Preserved T-Cell Immunity Despite Impaired Humoral Responses following SARS-CoV-2 Infection and Vaccination in Children with Profound B-cell Lymphopenia

Preserved T-Cell Immunity Despite Impaired Humoral Responses following SARS-CoV-2 Infection and Vaccination in Children with Profound B-cell Lymphopenia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Preserved T-Cell Immunity Despite Impaired Humoral Responses following SARS-CoV-2 Infection and Vaccination in Children with Profound B-cell Lymphopenia Sabryna Nantel, Samuel Sassine, Benoîte Bourdin, Margot Barbosa Da Torre, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8864462/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 16 You are reading this latest preprint version Abstract Understanding the mechanisms of vaccine-induced protection in children with humoral immunodeficiency is essential to guide prevention strategies and reduce COVID-19-related complications and mortality. Yet, comprehensive cellular, humoral and mucosal analyses are scarce in this high-risk population. We conducted a longitudinal evaluation of SARS-CoV-2 immunity at 1, 6 and 12 months after a primary series of the Pfizer-BioNTech mRNA vaccine (10 µg dose) in 27 children aged 5–11 years with primary or secondary antibody deficiencies and 48 age- and sex-matched healthy controls. Functional T-cell responses were quantified by interferon-gamma (IFN-γ) and IL-2 ELISpot, and SARS-CoV-2-specific B cells, CD4 + and CD8 + T-cell subsets were assessed using high-dimensional spectral cytometry. Systemic and mucosal antibody responses to spike (S) and receptor binding domain (RBD) were measured in serum and saliva, and neutralizing activity against ancestral and Omicron BA.5 strains variants was evaluated through microneutralization. Children with humoral immunodeficiency exhibited markedly impaired systemic antibody responses following two mRNA doses, despite SARS-CoV-2 infection, with restoration after a third vaccine dose. Those with severe B-cell lymphopenia were unable to mount neutralizing antibodies even after three doses and despite infection. Notwithstanding this profound humoral defect, they developed preserved, polyfunctional SARS-CoV-2-specific T-cell responses across multiple variants, which likely protected them from severe COVID-19. T-cell responses were higher in asymptomatic immunocompromised children, while all symptomatic infections were mild, supporting a potential contribution of cellular immunity to disease control in this population. These findings reveal a clear dissociation between humoral failure and preserved cellular immunity in B-cell–deficient children. They indicate that T-cell responses can act as alternate correlate of protection when neutralizing antibodies are absent, supporting timely vaccination in pediatric populations with profound B-cell deficiency. Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology Covid-19 inborn errors of immunity B-cell defects rituximab cell-mediated immune response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 KEY MESSAGES Our study demonstrates that a three-dose primary mRNA vaccination regimen is essential to strengthen humoral immunity in children with B-cell defects. While children with severe B-cell lymphopenia fail to generate neutralizing antibodies, they develop robust cell-mediated immune responses that likely protect them from severe infection. These findings highlight the central role of T cells in antiviral protection and underscore the importance of vaccine strategies that efficiently stimulate cellular responses in children with profound B-cell dysfunction. CAPSULE SUMMARY Vaccinated children with profound B-cell deficiency lack neutralizing antibodies yet remain protected from severe COVID-19 through robust SARS-CoV-2–specific T-cell immunity. RESEARCH IN CONTEXT Evidence before this study Children with humoral immunodeficiencies are considered at high risk for severe SARS-CoV-2 infection, yet data on their immune responses to mRNA vaccination remain limited. Prior studies have shown impaired antibody production in immunocompromised adults, but pediatric cohorts, particularly those with profound B-cell lymphopenia, are underrepresented. Existing reports rarely integrate systemic, mucosal, and functional immunity or combine variant-specific neutralization with high-dimensional cellular immune profiling. Thus, the relative contribution of cellular immunity to protection in B-cell–deficient children remains poorly defined. Added value of this study This study provides a longitudinal, mechanistically integrated assessment of SARS-CoV-2 vaccine immunity in children with humoral immunodeficiencies. Using high-dimensional flow cytometry, ELISpot, mucosal and serum immune profiling, and microneutralization against ancestral and Omicron BA.5 variants, we show that children with severe B-cell lymphopenia exhibit markedly impaired neutralizing antibody responses , even after three vaccine doses and despite frequent SARS-CoV-2 infection . In contrast to this profound humoral defect, they mount robust and polyfunctional SARS-CoV-2–specific CD4⁺ and CD8⁺ T-cell responses, particularly in the setting of asymptomatic infections. These findings reveal a dissociation between humoral failure and preserved cellular immunity in vaccinated, immunocompromised children. Implications of all the available evidence Our results suggest that T-cell responses can serve as complementary or alternative correlates of protection when neutralizing antibodies are absent, highlighting the limitations of antibody-centric measures of vaccine efficacy in children with profound B-cell dysfunction. They support a three-dose primary vaccination regimen for children with humoral immunodeficiencies and highlight the need for vaccine strategies that effectively stimulate cellular immunity in this high-risk population. Importantly, our findings support timely SARS-CoV-2 vaccination even in children with profound B-cell lymphopenia , as preserved cellular immunity may contribute to protection from severe disease . This work informs precision vaccination and underscores the potential importance of T-cell immunity when humoral responses are compromised. INTRODUCTION Immunocompromised (IC) children, whether due to primary immunodeficiencies arising from inborn errors of immunity (IEI) or secondary to immunosuppressive therapies, often exhibit impaired B‑ and/or T‑cell function, leading to compromised adaptive immunity.(1, 2) Consequently, they are disproportionately susceptible to infections such as SARS‑CoV‑2 and face increased risks of severe disease, complications, and even death compared to healthy children.(3, 4) Different degrees of immunosuppression are associated with distinct and graded clinical outcomes in COVID-19. Individuals who are severely IC have a markedly higher risk of adverse outcomes than those who are moderately IC,(5) while patients with isolated antibody deficiencies appear to be less vulnerable than those with combined immune defects or immune dysregulation.(6) Vaccination remains the most effective strategy to protect children from COVID-19 hospitalization and severe disease.(7, 8) In healthy pediatric populations, protection against symptomatic COVID-19 correlates with neutralizing antibody titers targeting the SARS-CoV-2 spike protein.(9) However, humoral responses to the standard two-dose regimen are often weak or undetectable in IC children, particularly in those with B-cell defects.(10-13) This has led to recommendations for an augmented three-dose primary series in both adult and pediatric IC individuals, although the durability and breadth of this protection is still not well defined.(14) Importantly, protection against severe COVID-19 does not rely solely on humoral immunity. T-cell responses play a critical and complementary role in limiting disease progression, as demonstrated in IC adults.(15, 16) Moreover, vaccine immunogenicity varies depending on the underlying condition or treatment received, underscoring the heterogeneity of this population and the need for tailored immunization approaches .(17, 18) In this study, we conducted a comprehensive, longitudinal evaluation of SARS-CoV-2 immunity in children with primary and secondary antibody deficiencies following two or three doses of the Pfizer–BioNTech mRNA vaccine. Building on evidence that humoral responses are frequently blunted in this population, we hypothesized that, even in the context of profound B-cell lymphopenia, vaccine-induced T-cell immunity may remain intact and could provide meaningful protection against severe COVID-19. To rigorously test this hypothesis, we integrated systemic and mucosal antibody profiling, variant-specific microneutralization, and high-dimensional T-cell analyses, and compared these responses to healthy pediatric controls receiving the standard two-dose regimen. We also assessed whether hybrid immunity enhances protection or compensates for humoral deficits in immunocompromised children. METHODS Study Design and Data Collection The “Immune Response in Young ImmunoSuppressed children to COVID-19 vaccination” (IRYIS) study cohort consists of 5-to-11-year-old children with primary or secondary antibody deficiency and healthy children, who were vaccinated following standard public health recommendations in Quebec. In total, 27 children with antibody deficiency were included in the study between Novembre 2021 and July 2022. They were compared to a total of 48 healthy children who were recruited between November 2021 and October 2022 through three different studies: the IRYIS study, the “Children and Older Teens Immune Response to SARS-CoV-2 in Montreal” (CHOIR) study, and the “Children and COVID-19 : Seroprevalence study” (EnCORE), which corresponds to the Montreal cohort of the Pediatric Network Expansion study “Understanding Immune Responses to SARS-CoV-2 Infection and Vaccination in Children Aged from Five to Eleven Years” study. The clinical characteristics of children with antibody deficiency and their diagnosis are reported in Supplemental Table S1 . Children with primary humoral immunodeficiency (n=15/27, 56%) had either a combined immunodeficiency or a predominant antibody deficiency (X-linked agammaglobulinemia, hypogammaglobulinemia or common variable immune deficiency) as defined by the 2019 International Union of Immunological Societies classification, or were patients receiving subcutaneous or intravenous immunoglobulins for recurrent infections, low memory B cells counts (less than 8% of total B cells) and/or dissociated vaccine responses with normal IgA and/or IgM levels (unclassified humoral immunodeficiency).(19) Of the children with secondary humoral immunodeficiency (n=12/27, 44%), ten received anti-CD20 therapy (n=10/12, 83%), a monoclonal antibody known as rituximab less than six months before receiving their first dose of COVID-19 vaccine or within the six months following the initiation of vaccination, with (6/10, 60%) or without (4/10, 40%) steroids or other immunosuppressive therapy. Two of these children (2/12, 17%) had agammaglobulinemia or hypogammaglobulinemia secondary to CAR-T cell therapy and/or hematopoietic stem cell transplantation. Participants were vaccinated between December 2021 and September 2022 through routine public health programs and following their physicians’ recommendations regarding the number of doses indicated in their situation. Participants all received the Comirnaty COVID-19 vaccine from Pfizer-BioNTech at a dosage of 10 µg per dose, as indicated for children aged from 5 to 11 years and were followed up to one year after vaccination. None of the subjects analyzed received booster vaccination after their recommended primary vaccination regimen. Immunological data of IC children prior to vaccination The immunological parameters of IC children vaccinated with two or three doses of vaccines are reported and compared in Supplemental Table S2. The immune characteristics of the subjects with or without severe B-cell lymphopenia are reported in Supplementary Table S3 . Cell lymphopenia was based on the absolute cell count lower that the 5 th percentile for age.(20) Determination of SARS-CoV-2 infection status SARS-CoV-2 infection status was evaluated using complementary clinical and immunological approaches. At each visit, documented SARS-CoV-2 infection confirmed by rapid antigenic testing was assessed, and the presence or absence of COVID-19-associated symptoms was documented. At each time point, immune responses to SARS-CoV-2 nucleocapsid (N) protein were evaluated using serum anti-N IgG titers and IFN-γ + T-cell responses to N peptide pools. Children were considered to have been infected with SARS-CoV-2 if they either had a positive COVID-19 test or demonstrated detectable anti-N IgG titer and/or T-cell response to N peptides, even in the absence of reported symptoms. Sample Collection and Processing Saliva and blood samples were collected from participants at multiple time points: one month after their second and third vaccine doses, as well as at six and twelve months post-vaccination (detailed timepoints are presented in Table 1 ). Saliva samples were collected using Salivette system (Sarstedt, Numbrecht, Germany). Blood samples were collected into serum separation tubes (SST TM , BD) and acid-citrate-dextrose tubes (ACD, BD). Following collection, saliva, serum, plasma, and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved according to standard operation procedures at the Mother-Child Institutional Biobank at the Sainte-Justine University Hospital and Research Center (CR-CHUSJ) as previously reported by our team.(21-23) PBMCs were stored in liquid nitrogen and all other samples were stored at -80°C. Assays were performed using all available samples for each participant at each visit. However, biological material was sometimes insufficient to perform every assay, resulting in variation in sample numbers across analyses. (23) Ethic Approvals The IRYIS and the CHOIR/EnCORE protocols were approved by the Research Ethics Board (REB) at the Sainte-Justine University Hospital and Research Center (CR-CHUSJ) under study numbers MP-21-2022-3909 and MP-21-2021-3105, and the CHOIR/EnCORE study was also approved by the University of Montreal REB under study number CERSES-20-103-P. Participants were enrolled after obtaining written informed consent from one of their parents or legal guardians. Cell-Mediated Immunity PBMCs isolated from blood samples were rapidly thawed and rested overnight. Each sample was assessed for viability. The number of assay(s) performed was determined based on the number of cells with adequate viability for each participant at each of their visits. Enzyme-linked immunospot assay (ELISpot) for the quantification of functional cellular immunity ELISpot assay was used to detect interferon-gamma (IFN-γ) or interleukin-2 (IL-2) secreting cells. PBMCs were stimulated for 20 hours with one µg/mL of spike (S) or nucleocapsid (N) mega pools of SARS-CoV-2 peptides from the ancestral strain, as well as the spike from Omicron subvariants BA.4/BA.5 and XBB.1.5 (JPT Peptide Technologies, JPT, Berlin, Germany) as previously described.(21-23) Culture media without peptide (AIM-V® Medium (1X), Thermo Fisher Scientific, Waltham, MA, USA) was used as negative control, and the number of spots detected in the negative well of each sample was subtracted from all stimulated wells of the same sample. CytoStim TM (Miltenyi Biotec, MA, USA) was used as positive control. CTL ImmunoSpot® SS UV Analyzer (Cellular Technology Ltd., OH, USA) was used to quantify spots with a detection limit of one secreting cell per 200 000. The positive threshold of 25 spot-forming units per million PBMCs was previously validated on pre-pandemic samples.