Human samples
Patient enrollment and consent
The IMPACC is a collaborative project developed by the NIAID and investigators from the Human Immunology Project Consortium (HIPC), the Asthma and Allergic Diseases, and the Cooperative Disease Research Centers (AADCRC). Drexel University collected 106 patient samples to be included in the IMPACC through the Tower Health Hospital network. Participants are enrolled within 48 hours of hospitalization where demographics, detailed medical history, and clinical data were taken. Consenting participants are enrolled within 48 hours of hospitalization under the IRB Protocols 2004007753 and 2102008337. Upon enrollment, demographics, COVID-19 symptom onset, detailed medical history (including comorbidities), and medical records were all recorded. Patients were confirmed positive with a SARS-CoV-2 polymerase chain reaction (PCR). Extensive clinical labs are taken during intake- and biological samples including blood, nasal swab, and endotracheal aspirates are collected. Clinical data and samples from days 4 and 7, representing patient admission to the hospital, were examined.
Biological Sample Processing
Blood samples and nasal swabs were collected at each timepoint and processed at Drexel University within 6 hours of collection according to the IMPACC standardized operating procedure [10]. Whole blood, nasal swabs, peripheral blood mononuclear cells (PMBCs), and plasma collected from each patient was processed at Drexel University and sent to IMPACC core facility sites for further analysis as previously reported [10, 11]. PBMCs were used to identify immune cell populations and changes in cell populations, gene expression, and activation markers. Plasma was used to characterize antibody titers, anti-RBD titers, antibody isotype, proteomics, and metabolomics. At Drexel, plasma was additionally used for ELISA antibody abundance analysis, Luminex cytokine and chemokine assays, and glycomic analysis. Whole blood was used in genome-wide association study (GWAS) and cytometry by the time-of-flight (CyTOF) and bulk RNA transcriptomics. Nasal Swabs were used for bulk RNAseq and viral load quantitaion.
PBMC Isolation: Patient blood samples were spun down at 1000 x g for 10 minutes at room temperature, and plasma was aliquoted. The remaining blood was diluted 1:2 with DPBS (Ca+ 2Mg+ 2 free) and slowly pipetted into a 50mL SepMate-50 tube (with 15mL Lymphoprep below the insert). Samples were spun at 800 x g for 20 minutes at 20ºC with brakes off. The top layer with PBMCs was transferred to a new tube and cells were washed at 400 x g for 5 minutes. Cells were resuspended in 20mL EasySep Buffer, then spun again at 300 x g for 10 minutes at room temperature. For RNASeq, cells were resuspended at 5 million per mL, and 50uL was aliquoted into CRYSTAL Gen tubes. Cells were spun at 500 x g for 5 minutes at room temperature and the excess media was removed. 200uL QIAGEN RLT Buffer with (BME) was added and vortex until the pellet was fully dissolved. Samples were stored at -80ºC for shipment. The remaining PBMCs were frozen down in FBS + DMSO for storage at Drexel University.
Anti-SARS-CoV-2 nucleocapsid IgA, IgG, and IgM quantitation
Monobind AccuBind® ELISA Anti-SARS-CoV-2 kits were used as a qualitative determination of Anti-SARS-CoV-2 specific IgA, IgG and IgM antibodies at Drexel’s IMPACC site. These kits utilize a sequential sandwich ELISA method. This test utilizes recombinant nucleocapsid protein (rNCP) from SARS-CoV-2 coated on microwells to capture antibodies in human plasma. Patient plasma was diluted 1:100 and added directly to the ELISA plate. Following incubation and washing, IgA, IgG or IgM labeled antibodies were added. After a second incubation and wash, reagent substrate is added to produce a measurable color through the reaction with enzyme and hydrogen peroxide. After the addition of a stop substrate, absorbance was read in each well at 450nm within 15 minutes of adding the stop solution.
