Coordinated innate and T-cell immune responses in mild COVID-19 patients from household contacts of COVID-19 cases during the first pandemic wave

Abstract

Objective: To better define the immunopathogenesis of COVID-19, the present study aims to characterize the early immune responses to SARS-CoV-2 infection in household contacts of COVID-19 cases. In particular, innate, T- and B-cell specific responses were evaluated over time.

Methods: Household contacts of COVID-19 cases screened for SARS-CoV-2 infection by nasopharyngeal swab for surveillance purposes were enrolled (T0, n=42). Of these, 28 subjects returned for a follow-up test (T1). The innate response was assessed by detecting a panel of soluble factors by multiplex-technology in plasma samples. Cell-mediated response was evaluated by measuring interferon (IFN)-γ levels by ELISA in plasma harvested from whole-blood stimulated with SARS-CoV-2 peptide pools, including spike (S), nucleocapsid (N) and membrane (M) proteins. The serological response was assessed by quantifying anti-Receptor-Binding-Domain (RBD), anti-Nucleocapsid (N), whole virus indirect immunofluorescence, and neutralizing antibodies.

Results: At T0, higher levels of plasmatic IFN-α, IL-1ra, MCP-1 and IP-10, and lower levels of IL-1β, IL-9, MIP-1β and RANTES were observed in subjects with positive swab compared to individuals with a negative one (p<0.05). Plasmatic IFN-α was the only cytokine detectable in subjects with positive SARS-CoV-2 swabs with high accuracy for swab score positivity (0.93, p<0.0001). Among subjects with positive swabs, significant negative correlations were found among the RT-PCR cycle threshold values reported for genes S and N and IFN-α or IP-10 levels. At T0, the IFN-γ T-cell specific response was detected in 50% (5/10) of subjects with positive swab, while anti-RBD/anti-N antibodies showed a positivity rate of 10% (1/10). At T1, the IFN-γ T-cell specific response was detected in most of the confirmed-infection subjects (77.8%, 7/9), whereas the serological response was still observed in a minority of them (44.4%, 4/9). Overall, the swab test showed a moderate concordance with the T-cell response (78.6%, k=0.467), and a scarce concordance with the serological one (72.9%, k=0.194).

Conclusions: Plasmatic IFN-α and the IFN-γ T-cell specific response appear early even in the absence of seroconversion, and show a greater positivity rate than the serological response in household contacts with positive swab.

Keywords: COVID-19; Interferon-alpha (IFN-α); Interferon-gamma (IFN-γ) release assay (IGRA); SARS-CoV-2; T-cell response; household contacts; spike protein; whole blood.

Figure 1

Flow chart of the enrolled household contacts of COVID-19 cases. Household contacts of COVID-19 cases (n=42) were enrolled and analyzed at the execution of the first nasopharyngeal swab (T0) and after 7-20 days (T1), at the end of the quarantine period. Twenty-eight of these subjects returned to follow-up. Footnote: COVID-19, COronaVIrus Disease 2019.

Figure 2

Plasmatic cytokines/chemokines modulated in household contacts at baseline. (A–H) Household contacts at T0 (n = 38) were stratified according to the swab result: positive (n = 10) and negative (n = 28). Plasma harvested from unstimulated blood samples were tested for the detection of 27 cytokines/chemokines using the Bio-Plex Pro Human Cytokine 27-plex Assay and for the detection of IFN-α and-β by means of an automatic ELISA. Red horizontal lines indicate medians. The green triangle identifies the subject with a positive swab only at T1. Statistical analysis was performed using Mann-Whitney U test to compare swab positive and negative subjects. A p < 0.05 was considered significant. IL, interleukin; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; IP, Interferon-gamma induced protein; RANTES, regulated on activation IFN, interferon.

Figure 3

T-cell response in household contacts of COVID-19 subjects. (A-C) Evaluation of IFN-γ levels in response to SARS-CoV-2 peptides in household contacts at T0 (n = 42) and T1 (n = 28) after whole-blood stimulation with 0.1 µg/mL of pools S (A) and M (C), and 1 µg/mL of pool N (B). Healthy donors (n=16) and COVID-19 patients (n = 53) were used as negative and positive control groups, respectively. The household contacts were stratified according to the swab result. The IFN-γ levels were assessed in plasma from stimulated whole-blood samples and reported by subtracting the background. The cut-off for each peptide pool was represented by a dashed line (pools S and N: 0.13 IU/mL; pool M: 0.19 IU/mL). Green triangle indicates the subject who scored positive only at T1. The red horizontal lines indicate the median. (D, E) Venn diagrams show the number of household contacts of COVID-19 cases at T0 and T1 with a positive response to the different SARS-CoV-2 peptides pools. (F) Venn diagrams show the number of confirmed hospitalized COVID-19 patients with a positive response to the different SARS-CoV-2 peptides pools, stratifying the results also with respect to days from symptom onset. The statistical comparison was done with the Kruskal-Wallis test and the Dunn’s multiple comparisons test, and p<0.05 was considered significant. IFN, interferon; COVID-19, COronaVIrus Disease 2019; S, spike; N, nucleocapsid; M, membrane.

Figure 4

Antibody response in household contacts of COVID-19 cases. Evaluation of SARS-CoV-2-specific anti-RBD (A), anti-N (B) and neutralizing (C) antibodies in household contacts at T0 (n=42) and T1 (n=28). Anti-RBD, anti-N and neutralizing antibodies were evaluated in sera samples and reported as Binding Antibody Units (BAU)/mL (A), Sample/Cutoff (S/CO) (B), and reciprocal of dilution (MNA₉₀) (C), respectively. Red dots indicate subjects with also a concomitant IFN-γ specific response as shown in the figure legend. Green triangle indicates the subject with positive swab only at T1. Dashed lines indicate the cut-off (anti-RBD: 7.1 BAU/mL; anti-N: 1.4 (S/CO); MNA₉₀: 8). The black horizontal lines indicate the median. (D, E) Venn diagrams show the number of household contacts of COVID-19 cases at T0 and T1 with an IFN-γ response and/or the serological one (anti-RBD and anti-N). Statistical analysis was performed using Fisher’s exact test with Bonferroni correction and p<0.01 was considered significant. IFN, interferon; COVID-19, COronaVIrus Disease 2019; RBD, receptor binding domain; N, nucleocapsid.