(21) Activation Induced Markers (AIM) assay and circulating T follicular helper cells (cT FH ) The AIM assay was used to detect activated CD4 + and CD8 + T cells in response to stimulation with ancestral SARS-CoV-2 S peptides. PBMCs were stimulated for 20 hours with one µg/mL of a mega pool of SARS-CoV-2 S peptides (JPT Peptide Technologies, Berlin, Germany). After stimulation, cells were stained with an AIM and cT FH (CXCR5 + PD1 hi /CD3 + CD4 + ) panel of fluorescent monoclonal antibodies as presented in Supplemental Table S4 . Data were acquired using the SONY Spectral Cell Analyzer ID7000. All data analysis was performed with FlowJo software version 10.10.0. Fluorescence minus one (FMO), unstimulated (negative control) and CytoStim TM (positive control) were used to set the gates. Results are represented as frequency of activated CD4 + or CD8 + T cells following peptide stimulation minus the frequency of activated cells detected in the unstimulated condition for each sample. B cell immunophenotyping PBMCs were stained directly after overnight resting to assess SARS-CoV-2 S-specific B cells. Miltenyi Biotec SARS-CoV-2 Spike B Cell Analysis Kit for human was used to detect B cells (CD19 + /CD3 - ) binding to the recombinant SARS-CoV-2 S protein of the ancestral strain. Additional antibodies were added to the Miltenyi Biotec panel to assess for lymphocytes (CD3), plasmablasts (CD27 + CD38 + /CD3 - CD19 + ) and IgD. The complete panel of fluorescent monoclonal antibodies used is described in Supplemental Table S4 . Data were acquired with the SONY Spectral Cell Analyzer ID7000. All data analysis was performed with FlowJo software version 10.10.0. FMO and unstained samples were used to set the gates. Results are presented as the number of S-specific B cells per million of PBMCs or as frequency of each subpopulation on total B cells. Mucosal Immunity Enzyme-linked immunosorbent assay on saliva As previously published by our team, enzyme-linked immunosorbent assays (ELISA) were performed to detect IgG and IgA binding to S and its RBD in saliva.(22, 24) Plates were coated with biotinylated S or RBD, incubated overnight and blocked subsequently. Samples were then added to each well for two hours. Horseradish peroxidase (HRP) conjugated IgG and IgA detection antibodies were then added for one hour. After a final wash, 3,3’,5,5’-Tetramethylbenzidine substrate (ThermoFisher) was added for color development. Optical density (OD) was read at a wavelength of OD450 using a Thermo Multiskan FC spectrophotometer. Samples were analyzed as previously described by subtracting the raw OD450 of phosphate-buffered saline (PBS) controls for each sample from their antigen specific OD450 at each dilution.(22, 24) These background-subtracted values were used to calculate the area under the curve (AUC) for each sample, which was then adjusted to a pre-selected standards, which consisted of pooled acute and convalescent COVID-19 patient saliva as described previously.(22, 24) This accounted for inter-plate variability. Detection range and positivity threshold for each antigen are presented in Supplemental Table S5 . Humoral Immunity Enzyme-linked immunosorbent assay on serum IgG and IgA binding antibody levels against the full-length SARS-CoV-2 S trimer, its RBD and N were measured in serum samples using chemiluminescent ELISA as previously described.(25) Briefly, LUMITRAC 600 high-binding white polystyrene 384-well microplates (Greiner Bio-One, Thermo Fisher Scientific, #78107425) were pre-coated overnight with 10 µL/well of antigen (50 ng of S, 20 ng of RBD, or 7 ng of N), all supplied by the National Research Council of Canada (NRC). Plates were centrifuged at 1000 rpm for one minute to ensure even coating, then incubated overnight at 4 °C. The next day, the assay was performed at room temperature with washing twice in 115 µL/well of PBS with Tween® detergent (PBS-T) before each of the following four steps. First, the wells were blocked for one hour in 115 µL/well of 5 % Blocker BLOTTO (ThermoFisher Scientific, #37530). Second, 10 µL of diluted serum in 1 % final Blocker BLOTTO in PBS-T was added and incubated for two hours. Serum samples were diluted 1:160 and 1:2560 to assess anti-N IgG antibody levels, at 1:160, 1:2560 and 1:40960 to assess anti-RBD and anti-S IgG antibody levels, and at 1:160 and 1:2560 to assess IgA antibody levels. Third, 10 µL of an anti-human IgG fused to HRP (IgG#5 by NRC, 0.9 ng/well) or anti-human IgA conjugated to HRP (Jackson ImmunoResearch, #109-035-011, 0.8 ng/well) diluted in 1% final Blocker BLOTTO in PBS-T was added, followed by one-hour incubation. Fourth, 10 µL/well of ELISA Pico Chemiluminescent Substrate (ThermoFisher Scientific, #37069, diluted 1:4 in MilliQ distilled H 2 O) was added, and the plates were centrifuged at 1000 rpm for one minute. After five minutes of incubation, chemiluminescence signals were read on an EnVision 2105 Multimode Plate Reader (Perkin Elmer) plate reader at 100 ms/well using an ultra-sensitive detector. Raw chemiluminescent values were normalized to a blank-subtracted reference point from the synthetic standard included on each assay plate, to create relative ratios. The synthetic standard used were as follows. For IgG, VHH72-Fc was used for S and RBD (supplied by NRC) and an anti-N IgG for N (clone HC2003, Genscript, #A02039). For IgA, anti-S CR3022 (Absolute Antibody, #Ab01680-16.0) and anti-N CR3018 (Absolute Antibody, #Ab01690-16.0) were used. For IgG and IgA results, relative ratios within the linear range of the assay were further converted to binding antibody units (BAU)/mL using the World Health Organization (WHO) International Standard 20/136, as previously described.(25) Linear ranges for the sample dilutions performed and positivity thresholds are presented in Supplemental Table S5 . Positivity thresholds were determined for the 1:160 dilution using three standard deviations from the mean of control samples (blanks, pre-pandemic negative sera, commercially purified IgG or IgA) as previously described or two standard deviations for anti-N IgG. Microneutralization assay Neutralizing antibody (NtAb) titers were measured through live SARS-CoV-2 microneutralization assay as previously described by our team.(21, 26) Two strains were used, the ancestral Wuhan-1-like SARS-CoV-2 isolated from a clinical sample in March 2020 in Québec City and the Omicron variant BA.5 provided by the National Microbiology Laboratory (NML), Public Health Agency of Canada. Heat-inactivated serum was diluted by two-fold starting with 1:20 dilution. Serum and virus were mixed at equal volumes and incubated for one hour at room temperature. The residual infectivity of the virus-serum mixture was assessed in quadruplicate wells of African green monkey kidney E6 cell line (Vero ATCC ® CRL-1586 TM ) for ancestral SARS-CoV-2 and in Vero cells overexpressing transmembrane protease serine 2 (TMPRSS2, JCRB) for Omicron BA.5. NtAb titers were defined as the reciprocal of the serum dilution that completely neutralized the infectivity of the 50% tissue culture infectious dose (TCID 50 ) of SARS-CoV-2 multiplied by 100, which was determined by the absence of cytopathic effect on cells after four days.(27) NtAb titers were then calculated using the Reed/Muench method.(28) These microneutralization assays were performed in the Containment Level 3 laboratory at the Centre Hospitalier Universitaire de Québec – Université Laval Research Center. Statistics Mann-Whitney or Kruskal-Wallis unpaired nonparametric tests were performed using Prism 9, version 9.2.0 (2021 GraphPad Software, LLC) to assess statistical significance. Simple linear regression is presented with Spearman correlation (R) and P-value (P). Significance was set as *P <.05, **P <.01, ***P <.001 and ****P <.0001. Time since infection is presented as mean ± SD, while experimental data are displayed as median [interquartile range, 25 th – 75 th percentile]. RESULTS Participant characteristics The IRYIS cohort consisted of children with primary or secondary humoral immunodeficiency. There were 27 children with a median age of nine years [6, 11] (63% male) that met the inclusion criteria and had samples suitable for analysis. They were compared to 48 healthy children with a median age of eight years [5,11] (50% male) who were recruited from three studies (IRYIS, CHOIR and EnCORE) ( Table 1 ). Ten children with humoral immunodeficiency (10/27, 37%) received only two doses of the COVID-19 vaccine as their primary series, while the remaining (17/27, 63%) received the recommended three-dose regimen. Immunologically, both IC subgroups where comparable at the onset of vaccination ( Supplementary Table S2 ). Many children included in the study reported a prior documented SARS-CoV-2 infection or were considered infected based on humoral or cellular anti-N assay results. In children with humoral immunodeficiency, 74% of participants were considered infected by SARS-CoV-2 before or at the time of vaccination compared to 50% of healthy controls ( Table 2 ). Most children in the humoral immunodeficiency (93%, 25/27) and healthy (88%, 42/48) groups were eventually infected with SARS-CoV-2 during the study and were therefore considered as having hybrid immunity by the end of the study. In both groups, 19% of children were reinfected a second time during the study period. Children who reported symptoms had mild disease requiring no hospitalisation (48 %), while the others had either asymptomatic or unreported infections (52%). Two mRNA vaccine doses fail to elicit robust humoral immunity in children with B-cell defects, whereas a third vaccine dose restores it To clearly delineate the impact of vaccine doses on immune responses in IC children, we divided children in subgroups and first analyzed children without evidence of prior SARS-CoV-2 infection based on diagnostic testing or N-specific responses at inclusion. In this infection-naïve subset, IC children mounted T-cell responses comparable to healthy controls after two and three doses, as shown by similar frequencies of IFN-γ- and IL-2-secreting T cells ( Fig 1A ). In contrast, humoral immunity was substantially reduced in IC children after two vaccine doses. Salivary anti-S and anti-RBD IgG levels were significantly lower than in healthy children ( Fig 1B ), while salivary IgA remained uniformly low across groups ( Fig S1A) , as expected after intramuscular vaccination(22, 24). Systemically, IC children showed reduced serum IgG to S and RBD (Fig 1C) , with a tendency towards reduced IgA titers ( Fig S1B ), whereas a third dose increased titers to values similar to healthy controls. Neutralizing antibody responses showed an even more pronounced deficit in IC children. Only 40% (4/10) of IC children had detectable NtAb after two doses, compared with 96% (24/25) of healthy controls. Following three doses, all IC children ( 5/5 ) generated detectable NtAb ( Fig 1D ). Collectively, these findings indicate that two doses are insufficient to elicit protective humoral immunity in children with antibody deficiencies and that a three-dose primary series is required to restore robust binding and neutralizing responses. Hybrid immunity only partially compensates for impaired humoral immunity in vaccinated children with B-cell defects Because most children were infected with SARS-CoV-2 during the study period, we next evaluated immune responses in those with hybrid immunity. As observed in infection-naïve participants, IC and healthy children mounted comparable functional T-cell responses , regardless of health status or number of vaccine doses ( Fig 2A ). Humoral immunity remained impaired in IC children, despite infection. In saliva, anti-S and anti-RBD IgG levels were significantly lower in IC participants ( Fig 2B ), whereas IgA responses remained uniformly low in most children and did not differ between groups ( Fig S2A ). Systemically, IC children who received two doses showed reduced serum IgG and IgA to S and RBD compared with healthy children ( Fig 2C and Fig S2B ). A third dose increased IgG to levels comparable to healthy controls, whereas IgA responses remained attenuated in the IC group. Importantly, hybrid immunity did not compensate for defective neutralizing responses in IC children who received only two doses ( Fig 2D ). Despite documented SARS-CoV-2 infection, only 38% (5/13) of IC children had detectable NtAb, with a median titer of 10 after two doses , compared to a titer of 226 in healthy children (P = 0.0002). A third vaccine dose increased NtAb titers to 50 , yet levels remained significantly lower than in healthy controls (P = 0.0043). Together, these data show that while hybrid immunity enhances overall responses, IC children continue to exhibit compromised humoral immunity and a limited capacity to generate functional neutralizing antibodies, even after natural infection and three vaccine doses. Impaired long-term humoral immunity in immunocompromised children We next examined the persistence of immunity six and twelve months after vaccination, acknowledging that the high incidence of SARS-CoV-2 infection across the cohort represents a major confounding factor for all long-term immune readouts ( Fig 3A ). By six months, most children, IC and healthy, had already acquired hybrid immunity, while by 12 months all except one healthy control had been infected. This implies that responses at later time points likely reflect the combined effects of vaccination and infection rather than vaccine-derived immunity alone. Within this context, T-cell responses in IC children appeared stable up to twelve months after vaccination, independently of the number of vaccine dose received, and remained reactive to the ancestral Spike and its Omicron BA.4/BA.5 and XBB.1.5 variants, secreting both IFN-γ and IL-2 ( Fig 3B and S3A ). Consistent with this, activation of S-specific CD4⁺ and CD8⁺ T cells remained comparable between groups ( Fig S3B ). These findings indicate that children with primary or secondary B-cell defects can maintain a functional memory T-cell pool over extended periods. In contrast, despite numerous infections, we observed persistently attenuated humoral responses after two vaccine doses in IC children compared to healthy controls. This was particularly evident from the markedly lower IgG and IgA levels to S and RBD at six months ( Fig 3C and S3C ), the substantially reduced pool of Spike-specific B cells ( Fig 3D ), and the near absence of neutralizing antibodies to the ancestral strain at this time point ( Fig 3E ). A third vaccine dose partially restored these humoral responses at both mucosal and systemic sites, increasing salivary IgG concentrations ( Fig S3D ), serum S- and RBD-specific IgG and IgA levels and neutralizing capacity against the ancestral and Omicron BA.5 strains ( Fig 3E ) toward levels observed in healthy children. Yet, S-specific B-cell frequencies remained lower in IC children at 6 months, and serum IgA responses declined by twelve months compared to healthy controls. Together, these findings demonstrate that, even in the setting of widespread hybrid immunity, IC children, especially if vaccinated only twice, fail to generate durable and fully functional humoral immunity , underscoring a persistent defect in their capacity to mount and maintain protective antibody responses. Impaired Memory and Antigen-Specific B-Cell Responses Underlie Defective Neutralization in IC Children To investigate the mechanisms underlying the profoundly impaired humoral responses observed in IC children, we performed extensive high-dimensional flow cytometry to phenotype and enumerate total and S-specific B cells, CD27⁺ memory B cells, plasmablasts and follicular helper T cells (Tfh), f ocusing on the 1-month post-vaccination time point to capture peak vaccine-induced responses while avoiding bias from repeated sampling across time . At this time point , IC children exhibited markedly diminished frequency of S-specific B cells and CD27⁺ memory B cells compared with healthy controls ( Fig 4A ), while total B-cell counts were unexpectedly comparable, and plasmablasts and Tfh frequencies were broadly equivalent between groups ( Fig S4A ). To determine which immune abnormalities best predicted defective antibody responses, we next assessed correlations between NtAb levels and multiple cellular parameters. Across the entire cohort, total B cell counts, and the frequency of memory and spike-specific B cells showed a moderate but statistically significant positive correlation with NtAb titers (Spearman R ≈ 0.40–0.44, p ≈ 0.005-0.010, n = 36, Fig 4B ), while plasmablast and Tfh frequencies did not correlate with neutralization capacity ( Fig S4B ). Importantly, when restricting the analysis to IC children