However, the underlying mechanisms are not fully understood. In this study, we conducted Single-cell RNA sequencing (scRNA-seq) and single-cell T cell receptor sequencing (scTCR-seq) analysis, which revealed a proliferative-exhausted MCM+FASLGlow CD8+ T cell phenotype in severe/critical COVID-19 patients. These CD8+ T cells were characterized by G2/M cell cycle arrest, downregulation of respiratory chain complex genes, and inhibition of mitochondrial biogenesis. CellChat analysis of infected lung epithelial cells and CD8+ T cells found that the galectin signaling pathway played a crucial role in CD8+ T cell reduction and dysfunction. To further elucidate the mechanisms, we established SARS-CoV-2 ORF3a-transfected A549 cells, and co-cultured them with CD8+ T cells for ex vivo experiments. Our results showed that epithelial galectin-3 inhibited the transcription of the mitochondrial respiratory chain complex III/IV genes of CD8+ T cells by suppressing the nuclear translocation of nuclear respiratory factor 1 (NRF1). Further findings showed that the suppression of NRF1 translocation was associated with ERK-related and Akt-related signaling pathways. Importantly, the galectin-3 inhibitor, TD-139, promoted nuclear translocation of NRF1, thus enhancing the expression of the mitochondrial respiratory chain complex III/IV genes and the mitochondrial biogenesis of CD8+ T cells. Our study provided new insights into the immunopathogenesis of COVID-19 and identified potential therapeutic targets for the prevention and treatment of severe/critical COVID-19 patients.Previous research suggested that the dramatical decrease in CD8 Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019, it has triggered a pandemic of acute respiratory disease known as “coronavirus disease 2019” (COVID-19). Emerging variants of SARS-CoV-2 are still ravaging many countries, causing breakthrough infections and repeated infections, which are costly to public health and global economics . The dys+ T cells. However, clinical studies of circulating immune cells and lung samples have documented a sharp decrease in CD8+ T cells in severe/critical COVID-19 patients [+ T cells from severe/critical COVID-19 patients exhibit impaired functions and a limited expansion phenotype compared with mild or moderate cases, despite highly expressed proliferation genes [Effective control of the acute viral infection requires the robust expansion of effector CD8patients ,6. Recenon genes ,8. The u+ T cells are activated, proliferated, and eventually expanded, and the cellular metabolism and mitochondrial function are the main determinants of this process [+ T cells rely on the enormous energy generated by oxidative phosphorylation and glycolysis to complete the cell cycle [+ T cells. Therefore, insufficient cellular metabolism and mitochondrial energy generation may be responsible for the impaired function and limited expansion of CD8+ T cells in severe/critical COVID-19.During viral infection, pathogen-specific CD8 process ,10. Durill cycle ,12. The + T cell phenotype with cell cycle arrest and impaired mitochondrial function was observed in severe/critical COVID-19 patients. Our ex vivo experiments further indicated that SARS-CoV-2 ORF3a induced the galectin-3 expression in lung epithelial cells and inhibited the transcription of mitochondrial complex genes and the mitochondrial biogenesis of CD8+ T cells, which was responsible for the cell cycle arrest and low expansion of CD8+ T cells. Our findings contributed to better understanding the mechanisms of immune dysregulation during severe SARS-CoV-2 infection and provided novel targets for developing COVID-19 treatments.In this study, through single-cell RNA sequencing (scRNA-seq) in combination with T cell receptor sequencing, a proliferative-exhausted CD8Ever since SARS-CoV-2 first appeared, researchers have been trying to understand how the immune system works during each stage of COVID-19. In this study, we first analyzed the scRNA-seq data of bronchoalveolar lavage fluid (BALF) to measure the T cell responses against SARS-CoV-2 and to investigate the ways by which COVID-19 patients developed into a severe or even critical situation. The Shared Nearest Neighbor (SNN) modular optimization-based clustering algorithm, implemented in Seurat v4, was utilized to identify clusters of BALF cells. Visualization of these identified clusters was achieved by Uniform Manifold Approximation and Projection (UMAP). A total of 27 distinct clusters were shown by clustering analysis, covering diverse cell types in the respiratory system A. Major + T cells and CD8+ T cells in severe/critical patients accounted for 32.04% and 34.63%, while CD4+ T cells and CD8+ T cells, respectively, accounted for 23.24% and 62.36% in moderate patients  C. For thpatients D. The prCD8+, cytotoxic signatures (GZMB and PRF1), and inflammatory proteins (S100A4 and S100A10), and mostly emerged at the stage of severe/critical COVID-19. Meanwhile, cluster 2 displayed high levels of proliferation markers   were also decreased in cluster 2, which revealed that the cellular function of cluster 2 was impaired. Notably, cluster 2 (CD8+MCM+FASLGlow cells) was a group of abnormal CD8+ T cells mostly from severe/critical patients and highly expressed proliferative genes, which conflicts with CD8+ T cell reduction in severe/critical patients. At the transcriptional level, MCM+FASLGlow CD8+ T cells were found to highly express exhausted genes such as HAVCR2 and LAG3  F. The leand LAG3 F.+ T cells from the BALF cells to further interrogate the key role of MCM+FASLGlow CD8+ T cells in severe/critical COVID-19 patients. The characteristic signatures of CD8+ T cells and other cells were identified, and the varying degree of the expression of signatures in specific cell clusters was visualized. Finally, 3870 CD8+ T cells displayed signatures of CD3 and CD8, and were isolated from BALF cells to perform subsequent analysis. These CD8+ T cells were clustered into 13 subpopulations and were clearly differentiated according to their sample origin  from severe/critical patients was much lower than that of corresponding moderate patients , UQCRB, UQCRQ, UQCRH, UQCR10 (encoding complex III), COX4I1, COX6A1, COX6C, and COX7A2 (encoding complex IV), but C10 had very low relative expression of these genes  were upregulated in C10 compared to C3  were infected with SARS-CoV-2 ORF3a lentivirus, and a stable cell line (termed A549-3a) was generated after puromycin selection. The production of the ORF3a protein in A549-3a cells was subsequently detected and confirmed by Western blot A. Next, 49 cells B. Moreov49 cells C. These + T cells ex vivo, we designed the A549-3a/CD8+ T cell co-culture system and the experimental procedure is shown in + T cells were harvested and detected. The qPCR results showed that after co-culture with A549-3a, the gene expression levels of the mitochondrial complex III/IV of CD8+ T cells were significantly reduced . This suggested that lung epithelial cells infected with SARS-CoV-2 ORF3a could downregulate mitochondrial complex-related gene expression and impair mitochondrial biogenesis in CD8+ T cells.To study the interaction between lung epithelial cells and CD8 reduced E,F. Amon+ T cells in severe/critical COVID-19. To investigate the role of galectin-3 in the impaired mitochondrial biogenesis of CD8+ T cells in COVID-19 patients, TD-139, a high-affinity inhibitor of galectin-3, was utilized in the A549-3a/CD8+ T cell co-culture system. As speculated, the transcriptional repression of mitochondrial complex III/IV genes  was differentially rescued with TD-139 treatment , a transcription factor involved in a wide range of gene expressions and mitochondrial biogenesis, has received considerable attention in recent years ,22,23. Hor group B,C. Take+ T cells in the lower chamber were collected and proteins were extracted and then examined. Quantification and statistical analysis of the results identified 25 differential proteins in CD8+ T cells, and ERK, GSK-3, and EGFR were the most significant downregulated proteins in the inhibitor-treated group .Naive CD8nfection . Our sin+ T cell clusters in severe/critical COVID-19 patients were less clonal than those in corresponding clusters in moderate patients, which indicated that these cells did not effectively proliferate to effector CD8+ T cells as they should. Moreover, CD8+ T cells derived from severe/critical patients showed an increased proportion of cells in the S and G2/M phases, which suggested a blockade of the cell cycle process. The pseudo-time analysis also demonstrated that these two populations were at the end of the developmental trajectory, implying that these “proliferative-exhausted” cells may represent a divergent destination for naive CD8+ T cells. Although C10 and C3 shared the same proliferative phenotype, C10 exhibited an obvious G2/M blockade with a high level of G2/M gene expression. Gene Set Enrichment Analysis (GSEA) revealed that several signaling pathways were involved in the negative regulation of the cell cycle in C10. These results suggested that these two CD8+ T cell clusters could be regulated differently and lead to different transcriptional activities.TCR immune repertoire analysis also revealed that these two CD8+ T cells rapidly change their metabolic energy supply from a resting state to increased mitochondrial metabolism [+ T cells with cell cycle arrest had impaired mitochondrial biogenesis. Genes encoding subunits of electron transport chain (ETC) complexes were downregulated at the transcriptional level, leading to an insufficient energy supply to meet the consumption required for cell expansion. This could explain why the specific CD8+ T cell subsets in severe/critical patients expressed proliferation-related signatures, but ultimately did not lead to T cell expansion: they could not complete mitosis.Energy metabolism plays a crucial role in the proliferation and expansion of immune cells. Upon stimulation, CD8tabolism . Our sin+ T cells. Galectin-3, a member of the β-galactoside-binding lectin family, was highly expressed by epithelial cells and had a strong effect on C10 of CD8+ T cells in patients with severe/critical COVID-19. ORF3a, an accessory protein of SARS-CoV-2, plays an important role in the immunopathogenesis of COVID-19. We transfected the lung epithelial cells with ORF3a (named A549-3a), and co-cultured A549-3a with CD8+ T cells. The ex vivo studies further verified that SARS-CoV-2 ORF3a expression upregulated various pro-inflammatory factors and proteins, including galectin-3 in A549-3a, and epithelial galectin-3 signaling downregulated mitochondrial biogenesis and promoted the G2/M cell cycle arrest of CD8+ T cells.Previous studies have reported that galectin-3 upregulation can serve as a prognostic biomarker in COVID-19 patients ,30, but + T cells were co-cultured with lung epithelial cells transfected with ORF3a, NRF1 activation and nuclear translocation were inhibited, and the expression of mitochondrial complex genes was downregulated in CD8+ T cells. In the co-culture system, TD-139, a galectin-3 inhibitor, promoted the NRF1 activation and nuclear translocation, and restored the expression of mitochondrial complex genes. Other galectin-3 inhibitors have also been reported to ameliorate mitochondrial damage in the hearts of obese rats [NRF1 is a nuclear respiratory factor that initiates the transcription of multiple mitochondrial respiratory chain-related genes ,34. Whenese rats . These f+ T cells, which was accompanied by promoting the transcription of mitochondrial complex genes. In contrast, Akt phosphorylation was upregulated in the galectin inhibitor-treated group. Akt phosphorylation promoted the activation of PGC-1α, an important regulator of mitochondrial biogenesis that enhances respiratory capacity and maintains homeostasis [+ T lymphocyte response to interferon signals and antiviral function.Our