key: cord-0884833-k5sqkkxw
authors: Törnell, Andreas; Grauers Wiktorin, Hanna; Ringlander, Johan; Arabpour, Mohammad; Nilsson, Malin R; Nilsson, Staffan; Kiffin, Roberta; Lindh, Magnus; Lagging, Martin; Hellstrand, Kristoffer; Martner, Anna
title: Rapid cytokine release assays for analysis of SARS-CoV-2-specific T cells in whole blood
date: 2022-01-12
journal: J Infect Dis
DOI: 10.1093/infdis/jiac005
sha: db8176cd8eaeac312ef728dc088349adea96221e
doc_id: 884833
cord_uid: k5sqkkxw

BACKGROUND: Waning of IgG antibodies against SARS-CoV-2 complicates diagnosis of past infection. Durability of T cell memory against SARS-CoV-2 remains unclear, and most current T cell protocols are unsuited for large-scale automation. METHODS: Whole blood samples from 31 patients with verified past COVID-19 and 46 controls, out of which 40 received SARS-CoV-2 vaccine were stimulated with peptides spanning the nucleocapsid (NC) or spike 1 (S1) regions of SARS-CoV-2 and analyzed for interferon-γ (IFN-γ) in supernatant plasma. Diagnostic accuracy of these assays was evaluated against serum anti-NC and anti-receptor-binding domain S1 IgG. RESULTS: Induction of IFN-γ in whole blood by NC or S1 peptides diagnosed past COVID-19 with high accuracy (AUC=0.93, AUC=0.95, respectively). In accordance with previous studies, NC-IgG levels rapidly waned with only 5/17 patients (29%) remaining seropositive >180 days after infection. By contrast, NC-peptide-induced T cell memory responses remained in 13/17 (76%) subjects >180 days after infection (P=0.012 vs. NC-IgG, McNemar test). After two vaccine doses, 18/18 donors exhibited S1-specific T cell memory. CONCLUSIONS: Cytokine release assays for the monitoring of T cell memory in whole blood may be useful for evaluation of complications following unverified past COVID-19 and for long-term assessment of vaccine-induced T cell immunity.

The detection of IgG antibodies in serum is the mainstay of the diagnosis of past COVID- 19 . Previous studies show that IgG antibodies commonly become detectable within two to three weeks after onset of symptoms, albeit with inter-individual variation [1] [2] [3] . In routine diagnostics, most laboratories utilize automated, platform-based immunoassays that detect IgG antibodies against nucleocapsid (NC) [4] and/or spike (S) proteins of SARS-CoV-2. A survey of the performance of platform-based IgG antibody tests, applied on serum derived from unvaccinated subjects after a previously verified infection, reported consistently high specificity, but lower sensitivity [5] . The sensitivity of these assays is even lower in patients with previous mild or asymptomatic COVID-19 [6] [7] [8] .

The diagnostic accuracy of serum IgG against SARS-CoV-2 is further limited by diminished antibody titers over time [9, 10] . Lau et al. estimated that neutralizing antibodies remain detectable for approximately 14 months in patients with symptomatic COVID-19 and for approximately six months in patients with asymptomatic infection [11] . The waning of serum antibodies against SARS-CoV-2 has been noted also in studies evaluating the durability of serum IgG analyzed using platform-based assays. Levels of antibodies against NC decline more rapidly than antibodies against S [12] , which may complicate the diagnosis of past natural COVID-19 in vaccinated subjects.

The shortcomings of antibody tests have spurred the development of tests that reflect SARS-CoV-2specific T cell immunity. A T cell assay may thus detect an immunological memory that is not captured by serum IgG. Protocols for detection of SARS-CoV-2-specific T cells typically comprise the isolation of peripheral blood mononuclear cells (PBMCs) followed by analysis of cell subsets using flow cytometry and are thus unsuited for large-scale routine diagnostics. Rapid cytokine-release A c c e p t e d M a n u s c r i p t 6 assays, based on the exposure of whole blood samples to antigens, are useful in the diagnosis of tuberculosis and other infections [13] and similar tests have been applied to detect specific T cell reactivity against SARS-CoV-2 antigens. Recent studies imply that results achieved in cytokinerelease assays for T cell reactivity correlate with seropositivity for IgG and that these assays capture T cell memory responses after vaccination [14, 15] .

