Severe Combined Immunodeficiency Patients Research Articles

PAX1 represses canonical Wnt signaling pathway and plays dual roles during endoderm differentiation

BackgroundPaired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing.MethodsThe repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1’s role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation.ResultsHere, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling.ConclusionsOur study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

Relevance of lymphocyte proliferation to PHA in severe combined immunodeficiency (SCID) and T cell lymphopenia

Severe combined immunodeficiency (SCID) is characterized by a severe deficiency in T cell numbers. We analyzed data collected (n = 307) for PHA-based T cell proliferation from the PIDTC SCID protocol 6901, using either a radioactive or flow cytometry method. In comparing the two groups, a smaller number of the patients tested by flow cytometry had <10% of the lower limit of normal proliferation as compared to the radioactive method (p = 0.02). Further, in patients with CD3+ T cell counts between 51 and 300 cells/μL, there was a higher proliferative response with the PHA flow assay compared to the 3H-T assay (p < 0.0001), suggesting that the method of analysis influences the resolution and interpretation of PHA results. Importantly, we observed many SCID patients with profound T cell lymphopenia having normal T cell proliferation when assessed by flow cytometry. We recommend this test be considered only as supportive in the diagnosis of typical SCID.

Heterogeneity in RAG1 and RAG2 deficiency: 35 cases from a single-centre.

Recombination activating genes (RAG)1 and RAG2 deficiency leads to combined T/B-cell deficiency with varying clinical presentations. This study aimed to define the clinical/laboratory spectrum of RAG1 and RAG2 deficiency. We retrospectively reviewed the clinical/laboratory data of 35 patients, grouped them as severe combined immunodeficiency (SCID), Omenn syndrome (OS), and delayed-onset combined immunodeficiency (CID) and reported nine novel mutations. The male/female ratio was 23/12. Median age of clinical manifestations was 1 months (mo) (0.5-2), 2 mo (1.25-5), and 14 mo (3.63-27), age at diagnosis was 4 mo (3-6), 4.5 mo (2.5-9.75), and 27 mo (14.5-70) in SCID (n = 25; 71.4%), OS (n = 5; 14.3%), and CID (n = 5; 14.3%) patients, respectively. Common clinical manifestations were recurrent sinopulmonary infections 82.9%, oral moniliasis 62.9%, diarrhea 51.4%, and eczema/dermatitis 42.9%. Autoimmune features were present in 31.4% of the patients; 80% were in CID patients. Lymphopenia was present in 92% of SCID, 80% of OS, and 80% of CID patients. All SCID and CID patients had low T (CD3, CD4, and CD8), low B, and increased NK cell numbers. Twenty-eight patients underwent hematopoietic stem cell transplantation (HSCT), whereas seven patients died before HSCT. Median age at HSCT was 7 mo (4-13.5). Survival differed in groups; maximum in SCID patients who had an HLA-matched family donor, minimum in OS. Totally 19 (54.3%) patients survived. Early molecular genetic studies will give both individualized therapy options, and a survival advantage because of timely diagnosis and treatment. Further improvement in therapeutic outcomes will be possible if clinicians gain time for HSCT.

Severe combined immunodeficiency diagnosis and genetic defects.

Severe combined immunodeficiency (SCID) is a rare and life-threatening genetic disorder that severely impairs the immune system's ability to defend the body against infections. Often referred to as the "bubble boy" disease, SCID gained widespread recognition due to the case of David Vetter, a young boy who lived in a sterile plastic bubble to protect him from germs. SCID is typically present at birth, and it results from genetic mutations that affect the development and function of immune cells, particularly T cells and B cells. These immune cells are essential for identifying and fighting off infections caused by viruses, bacteria, and fungi. In SCID patients, the immune system is virtually non-existent, leaving them highly susceptible to recurrent, severe infections. There are several forms of SCID, with varying degrees of severity, but all share common features. Newborns with SCID often exhibit symptoms such as chronic diarrhea, thrush, skin rashes, and persistent infections that do not respond to standard treatments. Without prompt diagnosis and intervention, SCID can lead to life-threatening complications and a high risk of mortality. There are over 20 possible affected genes. Treatment options for SCID primarily involve immune reconstitution, with the most well-known approach being hematopoietic stem cell transplantation (HSCT). Alternatively, gene therapy is also available for some forms of SCID. Once treated successfully, SCID patients can lead relatively normal lives, but they may still require vigilant infection control measures and lifelong medical follow-up to manage potential complications. In conclusion, severe combined immunodeficiency is a rare but life-threatening genetic disorder that severely compromises the immune system's function, rendering affected individuals highly vulnerable to infections. Early diagnosis and appropriate treatment are fundamental. With this respect, newborn screening is progressively and dramatically improving the prognosis of SCID.

