Background Toxic shock syndrome toxin-1 (TSST-1) is a superantigen produced by Staphylococcus aureus that causes the life-threatening toxic shock syndrome. The development of a safe and immunogenic vaccine against TSST-1 remains an unmet medical need. We investigated the safety, tolerability and immunogenicity of a recombinant TSST-1 variant vaccine (rTSST-1v) after 1-3 injections in healthy volunteers. Methods In this randomised, double-blind, adjuvant-controlled, parallel-group, phase 2 trial, healthy adults aged 18-64 were randomly allocated to undergo 1-3 injections of either 10 or 100 mu g rTSST-1v or Al(OH)3. The primary endpoint was safety and tolerability of rTSST-1v in the intention-to-treat population. The per-protocol population was used for the immunogenicity analysis. The trial is registered with EudraCT#: 2015-003714-24; ClinicalTrials.gov#: NCT02814708. Findings Between April and November 2017,140 subjects were enrolled and 126 completed the trial. rTSST-1v showed a good safety and tolerability profile. A total of 855 systemic adverse events occurred, 280 of which were suspected related adverse events, without dose dependency. Two participants were discontinued early because of allergic reactions. Seroconversion occurred in >81% of subjects within 3 months of the first immunisation which was sustained until 18 months after the third immunisation in over 70% of subjects in the pooled low-dose group and in over 85% in the pooled high-dose group. Interpretation rTSST-1v in cumulative doses of up to 300 mu g was safe, well-tolerated and highly immunogenic. Two immunisations with 100 mu g rTSST-1v provided the most persistent immune response and may be evaluated in future trials. Funding Biomedizinische Forschung & Bio-Produkte AG funded this study.

Background: Toxic shock syndrome toxin-1 (TSST-1) is a superantigen produced by Staphylococcus aureus that causes the life-threatening toxic shock syndrome. The development of a safe and immunogenic vaccine against TSST-1 remains an unmet medical need. We investigated the safety, tolerability and immunogenicity of a recombinant TSST-1 variant vaccine (rTSST-1v) after 1-3 injections in healthy volunteers. Methods: In this randomised, double-blind, adjuvant-controlled, parallel-group, phase 2 trial, healthy adults aged 18-64 were randomly allocated to undergo 1-3 injections of either 10 or 100µg rTSST-1v or Al(OH)3. The primary endpoint was safety and tolerability of rTSST-1v in the intention-to-treat population. The per-protocol population was used for the immunogenicity analysis. The trial is registered with EudraCT#: 2015-003714-24; ClinicalTrials.gov#: NCT02814708. Findings: Of 140 enrolled subjects, 126 completed the trial (per-protocol analysis). rTSST-1v showed a good safety and tolerability profile. A total of 855 systemic adverse events occurred, 280 of which were suspected related adverse events, without dose dependency. Two participants were discontinued early because of allergic reactions. Seroconversion occurred in >81% of subjects within 3 months of the first immunisation which was sustained until 18 months after the third immunisation in over 70% of subjects in the pooled low-dose group and in over 85% in the pooled high-dose group. Interpretation: rTSST-1v in cumulative doses of up to 300 µg was safe, well-tolerated and highly immunogenic. Two immunisations with 100 µg rTSST-1v provided the most persistent immune response and may be evaluated in future trials.Trial Registration: EudraCT 2015-003714-24, clinicaltrials.gov (NCT02814708).Funding: This research was funded by Biomedizinische Forschung & Bio-Produkte AG.Declaration of Interest: GG, CS, CF, MMS, NB, UD, AT, and BJ declare no competing interests. Martha M. Eibl was the owner of Biomedizinische Forschung & Bio-Produkte AG. DH, LS, NM, and AR are employees of the study funder Biomedizinische Forschung & Bio- Produkte AG, a biotechnology company engaged in the development of BioMed rTSST-1v.Ethical Approval: The study was approved by the local ethics committee (Ethics Committee of the Medical University of Vienna, European Research Council Number 1810/2015) and was conducted in compliance with the Declaration of Helsinki, the Good Clinical Practice guidelines and with the Note for Guidance on Clinical Evaluation of New Vaccines. Oral and written informed consent was obtained prior to any trial-related procedure.

Staphylococcus aureus is a human and animal pathogen as well as a commensal bacterium. It can be a causative agent of severe, life-threatening infections with high mortality, e.g., toxic shock syndrome, septic shock, and multi-organ failure. S. aureus strains secrete a number of toxins. Exotoxins/enterotoxins are considered important in the pathogenesis of the above-mentioned conditions. Exotoxins, e.g., superantigen toxins, cause uncontrolled and polyclonal T cell activation and unregulated activation of inflammatory cytokines. Here we show the importance of genomic analysis of infectious strains in order to identify disease-causing exotoxins. Further, we show through functional analysis of superantigenic properties of staphylococcal exotoxins that even very small amounts of a putative superantigenic contaminant can have a significant mitogenic effect. The results show expression and production of two distinct staphylococcal exotoxins, SEC and SEL, in several strains from clinical isolates. Antibodies against both toxins are required to neutralise the superantigenic activity of staphylococcal supernatants and purified staphylococcal toxins.