only, these associations were further strengthened (Spearman R ≈ 0.61–0.66, p ≈ 0.006–0.013), despite the smaller sample size (n = 16, Fig 4C), indicating that both the magnitude of the B-cell compartment and the ability to generate antigen-specific B cells are closely linked to functional neutralization in this subgroup. Together, these analyses indicate that both the overall size of the B-cell compartment and the ability to generate antigen-specific B cells, rather than defects in Tfh support or plasmablast differentiation, are closely linked with impaired neutralizing antibody responses in IC children. Children with severe B-cell lymphopenia show particularly impaired immune responses to SARS-CoV-2 Because total B-cell counts correlated with neutralizing capacity at the time point closest to vaccination, we next examined whether IC children with the most severely impaired humoral responses were those with the lowest circulating B-cell numbers at the time of vaccination. IC participants were stratified according to the presence (n=12) or absence (n=15) of profound B-cell lymphopenia, defined as a CD19⁺ lymphocyte count below the 5 th percentile for age, while other immune parameters were comparable between IC groups, except for IgM levels ( Supplementary Table S3 ). We then compared SARS-CoV-2–specific immunity across severely B-cell–lymphopenic children, B-cell–sufficient IC children, and healthy controls. Children with severe B-cell lymphopenia exhibited markedly diminished humoral immunity across all compartments. Serum IgG and IgA levels to S and RBD were profoundly reduced ( Fig 5A and 5SA ), while salivary IgG responses to S and RBD were substantially low ( Fig S5B ). While B-cell–sufficient IC children generated frequencies of S-specific B cells comparable to healthy controls, those with severe B-cell lymphopenia showed distinctly reduced induction of antigen-specific B cells ( Fig 5B ). Consistent with these deficits, neutralizing antibody titers against the ancestral strain were extremely low or undetectable in this subgroup, in contrast to both healthy children and B-cell–sufficient IC peers ( Fig 5C ). Furthermore, despite documented infection, children with severe B-cell lymphopenia failed to develop neutralizing activity against BA.5 , underscoring an intrinsic inability to mount adequate humoral immunity in the setting of profound B-cell deficiency. In contrast, children with severe B-cell lymphopenia exhibited heightened functional T-cell responses, measured by IFN-γ secretion in response to SARS-CoV-2 spike, particularly one month after vaccination ( Fig 5D ). Together, these results demonstrate that profound B-cell lymphopenia is the dominant determinant of impaired humoral immunity to SARS-CoV-2 in IC children, whereas preserved and potentially enhanced T-cell responses likely provide compensatory protection in the absence of functional B-cell immunity. Protection against COVID-19 symptoms is associated to potent T cells Vaccine efficacy is often reported in terms of its capacity to prevent severe infection and hospitalization. Although this small cohort of high-risk patients does not allow for proper statistical analysis of vaccine efficacy, we observed that although most IC children were infected during the study period (25/27), none reported severe disease nor were hospitalized ( Table 2 ). Indeed, children were either mildly symptomatic or were found to be asymptomatic. As T-cell responses have been shown to restrict COVID-19 symptoms,(15) we questioned if they played the same role in our IC pediatric cohort. We thus compared T-cell responses in children with or without symptoms, in both healthy and IC children. Interestingly, IC children that lacked symptoms had higher levels of IFN-g secreting cells in response to both the ancestral S peptides and the Omicron BA.4/BA.5 S peptides compared to IC children who had symptoms, while this difference was not present in healthy controls ( Fig 6A ). In contrast, no difference in NtAb levels to the ancestral spike or the Omicron BA.5 variant was noted based on symptomatology of the infection ( Figure 6B ). These findings suggest that in the absence of robust humoral immunity, potent S-specific functional T-cell responses may play a key role in limiting COVID-19 symptom severity in children with humoral immunodeficiency. DISCUSSION In this study, we provide a comprehensive analysis of adaptive immune responses to SARS-CoV-2 vaccination and infection in children with primary or secondary humoral immunodeficiencies. Our data reveal a clear dissociation between humoral and cellular immune responses in this high-risk pediatric population. While antibody responses are variably impaired depending on B-cell status, functional SARS-CoV-2–specific T-cell immunity is preserved and maintained through time across immunocompromised subgroups and is associated with protection from symptomatic COVID-19. These findings challenge the reliance on serum IgG levels and neutralizing antibody titers as universal correlates of protection and highlight T-cell immunity as an important determinant of clinical outcome in children with humoral immune defects. We show that a standard two-dose mRNA vaccination regimen is insufficient to induce robust and long-lasting humoral immunity in children with humoral immunodeficiency, regardless of prior or recent SARS-CoV-2 infection. In contrast, a three-dose primary series substantially increases binding and neutralizing antibody responses in immunocompromised children, reaching levels comparable to healthy controls when the B-cell compartment is preserved. This demonstrates that multiple vaccine doses are required to support B-cell differentiation in children with intrinsic or secondary B-cell defects. Importantly, despite recurrent vaccination and infection, children with severe B-cell lymphopenia remain unable to mount effective IgG and IgA antibody responses, both systemically and at mucosal infection sites. This aligns with immunocompromised adult studies showing stronger immunity after three doses, except in adults on anti-CD20 therapy.(29, 30) It clearly articulates that lack of B cells directly predict poor antibody responses and neutralization capacity. These findings collectively indicate that B-cell availability imposes a biological ceiling on vaccine-induced humoral immunity that cannot be overcome by additional antigen exposure. By integrating high-dimensional immune phenotyping with functional antibody analyses, we identify the cellular determinants underlying defective humoral immunity in IC children. Neutralizing antibody capacity correlated with total B-cell counts and with the frequency of memory B cells and SARS-CoV-2–specific B cells, but not with Tfh frequency or plasmablast abundance. These findings indicate that impaired neutralization results from intrinsic limitations in the generation and maintenance of antigen-specific B cells rather than from defective T-cell help or transient plasmablast responses. This mechanistic insight explains the heterogeneity in vaccine responsiveness observed among immunocompromised children and highlights B cell counts as an important biomarker and predictor of the quality of humoral responses after vaccination. In contrast to humoral immunity, SARS-CoV-2–specific T-cell responses to vaccination were induced and maintained up to 1-year after vaccination across IC children, including those with severe B-cell lymphopenia. These responses were functional, polyvariant, and characterized by IFN-γ and IL-2 secretion in response to ancestral and Omicron BA.4/BA.5 and XBB1.5 Spike antigens. Importantly, stronger functional T-cell responses were associated with asymptomatic or minimally symptomatic infection in children with humoral immunodeficiency, a relationship not observed in healthy controls. These results align with studies showing that strong T-cell responses correlate with milder disease and faster recovery in both healthy children and immunocompromised adults, particularly when neutralizing antibodies are low or absent.(30-33) Remarkably, none of our immunocompromised participants required hospitalization for COVID‑19, despite high infection rates (93 %) and humoral impairments. This is in contrast with adult cohort studies where antibody deficiencies, in association with comorbid risk factors, were associated with increased risk of death in patients over 40-years old especially.(34-37) Interestingly, children who remained asymptomatic showed significantly stronger IFN‑γ-secreting T‑cell responses to both ancestral and Omicron S peptides than those who developed symptoms. These heightened T-cell responses were also sustained in children with profound B-cell lymphopenia, likely contributing to the absence of severe disease in this subgroup despite high infection rates. Similar heightened T-cell responses have been described in adults treated with anti-CD20 antibodies, although its relationship with symptom severity was not described in these studies.(38, 39) Collectively, our observations support a model in which cellular immunity can independently mediate clinical protection when humoral immunity is compromised. These findings have direct implications for the clinical management of children with humoral immunodeficiencies. First, they support prompt vaccination with an augmented primary series, even in children with profound B-cell defects or undergoing treatment with B-cell depleting agents, as vaccination reliably induces functional T-cell immunity. Second, they argue against using serum and neutralizing antibody titers as the sole marker of vaccine efficacy or protection in this population. Instead, immune stratification based on B-cell immunophenotyping may help identify children who require additional interventions, such as closer monitoring, monoclonal antibody prophylaxis, or additional alternative vaccine strategies, including mucosal boosters. Importantly, our data suggest that vaccination should not be delayed based on the timing since B-cell–depleting therapies, as cellular immunity remains inducible and clinically relevant. This study has several limitations. The high incidence of infection during follow-up limited our ability to isolate vaccine-induced immunity, particularly for long-term analyses. Blood volume constraints restricted immune phenotyping to subsets of participants, and incomplete sampling at some time points reduced statistical power for certain analyses. Additionally, infection timing was often inferred retrospectively using anti-nucleocapsid responses, limiting precision in assessing immune kinetics. Future studies with prospective infection surveillance and standardized longitudinal sampling will be essential to refine correlates of protection in this population. In summary, our findings demonstrate that immune protection against COVID-19 in children with humoral immunodeficiency is mediated through distinct and dissociable pathways. While a three-dose mRNA vaccination regimen enables most children with preserved B-cell compartments to mount effective humoral responses, those with severe B-cell lymphopenia remain intrinsically unable to do so. In this context, preserved SARS-CoV-2–specific T-cell immunity emerges as an important correlate of protection, associated with mild or asymptomatic infection despite absent neutralizing antibodies. These results underscore the need to redefine correlates of protection and vaccination strategies for immunocompromised pediatric populations, with a renewed focus on inducing durable and functional cellular immunity. Abbreviations ACD Acid-citrate-dextrose AIM Activation induced markers AUC Area under the curve BAU Binding antibody units CAR-T cell Chimeric antigen receptor T cell CHOIR study Children and Older Teens Immune Response to SARS-CoV-2 in Montreal study COVID-19 Coronavirus Disease 2019 CR-CHUSJ Sainte-Justine University Hospital and Research Center ELISA Enzyme-linked immunosorbent Assay ELISpot Enzyme-linked immunospot EnCORE study Children and COVID-19 seroprevalence study FMO Fluorescence minus one HC Healthy controls HRP Horseradish peroxidase IC Immunocompromised IEI Inborn errors of immunity IFN-γ Interferon-gamma IL-2 Interleukin 2 IRYIS study Immune Response in Young Immunosuppressed children to COVID-19 vaccination study mRNA Messenger ribonucleic acid N Nucleocapsid NRC National Research Council of Canada OD Optical density PBMCs Peripheral blood mononuclear cells PBS Phosphate-buffered saline RBD Receptor binding domain SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2 S Spike SST Serum separation tubes TCID 50 50% tissue culture infectious dose WHO World Health Organization Declarations Author contributions Conceptualization : HD Methodology : SN, BB, HR, FQ, MDB, LS, LWard, SSM, GC, KC, YL, JMR, JG, ACG, MB, CQ, HD Investigation : HD Visualization : SN, BB Funding acquisition : HD, KZ Project administration : BB, MBDT, LWang, SNi, KA, ZL, LS Subject recruitment : HD, KZ, CQ Supervision : HD Writing – original draft : SN, SS, BB, HD Writing – review & editing : SN, SS, BB, GS, FQ, LWard, KC, MB, CQ, HD Acknowledgments We warmly thank all the participants and their parents/guardians who accepted to participate in the IRYIS, CHOIR and EnCORE studies. We also thank the nurses and research coordinators from each participating centers. We want to recognize the work of the Sainte-Justine University Hospital Mother Child Biobank personnel for processing and storing the samples from the studies. Special thanks to Jocelyne Ayotte, Jessie Beauchemin, Vanessa Truong, Annie Bilodeau, Amal Abdi and Guillaume Bourdel for their role in sample preparation. We also want to acknowledge the work of the EnCORE team in the realization of the study. We are grateful for Mélanie Desjardins, Laura Pierce, Adrien Saucier, Katia Charland and Carla Benea who had important roles in the CHOIR and EnCORE studies. Antigens, protein standards, and secondary antibodies for the serum ELISA were kindly provided by The Pandemic Response Challenge Program of the National Research Council of Canada (Dr. Yves Durocher). We would like to thank Tulunay R. Tursun, Martina Tersigni, and the Network Biology Collaborative Centre High-Throughput Screening Facility (RRID: SCR_025390) at the Lunenfeld-Tanenbaum Research Institute for assistance with serum ELISAs. The facility is supported by the Canada Foundation for Innovation (CFI) and the Government of Ontario. Declaration of AI and AI-assisted technologies in the writing process During the preparation of this work the author(s) used ChatGPT 5.1 for language editing. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Data Sharing The datasets generated during the current study are available from the corresponding author on request. Funding/Support The IRYIS study (HD, 2223-HQ-000213) was funded by the COVID-19 Immunity Task Force (CITF) and the Public Health Agency of Canada (PHAC). The CHOIR study was funded by the Canadian Institutes of Health Research (CIHR) (KZ, EG3-179445). The EnCORE study was funded by the Public Health Agency of Canada through the COVID-19 Immunity Task Force (KZ, 2021-HQ-000097). HD and SN are supported by the Fonds de Recherche du Québec – Santé through a Senior Clinical Research Scholar award (HD) and a doctoral research scholarship (SN). CQ is supported through the Canada Research Chair (Tier 1) in Infection Prevention: from hospital to the community (CRC-2019-00055). KZ is supported by the Fonds de Recherche du Québec – Santé through a Junior Researcher Scholar award and the Canada Research Chair (Tier 2) in Global Environmental Change and Infectious Diseases (CRC-2024-00035). MB is supported by the Canada Research Chair and the Sentinel North Research Chair at Université Laval which is funded by the Canada First Research Excellence Fund. The funders had no role in the study design, data collection or analysis, manuscript preparation or the decision to submit for publication. 