Phagocytic responses by effector cells to opsonized viruses have been recognized to play a key role in antiviral immunity. Limited data on coronavirus disease 2019 suggest that the role of Ab-dependent and -independent phagocytosis may contribute to the observed immunological and inflammatory responses; however, their development, duration, and role remain to be fully elucidated. In this study of 62 acute and convalescent patients, we found that patients with acute coronavirus disease 2019 can mount a phagocytic response to autologous plasma-opsonized Spike protein–coated microbeads as early as 10 d after symptom onset, while heat inactivation of this plasma caused 77–95% abrogation of the phagocytic response and preblocking of Fc receptors showed variable 18–60% inhibition. In convalescent patients, phagocytic response significantly correlated with anti-Spike IgG titers and older patients, while patients with severe disease had significantly higher phagocytosis and neutralization functions compared with patients with asymptomatic, mild, or moderate disease. A longitudinal subset of the convalescent patients over 12 mo showed an increase in plasma Ab affinity toward Spike Ag and preservation of phagocytic and neutralization functions, despite a decline in the anti-Spike IgG titers by >90%. Our data suggest that early phagocytosis is primarily driven by heat-liable components of the plasma, such as activated complements, while anti-Spike IgG titers account for the majority of observed phagocytosis at convalescence. Longitudinally, a significant increase in the affinity of the anti-Spike Abs was observed that correlated with the maintenance of both the phagocytic and neutralization functions, suggesting an improvement in the quality of the Abs.

Since emerging in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly worldwide, infecting >596 million individuals and causing >6.4 million deaths (1). A detailed characterization of the immune response against SARS-CoV-2 both during the acute phase and longitudinally is required, including in patients of varied ages and disease severity, to understand disease pathogenesis and immune correlates of protection.

Early polyfunctional T cell, B cell, and Ab responses against SARS-CoV-2 are associated with reduced clinical severity and better disease outcomes (2). At the beginning of the outbreak, there was much controversy over the protective capacity of Abs in coronavirus infections (3, 4). In rodent models, neutralizing Abs were demonstrated to block infection of SARS-CoV (5) and SARS-CoV-2 (6). However, neutralizing function alone may be insufficient for protection (7), while the combination of neutralizing Abs with Fc receptor–dependent effector functions may reduce disease severity (8, 9).

Limited data suggest that phagocytic responses by effector cells to Ab or complement-opsonized viruses may play a key role in the immunological (8, 10) and inflammatory responses in SARS-CoV-2 infections (1114). These include an association of Ab-dependent cellular phagocytosis (ADCP) with protection (8), correlation of earlier IgG class switch, and maintenance of Fc receptor binding properties with the reduction in disease severity (15). By contrast, phagocytosis mediated by classically activated complement-fixing IgM Abs has been associated with unfavorable clinical outcomes (16), and Ab-independent phagocytosis mediated by complements activated via the mannose-binding lectin pathway was associated with the hyperinflammatory state (11). However, the kinetics of development of these phagocytic responses, their duration, and their clinical significance remain to be fully elucidated. In particular, little is known about the phagocytic responses during acute disease and the long-term retention of these functions, and the associated predictive factors in convalescent patients are unknown beyond 5 mo (17). Importantly, studies to date used purified patient Abs or cloned mAbs to detect phagocytosis and so disregarded a potentially significant contribution of plasma proteins such as activated complements that are relevant in phagocytic responses against respiratory pathogens (11, 18, 19).

In this study, we found that patients with acute coronavirus disease 2019 (COVID-19) mounted phagocytic responses to autologous plasma-opsonized viral Spike protein–coated microbeads as early as 10 d after symptom onset (DPSs), independent of disease severity and despite variable levels of anti-Spike Ab titers. Heat inactivation of the plasma prior to use as opsonin caused 77–95% abrogation of the phagocytic response, and blocking of Fc receptors revealed 18–60% inhibition. These results suggest that COVID-19 can provoke early phagocytosis that is primarily driven by heat-liable components in the plasma with variable contribution from the anti-Spike Abs. In convalescent samples, there was an increase in phagocytosis that correlated significantly with higher anti-Spike IgG titers and with Ab-mediated neutralization. Older patients and patients with severe disease had significantly higher phagocytic responses when compared with younger patients and patients with asymptomatic, mild, or moderate disease. There was no significant difference in phagocytosis between men and women. This age- and disease severity–dependent difference in the anti-Spike Ab–dependent phagocytosis mirrored the anti-Spike neutralization function, suggesting synergistic functions, as previously reported (8, 9). Longitudinally, phagocytosis and neutralization functions were detectable at the 12-mo time point in parallel to a significant increase in affinity of the anti-Spike Abs despite a >90% decline of the end point Ab titers, suggesting this increase in affinity of the Abs was likely due to somatic hypermutation, which may play a major role in the preservation of these effector functions.

Stored frozen plasma samples from 6 hospitalized patients with acute COVID-19, 45 convalescent patients who were enrolled in an ongoing prospective cohort study (COSIN-Collection of Coronavirus COVID-19 Outbreak Samples in New South Wales) (20), and 11 convalescent patients from Central Adelaide Health Network were used in this study. For patients with acute disease, plasma was collected on days 10–25 after symptom onset, and for those in convalescence, plasma was collected on days 30–94 after symptom onset, except for one asymptomatic patient who did not recall the day of symptom onset, hence days after positive PCR test is reported (10 d). In the subset of the latter patients (n = 9), two additional follow-up samples collected on days 110–252 and 321–381 after symptom onset were used. This subset was chosen based on their availability and representation of the disease spectrum of severe, moderate, and mild. Infection with SARS-CoV-2 was confirmed in all patients by a validated quantitative RT-PCR used by diagnostic laboratories across all health services in New South Wales, Australia. Disease severity was classified according to the National Institutes of Health COVID-19 treatment guidelines (21).

Archival serum or plasma collected from 25 healthy donors with an age range of 24–73 y and male/female ratio of 1:2.4 was used as controls. Buffy coats from three healthy donors obtained from the Australian Red Cross (material transfer agreement no. 18-01NSW-06) were used as a source of blood monocytes for in vitro differentiation of primary macrophages. The control archival plasma samples were collected before 2019, and the serum, as well as the buffy coats, was collected before April 2020 when the local transmission was low in New South Wales and none of the donors were close contacts of patients with COVID-19.

The study protocol was approved by the Human Research Ethics Committees of the Northern Sydney Local Health District, the University of New South Wales, Sydney, NSW, Australia (ETH00520); Central Adelaide Health Network Human Research Ethics Committee, Adelaide, SA, Australia (Approval No. 13050); and the Women’s and Children’s Health Network Human research ethics (protocol HREC/19/WCHN/65), Adelaide, SA, Australia, which was conducted according to the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice guidelines and local regulatory requirements. Written informed consent was obtained from all participants before enrolment.

Monocytic cell line THP-1 was obtained from ATCC 202 TIB (22). THP-1 cells were cultured in RPMI 1640 supplemented with 2 mM l-glutamine (Life Technologies),10% FBS, 0.05 mM 2-ME, 10 mM HEPES, and 100 U/ml penicillin-streptomycin (Thermo Fisher Scientific) and passaged every 2 d. Expression of surface Fc receptors was assessed by flow cytometry using mAbs against FcγRI (CD64)-FITC, FcγRIII (CD16)-PE, CD14-PerCP (Becton Dickinson), and FcγRII (CD32)-ACP (Life Technologies), and the isotype and fluorochrome matched negative control mAbs (BD) and were used for the phagocytosis at passages 5–10 (23).

PBMCs were isolated from Buffy coats by gradient centrifugation (Lymphoprep Stem cell), resuspended in RPMI-1640 complete media containing 10% human AB serum (Sigma) at 2 × 106/ml, and seeded onto 24-well flat-bottom plates containing poly-l-lysine (Sigma)-coated glass coverslip inserts (Deckglaser). After a 1-h incubation in a humidified 37°C incubator with 5% CO2 air, nonadherent cells were removed by washing wells twice with prewarmed PBS, and the adherent monocytes (∼1 × 104/well) were differentiated to macrophages for 6 d using 50 ng/ml GM-CSF and 25 ng/ml IL-10 (Sigma) as described previously (24).

SARS-CoV-2 Wuhan-Hu-1 (GenPept: QJE37812) RBD protein (amino acid residues 319–541) and Spike protein (amino acid residues 15–1213) were cloned into pCEP4 mammalian expression vector containing N-terminal human Ig κ leader sequence and C-terminal Avi-tag and His-tag (Invitrogen). Expi293-Freestyle cells cultured at 37°C and 8% CO2 in growth medium containing Expi293 Expression Medium at 3 × 106/ml in 50 ml media were transfected overnight at 37°C with 50 µg of plasmid in 160 µl of ExpiFectamine plus 6 ml of OptiMEM-I (all from Invitrogen). The following day, 300 µl of ExpiFectamine Enhancer 1 and 3 ml of Enhancer 2 (Invitrogen) were added, and the secreted recombinant Spike or RBD protein in culture supernatant was harvested after 72 h and affinity purified using HisTrap HP Column (GE Healthcare, Sydney, Australia) as described previously (25). The purified Spike and RBD recombinant proteins were then buffer exchanged into sterile PBS by centrifuging at 4000 × g for 30 min at 4°C in a 10,000 MWCO Vivaspin centrifugal concentrator (Sartorius, Germany) and stored at −80°C. The Spike and RBD recombinant proteins were then biotinylated via the C-terminal Avi-tag using a labeling kit following the manufacturer’s instructions (Genecopeia) (20, 26).