Previous studies on immune responses following COVID-19 vaccination in patients with common variable immunodeficiency (CVID) were inconclusive with respect to the ability of the patients to produce vaccine-specific IgG antibodies, while patients with milder forms of primary antibody deficiency such as immunoglobulin isotype deficiency or selective antibody deficiency have not been studied at all. In this study we examined antigen-specific activation of CXCR5-positive and CXCR5-negative CD4+ memory cells and also isotype-specific and functional antibody responses in patients with CVID as compared to other milder forms of primary antibody deficiency and healthy controls six weeks after the second dose of BNT162b2 vaccine against SARS-CoV-2. Expression of the activation markers CD25 and CD134 was examined by multi-color flow cytometry on CD4+ T cell subsets stimulated with SARS-CoV-2 spike peptides, while in parallel IgG and IgA antibodies and surrogate virus neutralization antibodies against SARS-CoV-2 spike protein were measured by ELISA. The results show that in CVID and patients with other milder forms of antibody deficiency normal IgG responses (titers of spike protein-specific IgG three times the detection limit or more) were associated with intact vaccine-specific activation of CXCR5-negative CD4+ memory T cells, despite defective activation of circulating T follicular helper cells. In contrast, CVID IgG nonresponders showed defective vaccine-specific and superantigen-induced activation of both CD4+T cell subsets. In conclusion, impaired TCR-mediated activation of CXCR5-negative CD4+ memory T cells following stimulation with vaccine antigen or superantigen identifies patients with primary antibody deficiency and impaired IgG responses after BNT162b2 vaccination.

Background: Although previous studies described the production of IgG antibodies in a subgroup of patients with common variable immunodeficiency (CVID) following messenger RNA vaccinations with BNT162b2 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (CVID responders), the functionality of these antibodies in terms of avidity as measured by the dissociation rate constant (kdis) and the antibody response to booster immunization has not been studied. Objective: We sought to analyze in CVID responders and healthy individuals, the avidity of anti-SARS-CoV-2 serum antibodies and their neutralization capacity as measured by surrogate virus-neutralizing antibodies in addition to IgG-, IgM-, and IgA-antibody levels and the response of circulating (peripheral blood) follicular T-helper cells after a third vaccination with BNT162b2 SARS-CoV-2 messenger RNA vaccine. Methods: Binding IgG, IgA, and IgM serum levels were analyzed by ELISA in patients with CVID responding to the primary vaccination (CVID responders, n = 10) and healthy controls (n = 41). The binding avidity of anti-spike antibodies was investigated using biolayer interferometry in combination with biotin-labeled receptor-binding-domain of SARS-CoV-2 spike protein and streptavidin-labeled sensors. Antigen-specific recall T-cell responses were assessed by measuring activation-induced markers by flow cytometry. Results: After the third vaccination with BNT162b2, IgG-, IgM-, and IgA-antibody levels, surrogate virus-neutralizing antibody levels, and antibody avidity were lower in CVID responders than in healthy controls. In contrast, anti-SARS-CoV-2 spike protein avidity was comparable in CVID responders and healthy individuals following primary vaccination. Follicular T-helper cell response to booster vaccination in CVID responders was significantly reduced when compared with that in healthy individuals. Conclusions: Impaired affinity maturation during booster response provides new insight into CVID pathophysiology. (J Allergy Clin Immunol 2023;151:922-5.)

Mutations of the interleukin 2 receptor γ chain (IL2RG) result in the most common form of severe combined immunodeficiency (SCID), which is characterized by severe and persistent infections starting in early life with an absence of T cells and natural killer cells, normal or elevated B cell counts and hypogammaglobulinemia. SCID is commonly fatal within the first year of life, unless the immune system is reconstituted by hematopoietic stem cell transplantation (HSCT) or gene therapy. We herein describe a male infant with X-linked severe combined immunodeficiency (X-SCID) diagnosed at 5 months of age. Genetic testing revealed a novel C to G missense mutation in exon 1 resulting in a 3’ splice site disruption with premature stop codon and aberrant IL2 receptor signaling. Following the diagnosis of X-SCID, the patient subsequently underwent a TCRαβ/CD19-depleted haploidentical HSCT. Post transplantation the patient presented with early CD8 + T cell recovery with the majority of T cells (>99%) being non-donor T cells. Genetic analysis of CD4 + and CD8 + T cells revealed a spontaneous 14 nucleotide insertion at the mutation site resulting in a novel splice site and restoring the reading frame although defective IL2RG function was still demonstrated. In conclusion, our findings describe a spontaneous second-site mutation in IL2RG as a novel cause of somatic mosaicism and early T cell recovery following haploidentical HSCT.

X-linked lymphoproliferative disease (XLP1) is a combined immunodeficiency characterized by severe immune dysregulation caused by mutations in the SH2D1A/SAP gene. Loss or dysfunction of SH2D1A is associated with the inability in clearing Epstein-Barr-Virus (EBV) infections. Clinical manifestation is diverse and ranges from life-threatening hemophagocytic lymphohistiocytosis (HLH) and fulminant infectious mononucleosis (FIM) to lymphoma and antibody deficiency. Rare manifestations include aplastic anemia, chronic gastritis and vasculitis. Herein, we describe the case of a previously healthy eight-year old boy diagnosed with XLP1 presenting with acute non-EBV acute meningoencephalitis with thrombotic occlusive vasculopathy. The patient developed multiple cerebral aneurysms leading to repeated intracerebral hemorrhage and severe cerebral damage. Immunological examination was initiated after development of a susceptibility to infections with recurrent bronchitis and one episode of severe pneumonia and showed antibody deficiency with pronounced IgG1-3-4 subclass deficiency. We could identify a novel hemizygous SH2D1A point mutation affecting the start codon. Basal levels of SAP protein seemed to be detectable in CD8+ and CD4+ T- and CD56+ NK-cells of the patient what indicated an incomplete absence of SAP. In conclusion, we could demonstrate a novel SH2D1A mutation leading to deficient SAP protein expression and a rare clinical phenotype of non-EBV associated acute meningoencephalitis with thrombotic occlusive vasculopathy.

