key: cord-1047670-4mj2kq53
authors: Fedele, Giorgio; Russo, Gianluca; Schiavoni, Ilaria; Leone, Pasqualina; Olivetta, Eleonora; Perri, Valentina; Zingaropoli, Maria Antonella; Ciardi, Maria Rosa; Pasculli, Patrizia; Mastroianni, Claudio Maria; Stefanelli, Paola
title: Early IgG / IgA response in hospitalized COVID-19 patients is associated with a less severe disease.
date: 2021-09-12
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2021.115539
sha: 956244a6004688126b42a2377cb24c91a8b079fc
doc_id: 1047670
cord_uid: 4mj2kq53

We determined the kinetics of anti-SARS-CoV-2 antibody response in fifteen hospitalized COVID-19 patients. Patients were divided into mild/moderate (mild, n=1; moderate, n=4) or severe (n=10) and virus-specific anti-Nucleocapsid IgM, anti-Spike IgG and anti-Spike IgA were measured in serial serum samples collected 0-15 days after hospital admission. Surrogate neutralization assays were performed by testing inhibition of ACE-2 binding to Spike. In three patients (2 severe and 1 moderate case), serum antibodies and T-cell memory were monitored six months after baseline. Although IgM response tended to appear first, patients affected by less severe disease were more prone to an early IgG/IgA response. Neutralization of Spike binding to ACE2 correlated with anti-Spike IgG and IgA. IgG and IgA antibody response persisted at the six months follow-up. A recall T-cell response to the Spike antigen was observed in two out of three patients, not related to disease severity.

The immune response against SARS-CoV-2 remains unclear and many caveats in its knowledge are still present. A correlation of clinical severity with high titres of SARS-CoV-2 antibodies, mainly immunoglobulin (Ig)G, was shown (Zhao et The objective of the study was the characterization of the serological response to SARS-CoV-2 in patients hospitalized with confirmed SARS-CoV-2 infection. We aimed at comparing the kinetics of IgM, IgG, IgA, and virus neutralization activity in a group of patients affected by mild/moderate or severe SARS-CoV-2 infection. Six months after baseline, SARS-CoV-2 specific antibodies and T-cell responses were measured in a smaller number of individuals.

Fifteen patients hospitalized with confirmed COVID-19 at the Infectious Diseases division of Policlinico Umberto 1 st Hospital, Rome, Italy, were included in the study (Table1). The study was approved by local Ethic Committee and all participants have signed an informed consent.

At the hospital admission all patents were affected by COVID-19 related pneumonia and, according to the PaO 2 /FiO 2 ratio (partial oxygen pressure/oxygen flow), 10 patients were classified as having a severe disease (PaO2/FiO2 < 250), 4 having a moderate disease (PaO2/FiO2 > 250) in need of oxygen supply, and one mild disease only (PaO2/FiO2 > 250, no need of oxygen supply). Patients were re-tested for SARS-CoV-2 RNA 6 days after hospital admission and then every two days, until a negative NP swab result was obtained.

Serum samples were available at three different time-points: at hospital admission (T0), 5-10 days (T1), and 11-15 days (T2) after hospital admission. Immediately after collection, sera were shipped to the Department of Infectious Diseases, Istituto Superiore di Sanità (ISS); 0.5 ml aliquots were prepared and stored at -80 °C. Serology testing was performed at ISS by personnel blinded to specimen classification within one month from serum collection.

ELISA assays were used to measure Anti-Nucleocapsid IgM (Dia-Pro diagnostics Bioprobes, Sesto San Giovanni (MI), Italy), anti-Spike IgG and anti-Spike IgA (both from Euroimmun, Lübeck, Germany), according to manufacturers' instructions. A surrogate neutralization assay (GenScript, Leiden, The Netherlands) was used to detect the virus neutralizing ability of patients' sera. The assay detects and measures neutralizing antibodies blocking the Spike RBD-ACE2 interaction in vitro and was used according to manufacturer's instructions.