Anti–SARS-CoV-2 Spike and RBD IgG Ab in sera were quantified using modified direct ELISA (27). In brief, Nunc immunomicrotiter plates were coated with 250 ng of the recombinant SARS-CoV-2 RBD or 100 ng of SARS-CoV-2 Spike protein per well for 2 h at room temperature and washed three times with 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, 0.1% Tween 20 (wash buffer) to remove unbound protein. After blocking for 1 h at room temperature with 5% skim milk in wash buffer, plates were washed once, serially diluted serum (in 5% skim milk) was added to each well in duplicate and incubated for 1 h at room temperature, and plates washed twice with wash buffer. For the end point titer (EPT) of total anti-Spike or anti-RBD Abs, 50 µl of HRP-conjugated polyclonal affinity-purified whole IgG that recognizes both the H and L chains of all human IgG subclasses with minimum cross-reactivity to bovine, horse, or mouse serum proteins was added per well (209-035-088; Jackson Immunoresearch). Both EPT ELISAs are very sensitive whereby the anti-Spike EPT detected spike-specific IgG in sera at 1:1,000,000 dilutions and plateau at 1:10 dilutions, and the anti-RBD detected RBD-specific IgG in sera at 1:100,000 dilutions and plateau when using neat sera (i.e., 1 log less sensitive than the anti-Spike EPT).

For isotyping of the anti-Spike or anti-RBD Abs, 50 µl/well HRP-conjugated Ig subtype or IgG subclass-specific detection Abs was added. These include 1:6000 dilution of anti–total human IgG (Jackson Immunoresearch), 1:3000 dilution of anti–human IgA (α-chain-specific) (Sigma), or 1:3000 dilution of anti–human IgM (μ-chain-specific) (Sigma) Ab subtypes and 1:6000 dilution of anti–human IgG1, IgG2, IgG3, or IgG4 (Southern Biotech). After 1 h of incubation with the HRP-conjugated detection Abs at room temperature, wells were washed twice with wash buffer and incubated with 3,3′,5,5′-tetramethylbenzidine HRP substrate (50 µl/well) for 10 min; the colorimetric reaction stopped by adding 50 µl/well of 1 M HCl (Sigma) and OD at 450 nm measured using CLARIOstar microplate reader (BMG Labtech, Melbourne, Australia).

A total of 1.5 × 109 beads/ml streptavidin and Alexa 488 florescent-tagged 0.4-µm polyester microbeads (Sphereotech) were mixed with recombinant biotinylated SARS-CoV-2 Spike or RBD protein (at 50 µg/ml) in 1.5-ml Eppendorf tubes and incubated for 16 h at 4°C on a rotating chamber. Excess protein was removed by washing the beads twice with 1 ml LPS-minimized cold PBS and gentle centrifugation at 2292 × g for 5 min (Beckman coulter microfuge 20R). Aliquots (50 µl) of the SARS-CoV-2 Spike or RBD-coated beads were then opsonized for 2 h at 37°C with 10 µl of plasma obtained from patients with SARS-CoV-2 infection or healthy control donors and used immediately for the ADCP assay.

THP-1 cells in PBS (1 × 105 cells in 50 µl) were added onto 60 µl of the patient plasma opsonized microbeads in a 1.5-ml Eppendorf tube, mixed by gentle tapping, adjusted to 600 µl using RPMI 1640 containing 0.1% human serum and 100 mM HEPES, and transferred into 37°C, 5% CO2 incubator. After 2 h of incubation, cells were washed once with 1 ml of cold PBS containing 0.5% FBS and 0.005% of sodium azide and gentle centrifugation at 335 × g for 5 min at 4°C, fixed in 400 µl of 1% paraformaldehyde, and kept at 4°C in the dark until the acquisition of data using BD FACSCalibur Flow cytometer. A total of 2 × 104 events per tube were acquired from one sample tube of patients (n = 62) and healthy control subjects (n = 25). Relevant assay controls included the acquisition of 2 × 104 events per tube from cells incubated with no beads, Spike-coated nonopsonized beads, and RBD-coated nonopsonized beads. The proportions of cells that phagocytosed the beads (% of cells that took up the beads) and their fluorescent intensities (amounts of beads taken up per cell) were analyzed using BD FlowJo version 10.5.0 software. Phagocytosis scores (p-scores) were then calculated based on the proportion of cells that took up the opsonized beads denoting the number of positive cells and mean fluorescence intensity representing the average bead uptake by the positive cells as described previously (23). A positive p-score was defined as 3 SDs greater than the background mean p-score of healthy donors as described previously (23).

To assess whether the uptake of the microbeads opsonized with the plasma of patients with acute disease is via Fc receptor (Ab-dependent) and/or other heat-labile opsonins such as complements (e.g., C3b), either the Fc receptors on the THP-1 cells were preblocked using the universal Fc receptor blocking agent (Miltenyi, USA) (23) or the plasma heat inactivated at 56°C for 30 min as described previously (28).

In selected experiments, the intracellular uptake of the opsonized microbeads by effector cells was confirmed by confocal microscopy as described previously (23). In brief, primary macrophages (1 × 104 cells) on poly-l-lysine (Sigma)-coated glass coverslips were rinsed with PBS, resuspended in RPMI 1640 containing 0.1% human serum and 100 mM HEPES, and incubated with the Alexa 488–conjugated opsonized microbeads for 2 h at 37°C, 5% CO2. Cells were then washed twice with cold PBS containing 0.5% FBS and 0.005% of sodium azide, fixed with 1% paraformaldehyde for 5 min at room temperature, and rinsed twice with PBS. The fixed cells were blocked with 1% BSA in PBS, incubated with 1:1000 dilution of Alexa 555–conjugated Phalloidin (Sigma) for 30 min at room temperature, mounted in DAPI nuclear stain-containing media (Molecular Probes), and imaged using ZEISS LSM 880 confocal microscope (Carl Zeiss AG, Oberkochen, Germany), using 63×/1.4 Plan-Apochromat Oil Immersion objective, with Diode 405 nm (DAPI), Argon ion 488 nm (Alexa 488), and DPSS 561 nm (Alexa 555 phalloidin) laser excitation sources; emitted light was filtered using a combination of emission filters and imaged onto Airy detector array producing an effective lateral resolution of ∼100 nm. All the images were Airyscan processed with Zen Black Edition (Zeiss Software).

The microbead-based phagocytosis assay is widely used in many viral infections, including SARS-CoV-2 (29), dengue, HIV, and influenza (30), and is reported as a good proxy of the infectious HIV virions (31).

Retroviral SARS-CoV-2 Spike pseudovirus of the Wuhan-Hu-1 strain was generated in 293T cells by cotransfecting expression plasmids containing SARS-CoV-2 Spike and MLV gag/pol and luciferase vectors using the Calphos transfection kit (Takara Bio) as described by us (20). Pseudoviruses in culture supernatants were then harvested 48 h posttransfection, concentrated 10-fold using 100,000 MWCO Vivaspin centrifugal concentrators (Sartorius, Gottingen, Germany), and used for neutralization assays. In brief, the SARS-CoV-2 pseudovirus was incubated for 1 h with heat-inactivated (56°C for 30 min) patient serum before infecting 293T-ACE2 cells in quadruplicates (293T-ACE2 cells kindly provided by A/Prof. J. Bloom) by a 2-h spinoculation at 800 × g in 96-well white flat-bottom plates in triplicates (Sigma-Aldrich). Infected cells were incubated at 37°C in a humidified incubator with 5% CO2, replenished with fresh media, further incubated for 72 h, and then lysed with a lysis buffer (Promega). Relative luminescence unit in cell lysates was measured using CLARIOstar microplate reader (BMG Labtech), percentage neutralization of Spike pseudovirus was determined, and the 50% inhibitory was calculated using a nonlinear regression model (GraphPad Prism version 9.0) (A. M. Sholukh, A. Fiore-Gartland, E. S. Ford, Y. Hou, L. V. Tse, F. A. Lempp, H. Kaiser, R. Saint Germain, E. Bossard, J. J. Kee, et al., manuscript posted on medRxiv, DOI: 10.1101/2020.12.07.20245431). A positive 50% inhibitory cutoff was defined as 2 SDs greater than the mean background reading obtained from 10 healthy subjects.

Surface plasmon resonance was performed using a BIAcore T200 (Cytiva) to determine the binding characteristics of the anti-Spike polyclonal Abs in patient plasma to SARS-CoV-2 Spike Ag. In brief, 10 µg/ml C-terminal 6X-His-tag containing recombinant SARS-CoV-2 Spike protein was captured at a flow rate of 10 µl/min for 180 s onto carboxymethylated (CM5) dextran sensor chips immobilized with anti-His mAb using EDC/NHS amine coupling kit (GE Healthcare, Australia). The sensor chips were equilibrated with running buffer (HEPES buffer (HBS-EP+; 0.01 M HEPES [pH 7.4], 0.15 M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20) before the addition of plasma. Plasma from patients with SARS-CoV-2 infection or healthy control subjects diluted (1:100) in the running buffer was then injected over the immobilized flow cells at a rate of 20 µl/min for 120 s at 25°C. After each patient’s plasma, the chip was then regenerated using 10 mM glycine (pH 2) (Cytiva) for 60 s. Binding responses with plasma were double referenced-subtracted from nonspecific responses to an empty flow cell and blank injection (zero analyte concentration). Kinetic constants, including Ka, equilibrium constant (KD) (affinity), and dissociation constant (avidity), were calculated using BIAcore evaluation software version 4.1 (32).

