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Real-world data on vaccine-elicited neutralising antibody responses for two-dose AZD1222 in African populations are limited. We assessed baseline SARS-CoV-2 seroprevalence and levels of protective neutralizing antibodies prior to vaccination rollout using binding antibodies analysis coupled with pseudotyped virus neutralisation assays in two cohorts from West Africa: Nigerian healthcare workers (n = 140) and a Ghanaian community cohort (n = 527) pre and post vaccination. We found 44 and 28% of pre-vaccination participants showed IgG anti-N positivity, increasing to 59 and 39% respectively with anti-receptor binding domain (RBD) IgG-specific antibodies. Previous IgG anti-N positivity significantly increased post two-dose neutralizing antibody titres in both populations. Serological evidence of breakthrough infection was observed in 8/49 (16%). Neutralising antibodies were observed to wane in both populations, especially in anti-N negative participants with an observed waning rate of 20% highlighting the need for a combination of additional markers to characterise previous infection. We conclude that AZD1222 is immunogenic in two independent West African cohorts with high background seroprevalence and incidence of breakthrough infection in 2021. Waning titres post second dose indicates the need for booster dosing after AZD1222 in the African setting despite hybrid immunity from previous infection.

Introduction In early 2020, at first surge of the coronavirus disease 2019 (COVID-19) pandemic, many health care workers (HCW) were re-deployed to critical care environments to support intensive care teams looking after patients with severe COVID-19. There was considerable anxiety of increased risk of COVID-19 for these staff. To determine whether critical care HCW were at increased risk of hospital acquired infection, we explored the relationship between workplace, patient facing role and evidence of immune exposure to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a quaternary hospital providing a regional critical care response. Routine viral surveillance was not available at this time. Methods We screened over 500 HCW (25% of the total workforce) for history of clinical symptoms of possible COVID19, assigning a symptom severity score, and quantified SARS-CoV-2 serum antibodies as evidence of immune exposure to the virus. Results Whilst 45% of the cohort reported symptoms that they consider may have represented COVID-19, 14% had evidence of immune exposure. Staffs in patient facing critical care roles were least likely to be seropositive (9%) and staff working in non-patient facing roles most likely to be seropositive (22%). Anosmia and fever were the most discriminating symptoms for seropositive status. Older males presented with more severe symptoms. Of the 12 staff screened positive by nasal swab (10 symptomatic), 3 showed no evidence of seroconversion in convalescence. Conclusions Patient facing staff working in critical care do not appear to be at increased risk of hospital acquired infection however the risk of nosocomial infection from non-patient facing staff may be more significant than previous recognised. Most symptoms ascribed to possible COVID-19 were found to have no evidence of immune exposure however seroprevalence may underrepresent infection frequency. Older male staff were at the greatest risk of more severe symptoms.