Staphylococcal superantigen toxins lead to a devastating cytokine storm resulting in shock and multi-organ failure. We have previously assessed the safety and immunogenicity of a recombinant toxic shock syndrome toxin 1 variant vaccine (rTSST-1v) in clinical trials (NCT02971670 and NCT02340338). The current study assessed neutralizing antibody titers after repeated vaccination with escalating doses of rTSST-1v. At study entry, 23 out of 34 subjects (67.6%) had neutralizing antibody titers inhibiting T cell activation as determined by 3H-thymidine incorporation at a serum dilution of ≤1:100 with similar figures for inhibition of IL-2 activation (19 of 34 subjects, 55.9%) as assessed by quantitative PCR. After the first vaccination, numbers of subjects with neutralization titers inhibiting T cell activation (61.7% ≥ 1:1000) and inhibiting IL-2 gene induction (88.2% ≥ 1:1000) increased. The immune response was augmented after the second vaccination (inhibiting T cell activation: 78.8% ≥ 1:1000; inhibiting IL-2 induction: 93.9% ≥ 1:1000) corroborated with a third immunization months later in a small subgroup of subjects. Assessment of IFNγ, TNFα and IL-6 inhibition revealed similar results, whereas neutralization titers did not change in placebo participants. Antibody titer studies show that vaccination with rTSST-1v in subjects with no/low neutralizing antibodies can rapidly induce high titer neutralizing antibodies persisting over months.

Vasculitis can be a life-threatening complication associated with high mortality and morbidity among patients with primary immunodeficiencies (PIDs), including variants of severe and combined immunodeficiencies ((S)CID). Our understanding of vasculitis in partial defects in recombination activating gene (RAG) deficiency, a prototype of (S)CIDs, is limited with no published systematic evaluation of diagnostic and therapeutic modalities. In this report, we sought to establish the clinical, laboratory features and treatment outcome of patients with vasculitis due to partial RAG deficiency. Vasculitis was a major complication in eight (13%) of 62 patients in our cohort with partial RAG deficiency with features of infections and immune dysregulation. Vasculitis occurred early in life, often as first sign of disease (50%) and was complicated by significant end organ damage. Viral infections often preceded the onset of predominately non-granulomatous-small vessel vasculitis. Autoantibodies against cytokines (IFN-α, -ω and IL-12) were detected in a large fraction of the cases tested (80%), whereas the majority of patients were anti-neutrophil cytoplasmic antibodies (ANCA) negative (>80%). Genetic diagnosis of RAG deficiency was delayed up to 2 years from the onset of vasculitis. Clinical cases with sole skin manifestation responded well to first-line steroid treatment, whereas systemic vasculitis with severe end-organ complications required second-line immunosuppression and/or hematopoietic stem cell transplantation (HSCT) for definitive management. In conclusion, our data suggest that vasculitis in partial RAG deficiency is prevalent among patients with partial RAG deficiency and is associated with high morbidity. Therefore, partial RAG deficiency should be included in the differential diagnosis of patients with early-onset systemic vasculitis. Diagnostic serology may be misleading with ANCA negative findings, and search for conventional autoantibodies should be extended to include those targeting cytokines.

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In a recent search of the USIDNet database, vasculitis was found to be an uncommon complication (1.6%), however associated with CVID (38%). (Byram K. et al. AAAI meeting 2019) We also reported that hypomorphic RAG deficiency may occur in 1 in 500 patients with antibody deficiency, including CVID. The aim of this study was to establish the clinical, laboratory features and treatment outcome of vasculitis in RAG deficiency. In our cohort of 67 patients with hypomorphic RAG deficiency, we identified 7 patients (10.4%) with episodes of vasculitis. Median age was 5 years, with vasculitis occurred early in course of disease with a mean onset of 2 years compared to diagnosis of RAG deficiency with 3 years. Vasculitis was associate with a high mortality (5/7, 71%) and was complicated by significant end organ necrosis (n=3), stroke (n=1). Systemic vasculitis was refractory to first- or second-line therapy, including steroids and immunosuppression and required HSCT for definitive management. Although patients were mostly ANCA negative, anti-cytokine antibodies were detected in a large fraction of the cases (3/5, 60 %). Varicella or other viral infections preceded in the onset of disease in many of the patients (2/5, 40%). Our data suggest that vasculitis is a key component of morbidity among patients with hypomorphic RAG variants. Diagnostic serology may be misleading and may be extended to testing for anti-cytokine antibodies. Early escalated of treatment such as HSCT may improve therapeutic outcome. Evaluation for RAG deficiency should be considered among PID patients with antibody deficiency and vasculitis.

