Persistence and decay of human antibody responses to the receptor binding domain of SARS-CoV-2 spike protein in COVID-19 patients

Using an in-house enzyme linked immunosorbent assay (ELISA), we measured anti-RBD antibody responses in two cohorts: 1) symptomatic patients who tested positive for SARS-CoV-2 by PCR (n = 343) and 2) healthy (n = 1,515) and febrile controls (n = 33) collected prior to the SARS-CoV-2 pandemic. The majority of SARS-CoV-2 positive cases were severe (93% hospitalized, 53% requiring ICU level care, 13% died), male (62%), and older (median age: 59) (Table 1, Figure S1). Most pre-pandemic controls were younger (median age: 37) and female (66%). Plasma and/or serum was collected at multiple time points for most patients (63%; n=216), with 34% (n=118) having ≥ 4 samples. Forty-two percent of cases had a sample collected between 0-7 days after onset of symptoms (n=143), 55% had a sample between 8-14 days (n=189), 48% had a sample between 15-28 days (n=165), 35% had a sample between 29-45 days (n=121), 22% had a sample between 46-60 days (n=76), and 10% had a sample > 60 days (n=35). The last sample was collected 122 days post-symptom onset. Twenty-six (8%) cases were immunosuppressed (e.g., on methotrexate, rituximab, etc.), and we did not expect them to mount a robust immune response.

If followed for more than 14 days since symptom onset, most cases (92%) had at least one IgG measurement higher than seen among any pre-pandemic control (Fig. 1). From days 5 to 14, there was a sharp rise in RBD-specific antibodies of all isotypes, and IgG measurements continued to rise until day 25 after the onset of symptoms (Figure S2A). The population average IgA and IgM responses peaked less than a week earlier than IgG and then declined toward concentrations measured in pre-pandemic samples (Figure S2 and S3). IgG antibody responses also began to wane, but at a slower rate. Among 117 cases with ≥ 4 measurements, the individual peak IgM measurement often occurred before that of IgG (before: 55%, simultaneous: 38%) and simultaneously with that of IgA (before: 28%, simultaneous: 53%). Among hospitalized patients, the population average trajectory differed little between severity levels; the average IgG concentrations among hospitalized cases admitted to the ICU were higher than hospitalized cases not admitted to the ICU (Figure S2B). Concentrations of all isotypes were lower among immunosuppressed individuals (Figure S2C).

Each antibody isotype was indicative of infection, and the area under the receiver operating curve (AUC) for each antibody isotype increased to above 98% during the period of 15-28 days after symptom onset (Table 2). The AUC remained high for IgG (99%) and IgA (98%) after 28 days but began to fall for IgM (93%). Using test cutoffs set to ensure no false positives within the pre-pandemic samples (i.e., 100% within sample specificity), we found that the sensitivity of IgG antibodies rose from 7% (≤7 days) to 95% after 14 days of symptoms. The sensitivity of IgA and IgM rose to 90% and 81% 2-4 weeks post-symptom onset but dropped after 4 weeks to 66% and 44%, respectively. Through ten-fold cross-validation, we found that the mean specificity for each isotype was 99.9% (fold-specific range: 99.4 - 100%).

We found the accuracy of serologic identification of recent infections could be slightly improved by adding measurements of IgM and/or IgA to IgG at the earlier phases of infection (Table S2; Figure S4). Using random forest models to combine measurements of different isotypes, we estimated a cvAUC of 92% for IgG & IgM and 91% for IgG & IgA at 8-14 days post-symptom onset. These models provide an estimate of the contribution of each antibody isotype, as well as an approximation of the maximum predictive value of combined measures of anti-RBD IgG, IgA and IgM responses. While all isotypes contributed nearly equally to identifying recent infection antibody profiles in the early phase of illness, IgG responses were the most indicative of infection 8 or more days after the onset of symptoms (Figure S5). Using the pre-determined thresholds for seropositivity for each antibody isotype, out of the 357 samples collected during early infection (< 14 days post symptom onset), we were able to correctly identify an additional 19 (5%) cases among the IgG negative samples by adding IgM, 21 (6%) by adding IgA, and 33 (9%) by adding both IgM and IgA. When accounting for class imbalance in the random forest procedure, similar results were obtained (Figure S6, Figure S7).

Using the cutoffs defined earlier, we estimated the distribution of the time required to become seropositive (seroconversion) and return to becoming seronegative (seroreversion). Overall, 324 (94%) individuals had more than 1 measurement for every 28 days of follow-up. Of the 159 cases with samples after 20 days post-symptoms, most had evidence of seroconversion for all isotypes (IgG: 96%, IgM: 88%, IgA: 89%). The estimated median time to seroconversion from symptom onset was comparable across antibody isotype: 10.7 days (95% CI: 9.6-11.9) for IgG, 11.7 days (10.4-13.0) for IgA and 11.9 (10.5-13.4 days) for IgM (Fig. 2). On average, we estimated the median time to seroconversion among hospitalized patients to be over four days earlier as compared to nonhospitalized patients for all isotypes; men and those aged <65 years also seroconverted more quickly on average (Table S3).

