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Purpose of Review: Allergenic extracts are often standardized to control for potency, either by measuring concentrations of major allergens or "overall allergenicity" by competition for IgE in pooled sera from highly allergic subjects with a reference extract. Recent developments present an opportunity to use human mAb cloned from highly allergic subjects to define potency of allergenic extracts. Recent Findings: Two recent developments present an opportunity for revising potency measurements of allergen extracts: cloning allergen specific IgE from allergic subjects and extensive epitope mapping of major allergenic proteins. Summary: Because human IgE mAb recognize biologically relevant epitopes, they present a novel opportunity to determine the potencies of allergenic extracts and may contribute to the science base for allergen standardization. [ABSTRACT FROM AUTHOR]

We previously introduced a three-stage design and associated end-of-stage analyses for allergen immunotherapy (AIT) trials. End-of-stage differences alone may not provide a fuller picture of Stages 2 and 3 effects because they may depend upon stage-specific durations. Therefore, we introduce an additional trend analysis to evaluate the difference in progression curves of two groups over the entire stage. Results from such analysis are used to inform persistence of end-of-stage benefit and thus provide evidence for stagewise effects beyond the study periods. We jointly apply end-of-stage and trend analyses to support the enhanced three-stage design to determine treatment response over time and sustained response to AIT. A simulation study was performed to illustrate the statistical properties (bias and power) of trend analyses under varying statistical missing mechanisms and effect sizes. The extent of bias depended on the missing mechanism and magnitude. Powers were largely driven by effect and sample sizes as well as pre-specified success margins, particularly of relative trend. As an illustration, assuming relative treatment differences of 25–30%, stagewise dropout rate of 15%, and parallel outcome progressions, a sample size of 200 per group may achieve 97% power to demonstrate a treatment effect and 53% power to demonstrate a sustained effect post-treatment. Trend analysis supplements the end-of-stage analysis to enhance the statistical claims of stagewise effects. Inferential statistics support our proposed trend analysis for evaluating benefits of AIT over time and inform clinical understanding and decisions. [ABSTRACT FROM AUTHOR]

Background: Clinical trials of allergen immunotherapy (AIT) may require up to 5 years to complete. These lengthy trials may be complicated by high and potentially differential dropouts, especially among participants who perceive that they are receiving placebo. We propose a three‐stage design in which the placebo group in Stage 1 crosses over to receive active treatment in Stage 2. In Stage 3, AIT is discontinued to determine whether benefit is maintained post‐treatment. We apply inferential statistics to support the three‐stage design for clinical trials to determine clinical efficacy, treatment response over time, and sustained response to AIT. Methods: The proposed framework constitutes a series of hypothesis tests for comparing treatment responses at the end of each stage. A simulation study was performed to illustrate the statistical properties under varying statistical missing mechanisms and effect sizes. Results: The statistical properties in terms of bias and statistical power were consistent with what are expected from conventional analyses. Specifically, the extent of bias depended on the missing mechanism and magnitude. The statistical powers were largely driven by effect and sample sizes as well as prespecified success margins. As an illustration, assuming relative treatment differences of 25% and stagewise dropout rate of 15%, a sample size of 200 per group may achieve 93% power to demonstrate a treatment effect and 60% power to demonstrate a maintained response post‐treatment. Conclusions: Inferential statistics support our proposed study design for evaluating benefits of AIT over time and inform clinical understanding and decisions. [ABSTRACT FROM AUTHOR]