Reducing Mortality and Morbidity in Children with Severe Combined Immunodeficiency in Switzerland: the Role of Newborn Screening.

Newborn screening (NBS) for severe combined immunodeficiency (SCID) has been introduced in various countries with the aim of reducing morbidity and mortality. However, studies analyzing outcomes before and after the implementation of NBS programs remain limited. This study sought to compare the outcomes of SCID patients identified through Switzerland's national SCID NBS program, introduced in January 2019, with those of a historical cohort diagnosed between 2007 and 2019. The study included seven patients (32%) identified through NBS, and 15 (68%) born before NBS implementation and diagnosed based on clinical signs. Children in the NBS group were younger at diagnosis (median age 9days vs 9months, P = .002) and at hematopoietic stem cell transplantation (HSCT, median age 5months vs 11months, P = .003) compared to the clinical group. The NBS group had a lower incidence of infections before HSCT (29% vs 93%, P = .004). Although not statistically significant, the overall survival rate on last follow-up was higher in the NBS group (86% vs 67%, P = .62). Importantly, patients with active infections undergoing HSCT had a significantly lower overall survival probability compared to those without (P = .01). In conclusion, the introduction of NBS in Switzerland has led to earlier and often asymptomatic diagnosis of affected children, enabling timely intervention, infection prevention, and prompt treatment. These factors have contributed to higher survival rates in the NBS group. These findings underscore the critical importance of NBS for SCID, offering potential life-saving benefits through early detection and intervention.

Neurodevelopmental outcomes in two cases of artemis deficiency

Background: Severe Combined Immunodeficiency (SCID) is a category of inborn errors of immunity where there is impaired T and B cell development and/or function. Artemis SCID (Art-SCID) is characterized by dysfunctional Artemis protein, which is crucial for V(D)J recombination in T and B cell maturation. Art-SCID is fatal without management, and current definitive treatment involves hematopoietic stem cell transplantation (HSCT) or gene therapy. As the prognosis and survival of SCID patients improves, current research has begun unveiling long-term complications and morbidities. Previous literature has reported neurodevelopmental abnormalities in SCID patients, such as developmental delay and Autism Spectrum Disorder (ASD). However, it remains unknown whether these neurodevelopmental differences are linked to the SCID mutation, an adverse outcome of treatment and hospitalization, or comorbid social isolation and psychosocial challenges. Aims: In this case series, we discuss two cases of Art-SCID which presented with neurodevelopmental deficits following successful HSCT. Results: In both cases, SCID was detected on Newborn Screening (NBS), and Art-SCID was confirmed with genetic testing. Both patients were successfully treated with HSCT at 80 days of life, and followed up clinically well, with robust cell counts. Both patients later presented in toddlerhood with developmental, speech and language delay, however only one patient met diagnostic criteria for ASD. Conclusion: The definitive relationship between SCID, HSCT, and neurodevelopmental outcomes remain unclear, and warrants further study to allow for early intervention. We are currently working with colleagues across the country to further investigate and define this complex relationship. Statement of Novelty: We are investigating the complex relationship between SCID, HSCT, and potential neurodevelopmental outcomes. We present two cases of patients with Artemis SCID who were successfully treated with HSCT, and later presented in toddlerhood with developmental, speech, and language delay.

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations.

Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.

Abstract 4 Excellent Event-Free-Survival and Immunoglobulin Independence for Patients with Severe Combined Immunodeficiency Transplanted with Umbilical Cord Blood Stem Cells