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Characteristics of children included in the study Number (%) of children with humoral immunodeficiency (n = 27) Number (%) of healthy children (n = 48) Demographics Sex Male 17 (63) 24 (50) Female 10 (37) 24 (50) Age (Y) at vaccination Median [Min, Max] 9 [6, 11] 8 [5, 11] Number of COVID-19 vaccine doses received as primary vaccination regimen Two doses 10 (37) 48 (100) Three doses 17 (63) 0 (0) Time (M) since second vaccine dose at one-month analysis time point (n = 23) (n = 46) 2.2 ± 0.7 1.8 ± 0.7 Time (M) since third vaccine dose at one-month analysis time point (n = 16) 1.4 ± 0.5 N.A. Time (M) since last vaccine dose at six-months analysis time point (n = 24) (n = 38) 7.0 ± 1.0 6.2 ± 0.9 Time (M) since last vaccine dose at one-year analysis time point (n = 18) (n = 30) 13.8 ± 2.7 12.2 ± 1.0 Y : Year, M : Months, n : Number of samples available for analysis at each time point. N.A. : Not applicable Table 2. SARS-CoV-2 infections during the study Number (%) of children with humoral immunodeficiency (n = 27) Number (%) of healthy children (n = 48) SARS-CoV-2 Infection Status No history of infection 2 (7) 6 (12) History of infection 25 (93) 42 (88) Before/at vaccination 20 (74) 24 (50) After vaccination 5 (19) 18 (35) Number of SARS-CoV-2 infections 1 20 (74) 33 (69) 2 5 (19) 9 (19) Symptomatology during infection Symptomatic* 12 (48) 20 (48) Asymptomatic 1 (4) 7 (16) Unreported infection 12 (48) 15 (36) * Symptomatic children had mild disease requiring no hospitalization. Common COVID-19 symptoms included sore throat, cough, runny/stuffy nose, headache. Few participants had fever, intestinal symptoms, loss of smell/taste or fatigue/muscle pain. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8864462","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":598349319,"identity":"10296940-9c33-4982-9b6d-a1a9c69ab415","order_by":0,"name":"Sabryna Nantel","email":"","orcid":"","institution":"Sainte-Justine University Hospital and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Sabryna","middleName":"","lastName":"Nantel","suffix":""},{"id":598349325,"identity":"94556fea-4f05-48a6-9e21-f5a1cdd416c8","order_by":1,"name":"Samuel Sassine","email":"","orcid":"","institution":"Sainte-Justine University Hospital and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Sassine","suffix":""},{"id":598349326,"identity":"b63768dd-a7cd-4aab-88f0-afd775ffff45","order_by":2,"name":"Benoîte Bourdin","email":"","orcid":"","institution":"Sainte-Justine University Hospital and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Benoîte","middleName":"","lastName":"Bourdin","suffix":""},{"id":598349327,"identity":"d9af64eb-4877-4431-898f-fca60ebddd5b","order_by":3,"name":"Margot Barbosa Da Torre","email":"","orcid":"","institution":"Center for Public Health Research, University of Montreal","correspondingAuthor":false,"prefix":"","firstName":"Margot","middleName":"Barbosa Da","lastName":"Torre","suffix":""},{"id":598349328,"identity":"82dde0c6-05c7-4006-b865-075555d854dd","order_by":4,"name":"Gabrielle Sutton","email":"","orcid":"","institution":"Sainte-Justine University Hospital and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Gabrielle","middleName":"","lastName":"Sutton","suffix":""},{"id":598349332,"identity":"6c20d781-fbed-47b6-84e2-b64577c70d60","order_by":5,"name":"Henintsoa Rabezanahary","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Henintsoa","middleName":"","lastName":"Rabezanahary","suffix":""},{"id":598349333,"identity":"2937b92e-69a1-47bc-8af9-b2fee366e53f","order_by":6,"name":"Freda Qi","email":"","orcid":"","institution":"Lunenfeld-Tunenbaum Research Institute at Mount Sinai Hospital, Sinai Health","correspondingAuthor":false,"prefix":"","firstName":"Freda","middleName":"","lastName":"Qi","suffix":""},{"id":598349334,"identity":"1530b43a-2bf0-4093-a58e-ce79f2c9a1c6","order_by":7,"name":"Lesley A. Ward","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Lesley","middleName":"A.","lastName":"Ward","suffix":""},{"id":598349335,"identity":"fe723369-eb78-4237-9e43-da0645761765","order_by":8,"name":"Melanie Delgado-Brand","email":"","orcid":"","institution":"Lunenfeld-Tunenbaum Research Institute at Mount Sinai Hospital, Sinai Health","correspondingAuthor":false,"prefix":"","firstName":"Melanie","middleName":"","lastName":"Delgado-Brand","suffix":""},{"id":598349336,"identity":"e56a2c4f-5eb2-4760-aed5-c0470cf3d8a0","order_by":9,"name":"Kelsey Adams","email":"","orcid":"","institution":"Sainte-Justine University Hospital and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Kelsey","middleName":"","lastName":"Adams","suffix":""},{"id":598349337,"identity":"42b251b1-2510-4811-b0cf-6b32f9248f62","order_by":10,"name":"Salma Sheikh-Mohamed","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Salma","middleName":"","lastName":"Sheikh-Mohamed","suffix":""},{"id":598349338,"identity":"461df4b6-2829-449e-a6a7-f1be727dc71f","order_by":11,"name":"Louise Wang","email":"","orcid":"","institution":"Sainte-Justine University Hospital and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Louise","middleName":"","lastName":"Wang","suffix":""},{"id":598349339,"identity":"9c2d92f5-1958-4bcc-975e-9be7c1cd7151","order_by":12,"name":"Sylvie Nicholson","email":"","orcid":"","institution":"Sainte-Justine University Hospital and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Sylvie","middleName":"","lastName":"Nicholson","suffix":""},{"id":598349340,"identity":"c6cdd148-956c-427e-bae1-068c531d9b2e","order_by":13,"name":"Zineb Laghdir","email":"","orcid":"","institution":"Sainte-Justine University Hospital and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Zineb","middleName":"","lastName":"Laghdir","suffix":""},{"id":598349341,"identity":"9ae317c3-9126-4505-9393-5d38a5021772","order_by":14,"name":"Karen Colwill","email":"","orcid":"","institution":"Lunenfeld-Tunenbaum Research Institute at Mount Sinai Hospital, Sinai Health","correspondingAuthor":false,"prefix":"","firstName":"Karen","middleName":"","lastName":"Colwill","suffix":""},{"id":598349343,"identity":"0c6d70e0-b5f1-4c11-ad47-4bf98f4c309a","order_by":15,"name":"Gary Chao","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Gary","middleName":"","lastName":"Chao","suffix":""},{"id":598349344,"identity":"26891a5a-a2f7-4767-a6b2-e9ee83bdfed9","order_by":16,"name":"Laurie Seifried","email":"","orcid":"","institution":"Lunenfeld-Tunenbaum Research Institute at Mount Sinai Hospital, Sinai Health","correspondingAuthor":false,"prefix":"","firstName":"Laurie","middleName":"","lastName":"Seifried","suffix":""},{"id":598349346,"identity":"f6820667-fae2-443e-8fbd-8f1b2f34fa5d","order_by":17,"name":"Ying Liu","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Liu","suffix":""},{"id":598349348,"identity":"23e1fd2c-14ba-4d57-932e-7b795bc02808","order_by":18,"name":"James M. Rini","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"M.","lastName":"Rini","suffix":""},{"id":598349350,"identity":"cc462752-870c-4c63-951a-51870901c7ec","order_by":19,"name":"Jennifer Gommerman","email":"","orcid":"","institution":"University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Gommerman","suffix":""},{"id":598349351,"identity":"5229b49f-cbb5-4a0a-9d16-834f3d926809","order_by":20,"name":"Anne-Claude Gingras","email":"","orcid":"","institution":"Lunenfeld-Tunenbaum Research Institute at Mount Sinai Hospital, Sinai Health","correspondingAuthor":false,"prefix":"","firstName":"Anne-Claude","middleName":"","lastName":"Gingras","suffix":""},{"id":598349352,"identity":"4233fee4-eedb-4c54-89f3-dcb6fa96bfe0","order_by":21,"name":"Mariana Baz","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"","lastName":"Baz","suffix":""},{"id":598349353,"identity":"2b541350-36fa-4f8f-bf8d-8c426b0a7011","order_by":22,"name":"Kate Zinzser","email":"","orcid":"","institution":"Center for Public Health Research, University of Montreal","correspondingAuthor":false,"prefix":"","firstName":"Kate","middleName":"","lastName":"Zinzser","suffix":""},{"id":598349354,"identity":"756d79e2-2041-4369-a331-48e0dacc2bd4","order_by":23,"name":"Caroline Quach","email":"","orcid":"","institution":"Sainte-Justine University Hospital and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Quach","suffix":""},{"id":598349355,"identity":"38298f69-f037-4f14-b9e7-1c4b73a380dd","order_by":24,"name":"Hélène Decaluwe","email":"data:image/png;base64,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","orcid":"","institution":"Sainte-Justine University Hospital and Research Center","correspondingAuthor":true,"prefix":"","firstName":"Hélène","middleName":"","lastName":"Decaluwe","suffix":""}],"badges":[],"createdAt":"2026-02-12 17:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8864462/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8864462/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104168020,"identity":"8a1984d3-5e68-41b9-8c98-21f5ac1e1e2c","added_by":"auto","created_at":"2026-03-08 14:28:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1014937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTwo mRNA vaccine doses fail to elicit robust humoral immunity in children with B-cell defects, whereas a third vaccine dose restores it\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8864462/v1/bbec9c1fae266e13ff99ebcb.png"},{"id":104404236,"identity":"2f359a8a-35a2-4aa1-a372-30ce3ae2b87c","added_by":"auto","created_at":"2026-03-11 12:19:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":942524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHybrid immunity does not compensate for reduced humoral immunity in twice-vaccinated children with B-cell defects\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8864462/v1/aa2b9db85f00284b763c3a07.png"},{"id":104168017,"identity":"b3ede8eb-af04-498a-be49-8bf70804c724","added_by":"auto","created_at":"2026-03-08 14:28:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2319929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHumoral immunity remains impaired up to one year after vaccination in immunocompromised children, while cellular immunity is preserved\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8864462/v1/e34ad9abd61d127ad175318c.png"},{"id":104404850,"identity":"7093d5b6-ee7d-4592-912a-aa2e7179fb3d","added_by":"auto","created_at":"2026-03-11 12:21:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":962582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced antigen-specific and B-cell counts associate with limited neutralization capacity in immunocompromised children\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8864462/v1/aa82bc2b672828d4eb3fd25c.png"},{"id":104404736,"identity":"15890c55-2a61-4e3f-8def-f0dd5e3f766c","added_by":"auto","created_at":"2026-03-11 12:20:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1416773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSevere B-cell lymphopenia abrogates humoral responses to SARS-CoV-2 vaccination while T-cell responses are sustained\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8864462/v1/4c2e77940ebb2fadc560dc22.png"},{"id":104168021,"identity":"5e831bee-fd68-42e5-a283-7ede3ccb29f0","added_by":"auto","created_at":"2026-03-08 14:28:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":589788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2–specific T-cell responses associate with reduced COVID-19 symptom severity in children with humoral immunodeficiency\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8864462/v1/3b6aaaa33532297557ea6029.png"},{"id":104409394,"identity":"1480d0b4-74f6-430b-9d39-7455490309ca","added_by":"auto","created_at":"2026-03-11 12:45:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9935910,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8864462/v1/ac921f75-e0dd-4af2-9c4c-28b35acae5a2.pdf"},{"id":104168022,"identity":"2822260c-9c0c-4f6f-a4cc-3344d7a21c04","added_by":"auto","created_at":"2026-03-08 14:28:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16264438,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaldataSNNPJV.docx","url":"https://assets-eu.researchsquare.com/files/rs-8864462/v1/7fb7073cb033cb5cf67cefa4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preserved T-Cell Immunity Despite Impaired Humoral Responses following SARS-CoV-2 Infection and Vaccination in Children with Profound B-cell Lymphopenia","fulltext":[{"header":"KEY MESSAGES","content":"\u003cp\u003eOur study demonstrates that a three-dose primary mRNA vaccination regimen is essential to strengthen humoral immunity in children with B-cell defects. While children with severe B-cell lymphopenia fail to generate neutralizing antibodies, they develop robust cell-mediated immune responses that likely protect them from severe infection. These findings highlight the central role of T cells in antiviral protection and underscore the importance of vaccine strategies that efficiently stimulate cellular responses in children with profound B-cell dysfunction.\u003c/p\u003e"},{"header":"CAPSULE SUMMARY","content":"\u003cp\u003eVaccinated children with profound B-cell deficiency lack neutralizing antibodies yet remain protected from severe COVID-19 through robust SARS-CoV-2\u0026ndash;specific T-cell immunity.\u003c/p\u003e "},{"header":"RESEARCH IN CONTEXT","content":"\u003cp\u003e\u003cstrong\u003eEvidence before this study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChildren with humoral immunodeficiencies are considered at high risk for severe SARS-CoV-2 infection, yet data on their immune responses to mRNA vaccination remain limited. Prior studies have shown impaired antibody production in immunocompromised adults, but pediatric cohorts, particularly those with profound B-cell lymphopenia, are underrepresented. Existing reports rarely integrate systemic, mucosal, and functional immunity or \u003cstrong\u003ecombine variant-specific neutralization with high-dimensional cellular immune profiling.\u0026nbsp;\u003c/strong\u003eThus, the relative contribution of cellular immunity to protection in B-cell–deficient children remains poorly defined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdded value of this study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study provides a longitudinal, mechanistically integrated assessment of SARS-CoV-2 vaccine immunity in children with humoral immunodeficiencies. Using high-dimensional flow cytometry, ELISpot, mucosal and serum immune profiling, and microneutralization against ancestral and Omicron BA.5 variants, we show that children with severe B-cell lymphopenia \u003cstrong\u003eexhibit markedly impaired neutralizing antibody responses\u003c/strong\u003e, even after three vaccine doses and \u003cstrong\u003edespite frequent SARS-CoV-2 infection\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003eIn contrast\u003c/strong\u003e\u003c/strong\u003e to this profound humoral defect, they mount robust and polyfunctional SARS-CoV-2–specific CD4⁺ and CD8⁺ T-cell responses, particularly in the setting of asymptomatic infections. These findings reveal a dissociation between humoral failure and preserved cellular immunity in vaccinated, immunocompromised children.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImplications of all the available evidence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results suggest that T-cell responses can serve as complementary or alternative correlates of protection when neutralizing antibodies are absent, highlighting the limitations of antibody-centric measures of vaccine efficacy in children with profound B-cell dysfunction. They support a three-dose primary vaccination regimen for children with humoral immunodeficiencies and highlight the need for vaccine strategies that effectively stimulate cellular immunity in this high-risk population. \u003cstrong\u003eImportantly, our findings support timely SARS-CoV-2 vaccination even in children with profound B-cell lymphopenia\u003c/strong\u003e, as preserved cellular immunity\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003emay contribute to protection from severe disease\u003c/strong\u003e\u003c/strong\u003e. This work informs precision vaccination and underscores the potential importance of T-cell immunity when humoral responses are compromised.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eImmunocompromised (IC) children, whether due to primary immunodeficiencies arising from inborn errors of immunity (IEI) or secondary to immunosuppressive therapies, often exhibit impaired B‑ and/or T‑cell function, leading to compromised adaptive immunity.