comparative analysis of galectin-3 inhibitor-treated and untreated groups revealed multiple changes in different kinases, particularly in ERK- and Akt-related signaling pathways. ERK 1/2 is a master of transduction signals from surface receptors to the nucleus. In tumors and neurodegenerative diseases, ERK 1/2 inhibited mitochondrial biogenesis by phosphorylating different effector molecules ,37. Conseostasis . The reg+ T cell subset in severe/critical COVID-19 patients, which was characterized by abnormally high proliferative gene expression and cell cycle arrest. Galectin-3 produced by epithelial cells with a high expression of SARS-CoV-2 ORF3a inhibited the transcription of mitochondrial respiratory chain complex genes and resulted in mitochondrial biogenesis impairment and insufficient energy supply, eventually preventing the processes of CD8+ T cell proliferation and expansion. Blocking galectin-3 could be an effective solution to relieve the cell cycle arrest of CD8+ T cells in COVID-19 patients and restore their expansion ability and cell function. TD-139, a galectin-3 inhibitor, has been already available as a treatment for idiopathic pulmonary fibrosis (IPF) [In conclusion, our study identified a specific CD8HEK293T cell line and A549 cell line in our study were obtained from the American Type Culture Collection . The pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro plasmid was constructed by Nevan Krogan lab, and provided by Addgene, Watertown, MA, USA. Lentiviral particles were packaged by the 2nd Generation Packaging System, in which the packaging plasmid GAG and the envelope plasmid VSV-G were obtained from Addgene, USA.We obtained the single-cell RNA sequencing and TCR sequencing data of COVID-19 from GEO; the accession numbers were GSE145926 and GSE128033. These datasets provided single-cell sequencing data of BALF from four healthy subjects (HC1–HC4), three moderate COVID-19 patients (M1–M3), and six severe/critical infection patients (S1–S6), in which BALF samples from COVID-19 patients also underwent TCR sequencing . These Chttps://satijalab.org/seurat/, accessed on 5 May 2022). First, the cells were filtered based on the standard that the number of expressed genes was less than 200 or greater than 6000, and the percentage of mitochondrial genes was greater than 0.2. In addition, the filtered expression matrix was normalized using the ‘LogNormalize’ method and the scaling factor was set to 10,000. Using the “vst” method in the Seurat function “FindVariableFeatures”, the top 2000 variable features were identified. The expression values of all genes were transformed by z-score using the “ScaleData” function to obey the normal distribution. Then, principal component analysis (PCA) was performed through the top 2000 variable genes, and the data were dimensionality reduced according to the first 20 principal components. Next, based on the PCA-reduced data, the “FindClusters” function was used for cluster analysis, and the resolution was set to 0.6. Finally, the Unified Manifold Approximation and Projection (UMAP) analysis was performed using the first 20 principal components for visualization.We performed subsequent calculations on the count matrices using the R package Seurat (CD79A), CD4+ T cells (CD3E and CD4), CD8+ T cells , NK cells , macrophages , neutrophils (FCGR3B), and epithelial cells (KRT18). To ensure the accuracy of the cell-type assignments, the SingleR algorithm  was also performed independently. For single-cell immune repertoire data analysis, we utilized the scRepertoire package (v1.0.2) combined with the Seurat package to display the clonotype distribution of clusters defined by the scRNA-seq data.We initially annotated the cell clusters based on canonical cell markers as follows: B cells . Specifically, we focused on CD8+ T cells and identified different branches in the cell trajectory that distinguished various cell states. By considering relevant biological background, we determined the “root” starting point and inferred the order of cell population differentiation. Additionally, we employed Monocle 2 to further investigate the cluster and lineage relationships of two specific cell clusters in CD8+ T cells. To investigate the cell cycle arrest of T cells, we utilized the R package reCAT  and applied default and recommended parameters for the pipeline.Cell differentiation trajectories were reconstructed using the pseudo-temporal inference algorithm Monocle 3 . The fgsea results were initially screened against the criterion of adjusted p-value < 0.05. For specific analysis, the downregulated mitochondria-related pathways from the GSEA table were selected based on the normalized enrichment score < 0. In addition, the comparison results of related clusters were visualized via ggplot2 (v3.4.1).We performed gene set enrichment analysis (GSEA) using the R package fgsea and the C5, the Ontology Gene Sets from the MsigDB database  was performed to evaluate cell-cell interactions according to the expression of known ligand–receptor pairs in different cell types. Specifically, we added more than 100 related ligand–receptor pairs following official guidance from CellChat for further analysis of the galectin pathways. The results were visualized and displayed by relevant functions within the CellChat package.CellChat v1.5.0  were transfected into 293T cells and incubated for 48 h. Then we collected the supernatant containing lentiviral particles and filtered it through 0.22 µm filters. Lentiviral particles infected A549 cells with polybrene  at a final concentration of 8 μg/mL. The A549 cells expressing SARS-CoV-2 ORF3a (A549-3a in abbreviation) were selected with puromycin, and ORF3a expression in A549 was verified by Western blot assay.www.ncbi.nlm.nih.gov, accessed on 27 July 2022).We extracted RNA from cell samples using the RNAsimple RNA Kit  following the manufacturer’s instructions. The extracted RNA was subsequently reverse transcribed into cDNA by ABSScript II RT Mix . Real-time PCR was performed using 2 × Universal SYBR Green kit , and fluorescent signals were collected by CFX96 PCR instrument . Three runs were performed for each sample. The results were analyzed and examined as the relative mRNA levels based on cycle threshold (CT) values, which were converted to fold changes. The primers were designed using Primer-blast, NCBI  for 20 min on ice. Protein concentration was determined using the BCA protein concentration assay kit . Equal amounts of proteins (2 μg/μL) were loaded onto 10% gels and separated by SDS-PAGE. Subsequently, they were electrophoretically transferred to PVDF membranes. Non-specific areas on the PVDF membrane were blocked by 5% non-fat milk in PBST for 1 h. Next, we incubated the membranes with primary antibodies  overnight at 4 °C. We then incubated them with HRP-coupled goat anti-rabbit IgG secondary antibody for 1 h at room temperature. Finally, the protein bands were visualized using an ECL kit  and imaged on ChemiDoc XRS+ .+ T cells were positive selected from PBMC using magnetic beads coated with CD8 antibodies . These CD8+ T cells were placed into the Transwell lower chamber along with CD28 antibody  and IL-2 . A549 or A549-3a cells were seeded in Transwell inserts . For certain experiments, cells were treated with the galactin-3 inhibitor (TD-139) . The co-culture system was incubated for 48 h at 37 °C and 5% CO2.The 24-well plates were coated with CD3 antibody  overnight. PBMCs were isolated from the peripheral blood of healthy donors using Human PBMC Isolate kit . CD8+ T cells, we used a human phosphokinase array kit  following the manufacturer’s protocol. Firstly, cell lysates were prepared and incubated on membranes containing human phosphokinase antibodies overnight at 4 °C. Then, detection antibody cocktail and membranes were incubated at 4 °C. After washing, HRP-conjugated secondary antibody cocktail was incubated on membranes. Chemi Reagent Mix was subsequently added on membranes, and dot blots were visualized via chemiluminescence . Finally, we used Fiji software  to quantify spot signals.To explore important signaling in CD8+ T cells were seeded on climbing slides at a density of 106 cells/well. The cells were then fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 20 min, and blocked for 1 h. NRF1 antibody  as the primary antibody was added on slides and incubated overnight at 4 °C. Rhodamine (TRITC) conjugated goat anti-rabbit IgG (H + L) was then incubated for 1 h at room temperature. Slides were then counterstained for nuclei and sealed with antifade mounting medium with DAPI. Immunofluorescence staining was visualized using a laser scanning confocal microscope  with an excitation wavelength of 561 nm and a 100× silicone oil lens. Relative NRF-1 nuclear/cytosolic fluorescence ratios were quantified using Fiji software v1.4.CD8t-test (GraphPad Prism 7). To determine the statistical significance of the results, we used the following criteria: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Differences meeting these criteria were considered statistically significant, while those marked as ns indicated no significant difference.Single-cell RNA-sequencing and TCR-sequencing data analysis was performed using R (v4.0.2) for informatics analysis and graphics processing. All experiments were independently repeated at least three times with similar results. Statistical analysis was performed between the two groups using


Transcription activator-like effectors (TALEs) have been widely used for genome editing, transcriptional regulation, and locus-specific DNA imaging. However, TALEs are difficult to handle in routine laboratories because of their complexity and the considerable time consumed in TALE construction. Here, we described a simple and rapid TALE assembly method based on uracil-specific excision reagent (USER) cloning. Polymerase chain reaction was amplified with TALE trimer templates and deoxyuridine-containing primers. The products were treated with USER at 37°C for 30 min, followed by the treatment of T4 DNA Ligase at 16°C for 30 min. The TALE trimer unit could be rejoined hierarchically to form complete TALE expression vectors with high efficiency. This method was adopted to construct TALE-deaminases, which were used in combination with Cas9 nickases to generate efficient C-to-T or A-to-G base editing while eliminating predictable DNA off-target effects. This improved USER assembly is a simple, rapid, and laboratory-friendly TALE construction technique that will be valuable for DNA targeting. The transcription activator-like effector (TALE) and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 architecture have been used extensively for mastering and programming gene functions and applied to the advancement of agriculture and medicine. The DNA-binding domain of TALEs comprises an extended array of 34 amino acid (AA) repeats. Each repeat has one of four distinct repeat variable di-residues (RVDs) that differ only at AA positions 12 and 13 and confer nucleotide-binding specificity  [Although CRISPR/Cas9 is currently the most used system for gene editing due to its simplicity and convenience , 4, TALEWe recently generated TaC9-BE, the base editor with dual guides, including Cas9/sgRNA and TALE, which induced efficient C-to-T or A-to-G conversion without detectable predictable DNA off-target mutations , 20, proHere, we improved uracil-specific excision reagent (USER)-based cloning for TALE assembly. USER is composed of uracil DNA glycosylase, which catalyzes the excision of a uracil base and forms an apyrimidinic (AP) site, while endonuclease VIII breaks the phosphodiester backbone at the 3ʹ and 5ʹ sides of the AP site to release base-free deoxyribose. TALE trimers  were amp® Hot Start High-Fidelity DNA Polymerase . All PCR products with dU were excised with Thermolabile USER® II Enzyme  and assembled with T4 DNA Ligase  or Hi-T4 DNA Ligase . In constructing all the single-guide