Oligosymptomatic long-term carriers of SARS-CoV-2 display impaired innate resistance but increased high-affinity anti-spike antibodies

Abstract

The vast spectrum of clinical features of COVID-19 keeps challenging scientists and clinicians. Low resistance to infection might result in long-term viral persistence, but the underlying mechanisms remain unclear. Here, we studied the immune response of immunocompetent COVID-19 patients with prolonged SARS-CoV-2 infection by immunophenotyping, cytokine and serological analysis. Despite viral loads and symptoms comparable to regular mildly symptomatic patients, long-term carriers displayed weaker systemic IFN-I responses and fewer circulating pDCs and NK cells at disease onset. Type 1 cytokines remained low, while type-3 cytokines were in turn enhanced. Of interest, we observed no defects in antigen-specific cytotoxic T cell responses, and circulating antibodies displayed higher affinity against different variants of SARS-CoV-2 Spike protein in these patients. The identification of distinct immune responses in long-term carriers adds up to our understanding of essential host protective mechanisms to ensure tissue damage control despite prolonged viral infection.

Keywords: Health sciences; Immunology; Virology.

Graphical abstract

Figure 1

Overview of clinical features, viral loads, and mucosal immunity parameters of COVID-19 patients (A) SARS-CoV-2 positivity by qRT-PCR in nasopharyngeal samples, sex and age of non-infected controls (NI), non-persistent (NP) and persistent (P) COVID-19 patients. (B) Duration of symptoms of NP and P patients, represented as days after symptom onset (DSSO). (C) Quantification of the plasma damage markers D-dimer and Ferritin in NI (n = 14), NP (n = 26), and P (n = 24) at <10 DSSO by multiplex magnetics bead-based immunoassay. (D) Cycle threshold (Ct) values of qRT-PCR for SARS-CoV-2 target genes from nasopharyngeal samples from NP (n = 32) and P (n = 33) patients by DSSO. (E) SARS-CoV-2 positivity of NP (n = 32) and P (n = 33) patients by DSSO. (F) Quantification of IFNα, IL-29, IFNγ, IL-1b, IL-4, IL-13, IL-17, and IL-10 from URT samples in NP (n = 9) and P (n = 13) at <10 DSSO by multiplex immunoassay. Each dot represents a subject. Filled dots represent a positive qRT-PCR of nasopharyngeal samples for SARS-CoV-2, while empty dots represent a negative qRT-PCR. Statistical significance was calculated using Mann-Whitney test, Kruskal-Wallis analysis followed by Dunn post-test, or Holm-Sidak method. ∗∗p ≤ 0.01; and ∗∗∗p ≤ 0.001. DSSO, Days since symptom onset.

Figure 2

Long-term carriers display type 3-skewed immune responses (A) Longitudinal immunophenotyping of PBMCs from non-persistent (NP, blue, n = 28) and persistent (P, red, n = 22) patients depicting frequencies of CD14⁺CD16⁻ classical monocytes (cMono) and CD14⁻CD16⁺ non-classical monocytes (ncMono), CD14⁻CD16⁻CD304⁺ plasmacytoid cells (pDC) and CD3⁻CD56⁺ natural killer cells (NK). Filled dots represent individual samples longitudinally collected at different time points until resolution of infection from patients positive by qRT-PCR for SARS-CoV-2, while empty dots represent samples from the same patients at the convalescent phase, coinciding with the first negative qRT-PCR for SARS-CoV-2. (B and C) Weekly longitudinal quantification of plasma growth factors, cytokines and chemokines in non-persistent (NP, blue, n = 28) and persistent (P, red, n = 22) COVID-19 patients by multiplex immunoassay. Statistical significance was calculated using Kruskal-Wallis analysis followed by Dunn post-test or Mann-Whitney test (onset analysis), and indicated by ∗p ≤ 0.05; ∗∗p ≤ 0.01; and ∗∗∗p ≤ 0.001. DSSO, Days since symptom onset.

