SARS-CoV-2 Seroprevalence Study in Pediatric Patients and Health Care Workers Using Multiplex Antibody Immunoassays.

SARS-CoV-2 infection has become a global health problem specially exacerbated with the continuous appearance of new variants. Healthcare workers (HCW) have been one of the most affected sectors. Children have also been affected, and although infection generally presents as a mild disease, some have developed the Pediatric Inflammatory Multisystem Syndrome Temporally Associated with SARS-CoV-2 (PIMS-TS). We recruited 190 adults (HCW and cohabitants, April to June 2020) and 57 children (April 2020 to September 2021), of whom 12 developed PIMS-TS, in a hospital-based study in Spain. Using an in-house Luminex assay previously validated, antibody levels were measured against different spike and nucleocapsid SARS-CoV-2 proteins, including the receptor-binding domain (RBD) of the Alpha, Beta, Gamma, and Delta variants of concern (VoC). Seropositivity rates obtained from children and adults, respectively, were: 49.1% and 11% for IgG, 45.6% and 5.8% for IgA, and 35.1% and 7.3% for IgM. Higher antibody levels were detected in children who developed PIMS-TS compared to those who did not. Using the COVID-19 IgM/IgA ELISA (Vircell, S.L.) kit, widely implemented in Spanish hospitals, a high number of false positives and lower seroprevalences compared with the Luminex estimates were found, indicating a significantly lower specificity and sensitivity. Comparison of antibody levels against RBD-Wuhan versus RBD-VoCs indicated that the strongest positive correlations for all three isotypes were with RBD-Alpha, while the lowest correlations were with RBD-Delta for IgG, RBD-Gamma for IgM, and RBD-Beta for IgA. This study highlights the differences in antibody levels between groups with different demographic and clinical characteristics, as well as reporting the IgG, IgM, and IgA response to RBD VoC circulating at the study period.

Impact of JAK Inhibitors in Pediatric Patients with STAT1 Gain of Function (GOF) Mutations-10 Children and Review of the Literature.

INTRODUCTION: Since the first description of gain of function (GOF) mutations in signal transducer and activator of transcription (STAT) 1, more than 300 patients have been described with a broad clinical phenotype including infections and severe immune dysregulation. Whilst Jak inhibitors (JAKinibs) have demonstrated benefits in several reported cases, their indications, dosing, and monitoring remain to be established. METHODS: A retrospective, multicenter study recruiting pediatric patients with STAT1 GOF under JAKinib treatment was performed and, when applicable, compared with the available reports from the literature. RESULTS: Ten children (median age 8.5 years (3-18), receiving JAKinibs (ruxolitinib (n = 9) and baricitinib (n = 1)) with a median follow-up of 18 months (2-42) from 6 inborn errors of immunity (IEI) reference centers were included. Clinical profile and JAKinib indications in our series were similar to the previously published 14 pediatric patients. 9/10 (our cohort) and 14/14 patients (previous reports) showed partial or complete responses. The median immune deficiency and dysregulation activity scores were 15.99 (5.2-40) pre and 7.55 (3-14.1) under therapy (p = 0.0078). Infection, considered a likely adverse event of JAKinib therapy, was observed in 1/10 patients; JAKinibs were stopped in 3/10 children, due to hepatotoxicity, pre-HSCT, and absence of response. CONCLUSIONS: Our study supports the potentially beneficial use of JAKinibs in patients with STAT1 GOF, in line with previously published data. However, consensus regarding their indications and timing, dosing, treatment duration, and monitoring, as well as defining biomarkers to monitor clinical and immunological responses, remains to be determined, in form of international prospective multicenter studies using established IEI registries.

Primary Immune Regulatory Disorders With an Autoimmune Lymphoproliferative Syndrome-Like Phenotype: Immunologic Evaluation, Early Diagnosis and Management.