BACKGROUND: Although autoimmunity and hyperinflammation secondary to recombination activating gene (RAG) deficiency have been associated with delayed diagnosis and even death, our current understanding is limited primarily to small case series. OBJECTIVE: Understand the frequency, severity, and treatment responsiveness of autoimmunity and hyperinflammation in RAG deficiency. METHODS: In reviewing the literature and our own database, we identified 85 patients with RAG deficiency, reported between 2001 and 2016, and compiled the largest case series to date of 63 patients with prominent autoimmune and/or hyperinflammatory pathology. RESULTS: Diagnosis of RAG deficiency was delayed a median of 5 years from the first clinical signs of immune dysregulation. Most patients (55.6%) presented with more than 1 autoimmune or hyperinflammatory complication, with the most common etiologies being cytopenias (84.1%), granulomas (23.8%), and inflammatory skin disorders (19.0%). Infections, including live viral vaccinations, closely preceded the onset of autoimmunity in 28.6% of cases. Autoimmune cytopenias had early onset (median, 1.9, 2.1, and 2.6 years for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively) and were refractory to intravenous immunoglobulin, steroids, and rituximab in most cases (64.7%, 73.7%, and 71.4% for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively). Evans syndrome specifically was associated with lack of response to first-line therapy. Treatment-refractory autoimmunity/ hyperinflammation prompted hematopoietic stem cell transplantation in 20 patients. CONCLUSIONS: Autoimmunity/hyperinflammation can be a presenting sign of RAG deficiency and should prompt further evaluation. Multilineage cytopenias are often refractory to immunosuppressive treatment and may require hematopoietic cell transplantation for definitive management. (C) 2019 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology.

In X-linked agammaglobulinemia (XLA), a mutation in the gene coding for Bruton's tyrosine kinase (btk), which is involved in signaling through the B-cell antigen receptor, leads to absence of mature B cells and lack of antibodies of all immunoglobulin isotypes.1 Replacement therapy with human immunoglobulin derived from plasma donations has to be started as early as possible after diagnosis to prevent life-threatening infections and long-term disability. Infection with the hepatitis C virus (HCV) through contaminated immunoglobulin products has been described in the 1980s in patients with primary antibody deficiency, before blood and plasma donor screening became possible through the discovery of the HCV in 1989.