The pathways for differentiation of human CD[4.sup.+] T cells into functionally distinct subsets of memory cells in vivo are unknown. The identification of these subsets and pathways has clear implications for the design of vaccines and immune-targeted therapies. Here, we show that populations of apparently naive CD[4.sup.+] T cells express the chemokine receptors CXCR3 or CCR4 and demonstrate patterns of gene expression and functional responses characteristic of memory cells. The proliferation history and T cell receptor repertoire of these chemokine-[receptor.sup.+] cells suggest that they are very early memory CD[4.sup.+] T cells that have 'rested down' before acquiring the phenotypes described for 'central' or 'effector' memory T cells. In addition, the chemokine-[receptor.sup.+] 'naive' populations contain Th1 and Th2 cells, respectively, demonstrating that Th1/Th2 differentiation can occur very early in vivo in the absence of markers conventionally associated with memory cells. We localized ligands for CXCR3 and CCR4 to separate foci in T cell zones of tonsil, suggesting that the chemokine-[receptor.sup.+] subsets may be recruited and contribute to segregated, polarized micro-environments within lymphoid organs. Importantly, our data suggest that CD[4.sup.+] T cells do not differentiate according to a simple schema from nalve [right arrow] CD45R[O.sup.+] noneffector/central memory effector/effector memory cells. Rather, developmental pathways branch early on to yield effector/memory populations that are highly heterogeneous and multifunctional and have the potential to become stable resting cells. chemokines | immunologic memory | Th1/Th2 cells

Clinical studies demonstrate that efficacy and safety in allergen immunotherapy (AIT) are linked to a multiplicity of factors decisively influencing success or failure. In recent years, numerous trials were performed with correspondent study results published. Yet, the number of AIT products successfully obtaining licensure in the analogous time frame is comparably limited. Essential for licensure is that the AIT product investigated remains comparable in its qualitative and quantitative composition throughout the clinical development. Verification of efficacy is not solely demonstrated by a statistically significant difference between the test and control populations; it must also be shown to be clinically relevant. Choice of meaningful inclusion and end-point criteria is critical. Post hoc or subgroup analysis can be supportive but needs verification as predefined criteria in additional studies. Data analysis may be presented on varying analysis populations, while it should be based on the intention-to-treat population for regulatory review to allow objective assessment of the treatment effect on the overall study population. Apparently conflicting interpretations of clinical data between publications and regulatory review are frequently based on their inherently different objectives, with regulatory review taking into considerations the full data sets of all relevant clinical studies for the concerned AIT product to allow an informed decision on licensure. [ABSTRACT FROM AUTHOR]

Background: Mouse allergy is an important cause of indoor asthma and allergic rhinoconjunctivitis. The major mouse allergen, Mus m 1, is a complex of homologous pheromone‐binding lipocalins called major urinary proteins (MUPs). Methods: We analyzed the proteome of MUPs in mouse urine, commercial mouse epithelial extracts, and environmental samples using several approaches. These include as follows: two‐dimensional electrophoresis and immunoblotting; liquid chromatography‐high‐resolution mass spectrometry (LC/HRMS); multiple reaction monitoring (MRM) mass spectrometry; and LC/HRMS analysis of glycans at the N‐66 residue of MUP3. Results: Albumin is predominant in the extracts, while MUPs are predominant in urine. LC/HRMS of 4 mouse allergen extracts revealed surprising heterogeneity. Of 22 known mouse MUPs, only 6 (MUP3, MUP4, MUP5, MUP13, MUP20, and MUP21) could be identified with MRM using unique peptides. Assessment of MUP content in urine, extracts, and dust samples showed good correlation between MRM and other methods working with different detection principles. All 6 identifiable MUPs were found in electrophoretically separated urine bands, but only MUP3 and MUP20 were above LOQ in unseparated mouse urine, and only MUP3, MUP4, and MUP20 were found in mouse epithelial extracts. Glycan heterogeneity was noted among 4 individual inbred mice: of 13 glycan structures detected, 8 were unique to one mouse, and only 2 glycan modifications were present in all 4 mice. Conclusions: Using mass spectrometry and MRM, mouse allergen extracts and urine samples are shown to be complex and heterogeneous. The efficacy and safety of commercial mouse allergen extracts will be improved with better controls of allergen content. [ABSTRACT FROM AUTHOR]

It is shown that recombinant envelope proteins from macrophage-tropic human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), trigger a signal through CCR5 on CD4+ T cells, and that signal transduction induces chemotaxis of T cells. The response could contribute to HIV pathogenesis, by attracting CD4+ cells to viral replication sites.