RNA (sgRNA), two complementary oligonucleotides were synthesized by Guangzhou IGE Biotechnology Ltd., annealed via PCR , and cloned into the BbsI site of the sgRNA expression vector by using T4 DNA Ligase . All sgRNA and TALE target sequences are listed in TALE backbone with enhanced green fluorescent protein (EGFP) and the TALE Trimer Library were obtained from our previous research . All USEDelR153-mcherry was constructed on the basis of pCMV-YE1-BE4max-mcherry which was obtained from our previous research. All subcloning experiments were conducted with DH5α competent cells. All restriction endonucleases were purchased from Thermo Fisher Scientific.pCMV-ABE8E2. Then, they were seeded on 24-well plates (Greiner Bio-One) and transfected at approximately 90% density. After 24 h, 250 ng of sgRNA, 500 ng of plasmids with nCas9, and 300 ng of plasmids with TALE were transfected using 1 μl of Lipo8000 Transfection Reagent  per well. After 3 days of transfection, cells were collected and filtered through a 40 μm strainer , and the transfected cells were collected on a Beckman Coulter MoFlo Astrios Cell Sorter (laser option: blue 488 nm and red 561 nm). About 10 μl of NP40 Lysis Buffer was added to the collected cells, and the reaction was incubated at 56°C for 1 h. The products were used as a template for amplification.Human HEK293T and Rabbit fibroblasts cells were cultured in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% fetal bovine serum (Gibco) at 37°C under 5% COThe target sequence was amplified from the cell lysate by PCR  with specific primers. The products were then sent out for Sanger sequencing (Guangzhou IGE Biotechnology Ltd.). The PCR results of amplicon were quantified using EditR. All PCR primers are listed in p < 0.01.The GraphPad Prism software (version 8.4.3) was used for data analysis. The data are presented as the mean ± SEM. All tests conducted were two-way ANOVA-test, and the difference was considered significant at *A TALE repeat contains 34 AAs with RVD at positions 12 and 13 . We developed a TALE trimer library that contained 64 combinations for targeting NNNs , namely, U1-F/R, U2-F/R, U3-F/R, U4-F/R, U5-F/R, and U6-F/R, were generated, each with an 8–10 bp overlap at their 5ʹ ends before dU . The firACCCACCCCGCCCCGGCA) was presented as an example. The trimers of ACC, CAC, CCC, GCC, CCG, and GCA were amplified using dU primers U1-F/R, U2-F/R, U3-F/R, U4-F/R, U5-F/R, and U6-F/UR+3, respectively. The TALE vector was amplified with primer UT-F/R. The linearized backbone and six ordered trimers with dU were treated with USER and mixed for ligation. Four schemes were designed as follows (10), and Group 4 .The common TALE length is about 12–21 repeats. Here, the construction of 18 TALE repeats for targeting Protein Phosphatase 1 Regulatory Subunit 12C (PPP1R12C) site 2  in all five target sites  locus were tested with nCas9/guide RNA (gRNA) in the TaC9 system. The target site sequences are listed in ene loci . HEK293Tet sites . Similar3 system . TaC9-ABEDelR153 . To furt1-BE4max . The resDelR153, resulting in high-efficiency A-to-G conversion (60%) in all five sites  from a single polycistronic gene. The transfer RNA (tRNA) was engineered to separate each sgRNA . Then, tve sites . For C-tve sites . This reIn this study, we used iUSER cloning to construct either TALE or gRNA tandem vectors with a simple, rapid, and efficient process. Although USER cloning has been used in TALE assembly, it can only form pentamers, and thus, another step of Golden Gate cloning is required to finish only half of the whole TALE assembly . The twoThe USER cloning-rejoined TALE repeats formed pentamers in a ligation-independent manner similar to other ligation-independent cloning , 23. HowBoth TALE and CRISPR/Cas9 are powerful tools for genome editing. However, TALE technology has some advantages over CRISPR/Cas9 in certain aspects. Firstly, due to gRNA may tolerate to mismatched DNA targets, CRISPR/Cas9 could cause the potential off-targets, that cuts DNA at unintended loci. In contrast, TALE recognizes DNA targets through specific protein-DNA interactions, leading to a lower likelihood of off-target effects . SecondlIn summary, TALE technology has advantages over CRISPR/Cas9 in terms of target specificity, lower risk of off-target effects, and smaller size. Here, we developed a novel method to construct TALE with simple and rapid way, which would assist TALE for wildly using on gene targeting.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Rabies remains a great threat to public health worldwide. So far, the mechanism of rabies virus (RABV) infection is not fully understood, and there is no effective treatment for rabies. Identifying more host restriction factors of RABV will spur the development of novel therapeutic interventions against rabies. Accumulating studies suggest that tripartite motif-containing (TRIM) proteins have great effects on virus replication. TRIMs control the antiviral responses through either direct interaction with viral proteins or indirect regulation of innate immune signaling molecules in the host. The role of TRIM25 in rabies virus (RABV) infection is poorly understood. Using next-generation sequencing, we found that TRIM25 is upregulated during HEP-Flury infection. Knockdown of TRIM25 enhances HEP-Flury production, while overexpression of TRIM25 suppresses HEP-Flury replication. Knockdown of interferon α and interferon β weakens the anti-RABV response induced by TRIM25 overexpression, and potentiates RABV production. Furthermore, we found that TRIM25 regulates type-I interferon response by targeting retinoic acid-inducible gene I (RIG-I) during HEP-Flury infection. Knockdown of RIG-I weakens the anti-HEP-Flury response induced by TRIM25 overexpression, indicating that TRIM25 regulates RABV production via the RIG-I-IFN axis. In addition, we observed that TRIM25 does not directly interact with HEP-Flury structural proteins, suggesting that TRIM25 regulates HEP-Flury production indirectly. Taken together, our work identifies TRIM25 as a new host factor involved in HEP-Flury infection, which may be a potential target for the development of antiviral drugs against RABV. Rabies is an acute inflammation of the nervous system caused by Rabies virus (RABV) infection. The fatality rate is more than 99.9% once clinical symptoms appear. Worldwide, it is estimated that 59,000 people die from rabies annually . The RABThe host’s inherent immune responses are the primary defense against infections. Notably, the type I interferon (IFNα/β) is an important innate immune response factor for inhibiting RABV proliferation ,4,5,6. FUbiquitination is one of the forms of post-translational modification, which is important in the localization, metabolism, function and degradation of proteins. Ubiquitination modification is involved in the regulation of almost all life-essential cellular activities, such as cell proliferation, apoptosis, autophagy, gene expression, signal transduction, immune response, inflammatory response, and protein degradation/non-degradation modification . E3 ubiqIn order to identify more mechanisms regulating host infection by RABV, we performed transcriptome analysis of RABV-infected mouse neuroblastoma (NA) cells. We found that the expression of some Trim proteins was altered, especially Trim25, which was significantly changed after HEP-Flury infection. The focus of this study was to investigate the function of TRIM25 in HEP-Flury infection. Our findings suggest that TRIM25 inhibits HEP-Flury infection by activating the RIG-I-mediated type-I IFN response.p < 0.01) . The results showed that several ubiquitin-associated proteins, particularly some E3 ubiquitin ligases, were significantly upregulated or downregulated upon RABV infection compared with non-infection ( < 0.01) A. In ord < 0.01) B and pro < 0.01) C. The reTRIM25 is widely involved in the host’s resistance to viral infection ,35,36. BType I interferons play a key role in clearing RABV during viral infection ,17. SmalIt is well known that RIG-I plays a key role in the induction of metaphase interferon production during HEP-Flury infection . We wantRecent studies have shown that TRIM25 plays a role in the ubiquitination of RIG-I ,38. To fRABV infection causes almost 100% mortality, with no treatment being available thus far. RABV infection causes almost 100% mortality when no treatment is available. Therefore, it is very important to explore the host factors that regulate RABV infection. In the present study, we observed a strong upregulation of TRIM25 expression after HEP-Flury infection. Moreover, TRIM25 is a limiting factor for HEP-Flury infection. In fact, many members of the TRIM protein family exhibit certain antiviral effects. TRIM21 is upregulated both in vivo and in vitro during viral infection, significantly inhiting porcine reproductive and respiratory syndrome virus (PRRSV) replication . InfectiThe level of a protein can be regulated at transcriptional, translational, and post-translational levels . This stThe mechanism of antiviral activity caused by host factors is associated with either the restriction of viral replication, including binding, entry, synthesis, assembly and budding, or innate immune response. TRIM25 regulates innate immune response through several mechanisms in viral infection . Our stuType-I IFN production is regulated by TRIM25 through either RIG-I or MDA5 ,40,42,50Previous studies have indicated that TRIM25 delivers polyubiquitin chains to RIG-I, which induces RIG-I-mediated antiviral activity . In thisIn the “battle” between virus and host, the virus has developed a defense mechanism through interfering with host factors. Therefore, viral components may conversely target TRIM proteins to impact TRIM protein-mediated ubiquitination. For example, nucleocapsid proteins from severe acute respiratory syndrome coronavirus PRRSV (SARS-CoV) or SARS-CoV-2 interact with TRIM25 and affect TRIM25-mediated activation of RIG-I ,43,58. NIn summary, in this study, TRIM25 is identified as a novel host resistance factor for HEP-Flury infection. We demonstrated that TRIM25 inhibits HEP-Flury replication through an RIG-I-mediated type-I IFN response , rather RABV fixed HEP-Flury strain  was propagated in N2a cells. N2a cells  were cultured and passaged in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS) (Gibco). HEK 293T cells were cultured and passaged in DMEM medium containing 10% FBS.Antibodies to Myc (sc-40) and HA (sc-7392) were purchased from Santa Cruz Biotechnology ; antibodies to RIG-I (#4200S) and TRIM25 (#13773S) were purchased from Cell Signaling Technology ; Antibody to IFNβ (ab218229) andIFNα (ab191903) was purchased from abcam ; anti-Flag antibody (AF5051), normal mouse IgG (A7028) and anti-β-actin antibody (AA128) were purchased from Beyond Technology . Anti-HEP-Flury P antibody was from our laboratory. FITC-labeled anti-RABV nucleoprotein (N) antibody was from Fujirabio Diagnostics .The pCAGGS plasmid carrying the HA tag (pCAGGS-HA), the pCAGGS plasmid carrying the Flag tag (pCAGGS-Flag) and the ubiquitin plasmid carrying the Myc tag (pcDNA3.1-Myc-Ub) were purchased from Wuhan Miaoling Biotechnology Co., Ltd. . Cloning of the TRIM25 gene from N2a cells into pCAGGS-HA using primer TRIM25-F: 5′-AGCTCATCGATGGTACCCGGGCCATGGCGGAGCTGAATCCTCT-3′ and TRIM25-R: 5′-ATTAAGATCTGCTAGCTCGAGCTATTTGGAGCAGATAGAGAGGGTG-3′ for the RIG-I gene of N2a cells was amplified using primers and cloned into the pCAGGS-Flag plasmid:RIG-I-F: 5′-GATGACGACGATAAGGAATTCATGACAGCGGAGCAGCG-3′ and RIG-I-R: 5′-ATTAAGATCTGCTAGCTCGAGTCATACGGACATTTCTGCAGGATC-3′ for overexpression of RIG-I. The structural protein gene of HEP-Flury was amplified with the primers in Consistent with our previous method, the titer of HEP-Flury was detected by direct immunofluorescence . BrieflyAdd 80% acetone to N2a cells, and then fix at −20 °C for 20 min. After the fixation, discard the liquid and