DOI: 10.26190/unsworks/24454
title: Differentially expressed genes and pathways in SARS-CoV-2 specific memory B cells across disease severity at single cell resolution

description: This <i>dataset</i> was generated as a part of a study where we looked at the gene signatures and pathways in RBD-specific memory B cells across disease severity in SARS-CoV-2 infected individuals at convalacense. We identified that certain activated memory B cell signatures that were maintained over a period of 6 months in severe infected individuals, whereas declined in mild/moderate over time.
description: METHODOLOGICAL INFORMATION Metholodology have been discussed in thesis chapter 5 of "Utilizing single cell RNA sequencing tools to study the repertoire and transcriptomic dynamics of viral specific memory B cells" . Also as a publication in "High activation levels maintained in RBD-specific memory B cells in people with severe COVID-19". Data reporting: Study design, setting and participants. The COSIN (Collection of COVID-19 Outbreak Samples in NSW) study is an ongoing prospective cohort study evaluating the natural history of SARS-CoV-2 infection among adults and children in New South Wales, Australia. Children and adults diagnosed with SARS-CoV-2 infection confirmed by nucleic acid amplification test (NAT) were eligible for enrolment, irrespective of disease severity. Participants were enrolled through seven healthcare services (which provided both inpatient and community-based care) and their affiliated microbiology laboratories in New South Wales between 6th March 2020 and 17th September 2020. During this time, SARS-CoV-2 strains containing the Spike protein 614D variant were dominant in Sydney. Follow up visits were scheduled at one month (visit window: one to three months) and four months (visit window: four to six months) following symptom onset or date of diagnosis (whichever was first). At each follow up visit, clinical data and blood samples were collected. Disease severity was classified according to the NIH stratification (www.covid19treatmentguidelines.nih.gov). The following treatments were provided to the following patients while being treated as inpatients: 250002 - Telmisartan 80mg mane; Amlodipine 5mg mane; Sitagliptin 100mg mane; Allopurinol 1 tablet every 2 days; Paracetamol 1g QID PRN. 289036 - penrindopril; amlondopril; clotrimazole; prednisone; benzylpenicillin-ceftriaxone-doxycycline; rosuvastatin. 247004 – ibuprofen, paracetamol, nebulised saline. UC blood samples were collected in 2016 from Australian Red Cross Lifeblood. Ethics statement: The protocol was approved by the Human Research Ethics Committees of the Northern Sydney Local Health District and the University of New South Wales, NSW Australia (ETH00520) and was conducted according to the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice (ICH/GCP) guidelines and local regulatory requirements. Written informed consent was obtained from all participants before study procedures. RBD and Spike protein production: SARS-CoV-2 Spike RBD (residues 319-541), with a N-terminal human Ig kappa leader sequence and C-terminal Avi- and His-tags, was cloned into pCEP4 (Invitrogen). Expi293-Freestyle cells (ThermoFisher Scientific) were cultured at 37°C and 8% CO2 in growth medium containing Expi293 Expression Medium (ThermoFisher Scientific). The plasmid was transiently transfected into Expi293-Freestyle cells as follows: 1.5x108 42 total cells (50mL transfection) were mixed with 50 μg of plasmid, 160 μL of ExpiFectamine and 6 mL of OptiMEM-I and left overnight at 37°C in a shaking incubator. The following day 300 μL of ExpiFectamine Enhancer 1 and 3 mL of ExpiFectamine Enhancer 2 was added to the cells before the cells were left in culture for a further 48 hours. After a total of 72 hours in culture, the cell culture is collected and centrifuged for 20 minutes at 4000xg, 4°C. Cellular debris was clarified by passing the supernatant twice through a 0.22 μM filter. The His-tagged protein was then affinity purified from the cell supernatant using a HisTrap HP Column (GE Healthcare) and eluted with imidazole. The purified protein was then buffer exchanged and concentrated in sterile DPBS by centrifuging at 4000xg for 30 minutes at 4°C in a 10,000 MWCO Vivaspin centrifugal concentrator (Sartorius) and stored at –80°C. The recombinant RBD was biotinylated using the Avitag as described by the manufacturer (Genecopeia). Isolation of RBD specific memory B cells The tetramerization method was adapted from a previously published method used for hepatitis C virus tetramers in brief, biotinylated RBD was incubated with Streptavidin PE (SA-PE; Molecular probes; ThermoFisher Scientific) in a molar ratio of 4:1. The streptavidin dye was added step-wise in 1/10th volume increments to the biotinylated protein, for a total of 10 times with a 10 minute incubation at 4°C, in a rotating bioreactor, protected from light. Cryopreserved PBMCs were thawed rapidly in a 37°C waterbath and washed with prewarmed RPMI media supplemented with 2 mM L-glutamine, 50 IU/mL penicillin, 50 μg/mL