All data were analyzed with Prism Software (version 9.0; GraphPad). Unpaired nonparametric Mann–Whitney U test was used to compare p-scores (Spike/RBD), neutralization titer, and Ab EPT (Spike/RBD) between two patient groups/subgroups and/or healthy control subjects. To test the difference among three or more groups, we used parametric ANOVA or Kruskal–Wallis test followed by Dunn’s test, where appropriate, based on the distribution of the residual plot. Spearman correlation was used to compare Spike and RBD EPT against Spike p-score/RBD p-score/neutralizing titer. We used the Friedman test with pairwise Dunn’s test to compare the repeated measures of Spike p-score, Spike and RBD EPT, neutralization titer, and Ab affinity across the time points V1, V2, and V3.

To rank the variable of importance that is associated with Spike p-score (dependent variable), we first confirmed that all continuous variables met the assumption of normality and equal variance of residuals using the Kolmogorov–Smirnov test and by the residuals versus fitted value plot, respectively. We then performed multiple linear regression analyses using disease severity, age, gender, DPSs, anti-Spike/RBD end point Ab titer, anti-Spike/RBD Ab subtypes, and neutralization titer as independent variables. Comorbidity was not included as a covariate in the multiple regression because of insufficient information. We ranked the variables based on the change in R2 and by treating each variable as the last one that entered the regression model. This change in R2 represents the amount of unique variance that each variable explains that the other variables in the model cannot explain. We then performed a parameter covariance matrix to plot the associative relationship between the independent variable using the standardized β coefficient, where a score ≥1 is considered a positive relationship, 0 as no relationship, and <1 as a negative relationship.

Ab assays were performed on plasma and serum samples collected from 62 adult participants that included 6 patients with the acute disease (2 females; median age, 52.5 [range: 14–79] y) (Tables I and II) and 56 convalescent patients (25 females; median age, 53 [range: 19–94] y) (Tables I and II). For the participants with acute infection, samples were collected on days 10–25 after symptom onset (median: 20 DPSs) (Tables I and II). Convalescent samples were collected on days 10–99 after symptom onset (median: 59.5 DPSs) (visit 1) and in a subset of these (n = 9), additional samples were collected on days 110–252 after symptom onset (median: 150 DPSs) (visit 2) and on days 329–381 after symptom onset (median: 345 DPSs) (visit 3), respectively. Among the convalescent patients, 5 were asymptomatic, 16 had mild disease, 17 had moderate disease, and 18 had severe disease, and all groups had comparable median DPSs (Tables I and II). The median age for the patients with severe disease was higher (66 y), while for those with moderate disease was measurably lower (34 y) than the overall average of 53 y (Tables I and II). The male/female ratio (1.2:1) of the whole cohort was representative; however, in the severe disease group, males were overrepresented at a ratio of 2.6:1 and were underrepresented in patients with mild disease in a ratio of 1:2.2 (Tables I and II). This overrepresentation of severe disease in males is consistent with published data analysis of >3 million reported global cases of COVID-19 (33). At the time of collection of the 12-mo samples, Australia had limited community transmission after the first wave when these participants were infected (34), and none of these participants had received a vaccine. A detailed description of the individual acute and convalescent patients is shown in Tables III and IV, respectively.

Table I.

Summary of demographic profile of patients with acute SARS-CoV-2 infection

Disease ClassificationMildSevereTotal
No. of participants 
Median DPSs 19 21 20 
Median age, y (range) 52.5 (14–79) 62 (47–72) 52.5 (14–79) 
Male participants 
Female participants 
Male/female ratio 2:1 2:1 2:1 
Disease ClassificationMildSevereTotal
No. of participants 
Median DPSs 19 21 20 
Median age, y (range) 52.5 (14–79) 62 (47–72) 52.5 (14–79) 
Male participants 
Female participants 
Male/female ratio 2:1 2:1 2:1 
Table II.

Summary of demographic profile of convalescent patients with COVID-19 disease

Disease ClassificationAsymptomaticMildModerateSevereTotal
No. of participants 16 17 18 56 
Median DPSs 55 57 63 67 59.5 
Median age, y (range) 62 (19–73) 52.5 (20–82) 34 (19–73) 66 (23–94) 53 (19–94) 
<40 y old, n (sex) 1 (male) 5 (2 males, 3 females) 9 (4 males, 5 females) 3 (2 males, 1 female) 18 
40–59 y old, n (sex) 1 (male) 6 (2 males, 4 females) 3 (1 males, 2 females) 4 (4 males, 0 females) 14 
>60 y old, n (sex) 3 (2 males, 1 female) 5 (1 male, 4 females) 5 (4 males, 1 female) 11 (7 males, 4 females) 25 
Total male participants 13 31 
Total female participants 11 25 
Male/female ratio 4:1 1:2.2 1.3:1 2.6:1 1.2:1 
Disease ClassificationAsymptomaticMildModerateSevereTotal
No. of participants 16 17 18 56 
Median DPSs 55 57 63 67 59.5 
Median age, y (range) 62 (19–73) 52.5 (20–82) 34 (19–73) 66 (23–94) 53 (19–94) 
<40 y old, n (sex) 1 (male) 5 (2 males, 3 females) 9 (4 males, 5 females) 3 (2 males, 1 female) 18 
40–59 y old, n (sex) 1 (male) 6 (2 males, 4 females) 3 (1 males, 2 females) 4 (4 males, 0 females) 14 
>60 y old, n (sex) 3 (2 males, 1 female) 5 (1 male, 4 females) 5 (4 males, 1 female) 11 (7 males, 4 females) 25 
Total male participants 13 31 
Total female participants 11 25 
Male/female ratio 4:1 1:2.2 1.3:1 2.6:1 1.2:1 
Table III.

Demographic and clinical outcomes of patients with acute SARS-CoV-2 infection

Patient IDAge (y)SexSample Collection (DPSs)Disease Severity on AdmissionClinical OutcomeComorbidityTreatments
14 Female 10 Mild Recovered Crohn’s disease Infliximab 
45 Male 19 Mild Recovered Obese, Hashimoto’s disease Thyroxine 
79 Male 25 Mild Recovered Polymyalgia rheumatica Finasteride 
72 Male 16 Severe Died Arrhythmias, high cholesterol Duodart, Crestor, allopurinol, aspirin, metoprolol 
58 Female 22 Severe Recovered Current metastatic gastroesophageal junction—on chemotherapy currently, gout, high cholesterol On chemotherapy—temporarily ceased after diagnosis; Clexane, pantoprazole, allopurinol, rosuvastatin 
47 Male 21 Severe Recovered None Regular paracetamol, Livial daily 
Patient IDAge (y)SexSample Collection (DPSs)Disease Severity on AdmissionClinical OutcomeComorbidityTreatments
14 Female 10 Mild Recovered Crohn’s disease Infliximab 
45 Male 19 Mild Recovered Obese, Hashimoto’s disease Thyroxine 
79 Male 25 Mild Recovered Polymyalgia rheumatica Finasteride 
72 Male 16 Severe Died Arrhythmias, high cholesterol Duodart, Crestor, allopurinol, aspirin, metoprolol 
58 Female 22 Severe Recovered Current metastatic gastroesophageal junction—on chemotherapy currently, gout, high cholesterol On chemotherapy—temporarily ceased after diagnosis; Clexane, pantoprazole, allopurinol, rosuvastatin 
47 Male 21 Severe Recovered None Regular paracetamol, Livial daily 
Table IV.