BackgroundThere are no real world data on vaccine elicited neutralising antibody responses for the world’s most widely used vaccine, AZD1222, in African populations following scale up. Here, we measured i) baseline SARS-CoV-2 seroprevalence and levels of protective neutralizing antibodies prior to vaccination rollout using both flow cytometric based analysis of binding antibodies to nucleocapsid (N), coupled with virus neutralisation approaches and ii) neutralizing antibody responses to VOC prior to vaccination (January 2021) and after two-doses of AZD1222 vaccine administered with a 12 week interval in Lagos, Nigeria - a period when the Delta variant was circulating.MethodsHealth workers at multiple sites in Lagos were recruited to the study. For binding antibody measurement, IgG antibodies against SARS-COV-2 Wuhan-1 receptor-binding domain (RBD), trimeric spike protein (S), nucleocapsid protein (N) and Omicron S1 were measured using the Luminex-based SARS-CoV-2-IgG assay by flow cytometry. For plasma neutralising antibody measurement, SARS-CoV-2 lentiviral pseudovirus (PV) were prepared by transfecting 293T cells with Wuhan-614G wild type (WT), B.1.617.2 (Delta) and BA.1 (Omicron) plasmids in conjunction with HIV-1 expression vectors and luciferase encoding genome flanked by LTRs. We performed serial plasma dilutions from each time point and mixed plasma with PV before infecting HeLa-ACE2 cell lines, reading out luminescence and calculating ID50 (reciprocal dilution of sera required to inhibit 50% of PV infection).ResultsOur underlying study population receiving at least one dose of vaccine comprised 140 participants with a median age of 40 (interquartile range: 33, 48). 62/140 (44%) participants were anti-N IgG positive prior to administration of first vaccine dose. 49 had plasma samples available at baseline prior to vaccination and at two follow-up timepoints post two dose vaccination for neutralization assays. Half of the participants, 25/49 (51%) were IgG anti-N positive at baseline. Of the 24 individuals anti-N Ab negative at baseline, 12/24 had ID50 above the cut-off of 20. In these individuals, binding antibodies to S were also detectable, and neutralisation correlated with IgG anti-S, suggesting waning of N antibody after infection. Overall, neutralizing Ab titres to WT 1 month after second dose were 2579 and at 3 months post second-dose were 1695. As expected, lower levels of neutralization were observed against the Delta GMT 549 and Omicron variants 269 at 1 month. Positive anti-N IgG Ab status at baseline was associated with significantly higher titres of neutralizing antibodies following vaccination across all tested VOC. Those with anti-N Abs present at baseline did not experience waning of responses between months 1 and 3 post second dose. When data were analysed for negative anti-N IgG status at any timepoint, there was a significant decline in neutralization and binding antibodies between 1 month and 3 months post second-dose. The GMT in these individuals for Delta and Omicron was approximately 100, nearly a log lower in comparison to WT. We tested anti-N IgG in subjects who were anti-N IgG negative at baseline (n=78) and became positive between 1- and 3-months post second dose and found 7/49 (14%) with de-novo infection, with one additional participant demonstrating both reinfection and breakthrough infection to yield a total breakthrough rate of 8/49 (16%). Neutralising and binding Ab titres 1 month post vaccine, prior to breakthrough, were not associated with breakthrough infection. Neutralizing titres were higher at the last time point in individuals who had experienced vaccine breakthrough infection (with no evidence of infection prior to vaccine), indicating a boosting effect of infection in addition to vaccine. However, neutralisation and binding S antibodies against Omicron were low in those with either prior exposure or infection following two dose AZD1222.ConclusionsAZD1222 is immunogenic in this real world west African cohort with significant background seroprevalence and incidence of breakthrough infection over a short time period. Prior infection and breakthrough infection induced higher anti-SARS-CoV-2 Ab responses at 3 months post vaccine against all widely circulating VOC. However, responses to Omicron BA.1 were low at three months regardless of hybrid immunity from prior exposure or breakthrough infection. Booster doses after AZD1222 should be considered in the African setting, even after natural infection, as future variants may be more pathogenic as well as immune evasive in the context of waning immunity.

With the first 2020 surge of the COVID-19 pandemic, many health care workers (HCW) were re-deployed to critical care environments to support intensive care teams to look after high numbers of patients with severe COVID-19. There was considerable anxiety of increased risk of COVID19 for staff working in these environments.Using a multiplex platform to assess serum IgG responses to SARS-CoV-2 N, S and RBD proteins, and detailed symptom reporting, we screened over 500 HCW (25% of the total workforce) in a quaternary level hospital to explore the relationship between workplace and evidence of exposure to SARS-CoV-2.Whilst 45% of the cohort reported symptoms that they consider may have represented COVID-19, overall seroprevalence was 14% with anosmia and fever being the most discriminating symptoms for seropositive status. There was a significant difference in seropositive status between staff working in clinical and non-clinical roles (9% patient facing critical care, 15% patient facing non-critical care, 22% nonpatient facing). In the seropositive cohort, symptom severity increased with age for men and not for women. In contrast, there was no relationship between symptom severity and age or sex in the seronegative cohort reporting possible COVID-19 symptoms. Of the 12 staff screened PCR positive (10 symptomatic), 3 showed no evidence of seroconversion in convalescence.ConclusionThe current approach to Personal Protective Equipment (PPE) appears highly effective in protecting staff from patient acquired infection in the critical care environment including protecting staff managing interhospital transfers of COVID-19 patients. The relationship between seroconversion and disease severity in different demographics warrants further investigation. Longitudinally paired virological and serological surveillance, with symptom reporting are urgently required to better understand the role of antibody in the outcome of HCW exposure during subsequent waves of COVID-19 in health care environments.

