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Immune responses to SARS-CoV-2 infection in hospitalized pediatric and adult patients

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Science Translational Medicine  07 Oct 2020:
Vol. 12, Issue 564, eabd5487
DOI: 10.1126/scitranslmed.abd5487

Figures

  • Fig. 1 Clinical characteristics vary by age and patient outcome.

    Shown are clinical measurements at hospital admission for pediatric patients who did not require ventilation (group 1), patients with MIS-C (group 2), adults who did not require ventilation (group 3), adults who required mechanical ventilation or died (group 4), and pediatric patients who required mechanical ventilation (group 5). Clinical measurements included absolute lymphocyte counts (ALC) (A) and serum concentrations of CRP (B), peak D-dimer (C), and peak LDH (D). Data are presented as mean ± SD and compared by one-way ANOVA with Tukey post hoc comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (ALC, n = 41, 20, 33, 27, and 4 in groups 1 to 5; CRP, n = 34, 19, 20, 17, and 4 in groups 1 to 5; D-dimer, n = 25, 18, 17, 17, and 4 in groups 1 to 5; LDH, n = 27, 19, 26, 24, and 4 in groups 1 to 5).

  • Fig. 2 Serum cytokine concentrations vary by age and patient outcome.

    Cytokine concentrations in remnant serum samples obtained within 7 days of admission were determined using a multiplexed Luminex assay. (A) The concentrations in each group were compared by one-way ANOVA with Tukey’s post hoc comparison (mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Sample sizes for groups 1 to 5 are as follows: TNF, n = 34, 16, 26, 19, and 4; IP-10, n = 34, 16, 26, 19, and 4; IL-8, n = 34, 16, 26, 19, and 4; IL-6, n = 34, 16, 26, 19, and 4; IFN-γ, n = 26, 16, 13, 7, and 1; IL-17A, n = 22, 16, 22, 18, and 4. (B) Correlation between serum concentrations and age for IL-17A (n = 82) and IFN-γ (n = 63) as determined by Spearman’s test.

  • Fig. 3 CD4+ T cell responses to SARS-CoV-2 spike protein.

    PBMCs from 22 patients [group 1, n = 6; group 2, n = 5; group 3, n = 8; group 4, n = 3; healthy control (HC), n = 6] were incubated with or without intact SARS-CoV-2 spike protein for 24 hours. The CD4+ T cells were analyzed for intracellular IFN-γ (A and B) and CD25 (C). Representative flow cytometry plots of intracellular IFN-γ staining in CD4+ T cells before and after stimulation with spike protein are shown in (A). The data were analyzed by multiple regression with a mixed model for repeated measures with comparisons with and without spike protein (mean ± SD; **P < 0.01 and ***P < 0.001).

  • Fig. 4 Spike protein–specific antibody titers in patient serum samples.

    Anti–SARS-CoV-2 spike protein total IgG (A), IgA (B), IgG1 (C), and IgG3 (D) were measured by ELISA at a 1:50 serum dilution. Data are presented as ODUs in relation to the time serum was obtained after admission (n = 90) for IgG (Spearman r = 0.35, P = 0.0008), IgA (r = 0.35, P = 0.0008), IgG1 (r = 0.31, P = 0.01), and IgG3 (r = 0.24, P = 0.047). (E) Ratio of anti–SARS-CoV-2 spike protein–specific IgG1 to IgG3 in 71 patients with detectable IgG (group 1, n = 23; group 2, n = 10; group 3, n = 21; and group 4, n = 17) (*P < 0.05, **P < 0.01, and ***P < 0.0001, ANOVA with Tukey multiple comparison test). (F to H) Shown are serum IgG antibodies to other human CoVs: (F) HKU1, (G) NL63, and (H) 229E. In (F) to (H), n = 33, 9, 26, 22, and 3 in groups 1 to 5.

  • Fig. 5 Neutralizing antibody titers in patient serum vary by age.