Demographic and clinical details of the convalescent COVID-19 patients

Patient IDAge (y)SexSample Collection Date/DPSsDisease Severity on AdmissionClinical OutcomeComorbidityTreatments
2005-61247-050 73 Male 77 Asymptomatic Recovered Atrial fibrillation, chronic kidney disease, myelofibrosis, type 2 diabetes, emphysema Gabapentin, magnesium tablet, sevelamer, sotalol, warfarin, Aranesp 
2005-61247-052 45 Male 99 Asymptomatic Recovered None None 
2005-61289-002 62 Male 55 Asymptomatic Recovered None None 
2005-61289-003 19 Male 10a Asymptomatic Recovered None None 
2005-61213-014 67 Female 50 Asymptomatic Recovered None None 
2005-61213-003 57 Male 37 Mild Recovered None None 
2005-61247-003 23 Male 63 Mild Recovered None None 
2005-61247-017 22 Male 67 Mild Recovered None None 
2005-61250-005 51 Male 61, 110, 338 Mild Recovered None None 
50 VONI 75 Male 84 Mild Recovered Data not available Data not available 
2005-61213-005 53 Female 45 Mild Recovered None None 
2005-61216-005 63 Female 38 Mild Recovered None None 
2005-61250-001 73 Female 50 Mild Recovered Barrett's esophagus, hyperlipidemia, obstructive sleep apnea Aspirin, Celestone M, Claratyne, Diaformin, Lipidil, magnesium, Panadol, Somac, Vytorin, pembrolizumab 
2005-61250-003 53 Female 59, 150, 329 Mild Recovered Hypertension, obesity, asthma, psoriatic arthritis, gastroesophageal reflux Fluoxetine, folic acid, methotrexate, pantoprazole, perindopril-amlodipine, quetiapine 
2005-61250-004 23 Female 57 Mild Recovered None None 
2005-61250-006 52 Female 69 Mild Recovered None None 
2005-61250-008 21 Female 71, 169, 351 Mild Recovered Obesity, asthma, rheumatological (gout) Seretide and Ventolin, prednisone and mycophenolate mofetil, Panadeine forte 
2005-61250-009 82 Female 69 Mild Recovered Hypertension, asthma Ventolin, Seretide 
2005-61250-014 77 Female 56 Mild Recovered Hypertension None 
2005-61250-018 20 Female 45 Mild Recovered None None 
2005-61289-001 44 Female 48 Mild Recovered None None 
2005-61213-001 64 Male 30 Moderate Recovered None None 
2005-61213-002 72 Male 47 Moderate Recovered Hypertension, prostate cancer now in remission None 
2005-61213-004 30 Male 40 Moderate Recovered None None 
2005-61213-007 34 Male 67 Moderate Recovered None None 
2005-61213-009 25 Male 63 Moderate Recovered None Endone 
2005-61213-010 51 Male 64 Moderate Recovered None None 
2005-61216-002 67 Male 43, 129, 381 Moderate Recovered None None 
2005-61216-007 73 Male 54 Moderate Recovered Obesity, rheumatological (TNFR-associated periodic syndrome) None 
2005-61289-007 33 Male 85, 126, 340 Moderate Recovered None None 
2005-61213-006 32 Female 64 Moderate Recovered None None 
2005-61213-008 31 Female 57 Moderate Recovered None None 
2005-61213-011 47 Female 60 Moderate Recovered None None 
2005-61213-018 22 Female 66 Moderate Recovered None None 
2005-61216-003 67 Female 42, 134, 329 Moderate Recovered None None 
2005-61247-034 19 Female 71 Moderate Recovered None None 
2005-61250-012 33 Female 63 Moderate Recovered Hypertension, obesity Telmisartan 
37 LORE 50 Female 84 Moderate Recovered Data not available Data not available 
2005-61213-013 94 Male 55 Severe Recovered None Apixaban, esomeprazole, furosemide, ipratropium, macrogol, metoprolol, risedronate sodium, terbutaline 
2005-61216-001 42 Male 42, 252, 357 Severe Recovered None None 
2005-61247-004 23 Male 58 Severe Recovered None None 
2005-61247-044 71 Male 76 Severe Recovered Arrhythmias, high cholesterol Duodart, Crestor, allopurinol, aspirin, metoprolol 
2005-61250-002 72 Male 49 Severe Recovered Hypertension, obesity, diabetes, rheumatological (gout) Twinsta (telmisartan/amlodipine), metformin, alopurinol, prednisolone 
2005-61289-035 55 Male 90 Severe Recovered Hypertension, obesity, diabetes, prostate cancer on remission, kidney disease, transplant, anemia Thalidomide, metformin, aspirin 
22 MCBR 77 Male 56 Severe Recovered Data not available Data not available 
27 SMPE 53 Male 84 Severe Recovered Data not available Data not available 
36 LOED 50 Male 84 Severe Recovered Data not available Data not available 
41 FAPA 68 Male 84 Severe Recovered Data not available Data not available 
5 CRCO 70 Male 56 Severe Recovered Data not available Data not available 
60 MCSI 26 Male 84 Severe Recovered Data not available Data not available 
2005-61216-004 36 Female 39 Severe Recovered None None 
2005-61289-021 61 Female 94, 170, 345 Severe Recovered None None 
2005-61289-036 84 Female 87, 156, 359 Severe Recovered Lung disease, asthma Perindopril, benzylpenicillin, ceftriaxone, doxycycline, Spiriva, Crestor, Eutroxsig, amlodipine, clotrimazole and hydrocortisone, tiotropium, rosuvastatin 
35 LOHE 64 Female 56 Severe Recovered Data not available Data not available 
7 LAMO 71 Female 56 Severe Recovered Data not available Data not available 
17 CACA 70 Male 84 Severe Recovered Data not available Data not available 
Patient IDAge (y)SexSample Collection Date/DPSsDisease Severity on AdmissionClinical OutcomeComorbidityTreatments
2005-61247-050 73 Male 77 Asymptomatic Recovered Atrial fibrillation, chronic kidney disease, myelofibrosis, type 2 diabetes, emphysema Gabapentin, magnesium tablet, sevelamer, sotalol, warfarin, Aranesp 
2005-61247-052 45 Male 99 Asymptomatic Recovered None None 
2005-61289-002 62 Male 55 Asymptomatic Recovered None None 
2005-61289-003 19 Male 10a Asymptomatic Recovered None None 
2005-61213-014 67 Female 50 Asymptomatic Recovered None None 
2005-61213-003 57 Male 37 Mild Recovered None None 
2005-61247-003 23 Male 63 Mild Recovered None None 
2005-61247-017 22 Male 67 Mild Recovered None None 
2005-61250-005 51 Male 61, 110, 338 Mild Recovered None None 
50 VONI 75 Male 84 Mild Recovered Data not available Data not available 
2005-61213-005 53 Female 45 Mild Recovered None None 
2005-61216-005 63 Female 38 Mild Recovered None None 
2005-61250-001 73 Female 50 Mild Recovered Barrett's esophagus, hyperlipidemia, obstructive sleep apnea Aspirin, Celestone M, Claratyne, Diaformin, Lipidil, magnesium, Panadol, Somac, Vytorin, pembrolizumab 
2005-61250-003 53 Female 59, 150, 329 Mild Recovered Hypertension, obesity, asthma, psoriatic arthritis, gastroesophageal reflux Fluoxetine, folic acid, methotrexate, pantoprazole, perindopril-amlodipine, quetiapine 
2005-61250-004 23 Female 57 Mild Recovered None None 
2005-61250-006 52 Female 69 Mild Recovered None None 
2005-61250-008 21 Female 71, 169, 351 Mild Recovered Obesity, asthma, rheumatological (gout) Seretide and Ventolin, prednisone and mycophenolate mofetil, Panadeine forte 
2005-61250-009 82 Female 69 Mild Recovered Hypertension, asthma Ventolin, Seretide 
2005-61250-014 77 Female 56 Mild Recovered Hypertension None 
2005-61250-018 20 Female 45 Mild Recovered None None 
2005-61289-001 44 Female 48 Mild Recovered None None 
2005-61213-001 64 Male 30 Moderate Recovered None None 
2005-61213-002 72 Male 47 Moderate Recovered Hypertension, prostate cancer now in remission None 
2005-61213-004 30 Male 40 Moderate Recovered None None 
2005-61213-007 34 Male 67 Moderate Recovered None None 
2005-61213-009 25 Male 63 Moderate Recovered None Endone 
2005-61213-010 51 Male 64 Moderate Recovered None None 
2005-61216-002 67 Male 43, 129, 381 Moderate Recovered None None 
2005-61216-007 73 Male 54 Moderate Recovered Obesity, rheumatological (TNFR-associated periodic syndrome) None 
2005-61289-007 33 Male 85, 126, 340 Moderate Recovered None None 
2005-61213-006 32 Female 64 Moderate Recovered None None 
2005-61213-008 31 Female 57 Moderate Recovered None None 
2005-61213-011 47 Female 60 Moderate Recovered None None 
2005-61213-018 22 Female 66 Moderate Recovered None None 
2005-61216-003 67 Female 42, 134, 329 Moderate Recovered None None 
2005-61247-034 19 Female 71 Moderate Recovered None None 
2005-61250-012 33 Female 63 Moderate Recovered Hypertension, obesity Telmisartan 
37 LORE 50 Female 84 Moderate Recovered Data not available Data not available 
2005-61213-013 94 Male 55 Severe Recovered None Apixaban, esomeprazole, furosemide, ipratropium, macrogol, metoprolol, risedronate sodium, terbutaline 
2005-61216-001 42 Male 42, 252, 357 Severe Recovered None None 
2005-61247-004 23 Male 58 Severe Recovered None None 
2005-61247-044 71 Male 76 Severe Recovered Arrhythmias, high cholesterol Duodart, Crestor, allopurinol, aspirin, metoprolol 
2005-61250-002 72 Male 49 Severe Recovered Hypertension, obesity, diabetes, rheumatological (gout) Twinsta (telmisartan/amlodipine), metformin, alopurinol, prednisolone 
2005-61289-035 55 Male 90 Severe Recovered Hypertension, obesity, diabetes, prostate cancer on remission, kidney disease, transplant, anemia Thalidomide, metformin, aspirin 
22 MCBR 77 Male 56 Severe Recovered Data not available Data not available 
27 SMPE 53 Male 84 Severe Recovered Data not available Data not available 
36 LOED 50 Male 84 Severe Recovered Data not available Data not available 
41 FAPA 68 Male 84 Severe Recovered Data not available Data not available 
5 CRCO 70 Male 56 Severe Recovered Data not available Data not available 
60 MCSI 26 Male 84 Severe Recovered Data not available Data not available 
2005-61216-004 36 Female 39 Severe Recovered None None 
2005-61289-021 61 Female 94, 170, 345 Severe Recovered None None 
2005-61289-036 84 Female 87, 156, 359 Severe Recovered Lung disease, asthma Perindopril, benzylpenicillin, ceftriaxone, doxycycline, Spiriva, Crestor, Eutroxsig, amlodipine, clotrimazole and hydrocortisone, tiotropium, rosuvastatin 
35 LOHE 64 Female 56 Severe Recovered Data not available Data not available 
7 LAMO 71 Female 56 Severe Recovered Data not available Data not available 
17 CACA 70 Male 84 Severe Recovered Data not available Data not available 
a

Patient does not recall DPSs; thus, days after positive PCR test is reported instead of DPSs.