Primary immune regulatory disorders (PIRD) are associated with autoimmunity, autoinflammation and/or dysregulation of lymphocyte homeostasis. Autoimmune lymphoproliferative syndrome (ALPS) is a PIRD due to an apoptotic defect in Fas-FasL pathway and characterized by benign and chronic lymphoproliferation, autoimmunity and increased risk of lymphoma. Clinical manifestations and typical laboratory biomarkers of ALPS have also been found in patients with a gene defect out of the Fas-FasL pathway (ALPS-like disorders). Following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA), we identified more than 600 patients suffering from 24 distinct genetic defects described in the literature with an autoimmune lymphoproliferative phenotype (ALPS-like syndromes) corresponding to phenocopies of primary immunodeficiency (PID) (NRAS, KRAS), susceptibility to EBV (MAGT1, PRKCD, XIAP, SH2D1A, RASGRP1, TNFRSF9), antibody deficiency (PIK3CD gain of function (GOF), PIK3R1 loss of function (LOF), CARD11 GOF), regulatory T-cells defects (CTLA4, LRBA, STAT3 GOF, IL2RA, IL2RB, DEF6), combined immunodeficiencies (ITK, STK4), defects in intrinsic and innate immunity and predisposition to infection (STAT1 GOF, IL12RB1) and autoimmunity/autoinflammation (ADA2, TNFAIP3,TPP2, TET2). CTLA4 and LRBA patients correspond around to 50% of total ALPS-like cases. However, only 100% of CTLA4, PRKCD, TET2 and NRAS/KRAS reported patients had an ALPS-like presentation, while the autoimmunity and lymphoproliferation combination resulted rare in other genetic defects. Recurrent infections, skin lesions, enteropathy and malignancy are the most common clinical manifestations. Some approaches available for the immunological study and identification of ALPS-like patients through flow cytometry and ALPS biomarkers are provided in this work. Protein expression assays for NKG2D, XIAP, SAP, CTLA4 and LRBA deficiencies and functional studies of AKT, STAT1 and STAT3 phosphorylation, are showed as useful tests. Patients suspected to suffer from one of these disorders require rapid and correct diagnosis allowing initiation of tailored specific therapeutic strategies and monitoring thereby improving the prognosis and their quality of life.

Biallelic TRAF3IP2 variants causing chronic mucocutaneous candidiasis in a child harboring a STAT1 variant.

BACKGROUND: Inherited chronic mucocutaneous candidiasis (CMC) is often caused by inborn errors of immunity, impairing the response to, or the production of IL-17A and IL-17F. About half of the cases carry STAT1 gain-of-function (GOF) mutations. Only few patients have been reported with mutations of TRAF3IP2, a gene encoding the adaptor ACT1 essential for IL-17 receptor(R) signaling. We investigated a 10-year-old girl with CMC, carrying a heterozygous variant of STAT1 and compound heterozygous variants of TRAF3IP2. METHODS: By flow cytometry, STAT1 levels and phosphorylation (CD14+) as well as IL-17A, IL-22, IFN-γ, and IL-4 production (memory CD4+ T cells) were determined. ACT1 expression and binding to IL-17RA were assessed by Western blot and co-immunoprecipitation in HEK-293T cells transfected with plasmids encoding wild-type or mutant HA-tagged ACT1 and Flag-IL-17RA. We evaluated IL-17A responses by measuring luciferase induction under a NF-κB-driven reporter system in HEK-293T cells and Gro-α secretion in fibroblasts. RESULTS: A STAT1 variant (c.1363G>A/p.V455I) was identified by next-generation sequencing and classified as likely non-pathogenic as functional testing revealed normal STAT1 expression and phosphorylation upon IFN-γ. We also found compound heterozygous variants (c.1325A>G/p.D451G and c.1335delA/p.K454fs11*) of TRAF3IP2. By overexpression, despite normal protein expression, and impaired (K454fs11*) or normal (D451G) interaction with IL-17RA, both mutant alleles resulted in impaired NF-κB activation. Patient's fibroblasts displayed abolished GRO-α secretion upon IL-17A stimulation. Finally, ex vivo CD4+ T cells showed increased IL-17A, IL-22, and IL-4 and normal low IFN-γ expression upon stimulation. CONCLUSION: We identify novel compound heterozygous variants of TRAFP3IP2 causing autosomal recessive ACT1 deficiency in a child with CMC and provide a review of the current literature.