wash three times, then add FITC-labeled anti-RABV N protein antibody, and incubate at 37 °C for 1 h. Cells were then observed and photographed using a fluorescence microscope.Use LIPO3000  to transfect siRNA or recombinant plasmids into HEK 293T cells or N2a cells, and use NC and blank plasmids as controls. If multiple siRNAs or plasmids need to be transfected, perform the next round of transfection 12 h after the previous siRNA or plasmid transfection.The operation was performed according to the operating instructions of the Pierce™ Cross-Linked Magnetic IP/CO-IP Kit . After the cells were transfected with the corresponding plasmid for 36 h, the cells were collected, and the cell lysate was prepared. The corresponding antibody was coupled to the cross-linked magnetic beads, and then the plasma cell lysate was incubated with the cross-linked magnetic beads at 4 °C for 10 h, and the magnetic beads coupled with normal IGg were used as a control. The protein was eluted from the magnetic beads and detected by Western blot (WB) using the corresponding antibody.The operation of Western blotting was consistent with that described in our past studies . BrieflyThe cells were collected, and then the total RNA in the cells was extracted using TRIzol reagent . Total RNA was subjected to reverse transcription (RT) using Transcription First Strand cDNA Synthesis Kit . Real-time quantitative PCR was then carried out in a CFX connect real-time system  according to the operating instructions of SYBR Green Master Mix (Vazyme Biotech). According to the transcription level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, the gene to be detected was standardized and calibrated. Primer sequences for TRIM25 were used: forward, 5′-TCTGTAGGAGTCAAGGCTAAGGTG-3′, reverse, 5′-GTTGTGGGCGGTATTGTAGTCG-3′. The primer sequences for gRNA, GAPDH, IFNα, and IFNβ have been introduced in previous studies .t-test was used to analyze the experimental results. p < 0.05 was considered statistically significant.Experimental data were summarized and analyzed using GraphPad Prism 6 software . Student’s


Enterovirus A71 (EV-A71) infection is a major cause of severe hand, foot and mouth disease (HFMD) in young children. The characteristics of EV-A71 neutralizing antibodies in HFMD patients are not well understood. In this study, we identified and cloned EV-A71-neutralizing antibodies by single cell RNA and B cell receptor sequencing of peripheral blood mononuclear cells. From 145 plasmablasts, we identified two IgG1 monoclonal antibodies (mAbs) and six IgM mAbs that neutralized EV-A71. Four of the IgM mAbs harbor germline variable sequences and neutralize EV-A71 potently. Two genetically similar IgM antibodies from two patients have recurrent heavy chain variable domain gene usage and similar complementarity-determining region 3 sequences. We mapped the residues of EV-A71 critical for neutralization through selection of virus variants resistant to antibody neutralization in the presence of neutralizing mAbs. The residues critical for neutralization are conserved among EV-A71 genotypes. Epitopes for the two genetically similar antibodies overlap with the SCARB2 binding site of EV-A71. We used escape variants to measure the epitope-specific antibody response in acute phase serum samples from EV-A71 infected HFMD patients. We found that these epitopes are immunogenic and contributed to the neutralizing antibody response against the virus. Our findings advance understanding of antibody response to EV-A71 infection in young children and have translational potential: the IgM mAbs could potentially be used for prevention or treatment of EV-A71 infections. Hand, foot and mouth disease (HFMD) caused by enterovirus infections affects millions of young children annually. Enterovirus A71 (EV-A71) is a major causative pathogen of severe HFMD. EV-A71-specific antibody response is induced rapidly after illness onset, but the characteristics of primary antibody response to EV-A71 in young children are not well understood. Previous study demonstrated a dominant IgM producing EV-A71-specific antibody-secreting B cell response in children under 3 years old. However, no EV-A71-specific human IgM mAb has been reported. Here, we identified EV-A71 neutralizing mAbs, including two IgG1 and six IgM mAbs, from plasmablasts of EV-A71 infected children. IgM mAbs neutralize EV-A71 potently although possess few somatic mutations. We found sequence similarities between IgM antibodies from two patients. We selected EV-A71 variants in the presence of neutralizing mAbs and demonstrated that residues critical for neutralization are conserved among EV-A71 genotypes. We further explored epitope specific antibody responses in acute phase sera of EV-A71 infected patients and found that these neutralizing epitopes on the virus are immunogenic. Our findings advance understanding of antibody response in EV-A71 infected HFMD patients and can help inform the development of therapies against EV-A71 infections. Hand, foot and mouth disease (HFMD) is an infection that commonly affects children caused by one of various enteroviruses, most commonly enterovirus A71 (EV-A71), coxsackievirus A16, and coxsackievirus A6 ; HFMD epPicornaviridae, genus Enterovirus, species Enterovirus A. It is a non-enveloped virus containing a single-stranded, sense-strand, polyadenylated RNA of approximately 7400 nucleotides [EV-A71 belongs to the family leotides . Scavengleotides . Severalleotides .Humoral immunity is crucial for protection from EV-A71 infections. Maternal antibodies transferred across the placenta before birth and through breastfeeding may be protective against HFMD in young children –11. No aAntibody responses in HFMD patients with EV-A71 infections are not well understood. In the acute phase of EV-A71 infection, 80% of patients were found to have neutralizing antibodies against EV-A71 (≥ 1:8 antibody titer) one day after the onset of illness . No corrTwo publications have reported identification and characterization of mAb from EV-A71-infected children , 17. CheIn this study, we focused on the antibody response in young children during acute EV-A71 infection. This group has the highest risk of severe HFMD, but rarely has the antibody response been studied in this detail. We produced and characterized IgM and IgA mAbs in addition to IgG antibodies. Our objectives were to identify and characterize EV-A71 neutralizing mAbs, to map the epitopes on EV-A71 genotype C4 critical for neutralization, and to explore the epitope specific serologic response in young children during acute EV-A71 infection. We found potent neutralizing antibodies and determined the residues which were involved in the neutralization process. These residues are conserved among EV-A71 genotypes. Genetically similar neutralizing antibodies were identified from different patients, indicating the possible preferential usage of heavy chain variable gene segments in the initial response to EV-A71 infections in humans. Our results also indicate that these epitopes are immunogenic in EV-A71 infected HFMD patients.S1 Table). Three of them were severe HFMD patients who had central nervous system complications; the other three patients had mild HFMD without complication. All six patients recovered and discharged from hospital within 2 weeks. One PBMC sample was collected from each patient during hospitalization at one to five days after illness onset and was subjected to single-cell RNA sequencing (scRNA-seq) to obtain single-cell B cell receptor (BCR) sequences and gene expression data using the 10X Genomics Single Cell V(D)J Enrichment kit and Single Cell 5’ Library and Gel Bead kit. EV-A71 RNA is detectable by polymerase chain reaction in throat swabs or stool samples collected at the same day or one to two days after the collection of PBMC sample. It has been reported that EV-A71 viral load declines with increasing time after illness onset [Six children had hand, foot and mouth disease (HFMD) with laboratory-confirmed enterovirus A71 (EV-A71) infection were enrolled. The median age of participants was 25 months (range 7–35 months) , thus they were selected for further analyses of antibody responses. Plasmablasts are rapidly induced during acute infections and contribute to early antibody responses [S2 Fig) [S3A and S3B Fig). No obvious association was found between antibody isotype/subclass and disease severity. The heavy chain variable gene usage of these plasmablast antibodies was analyzed using the IMGT/V-QUEST tool [S3C and S3D Fig).Five of the six patients developed a neutralizing antibody response against EV-A71 at the time of PBMC sample collection  . A totalEST tool . The varFig 1A and 1B and S2 Table). Among the EV-A71 binding IgG1 antibodies, two neutralize EV-A71: M1-1 neutralizes C4b only, while M2-12 neutralizes both C4a and C4b (Table 1).We next sought to produce mAbs based on the BCR variable gene sequences of the plasmablasts. We firstly examined the BCR clonotype enrichment in the 228 plasmablasts. B cells that have the same variable gene usage and identical amino acids in the third complementarity determining region (CDR3) of both heavy and light chains were grouped into the same clonotypes. There are 201 unique clonotypes, including 78 IgG, 55 IgA, and 68 IgM. Among the 78 IgG clonotypes, 56 are IgG1. IgG1 is the predominant subclass of immunoglobulin in antiviral antibody responses . TherefoS4 Fig).To produce IgA and IgM mAbs in addition to IgG that may have neutralizing activity against EV-A71, we used two further screening strategies at single-cell level, rather than cluster level, to select cells from the plasmablasts defined above .The IgA/IgM containing HEK293F culture supernatants were used to measure binding and neutralization of EV-A71. Seven of the 28 (25%) IgA mAbs show weak binding activity with EV-A71 virion; none of them shows neutralizing activity. Twenty-five (67%) of the IgM mAbs are EV-A71 binding antibodies. Among them, we identified six (16%) EV-A71 neutralizing mAbs. M1-12 from donor M1 neutralizes C4b only, while M1-16, M1-17, M1-20, M1-24 from M1 and M3-7 from M3 neutralize both C4a and C4b . All antibodies from S1 had been screened before. We were able to make a further 13 IgM from M1, 6 IgM from M3, and 5 IgA antibodies from S3, from cells in the plasmablast cluster which were filtered out before by the two rules described above. Nine of the 19 IgM antibodies bind EV-A71, but no IgA mAb binds EV-A71. None of these 24 additional antibodies neutralizes EV-A71. Collectively, among the 56 IgM mAbs tested, six (10.7%) are neutralizing, while 28 (50%) are non-neutralizing EV-A71 binding antibodies . In summary, eight EV-A71 neutralizing antibodies, including two IgG1 and six IgM antibodies, were identified from 145 BCR clonotypes of five HFMD patients .Because we did not find any neutralizing antibodies from the selected plasmablasts from patients with severe HFMD, and we found potent neutralizing antibodies from the selected plasmablasts from M1 and M3, we generated and screened further antibodies from plasmablasts of cases M1, M3 and S3, not using the two selecting strategies described above . M1-12 was not included in these analyses as it does not neutralize C4a. Five of the IgM mAbs neutralize both C4a and C4b at similar concentrations, while the other one (M1-12) shows weak neutralizing activity against C4b but not against C4a (Table 1). EV-A71 specific mAbs were found in all five subjects; only the two severe HFMD patients not yielding any neutralizing mAb from plasmablast scRNA sequencing data.The neutralizing antibodies were purified and used to measure the binding and neutralizing titer. Binding of the neutralizing IgG1 and IgM mAbs with EV-A71 C4a virions was determined with ELISA assay and verified by immunoprecipitation assay . EV-A71-specific antibodies are found in five of the six heavy chain variable gene groups, except for V2 gene group. V5 genes