The topic of standardization in relation to allergen products has been discussed by allergists, regulators, and manufacturers for a long time. In contrast to synthetic medicinal products, the natural origin of allergen products makes the necessary comparability difficult to achieve. This holds true for both aspects of standardization: Batch-to-batch consistency (or product-specific standardization) and comparability among products from different manufacturers (or cross-product comparability). In this review, we focus on how the United States and the European Union have tackled the topic of allergen product standardization in the past, covering the early joint standardization efforts in the 1970s and 1980s as well as the different paths taken by the two players thereafter until today. So far, these two paths have been based on rather classical immunological methods, including the corresponding benefits like simple feasability. New technologies such as mass spectrometry present an opportunity to redefine the field of allergen standardization in the future. [ABSTRACT FROM AUTHOR]

The first approved COVID‐19 vaccines include Pfizer/BioNTech BNT162B2, Moderna mRNA‐1273 and AstraZeneca recombinant adenoviral ChAdOx1‐S. Soon after approval, severe allergic reactions to the mRNA‐based vaccines that resolved after treatment were reported. Regulatory agencies from the European Union, Unites States and the United Kingdom agree that vaccinations are contraindicated only when there is an allergy to one of the vaccine components or if there was a severe allergic reaction to the first dose. This position paper of the European Academy of Allergy and Clinical Immunology (EAACI) agrees with these recommendations and clarifies that there is no contraindication to administer these vaccines to allergic patients who do not have a history of an allergic reaction to any of the vaccine components. Importantly, as is the case for any medication, anaphylaxis may occur after vaccination in the absence of a history of allergic disease. Therefore, we provide a simplified algorithm of prevention, diagnosis and treatment of severe allergic reactions and a list of recommended medications and equipment for vaccine centres. We also describe potentially allergenic/immunogenic components of the approved vaccines and propose a workup to identify the responsible allergen. Close collaboration between academia, regulatory agencies and vaccine producers will facilitate approaches for patients at risks, such as incremental dosing of the second injection or desensitization. Finally, we identify unmet research needs and propose a concerted international roadmap towards precision diagnosis and management to minimize the risk of allergic reactions to COVID‐19 vaccines and to facilitate their broader and safer use. [ABSTRACT FROM AUTHOR]

Type I interferons (IFN-I) were first discovered as an antiviral factor by Isaacs and Lindenmann in 1957, but they are now known to also modulate innate and adaptive immunity and suppress proliferation of cancer cells. While much has been revealed about IFN-I, it remains a mystery as to why there are 16 different IFN-I gene products, including IFNβ, IFNω, and 12 subtypes of IFNα. Here, we discuss shared and unique aspects of these IFN-I in the context of their evolution, expression patterns, and signaling through their shared heterodimeric receptor. We propose that rather than investigating responses to individual IFN-I, these contexts can serve as an alternative approach toward investigating roles for IFNα subtypes. Finally, we review uses of IFNα and IFNβ as therapeutic agents to suppress chronic viral infections or to treat multiple sclerosis. [ABSTRACT FROM AUTHOR]

