High Frequency of Prior Severe Acute Respiratory Syndrome Coronavirus 2 Infection by Sensitive Nucleocapsid Assays

Abstract Prior infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is typically measured by nucleocapsid serology assays. In this study, we show that the Simoa serology assay and T-cell intracellular cytokine staining assay are more sensitive than the clinical Elecsys assay for detection of nucleocapsid-specific immune responses. These data suggest that the prevalence of prior SARS-CoV-2 infection in the population may be higher than currently appreciated.

Electrochemiluminescence assay (ECLA) plates (MesoScale Discovery [MSD] SARS-CoV-2 immunoglobulin G [IgG], catalog number K15359 U) were designed and produced with up to 10 antigen spots in each well including SARS-CoV-2 nucleocapsid, and assays were performed as previously described [3].The plates were blocked with 50 μL of Blocker A (1% bovine serum albumin in distilled water) solution for at least 30 minutes at room temperature shaking at 700 rpm with a digital microplate shaker.During blocking the serum was diluted to 1:5000 in Diluent 100.The calibrator curve was prepared by diluting the calibrator mixture from MesoScale Discovery (MSD) 1:9 in Diluent 100 and then preparing a 7-step, 4-fold dilution series plus a blank containing only Diluent 100.The plates were then washed 3 times with 150 μL of wash buffer (0.5% Tween in 1× phosphate-buffered saline) and blotted dry, and 50 μL of the diluted samples and calibration curve were added in duplicate to the plates and set to shake at 700 rpm at room temperature for at least 2 hours.The plates were again washed 3 times and 50 μL of SULFO-Tagged anti-Human IgG detection antibody diluted to 1× in Diluent 100 was added to each well and incubated, shaking at 700 rpm at room temperature for at least 1 hour.Plates were then washed 3 times, 150 μL of MSD GOLD Read Buffer B was added to each well, and the plates were read immediately afterward on a MESO QuickPlex SQ 120 machine.MSD titers for each sample were reported as relative light units, which were calculated using the calibrator.
Nine of 22 (41%) participants had a clinical history of prior SARS-CoV-2 infection diagnosed by reverse-transcription polymerase chain reaction (Table 1).Ten of 22 (45%) individuals were positive by the Roche Elecsys assay, including all individuals with a clinical history of diagnosed SARS-CoV-2 infection.In contrast, 14 of 22 (64%) participants were positive by the MSD ECLA assay, and 21 of 22 (95%) individuals were positive by the Simoa assay (Table 1).Participants who were positive by the MSD ECLA and Simoa assays included all those who were positive by the Roche assay as well as most individuals who were negative by the Roche assay.In a concurrent analysis, 0 of 22 (0%) prepandemic samples were positive by the Simoa assay (Supplementary Figure 1).These data show that the MSD ECLA and Simoa assays are more sensitive than the Roche assay for detection of anti-nucleocapsid antibodies.We next measured SARS-CoV-2 cellular immune responses in these individuals by intracellular cytokine staining assays [8].Total and central memory IFN-γ CD8 + and CD4 + T-cell responses were measured to membrane, nucleocapsid, envelope, and spike proteins, which are the dominant SARS-CoV-2 T-cell targets [6,9] (Figure 1).All participants demonstrated robust spike-specific central memory CD8 + and CD4 + T-cell responses, which reflected the combination of vaccine and natural immunity.All but 1 individual also showed nucleocapsid-specific central memory CD8 + and CD4 + T-cell responses.The participant with no detectable nucleocapsid T-cell responses was also the one who was negative by the Simoa nucleocapsid serology assay.The majority of participants also had membrane-and envelope-specific T-cell responses.

DISCUSSION
Our data demonstrate that 21 of 22 (95%) individuals in this cohort had evidence of prior SARS-CoV-2 infection by highly sensitive nucleocapsid serology and T-cell assays.These findings suggest that the clinically approved nucleocapsid serology assays may fail to detect all individuals with prior SARS-CoV-2 infection, particularly those with mild or asymptomatic infection, although a limitation of our study is the small sample size.Nevertheless, negative nucleocapsid serology by the approved assays may not reliably exclude all cases of prior SARS-CoV-2 infection.Our study did not address the durability of nucleocapsid antibodies, which likely also wane over time.
Epidemiologic studies based on current clinical nucleocapsid serology assays may underestimate the true prevalence of prior SARS-CoV-2 infection in the population.Moreover, clinical vaccine studies may not be able to identify or exclude all individuals with prior SARS-CoV-2 infection using the approved nucleocapsid assays.Our data further suggest that some SARS-CoV-2 infections may be asymptomatic, consistent with prior studies [10].Taken together, these data suggest that the actual prevalence of prior SARS-CoV-2 infection and the level of natural immunity in the population may be higher than currently appreciated.

Table 1 . Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Nucleocapsid Serology in 22 Participants
Table shows clinically diagnosed severe acute respiratory syndrome coronavirus 2 infection by reverse-transcription polymerase chain reaction and nucleocapsid serology by the approved Roche Elecsys immunoassay, the Mesoscale Discovery (MSD) electrochemiluminescence assay (ECLA), and the single-molecule array assay (Simoa).