Sequence and structural alignment analysis

To identify possible sources of primary infection responsible for αEp9 Ab generation, sequence and structural alignment with Ep9 residues and the SARS-CoV-2 NP was conducted. Alignment of Ep9 sequence with the orthologs from other human coronaviruses (hCoVs) such as SARS-CoV, MERS, HKU-1, NL63, 229E and OC43 was conducted using the Benchling sequence alignment tool [25] (https://benchling.com). To explore a wider range of human host pathogens pBLAST [11] (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to search for Ep9 homology in a database of non-redundant protein sequences; common human-host viruses were specified in the organism category. The queries were conducted with the blastp (protein-protein BLAST) program [11] with search parameters automatically adjusted for short input sequences. Alignments spanning >7 residues were included here. The Vector Alignment Search Tool (VAST) [12] (https://structure.ncbi.nlm.nih.gov/Structure/VAST/vast.shtml) was used to find structural alignment between SARS-CoV-2 Ep9 and proteins from other viral and bacterial human host pathogens. Alignment for NP from common hCoV were not further examined, as they had been included in sequence alignment analysis. The aligned sequences were sorted by the number of aligned residues as well as root-mean square deviation (RMDS). The top 50 structurally aligned proteins were then examined for structural homology in the Ep9 epitope region. Regions of proteins that aligned with the Ep9 region were selected for subsequent analysis.

Cloning

Predicted AIN epitopes were subcloned for phage display using the pM1165a phagemid vector [26] with an N-terminal FLAG-tag and a C-terminal P8 M13-bacteriophage coat protein. AIN constructs were subcloned using the Q5 site-directed mutagenesis kit (New England Biolabs, Ipswich, MA) as per manufacturer’s instructions. The above-mentioned cloned phagemids were then transformed into XL-1 Blue E. coli and spread on carbenicillin-supplemented (50 μg/ml) plates. Individual colonies of were then inoculated in 5 ml cultures, and shaken overnight at 37°C. The phagemid was isolated using the QIAprep spin miniprep kit (Qiagen, Germantown, MD) as per manufacturer’s instructions. Cloned sequences were verified by Sanger sequencing (Genewiz, San Diego, CA).

Phage propagation and purification

The Ep9 homologs were expressed as N-terminal fusions to the P8 coat protein of M13 bacteriophage. Plasmids were transformed into SS320 E. coli and spread onto carbenicillin-supplemented (50 μg/ml) LB-agar plates before overnight incubation at 37°C. A single colony was inoculated into a primary culture of 15 ml of 2YT supplemented with 50 μg/ml carbenicillin and 2.5 μg/ml of tetracycline and incubated at 37°C with shaking at 225 rpm until an optical density at 600 nm (OD₆₀₀) of 0.5 to 0.7 was reached. 30 μM isopropyl β-D-1-thiogalactopyranoside IPTG and M13KO7 helper phage at a multiplicity of infection (MOI) of 4.6 was added to the primary culture, and the culture was incubated for an additional 37°C with shaking at 225 rpm for 45 min. 8 ml of the primary culture was then transferred to 300 ml of 2YT supplemented with 50 μg/ml of carbenicillin and 20 μg/ml of kanamycin. The cultures were inoculated at 30°C with shaking at 225 rpm for around 19 h.

The phage propagation culture was centrifuged at 9,632 x g for 10 min at 4°C. The supernatant, containing the phage, was transferred into a separate tubes pre-aliquoted with 20% tube volume of phage precipitation buffer (20% w/v PEG-8,000 and 2.5 M NaCl), and incubated on ice for 30 min. The solution, containing precipitated phage, was centrifuged for 15 min at 4°C, and the supernatant was discarded. The precipitated phage was centrifuged a second time at 1,541 x g for 4 min at 4°C, and then dissolved in 20 ml of resuspension buffer (10 mM phosphate, 137 mM NaCl, pH 7.4–8.0 with Tween-20 0.05% v/v and glycerol 10% v/v). The resuspended pellet solution was divided into 1 ml aliquots, which were flash frozen with liquid nitrogen for storage in −80°C. Prior to use in ELISAs, the aliquoted phage-displayed constructs were re-precipitated in 0.2 ml of phage precipitation buffer after incubation for 30 min on ice. Aliquots were centrifuged at 12,298 x g for 20 min at 4°C and the supernatant was discarded. The phage pellets were re-centrifuged at 1,968 x g for 4 min at 4°C, and then resuspended in 1 ml of 10 mM phosphate, 137 mM NaCl, pH 7.4.