BackgroundImmune defects in chronic pulmonary aspergillosis (CPA) are poorly characterized. We compared peripheral blood cytokine profiles in patients with CPA versus healthy controls and explored the relationship with disease severity.MethodsInterferon-gamma (IFNγ), interleukin (IL)-17, tumor necrosis factor-α, IL-6, IL-12, and IL-10 were measured after in vitro stimulation of whole blood with lipopolysaccharide (LPS), phytohemagglutinin, β-glucan, zymosan (ZYM), IL-12 or IL-18, and combinations. Clinical parameters and mortality were correlated with cytokine production.ResultsCytokine profiles were evaluated in 133 patients (57.1% male, mean age 61 years). In comparison to controls, patients with CPA had significantly reduced production of IFNγ in response to stimulation with β-glucan + IL-12 (312 vs 988 pg/mL), LPS + IL-12 (252 vs 1033 pg/mL), ZYM + IL-12 (996 vs 2347 pg/mL), and IL-18 + IL-12 (7193 vs 12 330 pg/mL). Age >60 (hazard ratio [HR], 1.71; 95% confidence interval [CI], 1.00–2.91; P = .05) and chronic obstructive pulmonary disease (HR, 1.69; 95% CI, 1.03–2.78; P = .039) were associated with worse survival, whereas high IFNγ production in response to beta-glucan + IL-12 stimulation (HR, 0.48; 95% CI, .25–0.92; P = .026) was associated with reduced mortality.ConclusionsPatients with CPA show impaired IFNγ production in peripheral blood in response to stimuli. Defective IFNγ production ability correlates with worse outcomes. Immunotherapy with IFNγ could be beneficial for patients showing impaired IFNγ production in CPA.

Vaccines remain the cornerstone for containing the SARS-CoV-2 pandemic. mRNA vaccines provide protection in clinical trials using a two-dose approach, separated by a three to four week gap. UK policy in 2021 is to extend the dosing interval from three to twelve weeks and other countries are likely to follow suit given the demand for mRNA vaccines and ongoing uncontrolled transmission. There is a paucity of data in the elderly, even though these individuals are the first to receive vaccines due to risk of severe disease. Here we assessed real world immune responses following vaccination with mRNA-based vaccine BNT162b2. Median age was 81 years amongst 101 participants after the first dose of the BNT162b2 vaccine. Geometric mean neutralisation titres in participants over 80 years old after the first dose were lower than in younger individuals [83.4 (95% CI 52.0-133.7) vs 46.6 (95% CI 33.5-64.8) p 0.01]. A lower proportion of participants 80 years and older achieved adequate neutralisation titre of >1:20 for 50% neutralisation as compared to those under 80 (21% vs 51%, p 0.003). Binding IgG responses correlated with neutralisation. Sera from participants in both age groups showed significantly lower neutralisation potency against B.1.1.7 Spike pseudotyped viruses as compared to wild type. The adjusted ORs for inadequate neutralisation in the 80 years and above age group were 3.7 (95% CI 1.2-11.2) and 4.4 (95% CI 1.5-12.6) against wild type and B.1.1.7 pseudotyped viruses. We observed a trend towards lower somatic hypermutation in participants with suboptimal neutralisation, and elderly participants demonstrated clear reduction in class switched somatic hypermutation, driven by the IgA1/2 isotype. SARS-CoV-2 Spike specific T- cell IFN𝛾 and IL-2 responses were impaired in the older age group after 1 dose and although IFN𝛾 increased between vaccine doses, IL-2 responses did not significantly increase. There was a significantly higher risk of suboptimal neutralising antibody and T cell response following first dose vaccination with BNT162b2 in half of participants above the age of 80, persisting up to 12 weeks. These high risk populations warrant specific measures in order to mitigate against vaccine failure, particularly where SARS-CoV-2 variants of concern are circulating.