Plasma from all six participants with acute infection demonstrated detectable phagocytosis of microbeads coated with SARS-CoV-2 Spike protein as early as 10 DPSs regardless of disease severity, and despite two of six having no detectable anti-Spike Ab EPTs (Fig. 1A). The gating strategy used to determine the proportion of positive cells and the mean fluorescence intensity, as well as microscopic validation for intracellular uptake of the opsonized microbeads, is presented in Supplemental Fig. 1. In the patients (n = 4) with detectable anti-Spike Abs, the mean of EPTs was 11,015 (95% confidence interval [CI]: −6490, 28,500) with mean Spike p-score of 51.4 (95% CI: 18.2, 84.6). For the two of six patients with minimal EPTs, the Spike p-scores were markedly lower (2.38; 95% CI: 1.15, 3.61), whereas the background cutoff in samples from unexposed healthy control subjects was 0.9 (95% CI: 0.518, 1.28) (Fig. 1A). Two of the six patients with acute infection had high (patients 1 and 3), one patient had moderate (patient 5), and three had low levels (patients 2, 4, and 6) of nasopharyngeal viral loads (Fig. 1B). Interestingly, those three patients with low viral load had high p-scores and high anti-Spike EPTs, while those two with high viral load had low p-score and low anti-Spike EPTs regardless of their disease severity (Fig. 1C).

FIGURE 1.

Detection of heat-labile Spike phagocytosis in patients with acute SARS-CoV-2 infection. (A) Histograms showing positive Spike p-scores were detected in all patients with acute SARS-CoV-2 infection despite varying anti-Spike Ab EPTs (black dots) and differences in DPS onset and disease severity; positive phagocytosis is defined as 3 SDs greater than the mean p-scores of healthy control subjects (dotted line). (B) Nasopharyngeal viral load at the time of sample collection (DPS) among patients with acute infection where two of the six patients had high (patients 1 and 3), one patient had moderate (patient 5), and three patients had a low level (patients 2, 4, and 6) of nasopharyngeal viral loads. (C) Summary heatmap showing patients with low viral load had high p-scores and high anti-Spike EPTs, while those with high viral load had low p-score and low anti-Spike EPTs regardless of their disease severity. (D) Heat inactivation of the plasma caused a profound abrogation of Spike p-score by 77–95% in five of six of the patients with acute infection (mean Spike p-score, 5.7; 95% CI: −5.5, 16.9), suggesting major contribution by heat-liable components of the plasma. (E) Fc receptor blocking experiments with heat nonactivated plasma indicated that the Abs contributed to 18–60% (mean Spike p-score, 17.82; 95% CI: −0.58, 36.2) of the phagocytic function of all acute patients. (F) Surface plasmon resonance showing high-affinity binding of plasma obtained from patient 1 to recombinant Spike protein (KD = 4 × 10−12 M) and substantial Fc receptor–dependent phagocytosis (E) despite having minimal levels in anti-Spike Ab (A).

FIGURE 1.

Detection of heat-labile Spike phagocytosis in patients with acute SARS-CoV-2 infection. (A) Histograms showing positive Spike p-scores were detected in all patients with acute SARS-CoV-2 infection despite varying anti-Spike Ab EPTs (black dots) and differences in DPS onset and disease severity; positive phagocytosis is defined as 3 SDs greater than the mean p-scores of healthy control subjects (dotted line). (B) Nasopharyngeal viral load at the time of sample collection (DPS) among patients with acute infection where two of the six patients had high (patients 1 and 3), one patient had moderate (patient 5), and three patients had a low level (patients 2, 4, and 6) of nasopharyngeal viral loads. (C) Summary heatmap showing patients with low viral load had high p-scores and high anti-Spike EPTs, while those with high viral load had low p-score and low anti-Spike EPTs regardless of their disease severity. (D) Heat inactivation of the plasma caused a profound abrogation of Spike p-score by 77–95% in five of six of the patients with acute infection (mean Spike p-score, 5.7; 95% CI: −5.5, 16.9), suggesting major contribution by heat-liable components of the plasma. (E) Fc receptor blocking experiments with heat nonactivated plasma indicated that the Abs contributed to 18–60% (mean Spike p-score, 17.82; 95% CI: −0.58, 36.2) of the phagocytic function of all acute patients. (F) Surface plasmon resonance showing high-affinity binding of plasma obtained from patient 1 to recombinant Spike protein (KD = 4 × 10−12 M) and substantial Fc receptor–dependent phagocytosis (E) despite having minimal levels in anti-Spike Ab (A).

Close modal

The contribution of heat-liable components of plasma such as complement proteins to phagocytosis during acute infection was determined by heat inactivation of the plasma before use as an opsonin. Heat inactivation of the plasma caused a profound abrogation of phagocytosis by 77–95% in five of six of the patients with acute infection (mean Spike p-score, 5.7; 95% CI: −5.5, 16.9), while leading to a 95.6% enhancement in one of the patients (Fig. 1D), suggesting a major contribution of complement in five of the six patients. By contrast, Fc receptor blocking experiments with untreated plasma indicated that the Abs contributed to 18–60% (mean Spike p-score, 17.82; 95% CI: −0.58, 36.2) of the phagocytic function observed in all six patients (Fig. 1E). Interestingly, in one of the patients (patient 1) with the undetectable anti-Spike Abs (Fig. 1A), blocking of the Fc receptor showed 60.3% inhibition of phagocytosis, suggesting the presence of small amounts of effective anti-Spike Abs that were not detected by end point titration ELISA. To test this, we performed binding assays using surface plasmon resonance, and we found extremely high-affinity binding of the plasma to recombinant Spike protein (KD = 4 × 10−12 M) (Fig. 1F), supporting the notion of low-titer high-affinity Abs.

Measurement of anti–SARS-CoV-2 Ab EPT in sera for the 56 convalescent patients at 10–99 DPSs showed 100% had detectable anti-Spike IgG and 98% had anti-RBD IgG Abs (Fig. 2A, 2B). Further analysis of the IgG subclasses of the anti-Spike responses showed that the Abs were primarily IgG1 and IgG3 subtypes, but not IgG2 or IgG4 (Fig. 2C). There were also variable levels of anti-Spike IgA and IgM Abs (Fig. 2C). Functionally, 95% (53/56) of all patients had phagocytosis of Spike protein–coated microbeads (Fig. 2D), 84% (47/56) had phagocytosis of RBD protein–coated microbeads, albeit at lower p-scores (Fig. 2E), and 86% (48/56) could neutralize Spike pseudovirus (Fig. 2F).

FIGURE 2.

Disease severity–dependent differences in end point Ab titers, phagocytosis, and neutralization titer at convalescence. Disease severity–dependent increase in anti-Spike Ab EPTs in plasma of patients was significant in patients with severe disease when compared with moderate (difference of mean = 64,498.1; 95% CI: −7340, 66,700) and mild (difference of mean = 85,558.5; 95% CI: 1550, 15,600) (A). Anti-RBD Ab EPTs showing high-level expression in all patients, but there was no significant difference among the various patient groups (B). Analysis of the anti-Spike Ab classes and IgG subtypes showed progressive increase of IgA, IgM, IgG1, and IgG3 from asymptomatic to patients with severe disease, but only the increases in IgG1 were statistically significant; there were no detectable levels of IgG2 and IgG4 in all disease groups (C). Severe disease group has significantly higher Spike p-score compared with moderate (difference of mean = −454.9; 95% CI:−885.3, −24.38) and mild disease groups (difference of mean = −650.6; 95% CI: −1088, −213.3) (D). Anti-RBD Ab–mediated phagocytic response was significantly higher in the severe when compared with mild (difference of mean = 0.6; 95% CI: 4.34, 7.02) disease individuals (E), despite all patient groups having similar levels of anti-RBD Ab EPTs (B). Severe disease group has significantly higher neutralization titer compared with moderate (difference of mean = −440.8; 95% CI: −889.8, 8.122) and mild disease groups (difference of mean = −586.5; 95% CI: −1043, −130.4) (F). Dotted lines in each plot show median or mean values +3 SD of 25 healthy control samples (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 2.

Disease severity–dependent differences in end point Ab titers, phagocytosis, and neutralization titer at convalescence. Disease severity–dependent increase in anti-Spike Ab EPTs in plasma of patients was significant in patients with severe disease when compared with moderate (difference of mean = 64,498.1; 95% CI: −7340, 66,700) and mild (difference of mean = 85,558.5; 95% CI: 1550, 15,600) (A). Anti-RBD Ab EPTs showing high-level expression in all patients, but there was no significant difference among the various patient groups (B). Analysis of the anti-Spike Ab classes and IgG subtypes showed progressive increase of IgA, IgM, IgG1, and IgG3 from asymptomatic to patients with severe disease, but only the increases in IgG1 were statistically significant; there were no detectable levels of IgG2 and IgG4 in all disease groups (C). Severe disease group has significantly higher Spike p-score compared with moderate (difference of mean = −454.9; 95% CI:−885.3, −24.38) and mild disease groups (difference of mean = −650.6; 95% CI: −1088, −213.3) (D). Anti-RBD Ab–mediated phagocytic response was significantly higher in the severe when compared with mild (difference of mean = 0.6; 95% CI: 4.34, 7.02) disease individuals (E), despite all patient groups having similar levels of anti-RBD Ab EPTs (B). Severe disease group has significantly higher neutralization titer compared with moderate (difference of mean = −440.8; 95% CI: −889.8, 8.122) and mild disease groups (difference of mean = −586.5; 95% CI: −1043, −130.4) (F). Dotted lines in each plot show median or mean values +3 SD of 25 healthy control samples (*p < 0.05, **p < 0.01, ***p < 0.001).