(1, 2) Consequently, they are \u003cstrong\u003edisproportionately susceptible\u003c/strong\u003e to infections such as SARS‑CoV‑2 and face increased risks of severe disease, complications, and even death compared to healthy children.(3, 4) Different degrees of immunosuppression are associated with distinct and graded clinical outcomes in COVID-19. Individuals who are severely IC have a markedly higher risk of adverse outcomes than those who are moderately IC,(5)\u0026nbsp;while patients with \u003cstrong\u003eisolated antibody deficiencies\u003c/strong\u003e appear to be less vulnerable than those with combined immune defects or immune dysregulation.(6)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVaccination remains the most effective strategy to protect children from COVID-19 hospitalization and severe disease.(7, 8) In healthy pediatric populations, protection against symptomatic COVID-19 correlates with neutralizing antibody titers targeting the SARS-CoV-2 spike protein.(9) However, humoral responses to the standard two-dose regimen are often weak or undetectable in IC children, particularly in those with B-cell defects.(10-13) This has led to recommendations for an \u003cstrong\u003eaugmented three-dose primary series\u003c/strong\u003e in both adult and pediatric IC individuals, although the \u003cstrong\u003edurability and breadth\u003c/strong\u003e of this protection is still not well defined.(14) Importantly, protection against severe COVID-19 does not rely solely on humoral immunity. T-cell responses play a \u003cstrong\u003ecritical and complementary role\u003c/strong\u003e in limiting disease progression, as demonstrated in IC adults.(15, 16) Moreover, vaccine immunogenicity varies depending on the underlying condition or treatment received, underscoring the heterogeneity of this population and the need for \u003cstrong\u003etailored immunization approaches\u003c/strong\u003e.(17, 18)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we conducted a comprehensive, longitudinal evaluation of SARS-CoV-2 immunity in children with primary and secondary antibody deficiencies following two or three doses of the Pfizer–BioNTech mRNA vaccine. Building on evidence that humoral responses are frequently blunted in this population, we hypothesized that, even in the context of profound B-cell lymphopenia, vaccine-induced T-cell immunity may remain intact and could provide meaningful protection against severe COVID-19. To rigorously test this hypothesis, we integrated systemic and mucosal antibody profiling, variant-specific microneutralization, and high-dimensional T-cell analyses, and compared these responses to healthy pediatric controls receiving the standard two-dose regimen. We also assessed whether hybrid immunity enhances protection or compensates for humoral deficits in immunocompromised children.\u0026nbsp;\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eStudy Design and Data Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe “Immune Response in Young ImmunoSuppressed children to COVID-19 vaccination” (IRYIS) study cohort consists of 5-to-11-year-old children with primary or secondary antibody deficiency and healthy children, who were vaccinated following standard public health recommendations in Quebec. In total, 27 children with antibody deficiency were included in the study between Novembre 2021 and July 2022. They were compared to a total of 48 healthy children who were recruited between November 2021 and October 2022 through three different studies: the IRYIS study, the “Children and Older Teens Immune Response to SARS-CoV-2 in Montreal” (CHOIR) study, and the “Children and COVID-19 : Seroprevalence study” (EnCORE), which corresponds to the Montreal cohort of the Pediatric Network Expansion study “Understanding Immune Responses to SARS-CoV-2 Infection and Vaccination in Children Aged from Five to Eleven Years” study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe clinical characteristics of children with antibody deficiency and their diagnosis are reported in\u0026nbsp;\u003cstrong\u003eSupplemental Table S1\u003c/strong\u003e. Children with primary humoral \u0026nbsp; immunodeficiency (n=15/27, 56%) had either a combined immunodeficiency or a predominant antibody deficiency (X-linked agammaglobulinemia, hypogammaglobulinemia or common variable immune deficiency) as defined by the 2019 International Union of Immunological Societies classification, or were patients receiving subcutaneous or intravenous immunoglobulins for recurrent infections, low memory B cells counts (less than 8% of total B cells) and/or dissociated vaccine responses with normal IgA and/or IgM levels (unclassified humoral immunodeficiency).(19) Of the children with secondary humoral immunodeficiency (n=12/27, 44%), ten received anti-CD20 therapy (n=10/12, 83%), a monoclonal antibody known as rituximab less than six months before receiving their first dose of COVID-19 vaccine or within the six months following the initiation of vaccination, with (6/10, 60%) or without (4/10, 40%) steroids or other immunosuppressive therapy. Two of these children (2/12, 17%) had agammaglobulinemia or hypogammaglobulinemia secondary to CAR-T cell therapy and/or hematopoietic stem cell transplantation.\u003c/p\u003e\n\u003cp\u003eParticipants were vaccinated between December 2021 and September 2022 through routine public health programs and following their physicians’ recommendations regarding the number of doses indicated in their situation. Participants all received the Comirnaty COVID-19 vaccine from Pfizer-BioNTech at a dosage of 10 µg per dose, as indicated for children aged from 5 to 11 years and were followed up to one year after vaccination. None of the subjects analyzed received booster vaccination after their recommended primary vaccination regimen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunological data of IC children prior to vaccination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunological parameters of IC children vaccinated with two or three doses of vaccines are reported and compared in\u0026nbsp;\u003cstrong\u003eSupplemental Table S2.\u0026nbsp;\u003c/strong\u003eThe immune characteristics of the subjects with or without severe B-cell lymphopenia are reported in\u0026nbsp;\u003cstrong\u003eSupplementary Table S3\u003c/strong\u003e. Cell lymphopenia was based on the absolute cell count lower that the 5\u003csup\u003eth\u003c/sup\u003e percentile for age.(20)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of SARS-CoV-2 infection status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSARS-CoV-2 infection status was evaluated using complementary clinical and immunological approaches. At each visit, documented SARS-CoV-2 infection confirmed by rapid antigenic testing was assessed, and the presence or absence of COVID-19-associated symptoms was documented. At each time point, immune responses to SARS-CoV-2 nucleocapsid (N) protein were evaluated using serum anti-N IgG titers and IFN-γ\u003csup\u003e+\u003c/sup\u003e T-cell responses to N peptide pools. Children were considered to have been infected with SARS-CoV-2 if they either had a positive COVID-19 test or demonstrated detectable anti-N IgG titer and/or T-cell response to N peptides, even in the absence of reported symptoms. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Collection and Processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSaliva and blood samples were collected from participants at multiple time points: one month after their second and third vaccine doses, as well as at six and twelve months post-vaccination (detailed timepoints are presented in \u003cstrong\u003eTable 1\u003c/strong\u003e). Saliva samples were collected using Salivette system (Sarstedt, Numbrecht, Germany). Blood samples were collected into serum separation tubes (SST\u003csup\u003eTM\u003c/sup\u003e, BD) and acid-citrate-dextrose tubes (ACD, BD). Following collection, saliva, serum, plasma, and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved according to standard operation procedures at the Mother-Child Institutional Biobank at the Sainte-Justine University Hospital and Research Center (CR-CHUSJ) as previously reported by our team.(21-23) PBMCs were stored in liquid nitrogen and all other samples were stored at -80°C. Assays were performed using all available samples for each participant at each visit.\u0026nbsp;However, biological material was sometimes insufficient to perform every assay, resulting in variation in sample numbers across analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(23)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthic Approvals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe IRYIS and the CHOIR/EnCORE protocols were approved by the Research Ethics Board (REB) at the Sainte-Justine University Hospital and Research Center (CR-CHUSJ) under study numbers MP-21-2022-3909 and MP-21-2021-3105, and the CHOIR/EnCORE study was also approved by the University of Montreal REB under study number \u0026nbsp;CERSES-20-103-P. Participants were enrolled after obtaining written informed consent from one of their parents or legal guardians.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell-Mediated Immunity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePBMCs isolated from blood samples were rapidly thawed and rested overnight. Each sample was assessed for viability. The number of assay(s) performed was determined based on the number of cells with adequate viability for each participant at each of their visits.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEnzyme-linked immunospot assay\u0026nbsp;\u003c/em\u003e\u003cem\u003e(ELISpot) for the quantification of functional cellular immunity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eELISpot assay was used to detect interferon-gamma (IFN-γ) or interleukin-2 (IL-2) secreting cells. PBMCs were stimulated for 20 hours with one µg/mL of spike (S) or nucleocapsid (N) mega pools of SARS-CoV-2 peptides from the ancestral strain, as well as the spike from Omicron subvariants BA.4/BA.5 and XBB.1.5 (JPT Peptide Technologies, JPT, Berlin, Germany) as previously described.(21-23) Culture media without peptide (AIM-V® Medium (1X), Thermo Fisher Scientific, Waltham, MA, USA) was used as negative control, and the number of spots detected in the negative well of each sample was subtracted from all stimulated wells of the same sample. CytoStim\u003csup\u003eTM\u003c/sup\u003e (Miltenyi Biotec, MA, USA) was used as positive control. CTL ImmunoSpot® SS UV Analyzer (Cellular Technology Ltd., OH, USA) was used to quantify spots with a detection limit of one secreting cell per 200 000. The positive threshold of 25 spot-forming units per million PBMCs was previously validated on pre-pandemic samples.(21)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eActivation Induced Markers (AIM) assay and circulating T follicular helper cells (cT\u003csub\u003eFH\u003c/sub\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe AIM assay was used to detect activated CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in response to stimulation with ancestral SARS-CoV-2 S peptides. PBMCs were stimulated for 20 hours with one µg/mL of a mega pool of SARS-CoV-2 S peptides (JPT Peptide Technologies, Berlin, Germany). After stimulation, cells were stained with an AIM and cT\u003csub\u003eFH\u003c/sub\u003e (CXCR5\u003csup\u003e+\u003c/sup\u003ePD1\u003csup\u003ehi\u003c/sup\u003e/CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e) panel of fluorescent monoclonal antibodies as presented in \u003cstrong\u003eSupplemental Table\u003c/strong\u003e \u003cstrong\u003eS4\u003c/strong\u003e. Data were acquired using the SONY Spectral Cell Analyzer ID7000. All data analysis was performed with FlowJo software version 10.10.0. Fluorescence minus one (FMO), unstimulated (negative control) and CytoStim\u003csup\u003eTM\u003c/sup\u003e (positive control) were used to set the gates. Results are represented as frequency of activated CD4\u003csup\u003e+\u003c/sup\u003e or CD8\u003csup\u003e+\u003c/sup\u003e T cells following peptide stimulation minus the frequency of activated cells detected in the unstimulated condition for each sample. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eB cell immunophenotyping\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePBMCs were stained directly after overnight resting to assess SARS-CoV-2 S-specific B cells. Miltenyi Biotec SARS-CoV-2 Spike B Cell Analysis Kit for human was used to detect B cells (CD19\u003csup\u003e+\u003c/sup\u003e/CD3\u003csup\u003e-\u003c/sup\u003e) binding to the recombinant SARS-CoV-2 S protein of the ancestral strain. Additional antibodies were added to the Miltenyi Biotec panel to assess for lymphocytes (CD3), plasmablasts (CD27\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e+\u003c/sup\u003e/CD3\u003csup\u003e-\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e) and IgD. The complete panel of fluorescent monoclonal antibodies used is described in\u0026nbsp;\u003cstrong\u003eSupplemental Table S4\u003c/strong\u003e. Data were acquired with the SONY Spectral Cell Analyzer ID7000. All data analysis was performed with FlowJo software version 10.10.0. FMO and unstained samples were used to set the gates. Results are presented as the number of S-specific B cells per million of PBMCs or as frequency of each subpopulation on total B cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMucosal Immunity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEnzyme-linked immunosorbent assay on saliva\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs previously published by our team, enzyme-linked immunosorbent assays (ELISA) were performed to detect IgG and IgA binding to S and its RBD in saliva.(22, 24) Plates were coated with biotinylated S or RBD, incubated overnight and blocked subsequently. Samples were then added to each well for two hours. Horseradish peroxidase (HRP) conjugated IgG and IgA detection antibodies were then added for one hour. After a final wash, 3,3’,5,5’-Tetramethylbenzidine substrate (ThermoFisher) was added for color development. Optical density (OD) was read at a wavelength of OD450 using a Thermo Multiskan FC spectrophotometer. Samples were analyzed as previously described by subtracting the raw OD450 of phosphate-buffered saline (PBS) controls for each sample from their antigen specific OD450 at each dilution.(22, 24) These background-subtracted values were used to calculate the area under the curve (AUC) for each sample, which was then adjusted to a pre-selected standards, which consisted of pooled acute and convalescent COVID-19 patient saliva as described previously.(22, 24) This accounted for inter-plate variability. Detection range and positivity threshold for each antigen are presented in \u003cstrong\u003eSupplemental Table S5\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHumoral Immunity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEnzyme-linked immunosorbent assay on serum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIgG and IgA binding antibody levels against the full-length SARS-CoV-2 S trimer, its RBD and N were measured in serum samples using chemiluminescent ELISA as previously described.(25)\u0026nbsp;Briefly, LUMITRAC 600 high-binding white polystyrene 384-well microplates (Greiner Bio-One, Thermo Fisher Scientific, #78107425) were pre-coated overnight with 10 µL/well of antigen (50 ng of S, 20 ng of RBD, or 7 ng of N), all supplied by the National Research Council of Canada (NRC). \u0026nbsp; Plates were centrifuged at 1000 rpm for one minute to ensure even coating, then incubated overnight at 4\u0026nbsp;°C. \u0026nbsp;The next day, the assay was performed at room temperature with washing twice in 115 µL/well of PBS with Tween® detergent (PBS-T) before each of the following four steps. First, the wells were blocked for one hour in 115 µL/well of 5 % Blocker BLOTTO (ThermoFisher Scientific, #37530). \u0026nbsp; Second, 10 µL of diluted serum in 1 % final Blocker BLOTTO in PBS-T was added and incubated for two hours. \u0026nbsp;Serum samples were diluted 1:160 and 1:2560 to assess anti-N IgG antibody levels, at 1:160, 1:2560 and 1:40960 to assess anti-RBD and anti-S IgG antibody levels, and at 1:160 and 1:2560 to assess IgA antibody levels. \u0026nbsp;Third, 10 µL of an anti-human IgG fused to HRP (IgG#5 by NRC, 0.9 ng/well) or anti-human IgA conjugated to HRP (Jackson ImmunoResearch, #109-035-011, 0.8 ng/well) diluted in 1% final Blocker BLOTTO in PBS-T was added, followed by one-hour incubation. Fourth, 10 µL/well of ELISA Pico Chemiluminescent Substrate (ThermoFisher Scientific, #37069, diluted 1:4 in MilliQ distilled H\u003csub\u003e2\u003c/sub\u003eO) was added, and the plates were centrifuged at 1000 rpm for one minute. After five minutes of incubation, chemiluminescence signals were read on an EnVision 2105 Multimode Plate Reader (Perkin Elmer) plate reader at 100 ms/well using an ultra-sensitive detector. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRaw chemiluminescent values were normalized to a blank-subtracted reference point from the synthetic standard included on each assay plate, to create relative ratios. \u0026nbsp;The synthetic standard used were as follows. For IgG, VHH72-Fc was used for S and RBD (supplied by NRC) and an anti-N IgG for N (clone HC2003, Genscript, #A02039). For IgA, anti-S CR3022 (Absolute Antibody, #Ab01680-16.0) and anti-N CR3018 (Absolute Antibody, #Ab01690-16.0) were used. For IgG and IgA results, relative ratios within the linear range of the assay were further converted to binding antibody units (BAU)/mL using the World Health Organization (WHO) International Standard 20/136, as previously described.(25) Linear ranges for the sample dilutions performed and positivity thresholds are presented in \u003cstrong\u003eSupplemental Table S5\u003c/strong\u003e. \u0026nbsp;Positivity thresholds were determined for the 1:160 dilution using three standard deviations from the mean of control samples (blanks, pre-pandemic negative sera, commercially purified IgG or IgA) as previously described or two standard deviations for anti-N IgG.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicroneutralization assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNeutralizing antibody (NtAb) titers were measured through live SARS-CoV-2 microneutralization assay as previously described by our team.(21, 26) Two strains were used, the ancestral Wuhan-1-like SARS-CoV-2 isolated from a clinical sample in March 2020 in Québec City and the Omicron variant BA.5 provided by the National Microbiology Laboratory (NML), Public Health Agency of Canada. Heat-inactivated serum was diluted by two-fold starting with 1:20 dilution. Serum and virus were mixed at equal volumes and incubated for one hour at room temperature. The residual infectivity of the virus-serum mixture was assessed in quadruplicate wells of African green monkey kidney E6 cell line (Vero ATCC\u003csup\u003e®\u003c/sup\u003e CRL-1586\u003csup\u003eTM\u003c/sup\u003e) for ancestral SARS-CoV-2 and in Vero cells overexpressing transmembrane protease serine 2 (TMPRSS2, JCRB) for Omicron BA.5. NtAb titers were defined as the reciprocal of the serum dilution that completely neutralized the infectivity of the 50% tissue culture infectious dose (TCID\u003csub\u003e50\u003c/sub\u003e) of SARS-CoV-2 multiplied by 100, which was determined by the absence of cytopathic effect on cells after four days.(27) NtAb titers were then calculated using the Reed/Muench method.(28) These microneutralization assays were performed in the Containment Level 3 laboratory at the Centre Hospitalier Universitaire de Québec – Université Laval Research Center. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMann-Whitney or Kruskal-Wallis unpaired nonparametric tests were performed using Prism 9, version 9.2.0 (2021 GraphPad Software, LLC) to assess statistical significance. Simple linear regression is presented with Spearman correlation (R) and P-value (P). Significance was set as *P \u0026lt;.05, **P \u0026lt;.01, ***P \u0026lt;.001 and ****P \u0026lt;.0001. Time since infection is presented as mean\u0026nbsp;±\u0026nbsp;SD, while experimental data are displayed as median [interquartile range, 25\u003csup\u003eth\u003c/sup\u003e – 75\u003csup\u003eth\u003c/sup\u003e percentile].\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eParticipant characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe IRYIS cohort consisted of children with primary or secondary humoral immunodeficiency. There were 27 children with a median age of nine years [6, 11] (63% male) that met the inclusion criteria and had samples suitable for analysis. They were compared to 48 healthy children with a median age of eight years [5,11] (50% male) who were recruited from three studies (IRYIS, CHOIR and EnCORE) (\u003cstrong\u003eTable 1\u003c/strong\u003e). Ten children with humoral immunodeficiency (10/27, 37%) received only two doses of the COVID-19 vaccine as their primary series, while the remaining (17/27, 63%) received the recommended three-dose regimen. Immunologically, both IC subgroups where comparable at the onset of vaccination (\u003cstrong\u003eSupplementary Table S2\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMany children included in the study reported a prior documented SARS-CoV-2 infection or were considered infected based on humoral or cellular anti-N assay results. In children with humoral immunodeficiency, 74% of participants were considered infected by SARS-CoV-2 before or at the time of vaccination compared to 50% of healthy controls (\u003cstrong\u003eTable 2\u003c/strong\u003e). Most children in the humoral immunodeficiency (93%, 25/27) and healthy (88%, 42/48) groups were eventually infected with SARS-CoV-2 during the study and were therefore considered as having hybrid immunity by the end of the study. In both groups, 19% of children were reinfected a second time during the study period. Children who reported symptoms had mild disease requiring no hospitalisation (48 %), while the others had either asymptomatic or unreported infections (52%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTwo mRNA vaccine doses fail to elicit robust humoral immunity in children with B-cell defects, whereas a third vaccine dose restores it\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo clearly delineate the impact of vaccine doses on immune responses in IC children, we divided children in subgroups and first analyzed children without evidence of prior SARS-CoV-2 infection based on diagnostic testing or N-specific responses at inclusion. In this infection-naïve subset, IC children mounted \u003cstrong\u003eT-cell responses comparable to healthy controls\u003c/strong\u003e after two and three doses, as shown by similar frequencies of IFN-γ- and IL-2-secreting T cells (\u003cstrong\u003eFig 1A\u003c/strong\u003e). In contrast, \u003cstrong\u003ehumoral immunity was substantially reduced\u003c/strong\u003e in IC children after two vaccine doses. Salivary anti-S and anti-RBD IgG levels were significantly lower than in healthy children (\u003cstrong\u003eFig 1B\u003c/strong\u003e), while salivary IgA remained uniformly low across groups (\u003cstrong\u003eFig S1A)\u003c/strong\u003e, as expected after intramuscular vaccination(22, 24). Systemically, IC children showed \u003cstrong\u003ereduced serum IgG to S and RBD (Fig 1C)\u003c/strong\u003e, with a tendency towards reduced IgA titers (\u003cstrong\u003eFig S1B\u003c/strong\u003e), whereas a third dose increased titers to values similar to healthy controls. Neutralizing antibody responses showed an even more pronounced deficit in IC children. Only \u003cstrong\u003e40% (4/10)\u003c/strong\u003e of IC children had detectable NtAb after two doses, compared with \u003cstrong\u003e96% (24/25)\u003c/strong\u003e of healthy controls. Following three doses, \u003cstrong\u003eall\u003c/strong\u003eIC children (\u003cstrong\u003e5/5\u003c/strong\u003e) generated detectable NtAb (\u003cstrong\u003eFig 1D\u003c/strong\u003e). Collectively, these findings indicate that two doses are insufficient to elicit protective humoral immunity in children with antibody deficiencies and that a three-dose primary series is required to restore robust binding and neutralizing responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHybrid immunity only partially compensates for impaired humoral immunity in vaccinated children with B-cell defects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause most children were infected with SARS-CoV-2 during the study period, we next evaluated immune responses in those with hybrid immunity. As observed in infection-naïve participants, IC and healthy children mounted \u003cstrong\u003ecomparable functional T-cell responses\u003c/strong\u003e, regardless of health status or number of vaccine doses (\u003cstrong\u003eFig 2A\u003c/strong\u003e). Humoral immunity remained impaired in IC children, despite infection. In saliva, anti-S and anti-RBD IgG levels were significantly lower in IC participants (\u003cstrong\u003eFig 2B\u003c/strong\u003e), whereas IgA responses remained uniformly low in most children and did not differ between groups (\u003cstrong\u003eFig S2A\u003c/strong\u003e). Systemically, IC children who received two doses showed \u003cstrong\u003ereduced serum IgG and IgA to S and RBD\u003c/strong\u003e compared with healthy children (\u003cstrong\u003eFig 2C\u003c/strong\u003e and \u003cstrong\u003eFig S2B\u003c/strong\u003e). A third dose increased IgG to levels comparable to healthy controls, whereas \u003cstrong\u003eIgA responses remained attenuated\u003c/strong\u003e in the IC group. Importantly, hybrid immunity did \u003cstrong\u003enot\u003c/strong\u003e compensate for defective neutralizing responses in IC children who received only two doses (\u003cstrong\u003eFig 2D\u003c/strong\u003e). Despite documented SARS-CoV-2 infection, only \u003cstrong\u003e38% (5/13)\u003c/strong\u003e of IC children had detectable NtAb, with a median titer of \u003cstrong\u003e10 after two doses\u003c/strong\u003e, compared to a titer of \u003cstrong\u003e226\u003c/strong\u003e in healthy children (P = 0.0002). A third vaccine dose increased NtAb titers to \u003cstrong\u003e50\u003c/strong\u003e, yet levels remained significantly lower than in healthy controls (P = 0.0043). Together, these data show that while hybrid immunity enhances overall responses, \u003cstrong\u003eIC children continue to exhibit compromised humoral immunity\u003c/strong\u003e and a limited capacity to generate functional neutralizing antibodies, even after natural infection and three vaccine doses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpaired long-term humoral immunity in immunocompromised children \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next examined the persistence of immunity six and twelve months after vaccination, acknowledging that the high incidence of SARS-CoV-2 infection across the cohort represents a major confounding factor for all long-term immune readouts (\u003cstrong\u003eFig 3A\u003c/strong\u003e). By six months, most children, IC and healthy, had already acquired hybrid immunity, while by 12 months all except one healthy control had been infected. This implies that responses at later time points likely reflect the combined effects of vaccination and infection rather than vaccine-derived immunity alone. Within this context, T-cell responses in IC children appeared stable up to twelve months after vaccination, independently of the number of vaccine dose received, and remained reactive to the ancestral Spike and its Omicron BA.4/BA.5 and XBB.1.5 variants, secreting both IFN-γ and IL-2 (\u003cstrong\u003eFig 3B\u003c/strong\u003e and \u003cstrong\u003eS3A\u003c/strong\u003e). Consistent with this, activation of S-specific CD4⁺ and CD8⁺ T cells remained comparable between groups (\u003cstrong\u003eFig S3B\u003c/strong\u003e). These findings indicate that children with primary or secondary B-cell defects can maintain a functional memory T-cell pool over extended periods.\u003c/p\u003e\n\u003cp\u003eIn contrast, despite numerous infections, we observed persistently attenuated humoral responses after two vaccine doses in IC children compared to healthy controls. This was particularly evident from the markedly lower IgG and IgA levels to S and RBD at six months (\u003cstrong\u003eFig 3C\u003c/strong\u003e and\u003cstrong\u003e\u0026nbsp;S3C\u003c/strong\u003e), the substantially reduced pool of Spike-specific B cells (\u003cstrong\u003eFig 3D\u003c/strong\u003e), and the near absence of neutralizing antibodies to the ancestral strain at this time point (\u003cstrong\u003eFig 3E\u003c/strong\u003e). A third vaccine dose partially restored these humoral responses at both mucosal and systemic sites, increasing salivary IgG concentrations (\u003cstrong\u003eFig S3D\u003c/strong\u003e), serum S- and RBD-specific IgG and IgA levels and neutralizing capacity against the ancestral and Omicron BA.5 strains (\u003cstrong\u003eFig 3E\u003c/strong\u003e) toward levels observed in healthy children. Yet, S-specific B-cell frequencies remained lower in IC children at 6 months, and serum IgA responses declined by twelve months compared to healthy controls. Together, these findings demonstrate that, even in the setting of widespread hybrid immunity, \u003cstrong\u003eIC children, especially if vaccinated only twice, fail to generate durable and fully functional humoral immunity\u003c/strong\u003e, underscoring a persistent defect in their capacity to mount and maintain protective antibody responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpaired Memory and Antigen-Specific B-Cell Responses Underlie Defective Neutralization in IC Children\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the mechanisms underlying the profoundly impaired humoral responses observed in IC children, we performed extensive high-dimensional flow cytometry to phenotype and enumerate total and S-specific B cells, CD27⁺ memory B cells, plasmablasts and follicular helper T cells (Tfh), f\u003cstrong\u003eocusing on the 1-month post-vaccination time point to capture peak vaccine-induced responses while avoiding bias from repeated sampling across time\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003eAt this time point\u003c/strong\u003e\u003c/strong\u003e, IC children exhibited markedly diminished frequency of S-specific B cells and CD27⁺ memory B cells compared with healthy controls (\u003cstrong\u003eFig 4A\u003c/strong\u003e), while total B-cell counts were unexpectedly comparable, and plasmablasts and Tfh frequencies were broadly equivalent between groups (\u003cstrong\u003eFig S4A\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine which immune abnormalities best predicted defective antibody responses, we next assessed correlations between NtAb levels and multiple cellular parameters. Across the entire cohort, total B cell counts, and the frequency of memory and spike-specific B cells showed a \u003cstrong\u003emoderate but statistically significant\u003c/strong\u003e positive correlation with NtAb titers (Spearman R ≈ 0.40–0.44, p ≈ 0.005-0.010, n = 36, \u003cstrong\u003eFig 4B\u003c/strong\u003e), while plasmablast and Tfh frequencies did not correlate with neutralization capacity (\u003cstrong\u003eFig S4B\u003c/strong\u003e). \u003cstrong\u003eImportantly, when restricting the analysis to IC children only, these associations were further strengthened (Spearman R ≈ 0.61–0.66, p ≈ 0.006–0.013), despite the smaller sample size (n = 16, Fig 4C), indicating that both the magnitude of the B-cell compartment and the ability to generate antigen-specific B cells are closely linked to functional neutralization in this subgroup.\u0026nbsp;\u003c/strong\u003eTogether, these analyses indicate that both the overall size of the B-cell compartment and the ability to generate antigen-specific B cells, rather than defects in Tfh support or plasmablast differentiation, are closely linked with impaired neutralizing antibody responses in IC children.