are enriched in EV-A71 neutralizing antibodies, indicating biased variable gene usage . Several antibodies from V1, V3, and V4 gene groups bind EV-A71, but most of them have no neutralizing activity. On the contrary, a higher proportion of EV-A71 binding antibodies using V5 genes are neutralizing antibodies.A phylogenetic tree of heavy chain variable region was constructed, in which antibodies fall into groups by variable gene usage . Two neutralizing antibodies (M1-24 and M3-7) have the same heavy chain variable region gene segments and a VHCDR3 sequence identity of 83% , indicating a recurrent heavy chain variable region gene usage of antibodies induced by EV-A71 infection in different subjects, which has not been previously found in human antibodies induced by EV-A71 infection [Table 1).We further analyzed the Vnfection . M1-24 anfection –27. One Fig 4, five mAbs, M2-12, M1-17, M1-20, M1-24, and M3-7 inhibit EV-A71 infection at both pre-attachment and post-attachment stages with 8- to 53-fold higher concentrations of antibodies needed to inhibit EV-A71 infection at post-attachment stage compared with those for inhibition at pre-attachment stages. M1-16 inhibits EV-A71 infection at approximately similar concentrations at pre-attachment and post-attachment stages. Results of competition ELISAs for the EV-A71 neutralizing IgM antibodies using recombinant SCARB2 and PSGL1 show that M1-16, M1-17, M1-20, M1-24, M3-7 compete with SCARB2 for its binding with EV-A71, while only M1-16 competes with the binding of PSGL-1 .Antibodies with different neutralization mechanisms inhibit EV-A71 infection at different stages. Antibodies can neutralize EV-A71 by blocking receptor binding and inhibiting virus infection at the pre-attachment stage. Alternatively, EV-A71 can be neutralized by antibodies that block internalization or uncoating; such antibodies inhibit virus infection at both pre- and post-attachment stages , 29. NeuFig 5A, we successfully generated variants that escape neutralization of the other six antibodies. Each escaped virus contains an alteration at a single amino acid position on either the VP1 or VP2 proteins of EV-A71 . These amino acid alterations confer resistance to neutralization of the corresponding mAbs. Under the selection pressure of antibody M1-16, we identified two escape variants derived from C4a, which both contain single amino acid substitution, VP1 D94E and VP1 L97I. On the other hand, only one C4b variant containing VP1 L97I substitution was generated in the presence of M1-16. VP2 K149, the residue critical for the neutralization of M1-24, is located in the receptor binding site of EV-A71  [S7 Fig).We further explored the epitopes on the EV-A71 particle that are involved in neutralization by selection of escape variants in the presence of antibody. One EV-A71 subgenotype C4a strain and one C4b strain were used for selection. M1-1 and M1-12 have no neutralization activity against C4a; thus, they were not used for selection of C4a escape variants. We failed to generate any C4b variant viruses that could escape M1-1 and M1-12; this may be due to the fact that these antibodies partially neutralize and are unable to fully prevent virus infection even at the highest concentration tested. As shown in  . AnotherS8A Fig) [To cluster the epitopes critical for neutralization of human neutralizing antibodies against EV-A71, we tested the cross-reactivity among the mAbs and escape variants identified in this study and the neutralizing monoclonal antibodies and escape variants reported by Huang et al. . The humS8A Fig) . We testFig 5C). Epitope group I includes VP1 D94 and VP1 L97, which are located at the canyon northern rim and adjacent to the fivefold axis of EV-A71 particle . Epitope group II contains VP1 Y106, VP1 D164, and VP1 D110 at the canyon floor. Epitope groups III and IV, which include residues VP1 K218 and VP2 T141, VP1 N282, respectively, are at the canyon southern rim. Epitope group V, which contains VP2 D225, is at the threefold axis of symmetry. Epitope group VI, which contains VP2 K149, is at the twofold axis of symmetry. Four residues, VP1 D94, VP1 L97, VP1 Y106, and VP1 K218, were found to be involved in the neutralization of M1-16: substitution of any one of the four abolishes the neutralization activity of M1-16. Three of the epitopes have not been reported to be associated with human antibodies before, including VP1 K218 critical for neutralizing of M1-16, M1-17, and M3-7, and VP1 D94E and VP1 L97 for M1-16.Neutralizing mAbs are grouped according to the loss of neutralization that occurs with each escape variant (S5 table). Neutralizing antibody titers against each variant strain from each patient’s serum were plotted in parallel with titers against wild-type strain and linked . We also explored the correlation between age of patients and fold change of neutralization titer against variant/wild-type strains. The results show a weak, but statistically significant, negative correlation for variant VP1 D94E , in other words the component of serum neutralizing activity directed against VP1 D94 is greater with increasing age. No significant correlation was found for the other variants .The fold-change between neutralizing titer against wild-type strain and variants were compared between mild HFMD patients and severe HFMD patients; no significant difference was found for any of the variants  and protect against influenza virus challenge in mice , 37. NeuIgM antibodies can be secreted into mucosal surfaces and constitute the first-line defense against mucosal pathogens . The penThree groups of neutralizing epitopes critical for activity of human mAbs overlap with the binding site of SCARB2, the major receptor of EV-A71 . SCARB2 HCDR3 sequences. The mechanism of neutralization might be slightly different for M1-24 and M3-7 as different escape variants were identified. The difference in neutralizing mechanism might also be due to the differences in light chain sequences or lengths of VHCDR3 sequences. However, the key residues critical for neutralizing are close to each other, and overlap with the binding site of the EV-A71 receptor SCARB2. We also found a biased VH gene usage for EV-A71 neutralizing antibodies as compared with binding antibodies. We speculate that these results are consistent with the possibility that humans preferentially use these gene segments to generate the initial response to EV-A71 infection. This hypothesis remains to be confirmed in further studies.Recurrent variable region gene usage of human antibodies has been found for several pathogens but not EV-A71 before –27. We iOur study has several limitations. First, no neutralizing antibody was identified from severe HFMD patients. We included two severe patients in the screening of EV-A71 specific antibodies and identified some antibodies with EV-A71 binding activity, including two IgG1 antibody from S1, seven IgG1 and three IgA antibodies from S3. None of them have a clinical history of an immune compromising condition. Second, we did not measure the EV-A71 binding concentration of non-neutralizing IgA and IgM antibodies. The purification of IgA and IgM antibodies are still much more complicated than that of IgG, so we only purified those with neutralizing activity. Third, only two genetically similar antibodies were identified. There might be more antibodies of this class against EV-A71 to be explored; our findings are only the beginning in this area. Fourth, the structural mechanism of antibody neutralization, and fifth, the role of non-neutralizing binding antibodies in protection remains to be explored in future studies. Finally, no children with asymptomatic EV-A71 infections were included in our study as acute phase samples were unavailable from them; thus, our data may not represent the full collection of antibodies induced by EV-A71 infection.HFMD caused by enterovirus infection remains a major pediatric disease across the Western Pacific Region. Young children with primary EV-A71 infections have the highest risk of severe HFMD [The study was approved by the Institutional Review Boards of School of Public Health, Fudan University (IRB#2017-12-0654) and Henan Children’s Hospital (IRB#YZ-17-006). Written informed consent was obtained from parents or legal guardians of study participants on enrolment.HFMD inpatients admitted to department of infectious diseases and pediatric Intensive Care Unit (ICU) of Henan Children’s Hospital between February 15, 2017, and February 15, 2018, were recruited as previously described . BetweenPBMC suspensions were barcoded through the 10x Chromium Single Cell platform. The scRNA-seq libraries were constructed by using the 5’ Library Kit, and the single-cell BCR sequencing libraries were constructed by using the V(D)J Enrichment Kits, Human B Cell. The libraries were sequenced using a next-generation sequencing platform. Single-cell sequencing data were aligned and quantified using kallisto/bustools  against the GRCh38 human reference genome. A neighborhood graph was built and used to calculate a Uniform Manifold Approximation and Projection (UMAP). Clusters were annotated based on the expression of canonical marker genes. B cell subsets were then selected, sub-clustered and annotated.S4 Table) were cloned into expression vectors and transfected into HEK-293F cells  using polyethylenimine  and cultured in serum-free expression medium  [Heavy- and light-chain variable domain genes (M293TII) . For proM293TII) .50) was calculated using the Spearman-Karber method. For the neutralization assay, samples were heat inactivated at 56°C for 30 min, and twofold serial diluted. The diluted sample were incubated with 100 TCID50/50μl virus in 96-well plates at 37°C for two hours and then 1E4/100μl RD cells were added. The plate was incubated at 37°C in 5% CO2 for 5–7 days. Cytopathic effect was observed by microscopy. Serum samples and mAbs were tested in duplicates. Positive and negative serum controls and back titration were performed in each batch of test. Antibody titers were defined as the reciprocal of the highest dilution of each serum capable of inhibiting 50% of the cytopathic effect and calculated using the Spearman-Karber method.Neutralizing antibody titers against EV-A71 in serum samples from patients and cell culture supernatant containing mAbs or purified mAbs were measured using two EV-A71 strains, a subgenotype C4a strain , the major EV-A71 genotypes circulating in mainland China since 2007, and a C4b strain . Neutralization assays were performed with RD cells  maintained in Dulbecco’s modified Eagle’s medium . The titer of virus stock was determined by titration in RD cells. The 50% tissue culture infectious dose  according to the manufacturer’s instructions.In the pre-attachment neutralization assay, serially diluted mAbs were incubated with 100 TCIDEnzyme linked immunosorbent assay (ELISA) was used to test the binding of mAbs to EV-A71. EV-A71 virions were purified as previously described . The purIn the competition ELISA, EV-A71 pre-coated plates were blocked and incubated with purified IgM mAbs or isotype control. Recombinant SCARB2-hFc or PSGL-1-hFc fused with the Fc region of human IgG1  was then added. The bound SCARB2-hFc and PSGL-1-hFc was detected with biotinylated goat anti-human IgG Fc antibody and HRP-streptavidin conjugate. The percent reduction of receptor binding was calculated.50 multiply neutralization titer against 32 μg/ml mAb and incubated with an equal volume of mAb at a final concentration of 32 μg/ml for 1 hour at room temperature. After incubation, the mixture was added into RD cells and incubated for 4 days. Cells and supernatant were freeze-thawed three times, centrifugated to obtain virus containing supernatant. The virus was then plaque purified. The plaque purified escape variants were verified using neutralization assays, the P1 region was sequenced and compared with the sequence of the wild-type strain.EV-A71 variants were selected with mAbs as previously described . In brieEC50  and IC50  values were calculated using GraphPad Prism 7 software. To calculate the approximate EC50/IC50 values for poor binding/neutralizing antibodies .