (P = .18). However, on unadjusted analysis, Omicron BA.4/5 percentage neutralization was lower than in healthy controls (P = .02) at V1.For the overall cohort of patients with HM A, sVNT os, at V1 A, there analysis A. The pranalyzed . At V1, analyzed B increasral sVNT B was equanalysis  and at VC) or Omicron BA.4/5 (C) at baseline or V6 in patients who had either received T-C alone (n = 47) or had received T-C and then vaccination (n = 31). Patients who had received B-cell–depleting therapy in the last 12 months had significantly lower Omicron BA.4/5 percentage neutralization at baseline, V1, and V6 compared to patients who had not received any B-cell–depleting therapy (D). The effect of therapies received (C) and underlying disease type (D) is provided, with lower neutralization values noted in patients receiving B-cell–depleting therapies and those with aggressive or indolent lymphoma.There was no difference in percentage neutralization against ancestral C or Omicn BA.4/5 C at base therapy D. The efreceived C and undase type D is provTherefore, sVNT was improved against the ancestral but not Omicron BA.4/5 strain after T-C over the study period, without additional benefit of vaccination, and lower levels in those receiving B-cell–depleting therapy.The overall BT infection rate in patients with HM was 20.4% (19/93) and occurred at a median of 81 days  after T-C . Most paA and Supplementary Figure 3A and 3B). Blood sampling  occurred postdiagnosis of COVID-19 at a median of 28 days . On analysis of postinfection sera, only 4 of 16 (25%) patients had a reactive anti-nucleoprotein antibody. In comparison with 2-dose-vaccinated healthy controls with mild BT COVID-19  and Omicron BA.4/5 IgG RBD titers (P = .52) post–BT infection .Sixteen patients with confirmed BT COVID-19 had pre- and postinfection samples available for analysis  compared to pre–T-C levels (both P < .001) but did not change post–BT infection compared to post–T-C. This may be due to a lack of endogenous antibody production post–BT infection and/or because the median antibody level is already increased with receipt of T-C, and so no significant change was observed.In patients with HM, median ancestral and Omicron BA.4/5 RBD IgG titers were significantly raised post–BT infection B compareC) pre- and postinfection of ancestral (P = .06) or Omicron BA.4/5 (P = .86) sVNT. Individual change over these time points for IgG RBD titers and sVNT are demonstrated (A and 4B). In HM patients, Omicron BA.4/5 IgG RBD titers significantly correlated (C) with percentage neutralization of Omicron BA.4/5 . The proportion of patients with HM with a positive sVNT ≥30% to Omicron BA.4/5 remained comparable (13/16 [81.3%]) pre- and postinfection  and Omicron BA.4/5 strains (P = .73), compared with healthy controls post–BT infection (C).On adjusted median regression analysis , patientnfection C.+ and CD8+ T cells are presented for spike-specific T-cell responses (A). Patients with HM had a significant rise (P = .02) in the frequency of spike-specific activated CD134+CD137+CD4+ T cells post–BT infection (D) but not spike-specific activated CD69+CD137+CD8+ T cells . There was no significant influence of prior B-cell–depleting therapy in the pre- and post–BT infection frequency of activated CD4+ or CD8+ T cells (B and 5C).Representative FACS plots for CD4esponses A. Patiennfection D but notP = .77; D. There  T cells B and 5C.A and 4B), there was similar frequency of activated CD4+ T cells (P = .37) and CD8+ T cells (P = .67) post–BT infection in patients with HM. Healthy controls  also had similar frequency of activated T-follicular helper cells (Tfh) (P = .29) in comparison to patients with HM post–BT infection, despite a rise in the percentage of activated Tfh in patients with HM (P = .04). Comparing HM patients with BT infection to those without a history of COVID-19 or BT infection (n = 27), there was no difference in the frequency of activated CD4+ T cells and CD8+ T cells  at V1. There was also no difference in baseline frequency of activated CD4+ T cells and CD8+ T cells  in patients who went on to have BT infection compared with those who did not. Anti-RBD IgG titers and sVNT against Omicron BA.4/5 were not found to correlate with AIM frequency of CD4+ and CD8+ T cells post–BT infection in HM patients .On adjusted median regression analysis , in compFigure 4+ tetramer+ T cells but not spike-specific CD8+ tetramer+ T cells in 10 patients with BT infection by TAME assay (D). The representative AIM and TAME assay gating strategies are provided in Consistent with AIM assay results, there was a significant increase in spike-specific CD4ME assay D. The re+ T-cell and Tfh rise but not CD8+ T-cell rise, with no impact on prior receipt of B-cell–depleting therapy in the last 12 months. Our data show that vaccinated patients with HM who have received T-C exhibit robust epitope-specific T-cell