Close modal

Stratification of the convalescent patients by disease severity into asymptomatic (n = 5), mild (n = 16), moderate (n = 17), or severe (n = 18) disease showed a severity-dependent increase in anti-Spike Ab EPTs, which were largely IgG1 and IgG3 (Fig. 2A, 2C). A significant difference in anti-Spike Ab EPTs was observed between disease severity groups (Kruskal–Wallis test, H [3] = 12.61, p = 0.005), whereas in multiple comparisons, the severe group had significantly higher anti-Spike Ab EPT than the mild (Kruskal–Wallis test, p = 0.006) and moderate (Kruskal–Wallis test, p = 0.04) groups (Fig. 2A). We found no significant difference in anti-RBD Ab EPT among disease severity groups (Kruskal–Wallis test, p = 0.28) (Fig. 2B). Multiple comparisons of the anti-Spike Ab subtypes among disease severity groups showed higher anti-Spike IgG1 (Kruskal–Wallis test, mild versus severe: p = 0.04 and moderate versus severe: p = 0.03); however, other subtypes were not statistically significant (Fig. 3C). We found a significant difference in Spike p-score in between the disease severity groups [ANOVA, F (3, 52) = 5.695; p = 0.001], and on multiple comparisons we found that the severe disease group has significantly higher Spike p-score compared with moderate (mean difference, −454.9; 95% CI: −885.3 to −24.38; p = 0.03) and mild disease groups (mean difference, −650.6; 95% CI: −1088 to −213.3; p = 0.001) (Fig. 2D). Similarly, we found a significant difference in RBD p-score between severe and mild disease groups (Kruskal–Wallis test, p = 0.04) (Fig. 2E). The neutralization titer was also significantly different among disease severity groups [ANOVA, F (3, 52) = 4.545; p = 0.006] that on multiple comparison showed the severe disease group has significantly higher neutralization titer compared with the moderate (mean difference, −440.8; 95% CI: −889.8 to 8.122; p = 0.049) and mild disease groups (mean difference, −586.5; 95% CI: −1043 to −130.4; p = 0.006) (Fig. 2F).

FIGURE 3.

Age-dependent increase in end point Ab titers, phagocytosis, and neutralization titer at convalescence. Analysis of the age-dependent responses showing the patients >60 y of age with higher anti-Spike Abs EPTs than the 40- to 60-y-old (difference of mean = 78,911.2; 95% CI: 390, 20,200) and <40-y-old patients (difference of mean = 83,984; 95% CI: 1300, 9160) (A). However, there is no statistical difference in the anti-RBD EPT among the age groups (B). Higher anti-Spike IgG1 among the >60-y-old group was found when compared with <40-y-old (Kruskal–Wallis test, p = 0.0007) and significantly higher IgG3 among >60-y-old group when compared with 40- to 60-y-old group (Kruskal–Wallis test, p = 0.007) (C). Neither the Spike p-score nor RBD p-score was statistically different among the age groups (D and E). Neutralization titer was significantly higher in the >60-y-old patients when compared with the <40-y-old patients (difference of mean = 382.2; 95% CI: 58.4, 152) (F). Dotted lines in each plot show median or mean values +3 SD of 25 healthy control samples (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 3.

Age-dependent increase in end point Ab titers, phagocytosis, and neutralization titer at convalescence. Analysis of the age-dependent responses showing the patients >60 y of age with higher anti-Spike Abs EPTs than the 40- to 60-y-old (difference of mean = 78,911.2; 95% CI: 390, 20,200) and <40-y-old patients (difference of mean = 83,984; 95% CI: 1300, 9160) (A). However, there is no statistical difference in the anti-RBD EPT among the age groups (B). Higher anti-Spike IgG1 among the >60-y-old group was found when compared with <40-y-old (Kruskal–Wallis test, p = 0.0007) and significantly higher IgG3 among >60-y-old group when compared with 40- to 60-y-old group (Kruskal–Wallis test, p = 0.007) (C). Neither the Spike p-score nor RBD p-score was statistically different among the age groups (D and E). Neutralization titer was significantly higher in the >60-y-old patients when compared with the <40-y-old patients (difference of mean = 382.2; 95% CI: 58.4, 152) (F). Dotted lines in each plot show median or mean values +3 SD of 25 healthy control samples (*p < 0.05, **p < 0.01, ***p < 0.001).

Close modal

Analysis of the age-stratified groups of >60 y (n = 24), 40–60 y (n = 14), and <40 y (n = 18) showed that anti-Spike Ab EPTs were significantly different between the age groups of patients (Kruskal–Wallis test, H [2] = 13.6, p = 0.001), and multiple comparisons showed that patients >60 y of age had higher anti-Spike Ab EPTs compared with the 40- to 60-y-old (Kruskal–Wallis test, p = 0.02) and <40-y-old patients (Kruskal–Wallis test, p = 0.001) (Fig. 3A). By contrast, we did not find any difference in the anti-RBD Ab EPT among the age groups (Fig. 3B). Examination of the anti-Spike Ab classes and IgG subtypes revealed significant differences among the age groups (Kruskal–Wallis test, H [2] = 162.3, p = 0.0001). On multiple comparisons, we found higher anti-Spike IgG1 among the >60-y-old group when compared with the <40-y-old group (Kruskal–Wallis test, p = 0.0007) and significantly higher IgG3 among the >60-y-old group when compared with the 40- to 60-y-old group (Kruskal–Wallis test, p = 0.007), while anti-Spike IgG2 and IgG4 were not significantly different (Fig. 3C). We did not find a significant difference in Spike p-score or RBD p-score among the different age groups (Fig. 3D, 3E). Interestingly, neuralization titer was significantly different among the age groups (Kruskal–Wallis test, H [2] = 7.04, p = 0.029) that on multiple comparisons showed that neutralization titer was significantly higher in the >60-y-old patients when compared with the <40-y-old patients (Kruskal–Wallis test, p = 0.042) (Fig. 3F).

There were no significant differences in anti-Spike IgG EPTs, anti-RBD IgG EPTs, and Spike/RBD p-score between male and female patients (Supplemental Fig. 2A–C). However, neutralization function was significantly higher among males compared with females (Mann–Whitney U test, p = 0.01; difference of mean = 342.2) (Supplemental Fig. 2C).

Multiple linear regression analysis was performed to rank all the independent variables of importance that were associated with the dependent variable (Spike p-score), and we found that the anti-Spike EPT was the most important variable that can predict the Spike p-score (change in R2 = 0.2277) (Table V), while the rest of the variables are listed in descending order of importance in Table V. A parameter covariance matrix was plotted to determine the associative relationship between the noncategorical independent variable to yield the dependent variable Spike p-score, where we find that anti-Spike IgG1 and IgG3 were associated positively with most independent variables, whereas age and DPSs associated negatively or poorly with other variables (Fig. 4).

FIGURE 4.

A parameter Spearman covariance matrix to plot the associative relationship between the independent variable using the standardized β coefficient covariance scale (range −1 to +1), where the score >1 is considered a positive relationship, zero as no relationship, and <1 as negative relationship.

FIGURE 4.

A parameter Spearman covariance matrix to plot the associative relationship between the independent variable using the standardized β coefficient covariance scale (range −1 to +1), where the score >1 is considered a positive relationship, zero as no relationship, and <1 as negative relationship.

Close modal
Table V.

Multiple regression analysis of ranking of variables of importance for ADCP

VariableChange in R2 with Other Variables
Anti-Spike EPT 0.2277 
Anti-Spike-IgM 0.0519 
Anti-RBD-IgG1 0.0456 
Disease severity (severe) 0.0309 
DPSs 0.0297 
Anti-RBD EPT 0.0235 
Anti-RBD-IgM 0.0235 
Anti-Spike-IgG1 0.0234 
Neutralization titer 0.0189 
Anti-RBD-IgG2 0.0159 
Anti-Spike-IgG3 0.0127 
Anti-RBD-IgA 0.0115 
Age, y 0.0092 
Anti-Spike-IgG2 0.0027 
Anti-Spike-IgG4 0.002 
Anti -RBD-IgG3 0.0013 
Anti -RBD-IgG4 0.001 
Anti-Spike-IgA 0.0002 
Gender (female) 
VariableChange in R2 with Other Variables
Anti-Spike EPT 0.2277 
Anti-Spike-IgM 0.0519 
Anti-RBD-IgG1 0.0456 
Disease severity (severe) 0.0309 
DPSs 0.0297 
Anti-RBD EPT 0.0235 
Anti-RBD-IgM 0.0235 
Anti-Spike-IgG1 0.0234 
Neutralization titer 0.0189 
Anti-RBD-IgG2 0.0159 
Anti-Spike-IgG3 0.0127 
Anti-RBD-IgA 0.0115 
Age, y 0.0092 
Anti-Spike-IgG2 0.0027 
Anti-Spike-IgG4 0.002 
Anti -RBD-IgG3 0.0013 
Anti -RBD-IgG4 0.001 
Anti-Spike-IgA 0.0002 
Gender (female) 

Spike p-scores significantly correlated with anti-Spike EPTs (Fig. 5A) (Spearman r = 0.65, p = 0.0001) and neutralization titer (Spearman r = 0.56, p = 0.0001) among the convalescent individuals (n = 56) (Fig. 5B). Although neutralization titer significantly correlated with anti-Spike EPT (Spearman r = 0.64, p = 0.0001) and anti-RBD EPT (Spearman r = 0.34, p = 0.009) (Fig. 5C, 5D), it did not correlate with DPSs. Stratification of these patients by disease severity indicated that patients with moderate disease had the most significant correlation of the Spike p-score to the anti-Spike EPTs (Spearman r = 0.71, p = 0.001) and to the neutralization titer (Spearman r = 0.60, p = 0.0007), while individuals with severe disease had no significant correlation among the variables (Fig. 5E).

FIGURE 5.