We have developed rapid and refined cytokine-release based T cell assays in which whole blood samples were exposed ex vivo to antigens derived from the NC or the S1 portion of spike followed by analysis of interferon- (IFN-) in supernatant plasma. The assays demonstrated high accuracy in detecting previously verified COVID-19 as well as S1-specific T cell responses following vaccination.

Our results underscore that rapid T cell assays may be of value for more accurate diagnosis of past natural SARS-CoV-2 infection and for determining the durability of T cell reactivity after infection or vaccination.

This study was conducted between December 2020 and August 2021 at the Sahlgrenska University A c c e p t e d M a n u s c r i p t 7   Forty-six donors were regarded as naïve controls, as they had not undergone a confirmed COVID-19   infection, while 31 donors had undergone a RT-PCR confirmed COVID-19 infection (past-COVID) , at least 25 days prior to sampling. One case of COVID-19 was classified as severe (defined by hospitalization) while the remaining 30 subjects had mild infections. Blood was collected 3-5 weeks after vaccination from 40 of the 77 study participants. Detailed characteristics of at which time points samples were collected and the type of vaccines received is provided in Table 2 . The mean time between vaccine dose one and two was 39 days (range: 21-113 days), which is in accordance with the Swedish national guidelines.

Chemiluminescent microparticle immunoassays (CMIAs) were performed on serum using the Alinity system for the quantitation of IgG antibodies against the spike receptor-binding domain (RBD) for the S1 and NC IgG tests were 14 BAU/ml and 1.4 AU/ml, respectively. All values below LOD were set to 50% of LOD.

Peripheral venous blood was collected in BD vacutainer lithium-heparin tubes (BD, Plymouth, UK) and stored at room temperature for a maximum of 24 hours. One ml of whole-blood was stimulated in 10 ml-tubes (Sarstedt AB, Helsingborg, Sweden) with peptide pools from SARS-CoV-2 or no stimuli (negative control). The peptides were dissolved in PBS with 20% DMSO. Each sample was incubated A c c e p t e d M a n u s c r i p t 8 with 1 µg/ml/peptide of 15-mer peptides with 11-amino acid overlap spanning the complete sequence of the SARS-CoV-2 nucleocapsid phosphoprotein (amino acid (aa) 1-419, total 102 peptides; NC; 130-126-699, Miltenyi Biotec, Lund, Sweden), the N-terminal S1 domain of the SARS-CoV-2 surface glycoprotein (aa 1-692, total 170 peptides S1; 130-127-041, Miltenyi Biotec) or no stimuli. The samples were incubated with these peptides for approximately 48 hours at 37 o C and 5% CO 2 . Tubes were then centrifuged for five minutes at 1500 rpm and supernatant plasma was recovered and stored at -80 o C until analysis of IFN- content.

Whole blood samples obtained from ten unvaccinated donors were stimulated with S1 but not NC peptides and were thus only analyzed for antibodies and S1-Samples from seven of these donors were analyzed for NC- at later time-points. In four blood samples collected after the second vaccination, hemolysis occurred in the whole blood sample but not in the serum sample. These samples were therefore only analyzed for antibodies and not peptide-induced IFN-. Additionally, pre-vaccination samples were collected before and after infection for three individuals. All three donors were negative for S1 RBD-IgG, NC IgG, S1-and NC-γ before infection and became positive in these assays after COVID-19 infection (Supplementary figure 1A-D), the samples from before infection from these individuals were not used for any further analyses.

Plasma levels of IFN- from unstimulated and NC-or S1-stimulated whole blood was determined by IFN- ELISA (DY285B, R&D systems) according to the manufacturer's instructions. Plasma was diluted (1:2) in PBS containing 1% BSA and 10% rat or mouse serum (Stemcell Technologies and Invitrogen, respectively) to minimize unspecific reactivity. A reduced concentration of mouse serum in an additional standard curve was used to normalize the data in samples diluted in mouse serum as this A c c e p t e d M a n u s c r i p t 9 matrix interfered with the standard curve. Optical density was measured at 450 nm and 570 nm using a FLUOstar Omega plate reader (BMG, Ortenberg, Germany). Results are presented as peptideinduced IFN- obtained by subtracting levels of IFN- in unstimulated samples from peptidestimulated samples. Levels below LOD (<10 pg/ml) were set to 50% of LOD. 