Abstract Introduction An HLA matched related donor (MRD) is the preferred donor source for performing a hematopoietic stem cell transplant (HSCT) in patients with severe combined immunodeficiency (SCID) (Heimall 2017). However, many patients lack an MRD and thus an alternative donor, must be used. In these situations, an umbilical cord blood (UCB) represents a promising stem cell source (Gragert 2014, Martinez 2022). Objectives The primary outcome of interest was one-year event free survival (EFS) and the secondary outcome was time to immunoglobulin therapy independence based on donor source. Methods All patients diagnosed with SCID who underwent HSCT at Texas Children’s Hospital from 2003 to 2021 were included. EFS was defined as survival 365 days post-transplant without additional transplant, stem cell “top-off”, or donor lymphocyte infusion. The date of last immunoglobulin infusion was recorded to track time to immunoglobulin therapy independence. Results A total of 67 patients were included in the cohort. One-year EFS was 74.25% for all patients using the Kaplan-Meier method. When stratified by donor/stem cell source, EFS was 88.94% for UCB, 72.92% for MRD, 60.00% for URD, and 41.67% for haplo. There was statistical significance in EFS between URD UCB and the haploidentical group (p = 0.0006). The median time to IVIG independence was 8.6 months for all patients, 7.3 months for UCB, 23.4 months for URD, 26.6 months for haplo, and 32.5 months for MRD. When stratified by donor/stem cell source, cumulative incidence analysis showed UCB patients achieved Immunoglobulin independence earlier when compared to all other groups (UCB vs haplo p = 0.0335, UCB vs MRD p = 0.0012, UCB vs URD p = 0.0488). Discussion This single-center cohort of 67 patients demonstrates excellent survival for SCID patients undergoing HSCT, confirming prior studies (Pai 2014, Heimall 2017). EFS was highest in patients treated with an UCBT. In our analysis of immune reconstitution, we found that SCID patients transplanted with UCB, had a significantly shorter time to IVIG independence compared to other stem cell donors/sources. Given the high survival of UCB patients and this robust immune reconstitution, UCB may represent the best alternative stem cell source for SCID patients without a MRD option.

Whole exome sequencing reveals a missense mutation in the ADA gene causing severe combined immune deficiency in a Pakistani family ADA gene mutation causing SCID

BACKGROUND &amp; OBJECTIVE: Human adenosine deaminase deficiency, OMIM 102700 (ADA deficiency) is a genetic disorder which causes severe combined immunodeficiency (SCID). The enzyme adenosine deaminase is a housekeeping in nature and is involved in purine catabolism by catalyzing irreversible deamination of adenosine and deoxyadenosine. The SCID patients’ immune system is unable to fight off most of the bacterial and fungal infections due to profound lymphopenia (T-B-NK+). About 20% of the SCID patients are genetically homozygous for defective ADA gene. In our study we aimed to find out genetic variant in ADA gene in a family carrying severe combined immune deficiency. Objective in the current study is to unravel and characterize the molecular cause of the patient suffering from by birth SCID.&#x0D; METHODOLOGY: In the present study we enrolled a 6-month-old female SCID patient belonging to highly consanguineous Pakistani family. Patient clinical features included repeated chest infection with failure to thrive, fever and chronic diarrhea. Whole blood samples from patient, parents and healthy siblings were acquired in EDTA tubes. DNA was extracted from all the blood samples.&#x0D; RESULTS: Flowcytometry revealed lymphopenia (T-B-NK+) type of SCID. Whole Exome Sequencing (WES) identified a one nucleotide change (c.716G&gt;A) in ADA gene exon 8. The segregation of the identified variant in the family was confirmed through Sanger Sequencing.&#x0D; CONCLUSION: In this study, we presented detailed clinical and genetic description of patient suffering from severe combined immune deficiency. The immunological and genetic findings presented in this study will facilitate early diagnosis of the disease. Segregation of the identified variant in the family members will also aid in genetic counseling the family.

Clinical, immunological and molecular findings of 8 patients with typical and atypical severe combined immunodeficiency: identification of 7 novel mutations by whole exome sequencing.

Severe combined immunodeficiency (SCID) is one of the severe inborn errors of the immune system associated with life-threatening infections. Variations in SCID phenotypes, especially atypical SCID, may cause a significant delay in diagnosis. Therefore, SCID patients need to receive an early diagnosis. Here, we describe the clinical manifestations and genetic results of four SCID and atypical SCID patients. All patients (4 males and 4 females) in early infancy presented with SCID phenotypes within 6 months of birth. The mutations include RAG2 (p.I273T,p.G44X), IL7R (p.F361WfsTer17), ADA (c.780+1G>A), JAK3 (p.Q228Ter), LIG4 (p.G428R), and LAT (p.Y207fsTer33), as well as a previously reported missense mutation in RAG1 (p.A444V). The second report of LAT deficiency in SCID patients is presented in this study. Moreover, all variants were confirmed in patients and their parents as a heterozygous state by Sanger sequencing. The results of our study expand the clinical and molecular spectrum associated with SCID and leaky SCID phenotypes and provide valuable information for the clinical management of the patients.