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.

Identification and Characterization of Novel Compounds with Broad-Spectrum Antiviral Activity against Influenza A and B Viruses.

Influenza A (IAV) and influenza B (IBV) viruses are highly contagious pathogens that cause fatal respiratory disease every year, with high economic impact. In addition, IAV can cause pandemic infections with great consequences when new viruses are introduced into humans. In this study, we evaluated 10 previously described compounds with antiviral activity against mammarenaviruses for their ability to inhibit IAV infection using our recently described bireporter influenza A/Puerto Rico/8/34 (PR8) H1N1 (BIRFLU). Among the 10 tested compounds, eight (antimycin A [AmA], brequinar [BRQ], 6-azauridine, azaribine, pyrazofurin [PF], AVN-944, mycophenolate mofetil [MMF], and mycophenolic acid [MPA]), but not obatoclax or Osu-03012, showed potent anti-influenza virus activity under posttreatment conditions [median 50% effective concentration (EC50) = 3.80 nM to 1.73 µM; selective index SI for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, >28.90 to 13,157.89]. AmA, 6-azauridine, azaribine, and PF also showed potent inhibitory effect in pretreatment (EC50 = 0.14 µM to 0.55 µM; SI-MTT = 70.12 to >357.14) or cotreatment (EC50 = 34.69 nM to 7.52 µM; SI-MTT = 5.24 to > 1,441.33) settings. All of the compounds tested inhibited viral genome replication and gene transcription, and none of them affected host cellular RNA polymerase II activities. The antiviral activity of the eight identified compounds against BIRFLU was further confirmed with seasonal IAVs (A/California/04/2009 H1N1 and A/Wyoming/3/2003 H3N2) and an IBV (B/Brisbane/60/2008, Victoria lineage), demonstrating their broad-spectrum prophylactic and therapeutic activity against currently circulating influenza viruses in humans. Together, our results identified a new set of antiviral compounds for the potential treatment of influenza viral infections.IMPORTANCE Influenza viruses are highly contagious pathogens and are a major threat to human health. Vaccination remains the most effective tool to protect humans against influenza infection. However, vaccination does not always guarantee complete protection against drifted or, more noticeably, shifted influenza viruses. Although U.S. Food and Drug Administration (FDA) drugs are approved for the treatment of influenza infections, influenza viruses resistant to current FDA antivirals have been reported and continue to emerge. Therefore, there is an urgent need to find novel antivirals for the treatment of influenza viral infections in humans, a search that could be expedited by repurposing currently approved drugs. In this study, we assessed the influenza antiviral activity of 10 compounds previously shown to inhibit mammarenavirus infection. Among them, eight drugs showed antiviral activities, providing a new battery of drugs that could be used for the treatment of influenza infections.

Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines.

Influenza viruses cause annual, seasonal infection across the globe. Vaccination represents the most effective strategy to prevent such infections and/or to reduce viral disease. Two major types of influenza vaccines are approved for human use: inactivated influenza vaccines (IIVs) and live attenuated influenza vaccines (LAIVs). Two Master Donor Virus (MDV) backbones have been used to create LAIVs against influenza A virus (IAV): the United States (US) A/Ann Arbor/6/60 (AA) and the Russian A/Leningrad/134/17/57 (Len) H2N2 viruses. The mutations responsible for the temperature sensitive (ts), cold-adapted (ca) and attenuated (att) phenotypes of the two MDVs have been previously identified and genetically mapped. However, a direct comparison of the contribution of these residues to viral attenuation, immunogenicity and protection efficacy has not been conducted. Here, we compared the In vitro and in vivo phenotype of recombinant influenza A/Puerto Rico/8/34 H1N1 (PR8) viruses containing the ts, ca and att mutations of the US (PR8/AA) and the Russian (PR8/Len) MDVs. Our results show that PR8/Len is more attenuated in vivo than PR8/AA, although both viruses induced similar levels of humoral and cellular responses, and protection against homologous and heterologous viral challenges. Our findings support the feasibility of using a different virus backbone as MDV for the development of improved LAIVs for the prevention of IAV infections.