Recombination-activating gene (RAG) deficiency has an estimated disease incidence of 1:181,000, including severe combined immunodeficiency (SCID) at a rate of 1:330,000.1Kumanovics A. Lee Y.N. Close D.W. Coonrod E.M. Ujhazi B. Chen K. et al.Estimated disease incidence of RAG1/2 mutations: a case report and querying the Exome Aggregation Consortium.J Allergy Clin Immunol. 2017; 139: 690-692.e3Google Scholar, 2Kwan A. Abraham R.S. Currier R. Brower A. Andruszewski K. Abbott J.K. et al.Newborn screening for severe combined immunodeficiency in 11 screening programs in the United States.JAMA. 2014; 312: 729-738Google Scholar Complete or hypomorphic variants of SCID secondary to low recombinase activity (<5%) present early with severe infections and/or clinical signs of systemic inflammation, such as severe dermatitis, colitis, or both.3Schwarz K. Gauss G.H. Ludwig L. Pannicke U. Li Z. Lindner D. et al.RAG mutations in human B cell-negative SCID.Science. 1996; 274: 97-99Google Scholar, 4Felgentreff K. Perez-Becker R. Speckmann C. Schwarz K. Kalwak K. Markelj G. et al.Clinical and immunological manifestations of patients with atypical severe combined immunodeficiency.Clin Immunol. 2011; 141: 73-82Google Scholar Hypomorphic RAG1/2 mutations with more preserved residual V(D)J recombination activity (5% to 30%) result in a distinct phenotype of combined immunodeficiency with granuloma, autoimmunity, or both.1Kumanovics A. Lee Y.N. Close D.W. Coonrod E.M. Ujhazi B. Chen K. et al.Estimated disease incidence of RAG1/2 mutations: a case report and querying the Exome Aggregation Consortium.J Allergy Clin Immunol. 2017; 139: 690-692.e3Google Scholar, 2Kwan A. Abraham R.S. Currier R. Brower A. Andruszewski K. Abbott J.K. et al.Newborn screening for severe combined immunodeficiency in 11 screening programs in the United States.JAMA. 2014; 312: 729-738Google Scholar, 5Walter J.E. Rosen L.B. Csomos K. Rosenberg J.M. Mathew D. Keszei M. et al.Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency.J Clin Invest. 2015; 125: 4135-4148Google Scholar Beyond combined immunodeficiency, RAG deficiency has been found in patients with predominantly primary antibody deficiencies6Buchbinder D. Baker R. Lee Y.N. Ravell J. Zhang Y. McElwee J. et al.Identification of patients with RAG mutations previously diagnosed with common variable immunodeficiency disorders.J Clin Immunol. 2015; 35: 119-124Google Scholar, 7Geier C.B. Piller A. Linder A. Sauerwein K.M. Eibl M.M. Wolf H.M. Leaky RAG deficiency in adult patients with impaired antibody production against bacterial polysaccharide antigens.PLoS One. 2015; 10: e0133220Google Scholar and naive CD4+ T-cell lymphopenia in most cases. Currently, there is no published systematic evaluation for the presence of an underlying RAG deficiency in patients with primary antibody deficiencies. There is great variability among diagnostic modalities for evaluation and treatment for inflammatory lung disease in case reports of RAG deficiency with no standardized guidelines. Clinical features and lung disease for patients with late presentation of RAG deficiency have not been studied extensively. In addition, no studies have examined the prevalence of RAG deficiency in cohorts of adults with primary immunodeficiency (PID). Here we describe a cohort of 15 patients with late presentation of RAG deficiency. We also estimate the prevalence of RAG deficiency in adults with PID after genetic analysis in 2 separate large cohorts of patients with PID. We have analyzed the canonical regions of RAG1 and RAG2 in a total of 692 patients with PID from 2 separate cohorts, one from the United Kingdom (UK) and one from Austria (Vienna). The UK cohort is part of the National Institute for Health Research BioResource–Rare Diseases PID study, as previously described (Tuijnenburg et al8Tuijnenburg P. Lango Allen H. Burns S.O. Greene D. Jansen M.H. Staples E. et al.Loss of function NFKB1 variants are the most common monogenic cause of CVID in Europeans.J Allergy Clin Immunol. 2018 Feb 22; ([Epub ahead of print])https://doi.org/10.1016/j.jaci.2018.01.039Google Scholar). In the National Institute for Health Research BioResource–Rare Diseases PID cohort of 558 patients (299 adults) and the Vienna cohort of 134 patients (106 adults), we report a total of 5 newly identified cases of RAG deficiency. For details, see the Methods section and Tables E1 to E3 in this article's Online Repository at www.jacionline.org. Based on these findings, we estimate that the prevalence of RAG deficiency in adults with PID ranges from 1% to 1.9%. For all adult patients with PID currently registered with the UK Primary Immunodeficiency Network database (3294 patients older than age 18 years), we expect to find an additional 32.9 to 62.6 cases of RAG deficiency. Gene variants are shown in Fig 1, A. Cohort demographics are discussed in the Methods section in this article's Online Repository. Functional characterization of novel RAG variants is discussed in the Methods section in this article's Online Repository. The activity of mutant RAG1 and RAG2 proteins normally required for catalyzing V(D)J recombination events are shown in Table E2. In addition to the method previously described,9Lee Y.N. Frugoni F. Dobbs K. Walter J.E. Giliani S. Gennery A.R. et al.A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency.J Allergy Clin Immunol. 2014; 133: 1099-1108Google Scholar we also used a system to measure recombination activity in compound heterozygous cases by means of in vitro expression of murine RAG1 and RAG2. Both systems simulate the efficiency of protein expressed in patients in their ability to produce a diverse repertoire of T-cell receptor and B-cell receptor coding for immunoglobulins. More than half of the mutant proteins tested show almost complete loss of activity. All patients tested had an overall low combined RAG activity (6.4% to 28%). Immune phenotypes and clinical diagnoses are shown in Fig 1, B. Persistently low IgG and/or low IgA and IgM levels are seen in approximately 50% of cases (see Table E3). Dominant laboratory features were naive CD4+ T-cell lymphopenia with low absolute numbers and fraction of naive CD4+ cells (CD4+CD45RA+), and B-cell counts were variably low (see Table E3). ELISA was used to test for anti-cytokine antibodies (targeting IFN-α, IFN-ω, and IL-12) on plasma from 7 patients (data not shown). Four patients had positive results, which is comparable with our previously reported cohort (56%).5Walter J.E. Rosen L.B. Csomos K. Rosenberg J.M. Mathew D. Keszei M. et al.Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency.J Clin Invest. 2015; 125: 4135-4148Google Scholar Most adult RAG-deficient patients had inflammatory autoimmune complications (87%; see the Methods section and Fig E1, A, in this article's Online Repository at www.jacionline.org). Organ-specific manifestations were most common (73%) and similar to previously described reports of 48% to 77%.5Walter J.E. Rosen L.B. Csomos K. Rosenberg J.M. Mathew D. Keszei M. et al.Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency.J Clin Invest. 2015; 125: 4135-4148Google Scholar, 10Speckmann C. Doerken S. Aiuti A. Albert M.H. Al-Herz W. Allende L.M. et al.A prospective study on the natural history of patients with profound combined immunodeficiency: an interim analysis.J Allergy Clin Immunol. 2017; 139: 1302-1310.e4Google Scholar Granulomatous disease was seen in 40% of patients, with 5 of 6 patients showing granuloma localization within interstitial lung tissue. Other complications were also seen (see Fig E1, E). Similar to recent reports (21% to 77%),5Walter J.E. Rosen L.B. Csomos K. Rosenberg J.M. Mathew D. Keszei M. et al.Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency.J Clin Invest. 2015; 125: 4135-4148Google Scholar, 10Speckmann C. Doerken S. Aiuti A. Albert M.H. Al-Herz W. Allende L.M. et al.A prospective study on the natural history of patients with profound combined immunodeficiency: an interim analysis.J Allergy Clin Immunol. 2017; 139: 1302-1310.e4Google Scholar cytopenias occurred in 40% of patients: autoimmune hemolytic anemia (27%), immune thrombocytopenic purpura (20%), and autoimmune neutropenia in 1 patient. Progressive pulmonary diseases were the leading causes of morbidity and mortality (93%; see Fig E1, D), with pneumonia being the most common, followed by bronchiectasis, chronic bronchitis, granuloma, fibrosis, chronic obstructive pulmonary disease, and bronchiolitis (Fig 1, C). We observed a transition from acute infectious complications (pneumonia; mean onset, 14 years) to chronic inflammatory complications (mean age, 23 years; Fig 2, A). Progressive pulmonary diseases were also the leading concern for successful hematopoietic stem cell transplantation (HSCT). High-resolution computed tomographic imaging of the lung revealed bronchiectasis and granuloma. Histology of lung biopsy specimens (patients 1 and 3) revealed atypical lymphoid hyperplasia with granulomatous features and giant cell formation (Fig 2, B). Germinal center formations in patient 3 were comprised of CD3+ T cells and CD20+ B cells. Patient 1 had peribronchial fibrosis (Fig 2, B). Pulmonary lung function data revealed a median forced vital capacity of 79.55%, diffusing capacity of the lungs for carbon monoxide of 75%, and FEV1/forced vital capacity ratio of 78%. We performed a retrospective analysis of pulmonary function tests over 2 or more years to assess the decrease in respiratory function. Two of 4 patients had a significant decrease, indicating a variable degree of lung function in adult patients with RAG deficiency (see the Methods section and Fig E1, G, in this article's Online Repository). Thirteen (93%) of 14 patients received first-line immunoglobulin replacement therapy (see Fig E1, B, and Table E2). Fifty-seven percent received antibiotic prophylaxis, 21% received antiviral drugs, and 14% received disease-modifying antirheumatic drugs. Five (36%) patients were considered for HSCT. Comparisons of therapeutic approaches revealed no statistically significant difference in survival. Three of 8 patients who received only immunoglobulin replacement therapy were deceased. Among patients undergoing transplantation, the major mortality cause was infection after HSCT (see Fig E1, C). RAG1/2 are the most common defective genes associated with atypical SCID.10Speckmann C. Doerken S. Aiuti A. Albert M.H. Al-Herz W. Allende L.M. et al.A prospective study on the natural history of patients with profound combined immunodeficiency: an interim analysis.J Allergy Clin Immunol. 2017; 139: 1302-1310.e4Google Scholar Patients can survive into adulthood, and our findings suggest that the prevalence of such cases varies between 1% and 1.9% in adult PID cohorts. Total and naive CD4+ T-cell lymphopenia,4Felgentreff K. Perez-Becker R. Speckmann C. Schwarz K. Kalwak K. Markelj G. et al.Clinical and immunological manifestations of patients with atypical severe combined immunodeficiency.Clin Immunol. 2011; 141: 73-82Google Scholar, 10Speckmann C. Doerken S. Aiuti A. Albert M.H. Al-Herz W. Allende L.M. et al.A prospective study on the natural history of patients with profound combined immunodeficiency: an interim analysis.J Allergy Clin Immunol. 2017; 139: 1302-1310.e4Google Scholar autoimmunity, and progressive inflammatory lung disease should all prompt further investigations for RAG deficiency in adults with PIDs. The relative absence of RAG deficiency in the pediatric cohort of 216 patients suggests that milder forms of RAG deficiency might not be diagnosed as readily as a PID in childhood. In the era of whole-exome sequencing, the spectrum of RAG deficiency broadens further to include adults with autoimmune and inflammatory manifestations that can result in progressive decrease. Systemic analysis of PID-related genes9Lee Y.N. Frugoni F. Dobbs K. Walter J.E. Giliani S. Gennery A.R. et al.A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency.J Allergy Clin Immunol. 2014; 133: 1099-1108Google Scholar and functional in vitro assays that confirm decreased recombination activity are essential. Laboratory features of naive CD4+ T-cell lymphopenia and the presence of anti-cytokine antibodies can further support the diagnosis of partial RAG deficiency. Where RAG deficiency is confirmed, therapy can be adjusted based on the mechanistic understanding and might ultimately provide a targeted strategy for early intervention. This study was conducted in accordance with the Declaration of Helsinki. Patients provided written informed consent so that anonymized data could be included in a scientific publication. All results presented in this study were obtained as part of the routine medical care received by the patient. This study makes use of data generated by the NIHR BioResource–Rare Disease Consortium. A full list of the consortium members who contributed to the generation of the data is available in this article's Online Repository at www.jacionline.org. We thank Professor Christian J. Müller for providing pathology specimens and Dr Karl Waibel for providing pulmonary function testing. Download .docx (.22 MB) Help with docx files Online Repository text Download .pdf (1.28 MB) Help with pdf files Fig E1 Download .xlsx (.03 MB) Help with xlsx files Table E1 Download .xlsx (.03 MB) Help with xlsx files Table E2 Download .xlsx (.04 MB) Help with xlsx files Table E3 Download .docx (.1 MB) Help with docx files Appendix E1