Beyond Busulfan: Building a Better Conditioner for Bone Marrow Transplantation

Beyond Busulfan: Building a Better Conditioner for Bone Marrow Transplantation

A Herpes Simplex Virus 1 DNA Polymerase Multidrug Resistance Mutation Identified in a Patient With Immunodeficiency and Confirmed by Gene Editing.

Herpes simplex virus 1 can cause severe infections in individuals who are immunocompromised. In these patients, emergence of drug resistance mutations causes difficulties in infection management. Seventeen herpes simplex virus 1 isolates were obtained from orofacial/anogenital lesions in a patient with leaky severe combined immunodeficiency over 7 years, before and after stem cell transplantation. Spatial/temporal evolution of drug resistance was characterized genotypically-with Sanger and next-generation sequencing of viral thymidine kinase (TK) and DNA polymerase (DP)-and phenotypically. CRISPR/Cas9 was used to introduce the novel DP Q727R mutation, and dual infection-competition assays were performed to assess viral fitness. Isolates had identical genetic backgrounds, suggesting that orofacial/anogenital infections derived from the same virus lineage. Eleven isolates proved heterogeneous TK virus populations by next-generation sequencing, undetectable by Sanger sequencing. Thirteen isolates were acyclovir resistant due to TK mutations, and the Q727R isolate additionally exhibited foscarnet/adefovir resistance. Recombinant Q727R mutant virus showed multidrug resistance and increased fitness under antiviral pressure. Long-term follow-up of a patient with severe combined immunodeficiency revealed virus evolution and frequent reactivation of wild-type and TK mutant strains, mostly as heterogeneous populations. The DP Q727R resistance phenotype was confirmed with CRISPR/Cas9, a useful tool to validate novel drug resistance mutations.

Understanding of Severe Combined Immunodeficiency and Carrier Status in the U.S.-Based Irish Traveller Population

IntroductionThe Irish Travellers are an ethnically distinct minority community originating from Ireland, with substantial immigration to the U. S. resulting in a current subpopulation estimated around 20,000. Given the history of cultural endogamy in this community, there are known increased rates of severe combined immunodeficiency (SCID), with two recombinase activating gene 1 pathogenic variants (RAG1; p.Arg897*and p.Ser259Alafs*5) and one novel non-homologous end-joining 1 gene variant (NHEJ1; p.Gln224Argfs*27) detected in the US community. However, knowledge of rare genetic disorders, including how they are passed and the value of carrier testing, is limited among some members of this population. MethodsA multi-institutional collaboration developed an outreach project for SCID patients and their relatives in the U.S.-based Irish Traveller community. SCID patients were identified through newborn screening and pedigree interviews. Genetic testing for SCID variants was offered to kindred members, prioritizing members of child-bearing age. An online educational program was designed to identify knowledge gaps and disseminate information on SCID and implications of carrier status. Results and DiscussionBaseline knowledge and attitude assessments prior to educational intervention were obtained from 28 kindred members, all of whom were female and the majority (n = 23) were of child bearing age. Similar to their counterparts in Ireland, the majority (n = 21) had less than a high school diploma. 12 respondents identified three or more family members diagnosed with SCID. The majority of respondents (n ≥17) strongly agreed on the importance of knowing what diseases run in their family. However, only a minority (n ≤8) were able to correctly identify how autosomal dominant, autosomal recessive, and X-linked diseases were passed down. Open ended discussions with key members of this kindred also helped identify religious and cultural barriers which contributed to hesitancy around genetic testing in this underserved community. Early diagnosis of affected members, enabled by educational sessions and carrier screening within this high-risk population, is vital to increase preparedness for the birth of a child with SCID. This is especially important in the case of SCID caused by NHEJ1, as the radiosensitivity caused by this mutation necessitates treatment and conditioning protocols which differ from that of SCID caused by RAG1 mutations.