Of seroconverted cases with samples 46 days post-symptoms or after, most eventually had IgM (45/61) and IgA (30/64) seronegative measurements. The median time to seroreversion for IgM was 48.9 days (95% CI: 43.8 – 55.6), with the first 5% seroreverting by 23.7 days (95% CI: 21.6 – 26.0). We estimated a slightly later median seroreversion time for IgA of 70.5 days (95% CI: 58.5 - 87.5), with the first 5% seroreverting by 27.7 days (95% CI: 22.8–32.9, Fig. 2). Only 3 of 70 cases had evidence of seroreversion for IgG. All 3 patients who seroreverted for IgG required ICU level care, however 2 of the 3 did not have robust IgG responses (peak IgG measurement < 2 μg/mL, 1 of whom was immunosuppressed).

We measured pseudoneutralizing antibodies targeting the SARS-CoV-2 S protein in 88 samples from 15 individuals collected between 0 and 75 days post-symptoms (Fig. 3). Over the course of infection, all individuals tested developed detectable neutralizing antibodies (NAb). NAb titers were correlated with the concentration of anti-RBD IgG (r = 0.87). Of note, similar to anti-RBD IgG responses, NAb titers plateaued and remained detectable at later time points despite the more rapid decline of IgA and IgM responses.

We evaluated antibody responses to RBDs derived from spike proteins of endemic human coronaviruses (CoVs) (i.e., HKU1, 229E, OC43, and NL63), severe acute respiratory syndrome coronavirus (SARS-CoV-1) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) (Figure S8). Antibody responses to the endemic CoVs were comparable between pre-pandemic controls and individuals with COVID-19 at all phases of infection, demonstrating a lack of cross-reactivity. Although a few individuals with SARS-CoV-2 infection had increasing levels of antibodies to endemic CoVs over time, which could be explained by cross-reactive anamnestic responses/ immunologic memory, the majority stayed the same. Thus, overall, we did not observe a detectable cross-reactive response to the RBDs of the endemic human coronaviruses across the population of individuals infected with SARS-CoV-2. In contrast, we did observe significant cross-reactivity to SARS-CoV-1 RBD in individuals with COVID-19, but no significant cross-reactive responses to the MERS-CoV RBD. Of note, there were three pre-pandemic controls (samples collected prior to October 2019) with IgA cross-reactivity to SARS-CoV-1.

Since DBS could be used in large serosurveys where venous blood may be logistically challenging to collect and process, we also evaluated the assay with simulated dried blood spot eluates in a subset of patients (n= 20 at two timepoints; 40 samples) and pre-pandemic controls (n=20). The anti-RBD IgG DBS measurements had a high degree of linear correlation in both cases and control plasma (r = 0.99, Figure S9). While the classification of all samples was the same between DBS and plasma samples (100% classification concordance), values between the two sample types diverged more at low titer values.

We evaluated the magnitude and kinetics of the early human antibody response to the receptor binding domain of SARS-CoV-2 spike protein, with the additional objective of evaluating the specificity and sensitivity of these antibody responses for identifying individuals with recent infection. Thus, we measured antibody concentration in blood samples obtained from confirmed patients with symptomatic SARS-CoV-2 infection and from control individuals whose samples were collected prior to the pandemic. IgG, IgA and IgM antibody concentrations were measured by ELISA using recombinant SARS-CoV2 RBD in all samples. In a subset of samples, neutralizing antibody responses directed against the spike protein were also measured using a lentivirus pseudoneutralization model. From these data, we modeled the classification accuracy for each individual isotype and combinations of isotypes at different time points, and the temporal dynamics of seroconversion and seroreversion following the onset of symptoms.

We obtained plasma and/or serum samples, collected for routine clinical care, from individuals with PCR-confirmed SARS-CoV-2 infection presenting, with fever and/or viral respiratory symptoms from March to April 2020 and who met criteria for RT-PCR testing. Testing criteria for SARS-CoV-2 changed over time, but primarily included patients with severe symptoms requiring hospital admission, although those who had other risk factors for disease progression (e.g., were age 60 or older, had diabetes, or were immunocompromised), or who worked or lived in a setting where infection control requirements dictated a need for testing. Additional serum/plasma samples collected September 2015 to December 2019 prior to the SARS-CoV-2 pandemic included healthy adults seen at the MGH Immunization and Travel Clinic prior to travel, patients undergoing routine serology, and patients presenting with other known febrile illnesses. Plasma samples, except for the routine serology samples, were heat-inactivated at 56°C for one hour prior to analysis.). Patient demographic information, lab results, and clinical outcomes were extracted from the electronic medical record. Patients were considered immunosuppressed if they had underlying immunosuppressive condition (e.g., HIV with CD4 count less than 200) or were on an immunosuppressive/immunomodulating agent at the time of their admission (e.g., methotrexate, rituximab) All research was approved by the Institutional Review Board for Human Subjects Research at MGH.