responses post–BT infection.In summary, post–BT infection in vaccinated patients with HM, there was a significant CD4+ T cells in patients with HM after BT infection, similar to healthy controls, but no change in the frequency of activated or spike-specific CD8+ T cells post–BT infection.We highlight a modest rate of BT COVID-19 (20.4%) in a highly vaccinated cohort of patients with HM receiving T-C as preexposure prophylaxis during Omicron BA.5. This higher BT infection rate compared with earlier reports , 13, 28 Although Omicron BA.4 and BA.5 recombinant lineages  had started to emerge in Australia toward the end of the study period (November–December 2022), Omicron BA.5 was still the predominant sublineage being sequenced (39.4% of sequences of BA.5 compared with 14.0% recombinant lineages in the reporting period 21 November–18 December 2022) , 29. ThiThe increase in IgG titers to ancestral and Omicron BA.4/5 RBD with T-C and BT infection in patients with HM, but not Omicron BA.4/5 neutralization, may represent a rise in nonneutralizing antibodies. This is critical as neutralization has been put forward as an immune correlate of protection and underscores the importance of not assessing anti-RBD IgG titers in isolation . On unadOf concern is the consistently lower anti-RBD IgG and neutralization values against Omicron BA.4/5 in patients who had received B-cell–depleting therapy within 12 months, in comparison to other therapies. This likely reflects a lack of reserve in the SARS-CoV-2–specific B-cell responses induced by their prior vaccination, which cannot be fully compensated by the provision of T-C , 32. ThiThe lack of improvement in neutralization of Omicron BA.4/5 post–BT infection during circulation of Omicron BA.5 in patients with HM indicates an inadequate immune response to an autologous infection. This could also imply that a history of SARS-CoV-2 infection in vaccinated patients with HM may not confer protection to future infection, as is described in the general population . This ma+ T-cell activation may relate to the large majority of BT COVID-19 in our study being mild and/or the timing of blood sampling post–BT infection, given that CD8+ T cells are usually only activated for a few weeks. In immunocompetent individuals, the breadth and magnitude of T-cell responses were significantly higher in severe compared with mild cases [+ T cells, B cells, and humoral immunity, CD8+ T-cell responses were preserved, along with high activation of CD8+ T cells in patients with HM post–COVID-19 [+ T cells was associated with lower mortality and viral load [+ T-cell response may be a compensatory reaction to a blunted humoral response [+ T-cell activation was not required due to humoral immunity augmented by T-C as well as virologic control being supplemented by early antiviral therapy [+ T cells found post–BT infection in patients with HM in our cohort may represent an effort to coordinate the adaptive immune response and could be partly responsible for mild disease as previously described [+ and CD8+ T cells in HM patients with BT infection and those without infection. This agrees with findings from Nguyen et al and is reflective of a preserved T-cell response in a highly vaccinated cohort [The lack of increased CD8ld cases . Bange eCOVID-19 . Furtherral load . Their cral load . The autresponse . This is therapy . The sigescribed . Last, td cohort .Our study does have some limitations. We employed convenience sampling for the healthy controls based on available biobank samples. Healthy controls were younger and female predominant, and the sampling time postvaccination or post–BT infection varied compared to the HM patients. To account for these potentially confounding factors we performed adjusted regression analyses at the V0 timepoint. These factors should be considered when interpreting the comparative analyses between the V0 timepoint for the healthy controls and V1 timepoint for the HM patients. Although our study examined neutralization against Omicron BA.4/5, the predominant variant during the study period, we did not examine for neutralization ability against the latest Omicron subvariants. Given our findings and recently published clinical and in vitro data, we would expect the neutralization values to be lower than what we have reported , 13, 43.We provide a comprehensive and novel description of the dynamics of humoral and cell-mediated immune responses including for BT COVID-19 infection in patients with HM receiving T-C as preexposure prophylaxis. We identify priority groups, including patients receiving of B-cell–depleting therapy who may benefit from additional strategies, and absence of incremental immunological benefit of vaccination in the setting of T-C. This study provides proof-of-principle for this new prevention strategy and informs future use of monoclonal antibodies in development for this high-risk patient population.ofad550_Supplementary_DataClick here for additional data file.


