To date, children represent a small proportion of SARS-CoV-2 confirmed coronavirus disease (COVID-19) cases 1–3. Children are predominantly infected from symptomatic household adult contacts 4,5. Children have comparatively milder COVID-19 disease and up to one-third are asymptomatic 6. The immunological basis for milder paediatric disease is unclear, but may be relevant to other viral pandemics where striking age-related epidemiological differences were observed 7. In SARS-CoV-2 infection, reduced respiratory epithelial expression of the ACE2 receptor and trained innate immunity in children have been proposed 8,9. Investigating immune responses to SARS-CoV-2 across all age groups is key to understanding disease susceptibility, severity determinants, and vaccine candidates. Detailed investigations of immune responses during SARS-CoV-2 infection have been reported in adults 10–12, with exposure to SARS-CoV-2 causing specific T cell responses without seroconversion 13. Data on immune responses in children exposed to SARS-CoV-2 are limited.
Two parents (mother 38 years, and father 47 years) residing in Melbourne, Australia, attended a wedding inter-state without their children, in early March 2020. They returned home 3 days later and developed cough, coryza and subjective fevers, followed by lethargy and headache for a total of 14 (mother, A1) and 11 days (father, A2) (Fig. 1). Seven days after the onset of the parents’ symptoms, child one (male 9 years, C1) developed mild cough, coryza, sore throat, abdominal pain and loose stools, and child 2 (male 7 years, C2) developed mild cough and coryza. The third child (female 5 years, C3) was asymptomatic. Eight days after the onset of the parents’ symptoms, they were notified of an emerging outbreak of SARS-CoV-2 traced to the wedding. The parents were SARS-CoV-2 PCR positive on nasopharyngeal (NP) swabs taken the same day. Repeated NP swabs from the children were negative for SARS-CoV-2. Physical distancing precautions were not feasible in the household. Child 3 had particularly close contact, sleeping in the parents’ bed throughout the period both parents were unwell. All family members recovered fully without requiring medical care.
Serial samples, including blood, saliva, NP swabs, faeces and urine, were collected from all family members approximately every 2–3 days (Fig. 1). Nasopharyngeal swabs from the parents on days 8 and 12 were SARS-CoV-2 PCR positive. All NP, saliva and stool samples from the children were PCR negative for SARS-CoV-2. Nasopharyngeal swabs from the children were all positive for enterovirus by a multiplex respiratory viral panel on day 10.
We investigated the cellular immune response in peripheral blood mononuclear cells (PBMCs) from all family members on days 12, 37 and 88 by flow cytometry. Both parents and children had high proportions of CD8 T cells at day 12 that subsequently decreased (Fig. 2A), a decline associated with a corresponding increase in the proportion of CD4 T cells in all samples. Strikingly low proportions of monocytes were observed on day 12 in all family members, particularly in C3 (0.12%) relative to her siblings (average 0.5%) and parents (average 0.88%) (Fig. 2A). Monocytes returned to circulating proportions in all family members by day 37 (average 4.1%) and day 88 (average 2.5%). These signatures were also identified by unsupervised t-distributed stochastic neighbour embedding (tSNE) dimensionality reduction, where tSNE clusters corresponding to CD8 T, CD4 T and monocytes in parents and children showed identical sequential changes to those observed by manual gating (Fig. 2B). Low proportions of monocytes were observed in all circulating subsets with reductions in CD16+ subsets most evident (Fig. 2C). Both parents showed increases in central (TCM) and effector (TEM) memory CD8 T cells by day 88 (Fig. 2D), and CD8 T cell expression of the exhaustion marker PD1 increased in all family members over time (Fig. 2E). CD4 TEM cells reduced over time in the parents, and one parent (A2) had a marked decline in the CD4 effector (TEMRA) cell population (Fig. 2F).
The heterogeneous cellular immune responses observed in all family members at the first timepoint are consistent with emerging evidence on SARS-CoV-2 infection in adults 14. In addition to CD8 T cell viral responses, depletion of innate immune cell subsets, including CD16+ monocytes, is an emerging, unique signature of COVID-19 15. We observed further alterations in the myeloid compartment in our whole blood analysis. Low proportions of neutrophils were evident in all family members at day 12, particularly in C3 (5.1%) relative to her siblings (average 10.4%) and parents (average 15.5%) (Fig. 2G). Circulating neutrophils returned to an average of 30.5% in children and 45.4% in parents by day 88, a time point associated with the appearance of low-density immature neutrophils (SSChiCD16+CD14+/−) in PBMCs of all family members (Fig. 2B and 2H). Pre- and immature- neutrophils in PBMC fractions have been recently described in SARS-CoV-2 infected adults 16. In our study, parent A1 and all children had high proportions of eosinophils at all time points (Fig. 2G), in keeping with elevated eosinophils in SARS-CoV-2 infected patients during the recovery phase. Their role remains unclear 17.