A Bivalent Live-Attenuated Vaccine for the Prevention of Equine Influenza Virus.

Vaccination remains the most effective approach for preventing and controlling equine influenza virus (EIV) in horses. However, the ongoing evolution of EIV has increased the genetic and antigenic differences between currently available vaccines and circulating strains, resulting in suboptimal vaccine efficacy. As recommended by the World Organization for Animal Health (OIE), the inclusion of representative strains from clade 1 and clade 2 Florida sublineages of EIV in vaccines may maximize the protection against presently circulating viral strains. In this study, we used reverse genetics technologies to generate a bivalent EIV live-attenuated influenza vaccine (LAIV). We combined our previously described clade 1 EIV LAIV A/equine/Ohio/2003 H3N8 (Ohio/03 LAIV) with a newly generated clade 2 EIV LAIV that contains the six internal genes of Ohio/03 LAIV and the HA and NA of A/equine/Richmond/1/2007 H3N8 (Rich/07 LAIV). The safety profile, immunogenicity, and protection efficacy of this bivalent EIV LAIV was tested in the natural host, horses. Vaccination of horses with the bivalent EIV LAIV, following a prime-boost regimen, was safe and able to confer protection against challenge with clade 1 (A/equine/Kentucky/2014 H3N8) and clade 2 (A/equine/Richmond/2007) wild-type (WT) EIVs, as evidenced by a reduction of clinical signs, fever, and virus excretion. This is the first description of a bivalent LAIV for the prevention of EIV in horses that follows OIE recommendations. In addition, since our bivalent EIV LAIV is based on the use of reverse genetics approaches, our results demonstrate the feasibility of using the backbone of clade 1 Ohio/03 LAIV as a master donor virus (MDV) for the production and rapid update of LAIVs for the control and protection against other EIV strains of epidemiological relevance to horses.

Novel Approaches for The Development of Live Attenuated Influenza Vaccines.

Influenza virus still represents a considerable threat to global public health, despite the advances in the development and wide use of influenza vaccines. Vaccination with traditional inactivate influenza vaccines (IIV) or live-attenuated influenza vaccines (LAIV) remains the main strategy in the control of annual seasonal epidemics, but it does not offer protection against new influenza viruses with pandemic potential, those that have shifted. Moreover, the continual antigenic drift of seasonal circulating influenza viruses, causing an antigenic mismatch that requires yearly reformulation of seasonal influenza vaccines, seriously compromises vaccine efficacy. Therefore, the quick optimization of vaccine production for seasonal influenza and the development of new vaccine approaches for pandemic viruses is still a challenge for the prevention of influenza infections. Moreover, recent reports have questioned the effectiveness of the current LAIV because of limited protection, mainly against the influenza A virus (IAV) component of the vaccine. Although the reasons for the poor protection efficacy of the LAIV have not yet been elucidated, researchers are encouraged to develop new vaccination approaches that overcome the limitations that are associated with the current LAIV. The discovery and implementation of plasmid-based reverse genetics has been a key advance in the rapid generation of recombinant attenuated influenza viruses that can be used for the development of new and most effective LAIV. In this review, we provide an update regarding the progress that has been made during the last five years in the development of new LAIV and the innovative ways that are being explored as alternatives to the currently licensed LAIV. The safety, immunogenicity, and protection efficacy profile of these new LAIVs reveal their possible implementation in combating influenza infections. However, efforts by vaccine companies and government agencies will be needed for controlled testing and approving, respectively, these new vaccine methodologies for the control of influenza infections.