As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available PIs (nirmatrelvir and ensitrelvir) with cell-based and biochemical assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease-inhibitor-resistance mechanisms and show the relevance of specific mutations in the clinic, thereby informing treatment decisions.Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CL Understanding how SARS-CoV-2 could counter the antiviral drug nirmatrelvir and what it means for the future of COVID-19 treatment. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 (coronavirus disease-19), has established itself as a permanent human and animal pathogen worldwide. Vaccines, alongside monoclonal antibodies, have drastically reduced hospitalization and/or mortality, especially in immunocompromised individuals, the elderly, and people with pre-existing medical conditions –5.pro/Mpro, and is most relevant in the clinical setting, as it has been shown to decrease hospitalization, in-hospital disease progression and death   , pomotre(FB2001) , halted (FB2001) ), simnot(FB2001) , EDP-235(FB2001) , and HS-To date, several studies have addressed the issue of nirmatrelvir-resistant/escaping variants using different approaches: highlighting mutational hotspots , 15, in pro resistant variants in the Omicron-Mpro context by using SARS-CoV-2 replicons, circumventing potential safety issues for mutated transmissible variants. It is worth noting that the Omicron signature mutation (P132H) does not confer resistance to common inhibitors   . Replicapro that encodes an artificial, non-functional polyprotein (G-Mpro-L), and relies on Mpro activity for its replication  for subsequent selection experiments with the protease inhibitor nirmatrelvir , which was previously identified in selection experiments, was used. This mutation was of particular interest, because it was also found in resistance studies with authentic SARS-CoV-2  (Cterm-Mpro-LNterm)  ,D on the-LNterm) .pro variants and 47 distinct non-synonymous single- and double-mutated VSV-O-Mpro variants, as schematically shown by the unrooted phylogenetic tree , 4140, 4“rescue” ). This vpro variants, L167F/F305L and L167F/P168S, were chosen for further selection experiments. From VSV-O-Mpro, the variants O/A206T, O/R188W, O/Y54H, O/A210S, and O/A266T were chosen for further study. Among these variants, only O/A206T was selected for additional selection experiments. O/A206T did not fulfil the first two filtering criteria, but only the third (multiple occurrences), as it occurred 17 times independently in VSV-O-Mpro selection experiments. To confirm that this variant was not present in our VSV-Omicron-Mpro stocks already, Nanopore sequencing was performed on those stocks. We confirmed the absence of any VSV-O-Mpro variant subpopulation to the extent of sensitivity that Nanopore sequencing provides. Subsequently, the next round of selection experiments with the variants L167F/F305L and L167F/P168S was carried out, in which the triple-mutant variants L167F/F305L/F8L, L167F/F305L/S144A, L167F/F305L/P184S, and L167F/P168S/L57F were selected. From VSV-O/A206T-Mpro, the mutants O/A206T/F8L, O/A206T/K100N and O/A206T/T198I were selected. To assess whether mutants generated from the selection experiments and selected for further analyses were not detrimental for viral propagation, the Ultrafast Sample placement on Existing tRee (UShER) tool was applied. Phylogenetic analyses using sequences harbouring T21I  , two VSV-L167F-Ming T21I , T198I, ing T21I ,B were gry 2023)  has alsopro-On and 3CL/Mpro-Off (pro-tag (PNterm:Mpro:PCterm) or an INTERmolecular fusion protein made of the green fluorescent protein, Mpro and L (GFP-Mpro-L). The cells were then infected with VSV-ΔP or VSV-ΔL, respectively. The PNterm:Mpro:PCterm intramolecular tag in combination with VSV-ΔP-dsRed constitutes a gain-of-signal assay also called “Mpro-On”, whereas the artificial polyprotein (or fusion protein) GFP-Mpro-L in combination with VSV-ΔL-dsRed constitutes a loss-of-signal assay also called “Mpro-Off”.To quantify the resistance of the selected variants, the most interesting mutations were introduced into two previously described live-cell-based protease activity assays, namely 3CL/MMpro-Off  , because some variants were replicating slower, affecting final signal quantification  were also characterized in the dose-response assays. In ron-Mpro . In the pro-Off system. In Mpro-Off transfected cells, the signal increases over time in the absence of an inhibitor (50 (time maximum fifty), a value that represents the time required to reach the half-maximum value of the curve when the signal plateaus. The WT protease had a TM50 between 35–40 hpi . To investigate this unexpected result, viral replication was assessed over time using the Mnhibitor . Fluores5–40 hpi , and