Thirty-seven participants completed the 2 study visits assessing the immune response prior to and 3–4 weeks after receiving the 3rd COVID-19 vaccine dose. Of these participants, 17 had a solid organ transplant and 20 were receiving immunosuppressive medication for other chronic illnesses (Table 1). The mean age was 15.4 years, 62.2% male, with most participants prescribed a calcineurin inhibitor, prednisone, mycophenolate mofetil, and/or a tumor necrosis factor alpha (TNFα) blocking agent. Six participants self-reported COVID-19 illnesses prior to receiving the 3rd COVID-19 vaccine dose (Table 1).

Levels of binding antibodies to 3 SARS-CoV-2 spike protein subunits (S1, S2 and receptor-binding domain (RBD)) were detected in most participants after the 2-dose vaccine series with median MFI levels of 13,157 (95% CI: 6639–16,188), 13,158 (95% CI: 11,434–17,259), and 17,379 (95% CI: 13,838–19,540) for S1, S2, and RBD subunits, respectively (Fig. 1). These levels were significantly boosted after the 3rd vaccine dose for all three spike subunit proteins to median MFI levels of 19,741 (95% CI: 18,426–21,108), 18,797 (95% CI: 17,532–20,542), and 21,107 (95% CI 20,097–21,913) for S1, S2, and RBD subunits, respectively (p ≤ 0.0005) (Fig. 1). Five participants had low levels (<2000 MFI) for all spike protein subunits, which remained low even after the 3rd vaccine dose, suggesting a limited humoral immune response to the vaccine (Fig. 1).

Multiplex bead-based antibody binding assay that measures the IgG antibody response to SARS-CoV-2 spike subunit 1 (S1), spike subunit 2 (S2) and receptor-binding domain (RBD). Median Fluorescent Intensity (MFI) is shown and background well subtraction has been used to remove nonspecific signal. Each dot represents an individual (n = 37) after the two-dose vaccine series (Visit 1) and after the 3rd vaccine dose (Visit 2). P values were determined using Wilcoxon matched pairs signed rank test. Group median values are displayed above the graph.

The spike protein is the only SARS-CoV-2 viral component in the COVID-19 vaccine. Thus, antibody responses to other viral proteins, such as the nucleocapsid protein (NP), likely indicate prior infection with SARS-CoV2. As expected, most participants had low NP antibody levels before and after receiving the 3rd vaccine. Five of the six participants who had a self-reported COVID-19 illness prior to receiving the 3rd vaccine dose had elevated NP antibodies (>5000 MFI). Three of those participants had elevated NP antibodies after the 2-dose vaccine series (visit 1) and two additional participants developed elevated NP antibodies after receiving the 3rd vaccine dose (visit 2) (Fig. 2).

A multiplex bead-based antibody binding assay measured the IgG antibody response to SARS-CoV-2 nucleocapsid protein. Median Fluorescent Intensity (MFI) is shown and background well subtraction has been used to remove nonspecific signal. Each dot represents an individual (n = 37) after the two-dose vaccine series (Visit 1) and after the 3rd vaccine dose (Visit 2). P values were determined using Wilcoxon matched pairs signed rank test. Group median values are displayed above the graph.

As a surrogate to neutralizing antibody detection, the levels of antibodies that could block the human host receptor angiotensin converting enzyme 2 (ACE2) and viral RBD binding were measured. Blocking antibodies were measured against the original vaccine-matched strain (2019-nCoV) and the Omicron variant that emerged in the fall of 2021. Detectable blocking antibodies were found in 86.5% of participants before and after the 3rd vaccine dose against the 2019-nCoV strain. The five participants that had low levels of binding antibodies to the spike protein also had undetectable blocking antibodies before and after the 3rd vaccine dose (Fig. 3). The magnitude of blocking antibodies increased from a median of 89.91% (95% CI: 65.25–94.45%) after the 2-dose vaccine series to a median of 94.63% (95%CI: 94.51–96.37%) following the 3rd vaccine dose (p = 0.0001). On further evaluation of the 5 participants with no antibody response following the 3rd vaccine dose, 4 of these children were kidney transplant recipients and were prescribed mycophenolate mofetil, tacrolimus, and prednisone and 1 child had received rituximab for a rheumatologic disease 3 months prior to receiving the 3rd vaccine dose.

A neutralization antibody proxy assay determined the level of antibodies that block binding of the spike protein receptor-binding domain to the human host receptor angiotensin-converting enzyme 2 (ACE2). The assay threshold for positivity was 30% indicating the presence of neutralizing antibodies. RBD from the original 2019-nCoV or RBD from the SARS-CoV-2 Omicron variant were utilized. Plasma samples (n = 37) were obtained after 2-doses of the vaccine (Visit 1) and after the 3rd vaccine dose (Visit 2). The median percentage of surrogate neutralizing antibodies is displayed above the graph.

In contrast to the response to the vaccine-matched strain (2019-nCoV), only 15.2% of participants had surrogate neutralizing antibodies (median 14.34%, 95% CI: 0.00–16.82%) to the Omicron variant after 2 doses of the vaccine. After the 3rd vaccine dose, Omicron surrogate neutralizing antibodies were detected in 54.6% of participants (median 38%, 95% CI: 19.98–71.72%), but the magnitude remained lower than the response to the original vaccine-strain (p < 0.0001) (Fig. 3).

Most participants (86.5%) had a positive cellular response after the 2-dose vaccine series, which increased to 100% after the 3rd vaccine dose (p = 0.063). When characterized into CD4 and CD8 response, the CD4 response was identical to the overall cellular response. In contrast, 20 participants had a negative CD8 response after the 2-dose vaccine series with only 5 additional participants converting to a positive response after the 3rd vaccine dose; 11 participants had a positive response at both time points (Fig. 4). Of the 5 participants with initial negative cellular response, 3 were kidney transplant recipients, 2 had rheumatologic diseases, and 1 had received a stem-cell transplant. Only 1 of the 5 also had a negative humoral response.

Cellular immune response was measured via the SARS-CoV-2 inSIGHT™ T-cell immunity panel. The overall response (detected/not detected) was further categorized into CD4 and CD8 response. Samples (n = 37) were obtained after 2-doses of the vaccine (Visit 1) and after the 3rd vaccine dose (Visit 2). The proportion of participants with positive/negative results are displayed within the graph.