Seventy-two microliters of single donor, seronegative whole blood collected from sodium heparin tubes (Becton, Dickinson, NJ), was spiked with 8 μl heat-inactivated plasma (10% of the whole blood volume) to maintain the relative whole blood composition. Assuming plasma is 50% of the whole blood volume, the spiked plasma was 18.18% of the final plasma volume. Whole blood (40 μL) was spotted onto Whatman 903 Protein Saver cards (GE Healthcare, Cardiff, UK) in replicate and allowed to dry overnight at room temperature. Two 6-mm² punches from the DBS card (5 μL plasma per punch) were placed in 133 μL PBS-0.05% Tween 20 pH 7.4 (Sigma-Aldrich, St. Louis, MO) and incubated overnight at 4°C with gentle agitation eluates were then recovered after centrifugation. The total dilution of the spiked plasma in DBS eluate was assumed to be 1:73.15, which accounts for the initial dilution from spiking (1:5.5) and the further dilution during DBS elution (1:13.3)

The ELISA assays measured IgG, IgA, and IgM responses to the receptor binding domain of the spike protein (RBD) from SARS-CoV-2 [GenBank: MN975262], Middle East Respiratory Syndrome (MERS) virus [GenBank: AFY13307.1], SARS-CoV-1 [GenBank: AAP13441.1], and common cold coronaviruses HKU1 [GenBank: AAT98580.1], OC229E [GenBank: AAK32191], OC43 [GenBank:AAT84362], and NL63 [GenBank: AKT07952]. RBD sequences were cloned into pVRC vector followed by expression in mammalian cells Expi293 cells (ThermoFisher Scientific, Waltham,MA) with a C-terminal streptavidin-binding peptide (SBP)-His8X tag, and purified over TALON resin (Takara, Mountain View, CA) followed by size exclusion chromatography and cleavage of the His tag. RBD-specific antibody concentrations (μg/mL) were quantified using isotype-specific anti-RBD monoclonal antibodies. The RBDs were expressed in Expi293F suspension cells with a C-terminal SBP-His8X tag, and purified using affinity chromatography and then size exclusion chromatography prior to removal of the His tag as described previously (23). Briefly, 384 well Nunc MaxiSorp plates (Invitrogen, Carlsbad, CA) were coated by adding 50 μL of RBD in carbonate buffer (1 μg/mL) and incubating for 1 hour at room temperature (RT). Plates were then blocked for 30 min at RT with 5% nonfat milk in tris-buffered saline (TBS). Diluted samples (1:100 in TBS with 5% milk, 0.5% Tween) were added to the plate (25 μL/well) and incubated for 1 hour at 37°C with shaking. Serial 4-fold dilutions to 1:6400 were also included for individuals with high titers. At the end of incubation, samples were washed 5 times with 1X high salt TBS. Subsequently, goat anti-human IgA, IgG, and IgM- horseradish peroxidase conjugated secondary antibodies diluted (Jackson ImmunoResearch) at 1:10000 (IgG, IgM) or 1:5000 (IgA) in 5% milk in TBST were added to plates (25 μL/well) and incubated at RT with shaking for 30 min followed by 5X 1X high salt TBS washes and a last wash with 1X TBS. Bound secondaries were detected using 1-step Ultra TMB (tetramethylbenzidine; ThermoScientific, Waltham, MA, 25 μL/well). Plates were incubated at RT for 5 min in the dark before addition of 2 N sulfuric acid stop solution (25 μL/well). The optical density (OD) was read at 450 nm and 570 nm on a plate reader. OD values were adjusted by subtracting the 570 nm OD from the 450 nm OD. We used a standard curve of the anti-SARS-CoV-2 monoclonal, CR3022 (24), to calculate the concentration of anti-RBD IgG, IgA, and IgM expressed in μg/mL. Note: For the DBS and plasma comparisons the starting concentration was 1:200.

To determine the SARS-CoV-2 neutralization activity of our plasma samples, we used a lentivirus pseudoneutralization model as previously described (20), which is a strong correlate of protective immunity in challenged rhesus macaques (25). We expressed results from this assay as the antibody titer required to neutralize 50% of the SARS-CoV-2 pseudovirus (NT50).