the0 values , S9F,G. observed .pro mutants. Overall, mutations that have occurred in the Omicron-Mpro context, had a lower impact on viral replication rate than in the WT Mpro . Most nenotypes , Fig. 3BSome mutated residue side chains are within the ligand binding site, therefore close to the inhibitor, but do not directly interact with it, namely L167, P168, Y54, L141 and several others . FurtherFor example, Omicron/A206T that conferred strong resistance to drug treatment, was a frequently occurring mutation in our experiments and is distant from the catalytic site, prompting us to investigate the structure in more detail. Parental Omicron has a hydrophobic side chain alanine residue buried inside the protein at position 206, which is surrounded by the hydrophobic side chains of L253, P293, and V296 . The larpro assays, mutations F305L and F8L alone did not confer resistance. In addition, the F305L mutant showed a gain-of-function phenotype . Co. Copro a10 K* scores indicating stronger and lower Log10 K* scores weaker binding. An increase in the binding affinity of +9.3 in the F305L mutant was calculated compared to the WT, suggesting an increase of Mpro dimerization affinity and formation of the mature dimer. F8L, conversely, did not influence dimerization affinity  mutant. The Omicron A206T/F8L  also experienced a mild destabilization. For the mutant O/A206T/T198I we predicted an increased stability , indicating that the effects of this mutation might be mediated through a different mode of action.The predicted loss of stability was less severe for the M50 . The dimerization affinity was less affected than protease stability, with most mutants having a predicted Log10 K* score similar to the WT    simulatiCS) . In the pro were selected for further investigation and compared to WT Mpro-nirmatrelvir. Their structures, plus so-called rainbow blots, root-mean-square-deviations and interaction energy blots are depicted for the most resistant variants L167F/P168S/L57F and L167F/F305L/P184S/T21I  by altering the floor of the binding site, causing a drag effect on the ligand (Movie S1). The resistance mechanism of the L167F/F305L/S144A mutant seemed to be increased instability of the oxyanion loop (residues 138 to 145), which finally leads to the loss of the hydrogen bond with H163 subsequently resulting in the loss of interaction with residue Q189 (Movie S2). In L167F/F305L/P184S, the P184S mutation enhances the plasticity of the loop spanning from residue S184 to Q192, thereby reducing the ligand-pocket shape complementarity, which is essential for ligand positioning. At the same time, it did not alter the S1 pocket and the hydrogen bonds network within the central portion of the cavity (Movie S6).As can be observed in due Q189  . This mutation has been shown to not confer resistance to approved protease inhibitors, such as nirmatrelvir and ensitrelvir  has been shown to be the preferred residue instead of phenylalanine (F) (Movie S6).The triple mutant L167F/F305L/F8L is among our most resistant Mnine (F) . In factpro, we selected resistance mutations within and outside the catalytic site. The catalytic site mutant O/Y54H, which occurred 10 times independently, and O/R188W showed only mild resistance alone as well as in combination with R222L. O/Y54H/F305L showed an increased, selective resistance against ensitrelvir, with a 20-fold-change of the IC50 in the loss-of-signal assay (Mpro-Off). The mutants K100N, T198I, A210S, and A266T were frequent in GISAID, therefore we picked them for resistance characterization. Double mutants O/A206T/F8L and O/A206T/T198I showed strong selective resistance against nirmatrelvir (up to 100-fold-change), while being less effective for ensitrelvir. We speculate that in O/A206T/F8L, the contribution of F8L to resistance is the same as in mutant L167F/F305L/F8L (“wiper” effect). O/A206T and O/A206T/K100N also showed considerable resistance (~26-fold), and the computed destabilization correlated with resistance.In the Omicron-Mpro. We chose to characterize the A206T mutation due to the unusually high frequency in the selected VSV-O-Mpro pool of mutants. A206T only fits our third filtering parameter: frequent selection in our experiments, as it is far from the catalytic site and scarcely represented in the GISAID database. Likewise, some mutations may confer an advantage for viral replication only in the presence of a protease inhibitor, but would probably be unfavourable in an untreated individual. In a study on SARS-CoV Mpro polyprotein maturation  catalysed by domain III  of Mturation , the autmonomers  and 2 ca00–306 ofpro activity. The L167F/F305L/S144A mutant was among the slowest variants and the time to reach the plateau was 1.75 times longer than for the WT Mpro. However, mutations that were described as compensatory in the literature, such as T21I , and R222L after plaque purification of