Figure 3

T cell effector and memory responses of persistent patients (A) Frequencies of CD4⁺ T lymphocytes producing IFNγ, TNFα and IL-6 and CD8⁺ T lymphocytes producing IFNγ, TNFα and Granzyme B (Grz B) after polyclonal in vitro stimulation of PBMCs from NI, NP and P (≤10 DSSO) with anti-CD3/CD28 beads of PBMCs from (n = 12), NP (n = 11) and P (n = 23). (B) Longitudinal immunophenotyping of PBMC from NP and P depicting CD4⁺CD27⁺CD45RA⁺CCR7⁺ naive T cells (naive CD4), CD4⁺CD27⁺CD45RA⁻CCR7^- effector memory T cells (EM1) and CD8⁺CD27⁺CD45RA + ⁻CCR7⁺ naive T cells (naive CD8). Filled dots represent individual samples longitudinally collected until resolution of infection from NP and P patients positive by qRT-PCR for SARS-CoV-2. Empty dots represent samples from convalescent patients coinciding with the first negative qRT-PCR for SARS-CoV-2. (C and D) Immunophenotyping of SARS-CoV-2 reactive CD4 and CD8 T cells after in vitro stimulation of PBMC from NP (n = 11) and P (n = 9) after viral clearance with peptides spanning the Spike protein of the alpha variant of SARS-CoV-2 in the presence of IL-2. Percentage of (C) CD4⁺ and CD8⁺ cells expressing Ki67, (D) CD4⁺ cells expressing TNFα, CD8⁺ cells expressing IFNγ and Granzyme B (Grz B), and CD4⁺CD25⁺ cells expressing IL-10. Statistical significance was calculated using Kruskal-Wallis analysis followed by Dunn post-test, and indicated by ∗p ≤ 0.05; ∗∗p ≤ 0.01. DSSO, Days since symptom onset.

Figure 4

Early immune signature of persistent patients (A) Heatmap of cytokine concentration in serum from NI, NP and P at <10 DSSO measured by multiplex assay. K-means clustering was used to determine cytokine clusters 1–6. (B) Correlation matrix across PBMC immunophenotyping and cytokines concentrations from NI, NP and P at <10 DSSO. Only significant correlations (p < 0.05) are represented as dots. Pearson’s correlation coefficients from comparisons of cytokine measurements within the same patients are visualized by color intensity. (C) Fold change importance analysis between NP and P, calculated using the gtools package in R. IL-17A and MIP-1α have median equals zero in NP, resulting in an infinite fold change, which is represented by the arrow.

Figure 5

Humoral responses are altered in long-term carriers of SARS-CoV-2 (A) Longitudinal immunophenotyping of PBMC from NP and P depicting CD3⁺CD4⁺CXCR5⁺ follicular T (T_FH) cells. Filled dots represent individual samples longitudinally collected until resolution of infection from NP and P patients positive by qRT-PCR for SARS-CoV-2. Empty dots represent samples from convalescent patients coinciding with the first negative qRT-PCR for SARS-CoV-2. (B) Percentage of CD4⁺CXCR5⁺ T cells expressing IL-21 after polyclonal in vitro stimulation of PBMC from NP (n = 11) and P (n = 24) at <14 DSSO with anti CD3/CD28 beads. (C) Longitudinal immunophenotyping of PBMC from NP (n = 7) and P (n = 10) depicting CD27⁺CD38⁻ switched immunoglobulin (swIg) memory B cells (MBC) and plasmablast/plasma cells (PB/PC). (D) Longitudinal assessment of Spike-specific IgG and IgA antibodies in plasma from P and NP by ELISA assay. (E) Titers of high-affinity antibodies against Alpha S1, Beta S1, Gamma RBD, and Kappa RBD variants of SARS-CoV-2 in plasma from NP and P around 21 DSSO, and P by the time of viral clearance, by multiplex neutralization assay. Statistical significance was calculated using Mann-Whitney test or Kruskal-Wallis analysis followed by Dunn post-test and indicated by ∗p ≤ 0.05; ∗∗p ≤ 0.01; and ∗∗∗p ≤ 0.001. DSSO, Days since symptom onset.