Study Population

Immune responses to vaccination were evaluated in eight adult patients with NS in a cross-sectional study performed between January 2022 and August 2022 at the Erasmus University Medical Center Rotterdam, an acknowledged national center of expertise for NS. In all patients a genetic diagnosis was confirmed. The study was approved by the Medical Ethical Committee of the Erasmus University Medical Center (MEC-2022-0616; MEC-2020-0264, MEC-2022-0462, MEC- 2013-026 and MEC-2021-0050), Rotterdam, the Netherlands, and was performed in accordance with the Declaration of Helsinki. All patients and healthcare workers controls provided written informed consent.

Clinical Data Collection

Clinical data of NS patients was retrieved from electronic patient files, and included the following information: disease severity, medication use, medical history, comorbidities, and results of blood examinations performed within clinical care. Disease severity was retrospectively evaluated using previously collected medical photographs, which were scored according to the Ichthyosis Area and Severity Index (IASI) [22]. Intensity of erythema and scaling is scored at different pre-defined body parts, also taking into account body surface area involved. Pain and pruritus within the last 24 h were assessed by numerical rating scale (NRS). Patient report peak pruritus by scoring between 0 and 10 Clinical care blood examinations included measurement of serum immunoglobulin levels (i.e., IgE, IgM, IgA, IgG), and diagnostic vaccination responses to PPV23 and ActHiB. Medical history included the COVID-19 vaccination history, patients’ self-reported SARS-CoV-2 infections, and experienced symptoms during SARS-CoV2- infection. Warning signs for primary immunodeficiency disease (PID) in adults were evaluated according to the European Society for Immunodeficiencies (ESID) criteria [23].

Vaccination Response to PPV23 and ActHiB

Immune responses to a polysaccharide vaccine against Streptococcus pneumoniae (Pneumovax 23; PPV23) and a conjugate vaccine against Haemophilus influenza type b (ActHiB) were measured and evaluated as part of standard clinical work-up for suspicion of immunodeficiency. Patients were vaccinated with PPV23 and ActHiB and after 4 to 6 weeks, antigen-specific antibody concentrations against S. pneumoniae and H. Influenza type b were measured and compared to pre-vaccination concentrations. Using a Luminex assay, an increase in antibody concentration of at least 2-fold, reaching at least 1.00 µg/mL for a minimum of 9 out of 16 measured S. pneumoniae serotypes (1, 3, 4, 5, 6B, 7 F, 8, 9 V, 14, 15B, 18 C, 19 A, 19 F, 20, 23 F, 33 F) was determined as a normal response to vaccination according to the in-house ISO 15,189 accredited protocol [24, 25]. This protocol was adapted from Borgers et al. [26]. Antigen-specific antibody concentrations against H. Influenza type b were measured by ELISA. Antibody concentrations reaching at least 1.00 µg/mL were classified as a normal response to ActHiB vaccination [27].

Vaccination Response to mRNA-based COVID-19 Vaccination

All participants received an mRNA-based primary vaccination regimen against COVID-19 (mRNA-1273 or BNT162b2) via the Dutch vaccination program between March 2021 and June 2021. Participants received an additional COVID-19 mRNA-based (mRNA-1273 or BNT162b2) booster vaccination between December 2021 and February 2022. Serum and peripheral blood mononuclear cells (PBMC) were collected 4 to 8 weeks after booster vaccination. PBMC were stored in liquid nitrogen until further use.

