Daily Viral Kinetics and Innate and Adaptive Immune Response Assessment in COVID-19: a Case Series

Abstract

Viral shedding patterns and their correlations with immune responses are still poorly characterized in mild coronavirus (CoV) disease 2019 (COVID-19). We monitored shedding of viral RNA and infectious virus and characterized the immune response kinetics of the first five patients quarantined in Geneva, Switzerland. High viral loads and infectious virus shedding were observed from the respiratory tract despite mild symptoms, with isolation of infectious virus and prolonged positivity by reverse transcriptase PCR (RT-PCR) until days 7 and 19 after symptom onset, respectively. Robust innate responses characterized by increases in activated CD14⁺ CD16⁺ monocytes and cytokine responses were observed as early as 2 days after symptom onset. Cellular and humoral severe acute respiratory syndrome (SARS)-CoV-2-specific adaptive responses were detectable in all patients. Infectious virus shedding was limited to the first week after symptom onset. A strong innate response, characterized by mobilization of activated monocytes during the first days of infection and SARS-CoV-2-specific antibodies, was detectable even in patients with mild disease.IMPORTANCE This work is particularly important because it simultaneously assessed the virology, immunology, and clinical presentation of the same subjects, whereas other studies assess these separately. We describe the detailed viral and immune profiles of the first five patients infected by SARS-CoV-2 and quarantined in Geneva, Switzerland. Viral loads peaked at the very beginning of the disease, and infectious virus was shed only during the early acute phase of disease. No infectious virus could be isolated by culture 7 days after onset of symptoms, while viral RNA was still detectable for a prolonged period. Importantly, we saw that all patients, even those with mild symptoms, mount an innate response sufficient for viral control (characterized by early activated cytokines and monocyte responses) and develop specific immunity as well as cellular and humoral SARS-CoV-2-specific adaptive responses, which already begin to decline a few months after the resolution of symptoms.

Keywords: COVID-19; SARS-CoV-2; antibody response; cytokines; immunity; viral load.

FIG 1

Kinetics of viral load and inflammatory markers. (A) Viral kinetics of nasopharyngeal swabs (NPS) and oropharyngeal swabs (OPS). Filled symbols, isolation of infectious virus in cell culture; open symbols, infectious virus could not be isolated in cell culture. (B) Cytokine and chemokine dynamics. Individual concentrations in picograms per milliliter for each marker were plotted at different days POS for each patient. The dotted line represents the limit of detection for each marker. Samples with undetectable concentrations were arbitrarily given a value of 50% of the last standard dilution value. (C) C-reactive protein (CRP) dynamics. Concentrations in milligram per liter were plotted at different days POS for each patient.

FIG 2

Early increase in intermediate monocytes after SARS-CoV-2 infection. (A) Gating strategy for the identification of different monocyte subsets, classical monocytes (CM), intermediate monocytes (IM), and nonclassical monocytes (NCM), by flow cytometry after gating out dead cells and lineage (CD3, CD20) cells and gating on HLA-DR⁺ cells. Early time point flow cytometry counterplots from P1 to P5 as well as that of a healthy control (HC) are shown. (B) Percentages of CM, IM, and NCM calculated from the lymphocyte live gate for each patient at different days POS. The dotted line represents one healthy donor. (C) Geometric mean (GM) fluorescent intensity (MFI) values for different activation and migration markers in intermediate monocytes. The dotted line in red shows the mean of the values for the isotype controls for each marker.

FIG 3

Antibody responses to SARS-CoV-2. (A) IgG and IgA antibody kinetics for P1 to -5 using ELISAs with either SARS-CoV-2 full-S (only IgG, left graph) or the S1 domain (IgG and IgA, middle and right graphs) as the antigen. For the full-S ELISA, endpoint titers were determined (starting dilution, 1:100), whereas for S1-based ELISAs, the ratio of sample to calibrator was measured (dilution, 1:100). (B) Antibody kinetics using a recombinant full-S-based immunofluorescence assay (rIFA) for IgG, IgM, and IgA isotypes using a starting dilution of 1:40 (IgG and IgA) or 1:10 (IgM). The left panel shows representative rIFA staining patterns for IgG and IgM for two patients and a negative-control serum. (C) Kinetics of neutralizing antibody endpoint titers using a pseudotyped VSV neutralization assay (starting dilution, 1:10).

FIG 4

Kinetics of T-cell immune responses. (A) Graphs show the frequencies of total lymphocytes and of CD8 T cells in Giga/liter (G/l). (B) The ratio of CD4 to CD8 T cells was determined by flow cytometry as the ratio of the frequencies of CD4 and CD8 T cells (percentage of CD3 T cells). (C) Representative flow plots are shown for CD8 T-cell activation markers CD38 and HLA-DR. Histograms are shown for the expression of Ki67 (middle) and granzyme B (lower) of activated (blue) and naive (gray) CD8 T cells (gated on CD45RA⁺ CCR7⁺ CD8 T cells). The summary graph depicts the kinetics of HLA-DR⁺ CD38⁺ CD8 T cells at different time points after symptom onset. The bold dotted line is the mean of results for three healthy controls, with its range indicated as a thin dotted line; open circles identify the time points that are shown as flow plots. (D) Similar to panel C; representation of flow plots for HLA-DR⁺ CD38⁺ CD4 T cells (red), expressing the proliferation marker Ki67, with summarized data in the right graph.