B cell activation via the B cell receptor (BCR) signalosome involves participation of signaling molecules such as BTK and BLNK. Genetic defects in these molecules are known to impair B cell differentiation and subsequently lead to agammaglobulinemia. Here we identified novel mutations in BTK and BLNK in two unrelated patients that perturb the intrinsic B-cell receptor signaling pathway and lead to selective IgM deficiency, whereas production of other immunoglobulin isotypes and IgG antibody response remain intact. Currently it is unknown how BCR signaling strength affects mature B cell development in humans. Both patients show reduced levels of BCR signalosome phosphorylation as well as impaired BCR-dependent Ca2+ influx, which was accompanied by a marked decrease in IgD+IgM+CD27+ MZ-like B-cells. We further describe reduced expression of essential B cell differentiation factors such as BAFF-R and T-Bet in the patients' B-cells, which might contribute to the observed deficiency of MZ-like B cells. MZ-like B cells are known to produce natural IgM antibodies that play an essential role in immune homeostasis. By using surface plasmon resonance (SPR) technology and a synthetic blood group A trisaccharide as antigen we were able to show that both patients lack the presence of anti-blood group A IgM considered to be prototypical natural antibodies whereas IgG levels were normal. Antibody binding dynamics and binding affinity of anti-blood group A IgG were comparable between patients and healthy controls. These results indicate that human IgM deficiency can be associated with signaling defects in the BCR signalosome, defective production of natural IgM antibodies in the blood group A/B/0 system and abnormalities in B cell development.