Correlation of Spike p-score to end point Ab titer and neutralization titer in patients with varying disease severity. Spearman correlation studies of all 56 convalescent patients showed significant positive correlation of Spike p-scores with anti-Spike Ab EPTs (A) and the Spike pseudovirus neutralization (B). Stratification of the patients by disease severity indicated that patients with moderate disease had the most significant correlation of the Spike phagocytosis to the anti-Spike Ab EPTs and to the Spike pseudovirus neutralization, while patients at both ends of the spectrum had the least significant correlation. Patients with moderate disease also displayed significant positive correlation of the neutralization titer with the anti-Spike Ab EPTs (C), anti-RBD Ab EPTs (D), and DPSs (E). r and p values are shown in the text and in Supplemental Table I.

FIGURE 5.

Correlation of Spike p-score to end point Ab titer and neutralization titer in patients with varying disease severity. Spearman correlation studies of all 56 convalescent patients showed significant positive correlation of Spike p-scores with anti-Spike Ab EPTs (A) and the Spike pseudovirus neutralization (B). Stratification of the patients by disease severity indicated that patients with moderate disease had the most significant correlation of the Spike phagocytosis to the anti-Spike Ab EPTs and to the Spike pseudovirus neutralization, while patients at both ends of the spectrum had the least significant correlation. Patients with moderate disease also displayed significant positive correlation of the neutralization titer with the anti-Spike Ab EPTs (C), anti-RBD Ab EPTs (D), and DPSs (E). r and p values are shown in the text and in Supplemental Table I.

Close modal

Interestingly, patients with moderate disease also displayed a significant positive correlation between the neutralization titer and the anti-Spike EPT (Spearman r = 0.7, p = 0.0005), as well as with anti-RBD EPT (Spearman r = 0.6, p = 0.01), while a negative but significant correlation with DPSs was observed (Spearman r = −0.6, p = 0.007) (Fig. 5C–E). When the participants were stratified into different age groups (<40, 40–60, and >60 y of age), we did not find any correlation between Spike p-score and anti-Spike EPTs in any of these age groups (Fig. 6A). We found that only neutralization titer significantly correlates with Spike p-score and anti-Spike EPT across the <40, 40–60, and >60 y age groups (Fig. 6B, 6C). Interestingly, only in the 40- to 60-y-old patients’ group did we find a significant correlation between the neutralization titer and anti-RBD EPT (Fig. 6D).

FIGURE 6.

Correlation of Spike p-score to end point Ab titer and neutralization titer in different age group patients. Stratification of the convalescent patients by age into <40, 40–60, and >60 y of age showed no significant correlation of the Spike phagocytosis to anti-Spike Ab EPTs in any of the age groups (A); however, there was significant but variable correlation of the Spike phagocytosis to the Spike neutralization titer in each age group (B). All age groups showed significant correlation of their neutralization titer to the anti-Spike EPTs (C), but only the 40- to 60-y-old patients showed significant correlation of the neutralization titer to both anti-Spike and anti-RBD Ab EPTs (D). r and p values are shown in the text and in Supplemental Table I.

FIGURE 6.

Correlation of Spike p-score to end point Ab titer and neutralization titer in different age group patients. Stratification of the convalescent patients by age into <40, 40–60, and >60 y of age showed no significant correlation of the Spike phagocytosis to anti-Spike Ab EPTs in any of the age groups (A); however, there was significant but variable correlation of the Spike phagocytosis to the Spike neutralization titer in each age group (B). All age groups showed significant correlation of their neutralization titer to the anti-Spike EPTs (C), but only the 40- to 60-y-old patients showed significant correlation of the neutralization titer to both anti-Spike and anti-RBD Ab EPTs (D). r and p values are shown in the text and in Supplemental Table I.

Close modal

Longitudinal study of nine convalescent patients showed a significant decline of anti-Spike IgG EPT (χ2 [2] = 18, p = 0.0001), Spike p-score (χ2 [2] = 12.6, p = 0.0007), and neutralization function (χ2 [2] = 8, p = 0.01) from visit 1 (1 mo after symptom onset) to visit 3 (12 mo after symptom onset) regardless of disease severity (Fig. 7A–C). The anti-Spike EPT significantly decreased from visit 1 to visit 3 (Friedmann test, visits 1–3, p = 0.0001) (Fig. 4A). Similarly, the Spike p-score significantly decreased from visit 1 to visit 3 (Friedmann test, visits 1–3, p = 0.001) (Fig. 7B). The neutralization titer also significantly decreased from visit 1 to visit 3 (Friedmann test, visits 1–3, p = 0.01) (Fig. 7C). Although the anti-Spike EPT declined by an average of 91.2% (SEM: 1.43) from visit 1 to visit 3, there was better maintenance of the Spike p-score and neutralization titer, which showed 65.3% ± 9.27% and 41.8% ± 39.2% SEM decline on average, respectively (Fig. 7D). Interestingly, the surface plasmon resonance studies showed up to an 840-fold increase in the affinity (KD) of the anti-Spike Abs in the patient plasma to Spike protein in eight of nine patients (mean = 150-fold increase; range, 3.3–836.8) (Fig. 7E, 7F). Conversely, the binding avidity showed a significant decline in eight of nine patients by 115.8-fold (range, 5.1–515.8) (Supplemental Fig. 3). These results suggest that an improvement in the quality of the Abs over time may have contributed to the retention of the phagocytic and neutralization functions, despite the substantial decrease in the EPTs.

FIGURE 7.

Longitudinal study of anti-Spike Abs EPTs, Spike phagocytosis, Spike neutralization titer, and affinity of plasma anti-Spike Abs. Longitudinal study of nine convalescent patients with mild, moderate, or severe disease (n = 3 each) showing significant time-dependent decline of anti-Spike IgG Abs EPTs (A), Spike phagocytosis (B), and anti-Spike neutralization titer (C) over a 12-mo period regardless of their disease severity. Although the anti-Spike EPT declined by on average 91.2% (SEM: 1.43) from visit 1 to visit 3, there was substantial retention of the Spike p-score and neutralization titer, which showed 65.3% ± 9.27% and 41.8% ± 39.2% SEM decline on average, respectively (D). Surface plasmon resonance showing increase in the affinity (KD) of the anti-Spike Abs in patient plasma to recombinant Spike protein in eight of nine patients between visits 1 and 3 (E) by up to 840-fold (mean = 150-fold increase; range, 3.3–836.8) (F). These results suggest improvement in the quality of the anti-Spike Abs over time may have contributed to the retention of the phagocytosis and neutralization titer. Dotted lines in each plot show median or mean values +3 SD of 25 healthy control samples (*p < 0.05, **p < 0.01).

FIGURE 7.

Longitudinal study of anti-Spike Abs EPTs, Spike phagocytosis, Spike neutralization titer, and affinity of plasma anti-Spike Abs. Longitudinal study of nine convalescent patients with mild, moderate, or severe disease (n = 3 each) showing significant time-dependent decline of anti-Spike IgG Abs EPTs (A), Spike phagocytosis (B), and anti-Spike neutralization titer (C) over a 12-mo period regardless of their disease severity. Although the anti-Spike EPT declined by on average 91.2% (SEM: 1.43) from visit 1 to visit 3, there was substantial retention of the Spike p-score and neutralization titer, which showed 65.3% ± 9.27% and 41.8% ± 39.2% SEM decline on average, respectively (D). Surface plasmon resonance showing increase in the affinity (KD) of the anti-Spike Abs in patient plasma to recombinant Spike protein in eight of nine patients between visits 1 and 3 (E) by up to 840-fold (mean = 150-fold increase; range, 3.3–836.8) (F). These results suggest improvement in the quality of the anti-Spike Abs over time may have contributed to the retention of the phagocytosis and neutralization titer. Dotted lines in each plot show median or mean values +3 SD of 25 healthy control samples (*p < 0.05, **p < 0.01).

Close modal

The studies reported in this article demonstrated that patients with acute SARS-CoV-2 infection can mount phagocytic responses mediated by heat-liable components of the plasma as early as 10 DPSs, regardless of their disease severity and levels of their anti-Spike end point Ab titers. We also discovered that COVID-19 severity increases the phagocytic responses in patients who recovered from COVID-19. Importantly, we discovered for the first time, to our knowledge, that the affinity of the anti-Spike Abs in convalescent patients increased over a 12-mo period leading to retention of their phagocytic and neutralization despite a decline in the Ab titers by >90%.

Studies to date used purified patient Abs or cloned mAbs to detect phagocytosis, which represents narrow Ab-dependent phagocytosis that does not recapitulate the potential clinically relevant contribution of plasma/serum proteins such as activated complements that are critical in phagocytic responses against respiratory pathogens (11, 18, 19). It is therefore imperative to design experiments that represent all mediators in the plasma/serum that contribute to the phagocytic functions. In this study, we used nonpurified native patient plasma as a true clinical representation. By doing so we discovered that the early phagocytic response in this disease is primarily driven by heat-liable components in the plasma with contribution from Fc receptor–dependent Ab functions, and patients with the acute disease can mount anti-Spike Ab–mediated phagocytosis despite having undetectable anti-Spike Ab titers. The most important heat-liable components of plasma that play a major role in viral phagocytosis are the classically activated complements that on interaction with complement-fixing IgM and/or IgG1 can lead to enhanced phagocytosis through the collaboration of the Fc and complement receptors (8, 15) or direct opsonization of viruses by complements activated via the mannose-binding lectin pathway (1114, 18).