Cytokine and chemokine analysis
Patient plasma was analyzed for chemokine/cytokine levels using the human immune monitoring 65-Plex ProcartaPlex™ Panel (Invitrogen™). This kit was used to determine the levels of 65 cytokines, chemokines, growth factors, and soluble receptors produced at the designated time points at the Drexel IMPACC site. The following human chemokine/cytokine premixed panel was used according to the manufacturer’s protocol: G-CSF (CSF-3), GM-CSF, IFN alpha, IFN-g, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-16, IL-17A (CTLA-8), IL-18, IL-20, IL-21, IL-22, IL-23, IL-27, IL-31, LIF, M-CSF, MIF, TNF-a, TNF-b, TSLP, BLC (CXCL13), ENA-78 (CXCL5), Eotaxin (CCL11), Eotaxin-2 (CCL24), Eotaxin-3 (CCL26), Fractalkine (CX3CL1), Gro-alpha (CXCL1), IP-10 (CXCL10), I-TAC (CXCL11), MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MDC (CCL22), MIG (CXCL9), MIP-1a (CCL3), MIP-1b (CCL4), MIP-3a (CCL20), SDF-1a (CXCL12), FGF-2, HGF, MMP-1, NGF-b, SCF, VEGF-A, APRIL, BAFF, CD30, CD40L (CD154), IL-2R (CD25), TNF-RII, TRAIL (CD253), TWEAK. Data was acquired on a Luminex™ FLEXMAP 3D™ System using bead regions defined in the protocol and analyzed using Belysa Curve Fitting Software (Sigma Aldrich). Standard curves were generated, and sample concentrations were calculated in pg/mL.
Nasal viral PCR, host transcriptomics, and metagenomics
RNA preparation
Inferior nasal turbinate swabs were collected and placed in 1ml of Zymo-DNA/RNA shield reagent (Zymo Research). RNA was extracted from 250 µL of sample and eluted into a volume of 50ul using the KingFisher Flex sample purification system (ThermoFisher) and the quick DNA-RNA MagBead kit (Zymo Research) following the manufacturer’s instructions. Each sample was extracted twice in parallel. The 2 eluted RNA samples were pooled and aliquoted into 20 µL aliquots using a Rainin Liquidator 96 pipettor for downstream RT-qPCR, RNA-sequencing, and viral sequencing.
RealTime Quantitative Polymerase Chain Reaction: Master mixes containing nuclease-free water, combined primer/probe mixes, and One-Step RT675 qPCR ToughMix (Quantabio) were prepared on ice, and 15 µL was dispensed in each well of a 384-reaction plate (Thermofisher) CoV2 was quantitated using the CDC qRT-PCR assay (primers and probes from IDT). Briefly, this comprises two reactions targeting the CoV2 nucleocapsid gene (N1 and N2) and one reaction targeting RPP30 (RP). Each batch included positive controls of plasmids containing N1/N2 and RP target sequence (2019-nCoV_N_Positive Control and Hs_RPP30 Positive Control, IDT) to allow quantitation of each transcript. Primer/probe sequences were: 2019-nCOV_N1-F GAC CCC AAA ATC AGC GAA AT, 2019-nCOV_N1-R TCT GGT TAC TGC CAG TTG AAT CTG, 2019-nCOV_N1-P ACC CCG CAT TAC GTT TGG TGG ACC, 2019-nCOV_N2-F TTA CAA ACA TTG GCC GCA AA, 2019-nCOV_N2-R GCG CGA CAT TCC GAA GAA, 2019-nCOV_N2-P ACA ATT TGC CCC CAG CGC TTC AG, RP-F AGA TTT GGA CCT GCG AGC G, RP-R GAG CGG CTG TCT CCA CAA GT and RP-P TTC TGA CCT GAA GGC TCT GCG CG. After RNA extracts were gently vortexed and added 5 µL per sample. Plates were centrifuged for 30 seconds at 500 x g, 4C. The quantitative polymerase chain reaction was performed using a Quantstudio5 (Thermo Fisher) with cycling conditions: 1 cycle 10 min at 50°C, followed by 689 3 min at 95°C, 45 cycles 3 sec at 95°C, followed by 30 sec at 55.0°C.