Expression and Purification of eGFP fusion peptides

pET28c plasmids encoding eGFP fusions to C-terminal Ep9-FLAG, EpNeu-FLAG, EpPred-FLAG and FLAG (negative control) and N-terminal His₆ peptide epitopes, were transformed into BL21DE3 Star E. coli chemically competent cells. Transformants were spread on carbenicillin-supplemented (50 μg/ml) LB-agar plates and incubated at 37°C overnight. Single colonies of each construct were selected to inoculate 25 ml LB media supplemented with carbenicillin (50 μg/ml). After incubation at 37°C with shaking at 255 rpm overnight, 5 ml of seed cultures were used to inoculate 500 ml of LB media supplemented with carbenicillin (50 μg/ml). Expression cultures were incubated at 37°C with shaking at 225 rpm until an OD₆₀₀ of ~0.5 was reached. The cultures were induced with 0.5 mM IPTG and incubated at 25°C for 18 h. The cells were pelleted by centrifugation at 9,632 x g for 20 min and resuspended in Tris-HCl lysis buffer (20 mM Tris-HCl, 250 mM NaCl, pH 8). Cells were lysed by sonication and the insoluble fractions were pelleted by centrifugation at 24,696 x g. The supernatant was affinity-purified using Profinity™ IMAC (BioRad, Hercules, CA) resin charged with nickel sulfate. The protein lysate was batch bound overnight to the IMAC resin and purified using gravity columns. Columns were washed with lysis buffer supplemented with 20 mM imidazole, and the elution fractions were collected from lysis buffer containing 250 mM imidazole. The elution fractions were then buffer-exchanged with lysis buffer lacking imidazole using Vivaspin® 20 Ultrafiltration Units (Sartorius, Goettingen, Germany) with a molecular weight cutoff of 10 kDa. The final buffer imidazole concentrations were calculated to be ~0.1 mM. Purified and buffer-exchanged protein fractions were then visualized using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie dye staining.

Phage ELISAs

As described in previous reports [10], pooled plasma from five αEp9(+) patients, five αEp9(−) patients, or healthy individuals (Sigma-Aldrich, Saint Louis, MO) were separately prepared in coating buffer (50 mM Na₂CO₃, pH 9.6); the plasma was diluted 100-fold during this step. Plasma samples were then immobilized in 96 well microtiter plates by shaking the plasma solutions at 150 rpm at room temperature (RT) for 30 min. After aspiration and washing by plate washer (BioTek, Winooski, VT), each well was blocked with 100 μL of ChonBlock Blocking Buffer (CBB) (Chondrex, Inc., Woodinville, WA) for 30 mins, shaking at 150 rpm at RT. Wells were subsequently washed three times with PBS-T (0.05% v/v Tween-20 in PBS). Next, 1 nM phage-displayed candidate “primary” epitopes and controls prepared in CBB was incubated in microtiter wells for 1 h at RT with shaking at 150 rpm. Unbound phage were aspirated and removed using three washes with PBS-T. The peroxidase-conjugated detection antibody, αM13-HRP (Creative Diagnostics, Shirley, NY), was diluted 1,000-fold in Chonblock Secondary Antibody Dilution (Chondrex, Inc., Woodinville, WA) buffer; 100 μl of this solution was added to each well before incubation for 30 min at RT with shaking at 150 rpm. Following aspiration and three washes (100 μl each), 1-Step Ultra 3,3′,5,5′-Tetramethylbenzidine TMB-ELISA Substrate Solution (ThermoScientific, Carlsbad, CA) was added (100 μl per well). Absorbance of TMB substrate was measured twice at 652 nm by UV-Vis plate reader (BioTek Winooski, VT) after 5 and 15 min of incubation. The measurement at 5 mins ensured that ELISAs with strong signals were quantified before oversaturation, and the second measurement at 15 mins was collected to enhance any wells with lower signal levels; the approach ensures that no comparable signals were observed in the negative controls. In ELISAs without oversaturated signals, the measurements at 15 mins were used for data analysis. The experiment was repeated three times using plasma from different αEp9(+) and αEp9(−) patients for each experiments, using a total of 15 patients for each group. Each experiment was conducted in technical duplicate.