Humoral Immune Response to mRNA-based COVID-19 Vaccination

Spike (S)-specific binding antibodies were measured by Liaison SARS-CoV-2 TrimericS immunoglobulin G (IgG) assay (DiaSorin) as previously described [28]. The lower limit of detection (LLoD) and the responder cutoff were defined as 4.81 BAU/ml and 33.8 BAU/ml, respectively. The presence of neutralizing antibodies against the ancestral SARS-CoV-2 (D614G) was measured in a plaque reduction neutralization test (PRNT) as previously described [28, 29]. Briefly, ancestral SARS-CoV-2 was cultured from clinical material and sequence was confirmed (GISAID: hCov-19/Netherlands/ZH-EMC-2498). The human airway Calu-3 cell line (ATCC HTB-55) was used to grow virus stocks and for PRNT. Calu-3 cells were cultured in OptiMEM supplemented with GlutaMAX (Gibco), penicillin (100 units/mL, Capricorn Scientific), streptomycin (0.1 mg/mL, Capricorn Scientific), and 10% fetal bovine serum (FBS; Sigma). Two-fold dilution series of heat-inactivated sera were prepared in OptiMEM without FBS. The dilution range of a sample was dependent on its S-specific binding antibody titers (< 1500 BAU/mL: 1:10–1:1280; 1500–6000 BAU/mL: 1:80–10240; >6000 BAU/mL: 1:640–1:81920). Following the addition of 400 plaque-forming units (PFU) in an equal volume of OptiMEM, the virus-serum mix was incubated for 1 h at 37 °C before transfer of 100 µL to confluent Calu-3 and incubation for 8 h at 37 °C. Next, the cells were fixed with 10% neutral-buffered formalin for 30 min, permeabilized in 70% ethanol, and plaques were stained with a polyclonal rabbit anti-SARS-CoV-2 nucleocapsid antibody (Sino Biological) and a peroxidase-conjugated goat anti-rabbit secondary antibody (Dako). The signals were developed using a TMB substrate (TrueBlue; SeraCare/KPL) and the number of plaques was quantified using an ImmunoSpot Image Analyzer (CTL Europe GmbH). The dilution at which a 50% reduction of infected plaques (PRNT50) was reached compared to the infection control was estimated by determining the proportionate distance between two dilutions from which an endpoint titer was calculated. When no neutralization was observed, the PRNT50 was given a value of 10. Humoral immune responses from Netherton patients were compared to a control cohort of 18 healthcare workers (HCW), which was matched for age, sex, and infection status (age range: 24–51 years; 12 men and 6 women) The HCW study was approved by the institutional review board of the Erasmus MC (medical ethical committee, MEC-2020-0264).

T-cell Response to mRNA-based COVID-19 Vaccination

SARS-CoV-2-specific T-cell responses were assessed in PBMC collected 4–8 weeks after booster as described previously [30]. Briefly, 1 × 106 PBMC were stimulated in 200µL RPMI1640 medium supplemented with Glutamax (Gibco), penicillin (100 units/mL, Capricorn Scientific), streptomycin (0.1 mg/mL, Capricorn Scientific), and 10% human serum (R10H) with an overlapping peptide pool (15-mers) covering the S protein of the ancestral, Omicron BA.1, or Omicron BA.5 variants. A CEFX super stimulation pool containing immunogenic peptides from common human pathogens and commensals was included as a positive control. Cells were stimulated with an equimolar concentration of DMSO as negative control. PBMC were incubated at 37℃ for 20 h prior to staining for phenotypic lymphocyte and activation markers. Cells were stained following ex vivo stimulation at 4 °C for 30 min with the following antibodies: anti-CD3PerCP (Clone SK7, BD, 1:25), anti-CD4V450 (Clone L200, BD, 1:50), anti-CD8FITC (Clone DK25, Dako, 1:25), anti-CD45RAPE-Cy7 (Clone L48, BD, 1:50), anti-CCR7BV711 (Clone 150503, BD, 1:25), anti-CD69APC-H7 (Clone FN50, BD, 1:50), anti-CD137PE (Clone 4B4-1, Miltenyi, 1:50), and anti-OX40BV605 (Clone L106, BD, 1:25). LIVE/DEAD™ Fixable Far Red Cell staining was included (APC, Invitrogen, 1:1000) for exclusion of dead cells. T-cells were gated as LIVE CD3 + cells and subdivided into CD4 + or CD8 + subsets. Within the CD4 + and CD8 + sub-population, memory cells were gated by the exclusion of CCR7 + CD45RA + naïve T-cells. Within the memory population, SARS-CoV-2-specific T-cells were identified as activation induced marker (AIM) positive as CD137 + OX40 + for CD4 + or CD137 + CD69 + for CD8 + T-cells. On average 175,000 cells were acquired on a FACSLyric (BD) after data clean-up in the time gate. NS patient 2, was excluded from further analysis because of low cell counts. AIM + T-cells percentages from NS patients were compared to an age-matched cohort of 16 healthcare workers (MEC-2022-0462; age-matched; age range: 29–59; 4 men and 12 women).response. AIM expression was analyzed with FlowJo software version 10.8.1; the gating strategy is depicted in Figure S1. Data is presented as the percentage of double positive (AIM+) CD4 and CD8 T-cells.

Analysis

Characteristics of NS patients, and results of the vaccination responses to PPV23 and ActHiB were analyzed descriptively. Median number of days between COVID-19 mRNA-based booster vaccination and antibody response measurement are presented as median (interquartile range (IQR)). Levels of SARS-CoV-2 S-specific binding and neutralizing antibodies, and AIM + T-cells percentages are reported as geometric mean titer (GMT) or geometric means (GM), respectively, with 95% CI, and compared between NS patients and the control group by Mann-Whitney U test. P-values less than 0.05 were considered statistically significant. Statistical analyses were performed and graphs were prepared with Graphpad PRISM version 10 (San Diego, CA, USA).