streptomycin and 10% heat inactivated fetal calf serum (Brown and Essigmann). The cells were resuspended in DPBS and counted. All subsequent incubations were performed protected from light. A maximum of 1 × 107 cells were stained with Fixable Viability Stain 700 (FVS700) (BD Bioscience in a 1:1000 dilution) and incubated at 4°C for 20 minutes, to differentiate the live cells from dead. Cells were washed twice with FACS wash buffer (DPBS + 0.1% BSA), followed by incubation with 5 μL Human Fc block (BD) per 2 × 106 cells at room temperature for 10 minutes, to block non-specific antibody binding. SARS-CoV-2-specific B cells were identified by staining with 1 μg/mL of RBD tetramer at 4°C for 30 minutes. All consecutive steps were done either at 4°C or on ice and washed twice. The cocktail for staining contained 50 μL stain brilliant buffer and the titrated combination of antibodies: 5 μL each of CD21 BV421, IgD BV510, CD10 BV605, CD19 BV711 and CD20 APC-H7, 10 μL of IgG BV786, 2 μL each of CD27 PE-CF594 and CD38 PE-Cy7, 2.5 μL HLA-DR BB515 and 0.5 μL CD3 BB700. All the reagents were from BD Bioscience. The cells were incubated with the staining cocktail at 4°C for 30 minutes. They were washed and resuspended in FACS wash buffer. A BD FACSAria™ III sorter was used to phenotype and single cells were sorted (index sorted) into a 96 well PCR plate containing 2 μL of cold buffer. The buffer was made with lysis buffer containing 0.95 μL 0.2% Triton X-100 solution in nuclease free water and 0.05 μL recombinant RNAse inhibitor (Clontech) along with 0.5 μL 10mM dNTP mix(Promega), and 0.5 μL 5 μM vir70 primer (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3', Sigma). The plates were stored in a -80°C freezer. The index data analysis was performed using FlowJo version 10.7.1 (TreeStar). Sequencing of RBD specific memory B cells The samples were then RT-PCR amplified with the SMARTSeq2 approach and sequenced with Illumina 2 × 150 PE Nextera XT Library Preparation Kit as previously described (Rizzetto et al., 2018; Wu et al., 2019). Single Cell RNA sequencing data analysis Paired-end reads from SMARTseq2 were aligned with STAR (version 2.7.1a) using the GRCh38 human reference genome and Transcripts per Million (TPM) were calculated by RSEM (version 1.2.28) using the rsem-calculate-expression command on HPC clusters. Inhouse script was used for initial data cleaning and quality control. Genes expressed in none of the cells were removed. Cells with less than 400 expressed genes and more than 30 percent expressed mitochondrial genes were removed from the matrix. Gene expression matrix was normalized, and batch corrected using Seurat integration implemented in R. Seurat (v4.0.1) (Stuart et al., 2019) was used to loading gene matrix (730 x12969) for identification of clusters using FindNeighbors() and FindClusters() functions with a resolution of 0.5. Differential gene expression analysis between groups (clusters, UC vs S group, UC vs M group, S vs M group) of only IgD- RBD-specific MBCs were identified using FindAllMarkers with Benjamini-Hochberg-adjusted p-value &lt;0.05 and log2 fold change &gt;0.1. MAST method was utilized for testing the differential gene expression between the groups in Seurat. Gene Set Enrichment Analysis Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005) analysis is performed using inhouse scripts that utilizes Fast Gene Set Enrichment Analysis (fgsea) version 1.14.0 in R to identify pathways related to genes. The permutations were set to 1000. The databases used to identify pathways in molecular signatures reference platform were Gene Ontology (Biological Processes, Cellular Components, Molecular Function) and Hallmark. Gene signature pathways with p-value &lt;0.05 and NES &gt;0 have been significantly upregulated. BCR reconstruction The sequenced samples were used to reconstruct full-length B cell Receptor from VDJpuzzle 2.0 (Rizzetto et al., 2018) by aligning reads with GRCh38 reference genome. V(D)J genes and FWRs/CDRs regions were classified as per Igblastn alignments with heavy and light chain database present in IMGT database as part of VDJpuzzle algorithm. Isotypes were determined as per VDJpuzzle algorithm by aligning constant region with the germline constant region sequences from IMGT database (Lefranc et al., 2009). For IgD+ B cells that reconstructed both IgD and IgM contigs were annotated as “IgDM” and only highly expressed contigs were considered for the analysis. Change-O command from Immcantation was used to determine mutations from germline along with Clones. Gene Usage analysis and physicochemical properties of CDR3H amino acids were calculated using countGenes() and aminoacidProperties() command in alakazam packages version 1.0.2 in Rv3.6 (Gupta et al., 2015). Phylogenetic tree was reconstructed using full-length nucleotide sequences of B cell receptor in Clustal Omega for multiple sequence alignment and Maximum parsimony tree was reconstructed (https://www.ebi.ac.uk/Tools/msa/clustalo/) (Sievers et al., 2011). Itolv6 (Letunic and Bork, 2019) was used to visualize the phylogenetic tree.