(TIF)Click here for additional data file.S2 Fig(A) UMAP and clustering for all PBMCs. (B) Dot plot of markers of PBMC clusters. (C) Sub-clustering of B cells. (D) Dot plot of B cell sub-clusters.(TIF)Click here for additional data file.S3 FigNumbers (A) and percentages (B) of IgM, IgA, and IgG antibodies in plasmablasts from each study subject. Antibody heavy chain variable gene usage in plasmablasts from mild HFMD patients (C) and severe HFMD patients (D), the values of each plot sum up to 100%.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigVariable gene usage was determined using IMGT/V-Quest tool.(TIF)Click here for additional data file.S6 FigInhibition of EV-A71 (C4a) infection at pre-attachment (red) and post-attachment (black) stages were determined by neutralization assay, cell viability was determined using Cell Counting Kit-8. (A) mAbs neutralizing EV-A71 at post-attachment stage at much higher concentrations than at pre-attachment stage. (B) mAb neutralized EV-A71 at similar concentrations at two stages. The curves were fit by nonlinear regression. Half-maximal inhibitory concentrations (IC50) were calculated using GraphPad Prism 7 software. (C) Receptor competition of IgM mAbs were tested by using ELISA assay. Plates pre-coated with purified mature EV-A71 virion were incubated with mAbs and then recombinant receptors were added. Numbers indicate the percent reduction of binding of receptors.(TIF)Click here for additional data file.S7 FigAmino acid sequences of representative strains of genotypes and subgenotypes of EV-A71 were aligned. Residues vary among these strains are shown. The six residues critical for neutralization activity of mAbs identified in this study are conserved among EV-A71 genotypes.(TIF)Click here for additional data file.S8 Fig(A) Neutralizing antibodies reported by Huang et al. were used for selection of EV-A71 C4a and EV-A71 C4b escape variants. (B) Mapping of residues critical for neutralization of mAbs in this study and those from Huang et al.(TIF)Click here for additional data file.S9 FigA to J, neutralizing titer of serum samples against wild-type EV-A71 and variant strains, as grouped by disease severity. K to T, comparison of fold changes in neutralizing titers against wild-type EV-A71 and variant strains between patients with mild and severe HFMD. The ratio of neutralizing antibody titer against variant vs. wild-type was calculated using raw data and compared between patients with mild and severe HFMD using the Wilcoxon rank sum test.(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Breakthrough infections in SARS-CoV-2 vaccinated individuals are an ideal circumstance for the simultaneous exploration of both the vaccine-induced memory reaction to the spike (S) protein and the primary response to the membrane (M) and nucleocapsid (N) proteins generated by natural infection. We monitored 15 healthcare workers who had been vaccinated with two doses of Pfizer BioNTech BNT162b2 and were then later infected with the SARS-CoV-2 B.1.617.2. (Delta) variant, analysing the antiviral humoral and cellular immune responses. Natural infection determined an immediate and sharp rise in anti-RBD antibody titres and in the frequency of both S-specific antibody secreting cells (ASCs) and memory B lymphocytes. T cells responded promptly to infection by activating and expanding already at 2–5 days. S-specific memory and emerging M- and N-specific T cells both expressed high levels of activation markers and showed effector capacity with similar kinetics but with different magnitude. The results show that natural infection with SARS-CoV-2 in vaccinated individuals induces fully functional and rapidly expanding T and B lymphocytes in concert with the emergence of novel virus-specific T cells. This swift and punctual response also covers viral variants and captures a paradigmatic case of a healthy adaptive immune reaction to infection with a mutating virus. The extraordinary efforts put into the development of vaccines and their subsequent distribution to the whole world have saved millions of lives from SARS-CoV-2 associated coronavirus disease 2019 (COVID-19)  SARS-CoV-2 vaccines reduce the incidence and mortality of COVID-19 [The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has resulted in almost 7 million deaths all around the globe and in 770 million confirmed cases as of September 2023 . Antibody levels were quantified using a Cobas 601 modular analyser, also from Roche Diagnostics, Switzerland, with a predefined threshold of 0.8 U/mL. The measurements in Elecsys Anti-SARS-CoV-2 S U/mL correspond to Binding Arbitrary Units per millilitre (BAU/mL). Before analysis, certain samples were diluted at a 1:4 ratio.Peripheral blood mononuclear cells (PBMCs) were labelled with an 18-color flow cytometry panel comprising S- and RBD-fluorescent tetramers and antibodies against surface markers for the definition of different B cell subpopulations . TetramePBMCs were isolated from freshly obtained whole blood with Ficoll–Paque density gradient centrifugation. Cells were seeded in U-bottom 96-well plates and stimulated for 18 h using PepTivator peptide pools  consisting of lyophilised 15 mer peptide sequences with 11 amino acids overlap, spanning the entire sequence of the S, M and N proteins of the SARS-CoV-2 Wuhan wild-type virus . Purified αCD40  was added at the culture start.PBMCs were collected and placed in V-bottom 96-well plates. They were subsequently resuspended in 30 μL of previously titrated antibodies  were introduced into the cell cultures. After 17 h, the cells were initially stained for surface markers as previously described, followed by a thorough washing and fixation step in 3.6% formaldehyde at 4 °C for 5 min. Intracellular cytokine staining (ICS) was then carried out by incubating the cells in 30 µL of antibodies, which were appropriately diluted in a 0.5% p-value was less than 0.05.Statistical analyses were executed using Prism 10 . Specific information about the statistical tests employed for each experiment can be found in the corresponding figure. Time points were assessed through Kruskal–Wallis, Friedman, or Mann–Whitney tests, and post hoc Dunn’s tests were applied as necessary. Statistical significance was inferred when the To assess the immune response to SARS-CoV-2 breakthrough infection in vaccinated individuals, we measured anti-RBD antibody titres in serum samples collected on the day of the first positive COVID-19 test (day 0) and 2–5, 6–9 and 15–17 days later until negativisation . During We then fully characterised the phenotype of circulating S- and RBD-specific (tetramer+) B cell subpopulations discriminating between seven distinct B cell subsets . During We then focused on the subsets, which showed the most significant variations in frequency following infection, namely S-specific cmemSW, S-specific ASC and RBD-specific B cells. We measured the surface expression of CD11c, CD80, CD86, CD95, CXCR5 and CD73 . CD11c iWe then assessed the antigen-specific T responses against S and non-S peptides  and absoTo fully capture the features of the virus-specific T cell response, we also performed intracellular cytokine staining (ICS) after overnight exposure to peptide pools and assessed cytokine production by antigen-specific T cells. At day 0, we detected a substantial fraction of memory S-specific IFN-γ+ CD4+ and CD8+ T cells together with a small fraction of M-specific IFN-γ+ CD4+ and CD8+ T cells, while those specific for the N protein were virtually absent . Thus, bAs our donors were all vaccinated at the time of infection with SARS-CoV-2, we had the intriguing opportunity to compare the features of recall and primary immune responses. Thus, we deepened the characterisation of S- (vaccine-induced), M- and N-specific (first encounter) CD4+ T cells at all time points only in subjects with clearly detectable CD4+ T cell responses. First, we assessed the differentiation status of both AIM+ and total CD4+ cells for each peptide stimulation and time point through the detection of CD45RA and CCR7 expression a,b. In tBreakthrough infection with SARS-CoV-2 and its variants has been shown to induce rapid recovery of memory and de novo adaptive immune cell responses ,21,22,23Phenotypic analysis of B cells showed an increase in the fraction of the cmemSW subset, which represented 6% (range: 2.44–23.48%) of the total B cells and were 5-fold enriched in the vaccine-induced S-specific subsets. Importantly, at the last time point, the cmemSW subset was the most represented within S-specific B cells and showed an activated phenotype, a progressive loss of CXCR5 expression, likely accounting for their recent release from lymphoid follicles, and a steady increase in CD11c. In addition, as a surrogate marker of affinity maturation having occurred in the germinal centre, S-specific-cmemSW B cells increased the expression levels of CD27 ,26, furtDN B cells are thought to derive from the extrafollicular pathway and do not undergo somatic hypermutation; they are divided into DN1, defined as low affinity , and DN2This S-specific B cell activation was paralleled by the concomitant activation of SARS-CoV-2-specific T cell responses. Recall T cell response to the S protein was characterised by a rapid expansion of antigen-specific memory T cells with SI > 30 and expressing high levels of activation markers in addition to markers of functional differentiation. S-specific T cells were mostly EM and CM with a very minor fraction presenting the terminally differentiated EMRA phenotype. Also, S-specific T cells demonstrated a strong effector capacity (such as IFN-γ and IL-2 production). Early after infection, we also observed the presence of polyfunctional memory CD4+ T cells capable of producing, simultaneously, IL-2 and IFN-γ. The T cell subset expressing high levels of CXCR5, PD-1 and ICOS is specialised in aiding B cell affinity maturation and antibody production. These T follicular helper cells increased jointly with B cell activation, as already reported ,35. We aM- and N-specific T cells showed the features of a blossoming primary response following kinetics akin to S-specific T cells but of a smaller magnitude. Responses to the M protein were detectable already at the first time point, whilst N-specific T cells were barely detectable. This may be explained by cross-reactivity of M-specific T cells due to T cell receptor (TCR) degeneracy, which allows for the recognition of several similar peptides . For insThese data provide further evidence that exposure of vaccinated individuals to viral antigens during natural infection with SARS-CoV-2 variants induces a paradigmatic immune response carried out by both a legion of vaccine-induced, antigen-experienced memory B and T cells and by newly emerging virus-specific cells.Our study demonstrates that SARS-CoV-2 breakthrough infections and its variants trigger rapid recovery of memory adaptive immune cell responses. In our cohort, we monitored the persistence of S-specific B and T cell recall responses in BNT162b2-vaccinated individuals experiencing breakthrough infections during the SARS-CoV-2 Delta wave. Infection led to a rapid increase in anti-RBD neutralising antibodies, and a detailed analysis of S-specific B cells revealed a surge in vaccine-derived memory B lymphocytes and activated S-specific antibody-secreting cells. Additionally, SARS-CoV-2-specific T cell responses, particularly to the S protein, showed a robust and diverse effector capacity, and the presence of polyfunctional memory CD4+ T cells. These findings shed light on the robust and multifaceted immune response in vaccinated individuals facing SARS-CoV-2 variants during natural infection.