αEp9 IgG and IgM ELISA

Plasma from 46 patients, previously tested for the presence of αEp9 Abs using phage ELISAs [10], were used to test levels of αEp9 IgGs and IgMs. Due to low sample availability, the plasmas from 29 patients were then used to compare IgG binding to the Ep9 and the EpNeu epitopes. 2 μM eGFP-Ep9 or eGFP-FLAG in PBS pH 8.0 were immobilized onto 96 well microtiter plates via overnight incubation with shaking at 150 rpm at 4°C. Excess protein was aspirated and removed with three consecutive PBS-T washes. Wells were blocked by adding CBB (100 μl) before incubation at 30 min at RT with shaking at 150 rpm. Next, αEp9(+) patient plasma, diluted 1:100 in CBB (100 μl), was added to duplicate wells before incubation at RT for 1 h with shaking at 150 rpm. The solutions were discarded and sample wells were washed with PBS-T three times. αEp9 Abs binding to the potential epitopes was detected using horse radish peroxidase (HRP) conjugated αHuman Fc IgG (Thermo Fisher Scientific, Waltham MA) or αIgM μ-chain specific (Millipore Sigma, Temecula, CA) Abs diluted 1:5,000 in ChonBlock Sample Antibody Dilution buffer. 100 μl of detection Abs were added to each sample well, and incubated for 30 min at RT with shaking at 150 rpm. Sample wells were aspirated and washed three times in PBS-T, and the binding signal was detected after addition of TMB substrate (100 μl per well).

Bivalent Abs binding ELISA

eGFP-Ep9 or eGFP-FLAG was serially diluted (120 nM, 40 nM, 13 nM and 4 nM) in PBS pH 8.0, and added to the appropriate wells in 96 well microtiter plates, followed by shaking overnight at 150 rpm at 4°C. Excess unbound protein was removed, and the plate was washed three times in PBS-T. Wells were then blocked in CBB and incubated for 30 min at RT. After blocking, pooled plasma (100 μl per well) from either five αEp9(+) patients, or five non-αEp9, αNP(+) patients, or healthy individuals was added to the appropriate wells. Plasma from pooled patients was diluted 100-fold in CBB. As a positive control αFLAG Ab was used as a 1:2,000 dilution in CBB. Samples were incubated for 1 h at RT with 150 rpm shaking. The solution was removed by aspiration, and the plate and washed three times with PBS-T. Then 1 nM EpNeu displaying phage or the phage negative control with no epitopes displayed was diluted in CBB. 100 μl phage solution was added to microtiter wells and incubated for 30 min at RT with shaking at 150 rpm. After aspirating and washing off unbound phage, binding of phage-displayed EpNeu to plasma αEp9 Abs was visualized using αM13-HRP Ab diluted 1:10,000 in ChonBlock Sample Antibody Dilution buffer. Samples were incubated for 30 min at RT with 150 rpm shaking, and unbound Abs were removed through washing with PBS-T three times before addition of TMB substrate (100 μl). Experiments were conducted in technical triplicates and repeated three times with different αEp(+) and αEp(−) patient samples.

