A case report describing the immune response of an infant with congenital heart disease and severe COVID-19

Abstract

Background: Children with SARS-CoV-2 infection generally present with milder symptoms or are asymptomatic in comparison with adults, however severe disease occurs in a subset of children. To date, the immune correlates of severe COVID-19 in young children have been poorly characterised.

Methods: We report the kinetics of immune responses in relation to clinical and virological features in an infant with acute severe COVID-19 using high-dimensional flow cytometry and multiplex cytokine analysis.

Results: Systemic cellular and cytokine profiling show an initial increase in neutrophils and monocytes with depletion of lymphoid cell populations (particularly CD8 + T and NK cells) and elevated inflammatory cytokines. Expansion of memory CD4 + T (but not CD8 + T) cells occurred over time, with a predominant Th2 bias. Marked activation of T cell populations observed during the acute infection gradually resolved as the child recovered. Substantial in vitro activation of T-cell populations and robust cytokine production, in response to inactivated SARS-CoV-2 stimulation, was observed 3 months after infection indicating durable, long-lived cellular immune memory.

Conclusions: These findings provide important insights into the immune response of a young infant with severe COVID-19 and will help to inform future research into therapeutic targets for high-risk groups.

Keywords: Paediatric research; Viral infection.

Fig. 1. 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.

Fig. 2. 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/µL 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, γδVδ2+ 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+, γδVδ2+ 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.

Fig. 3. 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.