RNA-sequencing cDNA Library Production: From each nasal RNA sample, 10ul was aliquoted to a library construction plate using the Perkin 692 Elmer Janus Workstation (Perkin Elmer, Janus II). Ribosomal depletion, cDNA synthesis, and library construction steps were performed using the Total Stranded RNA Prep with Ribo-Zero Plus kit, following the manufacturer’s instructions (Illumina). All steps were automated on the Perkin Elmer Sciclone NGSx Workstation to reduce batch-to-batch variability and increase sample throughput. Final cDNA libraries were quantified using the Quant-it dsDNA High Sensitivity assay, and library insert size distribution was checked using a fragment analyzer (Advanced Analytical; kit ID DNF474). Samples, where adapter dimers constituted more than 4% of the electropherogram area, were failed before sequencing. Technical controls (K562, Thermo Fisher Scientific, cat# AM7832) were compared to expected results to ensure that batch-to-batch variability was minimized. Successful libraries were normalized to 10nM for sequencing.
RNA-sequencing Clustering and Sequencing
Barcoded libraries were pooled using liquid handling robotics prior to loading. Massively parallel sequencing-by-synthesis with fluorescently labeled, reversibly terminating nucleotides was carried out on the NovaSeq 6000 sequencer using S4 flowcells with a target depth of 50 million 100 base-pair paired-end reads per sample (25 million read pairs).
Total IgG isolation
Total IgG was isolated from 20µL of plasma using a Protein G spin plate as described by the manufacturer (ThermoFisher, MA). Four 200µL 1X PBS washes removed unbound plasma protein using a vacuum manifold apparatus. Next, IgG was eluted by incubating 150µL of 0.1M glycine HCl pH 2–3 for 5 minutes at room temperature. The eluate was collected into a 96-well 2mL collection plate pre-loaded with 15µL of 1.5M Tris pH 8 to neutralize the glycine elution buffer. The wash process was repeated a second time to ensure a high yield of IgG. The resulting 315µL of the neutralized eluate was concentrated and buffer-exchanged to 20µL of 1X PBS using Amicron Ultra-0.5 centrifugal Filter 10 kDa MWCO (Millipore) following the manufacturer’s instructions. NanoDrop 1000 spectrophotometer readings monitored protein yield through the isolation process.
Total IgM isolation
Total IgM was isolated from plasma by incubating 80µL of goat anti-IgM agarose-conjugated agarose beads (A9935, Millipore Sigma, MA) with 80µL plasma and 100µL 1X PBS for 2 hours at room temperature. Following the incubation, the solution was transferred to a 1.2um MultiScreen HTS 96-well filter plate. Four 200µL 1X PBS washes removed unbound plasma protein using a vacuum manifold apparatus. Next, IgM was eluted by incubating 150µL of 0.1M glycine HCl pH 2–3 for 5 minutes at room temperature. The eluate was collected into a 96-wel 2mL collection plate pre-loaded with 15µL of 1.5M Tris pH 8 to neutralize the glycine elution buffer. The wash process was repeated a second time to ensure a high yield of IgM. The resulting 315µL of the neutralized eluate was concentrated and buffer-exchanged to 20µL of 1X PBS using Amicron Ultra-0.5 centrifugal Filter 10 kDa MWCO (Millipore) following the manufacturer’s instructions. NanoDrop 1000 spectrophotometer readings monitored protein yield through the isolation process
Immunoglobulin N-glycan analysis
N-glycans from IgG and IgM were released, labeled, and analyzed as described previously using the Waters GlycoWorks RapiFluor MS kit, adapted for PCR tubes [47]. Briefly, samples were denatured using the RapiGest reagent for 5 minutes at 95°C using a PCR thermocycler. Next, glycoprotein samples were deglycosylated using PNGase F for 6 minutes at 60°C using a PCR thermocycler. Afterward, samples were labeled with RapiFluor label (RFMS) for 5 minutes at room temperature. A solid-phase extraction (SPE) clean-up module isolated RFMS labeled N-glycans which were then eluted into a 96-well 2mL Waters ANSI plate capped with a PFTE 96-well membrane top for high-throughput N-glycan analysis. An ACQUITY Premier UPLC System was used following the setting and protocol described previously [47]. Briefly, a ACQUITY UPLC BEH Amide Column, 130Å, 1.7 µm, 2.1 mm X 50 mm column (Waters, MA) was used to chromatographically separate N-glycans during the 18.3 min run employing a gradient of 50mM Ammonium Formate pH 4.4 (Waters) made with LC-MS Water (Millipore), LC-MS ACN (VWR, Honeywell) 25%-75% gradient transitioning over 12 min to 60%-40%. N-glycans separated by charge and stereochemistry were quantitated using Waters AQUITY Fluorescent detector set to 265/425 em/ex, 10Hz using Empower 3 software. Lastly, N-glycan identity was confirmed using a Waters AQUITY QDa Mass spectrometer. The resulting UPLC fluorescent trace was analyzed with Empower v3.3.1 software, UPLC trace percent-area was combined with collected MS-spectra to identify eluted peaks as described previously [47]. Pooled N-glycans labeled with the RapiFluor tag were digested with Neuraminidase S (New England BioLabs, MA, P0743L) or Neuraminidase (New England BioLabs, MA, P0720S) for 12 hours at 32°C following the manufacturer’s instructions. Digested N-glycans were cleaned up using Water’s SPE kit and analyzed using the UPLC detailed above.