Background Vaccines remain the cornerstone for containing the SARS-CoV-2 pandemic. mRNA vaccines provide protection in clinical trials using a two-dose approach, separated by a three to four week gap. UK policy in 2021 is to extend the dosing interval from three to twelve weeks. There is a paucity of data in the elderly, even though these individuals are the first to receive vaccines due to risk of severe disease. Here we assessed real world immune responses following vaccination with mRNA-based vaccine BNT162b2. Methods We did a prospective cohort study of individuals presenting for first dose vaccination. Following the first and second doses of the BNT162b2 vaccine, we measured IFNγ T cell responses, as well as binding antibody (IgA, IgG and IgG1-4) responses to Spike and Spike RBD. We also measured neutralising antibody responses to Spike in sera using a lentiviral pseudotyping system. We correlated age with immune responses and compared responses after the first and second doses. Findings Median age was 63.5 years amongst 42 participants. Three weeks after the first dose a lower proportion of participants over 80 years old achieved adequate neutralisation titre of >1:20 for 50% neutralisation as compared to those under 80 (8/17 versus 19/24, p=0.03). Geometric mean neutralisation titres in this age group after the first dose were lower than in younger individuals (p Interpretation A high proportion of individuals above the age of 80 have suboptimal neutralising antibody responses following first dose vaccination with BNT162b2, cautioning against extending the dosing interval in this high risk population. Research in Context Evidence before this study We searched PubMed for research articles published from June 1st 2020 until February 8th 2021. We limited our search to English language papers. We used the following terms: “SARS-CoV-2” AND “vaccine” OR “BNT162b2” OR “Pfizer/BioNTech”. We identified only one paper. It showed lower neutralising antibody responses following the first dose of BNT162b in a small group of 12 individuals over 65 compared to those under 65. There were no data for patients above 82 years of age and no data on T cell responses by age. We did not find pre-prints on age related heterogeneity in individuals immunised with the Pfizer/BioNtech mRNA vaccine. What this study adds We show real world immune responses in forty two individuals to BNT162b2, spanning both T and B cell arms. We show that a high proportion of individuals above the age of 80 have suboptimal neutralising antibody responses following first dose vaccination with BNT162b2. The second dose generates robust responses in these poor responders. We quantify SARS-CoV-2 Spike and receptor binding domain (RBD) IgA and IgG isotypes as well as IgG subclasses. Finally we show that SARS-CoV-2 specific T cell responses are robustly induced by first dose vaccination and are not impacted by age. Implications of all the available evidence These data caution against extending the dosing interval of BNT162b2 in the elderly population, particularly during periods of high transmission, and also where there is risk of infection with variants that are less susceptible to vaccine-elicited neutralising antibodies.

SARS-CoV-2 transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 mutations found in the B.1.1.7 Spike protein. The vaccine sera exhibited a broad range of neutralizing titres against the wild-type pseudoviruses (

The majority of SARS-CoV-2 vaccines in use or in advanced clinical development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described that act against both open and closed conformations. The long-term success of vaccination strategies will depend upon inducing antibodies that provide long-lasting broad immunity against evolving, circulating SARS-CoV-2 strains, while avoiding the risk of antibody dependent enhancement as observed with other Coronavirus vaccines. Here we have assessed the results of immunization in a mouse model using an S protein trimer that is arrested in the closed state to prevent exposure of the receptor binding site and therefore interaction with the receptor. We compared this with a range of other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induce a long-lived, strongly neutralizing antibody response as well as T-cell responses. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralising responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, virus-inhibiting immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines.