There is ample evidence that supports the association between complement activation through the classical, lectin, and/or alternate pathways and clinical severity in COVID-19, including a strong link to worsening disease severity and/or systemic inflammation (3538). Interestingly, it has also been shown that anti-Spike Abs from patients with acute COVID-19 can initiate complement tissue deposition that may initiate tissue injury (39). Our finding showing blocking of the Fc receptors can only partially reduce the phagocytic response supports the proposal of collaborative effects between complements and the Fc receptor–specific Ab functions. We propose that this collaborative response may have contributed to the low viral load observed in the patients with a high p-score by causing a more effective viral elimination with a favorable clinical outcome as observed by Defendi et al. (11). In contrast, enhancement of phagocytosis by complement may trigger Fc receptor and/or complement receptor–mediated excessive activation of phagocytic effector cells leading to a hyperinflammatory state that may explain the association between increased phagocytic responses and severe disease observed in our cohort. Interestingly, one of the patients with acute infection with severe disease showed enhancement of phagocytosis on heat inactivation of his plasma. Mechanisms for this enhancement remain to be elucidated. We propose that a potential mechanism may include heat-induced breaking up of immune complexes and retrieval of free Spike protein-binding Abs (e.g., by eliminating complement-mediated interferences) that were made available to opsonize the Spike-coated microbeads leading to increased function. This proposal is supported by a previous study showing significantly enhanced detection of IgM and IgG Abs against West Nile Virus Envelop Ag in heat-inactivated serum compared with non–heat-inactivated serum (40). However, a proportion of their circulating anti-Spike Abs may not have been in immune complex forms but were free to opsonize the Spike-coated microbeads that were readily blocked by the FcR Abs.

Our observations in the acute patients are potentially significant; however, the limited numbers preclude us from drawing definitive conclusions. By contrast, in our convalescent patients where we had a sufficiently powered sample size, at early time points we found a strong positive correlation between phagocytosis and end point Ab titers, as well as with neutralization functions, which was strongly associated among individuals with severe and moderate disease. Multiple regression analysis of 19 relevant variables showed that the most important variable required for high phagocytic response in convalescent patients during early time points was the anti-Spike Ab EPT. We also report that older patients mounted significantly higher neutralization function and had higher Ab EPTs, while gender played a minimal role in the phagocytic Ab effector function. These results are consistent with recent studies in which disease severity and age were identified as major contributors to the end point Ab titers and corresponding Ab-dependent functions, including neutralization, but our study showed no significant difference of phagocytosis among the age groups, albeit marginally higher p-scores in the older patients (Refs. 39, 41, and A. S. Iyer, F. K. Jones, A. Nodoushani, M. Kelly, M. Becker, D. Slater, R. Mills, E. Teng, M. Kamruzzaman, W. F. Garcia-Beltran, et al., manuscript posted on medRxiv, DOI: 10.1101/2020.07.18.20155374). The effect of aging on the phagocytic function of primary macrophages in humans is conflicting (42, 43); thus, future studies using autologous macrophages from each patient might be required when comparing phagocytosis among the different age groups to allow drawing definitive conclusions. Although comorbidities may have contributed to disease severity in the older patients, pre-existing cross-reactive Abs against other common coronaviruses may have led to their consistently higher anti-Spike Ab titers (6) that in turn may explain their higher neutralization function.

Analysis of the anti-Spike IgG subclasses of the convalescent patients showed a progressive increase of IgG1 and IgG3 from asymptomatic to severe disease. This result agrees with a recent study showing that IgG1 and IgG3 dominate the Ab pool of convalescent patients and levels of IgG3 positively correlated with disease severity (44). Interestingly, we observed that the relative quantities of IgG3 were higher than IgG1 in the convalescent patients; this is consistent with a very recent report by Kober et al. (45). However, our multiple regression analysis showed that IgG1 is ranked higher than IgG3 as a variable that can predict phagocytic activity, despite IgG3 having a higher binding affinity to FcγRIIa than IgG1 and having comparable affinities to FcγRI (46), the two major receptors expressed in the effector cells (THP-1) used in our assay (23). Although IgG3 is proposed to be involved in an enhanced phagocytic activity in HIV infection (31, 47), our results suggest that high IgG3 levels in convalescent patients with COVID-19 may primarily contribute to the neutralizing function. This proposal is supported by the observation that IgG3, together with IgM, was identified as key to SARS-CoV-2 neutralization (45).

Circulating IgA has been detected in patients with COVID-19 (29, 48, 49). Moreover, virus-specific circulating IgA has been shown to contribute to sustained neutralization of the virus despite a drop in IgG and IgM levels (49), but its contribution to ADCP is not elucidated. In this study, low IgA levels tracked asymptomatic and mild diseases like a previously reported study (29). We also demonstrated for the first time, to our knowledge, that low IgA also tracked younger age, and we found that IgA level was ranked at the bottom of the variables of importance to the p-score. The minimal contribution of IgA to the phagocytic activity observed in this study is unlike its reported substantial contribution to a sustained neutralization function (49).

To date, phagocytic activity in SARS-CoV-2 has been reported to be maintained for 5 mo postinfection (17); our longitudinal study of a subset of our convalescent patients indicated retention of both phagocytic and neutralization functions for >12 mo, despite a progressive decline of their anti-Spike Ab EPTs. Although the progressive decline of the Ab titers is consistent with previous studies (5052), we show in this study, a significant increase in the anti-Spike Ab affinity over time. Importantly, we found detectable Spike phagocytosis and neutralization titer, suggesting improvement of the quality of these Abs over time may have contributed to the retention of the effector functions despite the decay of the EPTs to baseline. This fits with longitudinal B cell studies that have reported ongoing affinity maturation and improved affinity of the memory B cell receptors up to 12 mo (53), but this is the first study, to our knowledge, to demonstrate Abs in the circulation showing functional improvement that correlated with the functional characteristics observed in the memory B cell compartment. Currently, quantification of Ab titers over time together with neutralization function is considered the gold standard to assess the level of immune protection after COVID-19 infection and/or immunization. However, our data indicate that the quality rather than the quantity of the Abs is a better measure of function, hence we recommend that measurement of the affinity and avidity of Abs should be incorporated into the current clinical assessments of immunological protection.

The authors thank the study participants for their contribution to the research.

The Kirby Institute was supported by the Australian Government Department of Health and Ageing. Research reported in this publication was supported by Snow Medical Foundation as an investigator-initiated study. R.A.B. (grant 1195720), M. Martinello (grant 180135), C.R. (grant 1173666), and A.R.L. (grant 1137587) are fellows supported by the National Health and Medical Research Council.

The views expressed in this publication do not necessarily represent the position of the Australian Government. The content is solely the responsibility of the authors.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • ADCP

    Ab-dependent cellular phagocytosis

  •  
  • CI

    confidence interval

  •  
  • COVID-19

    coronavirus disease 2019

  •  
  • DPS

    day after symptom onset

  •  
  • EPT

    end point titer

  •  
  • KD

    equilibrium constant

  •  
  • p-score

    phagocytosis score

  •  
  • SARS-CoV-2

    severe acute respiratory syndrome coronavirus 2

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The Kirby Institute Rowena A. Bull1 (Co-chair), Marianne Martinello1 (Co-chair), Andrew R. Lloyd,1 John Kaldor,1 Greg Dore1

Marie Bashir Institute Tania Sorrell2

New South Wales Health Pathology William Rawlinson3 and Dominic Dwyer3

Prince of Wales Hospital Jeffrey J. Post4

Royal North Shore Hospital Bernard Hudson5

Sydney Children’s Hospital Adam Bartlett6 and Daniel Lemberg 6UNSW Sarah C. Sasson7 and Nick Di Girolamo7

The Kirby Institute Rowena A. Bull1 (Coordinating Principal Investigator), Marianne Martinello1 (Coordinating Principal Investigator), Marianne Byrne,1 Mohammed Hammoud,1 Andrew R. Lloyd,1 and Roshana Sultan1

Prince of Wales Hospital Jeffrey J. Post4

Northern Beaches Hospital Michael Mina8

Royal North Shore Hospital Bernard Hudson5

Westmead Hospital Nicky Gilroy9

New South Wales Health Pathology William Rawlinson3

St George Hospital Pam Konecny10

Blacktown Mt Druitt Hospital Marianne Martinello11

Sydney Children’s Hospital Adam Bartlett6

St Vincent’s Hospital Gail Matthews12

Blacktown Mt Druitt Hospital Dmitrii Shek11 and Susan Holdaway11

Royal North Shore Hospital Katerina Mitsa-kos5

Prince of Wales Hospital Dianne How-Chow4 and Renier Lagunday4

St George Hospital Sharon Robinson10

Northern Beaches Hospital Lenae Terrill8

Westmead Hospital Neela Joshi,9 (Lucy) Ying Li,9 and Satinder Gill9

St Vincent’s Hospital Alison Sevehon12 1. The Kirby Institute, UNSW Sydney, Sydney, Australia. 2. Marie Bashir Institute, University of Sydney, Sydney, NSW, Australia. 3. New South Wales Health Pathology, Sydney, NSW, Australia. 4. Prince of Wales Hospital, Sydney, NSW, Australia. 5. Royal North Shore Hospital, Sydney, NSW, Australia. 6. Sydney Children’s Hospital, Sydney, NSW, Australia. 7. UNSW, Sydney, NSW, Australia. 8. Northern Beaches Hospital, Sydney, NSW, Australia. 9. Westmead Hospital, Sydney, NSW, Australia. 10. St George Hospital, Sydney, NSW, Australia. 11. Blacktown Mt Druitt Hospital, Blacktown, NSW, Australia. 12. St Vincent’s Hospital, Sydney, NSW, Australia.

Supplementary data