Antigen-specific complement deposition assay
Antibody-specific complement deposition against the RBD and Spike S1 antigens were assayed following the previously developed protocol [48]. Briefly, 20µL FluoSpheres™ NeutrAvidin™-Labeled Microspheres (ThermoFisher) were incubated with 20µg RBD (aa319-541, Invitrogen) (biotinylated in-house using the EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit) or 20µg biotinylated SARS-CoV-2 (2019-nCoV) Spike S1-His Recombinant Protein, Biotinylated (SinoBiological) antigen for 4 hours at 37°C. After washing twice with 200µL 1X PBS, the antigen-bound beads were blocked with 200µL 5% BSA in 1X PBS for 1 hour at 37°C. Next, the beads were washed twice with 500µL of 0.1% BSA in 1X PBS and diluted 1:100 in 1X PBS. A subset of plasma and purified IgM samples were treated with either a Mannosidase (New England BioLabs, MA, P0768S) or Neuraminidase (New England BioLabs, MA, P0720S) for 12 hours at 32°C prior to antigen-specific complement deposition analysis following the manufacturer’s instructions. Next, 15µL of the 1:100 bead solution was transferred to low-binding 1.5mL tubes (Corning) and incubated with 20µL of 1:10 1X PBS diluted pooled severe or nonsevere plasma or 5µg of IgM isolated from pooled severe or nonsevere plasma for 2 hours at 37°C. Next, the immune-complexed beads were incubated for 15 minutes with freshly resuspended Guinea pig complement (Cedarlane, CL4051) and diluted 1:50 in Gelatin Veronal Buffer with Mg2+ & Ca2+ (GVB++) at 37°C. The complement deposition was halted with two washes of 200µL 15mM EDTA. Next, 50µL of a 1:100 diluted FITC labeled Goat anti-Guinea pig Complement C3 antibody (MP Biomedicals, 085538) was incubated for 30 minutes with the immune-complexed beads. Lastly, two 200µL 1X PBS washes removed unbound FITC labeled anti-C3 antibody. Washed samples were re-suspended in 100uL and analyzed using a Fortessa Flow Cytometer (BD). Beads were gated for the presence or absence of the FITC antibody, and the MFI of the bead content was divided by the total number of beads to determine the rate of complement deposition in each sample. The gating strategy is displayed in Fig. 4B. Flow Minus One (FMO) control samples were run with the same protocol to confirm a low background signal and inform the gating cut-off strategy.
Statistical analysis
A biomarker was removed from analysis if its overall number of missing values was greater than 3 (13.6% of 22 patients) to reduce potential bias [49–51]. Data analysis was performed using R and GraphPad Prism 8. COVID-19 trajectory groups were categorized as “1–3” and “4–5” for the averages of measured transcriptomic, proteomic, Luminex, and clinical data. Gender and COVID-19 trajectory group categories were summarized as counts and percentages, continuous variables were summarized as the median and interquartile range (IQR) overall and by trajectory group category. For transcriptomic data, raw counts were normalized to counts per million (CPM), then values were log2 transformed for statistical analysis. A pseudo-count of 2 was added to all count data prior to log transformation because zero cannot be ‘logged’ [52–54]. Mann-Whitney U test was used to test the significance of continuous variables between trajectory group categories. A chi-square test was used to test the association between gender and trajectory group category. Associations between IgM Mannosylated or total S2 and other variables were tested using simple linear regression. Raw trajectory group values were used in simple linear regression. Coefficient of determination R2 was obtained from linear regression. p < 0.05 was considered statistically significant for all tests.