    (A) VSV-S was incubated with serial twofold dilutions of patient serum or culture media as a control for 1 hour at 37°C and subsequently was added to cultured Vero cell monolayers. Neutralization of VSV-S by antibody was measured after 48 hours by comparing reduction in plaque number relative to control wells. (B) A comparison of AUC for neutralizing antibody data in (A) for pediatric and adult patients. (C to E) Correlations between neutralizing antibody AUC and age in years (C) or serum concentrations of IFN-γ (D) and IL-17A (E) and age in years are presented. In (A), n = 8 per group; in (B), n = 16 per group. Data in (A) and (B) are presented as mean ± SD. Data in (B) were analyzed by unpaired Student’s t test. Correlations in (D) to (F) were determined by Spearman’s nonparametric correlation. *P < 0.05.

  • Fig. 6 Antibody effector functions vary by clinical outcome.

    (A) ADCC activity of serum antibody was measured by an FcγRIIIa bioreporter assay and expressed as fold induction. (B and C) ADCP was measured as the percent of THP-1 cells internalizing spike protein–coated beads in the presence of COVID-19 patient serum after 16 hours of culture. ADCP was compared between pediatric and adult patient serum samples (B) and by group (C). (D and E) ADCP after (D) an Fc-blocking antibody (2 μg per sample) or (E) an ACE2-blocking antibody (2 μg/ml) was added to a subset of serum samples to assess the role of Fc receptors and ACE2, respectively, in bead internalization by THP-1 cells. HC, healthy control sera obtained before 2020. NT, no treatment. In (B), P < 0.0001 for comparisons between healthy control serum samples and all other groups. Statistics were calculated by unpaired Student’s t test (B), one-way ANOVA (C), or paired Student’s t test (D). *P < 0.05, **P < 0.01, and ****P < 0.0001. Data are presented as mean ± SD (A and B). [(A), n = 4, 13, 16, 11, and 13 in HC and groups 1 to 4; (B), n = 29 and 28 for pediatric and adult serum samples, respectively; (C), n = 5, 14, 15, 14, and 14 in HC and groups 1 to 4; (D), n = 15; (E), n = 5].

Tables

  • Table 1 Demographics and clinical features of children and adults with COVID-19.

    Results are presented as means ± SD. n, number; COPD, chronic obstructive pulmonary disease.

    Age <24 (n = 65)*Age >24 (n = 60)P value
    Age13.34 ± 6.0961.05 ± 12.96<0.0001
    Male:female (n)41:2434:260.47
    Black:white:other/unknown (n)25:5:3530:7:230.22
    Hispanic (n, %)26 (40%)15 (25%)0.09
    BMI27.19 ± 14.0929.78 ± 5.610.198
    Underlying medical conditions (n)
    Obesity (BMI > 30)18210.44
    Diabetes mellitus8200.0056
    Asthma or COPD18120.40
    Hypertension335<0.0001
    Treatment (n)
    Hydroxychloroquine947<0.0001
    Remdesivir840.37
    Systemic corticosteroid1480.25
    IVIG100<0.0001
    Other biologics45>0.99
    Outcome
    LOS (days)§6.37 ± 5.9114.77 ± 16.68<0.0001
    Mechanical ventilation (n, %)5 (7.7%)22 (36.7%)<0.0001
    Deaths (n, %)2 (3.1%)17 (28.3%)0.0001

    *Includes 20 patients with MIS-C.

    †Continuous variables were compared by Student’s t test; categorical variables were compared by Fisher’s exact test.

    ‡Other biologics include tocilizumab, sarilumab, and anakinra.

    §LOS excludes patients who died.

    • Table 2 Clinical and laboratory findings in subgroups of pediatric and adult patients with COVID-19.

      Data are mean ± SD. ANC, absolute neutrophil count; Gp, group; WBC, white blood cell; NS, not significant.

