*3.1. Immunological Studies*

The immunological features of the patient are shown in Table 1. The serum level of IgM was normal, whereas no IgA was detected. The IgG level was normal due to the immunoglobulin treatment. However, in the diagnostic sample, IgG was absent. The study of lymphocyte subpopulations showed a marked reduction in B cells, a reversed CD4/CD8 T-cell ratio, and normal NK cell levels. The B-cell immunophenotyping highlighted the absence of transitional B cells, plasmatocytes, and switched-memory B cells, whereas the percentages of unswitched-memory B cells, marginal-zone B cells, and CD21*low* B cells were elevated.

The study of the complete COVID-19 vaccination program showed a very low antibody response after the second dose of the AstraZeneca vaccine, with a decrease in antibody level of −23% at 3 months and a stronger response after the booster dose with the Moderna vaccine. The cellular immunity study by the quantification of the SARS-CoV-2-specific IFN-γ production showed a positive cellular response one month after the second dose, which, as in the case of antibodies, was lost at 3 months, with subsequent recovery of T-cell-mediated responses after the booster dose. All immunological values of the humoral and cellular responses are shown in Table 2. After the booster dose of the vaccine, the lymphocyte subpopulations remained stable, but we detected an increase in the percentage of switched-memory B cells (5.8%) and a decrease in unswitched-memory B cells (17%) in the immunophenotyping of B cells.


**Table 1.** Immunological features of the CVID patient in current treatment with intravenous immunoglobulins previous to the SARS-CoV-2 vaccination. Ig: immunoglobulin.

**Table 2.** Humoral and cellular responses to the SARS-CoV-2 vaccine in the CVID patient. We made the measurements one month and three months after the second dose of the vaccine and one month after the booster dose.


#### *3.2. Genetic Study*

The NGS test revealed a heterozygous substitution of guanine to adenine at position 485 of exon 5 of the *IKZF1* gene (chromosome 7p12.2) in the patient (c.485G>A) that causes a codon change of "CGG" to "CAG", which mean a non-synonymous switch from arginine to glutamine in the 162 protein residue (Arg162Gln). This variant was not identified in the DNA of the blood samples from the patient's parents by Sanger sequencing (Figure 2A–C).
