PMC9053208_01
Clinical and laboratory characteristics of an infant with severe COVID-19.. A Clinical timeline showing immuno-modulatory therapies in relation to clinical course. B Plain chest film performed on admission illustrating bilateral interstitial infiltrates with left lower lobe collapse and consolidation. C Virologic findings illustrating SARS-CoV-2 Cycle threshold Ct values in naso-/oropharyngeal and urine specimens that reduce in association with clinical improvement. D Serum IgG, IgM, and IgA to S1 using in-house ELISA modified from Mount Sinai Laboratories, USA; and microneutralisation assay demonstrating a rapid rise in neutralising antibody titres. E Elevated ferritin and LDH with initial hyper-inflammatory picture and rapid reduction in ferritin coinciding with clinical improvement. F Persistent lymphopaenia; initial high IL-6 with reduction after administration of tocilizumab. IVIG intravenous immunoglobulin, LDH lactate dehydrogenase, NPA nasopharyngeal aspirate, EU ELISA units, Ct cycle threshold.
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Fig1
A day prior to presentation he developed low-grade fevers, cough, and increased work of breathing (Fig. 1A, Day-2). At his local hospital, he was hypoxic (initial oxygen saturation was 70%) and was commenced on low-flow nasal prong oxygen. He was intubated and ventilated and transferred to the intensive care unit for further management (Fig. 1A, Day 0). A plain chest film showed peri-hilar interstitial and ground-glass opacities with left lower lobe collapse and small bilateral pleural effusions (Fig. 1B). Based on his cardiorespiratory deterioration and elevated inflammatory markers, therapies targeting the COVID-19 inflammatory response were administered, including tocilizumab 8 mg/kg and dexamethasone 0.15 mg/kg (twice daily for 5 days, Fig. 1A). SARS-CoV-2 in upper airway swabs were initially low (indicative of high viral load) with detectable viral RNA present until day 25. SARS-CoV-2 RT-PCR was positive in urine at day 3 of admission (Fig. 1C); stool was negative at all time-points. Serology for SARS-CoV-2 S1 IgG, IgM, and IgA was initially negative (day 3) with seroconversion occurring by day 5 and IgG and IgA remaining positive until day 84 (Fig. 1D) indicating durable humoral immunity. Neutralising antibodies showed an early rise (days 3-5) with persistently high titres from day 10 through 28 (Fig. 1D). A rapid decline in inflammatory markers (e.g., ferritin and IL-6, Fig. 1E, F) was observed in association with clinical improvement and extubation occurred on day 5. Lymphocyte counts remained below normal reference ranges throughout admission and follow-up (Fig. 1F), a pattern also observed in adults with severe COVID-19 .
Immune cell profiling in whole blood and PBMCs.. A Flow cytometry was performed on whole blood samples collected at 3, 5, 10, 28, and 84 days following admission. Neutrophils, eosinophils, CD4 T cells, CD8 T cells, B cells, Natural Killer (NK) cells, monocytes (classical, intermediate, and non-classical), and dendritic cells were classified by manual gating and expressed as cells/microL using counting beads. B Unsupervised clustering and dimensionality reduction were performed using FlowSOM and UMAP on a concatenated file containing 150,000 live single cells (30,000 randomly selected cells from each time point). The UMAP plots at each time point are coloured according to the generated FlowSOM clusters. C Flow cytometry was performed on peripheral blood mononuclear cells collected on days 3, 5, 10, 28, and 84 following admission. CD4, CD8, gammadeltaVdelta2+ T-cells, and MAIT cells were classified by manual gating and expressed as a frequency of CD3+ T-cells. These results were compared to an age/sex-matched control, obtained at a single time-point. D Subsets of CD4+ and CD8+ T-cells were further categorised by flow cytometry to determine the frequency of Th1, Th2, Th17, Treg, and memory subsets. These were expressed as the frequency of CD4+ or CD8+ T-cells. E Flow cytometry was performed to assess the activation status of T-cells. CD69+ was expressed as a proportion of total CD4+, CD8+, gammadeltaVdelta2+ T-cells, and MAIT cells. F Cytokines were quantified in the serum using a multiplex cytokine assay and visualised in a heatmap containing log2 transformed values and clustered according to peak expression at day 3 (cluster 1), day 5 (cluster 2), day 10 (cluster 3), and day 28 (cluster 4). IL-18 levels at days 3, 5, 10, 28, and 84 were quantified by ELISA and expressed as pg/ml. UMAP Uniform Manifold Approximation and Projection, MAIT mucosal-associated invariant T cells.