Recent studies identified an emerging role of group 2 and 3 innate lymphoid cells (ILCs) as key players in the generation of T‐dependent and T‐independent antibody production. In this retrospective case‐control study, CD117+ ILCs (including the majority of ILC2 and ILC3) were reduced in patients with common variable immunodeficiency (CVID). The reduction in CD117+ ILCs was distinctive to CVID and could not be observed in patients with X‐linked agammaglobulinemia. Patients with a more pronounced reduction in CD117+ ILC numbers showed significantly lower numbers of peripheral MZ‐like B cells and an increased prevalence of chronic, non‐infectious enteropathy. Subsequent phenotyping of ILC subsets in CVID revealed that the reduction in CD117+ ILC numbers is due to a reduction in ILC2 numbers. In vitro expansion of CVID ILC2 in response to IL‐2, IL‐7, IL‐25 and IL‐33 was impaired. Furthermore, upregulation of MHCII and IL‐2RA in response to IL‐2, IL‐7, IL‐25 and IL‐33 was impaired in CVID ILC2. Thus, our results indicate a dysregulation of ILC subsets with a reduction in ILC2 numbers in CVID, however, further studies are needed to explore whether ILC abnormalities are a primary finding or secondary to disease complications encountered in CVID.

Over the past decades, a pleiotropic spectrum of B-cell intrinsic defects leading to early onset agammaglobulinemia and absent B cells has been described. Herein we report terminal 14q32.33 deletion as a novel cause of agammaglobulinemia. We describe a 20-year old man with a 1MB terminal 14q32.33 deletion resulting in a loss of the entire Immunoglobulin heavy chain gene region of chromosome 14. The patient presented with absent serum immunoglobulin levels and absent circulating B cells since age 2. The clinical picture was dominated by severe episodes of recurrent upper respiratory tract infections. In the literature, the most prevalent features of terminal 14q32.33 deletions include mental disability, facial malformation, hypotonia, seizures, and visual problems with retinal abnormalities. Neither increased susceptibility to infections nor agammaglobulinemia have been described as a manifestation of terminal 14q32.33 deletion. Thus, our findings expand the known clinical spectrum of terminal 14q32.33 deletion to include susceptibility to infections.

Staphylococcus aureus is a frequent cause of life-threatening diseases [1,2]. Being part of the commensal flora, and colonizing at least intermittently up to 60% of the population [3], staphylococci may become pathogenic and cause local infections such as wound infections, osteomyelitis, pneumonia, and systemic diseases such as the toxic shock syndrome and septic shock, leading to multiorgan failure [4]. Despite great efforts, no effective vaccine has yet become available. There are numerous examples of secreted and membraneassociated proteins and polysaccharides that were suggested to contribute to the virulence of S. aureus [5]. This arsenal includes pore-forming hemolysins (e.g. α/γ toxins), hydrolytic enzymes (e.g. β/δ toxins, ETs), adhesins for elastin, collagen, fibronectin, and fibrinogen (e.g. Efb, Cna, Ebps, ClfAB, FnbAB), proteins that bind antimicrobial peptides, complement factors, or antibodies (e.g. SCIN, CHIPS, Eap, Spa), cation-binding proteins (e.g. IsdB), and a vast number of secreted immune system modulating toxins (e.g. SAGs, SSLs) [6,7]. The majority of genes encoding virulence factors are located in the core genome of all clinical isolates, which renders them more interesting for vaccination strategies. However, while clinical trials are ongoing, studies have been reported suggesting that certain virulence factors are not produced for unknown reasons, or due to mutations in the gene or in genes of transcription factors, either in single isolates or whole clonal complexes [8–10]. In 2013, at a symposium held by the National Institute of Allergy and Infectious Diseases (NIAID), reasons for the lack of an effective vaccine against S. aureus infections were discussed with representatives of the three largest companies present. The first vaccine, StaphVAX developed by Nabi and presented by GSK at the symposium, contained the two most important staphylococcal capsular determinants, type 5 and 8 capsule polysaccharides, conjugated to the nontoxic recombinant Pseudomonas aeruginosa exotoxin A. In a first phase III efficacy trial, application of this vaccine did not show significant decrease of S. aureus bacteremia after a 1-year follow-up. Early analysis of the first phase III study suggested trends for efficacy in the first 10 months after vaccination. A confirmatory phase III trial in 3600 hemodialysis patients revealed no significant differences between vaccinated and placebo participants for the decrease of bacteremia, which might have also been influenced by the study population [11]. A phase III trial by Merck, including 8000 participants, tested the candidate vaccine V710, codeveloped by Intercell AG and Merck. It contained the non-adjuvanted iron regulated surface determinant B factor (IsdB). The cohort comprised patients undergoing elective cardiac surgery. The primary end points were prevention of postoperative bacteremia and/or deep sternal wound infection. The study was terminated at the recommendation of the independent data monitoring committee for safety and efficacy reasons. A multiple antigen approach was presented by Pfizer. SA4Ag included the adhesion molecule ClfA, the manganese transporter MntC, and the above-mentioned capsule polysaccharides type 5 and 8. However, they were conjugated to the carrier protein CRM197, a nontoxic mutant of the diphtheria toxin. A phase I/IIa study among Japanese adults to assess safety and immunogenicity was completed (NCT02492958). It is predicted to provide a broad cellular and humoral immune response. It is now in a phase IIb trial, evaluating the prevention of postoperative S. aureus infections upon elective spinal fusion surgery (NCT02388165). These clinical trials with vaccines designed against bacterial constituents and virulence factors were based on experience from prior clinical studies, often with single antigens. Presently, the development of multi-component vaccines is favored [12]. Several experts prefer vaccines to prevent infection [13]. The induction of T cell and B cell activation and broad immunity are thought to be most important. RA Proctor emphasized in a recent review that there is a consensus on the importance of T cell immunity for the protection of humans [14]. The aims of vaccine development may be prevention of severe infection or prevention of disease with complications. Prevention of disease mainly focuses on secreted toxins as primary targets. Secreted staphylococcal exotoxins are thought to play a central role in the severity of systemic disease. Toxic shock syndrome is well characterized as a systemic inflammatory disease with massive immune dysregulation, which can be lethal because of multiorgan failure. There is no specific therapy. The superantigen toxin TSST-1 has been verified as being the main causative agent. Two forms of TSS are diagnosed: menstrual (tampon associated) and non-

