key: cord-282821-qvtvpnrr
authors: Thijsen, Steven; Heron, Michiel; Gremmels, Hendrik; van der Kieft, Robert; Reusken, Chantal; Kremer, Kristin; Limonard, Gijs; Bossink, Ailko
title: Elevated nucleoprotein-induced interferon-γ release in COVID-19 patients detected in a SARS-CoV-2 enzyme-linked immunosorbent spot assay
date: 2020-06-12
journal: J Infect
DOI: 10.1016/j.jinf.2020.06.015
sha: 
doc_id: 282821
cord_uid: qvtvpnrr

nan

Various studies suggest a suppressed T-cell immunity in patients with severe COVID-19 based on decreased T-cell numbers or abnormal interferon gamma (IFNγ) expression by T-lymphocytes detected by flowcytometry. (2) (3) (4) The objective of the present study was to determine the functional T-cell responses to SARS-CoV-2 antigens (mosaic surface protein and nucleoprotein), by using an enzyme-linked immunosorbent spot (ELISpot) interferon-γ release assay, in patients with RT-PCR confirmed COVID-19 (n=27) and healthy controls (n=16). Of the 27 COVID-19 patients, 9 were included from the ICU and 18 from the pulmonary ward.

The moment of sampling varied from 6 to 32 days post onset of symptoms (dps). In addition, the concomitant humoral immune response was assessed by detection of SARS-CoV-2 specific IgA and IgG antibodies directed against the structural protein (S1 domain) of SARS-CoV-2 with a commercial ELISA (EUROIMMUN Medizinische Labordiagnostika AG, Lϋbeck, Germany).

Our results show that the SARS-CoV-2-specific T-cell response measured in the ELISpot versus the dps induced by the mosaic surface protein and the nucleoprotein showed different patterns. In all but one of the 27 COVID-19 cases the T-cell response against the mosaic surface protein was absent or weak, as shown by the ELISpot results which were lower than 20 spot forming cells (SFC). The outlier was recruited from the pulmonary ward and exhibited 45 SFC (Fig. 1a) . In contrast, the Tcell response against the nucleoprotein measured by the ELISpot assay was elevated (10-150 SFC) in 12 of 19 patients (63%) that were sampled at ≥14 dps ( Fig.   1b) . A subgroup of 9 (Fig. 1b, red oval) showed a delayed or reduced T-cell response against the nucleoprotein, compared to the other patients. Five of these showed practically no response, and four showed a weak response (10-20 SFC) at 18-32 dps. Absolute lymphocyte numbers loaded in the SARS-CoV-2 ELISpot were not significantly different between the normal and reduced responders (data not shown).

However, the number of spot forming cells by using the mitogen control stimulant was also significant lower in the delayed or reduced responders (P<0.001) (Fig. 3) .

Moreover, SARS-CoV-2 IgA and IgG antibody levels did not differ between the normal and delayed or reduced responders (data not shown).

Serology showed a sigmoidal pattern, with a sharp increase in specific IgA (Fig. S1) and IgG antibodies (Fig. 1c) against the structural protein (S1 domain) of SARS-CoV-2 around 14 and 15 dps, respectively. Most of the healthy controls showed antibody levels below the cut-off. Except 4 controls, who showed detectable anti-SARS-CoV-2

IgA antibody levels, while anti-SARS-CoV-2 IgG antibodies were negative (Fig. S2) . As none of the healthy controls had more than nine SFC specific for the SARS-CoV-2 nucleoprotein, 10 or more spots was determined to be indicative for COVID-19 disease. Prolonged illness, when sampled more days post onset of symptoms, increased the chance of finding higher number of spots. ROC analysis was performed for the nucleoprotein ELISpot results and >7, >14 and >21 dps. All ROC analyses showed significant areas under the ROC curve, respectively 0.77 (p=0.004), 0.82 (p=0.001) and 1 (p=0.002) for detection of COVID-19 disease (Fig.   1f) .

Interestingly, in a recent published study by Grifoni et al.(5) SARS-CoV-2 epitope pools were used to probe CD4+ T cell responses. They found that M, spike and N proteins were co-dominant each recognized by 100% of COVID-19 cases studied.

With respect to SARS-CoV-2 CD8+ T cell responses, the spike protein was even less dominant, significant reactivity was noted for M, N and other antigens. In agreement, in our study T-cell reactivity was detected with SARS-CoV-2 ELISpot against the N protein. In contrast however, the mosaic surface protein, consisting of exposed healthy controls (green symbols). The broken line represents the cut-off for the SARS-CoV-2 antibody ELISA. Figure 1f depicts the ROC analyses of the SARS-CoV-

Dynamic profile for the detection of anti-SARS-CoV-2 antibodies using four immunochromatographic assays

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