VSV-O-R188W-Mpro. T21I and R222L are likely compensating for substitutions that occur in the catalytic site. For example, T21I seems to compensate for the loss of replicative fitness of the previously described, highly resistant Mpro-E166V variant (st of June 2023). From mutations generated in the VSV-O-Mpro variant, viral replication kinetics exhibited a different behaviour in comparison to VSV-L167F-Mpro variants. Overall, mutations related to the Omicron-Mpro did not decrease the viral replication rate to the same extent as in the L167F-Mpro. The slowest variant was O/A206T/F8L, with a 1.4 fold-change (increase of ~16 hours) to reach the plateau. As expected, most mutations that have been computationally predicted to decrease protease stability indeed had a negative impact on viral replication, with higher TM50 values related to Mpro variants bearing such mutations. Furthermore, they were less represented in isolates in the GISAID repository (1 or 2 digit entries), whereas those that had similar TM50 values compared to the WT such i.e. F8L, P184S or K100N and T198I alone, were more frequent (3 or 4 digit entries). These correlations support the validity of computational predictions.In kinetic studies, we observed that the gain in signal was slower for some mutants, which might be an indirect read-out of M as T21I , could p variant . Furtherpro, some of which aligned exactly with those identified in SARS-CoV-2 gain-of-function experiments reported in the literature  and VSV polymerase L. It has been reported in the literature that the SARS-CoV-2 polymerase can proofread . Resistance phenotypes were reproduced at least twice, usually more often and in different combinations. Representative measurements were chosen to compile graphs and figures.The overall rationale of the study was to use a previously developed mutation selection tool based on VSV to select and characterize a comprehensive collection of SARS-CoV-2 main protease inhibitor escape mutants. The study was performed on cell lines, in biochemical settings and in-silico, and no animal husbandry or human participants were involved. Human cell lines with replicating BSL-1 and −2 viruses were treated with protease inhibitors to observe resistance phenotypes in appropriate facilities. Viral titres were determined using TCIDpro instead of the intergenic region between G and L was cloned as previously described  was cloned as follows: N-terminal fragment comprising part of G and Mpro was amplified by PCR using primers 33n-before-KpnI-for (Q195) and Omicron rev VSV-G-Mpro-L as a template, and the C-terminal fragment comprising the remaining part of Mpro and part of L was amplified by PCR using primers Omicron for and 33n-after-HpaI-rev (Q176). N- and C-terminal fragments were ligated together by Gibson assembly in a VSV-backbone vector digested with KpnI and HpaI. VSV-G-Mpro-L L167F (VSV-L167F-Mpro) was plaque purified as it was generated in a previous work.The chimeric VSV variant with Mescribed . VSV-G-Mpro-Off and -On point mutants were generated by point directed mutagenesis on parental plasmids  with mutation primers and the Herculase II Fusion DNA Polymerase. Herculase is a polymerase that can overcome difficult (GC-rich) sequences and amplify large plasmids. However, primers could not be chosen for optimal design because the mutation site was fixed. For this reason, this simple point directed mutagenesis did not work for each construct. For Mpro-On and Mpro-Off plasmids, where point-directed mutagenesis did not work, we used mutagenic Gibson assembly as previously described  were cultured in Glasgow Minimum Essential Medium (GMEM) (Lonza) supplemented with 10 % fetal calf serum (FCS), 5 % tryptose phosphate broth, and 100 units/ml penicillin and 0.1 mg/ml streptomycin (P/S) (Gibco). 293T cells , and 293-VSV   were culpro-L WT and a few mutants were rescued in 293T cells by CaPO4 transfection of whole-genome VSV plasmids together with T7-polymerase, N-, P-, M-, G- and L expression plasmids as helper plasmids (pro-L was fully replication competent and produced on BHK-21 cells.VSV-G-Mplasmids . Briefly4 BHK-21 cells/well were seeded in a 96-well plate one day before infection with the chimeric VSV-Mpro viruses. VSV-L167F-Mpro and VSV-O-Mpro infected cells (MOI = 0.01) were treated with nirmatrelvir with concentration ranging from 10 to 100 μM. The resistant variants VSV-L167F/P168S-Mpro and VSV-L167F/F305L-Mpro at MOI = 0.01 were passaged 5 times, and with increasing concentrations of inhibitor (from 50 to 100 μM). VSV-O-Mpro and VSV-O/A206T-Mpro at MOI ranging from 0.001 and 0.01 were passaged only once with increasing concentrations of inhibitor (from 10 to 100 μM). Each virus variant occupied from 24 wells to 96 of the 96-well plate. Supernatants from wells that displayed cytopathic effect after 48–72 hours were collected for downstream viral RNA isolation, cDNA synthesis, PCR