      Group 1Group 2Group 3Group 4Group 5P value*
      Age <24 (no
      mechanical
      ventilation)
      (n = 41)
      MIS-C (n = 20)Adult (no
      mechanical
      ventilation)
      (n = 33)
      Adult (mechanical
      ventilation or
      death) (n = 27)
      Age <24
      (mechanical
      ventilation or
      death) (n = 4)
      Age (years)14.90 ± 5.619.15 ± 5.2859.42 ± 14.8963.04 ± 10.0518.25 ± 3.30<0.0001
      Days since onset of
      symptoms
      4.51 ± 4.57 (n = 33)4.06 ± 1.47 (n = 18)5.00 ± 3.04 (n = 31)4.70 ± 2.91 (n = 23)3.75 ± 2.75 (n = 4)NS
      BMI30.45 ± 15.48 (n = 36)19.04 ± 5.81 (n = 19)29.61 ± 5.77 (n = 31)29.99 ± 5.52 (n = 26)28.00 ± 9.93 (n = 4)<0.01 (Gp 2 versus 1,
      3, and 4) and <0.05
      (Gp 2 versus 5)
      LOS4.84 ± 5.36 (n = 38)8.10 ± 4.05 (n = 20)7.88 ± 6.84 (n = 33)37.50 ± 19.60 (n = 10)21.00 ± 9.90 (n = 2)<0.0001 (Gp 4
      versus 1, 2, and 3)
      Mortality (n)000172<0.0001 (Gp 4 versus 1, 2, and 3)
      WBC (×10−3 cells/μl)9.89 ± 6.639.39 ± 3.787.15 ± 4.578.29 ± 2.6211.15 ± 4.01NS
      Hemoglobin (g/dl)13.22 ± 2.8611.23 ± 1.8312.65 ± 2.0212.89 ± 2.3212.33 ± 1.24NS
      Platelets
      (×10−3 cells/μl)
      265.29 ± 144.25174.95 ± 98.23206.62 ± 80.29214.85 ± 823.28181.25.00 ± 538.84NS
      ANC (cells/μl)6878.05 ± 5664.747750.00 ± 3623.535339.39 ± 4224.776533.33 ± 2344.558900.00 ± 2892.52NS
      ALC (cells/μl)2092.68 ± 1443.501105.00 ± 592.471069.70 ± 470.691129.63 ± 685.461450.00 ± 925.56<0.01 (Gp 1 versus
      2, 3, and 4)
      CRP (mg/dl)4.56 ± 7.11 (n = 34)18.80 ± 13.57
      (n = 19)
      12.83 ± 7.75 (n = 20)19.50 ± 12.83
      (n = 17)
      18.95 ± 11.37 (n = 4)<0.0001 (Gp 1 versus
      2 and 4) and <−0.05
      (Gp 1 versus 3)
      CRP (peak) (mg/dl)9.66 ± 10.67 (n = 31)24.49 ± 10.95
      (n = 18)
      14.10 ± 8.90 (n = 25)31.06 ± 17.94
      (n = 23)
      24.13 ± 9.98 (n = 4)<0.0001 (Gp 1 and 3
      versus 4) and <0.01
      (Gp 1 versus 2)
      Ferritin (peak)
      (ng/ml)
      969.52 ± 1155.33
      (n = 27)
      1893.16 ± 3144.32
      (n = 19)
      1276.11 ± 1551.25
      (n = 9)
      4158.47 ± 5192.46
      (n = 17)
      1257.25 ± 1076.43
      (n = 4)
      NS
      LDH (peak) (U/liter)415.78 ± 255.56
      (n = 27)
      509.74 ± 434.86
      (n = 19)
      460.00 ± 165.62
      (n = 26)
      770.88 ± 553.94
      (n = 24)
      797.25 ± 233.62
      (n = 4)
      <0.01 (Gp 1 versus
      4) and <0.05 (Gp 3
      versus 4)
      Troponin (peak)
      (ng/ml)
      0.01 ± 0.005 (n = 25)0.09 ± 0.16 (n = 18)0.12 ± 0.56 (n = 30)0.07 ± 0.10 (n = 26)0.10 ± 0.12 (n = 4)NS
      CPK (peak) (U/liter)645.65 ± 1150.71
      (n = 20)
      517.47 ± 438.42
      (n = 17)
      537.93 ± 675.00
      (n = 29)
      757.29 ± 1307.40
      (n = 24)
      1088.25 ± 690.81
      (n = 4)
      NS
      D-dimer (peak)
      (μg/ml)
      1.43 ± 1.52 (n = 25)11.14 ± 7.00 (n = 18)2.58 ± 1.72 (n = 17)12.57 ± 9.46 (n = 17)9.30 ± 5.96 (n = 4)<0.001 (Gp1 versus
      2; 2 versus 3), <0.01
      (Gp 1 versus 4), and
      <0.05 (Gp 3 versus 4)

      *Groups 1 to 4 were compared by ANOVA with Tukey’s multiple comparison.

      †Laboratory values are from the time of admission unless indicated as peak.

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