Background: Vaccines remain the cornerstone for containing the SARS-CoV-2 pandemic. mRNA vaccines provide protection in clinical trials using a two-dose approach, separated by a three to four week gap. UK policy in 2021 is to extend the dosing interval from three to twelve weeks. There is a paucity of data in the elderly, even though these individuals are the first to receive vaccines due to risk of severe disease. Here we assessed real world immune responses following vaccination with mRNA-based vaccine BNT162b2. Methods: We did a prospective cohort study of individuals presenting for first dose vaccination. Following the first and second doses of the BNT162b2 vaccine, we measured IFNγ T cell responses, as well as binding antibody (IgA, IgG and IgG1-4) responses to Spike and Spike RBD. We also measured neutralising antibody responses to Spike in sera using a lentiviral pseudotyping system. We correlated age with immune responses and compared responses after the first and second doses. Findings: Median age was 63.5 years amongst 42 participants. Three weeks after the first dose a lower proportion of participants over 80 years old achieved adequate neutralisation titre of >1:20 for 50% neutralisation as compared to those under 80 (8/17 versus 19/24, p=0.03). Geometric mean neutralisation titres in this age group after the first dose were lower than in younger individuals (p

SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2, and amino acid variation in Spike is increasingly appreciated. Given both vaccines and therapeutics are designed around Wuhan-1 Spike, this raises the theoretical possibility of virus escape, particularly in immunocompromised individuals where prolonged viral replication occurs. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences by both short and long read technologies over 23 time points spanning 101 days. Although little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days, N501Y in Spike was transiently detected at day 55 and V157L in RdRp emerged. However, following convalescent plasma we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro, the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility, but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type and appeared to compensate for the reduced infectivity of D796H. Consistent with the observed mutations being outside the RBD, monoclonal antibodies targeting the RBD were not impacted by either or both mutations, but a non RBD binding monoclonal antibody was less potent against ΔH69/ΔV70 and the double mutant. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with reduced susceptibility to neutralising antibodies.

The response to the coronavirus disease 2019 (COVID-19) pandemic has been hampered by lack of an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral therapy. Here we report the successful use of remdesivir in a patient with COVID-19 and the prototypic genetic antibody deficiency X-linked agammaglobulinaemia (XLA). Despite evidence of complement activation and a robust T cell response, the patient developed persistent SARS-CoV-2 pneumonitis, without progressing to multi-organ involvement. His unusual clinical course identifies a key role for SARS-CoV-2 antibodies in both viral clearance and progression to severe disease. In the absence of these confounders, we took an experimental medicine approach to examine the in vivoutility of remdesivir. Over two independent courses of treatment, we observed a dramatic, temporally correlated clinical and virological response, leading to clinical resolution and viral clearance, with no evidence of acquired drug resistance. We therefore provide unambiguous evidence for the antiviral efficacy of remdesivir in vivo, and its potential benefit in selected patients.

Increased extracellular sodium activates Th17 cells, which provide protection from bacterial and fungal infections. Whilst high salt diets have been shown to worsen autoimmune disease, the immunological consequences of clinical salt depletion are unknown. Here, we investigate immunity in patients with inherited salt-losing tubulopathies (SLT). Forty-seven genotyped SLT patients (with Bartter, Gitelman or EAST Syndromes) are recruited. Clinical features of dysregulated immunity are recorded with a standardised questionnaire and immunological investigations of IL-17 responsiveness undertaken. The effects of altering extracellular ionic concentrations on immune responses are then assessed. Patients are hypokalaemic and hypomagnesaemic, with reduced interstitial sodium stores determined by 23Na-magnetic resonance imaging. SLT patients report increased mucosal infections and allergic disease compared to age-matched controls. Aligned with their clinical phenotype, SLT patients have an increased ratio of Th2:Th17 cells. SLT Th17 and Tc17 polarisation is reduced in vitro, yet STAT1 and STAT3 phosphorylation and calcium flux following T cell activation are unaffected. In control cells, the addition of extracellular sodium (+40 mM), potassium (+2 mM), or magnesium (+1 mM) reduces Th2:Th17 ratio and augments Th17 polarisation. Our results thus show that the ionic environment typical in SLT impairs IL-17 immunity, but the intracellular pathways that mediate salt-driven Th17 polarisation are intact and in vitro IL-17 responses can be reinvigorated by increasing extracellular sodium concentration. Whether better correction of extracellular ions can rescue the immunophenotype in vivo in SLT patients remains unknown., Salt levels in culture affect the polarisation of Th17 cells, which normally protect the host from fungal and bacterial infections. Here, the authors study patients with salt-losing tubulopathies (SLT) to find that, while Th17 immunity is dampened in SLT patients, their Th17-inducing signaling pathways are intact and can be reinvigorated by exogenous salt.