Dose-dependent ELISA

Wells of microtiter plates were coated with serially diluted concentration of eGFP-Ep9, EpNeu and EpPred or eGFP-FLAG, and incubated overnight at 4°C before blocking as described above. Next, pooled plasma (100 μl per well) from either five αEp9(+) patients, or five αEp9(−) patients, or healthy individuals at 1:100 total plasma dilution in CBB was added to the appropriate wells. Samples were incubated for 1 h at RT with shaking at 150 rpm. After incubation, unbound solution was removed, and the plates were washed three times with PBS-T. αEp9 IgG levels were detected by adding αFc IgG-HRP diluted 1:5,000 in ChonBlock Sample Dilution buffer, followed by incubation for 30 min at RT with shaking at 150 rpm, followed by addition of TMB substrate (100 μl per well). Experiments were conducted in technical triplicates and repeated three times with different αEp(+) and αEp(−) patient samples.

Linear B-cell epitope prediction

Linear epitopes from the Influenza A/Para/128982-IEC/2014(H3N2) neuraminidase protein were predicted using the partial sequence with Accession AIX95025.1 from the National Center for Biotechnology Information’s GenBank and the linear B-cell epitope prediction tool, Bepipred 2.0 [16] (http://www.cbs.dtu.dk/services/BepiPred-2.0/). The prediction thresholds were set to 0.5. The specificity and sensitivity of epitope prediction at this threshold is 0.572 and 0.586, respectively.

Structure-based B-cell epitope prediction

The structure of Influenza A/Para/128982-IEC/2014(H3N2) neuraminidase protein was modelled using Swiss-Model [14, 19, 28–31] (https://swissmodel.expasy.org/interactive). Using the ProMod3 3.2.0 tool [19], a structural model was generated based on the crystal structure (2.35Å, PDB 4GZS 1.A) of a homologous H3N2 neuraminidase with 96.39% sequence identity. Modelling methods and quality assessments are further detailed in the report below.

The structural model of Influenza A/Para/128982-IEC/2014(H3N2) neuraminidase was used to predict structure-based epitopes. Using the in silico online platform Discotope 2.0 [17] (http://www.cbs.dtu.dk/services/DiscoTope-2.0/), structure-based epitope propensity scores were calculated to predict likely B-cell epitope residues. The score of −3.7 was set as the threshold for epitope prediction, which estimates a specificity and sensitivity of 0.75 and 0.47, respectively (S5 Fig).

Statistical analysis

The ELISA data were analyzed in GraphPad Prism 9 (https://www.graphpad.com). Since the ELISA assays of 21 potential AIN epitopes were conducted over several microtiter plates for repeated experiments, the raw absorbance values for every patient sample were normalized and represented as the ratio of phage negative control to the signal. For heatmaps, two-way Analysis of variance (ANOVA) with a Tukey adjustment for multiple comparisons tests were conducted for the entire dataset of epitopes. For column comparisons of two groups, for example IgM levels and IgG levels in the αEp(+) patients, unpaired, two-tailed, parametric t-tests were applied. Additionally, for column comparisons between more than two groups, for example IgM or IgG levels groups by weeks PSO, One-way ANOVA with a Tukey adjustment for multiple comparisons tests were used. Where indicated, an ANOVA with a Dunnett’s adjustment were performed to compare results to healthy Abs interactions to αEp9(+) patient results. Graphs represent SD error bars for technical replicates, defined as replicates of the same conditions in multiple wells of the same plate. Whereas error bars are shown as SEM when an experiment is repeated with different patient sample sets. Correlations between Ep9 and EpNeu levels in patients were determined by plotting normalized values on an XY graph and performing a linear Pearson’s correlation coefficient test, where a r coefficient between 1.0–0.7 were considered strong correlations, values between 0.7 and 0.5 were considered a moderate correlation, and values below 0.5 were considered a weak correlation [32]. The significance of the correlation was evaluated based on p-value <0.05.