Characterization of expanded γδ T cells from patient with atypical X-linked severe combined immunodeficiency reveals preserved function and IL2RG-mediated signaling

Abnormally high γδ T cell numbers are common among atypical severe combined immunodeficiency (SCID) patients but detailed immunophenotyping and functional characterization of these expanded γδ T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG c.172C > T;p.(Pro58Ser) variant causing failure in IL2RG (CD132) plasma membrane targeting leading to impaired IL-2 responses in CD4 and CD8 T cells. Our aim was to investigate the phenotype and function of the index patient's abnormally large peripheral γδ T cell population.We found that in contrast to his αβ T cells, the patient's γδ T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. Vδ2+ population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's γδ T cells showed enhanced cytotoxicity towards A549 cancer cells. TCRvγ repertoire was versatile but sequencing of IL2RG revealed a novel c.534C>A; p.(Phe178Leu) somatic missense variant restricted to γδ T cells. Over time this variant became predominant in patient's γδ T cells and was enriched during in vitro expansion of patient's γδ T cells, indicating that the clones with this somatic variant may have growth or survival advantage. In silico analysis with DynaMut2 tool indicated that the overall effect of Phe178Leu substitution for IL2RG was predicted to be stabilizing and IL2RG-Pro58Ser/Phe178Leu to be more stable than IL2RG-Pro58Ser variant alone. Furthermore, IL2RG-Pro58Ser/Phe178Leu showed significantly increased cell surface expression when compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic IL2RG p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline IL2RG p.(Pro58Ser) variant.In conclusion, our results indicate that expansion of γδ T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells. (Tuovinen et al., Journal of Clinical Immunology 2022)

Late-onset enteric virus infection associated with hepatitis (EVAH) in transplanted SCID patients

Allogenic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are potentially curative treatments for severe combined immunodeficiency (SCID). Late-onset posttreatment manifestations (such as persistent hepatitis) are not uncommon. We sought to characterize the prevalence and pathophysiology of persistent hepatitis in transplanted SCID patients (SCIDH+) and to evaluate risk factors and treatments. We used various techniques (including pathology assessments, metagenomics, single-cell transcriptomics, and cytometry by time of flight) to perform an in-depth study of different tissues from patients in the SCIDH+ group and corresponding asymptomatic similarly transplanted SCID patients without hepatitis (SCIDH-). Eleven patients developed persistent hepatitis (median of 6 years after HSCT or GT). This condition was associated with the chronic detection of enteric viruses (human Aichi virus, norovirus, and sapovirus) in liver and/or stools, which were not found in stools from the SCIDH- group (n= 12). Multiomics analysis identified an expansion of effector memory CD8+ Tcells with high type I and II interferon signatures. Hepatitis was associated with absence of myeloablation during conditioning, split chimerism, and defective B-cell function, representing 25% of the 44 patients with SCID having these characteristics. Partially myeloablative retransplantation or GT of patients with this condition (which we have named as "enteric virus infection associated with hepatitis") led to the reconstitution of T- and B-cell immunity and remission of hepatitis in 5 patients, concomitantly with viral clearance. Enteric virus infection associated with hepatitis is related to chronic enteric viral infection and immune dysregulation and is an important risk for transplanted SCID patients with defective B-cell function.

P334: Not all severe combined immunodeficiency patients are created equal and when rapid genetic testing matters

P334: Not all severe combined immunodeficiency patients are created equal and when rapid genetic testing matters

Effect of Early Bacillus Calmette-Guerin Vaccination of Pediatric Severe Combined Immunodeficiency Patients on the Outcome of Hematopoietic Stem Cell Transplantation Using a Reduced-Intensity Conditioning Regimen

The eminence of Bacillus Calmette-Guerin (BCG) vaccine in newborn vaccination programs has been conspicuous throughout the years, especially in low-income developing countries where tuberculosis is prevalent; however, application of the BCG vaccine is not without constraints, especially in patients afflicted with immunodeficiency diseases, such as severe combined immunodeficiency (SCID). The present study aimed to evaluate whether the administration of BCG vaccine at birth could improve the outcomes of hematopoietic stem cell transplantation (HSCT) in pediatric patients with SCID. In this study, 30 SCID patients who underwent HSCT using a reduced-intensity conditioning regimen (RIC) were followed-up for 2 years post-HSCT. The outcomes of HSCT were evaluated in both non-BCG-vaccinated patients (n = 12) and BCG-vaccinated patients (n = 18). Our results show a higher incidence of acute graft-versus-host disease (aGVHD), but not of chronic GVHD, in the BCG-vaccinated patients, and a similar overall survival (OS) rate in the 2 groups. We speculate that the similar OS rate in the 2 groups, despite the risk of BGC vaccination, was because this group received an RIC conditioning regimen. There was no other difference between the 2 groups. Considering the effect of the BCG vaccine on HSCT outcome, we suggest that the administration of BCG vaccine be deferred until age 3 months so that APT testing without the interference of maternal antibodies can be performed. However, this study could benefit from a larger cohort to further validate our findings, as the possible reason for some factors not being statistically significant was our small sample size.