Autoantibodies against cytokines have been associated with immunodeficiency, susceptibility to infectious diseases, autoimmunity and inflammation in humans, but have not yet been investigated in the Veterinary field so far. The aim of the current study was to determine the presence of anti-cytokine autoantibodies in canines suffering from various conditions including recurrent infections, autoimmune diseases and cancer in comparison to healthy controls. This is the first report of the presence of autoantibodies against cytokines in dogs. A total of 101 serum samples (51 patients and 50 clinically healthy dogs) from the state of Mexico and surroundings were analysed using a multiplex bead-based flow cytometry assay. Results show significant levels of various anti-cytokine autoantibodies in diseased dogs but not in healthy controls. In addition we show distinct associations of various disease types to the specificity of anti-cytokine autoantibodies and to response complexities. Apart from the direct functional/causal implication of anti-cytokine auto-antibodies on disease processes, this findings point to the possibility to use anti-cytokine response patterns as diagnostic tools.

Humans as models of human disease Mice are a convenient model for exploring the functions of cellular signaling pathways. Occasionally, however, an “experiment of nature” highlights the perils of overreliance on mice. RIPK1 is a well studied protein kinase that regulates cell death. Mice deficient in RIPK1 die soon after birth because of the protein's widespread role in multiple tissues and organs. Cuchet-Lourenço et al. studied patients with inherited immunodeficiency of unknown cause (see the Perspective by Pasparakis and Kelliher). They identified inactivating mutations in the RIPK1 gene in four individuals. Unlike what has been seen in mice, the deleterious effects of RIPK1 loss in humans were confined to the immune system, a finding with potential therapeutic implications. Science , this issue p. 810 ; see also p. 756

RIPK1 (receptor-interacting serine/threonine kinase 1) is a master regulator of signaling pathways leading to inflammation and cell death and is of medical interest as a drug target. We report four patients from three unrelated families with complete RIPK1 deficiency caused by rare homozygous mutations. The patients suffered from recurrent infections, early-onset inflammatory bowel disease, and progressive polyarthritis. They had immunodeficiency with lymphopenia and altered production of various cytokines revealed by whole-blood assays. In vitro, RIPK1-deficient cells showed impaired mitogen-activated protein kinase activation and cytokine secretion and were prone to necroptosis. Hematopoietic stem cell transplantation reversed cytokine production defects and resolved clinical symptoms in one patient. Thus, RIPK1 plays a critical role in the human immune system.

BACKGROUND The genetic analysis of human primary immunodeficiencies has defined the contribution of specific cell populations and molecular pathways in the host defense against infection. Disseminated infection caused by bacille Calmette-Guerin (BCG) vaccines is an early manifestation of primary immunodeficiencies, such as severe combined immunodeficiency. In many affected persons, the cause of disseminated BCG disease is unexplained. METHODS We evaluated an infant presenting with features of severe immunodeficiency, including early-onset disseminated BCG disease, who required hematopoietic stem-cell transplantation. We also studied two otherwise healthy subjects with a history of disseminated but curable BCG disease in childhood. We characterized the monocyte and dendritic-cell compartments in these three subjects and sequenced candidate genes in which mutations could plausibly confer susceptibility to BCG disease. RESULTS We detected two distinct disease-causing mutations affecting interferon regulatory factor 8 (IRF8). Both K108E and T80A mutations impair IRF8 transcriptional activity by disrupting the interaction between IRF8 and DNA. The K108E variant was associated with an autosomal recessive severe immunodeficiency with a complete lack of circulating monocytes and dendritic cells. The T80A variant was associated with an autosomal dominant, milder immunodeficiency and a selective depletion of CD11c+CD1c+ circulating dendritic cells. CONCLUSIONS These findings define a class of human primary immunodeficiencies that affect the differentiation of mononuclear phagocytes. They also show that human IRF8 is critical for the development of monocytes and dendritic cells and for antimycobacterial immunity. (Funded by the Medical Research Council and others.).