The impairment of cell–cell fusion and the enhanced immunogenicity profile of this HERV-W vaccine strengthens the prospects of obtaining a meaningful immune response against HERV-W in patients with HERV-W-overexpressing cancers. Human endogenous retrovirus type W (HERV-W) is expressed in various cancers. We previously developed an adenovirus-vectored cancer vaccine targeting HERV-W by encoding an assembled HERV-W group-specific antigen sequence and the HERV-W envelope sequence Syncytin-1. Syncytin-1 is constitutively fusogenic and forms large multinucleated cell fusions when overexpressed. Consequently, immunising humans with a vaccine encoding Syncytin-1 can lead to the formation of extensive syncytia, which is undesirable and poses a potential safety issue. Here, we show experiments in cell lines that restoring an evolutionary lost cleavage site of the fusion inhibitory R-peptide of Syncytin-1 inhibit cell fusion. Interestingly, this modification of the HERV-W vaccine’s fusogenicity increased the expression of the vaccine antigens in vitro. It also enhanced Syncytin-1-specific antibody responses and CD8 ERVWE1 provirus encodes the envelope (Env) protein Syncytin-1, which has been co-opted to play crucial roles in placentation [Human endogenous retrovirus (HERV) genes originate from now-extinct exogenous retroviruses that integrated into the genome of our ancestors. HERV type W (HERV-W) proviruses were acquired approximately 30–40 million years ago; subsequently, HERV-W genes spread throughout the genome via reinfections and retro-transpositions . Since tentation . Syncytientation ; furtherentation ,8. The eentation ,10, and entation ,12. Therentation ,14,15,16In a recent study, we aimed to make a HERV-W-targeting cancer vaccine based on the virus-like-vaccine concept . In a reThe HERV-W Env Syncytin-1 is, however, an atypical vaccine antigen as it is constitutively fusion-competent. When expressed on a cell surface, Syncytin-1 can induce cell–cell fusion via interaction with either of the identified receptors, human ASCT1 and ASCT2, and when Syncytin-1 is overexpressed, these interactions can form large, multinucleated cells. The immunisation of humans with a vaccine encoding the constitutively fusogenic Syncytin-1 would not only result in an immune attack of the HERV-W vaccine-infected cells but also adjacently fused non-transduced cells, worsening the local vaccine-induced tissue damage. To avoid this effect, we were interested in developing an immunogenic but non-fusogenic HERV-W vaccine. 6CC disulfide motif, which is located in the extracellular part of the transmembrane subunit (TU) of Syncytin-1, impaired disulfide binding to a CXXC motif in its surface subunit (SU). This mutation inhibited cell–cell fusion but maintained Syncytin-1 surface expression [In the functional characterisation of Syncytin-1, mutagenesis studies found that Syncytin-1 fusion can be abolished by different mutations in both the extracellular and intracellular parts of Syncytin-1 while still enabling its surface expression ,20,21. Opression .6CC motif-mutated and LQMV-reconstituted Syncytin-1 Env proteins as possible non-fusogenic HERV-W Env candidates in our vaccine constructs, and we explored the fusion capacity and surface expression of these constructs in human cell lines. Furthermore, we encoded the LQMV mutant in an adenovirus-vectored vaccine and examined its immunogenicity compared to the wt HERV-W vaccine in mice [+ T-cell responses towards subdominant antigens and increased antibody responses towards cancer cells expressing the native Syncytin-1 protein compared to the corresponding fusion-competent vaccine. The fusogenic and non-fusogenic HERV-W vaccines increased the survival of tumour-challenged mice to the same extent, possibly reflecting similar dominant epitope-specific responses. Thus, these results imply that we can avoid vaccine-induced cell–cell fusions while obtaining quantitatively higher antigen-specific immune responses by inserting LQMV into the CT of Syncytin-1. In the present study, we aimed to construct a non-fusogenic HERV-W vaccine without compromising the immunogenicity of the vaccine. We tested CX in mice . We show2.The human T24 urinary bladder carcinoma cell line  and the human HEK293 epithelial kidney cell line  were cultured in DMEM GlutaMAX. The murine RenCa renal cortical adenocarcinoma cell line from a male BALB/c mouse  and the human A549 lung carcinoma cell line  were cultured in RPMI 1640 GlutaMAX and Ham’s F12 Nutrient Mix GlutaMAX media , respectively. All media were supplemented with 10% FBS, 100 units/mL of penicillin–streptomycin (pen/strep) , and 1 mM sodium pyruvate . All cells were maintained at 37 °C and 5% COWT), one encoding Syncytin-1 with four amino acids inserted into the CT region and HERV-W Gag (HERV-WLQMV), and one encoding Syncytin-1 with a cysteine-to-alanine exchange in the ectodomain and HERV-W Gag (HERV-WC>A).The sequences of HERV-W Gag and the wt HERV-W Env, Syncytin-1, were described in our recent study . The HERCYYVNQSGIVTEKVKEIRDRIQRRAEELRNTGPWGLLSQWMPWILPFLGPLAAIILLLLFGPCIFNLLVNFVSSRIEAVKLQMVLQMEPKMQSKTKIYRRPLDRPASPRSDVNDIKGTPPEEISAAQPLLRPNSAGSS*.The Syncytin-1 sequence, incl. the above-mentioned mutated variants in bold and/or underlined, is as follows: MALPYHIFLFTVLLPSFTLTAPPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSLSKGTPTFTAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQTGMSDGGGVQDQAREKHVKEVISQLTRVHGTSSPYKGLDLSKLHETLRTHTRLVSLFNTTLTGLHEVSAQNPTNCWICLPLNFRPYVSIPVPEQWNNFSTEINTTSVLVGPLVSNLEITHTSNLTCVKFSNTTYTTNSQCIRWVTPPTQIVCLPSGIFFVCGTSAYRCLNGSSESMCFLSFLVPPMTIYTEQDLYSYVISKPRNKRVPILPFVIGAGVLGALGTGIGGITTSTQFYYKLSQELNGDMERVADSLVTLQDQLNSLAAVVLQNRRALDLLTAERGGTCLFLGEECIn all three plasmids, the sequences coding for the Env and Gag proteins were separated by a self-cleavable P2 A peptide sequence, and a CMV promoter controlled the antigen expression. A control plasmid for transfection encoded a copGFP sequence. GenScript Biotech  synthesised all plasmids. WT and HERV-WLQMV constructs were further cloned into a shuttle vector in E. coli and hereafter into a BAC vector containing the backbone of a replication-deficient human adenoviral vector type 19a/64 (hAd19a/64) (lacking E1 and E3 genes) [The HERV-W3 genes) . A negatThe hAd19a/64 vaccines were produced by Sirion Biotech, following the procedure described in . In brieWT, HERV-WLQMV, or HERV-WC>A plasmids using PEI and Opti-MEM  in complete DMEM media without pen/strep. Transfection with PEI and Opti-MEM was carried out in the ratios of DNA to PEI, 1:3, and DNA to Opti-MEM, 1:100, meaning 3 µg of DNA to 9 µL (1 mg/mL) of PEI to 300 µL of Opti-MEM. Each condition was performed in duplicates. The cells were incubated for 24 h prior to cell surface staining. HEK293 and T24 cells were transfected with either HERV-WWT, HERV-WLQMV, or an empty vaccine (Neg. ctrl vaccine) at a multiplicity of infection (MOI) of 10. Each condition was performed in triplicate, and the cells were stained after 24 h of incubation. Human A549 cells were transduced with the hAd19a/64 HERV-W3. For the cell surface staining of the HERV-W Env surface subunit, the cells were incubated with 15 µg/mL of primary rabbit anti-human HERV polyclonal antibody  for 1 h at 4 °C. Following this, the cells were stained with secondary PE donkey anti-rabbit IgG antibody  and eBioscience™ Fixable Viability Dye eFlour™ 780  for 30 min at 4 °C. Next, the cells were fixated in 1% paraformaldehyde (PFA) for 15 min at 4 °C, and flow cytometry was performed using either the LSRFortessa™ 3-laser or 5-laser cell analyser . The flow cytometry data were analysed with FlowJo™ v10 analysis software and GraphPad Prism 9. The following antibody staining of the transfected and transduced cells was carried out in a FACS buffer consisting of PBS with 1% BSA and 0.1% NaNT24 and A549 cells were seeded on Thermanox coverslips  in 24-well plates and hereafter transfected or transduced as described above except in the case of the A549 cells, which were transduced with 50MOI. Prior to cell seeding, the coverslips for the A549 cells were pre-coated with poly-L-lysine. Following 24 h of incubation, the cells were fixed with 2% glutaraldehyde in a 0.05 M sodium phosphate buffer (pH 7.2). As a control, T24 cells were transfected with a plasmid encoding a copGFP sequence and incubated for 48 h. TEM was performed by the Core Facility for Integrated Microscopy at the University of Copenhagen .WT, HERV-WLQMV, or Neg. ctrl vaccine were obtained.Using a ZOE Cell Imager , light microscopy pictures of unstained and non-fixed T24 cells 24 h after transduction with 25MOI of the hAd19a/64 HERV-WWT vaccine or the HERV-WLQMV vaccine. Denatured samples were run on a NuPAGE™ Bis-Tris Mini Gel  under reducing conditions and transferred to a nitrocellulose membrane . The membrane was incubated overnight at 4 °C with the primary antibodies: the anti-T2A-antibody  to detect the P2A peptide on HERV-W Gag and the housekeeping control protein anti-GAPDH antibody . The bound primary antibody was detected after 1 h of incubation with the secondary polyclonal goat anti-rabbit IgG antibody , using LumiGlo Chemiluminescent  or SuperSignal West Femto Maximum Sensitivity Substrate . The relative expression differences in the protein bands were analysed with iBright analysis software, V5.1.0. HERV-W Gag expression was evaluated via Western blotting, as described previously in . In brieBMDCs derived from the femurs of two BALB/c mice were isolated and matured based on a study published by Jin et al. . DetaileFemale BALB/c mice from Envigo were obtained at 6–8 weeks of age and housed at the Panum Institute, University of Copenhagen. The mice were acclimatised for at least one week prior to any experiments, and all the experiments were performed in accordance with the national guidelines. The experimental procedures were approved by the National Animal Experiments Inspectorate . 7 infectious units (IFU) diluted in 1 × PBS. At the end of each experiment, the mice were euthanised via cervical dislocation. Prior to subcutaneous (s.c.) vaccinations, the mice were anaesthetised with isoflurane. The mice were vaccinated s.c. in the lower right limb with 30 µL of the relevant vaccine, which contained 2 × 10g for 8 min at 8 °C.Blood samples were collected prior to and on the last day of the vaccination studies. Serum was isolated from the blood samples via two centrifugations at 800× 6 RenCa cells modified to express the HERV-W Env Syncytin-1 (see previous study [7 IFU of the HERV-WWT vaccine (n = 15), HERV-WLQMV vaccine (n = 15), or the Neg. ctrl vaccine (n = 10). The mice were continuously monitored, and when humane endpoints were reached  the mice were euthanised via cervical dislocation. The lungs were collected and evaluated for the presence of tumour nodules. One mouse in the HERV-WWT vaccinated group and two in the HERV-WLQMV group showed no signs of sickness throughout the experiment. When evaluating the presence of tumour nodules in the lungs of these remaining mice, the HERV-WWT-vaccinated mouse showed one minor nodule. In contrast, no nodules were visible in the HERV-WLQMV-vaccinated mice. The probability of survival was calculated between groups two-and-two using a log-rank Mantel-Cox test in GraphPad Prism 9 with a statistical significance level defined as * = p < 0.05. Intravenous (i.v.) tumour inoculation was performed as described previously in Skandorff et al. . Each mous study ). These + Renca cells for 1 h at 4 °C. Following this, the cells were stained with PE goat anti-mouse IgG  and eBioscience™ Fixable Viability Dye eFlour™ 780  for 30 min at 4 °C. Finally, the cells were fixated in 1% PFA, and flow cytometry was performed on an LSRFortessa™ 3-laser flow cytometer . FlowJo™ v10 and GraphPad Prism 9 were used to analyse the antibody responses. Statistical significance levels were calculated using the Mann–Whitney t-test (* = p < 0.05 and ** = p < 0.01). Antibodies, from serum isolated from BALB/c mice prior to and at the end of the vaccination, were evaluated via flow cytometry for binding to RenCa cells modified to stably express HERV-W Env. Pre- and end-bleed sera were diluted 1:20 and added to the HERV-W EnvT-cell responses were evaluated using splenocytes isolated from the spleens of the vaccinated mice, as described previously . The spl®, San Diego, CA, USA), PE-Cy7 rat anti-mouse CD4 , PerCP-Cy5.5 