Our analyses highlighted that active cellular immune responses in the family members were not accompanied by a corresponding increase in plasma cytokine levels, consistent with mild or absence of symptoms. We quantified 18 plasma cytokines using a custom multiplex bead array and only IL-8, MCP-1 and CCL5 (RANTES) were detectable (Fig. 2I), with levels remaining constant over time, excluding C1 and C2 who had a ~ 2-fold increase in RANTES levels at day 37 (Fig. 2I). A case of mild adult COVID-19 disease reported an identical plasma cytokine signature to that observed in our family members 12.
To explore SARS-CoV-2 specific humoral immune responses, we first quantified salivary and plasma antibodies against the S1 protein by ELISA. Saliva from all family members tested positive for IgA antibodies against the S1 protein at all timepoints (Fig. 3A). A2 had an increase in salivary anti-S1 IgA at day 12, one day after symptom resolution. C1 and C2 also had increased anti-S1 salivary IgA (Fig. 3A; day 25 and day 18 samples, respectively), coincident with symptom resolution. Anti-S1 IgM and IgG were present in most salivary samples, but with a less consistent pattern in family members. Both parents and C3 had detectable levels of plasma IgG and IgM to SARS-CoV-2 S1 protein at all timepoints (Fig. 3B). IgG levels increased between timepoints for parent A2; those for parent A1 remained stable. Levels of S1-specific IgA in plasma were only detected in A1. Finally, A1 had a robust neutralising antibody response on days 12, 37 and 88 (titers 403, 226 and 160, respectively) (Fig. 3C). A2 and C3 had low level but detectable neutralizing antibody activity in sera on days 12 and 37, respectively.
To further characterise whether the children had serological evidence of SARS-CoV-2 immunity despite being PCR negative, we undertook a systems serology analysis using a CoV-specific multiplex panel with the inclusion of additional aged-matched pre-pandemic healthy individuals. All family members, including the children, exhibited SARS-CoV-2-specific antibody features that differed from pre-pandemic controls (Fig. 4). This included serological signatures against the S1 protein, as well SARS-CoV-2 Trimer S, receptor binding domain (RBD) and S2. In addition, both parents, but not the children, had serological responses to other non-SARS-CoV-2 coronaviruses (Fig. 4C). Unsupervised hierarchical clustering analysis revealed that C3 clustered closest to her parents in all responses. C1 and C2, who had no evidence of a serologic response, clustered closest to the healthy controls whilst still exhibiting a SARS-COV-2 positive signature (Fig. 4C).
Our combined salivary and serological findings show that, despite having no virological evidence of infection, all three children developed antibody responses against various SARS-CoV-2 epitopes. Of the three children, C3, who remained asymptomatic throughout, demonstrated the most robust antibody response. We also observed that symptom resolution in A2, C1 and C2 coincided with a spike in salivary anti-S1 IgA, but not IgG. SARS-CoV-2 likely infects the salivary glands and is detectable in saliva 18. Our observation therefore provides the first evidence that control of SARS-CoV-2 at the site of infection may be mediated by a mucosal IgA antibody response. This potential key role for mucosal antibodies in protection warrants confirmation in larger studies. Whilst enterovirus was identified in the children’s respiratory panel, this is a common finding at our hospital and reflects recent exposure. The SARS-CoV-2 specific response identified in the saliva and serum would not be explained by this finding.
This in-depth family case study provides novel insights into immunological responses in children exposed to SARS-CoV-2. Despite close contact with infected parents, PCR testing for SARS-CoV-2 was repeatedly negative in all children, who developed minimal or no symptoms. However, the children had similar cellular and SARS-CoV-2 specific antibody-mediated immune responses to their parents, suggesting that the children were infected with SARS-CoV-2 but, unlike the adults, mounted an immune response that was highly effective in restricting virus replication. Whether this family will be protected from reinfection with SARS-CoV-2 is uncertain, as only one parent demonstrated a robust neutralising antibody response. The discordance between the virological PCR results and clinical serological testing, despite an evident immune response, highlights limitations to the sensitivity of nasopharyngeal PCR and current diagnostic serology in children. Our findings emphasise the need for further detailed investigation of the immune response to SARS-CoV-2 to advance our understanding of exposure and protective immunity in children.