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Fig2
A day prior to presentation he developed low-grade fevers, cough, and increased work of breathing (Fig. 1A, Day-2). At his local hospital, he was hypoxic (initial oxygen saturation was 70%) and was commenced on low-flow nasal prong oxygen. Flow cytometry was performed on whole blood collected 3, 5, 10, 28, and 84 days following admission (Fig. 2). Neutrophil and eosinophil proportions remained stable over the first 10 days, although a substantial increase in both cell types was observed at day 28, with a 3.5-fold increase in neutrophils and a 12.8-fold increase in eosinophils (Fig. 2A). COVID-19, cells of lymphoid origin, including CD4 + T cells, CD8 + T cells, B cells, and natural killer (NK) cells were markedly reduced at day 10, subsequently increasing (Fig. 2A). Classical (CD14+CD16-) monocytes changed substantially over time, with marked increases observed at days 5, 10, 28, and 84 days after admission. Non-classical (CD14lowCD16+) monocytes were essentially absent at the first three time points, returning to the circulation by day 84 (Fig. 2A). The frequency of clusters at each time point revealed identical sequential changes to that observed by manual gating and identified the presence of a CD16low immature granulocyte cluster at day 3 and day 5 (Fig. 2B and Supplementary Fig. 1). For comparison, PBMCs from an age/sex-matched infant who had undergone correction for a tetralogy of Fallot was obtained at a single time-point). gammadeltaTCR+Vdelta2+ T-cells, which were much lower at day 3 compared to the control, increased more than ten-fold (0.22% at day 3 to 2.83% at day 84) (Fig. 2C). T-cell (MAIT) frequencies had increased (0.2-0.35% from day 3 to day 84 respectively, Fig. 2C). The proportion of Th2 cells, which were very low in the control child (2.36%, consistent with the previous reports), increased in the COVID-patient from 9.32% at day 3 to 28.2% at day 84, while the proportion of Th1, Th17, or Treg populations varied little over time (Fig. 2D). CD4+ T-cell subsets expanded from day 3 onwards, however, this was not seen for CD8+ T-cells (Fig. 2D). CD69, a marker used to define activated T cells, was minimally expressed on the control PBMC sample (Fig. 2E). CD69 expression was observed on CD4+ T cells, CD8+ T cells, gammadeltaTCR+Vdelta2+ T-cells, and MAIT cells at day 3 (Fig. 2E). CD69 expression on these T-cell subsets gradually declined after day 3 to levels akin to the control, although CD69+ expression on MAIT cells increased again by day 84 (Fig. 2E). Multiplex analysis of serum samples revealed a broad array of cytokines produced throughout the course of infection (Fig. 2F). TNFalpha and the chemokine IP-10 (CXCL10) at day 3 (Fig. 2F), consistent with previous data from severe COVID-19 disease in adults. In addition, IFNgamma, G-CSF, Eotaxin, MIP-1alpha, and MCP-1 were also highly elevated at this time-point compared to later time-points (Fig. 2F), indicative of a potent pro-inflammatory immune response and enhanced cell migration. The level of IL-6 was markedly reduced by day 5 after tocilizumab (anti-IL-6) therapy and remained low at subsequent time-points (Fig. 2F). High levels of PDGF-BB, IL-1beta, IL-2, IL-7, IL-15, and IL-17 were also observed at day 10 coinciding with disease resolution (Fig. 2F). By day 28, high levels of IL-1beta and IL-15 were observed, while day 84 was characterised by increased IL-9, MIP-1beta, and RANTES (Regulated on Activation, Normal T cell expressed and Secreted, Fig. 2F). MAIT cells (Fig. 2E). Moreover, whilst IL-12 was predominantly detected at day 10, both IL-18 and IL-12 were detected at day 84 (Fig. 2F) correlating with high CD69 expression on MAIT cells at this time-point. Innate-like T-cells, such as Vdelta2+ gammadelta T cells and MAIT cells have been implicated in anti-viral immunity.
Immune response to inactivated SARS-CoV-2.. A Flow cytometry was performed on PBMCs following 4-day stimulation with inactivated SARS-CoV-2 to observe T-cell activation status. CD69+ was expressed as a proportion of total CD4+ or CD8+ T-cells. B Cytokines were quantified in PBMC supernatants following 4-day stimulation with inactivated SARS-CoV-2 by multiplex cytokine assay to assess memory T-cell responses.
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Fig3
To examine the memory T cell response, PBMCs from the control infant and COVID-infected infant (day 84) were stimulated with inactivated SARS-CoV-2 (Fig. 3). T-cells compared to the age/sex-matched control (<5%) (Fig. 3A). This was accompanied by robust production of both pro- and anti-inflammatory cytokines and chemokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IL-17, IFNgamma, IP-10, TNFalpha) in PBMC supernatants, which was not observed in our control patient (Fig. 3B and Supplementary Fig. 2). These data reveal a strong memory T cell response to SARS-CoV-2, suggestive of long-lasting protective T cell-mediated immunity.