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChildren with severe B-cell lymphopenia show particularly impaired immune responses to SARS-CoV-2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause total B-cell counts correlated with neutralizing capacity at the time point closest to vaccination, we next examined whether IC children with the most severely impaired humoral responses were those with the lowest circulating B-cell numbers at the time of vaccination. IC participants were stratified according to the presence (n=12) or absence (n=15) of profound B-cell lymphopenia, defined as a CD19⁺ lymphocyte count below the 5\u003csup\u003eth\u003c/sup\u003e percentile for age, while other immune parameters were comparable between IC groups, except for IgM levels (\u003cstrong\u003eSupplementary Table S3\u003c/strong\u003e). We then compared SARS-CoV-2–specific immunity across severely B-cell–lymphopenic children, B-cell–sufficient IC children, and healthy controls.\u003c/p\u003e\n\u003cp\u003eChildren with severe B-cell lymphopenia exhibited \u003cstrong\u003emarkedly diminished humoral immunity\u003c/strong\u003e across all compartments. Serum IgG and IgA levels to S and RBD were profoundly reduced (\u003cstrong\u003eFig 5A\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;5SA\u003c/strong\u003e), while salivary IgG responses to S and RBD were substantially low (\u003cstrong\u003eFig S5B\u003c/strong\u003e). While B-cell–sufficient IC children generated frequencies of S-specific B cells comparable to healthy controls, those with severe B-cell lymphopenia showed \u003cstrong\u003edistinctly reduced induction of antigen-specific B cells\u003c/strong\u003e (\u003cstrong\u003eFig 5B\u003c/strong\u003e). Consistent with these deficits, neutralizing antibody titers against the ancestral strain were extremely low or undetectable in this subgroup, in contrast to both healthy children and B-cell–sufficient IC peers (\u003cstrong\u003eFig 5C\u003c/strong\u003e). Furthermore, despite documented infection, children with severe B-cell lymphopenia \u003cstrong\u003efailed to develop neutralizing activity against BA.5\u003c/strong\u003e, underscoring an intrinsic inability to mount adequate humoral immunity in the setting of profound B-cell deficiency. In contrast, children with severe B-cell lymphopenia exhibited heightened functional T-cell responses, measured by IFN-γ secretion in response to SARS-CoV-2 spike, particularly one month after vaccination (\u003cstrong\u003eFig 5D\u003c/strong\u003e). Together, these results demonstrate that profound B-cell lymphopenia is the dominant determinant of impaired humoral immunity to SARS-CoV-2 in IC children, whereas preserved and potentially enhanced T-cell responses likely provide compensatory protection in the absence of functional B-cell immunity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtection against COVID-19 symptoms is associated to potent T cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVaccine efficacy is often reported in terms of its capacity to prevent severe infection and hospitalization. Although this small cohort of high-risk patients does not allow for proper statistical analysis of vaccine efficacy, we observed that although most IC children were infected during the study period (25/27), none reported severe disease nor were hospitalized (\u003cstrong\u003eTable 2\u003c/strong\u003e). Indeed, children were either mildly symptomatic or were found to be asymptomatic. As T-cell responses have been shown to restrict COVID-19 symptoms,(15) we questioned if they played the same role in our IC pediatric cohort. We thus compared T-cell responses in children with or without symptoms, in both healthy and IC children. Interestingly, IC children that lacked symptoms had higher levels of IFN-g\u0026nbsp;secreting cells in response to both the ancestral S peptides and the Omicron BA.4/BA.5 S peptides compared to IC children who had symptoms, while this difference was not present in healthy controls (\u003cstrong\u003eFig 6A\u003c/strong\u003e). In contrast, no difference in NtAb levels to the ancestral spike or the Omicron BA.5 variant was noted based on symptomatology of the infection (\u003cstrong\u003eFigure 6B\u003c/strong\u003e). These findings suggest that in the absence of robust humoral immunity, potent S-specific functional T-cell responses may play a key role in limiting COVID-19 symptom severity in children with humoral immunodeficiency.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we provide a comprehensive analysis of adaptive immune responses to SARS-CoV-2 vaccination and infection in children with primary or secondary humoral immunodeficiencies. Our data reveal a clear dissociation between humoral and cellular immune responses in this high-risk pediatric population. While antibody responses are variably impaired depending on B-cell status, functional SARS-CoV-2–specific T-cell immunity is preserved and maintained through time across immunocompromised subgroups and is associated with protection from symptomatic COVID-19. These findings challenge the reliance on serum IgG levels and neutralizing antibody titers as universal correlates of protection and highlight T-cell immunity as an important determinant of clinical outcome in children with humoral immune defects.\u003c/p\u003e\n\u003cp\u003eWe show that a standard two-dose mRNA vaccination regimen is insufficient to induce robust and long-lasting humoral immunity in children with humoral immunodeficiency, regardless of prior or recent SARS-CoV-2 infection. In contrast, a three-dose primary series substantially increases binding and neutralizing antibody responses in immunocompromised children, reaching levels comparable to healthy controls when the B-cell compartment is preserved. This demonstrates that multiple vaccine doses are required to support B-cell differentiation in children with intrinsic or secondary B-cell defects.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImportantly, despite recurrent vaccination and infection, children with severe B-cell lymphopenia remain unable to mount effective IgG and IgA antibody responses, both systemically and at mucosal infection sites. This aligns with immunocompromised adult studies showing stronger immunity after three doses, except in adults on anti-CD20 therapy.(29, 30) It clearly articulates that lack of B cells directly predict poor antibody responses and neutralization capacity. These findings collectively indicate that B-cell availability imposes a biological ceiling on vaccine-induced humoral immunity that cannot be overcome by additional antigen exposure.\u003c/p\u003e\n\u003cp\u003eBy integrating high-dimensional immune phenotyping with functional antibody analyses, we identify the cellular determinants underlying defective humoral immunity in IC children. Neutralizing antibody capacity correlated with total B-cell counts and with the frequency of memory B cells and SARS-CoV-2–specific B cells, but not with Tfh frequency or plasmablast abundance. These findings indicate that impaired neutralization results from intrinsic limitations in the generation and maintenance of antigen-specific B cells rather than from defective T-cell help or transient plasmablast responses. This mechanistic insight explains the heterogeneity in vaccine responsiveness observed among immunocompromised children and highlights B cell counts as an important biomarker and predictor of the quality of humoral responses after vaccination.\u003c/p\u003e\n\u003cp\u003eIn contrast to humoral immunity, SARS-CoV-2–specific T-cell responses to vaccination were induced and maintained up to 1-year after vaccination across IC children, including those with severe B-cell lymphopenia. These responses were functional, polyvariant, and characterized by IFN-γ and IL-2 secretion in response to ancestral and Omicron BA.4/BA.5 and XBB1.5 Spike antigens. Importantly, stronger functional T-cell responses were associated with asymptomatic or minimally symptomatic infection in children with humoral immunodeficiency, a relationship not observed in healthy controls. These results align with studies showing that strong T-cell responses correlate with milder disease and faster recovery in both healthy children and immunocompromised adults, particularly when neutralizing antibodies are low or absent.(30-33)\u003c/p\u003e\n\u003cp\u003eRemarkably, none of our immunocompromised participants required hospitalization for COVID‑19, despite high infection rates (93 %) and humoral impairments. This is in contrast with adult cohort studies where antibody deficiencies, in association with comorbid risk factors, were associated with increased risk of death in patients over 40-years old especially.(34-37) Interestingly, children who remained asymptomatic showed significantly stronger IFN‑γ-secreting T‑cell responses to both ancestral and Omicron S peptides than those who developed symptoms. These heightened T-cell responses were also sustained in children with profound B-cell lymphopenia, likely contributing to the absence of severe disease in this subgroup despite high infection rates. Similar heightened T-cell responses have been described in adults treated with anti-CD20 antibodies, although its relationship with symptom severity was not described in these studies.(38, 39) Collectively, our observations support a model in which cellular immunity can independently mediate clinical protection when humoral immunity is compromised.\u003c/p\u003e\n\u003cp\u003eThese findings have direct implications for the clinical management of children with humoral immunodeficiencies. First, they support prompt vaccination with an augmented primary series, even in children with profound B-cell defects or undergoing treatment with B-cell depleting agents, as vaccination reliably induces functional T-cell immunity. Second, they argue against using serum and neutralizing antibody titers as the sole marker of vaccine efficacy or protection in this population. Instead, immune stratification based on B-cell immunophenotyping may help identify children who require additional interventions, such as closer monitoring, monoclonal antibody prophylaxis, or additional alternative vaccine strategies, including mucosal boosters. Importantly, our data suggest that vaccination should not be delayed based on the timing since B-cell–depleting therapies, as cellular immunity remains inducible and clinically relevant.\u003c/p\u003e\n\u003cp\u003eThis study has several limitations. The high incidence of infection during follow-up limited our ability to isolate vaccine-induced immunity, particularly for long-term analyses. Blood volume constraints restricted immune phenotyping to subsets of participants, and incomplete sampling at some time points reduced statistical power for certain analyses. Additionally, infection timing was often inferred retrospectively using anti-nucleocapsid responses, limiting precision in assessing immune kinetics. Future studies with prospective infection surveillance and standardized longitudinal sampling will be essential to refine correlates of protection in this population.\u003c/p\u003e\n\u003cp\u003eIn summary, our findings demonstrate that immune protection against COVID-19 in children with humoral immunodeficiency is mediated through distinct and dissociable pathways. While a three-dose mRNA vaccination regimen enables most children with preserved B-cell compartments to mount effective humoral responses, those with severe B-cell lymphopenia remain intrinsically unable to do so. In this context, preserved SARS-CoV-2–specific T-cell immunity emerges as an important correlate of protection, associated with mild or asymptomatic infection despite absent neutralizing antibodies. These results underscore the need to redefine correlates of protection and vaccination strategies for immunocompromised pediatric populations, with a renewed focus on inducing durable and functional cellular immunity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcid-citrate-dextrose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAIM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eActivation induced markers\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArea under the curve\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBAU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBinding antibody units\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAR-T cell\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChimeric antigen receptor T cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCHOIR study\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChildren and Older Teens Immune Response to SARS-CoV-2 in Montreal study\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOVID-19\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCoronavirus Disease 2019\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCR-CHUSJ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSainte-Justine University Hospital and Research Center\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-linked immunosorbent Assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISpot\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-linked immunospot\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEnCORE study\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChildren and COVID-19 seroprevalence study\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFMO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluorescence minus one\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHealthy controls\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHorseradish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunocompromised\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIEI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInborn errors of immunity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIFN-γ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterferon-gamma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIRYIS study\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmune Response in Young Immunosuppressed children to COVID-19 vaccination study\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMessenger ribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNucleocapsid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNRC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNational Research Council of Canada\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBMCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeripheral blood mononuclear cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRBD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReceptor binding domain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSARS-CoV-2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSevere Acute Respiratory Syndrome Coronavirus 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSpike\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSerum separation tubes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCID\u003csub\u003e50\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e50% tissue culture infectious dose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWHO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWorld Health Organization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization : HD\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethodology : SN, BB, HR, FQ, MDB, LS, LWard, SSM, GC, KC, YL, JMR, JG, ACG, MB, CQ, HD\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInvestigation : HD\u003c/p\u003e\n\u003cp\u003eVisualization : SN, BB\u003c/p\u003e\n\u003cp\u003eFunding acquisition : HD, KZ\u003c/p\u003e\n\u003cp\u003eProject administration : BB, MBDT, LWang, SNi, KA, ZL, LS\u003c/p\u003e\n\u003cp\u003eSubject recruitment : HD, KZ, CQ\u003c/p\u003e\n\u003cp\u003eSupervision : HD\u003c/p\u003e\n\u003cp\u003eWriting – original draft : SN, SS, BB, HD\u003c/p\u003e\n\u003cp\u003eWriting – review \u0026amp; editing : SN, SS, BB, GS, FQ, LWard, KC, MB, CQ, HD\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe warmly thank all the participants and their parents/guardians who accepted to participate in the IRYIS, CHOIR and EnCORE studies. We also thank the nurses and research coordinators from each participating centers. We want to recognize the work of the Sainte-Justine University Hospital Mother Child Biobank personnel for processing and storing the samples from the studies. Special thanks to Jocelyne Ayotte, Jessie Beauchemin, Vanessa Truong, Annie Bilodeau, Amal Abdi and Guillaume Bourdel for their role in sample preparation. We also want to acknowledge the work of the EnCORE team in the realization of the study. We are grateful for Mélanie Desjardins, Laura Pierce, Adrien Saucier, Katia Charland and Carla Benea who had important roles in the CHOIR and EnCORE studies.