We evaluated a patient with disseminated Mycobacterium tuberculosis and Mycobacterium chelonae infection, of which he died. He also developed autoimmune (type I) diabetes and primary hypothyroidism. His serum contained a high titer of immunoglobulin G autoantibody to interferon-gamma (IFN-gamma) capable of blocking in vitro responses to this cytokine by peripheral blood mononuclear cells from normal donors. These results suggest that autoantibodies to IFN-gamma can induce susceptibility to disseminated mycobacterial infection, which may be refractory to chemotherapy.

Background Anti-cytokine autoantibodies (ACAAs) are pathogenic in a handful of rare immunodeficiencies. However, the prevalence and significance of other ACAAs across immunodeficiencies have not yet been described. Objective We profiled ACAAs in a diverse cohort of serum samples from patients with immunodeficiency and assessed the sensitivity and specificity of protein microarrays for ACAA identification and discovery. Methods Highly multiplexed protein microarrays were designed and fabricated. Blinded serum samples from a cohort of 58 immunodeficiency patients and healthy control subjects were used to probe microarrays. Unsupervised hierarchical clustering was used to identify clusters of reactivity, and after unblinding, significance analysis of microarrays was used to identify disease-specific autoantibodies. A bead-based assay was used to validate protein microarray results. Blocking activity of serum containing ACAAs was measured in vitro . Results Protein microarrays were highly sensitive and specific for the detection of ACAAs in patients with autoimmune polyendocrine syndrome type I and pulmonary alveolar proteinosis, detecting ACAA levels consistent with those reported in the published literature. Protein microarray results were validated by using an independent bead-based assay. To confirm the functional significance of these ACAAs, we tested and confirmed the blocking activity of select ACAAs in vitro . Conclusion Protein microarrays are a powerful tool for ACAA detection and discovery, and they hold promise as a diagnostic for the evaluation and monitoring of clinical immunodeficiency.

Selenium, a trace element that is fundamental to human health, is incorporated into some proteins as selenocysteine (Sec), generating a family of selenoproteins. Sec incorporation is mediated by a multiprotein complex that includes Sec insertion sequence-binding protein 2 (SECISBP2; also known as SBP2). Here, we describe subjects with compound heterozygous defects in the SECISBP2 gene. These individuals have reduced synthesis of most of the 25 known human selenoproteins, resulting in a complex phenotype. Azoospermia, with failure of the latter stages of spermatogenesis, was associated with a lack of testis-enriched selenoproteins. An axial muscular dystrophy was also present, with features similar to myopathies caused by mutations in selenoprotein N (SEPN1). Cutaneous deficiencies of antioxidant selenoenzymes, increased cellular ROS, and susceptibility to ultraviolet radiation-induced oxidative damage may mediate the observed photosensitivity. Reduced levels of selenoproteins in peripheral blood cells were associated with impaired T lymphocyte proliferation, abnormal mononuclear cell cytokine secretion, and telomere shortening. Paradoxically, raised ROS in affected subjects was associated with enhanced systemic and cellular insulin sensitivity, similar to findings in mice lacking the antioxidant selenoenzyme glutathione peroxidase 1 (GPx1). Thus, mutation of SECISBP2 is associated with a multisystem disorder with defective biosynthesis of many selenoproteins, highlighting their role in diverse biological processes.

The case of a previously healthy HIV seronegative woman with disseminated Mycobacterium tuberculosis infection and markedly reduced interferon gamma production is reported here. Complete healing of her disseminated lesions was seen only after addition of subcutaneous interferon gamma to her tuberculosis treatment.