amplification and Sanger or Nanopore sequencing as described in this section.10pro-L RNA was isolated either by using the E.Z.N.A. Viral RNA Kit (Omega Bio-Tek Inc.), the NucleoSpin RNA Virus (Macherey-Nagel GmbH) or by semiautomated RNA extraction using the Easymag (BioMerieux™). BHK-21 cells were infected with the respective VSV-G-Mpro-L variant in 96-well plates as described above. Virus-containing supernatants were collected from individual 96-wells and the RNA was purified from the supernatants according to manufacturers’ instructions (Easymag). Then, cDNA was synthesized from isolated viral RNA by RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific). GCterm-Mpro-LNterm fragment sequence was amplified by PCR with primers (primer_for: CTCAGGTGTTCGAACATCCTCAC and primer_rev: GATGTTGGGATGGGATTGGC) and either sent for Sanger sequencing (MicroSynth AG) or sequenced using Nanopore (as described below). Obtained sequences were mapped to the L167F-Mpro or Omicron-Mpro reference sequences in Geneious Prime 2023.0.1 build 2022–11-28 and examined for mutations.VSV-G-MCterm-Mpro-LNterm fragments, as described above, were used. The sequencing libraries were prepared using the Rapid Barcoding Kit SQK-RBK110.96 , and up to 96 samples were multiplexed and sequenced on a MinION Mk1B sequencer with R9.4.1 flowcells (ONT). Raw data in the form of electrical signals are translated into nucleotide sequences  and saved in fastq files. Basecalling using the super high accuracy model, demultiplexing, as well as barcode and adapter sequence trimming, was performed in Guppy .After the first two selection experiments where we sequenced our samples with Sanger sequencing (MicroSynth AG), we used Nanopore sequencing. First, PCR amplification products of the GRaw reads were filtered to a PHRED quality score of ≥ Q15 and length between 200 bp and 1800 bp using SeqKit (version 2.4.0) , alignedThe resulting VCF files were imported to Geneious Prime 2023.0.1 and called variants manually checked for plausibility.http://www.phylogeny.fr/ with standard parameters. The output as .newick file was imported to Geneious Prime 2023.0.1, visualized and exported as PDF. Graphic rearrangements and overhauls were made using Inkscape version 1.1.Mutations had to be manually inserted into the main protease sequence for each variant. The phylogenetic tree was generated using the online tool st June 2023) was used; specifically, the àll_mutations` field from a JSON dump obtained with GISAID credentials. Using Python 3.9 and the pyarrow and pandas’ libraries, sequences were classified into WT  and Omicron . Then, for both these groups of sequences, the counts of 95 nsp5 mutations of interest were aggregated.To obtain updated nsp5 frequencies, metadata from the EpiCoV database  and incubated 8–9 hours for Mpro-On and 10 hours for Mpro-Off assays. Then, cells were seeded into a 96-well plate with 2 × 104 cells per well in 50 μl complete growth medium. Directly after seeding, compounds and virus (MOI 0.1) were added in 50 μl complete growth medium to wells. After 48 to 72 hours, supernatants were removed, and fluorescent spots counted in a Fluoro/ImmunoSpot counter (CTL Europe GmbH). For experiments where FluoroBrite™ medium was used, supernatants were not removed and the signal was read out every 12 hours. The manufacturer-provided software CTL switchboard 2.7.2. was used to scan 90% (70% when FluoroBrite™ medium was used) of each well area concentrically to exclude reflection from the well edges, and counts were normalized to the full area. Automatic fiber exclusion was applied while scanning. The excitation wavelength for dsRed was 570 nm, and the D_F_R triple band filter was used to collect fluorescence. To increase comparability between Mpro-On and -Off signals, we normalized dsRed events with the following strategies. In Mpro-On, the highest compound concentrations would not reach the same value due to the different responses of each mutant. Therefore, we normalized to the highest mean of the experiment. In Mpro-Off, we normalized the signal to each individual’s highest mean of the construct. For both assays, fluorescent spot count (y-axis) data were plotted against PI concentration (x-axis) and IC50 values were extrapolated as described later in this section.3 × 10pro, Omicron and mutants, we adapted the 3CL/Mpro-Off assay to enable multiple read-outs over time every 12 hours. We exchanged the standard DMEM supplemented with 10 % FCS, P/S, 2 % glutamine, 1x sodium pyruvate and 1x non-essential amino acids (Gibco), with FluoroBrite™ DMEM equally supplemented. FluoroBrite™ DMEM has the same composition as DMEM, except it lacks phenol red which interferes with spot recognition. We experimented as described in the previous paragraph. After seeding 2 × 104 transfected cells in 50 μl of FluoroBrite™ complete medium into 96-well plates, only virus (MOI 0.1) was added (50 μl). The signal was read out at 12 hours intervals. As the virus starts replicating, dsRed is expressed by the infected cells, and the expression of dsRed is correlated with the amount of viral progeny and therefore with the number of spot count. We generally stopped signal acquisition after 84 up to 108 hpi, because the cells die due to virus-induced cytopathy, and the spot count decreases. The manufacturer-provided software CTL switchboard 2.7.2. was used as described above. Replication kinetic curves were normalized individually to the highest mean of the construct. We normalized fluorescent spot count data by the highest mean of each dataset. Then, we plotted the fluorescent dsRed signal (spot count) against time (hpi). We then performed non-linear regression analyses using the built-in function “Sigmoidal 4PL, X is concentration” in GraphPad Prism 9.5, where concentration was replaced with “hours” and extrapolated the TM50 (time-maximum fifty) value, which represents the time required to achieve half of the maximum signal/plateau.For replication kinetic experiments with WT Mpro with a C-terminal Hexahistdine tag (Mpro-6H) was expressed in E.coli followed by a tag removal with the human rhinovirus 14 3C protease (HRV3C protease).Mpro-6H construct was codon optimized for the expression in E. coli and ordered from Thermo Scientific . Point mutations to generate the mutant variants were introduced using the Q5® Site-Directed Mutagenesis Kit purchased from New England Biolabs . Primers were acquired from Sigma Aldrich .WT Mpro-6H variants were transformed into electrocompetent E. coli BL21(DE3) cells. An overnight culture was inoculated 1:50 in 1L TB medium and cells were grown at 37°C, 220 rpm to an OD600nm of 1. After induction with 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) temperature was lowered to 25°C for 4 hours. Cells were isolated by centrifugation at 3200 g, 20 minutes at 4°C, and cell pellets frozen at −20°C.MFor purification, frozen pellets were resuspended in 50 mM Tris/50mM NaCl, pH 7.5 buffer and disrupted with a French press (Thermo Scientific). Lysed cells were centrifuged at 20000 g, 4°C, 20 min and the clarified supernatant was adjusted to 50 mM Tris/300 mM NaCl/20 mM Imidazole. IMAC purification was carried out with a ÄKTApurifier  and a HisTrap FF Crude 1 mL  column. Unbound proteins washed out using 4 column volumes (CV) 50 mM Tris/300 mM NaCl/20 mM Imidazole, pH 7.5 and 1 CV 50 mM Tris/300 mM NaCl/40 mM Imidazole, pH 7.5. The protein-containing fractions were pooled, and the buffer was changed to 50 mM Tris/50mM NaCl, pH 7.5 using a HiTrap Desalting 5 mL  column. Protein concentration was determined with a NanoDrop ND-1000 Spectrometer (Thermo Scientific).pro. Mpro-6H was incubated together with NT*-HRV3protease at 4°C overnight followed by negative IMAC purification with a manually packed Ni-NTA column. The flowthrough yields the mature Mpro with native N- and C-termini without His tag, because the His tag as well as the NT*-HRV3C protease are bound to the Ni-NTA resin.The highly soluble Hexahistidine tagged NT*-HRV3Cprotease was expressed as previously described  and puripro activity assay from BPS Biosciences, catalog number #78042–2. The Mpro in the kit was replaced by an in-house produced Mpro and mutants thereof , as described in the Mpro purification paragraph. Solutions of WT and mutant Mpro variants – 84.5 ng/reaction in 30 μl buffer –  were prepared according to the kit’s manual, to reach a final concentration of 50 nM in 50 μl. 10 μl of five-fold excess to tested nirmatrelvir concentrations (180 μM) were added to the 30 μl of Mpro solution and incubated for 30 minutes. Then, 10 μl of a fluorescent substrate (Ac-Abu-Tle-Leu-Gln-MCA) were added to get a final concentration of 40 μM. This generates a 1:5 dilution of the excess of protease inhibitors – and therefore final concentrations – and the reactions were incubated for 4 hours. Fluorescence was measured by excitation at 365 nm and read-out at 415–445 nm emission with a Glomax Explorer fluorometer (Promega).The biochemical assay used to confirm mutations was based on the 3CLpro cutting reporter: Oligonucleotides for the “short” (GCAGTGCTCCAAAGCGGATTTCGC) and the “long” (ATCACGAGTGCAGTGCTCCAAAGCGGATTTCGCAAAATGGCC) cleavage sequence of Mpro (nsp4/nsp5 autocleavage site), flanked by N-terminal AgeI and a C-terminal HpaI restriction enzyme sites were ordered at Eurofins. The annealed oligos were ligated into a vector presenting N-terminally the fragment 1 (F[1]) and C-terminally the fragment 2 (F[2]) of the Renilla luciferase harboring the corresponding restriction enzyme sites (pcDNA3.1 backbone vector) as previously described  were pre-incubated with inhibitor (10μM) or DMSO for 15 minutes at room temperature (R.T.). After that, 25 μl of cell suspension were added and the mixture was incubated for 3 hours at R.T. The cleavage reaction