\u0026nbsp;Antigens, protein standards, and secondary antibodies for the serum ELISA were kindly provided by The Pandemic Response Challenge Program of the National Research Council of Canada (Dr. Yves Durocher). \u0026nbsp;We would like to thank Tulunay R. Tursun, Martina Tersigni, and the Network Biology Collaborative Centre High-Throughput Screening Facility (RRID: SCR_025390) at the Lunenfeld-Tanenbaum Research Institute for assistance with serum ELISAs. The facility is supported by the Canada Foundation for Innovation (CFI) and the Government of Ontario.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;During the preparation of this work the author(s) used ChatGPT 5.1 for language editing. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Sharing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available from the corresponding author on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding/Support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe IRYIS study (HD, 2223-HQ-000213) was funded by the COVID-19 Immunity Task Force (CITF) and the Public Health Agency of Canada (PHAC). The CHOIR study was funded by the Canadian Institutes of Health Research (CIHR) (KZ, EG3-179445). The EnCORE study was funded by the Public Health Agency of Canada through the COVID-19 Immunity Task Force (KZ, 2021-HQ-000097).\u0026nbsp;HD and SN are supported by the Fonds de Recherche du Québec – Santé through a Senior Clinical Research Scholar award (HD) and a doctoral research scholarship (SN). CQ is supported through the Canada Research Chair (Tier 1) in Infection Prevention: from hospital to the community (CRC-2019-00055). KZ is supported by the Fonds de Recherche du Québec – Santé through a Junior Researcher Scholar award and the Canada Research Chair (Tier 2) in Global Environmental Change and Infectious Diseases (CRC-2024-00035). MB is supported by the Canada Research Chair and the Sentinel North Research Chair at Université Laval which is funded by the Canada First Research Excellence Fund.\u0026nbsp;The funders had no role in the study design, data collection or analysis, manuscript preparation or the decision to submit for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang JJF, Dhir A, Hildebrand KJ, Turvey SE, Schellenberg R, Chen LYC, et al. Inborn errors of immunity in adulthood. Allergy Asthma Clin Immunol. 2024;20(1):6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePourshahnazari P, Betschel SD, Kim VHD, Waserman S, Zhu R, Kim H. Secondary Immunodeficiency. Allergy Asthma Clin Immunol. 2025;20(Suppl 3):80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConnelly JA, Chong H, Esbenshade AJ, Frame D, Failing C, Secord E, et al. Impact of COVID-19 on Pediatric Immunocompromised Patients. 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American Journal of Epidemiology. 1938;27:493\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee A, Wong SY, Chai LYA, Lee SC, Lee MX, Muthiah MD, et al. Efficacy of covid-19 vaccines in immunocompromised patients: systematic review and meta-analysis. Bmj. 2022;376:e068632.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eApostolidis SA, Kakara M, Painter MM, Goel RR, Mathew D, Lenzi K, et al. Cellular and humoral immune responses following SARS-CoV-2 mRNA vaccination in patients with multiple sclerosis on anti-CD20 therapy. Nat Med. 2021;27(11):1990\u0026ndash;2001.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong Y, Kang AYH, Tay CJX, Li HE, Elyana N, Tan CW, et al. Correlates of protection against symptomatic SARS-CoV-2 in vaccinated children. Nat Med. 2024;30(5):1373\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMizera D, Dziedzic R, Drynda A, Gradzikiewicz A, Jakieła B, Celińska-L\u0026ouml;wenhoff M, et al. Cellular immune response to SARS-CoV-2 in patients with primary antibody deficiencies. Frontiers in immunology. 2023;14:1275892.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBange EM, Han NA, Wileyto P, Kim JY, Gouma S, Robinson J, et al. CD8(+) T cells contribute to survival in patients with COVID-19 and hematologic cancer. Nat Med. 2021;27(7):1280\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBucciol G, Tangye SG, Meyts I. Coronavirus disease 2019 in patients with inborn errors of immunity: lessons learned. Curr Opin Pediatr. 2021;33(6):648\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLindahl H, Kahn F, Nilsdotter-Augustinsson \u0026Aring;, Fredrikson M, Hedberg P, Killander M\u0026ouml;ller I, et al. Inborn errors of immunity are associated with increased COVID-19-related hospitalization and intensive care compared to the general population. The Journal of allergy and clinical immunology. 2025;155(2):387\u0026thinsp;\u0026ndash;\u0026thinsp;97.e6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeyts I, Bucciol G, Quinti I, Neven B, Fischer A, Seoane E, et al. Coronavirus disease 2019 in patients with inborn errors of immunity: An international study. The Journal of allergy and clinical immunology. 2021;147(2):520\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSonmez G, Gunduz G, Esenboga S, Cagdas D. Lessons From COVID-19 on Inborn Errors of Immunity: A Five-Year Narrative Review. Scand J Immunol. 2025;102(5):e70064.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadelon N, Lauper K, Breville G, Sabater Royo I, Goldstein R, Andrey DO, et al. Robust T-Cell Responses in Anti-CD20-Treated Patients Following COVID-19 Vaccination: A Prospective Cohort Study. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2022;75(1):e1037-e45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiise J, Meyer S, Blaas I, Chopra A, Tran TT, Delic-Sarac M, et al. Rituximab-treated patients with lymphoma develop strong CD8 T-cell responses following COVID-19 vaccination. British journal of haematology. 2022;197(6):697\u0026ndash;708.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Characteristics of children included in the study\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"649\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNumber (%) of children with humoral immunodeficiency\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 27)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNumber (%) of\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehealthy children \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 48)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDemographics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Sex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Male\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e17 (63)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e24 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10 (37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e24 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Age (Y) at vaccination\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Median [Min, Max]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e9 [6, 11]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e8 [5, 11]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of COVID-19 vaccine doses received as primary vaccination regimen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Two doses\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10 (37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e48 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Three doses\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e17 (63)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime (M) since second vaccine dose at one-month analysis time point\u0026nbsp;\u003c/strong\u003e(n = 23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;(n = 46)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2.2 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.8 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime (M) since third vaccine dose at one-month analysis time point\u0026nbsp;\u003c/strong\u003e(n = 16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.4 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN.A.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime (M) since last vaccine dose at six-months analysis time point\u0026nbsp;\u003c/strong\u003e(n = 24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;(n = 38)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e7.0 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e6.2 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime (M) since last vaccine dose at one-year analysis time point\u0026nbsp;\u003c/strong\u003e(n = 18)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;(n = 30)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e13.8 \u0026plusmn; 2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12.2 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003eY : Year, M : Months, n : Number of samples available for analysis at each time point.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003eN.A. : Not applicable\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. SARS-CoV-2 infections during the study\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNumber (%) of children with humoral immunodeficiency\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 27)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNumber (%) of healthy children\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 48)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSARS-CoV-2 Infection Status\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;No history of infection\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e6 (12)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;History of infection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e25 (93)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e42 (88)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Before/at vaccination\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e20 (74)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e24 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;After vaccination\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e5 (19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e18 (35)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of SARS-CoV-2 infections\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e20 (74)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e33 (69)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e5 (19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e9 (19)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSymptomatology during infection\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Symptomatic*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12 (48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e20 (48)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Asymptomatic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e7 (16)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Unreported infection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12 (48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e15 (36)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\"\u003e\n \u003cp\u003e* Symptomatic children had mild disease requiring no hospitalization. Common COVID-19 symptoms included sore throat, cough, runny/stuffy nose, headache. Few participants had fever, intestinal symptoms, loss of smell/taste or fatigue/muscle pain.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Covid-19, inborn errors of immunity, B-cell defects, rituximab, cell-mediated immune response ","lastPublishedDoi":"10.21203/rs.3.rs-8864462/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8864462/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding the mechanisms of vaccine-induced protection in children with humoral immunodeficiency is essential to guide prevention strategies and reduce COVID-19-related complications and mortality. Yet, comprehensive cellular, humoral and mucosal analyses are scarce in this high-risk population. We conducted a longitudinal evaluation of SARS-CoV-2 immunity at 1, 6 and 12 months after a primary series of the Pfizer-BioNTech mRNA vaccine (10 \u0026micro;g dose) in 27 children aged 5\u0026ndash;11 years with primary or secondary antibody deficiencies and 48 age- and sex-matched healthy controls. Functional T-cell responses were quantified by interferon-gamma (IFN-γ) and IL-2 ELISpot, and SARS-CoV-2-specific B cells, CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T-cell subsets were assessed using high-dimensional spectral cytometry. Systemic and mucosal antibody responses to spike (S) and receptor binding domain (RBD) were measured in serum and saliva, and neutralizing activity against ancestral and Omicron BA.5 strains variants was evaluated through microneutralization. Children with humoral immunodeficiency exhibited markedly impaired systemic antibody responses following two mRNA doses, despite SARS-CoV-2 infection, with restoration after a third vaccine dose. Those with severe B-cell lymphopenia were unable to mount neutralizing antibodies even after three doses and despite infection. Notwithstanding this profound humoral defect, they developed preserved, polyfunctional SARS-CoV-2-specific T-cell responses across multiple variants, which likely protected them from severe COVID-19. T-cell responses were higher in asymptomatic immunocompromised children, while all symptomatic infections were mild, supporting a potential contribution of cellular immunity to disease control in this population. These findings reveal a clear dissociation between humoral failure and preserved cellular immunity in B-cell\u0026ndash;deficient children. They indicate that T-cell responses can act as alternate correlate of protection when neutralizing antibodies are absent, supporting timely vaccination in pediatric populations with profound B-cell deficiency.\u003c/p\u003e","manuscriptTitle":"Preserved T-Cell Immunity Despite Impaired Humoral Responses following SARS-CoV-2 Infection and Vaccination in Children with Profound B-cell Lymphopenia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 14:28:25","doi":"10.21203/rs.3.rs-8864462/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-20T14:40:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T11:21:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T11:57:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T10:00:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-10T15:17:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179545399451376055932166174650864399384","date":"2026-03-04T09:23:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-04T05:55:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86413121714417037120534256961429790617","date":"2026-03-04T00:50:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167121096965049883305859079059978899624","date":"2026-03-03T08:52:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102692340099578741561518332173008242384","date":"2026-02-27T17:25:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326756757621327728317129846966702173455","date":"2026-02-26T17:11:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272787516942624956026679242883794989148","date":"2026-02-26T00:26:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-25T16:20:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-25T16:11:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-17T04:30:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2026-02-12T16:56:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b01eb052-26e9-4e40-adde-1117f19f7c25","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63678096,"name":"Health sciences/Diseases"},{"id":63678097,"name":"Biological sciences/Immunology"},{"id":63678098,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-11T17:53:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 14:28:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8864462","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8864462","identity":"rs-8864462","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}