rat anti-mouse CD45R/B220 , and FITC rat anti-mouse CD44 . The splenocytes were fixated in 1% PFA and permeabilised with saponin before they were stained with two intracellular fluorophore-conjugated antibodies: APC rat anti-mouse IFNγ  and PE rat anti-mouse TNFα . Finally, flow cytometry was carried out on an LSRFortessa-3 flow cytometer , and the data were analysed using FlowJo™ v10 and GraphPad Prism 9. Following peptide stimulation, the splenocytes were stained with the following cell surface fluorophore-conjugated antibodies: BV421 rat anti-mouse CD8b antibody  was subtracted from the percentage of responses measured in the stimulated samples of the corresponding mouse. As a lower limit of positive geometric mean fluorescent intensity (MFI) IFNγ T-cell responses, the minimum response was defined as a response above the mean plus two times the standard deviation of the MFI of the splenocytes that were not stimulated with peptides (Neg. ctrl) . However, when calculating response differences between the two vaccine groups, all responses were included. Response differences were calculated using the non-parametric Mann–Whitney test, with statistical significance levels defined as * = LQMV) , which was previously shown to abrogate the disulfide bond between the TU and SU [C>A)  A. Other U and SU . Again, SU [C>A) A. The exWT) . T24 cells are highly fusion-permissive when expressing Syncytin-1, which results in large, multinuclear cells, as seen when transfected with plasmids containing the wt Syncytin-1 together with the assembled HERV-W Gag sequence (HERV-WWT) . In cont control B. C>A mutant was lower than for HERV-WWT in the T24 cells and almost absent in the HEK293 cells [LQMV mutant than the HERV-WWT in transfected HEK293 cells   vaccines encoding the antigens from the HERV-W vaccine . Similared cells F. By exavaccine) . WT vaccine, we further explored whether encoding HERV-W Gag and Env could give rise to Env-covered extracellular virus-like particles (VLPs) [WT or HERV-WLQMV vaccine (In a recent study of the HERV-Ws (VLPs) . However vaccine . WT or HERV-WLQMV and compared it to a lack of transduction (Neg. ctrl) or an empty vectored hAd19a/64 vaccine (Neg. ctrl vaccine) (LQMV than HERV-WWT (+ cells expressing MHC class II (MHC-II) and CD86 on their surfaces were similar between the two vaccines  ,25. In tvaccine) . RegardiHERV-WWT A, while HERV-WWT B. No celvaccines . HoweverHERV-WWT C. We als vaccine . Overall+ and CD4+ T-cells via intracellular staining after peptide stimulation with two identified HERV-W Env-responding 9-mer peptides (peptide 28 and peptide 34), as well as three pools of 16-mer peptides overlapping by 11 amino acids  and evaluated their cellular and humoral immune responses after 14 days (the expected peak of T-cell responses) and 28 days (early memory responses) A. Using LQMV vaccine were generally higher than the responses to the HERV-WWT vaccine. The percentage of IFNγ and TNFα CD8+ T-cell responses to peptide 34 from the TU ectodomain and the SU peptide pool were significantly higher for the HERV-WLQMV vaccine group compared to the HERV-WWT vaccine group on day 14 (+ T-cell responses on day 14 (+TNFα+) and single-positive (IFNγ+) CD8+ T-cell responses elicited to peptide 28 were generally the highest of all the peptide conditions [Interestingly, the T-cell responses to the HERV-Wn day 14 C. The san day 14 D and forn day 14 , and witn day 14 . Consistnditions . However+ T-cell responses were generally quite low in the BALB/c mice. However, we could still observe IFNγ+TNFα+-producing CD4+ T-cells responding to the peptide pools of the SU region and the ectodomain on day 14 and day 28; in most cases, these responses were the strongest for the HERV-WLQMV vaccine group, but the difference between the groups did not achieve statistical significance  and on the day of the splenocyte isolation (end-bleed): day 14 or day 28 after vaccination A. We isocination .LQMV-vaccinated mice than the HERV-WWT-vaccinated mice, we were curious whether this could also reflect improved tumour protection against HERV-W Env-expressing tumours. The BALB/c mice were injected intravenously (i.v.) in the tail with the HERV-W Env-expressing murine RenCa tumour cells. The mice were randomised, and four days after the tumour challenge, the mice were vaccinated with the HERV-WWT (n = 15), the HERV-WLQMV (n = 15), or the Neg. ctrl vaccine (n = 10) (Since both T-cell and IgG responses were higher for the HERV-W(n = 10) C. Togeth(n = 10) D and ind(n = 10) E,F, the (n = 10) G. LQMV mutant increased Env cell surface expression in HEK293 cells and was clearly expressed on the cell surfaces of T24 cells. Furthermore, mutating the disulfide bond might affect protein folding; therefore, we proceed with the HERV-WLQMV variant. Interestingly, when encoded in an hAd19a/64 vector, the HERV-WLQMV mutant also induced higher levels of Env cell surface expression than the HERV-WWT vaccine in both murine BMDCs and human A549 cells. In a recent study, we generated a potential cancer vaccine targeting HERV-W which encoded the sequence for the fusogen Syncytin-1 and an assembled HERV-W Gag sequence . FollowiWT and HERV-WLQMV vaccines by measuring the expression of cell surface activation markers and secreted proinflammatory biomarkers of transduced murine BMDCs. Most metrics were similar between the two vaccines, but we observed a reduction in CD40 cell surface expression concomitant with an increase in TNFα in the supernatant of HERV-WLQMV transduced BMDCs. It was not possible to pinpoint a more or less activated phenotype of these transduced BMDCs, but as previously mentioned, the BMDCs confirmed the HERV-WLQMV vaccine-induced increase in Env protein cell surface expression. Next, we assessed potential differences in innate immunogenicity between the HERV-WLQMV vaccine resulted in significantly higher CD8+ T-cell responses to the HERV-WLQMV vaccine compared to HERV-WWT vaccine at 14 and 28 days after vaccination for the less-responsive epitopes and peptide pools, such as peptide 34 and the SU peptide pool. In contrast, responses to the most immunogenic peptide, peptide 28, were unchanged.Increased antigen expression has previously been found to increase T-cell responses to adenovirus-vector-encoded antigens . InteresLQMV vaccine on day 14 and day 28 after vaccination compared to the HERV-WWT vaccine. Both vaccines improved the overall probability of survival of the mice subjected to distantly injected lung tumours expressing HERV-W Env. However, there was no difference between the two vaccines, likely reflecting similar responses to major epitopes, such as peptide 28. Furthermore, we observed significantly higher humoral IgG responses to the HERV-W+ T-cell responses and antibody responses to the HERV-WLQMV vaccine compared to the HERV-WWT vaccine. While it may seem likely that increased antigen expression can contribute to increased immunogenicity, we cannot be sure that this is the main reason for the observed differences. Further, we have no explanation for why we observe this, nor why this profile was inverted in T24 cells. To our knowledge, no other study has characterised the immunogenicity of the LQMV mutant of Syncytin-1 nor assessed its expression in multiple cell lines and primary cells. We were consequently surprised by the findings of higher levels of cell surface expression in most cells, as well as higher and broader Env-specific CD8LQMV Env cell surface expression via transfection in HEK293 cells and transduced murine BMDCs, as well as the increase in the expression levels of both Env and Gag of transduced A549 cells, could be attributed to changes in protein homeostasis, such as increased production, increased surface accumulation, and decreased breakdown. Alternatively, cell stress can induce increased transactivation of the CMV promoter through which the expression of the HERV-W antigens is determined [LQMV mutant induced more cell stress than the HERV-WWT. This could explain why we see higher levels of expression of HERV-W Gag and Env in HERV-WLQMV-transduced A549 cells compared to HERV-WWT. Furthermore, the increased transcription and expression of antigens caused by cell stress would be expected to affect viability. However, potential differences in cell stress were not reflected in cell viability, which was equal between the groups in both murine BMDCs . Thus, Two fusion receptors for Syncytin-1 have been identified in humans: the sodium-dependent transporters ASCT1 and ASCT2 ,31. MiceWT-transduced murine BMDCs (data not shown) in this study, nor in a similar experimental setup with the transduction of human PBMC-derived dendritic cells (data not shown). This could indicate that cell–cell fusion is highly regulated in antigen-presenting cells. However, in vivo vaccination studies in mice involve many different cell types for which cell–cell fusion induced by Syncytin-1 is possible. Still, cell–cell fusion induced by Syncytin-1 would most certainly be more pronounced in humans. Therefore, we might observe greater differences in cellular and humoral responses between the two HERV-W vaccines if tested in humans. Accordingly, the HERV-WLQMV would not only be a more desired vaccine with respect to fusion-related side effects but it would also have a higher potential to break immunological tolerance in humans. These differences in the mechanism of action in humans versus mice gain further importance when considering that HERV-W is a self-antigen in humans and it is thus likely that there will be a reduced frequency of immune precursors recognising HERV-W with high affinity. Here, it is highly encouraging to observe an enhancement of subdominant antigen T-cell responses by the HERV-WLQMV vaccine, demonstrated by a broadening of the anti-HERV-W Env T-cell response. Indeed, vaccines designed for increased MHC-I- and MHC-II-restricted antigen presentation have demonstrated a wider efficacy gap to non-modified antigens when targeting cancers through their dominant epitopes in tolerant models [LQMV vaccine may outcompete the HERV-WWT vaccine in the treatment of human HERV-W-expressing cancers, despite eliciting similar levels of dominant epitope immunogenicity and anti-cancer efficacy against HERV-W-expressing cancers in the highly immunogenic murine system. Additionally, the immune responses to the HERV-WLQMV vaccine would, at the very least, be less sensitive to immune escape.We did not observe cell–cell fusions of HERV-Wt models . TherefoLQMV immunity will translate into anti-cancer efficacy in humans relies, first of all, on breaking the immunological tolerance to the endogenous (self-antigen) target in the tumour, here HERV-W, without inducing autoimmune reactions to other tissues potentially expressing HERV-W antigens at low levels. Breaking tolerance and avoiding autoimmunity remain major challenges in cancer immunotherapy. Innate and adaptive immune responses to HERV-W in pathological conditions, such as multiple sclerosis and type 1 diabetes, indicate an incomplete tolerance which can be broken in inflamed conditions [Whether or not the improvements in HERV-Wnditions ,35,36,37+ T-cells [To test the claim that subtle differences in antigen display and immunogenicity translate into wide differences in a tolerant system, we explored vaccines against a murine endogenous melanoma-associated retrovirus (MelARV). The Env of MelARV is a murine self-antigen overexpressed in many cancer models . In a pr T-cells . These d T-cells .LQMV mutant here represents a fortunate and surprising strategy for increasing the HERV-W vaccine immunogenicity. Our current HERV-W vaccines are also encoded in adenoviral vectors, but in contrast to the MelARV study, a mutation of the ISD is unnecessary because the ISD of the HERV-W Env Syncytin-1 is natively non-immunosuppressive ,40. NotaLQMV vaccine significantly improved survival in mice with HERV-W Env-positive cancers compared to the control vaccine. Relative to the wt HERV-W vaccine, the HERV-WLQMV vaccine increased specific immunogenicity, which enhances the prospects for developing immunotherapies for patients with HERV-W-overexpressing cancers. In this study we generated a non-fusogenic HERV-W cancer vaccine capable of inducing cellular and humoral immune responses without the risk of causing undesired and potentially dangerous vaccinate-induced cell–cell fusions. We found that the HERV-W