PBMCs were thawed and cultured at 2.5-5 × 10 6 cells/ml in round-bottom 96-well plates in the presence of 1 µg/ml/peptide of SARS-CoV-2 NC-or S1-spanning peptides overnight at 37 o C and 5% 

In a first set of experiments, we aimed to optimize assay conditions and noted that a SARS-CoV-2 peptide concentration of 1 µg/ml/peptide and an incubation time of 48 hours yielded strong IFN- A c c e p t e d M a n u s c r i p t 11 formation in whole blood from previously SARS-CoV-2-infected patients but not in uninfected patients ( Supplementary Figure 2A-B) . In further experiments, peptides spanning the NC or S1 regions of SARS-CoV-2 were thus added at 1 µg/ml/peptide to whole blood specimens from uninfected control subjects (n=46) or patients with past COVID-19 previously confirmed by detection of SARS-CoV-2 RNA by PCR (n=31). After 48 hours of incubation plasma supernatants were harvested and analyzed for content of IFN-. Figure 1B ) between these cohorts.

In samples collected from previously infected patients, 23/26 (88%) were reactive in the NC- test whereas 16/31 (52%) were seropositive for NC-IgG. Additionally, 2/46 (4.3%) samples from uninfected subjects were seropositive for NC-IgG but were negative for NC- whereas 5/41 (12%) samples showed reactivity in the NC- test but were seronegative for NC-IgG. While the difference was not significant, the NC- test thus tended to show a higher reactivity among subjects who had not undergone a verified COVID-19 infection.

We also performed analyses of S1-induced IFN- (S1- test) in plasma supernatants from the whole blood cultures. The S1- test as well as presence of IgG antibodies against S1 (anti-RDB S1-IgG) diagnosed past COVID-19 (median 179 days from infection, range 25-303) with high accuracy ( Figure   1C -D). All but two patients with previous infection, 24/26 (92%), were positive in the S1-assay, while 21/26 (81%) were seropositive for anti-RBD S1-IgG. Among uninfected subjects 5/42 (12%) were positive in the S1-assay, while no one, 0/42 (0%), was seropositive for anti-RDB S1-IgG.

A c c e p t e d M a n u s c r i p t 12 However, out of the five S1-responsive donors without confirmed COVID-19 infection, two were responsive also in the NC-assay and another one had NC-IgG antibodies. Hence, previous asymptomatic infections cannot be excluded. The sensitivity and specificity of each test is listed in Table 3 . Among previously infected patients, there was a trend towards correlation between NC-and NC-IgG and there was a significant correlation between levels of S1-and anti-RDB S1-IgG in infected donors ( Figure 1B and D, right panels) .

We aimed to determine the T cell subset producing IFN-γ in parallel experiments where PBMC were isolated from control subjects and patients with previously verified COVID-19. The PBMC were incubated with peptides spanning the NC or S1 regions of SARS-CoV-2, thus mimicking the induction protocol applied for whole blood specimens. After overnight incubation, intracellular IFN-γ in gated CD4 + or CD8 + T cells was detected by flow cytometry. In NC peptide-stimulated PBMC, the induced IFN-γ by CD4 + and CD8 + T cells significantly discriminated previously infected patients from controls with a similar trend for S1 peptide-stimulated PBMC (Figure 2) . For the small subset of infected donors analyzed by both flow cytometry and by the whole blood IFN-γ releaseassay, there was no significant correlation between intracellular and extracellular IFN-γ production. Concordance between SARS-CoV-2 peptide induced intracellular IFN-γ in CD4 + and CD8 + T cell subsets and extracellularly released IFN-γ has however been demonstrated previously [16] .

We aimed to determine the durability of immune reactivity to SARS-CoV-2 after COVID-19 infection and thus compared seropositivity and results obtained in the whole blood NC- test over time. This analysis was restricted to patients for whom samples for both of these tests were available on the A c c e p t e d M a n u s c r i p t 13 same day. Samples within each interval after confirmed infection are from unique individuals, and the latest collected sample was chosen where multiple samples were available within the same interval. In agreement with earlier studies [12, [17] [18] [19] , only 5/17 (29%) of patients remained NC-IgG seropositive when analyzed at >180 days after verified COVID-19 (median 333 days, range 237-408).

In contrast, 13/17 (76%) previously infected patients with samples taken >180 days after PCR confirmed SARS-CoV-2 infection showed reactivity in the NC- whole blood test (P=0.012 vs NC-IgG, McNemar test, Table 4 ), thus implying that the T cell response to NC is more long-lasting than antibodies against NC.