Clinical and virological characteristics of viral shedding in children with norovirus gastroenteritis.

The correlation between the clinical manifestations and fecal viral load of norovirus (NoV) infection remains unknown. We established a SYBR® Green-based real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method to quantify NoV and then sequenced its genomes from the feces of patients admitted at the Chang Gung Memorial Hospital from 2017 to 2018. NoV GII.4 Sydney (n=21, 36.2%) and GII.P16-GII.2 (n=19, 32.8%), the two predominant genotypes found among 58 isolates, were closely related to the Taiwan variant 2012a cluster in the VP1 region and genotypes of China strain. An increase in viral load could be observed on Day 3 following the onset of NoV infection. The viral load then declined rapidly from days 10-15 but remained high for >1 month in a severe combined immunodeficiency patient. Significantly longer shedding was found in patients with fever (p=0.03) or infected by the GII.4 Sydney strain (p<0.01). The qRT-PCR-mediated method proposed in this work could quantify the viral load in patients with NoV infection. Significant viral shedding over a period of 2 weeks in children with acute gastroenteritis and >1 month in an immunodeficient patient was observed. Significantly longer shedding could be correlated with infection by the GII.4 Sydney strain and febrile patients.

Aberrant T-cell exhaustion in severe combined immunodeficiency survivors with poor T-cell reconstitution after transplantation

Severe combined immunodeficiency (SCID) comprises rare inherited disorders of immunity that require definitive treatment through hematopoietic cell transplantation (HCT) or gene therapy for survival. Despite successes of allogeneic HCT, many SCID patients experience incomplete immune reconstitution, persistent T-cell lymphopenia, and poor long-term outcomes. We hypothesized that CD4+ T-cell lymphopenia could be associated with a state of T-cell exhaustion in previously transplanted SCID patients. We analyzed markers of exhaustion in blood samples from 61 SCID patients at a median of 10.4 years after HCT. Compared to post-HCT SCID patients with normal CD4+ T-cell counts, those with poor T-cell reconstitution showed lower frequency of naive CD45RA+/CCR7+ T cells, recent thymic emigrants, and TCR excision circles. They also had a restricted TCR repertoire, increased expression of inhibitory receptors (PD-1, 2B4, CD160, BTLA, CTLA-4), and increased activation markers (HLA-DR, perforin) on their total and naive CD8+ T cells, suggesting T-cell exhaustion and aberrant activation, respectively. The exhaustion score of CD8+ T cells was inversely correlated with CD4+ T-cell count, recent thymic emigrants, TCR excision circles, and TCR diversity. Exhaustion scores were higher among recipients of unconditioned HCT, especially when further in time from HCT. Patients with fewer CD4+ T cells showed a transcriptional signature of exhaustion. Recipients of unconditioned HCT for SCID may develop late post-HCT T-cell exhaustion as a result of diminished production of T-lineage cells. Elevated expression of inhibitory receptors on their T cells may be a biomarker of poor long-term T-cell reconstitution.

Incidence of Poliovirus Infection in Patients with Severe Combined Immunodeficiency (SCID)

Background: Severe combined immunodeficiency (SCID) is a group of disorders with impairment in function of T, B and sometimes NK cells that could lead to high susceptibility to infectious diseases and premature death. Live-attenuated vaccines are contraindicated in SCID patients. However, in regions without screening programs for SCID, oral polio vaccine (OPV) could lead to vaccine-derived polioviruses (VDPVs) which are reverted to the neurovirulent virus types and could cause outbreaks of vaccine-associated paralytic poliovirus (VAPP) in communities with inadequate vaccine coverage.&#x0D; Method: 20 SCID patients registered in Iranian national registry for Primary immunodeficiency (PID) disorders were tested for polio virus stool shedding. The demographic data, clinical presentation, polio test results, laboratory data and whole exome sequencing (WES) of the patients were available.&#x0D; Results: Among the 20 SCID patients enrolled in the study, 6 patients tested positive for immunodeficiencyrelated VDPVs (iVDPV) shedding. Four patients (20%) had iVDPV type 2, one patient (5%) had iVDPV type 1 and one patient (5%) had iVDPV type 3.&#x0D; Conclusion: Due to the high possibility of asymptomatic and long-term iVDPV shedding in SCID patients, the enhancement of screening of PID patients for poliovirus and iVDPV excretion is strongly needed.