Applying lessons learned from cytomegalovirus infection in transplant patients to vaccine design.

Studies in transplant recipients over the past decade aiming to characterize the immune response to cytomegalovirus (CMV) replication have provided insights that can be used to guide CMV vaccine development. These studies have characterized multiple aspects of the immune response to virus infection in humans, and have identified immunologic variables that correlate with the ability to control virus replication. These findings can be used to guide vaccine development by informing decisions regarding antigen selection and the type of immune response that must be elicited by these antigens to promote protective immunity. In addition, these studies have provided information that could aid in the identification of immunologic endpoints in vaccine clinical trials, thus facilitating their design and implementation.

Role of cytomegalovirus (CMV)-specific polyfunctional CD8+ T-cells and antibodies neutralizing virus epithelial infection in the control of CMV infection in an allogeneic stem-cell transplantation setting.

The role of cytomegalovirus (CMV)-specific polyfunctional CD8+ T-cells and that of antibodies neutralizing virus epithelial infection (AbNEI) in the control of CMV DNAemia were investigated in 39 CMV-seropositive allogeneic stem-cell transplant (Allo-SCT) recipients with (n = 24) or without (n = 15) CMV DNAemia. AbNEI levels were monitored prospectively by means of a neutralization assay employing retinal epithelial cells (ARPE-19) and the recombinant CMV strain BADrUL131-Y4. Quantification of CMV-specific polyfunctional CD8+ T-cells (expressing two or three of the following markers: IFN-γγ, TNF-α and CD107a) in whole blood was performed by flow cytometry for intracellular cytokine staining. We found no differences in the dynamic pattern of AbNEI in patients with or without subsequent CMV DNAemia. Baseline and peak AbNEI titres were not predictive of the dynamics of CMV replication within episodes. No correlation was found between CMV DNA loads and AbNEI levels during episodes of CMV DNAemia (ρ = 0.09; 95 % confidence interval - 0.52 to 0.64; P = 0.78). The detection of pp65/IE-1 CMV-specific polyfunctional CD8+ T-cells was associated with low-level virus replication within subsequent episodes of CMV DNAemia. Interestingly, the presence of AbNEI titres (inverse) >4.7 log2 was predictive of the occurrence of CMV DNAemia (sensitivity, 83 %; specificity, 80 %). Our findings provide an insight to the role of humoral and cellular immunity in the control of CMV infection in an Allo-SCT setting.

Viral load, CMV-specific T-cell immune response and cytomegalovirus disease in solid organ transplant recipients at higher risk for cytomegalovirus infection during preemptive therapy.

Despite advances in prevention, cytomegalovirus (CMV) recurrence is an important challenge in high-risk organ recipients. The present study prospectively evaluates the impact of CMV-specific T-cell immune response and secondary prophylaxis on the risk of recurrence in a cohort of CMV high-risk organ recipients and whether it is possible to determine a safe standardized viral load value below which CMV disease is unlikely. Thirty-nine recipients were included. Thirty-six had primary infections, and 88.9% recurred. Rate and duration of recurrent CMV infection was similar in patients with and without secondary prophylaxis: 57.9% vs. 53.6%, P = 0.770 and 16 vs. 15 days, P = 0.786, respectively. The only factor independently associated with no episodes of CMV recurrence was the acquisition of CMV-specific T-cell immune response (OR: 0.151, 95% CI: 0.028-0.815; P = 0.028). Cytomegalovirus diseases (N = 5) occurred in patients with CMV viral load above 1500 IU/ml who did not follow the planned monitorization schedule. Our observations suggest that episodes of recurrent CMV infection are common after preemptive therapy despite secondary prophylaxis and that CMV-specific T-cell immune response is associated with a decreased risk of recurrent infections. Preemptive therapy may be safe in patients at high risk for CMV infection with strict close monitoring of the CMV viral load.