We obtained samples from 36 subjects after the first dose of vaccination against COVID-19, and from 22 subjects after the second dose of vaccination. Vaccination triggered a strong induction of S1specific T cells in whole blood along with S1-IgG in serum four weeks after vaccination (range three to five weeks) (Figure 3) . When comparing T cell responses after the first dose of vaccine in subjects with or without previous COVID-19, whole blood from vaccinated individuals with previous infection produced significantly higher levels of IFN-in the S1- assay (P<0.01, permutation test, Figure 3A) , with a similar difference when comparing S1 IgG levels in vaccinated donors with and without previous infection (P<0.0001, permutation test, Figure 3B ). As expected, vaccination did not alter levels of NC-induced IFN- or NC-IgG (Supplementary Figure 3) . In this small cohort, we observed no differences in immune responses among recipients across different vaccines, nor significant associations between vaccine responsiveness, age or gender (data not shown).

A c c e p t e d M a n u s c r i p t 14 

We report that ex vivo stimulation of whole blood from previously SARS-CoV-2-infected patients with peptides spanning the NC and S1 regions of SARS-CoV-2 stalwartly evoked the formation of IFN-, likely indicative of antigen-specific T cell memory. ROC analyses suggested that the sensitivity of these whole blood assays in terms of identifying previously infected patients were higher than analysis of antigen-specific IgG levels in serum. A higher number of control subjects tested positive in the NC and S1 IFN- tests suggesting lower specificity compared with antibody tests, although previous undiagnosed COVID-19 or cross-reactivity from infection with other coronaviruses cannot formally be excluded in these patients. Our results also imply that SARS-CoV-2-specific T cell reactivity is significantly more durable than NC-IgG. Thus, NC- responses in blood remained detectable for >six months in >70% of previously infected patients despite that <30% of these patients had detectable antibodies against NC. We observed T cell responses to S1 in whole blood samples from vaccinated subjects that coincided with the appearance of anti-RBD S1-IgG in serum.

Our results thus confirm and extend results from earlier studies of evoked T cell memory in whole blood from SARS-CoV-2-infected and vaccinated subjects [14, 15] .

In vaccinated subjects, assays of anti-RDB S1-IgG or T cell reactivity against spike proteins obviously cannot be used to diagnose past COVID-19. The NC- test displayed superior sensitivity in detecting past COVID-19 compared with the detection of NC-IgG antibodies. We thus propose that the NC- test, or similar assays using whole blood samples, may be valuable in identifying patients with previous infection that was not captured by the detection of virus or antigen during active viral replication. The NC- test may thus be instrumental in the evaluation of suspected long-term morbidity from COVID-19 among patients with fading or undetectable NC-IgG, but also in monitoring the durability of T cell immunity after natural infection in the post-vaccine era. Earlier studies A c c e p t e d M a n u s c r i p t 15 suggest that NC-specific T cell memory, analyzed in PBMCs by OX40/4-1BB expression on CD4 + T cells using flow cytometry, persists for at least eight months after COVID-19 [20] , and our results thus support the longevity of T cell memory although further follow-up from infection is warranted.

In this smaller series of patients, the S1- test showed slightly higher sensitivity in diagnosing past COVID-19 compared with anti-RBD S1-IgG. The S1- test may, hence, provide a means to monitor the durability of T memory responses after vaccination. Similar to the NC-IgG test, previous studies also report reduction of S1-IgG over time after natural infection [21] . A recent study thus reported waning of S1-IgG also after two SARS-CoV-2 vaccine doses [9] . Long-term studies are required to clarify whether or not the S1- test, or similar whole blood-based assays, are helpful in monitoring the efficiency of vaccine-induced T cell immunity and if presence of SARS-CoV-2 specific T cells provides clinically meaningful protection against COVID-19 also in the absence of S1-specific antibodies.