was stopped by the addition of 5x SDS-Loading buffer. Before the SDS-Page the samples were cooked at 95°C for 10 minutes. Western blot images were obtained using the ChemiDoc MP from Bio-Rad.The cells expressing the M4 BHK-21 cells per well were seeded in 48-well plates one day before infection. Cells were infected in duplicates with a MOI of 0.05 of VSV-Mpro WT or VSV-O-Mpro and indicated concentrations of nirmatrelvir were added to the wells. After 48 hours, supernatants were collected and titrated with TCID50. For quantification, the 50 % tissue culture infective dose (TCID50) assay was performed as described previously  for the gain-of-signal assay and minus 3 or higher (negative slope) for the loss-of signal assay.However, under some circumstances, slightly bell-shaped curves arise due to an initial signal in- or decrease , followed by a signal peak and then a de- or increase. In the On assay, the decrease at the end occurs because of inhibitor toxicity at high concentrations. In the Off assay, the initial increase can be attributed to the fast death of cells in the absence of any inhibitor, whereas cells die slower at small doses, allowing for more cell division and thereby more “substrate” (cells and virus) generating dsRed. The bell-shaped curves can cause overfitting with the sigmoidal models, leading to extremely steep de- or increasing curves. Although in general, this does not lead to important changes in ICMost of the molecular modeling operations, such as the structure preparation, the setup for molecular dynamics (MD) simulations, and trajectory analyses, were performed on a Linux workstation, equipped with a 20 cores Intel Core i9-9820X 3.3 GHz processor, running the Ubuntu 20.04 operating system. Molecular dynamics simulations were carried out on an in-house Linux GPU cluster composed of 20 NVIDIA devices ranging from GTX1080Ti to RTX3090.pro mutations on Nirmatrelvir resistance. In detail, a recently developed enhanced sampling molecular dynamics method named Thermal Titration Molecular Dynamics (TTMD)  , 54 was s (TTMD) . The toppro complex were retrieved from the Protein Data Bank (PDB)   and prepnk (PDB) . Particupro proteases was considered in the simulated systems.The catalytically competent dimeric conformation of the protease was reconstructed by applying symmetric crystallographic transformations. Inconsistencies in the experimental structure were checked and fixed through the “structure preparation” tool. Missing hydrogen atoms were added according to the predominant tautomeric and protomeric state of each residue at pH = 7.4 through the “Protonate3D” module. Finally, each water molecule was removed before storing the prepared complex structure for subsequent calculations. In the end, a 1:1 protein-ligand non-covalent complex between nirmatrelvir and various MEach complex obtained in the previous step was prepared for MD simulation making use of various tools provided within Visual Molecular Dynamics 1.9.2 (VMD)  and the Before the production phase, a two-step equilibration process was performed. First, 0.1 ns of simulation in the canonical ensemble were carried out, imposing harmonic positional restraints on both the protein and ligand atoms. Then, 0.5 ns of simulation in the isothermal-isobaric ensemble were performed, with harmonic positional restraints applied only on the protein backbone and ligand atoms. In each case, a 5 kcal mol−1 Å−2 force constant was imposed on each restrained atom for the whole duration of the simulation, and the temperature was maintained at a constant value of 300 K through a Langevin thermostat . In the All MD simulations were conducted exploiting the ACEMD16 3.5 engine, which is based upon the open-source Python library for biomolecular simulations OpenMM . A 2 fs As thoroughly described in the work of Pavan et al. , Thermalgithub.com/molecularmodelingsection/TTMD.The TTMD workflow relies on a series of short classic molecular dynamics simulations, defined as “TTMD-steps”, performed at progressively increasing temperatures. The temperature increase is used to augment the kinetic energy of the system, thus shortening the simulation time required to observe protein-ligand unbinding events compared to classic MD simulations. To monitor the progress of the simulation, the conservation of the native binding mode is evaluated through a protein-ligand interaction fingerprint (PLIF) based scoring function . The proIn detail, the user must define a “temperature ramp”, i.e., the number, the temperature, and the length of each “TTMD-step”. Consistently with previous works on the target , 54, in The PDB entries 7ALI , 8DZ2 4, and 7TLThe stability of all the mutations was calculated using the default settings of the residue_scanning_backend.py module in Bioluminate –47 releahttps://github.com/donaldlab/OSPREY3.For the affinity predictions in Osprey version 3.3 , 98–100 Supplement 1