Background Staphylococcal toxic shock syndrome is a superantigen-driven potentially life-threatening disease affecting mainly young and otherwise healthy individuals. Currently, no specific treatment or preventive measure is available. We aimed to assess the safety, tolerability, and immunogenicity of a recombinant detoxified toxic shock syndrome toxin-1 variant (rTSST-1v) vaccine in adult volunteers.Methods In this randomised, double-blind, adjuvant-controlled, dose-escalation first-in-human trial, healthy adults aged 18-64 years were enrolled from the Medical University of Vienna, Austria. Participants were randomly assigned (2:1 and 3:1) by block randomisation (block sizes of three and 12) to receive increasing doses of rTSST-1v (100 ng to 30 mu g) or the adjuvant comparator aluminium hydroxide (Al(OH)(3)) (200 mu g, 600 mu g, or 1 mg). Investigators and participants were masked to group allocation. The per-protocol population received a booster immunisation 42 days after the first vaccination. The primary endpoint was safety and tolerability of rTSST-1v. The per-protocol population included all participants who had adhered to the study protocol without any major protocol deviations. The per protocol population was the primary analysis population for immunogenicity. The trial is registered with EudraCT, number 2013-003716-50, and ClinicalTrials.gov, number NCT02340338.Findings Between Aug 19, 2014, and April 14, 2015, 46 participants were enrolled (safety population), of whom three were assigned to cohort 1 (two to receive 100 ng rTSST-1v and one to receive 200 mu g Al(OH)(3)), three to cohort 2 (two to receive 300 ng rTSST-1v and one to receive 600 mu g Al(OH)(3)), four to cohort 3 (three to receive 1 mu g rTSST-1v and one to receive 1 mg Al(OH)(3)), 12 to cohort 4 (nine to receive 3 mu g rTSST-1v and three to receive 1 mg Al(011),), 12 to cohort 5 (nine to receive 10 mu g rTSST-1v and three to receive 1 mg Al(OH),), and 12 to cohort 6 (nine to receive 300 mu g rTSST-1v and three to receive 1 mu g Al(OH)(3)). 45 participants (98%) were included in the per-protocol population. rTSST-1v had a good safety profile, and no vaccination-related severe or serious adverse events occurred. Adverse event rates were similar between participants who received rTSST-1v and those who received placebo (26 [76%] vs 10 [83%]; p=0.62) independent of pre-existing TSST-1 immunity.Interpretation rTSST-1v was safe, well-tolerated, and immunogenic. This study represents an important step in vaccine development to prevent or treat a potentially lethal disease.

Background: Nosocomial infections caused by the bacterial pathogen Staphylococcus aureus can lead to serious complications due to the varying presence of secreted toxins. Comparative studies of genomic information and production rates are needed to assess the pathogenic potential of isolated strains. Genotypic and phenotypic profiling of clinical and colonising isolates of S. aureus was used to characterise the release of exotoxins. Blood isolates were compared with colonisation strains to determine similarities and differences of single strains and clusters.Results: Fifty-one fresh isolates obtained from colonised individuals (n = 29) and S. aureus bacteremia (SAB) patients (n = 22) were investigated. The prevalence of genes encoding for three cytolysins (alpha/beta/gamma toxin) and twenty-four superantigens (SEA-SElX) was determined. Isolates exhibited eighteen distinct combinations of superantigens. Sequence analysis identified mutated open reading frames in hla in 13.7 % of all strains, in selw (92.2 %) and in selx (15.7 %). All corrupted genes were associated with specific clonal complexes. Functional assessment of alpha toxin activity by a rabbit erythrocyte lysis assay revealed that supernatants lacking alpha toxin still displayed hemolysis. This was due to the presence of gamma toxin, as proven by inhibition experiments using antisera raised against the respective recombinant proteins. Alpha toxin, SEC, and TSST1 production was quantified by enzyme-linked immunosorbent assays on supernatants of all hla, sec, and tst positive isolates. Blood isolates and colonising strains showed comparable amounts of secreted proteins within a wide range. Agr types I to IV were identified, but did not allow a prediction of high or low production rates. In contrast, alpha toxin production rates between distinct clonal complexes clearly differed. Spa typing was performed and revealed thirty-two unique spa gene patterns and eight small clusters comprising nineteen isolates. Recognised spa-typing clusters displayed highly similar production rates.Conclusion: Production rates of the three most prevalent exotoxins varied within both groups of blood isolates and colonising strains. By comparing genotypes and secretion, we found that identical complex gene patterns did not allow predictions of toxin production and function. However, identification of spa typing clusters was suitable to predict similar quantities of released exotoxins.