In addition to whole blood assays of SARS-CoV-2 peptide-induced IFN-, we utilized a similar induction protocol for the assessment of T cell activation in Ficoll-separated PBMC analyzed by flow cytometry. These experiments suggested that CD4 + and CD8 + T cells from infected patients accumulated intracellular IFN- after exposure to SARS-CoV-2 peptides in accordance with previous reports [16, 22] , implying that both T cell subsets contributed to the formation of IFN- in whole blood assays. For the small subset of infected donors analyzed by both flow cytometry and by the whole blood IFN-γ releaseassay, there was no significant correlation between intracellular and extracellular IFN-γ production. Concordance between SARS-CoV-2 peptide induced intracellular IFNγ in CD4 + and CD8 + T cell subsets and extracellularly released IFN-γ has however been demonstrated previously [16] . with low IFN- or IgG antibody responses. A few subjects in the control group showed reactivity in more than one of the NC-, S1- or NC-IgG assays, suggesting the possibility of previous undiagnosed SARS-CoV-2 infections. Another possible explanation is that the background levels of IFN-observed in S1-or NC-stimulated control subjects stem from cross-reactivity from previous common cold coronavirus infections, as has been suggested to occur in other studies [23, 24] . However, the prevalence of common cold coronaviruses is high [25] while the frequency of reactivity among the controls was low, which speaks against cross-reactivity induced by other coronaviruses.

Nevertheless, if the background reactivity in the controls stem from a previous infection, this is congruent with the higher number of controls being reactive in the IFN-assays compared with the IgG assays, as the T cell responses appear more durable.

In conclusion, whole blood assays for analysis of T cell memory against SARS-CoV-2 may be useful in determining the duration of T cell immunity after natural infection or vaccination, and in particular in diagnosing past COVID-19 in patients with waning antibody titers. This study recruited hospital staff members with minimal co-morbidity, and the results therefore reflect T cell immunity in a largely healthy population. The potential utility of whole blood-based assessment of T cell memory warrants further evaluation in subjects at risk for severe COVID-19, including immunosuppressed patients. M a n u s c r i p t 24 A c c e p t e d M a n u s c r i p t 25 

WHO International Standard for anti-SARS-CoV-2 immunoglobulin

Robust neutralizing antibodies to SARS-CoV-2 infection persist for months

Humoral Immune Response to SARS-CoV-2 in Iceland

Performance Characteristics of the Abbott Architect SARS-CoV-2 IgG Assay and Seroprevalence in

Evaluation of 11 SARS-CoV-2 antibody tests by using samples from patients with defined IgG antibody titers

Longitudinal antibody repertoire in "mild" versus "severe" COVID-19 patients reveals immune markers associated with disease severity and resolution

COVID-19-neutralizing antibodies predict disease severity and survival

Dynamic changes in anti-SARS-CoV-2 antibodies during SARS-CoV-2 infection and recovery from COVID-19

Spike-antibody waning after second dose of BNT162b2 or ChAdOx1

Dynamics of Neutralizing Antibody Titers in the Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection

Neutralizing antibody titres in SARS-CoV-2 infections

SARS-CoV-2 Seroprevalence Survey Estimates Are Affected by Anti-Nucleocapsid Antibody Decline

Interferon-gamma release assays or tuberculin skin test for detection and management of latent tuberculosis infection: a systematic review and metaanalysis

Broad T Cell Targeting of Structural Proteins After SARS-CoV-2 Infection: High Throughput Assessment of T Cell Reactivity Using an Automated Interferon Gamma Release Assay

A whole blood test to measure SARS-CoV-2-specific response in COVID-19 patients

IFN-γ(+) cell response and IFN-γ release concordance after in vitro SARS-CoV-2 stimulation

The Duration, Dynamics, and Determinants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Responses in Individual Healthcare Workers

Analysis of Humoral Immune Responses in Patients With Severe Acute Respiratory Syndrome Coronavirus 2 Infection

Longitudinal Serological Analysis and Neutralizing Antibody Levels in Coronavirus Disease 2019 Convalescent Patients

Longitudinal Analysis of Human Memory T-Cell Response According to the Severity of Illness up to 8 Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection

Evolution of antibody immunity to SARS-CoV-2

SARS-CoV-2 specific memory T cell epitopes identified in COVID-19-recovered subjects

Low-Avidity CD4(+) T Cell Responses to SARS-CoV-2 in Unexposed Individuals and Humans with Severe COVID-19

Identification of TCR repertoires in functionally competent cytotoxic T cells cross-reactive to SARS-CoV-2

Coronavirus Occurrence and Transmission Over 8 Years in the HIVE Cohort of Households in Michigan

We are grateful to staff members at the Departments of Virology and Gynecology for their participation in this study.

A c c e p t e d M a n u s c r i p t 18 A c c e p t e d M a n u s c r i p t