Additional blood samples from patients #4, #11 and #15 were collected six months after 

Fifteen patients were included in the study ( Anti-Spike IgG at T0 were above the positivity threshold in 5 patients (13.3 %). At T1 a positive IgG response was measured in 13 patients (86.7 %); at T2 14 patients (93.3%) had a positive IgG response. When patients were classified according to disease severity, we found that at T0 the median IgG response was higher in mild/moderate patients, although this difference was not statistically significant. IgG levels were higher among severely affected patients at T1 and T2 (Figure 1 ).

Anti-Spike IgA at T0 were above the positivity threshold in 11 out of 15 patients (73.3 %). At T1 and T2 a positive IgA response was measured in all the patients. Higher IgA levels were found at T0 in patients with a mild/moderate disease (Figure 1 ).

At hospital admission Spike RBD-ACE2 neutralization activity was measured in 13 patients (86.7 %). At T1 and T2 a neutralizing response was measured in all the patients. Patients with mild/moderate disease presented higher neutralizing antibody levels at hospital admission as compared to severe patients, although this was not statistically significant (Figure 1) .

A statistically significant positive correlation of in vitro neutralization of Spike RBD-ACE2 binding with IgG and IgA serum levels at all the time-points was found, whereas no correlation was evident between anti-Nucleocapsid IgM and Spike RBD-ACE2 neutralizing activity ( Figure 2 ).

A 6-month follow-up analysis was performed on 3 out of 15 patients. We did not observe any decline in anti-Spike IgG levels (T2 mean±SD: 6.53± 0.27; 6-month mean±SD: 6.21± 0.9).

Anti-Spike IgA levels declined but were still above the positivity threshold (T2 mean±SD: 11.70± 0.56; 6-month mean±SD: 4.13± 2.16). Sera retained surrogate neutralization activity (data not shown). Two patients (one moderate and one severe case) were able to mount a virus-specific T-cell response, marked by the production of TNF- and IFN- by CD4 and CD8 T cells (Figure 3 ), while one patient was unresponsive. Profiles of T-cell response were rather different. A higher frequency of cytokine positive CD4 + vs. CD8 + T cells (0.91% vs 0.56%) was found in patient #10 (moderate), while in patient #15 (severe) this ratio was inverted (0.78% vs 0.96%). The proportion of CD4 and CD8 cells producing one, two or three cytokines was also different in the two responsive patients.

The correlation of antibody responses to SARS-CoV-2 with COVID-19 disease severity is still unclear. Our data suggest that individuals with an earlier appearance of virus-specific IgG and IgA potentially have a better outcome. In patients with a mild/moderate disease, IgG responses tended to occur simultaneously with IgM or even slightly earlier, while in patients with severe disease IgG and IgA were produced later during the course of the disease. Both IgG and IgA levels were correlated to serum neutralizing activity.

Our observations agree with a recent study by Carsetti and co-workers (2020) who found that IgA and IgG produced relatively late in the course of the infection characterize severe disease (Carsetti et al, 2020) . Moreover, it has been shown that patients with more severe illness display higher antibody titers than those with milder disease (Long et 

Before drawing conclusions, limitations of the study should be mentioned. The number of patients enrolled is small and probably not sufficient for making strong conclusions. Only 3 patients were used in the cellular immunity studies at 6 months. Although incomplete and preliminary, the 6-month follow-up showed the persistence of serological response in three patients. Data on T-cell response suggested the possibility of differential responses that could reflect asymptomatic exposure.

In conclusion, our data confirm the notion that an early IgG/IgA response focused on the 

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LEGEND TO THE FIGURES

SARS-CoV-2 specific antibody response in mild/moderate and severe COVID-19

IgA antibodies were tested by enzyme-linked immunosorbent assay. Sera neutralization activity was assessed by surrogate virus neutralization test based on antibody-mediated blockage of ACE2-Spike protein-protein interaction

Signal to Cut-off (S/Co) ratio

Serum IgG and IgA levels correlate with inhibition of SARS-CoV-2-ACE2 binding. Linear regression correlating serum levels of anti-Nucleocapsid IgM (top), anti-Spike IgG (middle) and, anti-Spike IgA (bottom) antibodies with Spike-ACE2 binding neutralization

numerical values along horizontal axis represent Signal to Cut-off ratios for each serological assay

The authors wish to thank the ISS working group on COVID-19: Stefano Fiore, Concetta 

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The authors have nothing to disclose. 

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