Background: Allergen extracts are the primary tool for diagnosis and treatment of allergic diseases. In the United States, most allergen extracts are non‐standardized. More sophisticated analytical approaches are needed to characterize these products and enable manufacturers and regulators to better determine potency. Objective: To expand the multiple reaction monitoring (MRM) assay for an in‐depth characterization of German cockroach (GCr; Blattella germanica) allergen extracts. Methods: We applied advanced liquid chromatography (LC) and mass spectrometry (MS) techniques including MRM. The expanded LC/MRM‐MS method was optimized to measure known GCr allergens and their isoforms/variants in commercial extracts and environmental samples. We performed isoform‐specific allergen measurements in multiple extracts from four commercial sources and extracts prepared using environmental samples from urban homes. To investigate causes of heterogeneity, we examined over 30 extraction process variables. Results: Evaluation of the commercial extracts confirmed the variability of production lots and commercial sources. Commonly used defatting and extraction protocols yielded extracts with comparable allergen profiles and content. However, the identity and quality of source materials was a major contributor to variability. In comparing commercial GCr extracts to environmental samples, relative quantities of Bla g 1, Bla g 2, Bla g 3, Bla g 4 and Bla g 11 were similar, while Bla g 5, Bla g 6, Bla g 7 and Bla g 8 were present in the environmental samples but largely absent for the commercial extracts. Conclusions and Clinical Relevance: LC/MRM‐MS can be used to measure all known GCr allergens in commercial allergen extracts and environmental samples. Significant differences exist between allergen profiles of commercial extracts and the profiles of environmental samples from dwellings. This analytical platform can serve as a template to achieve better product characterization of similarly complex products. [ABSTRACT FROM AUTHOR]

The seventh "Future of the Allergists and Specific Immunotherapy (FASIT)" workshop held in 2019 provided a platform for global experts from academia, allergy clinics, regulatory authorities and industry to review current developments in the field of allergen immunotherapy (AIT). Key domains of the meeting included the following: (a) Biomarkers for AIT and allergic asthma; (b) visions for the future of AIT; (c) progress and data for AIT in asthma and the updates of GINA and EAACI Asthma Guidelines (separated for house dust mite SCIT, SLIT tablets and SLIT drops; patient populations) including a review of clinically relevant endpoints in AIT studies in asthma; (d) regulatory prerequisites such as the "Therapy Allergen Ordinance" in Germany; (e) optimization of trial design in AIT clinical research; (f) challenges planning and conducting phase III (field) studies and the future role of Allergen Exposure Chambers (AEC) in AIT product development from the regulatory point of view. We report a summary of panel discussions of all six domains and highlight unmet needs and possible solutions for the future. [ABSTRACT FROM AUTHOR]

Viral defense at mucosal sites depends on interferons (IFN) and IFN stimulated genes (ISGs), either of which may be constitutively expressed to maintain an "antiviral state" (AVS). However, the mechanisms that govern the AVS are poorly defined. Using a BEAS-2B respiratory epithelial cell line deficient in IRF1, we demonstrate higher susceptibility to infection with vesicular stomatitis virus (VSV) and influenza virus. IRF1-mediated restriction of VSV is IFN-independent, as blockade of types I and III IFNs and JAK-STAT signaling before infection did not affect VSV infection of either parent or IRF1 KO cells. Transcriptome analysis revealed that IRF1 regulates constitutive expression of ~300 genes, including antiviral ISGs: OAS2, BST2 , and RNASEL and knockdown of any of these IRF1-dependent genes increased VSV infection. Additionally, IRF1 enhances rapid expression of IFNβ and IFNλ after stimulation with poly I:C and also regulates ISG expression. Mechanistically, IRF1 enhances recruitment of BRD4 to promotor-enhancer regions of ISGs for rapid expression and maintains levels of histone H3K4me1 for optimal constitutive expression. Finally, IRF1 also regulates constitutive expression of TLR2 and TLR3 and promotes signaling through these pattern recognition receptors (PRR). These data reveal multiple roles for IRF1 toward effective anti-viral responses by maintaining IFN-independent constitutive expression of anti-viral ISGs and supporting early IFN-dependent responses to PRR stimulation. [ABSTRACT FROM AUTHOR]

Type I interferons (IFNs) signal by forming a high affinity IFN-IFNAR2 dimer, which subsequently recruits IFNAR1 to form a ternary complex that initiates JAK/STAT signaling. Among the 12 IFNα subtypes, IFNα1 has a uniquely low affinity for IFNAR2 (<100 × of the other IFNα subtypes) and commensurately weak antiviral activity, suggesting an undefined function distinct from suppression of viral infections. Also unique in IFNα1 is substitution of a serine for phenylalanine at position 27, a contact point that stabilizes the IFNα:IFNAR2 hydrophobic interface. To determine whether IFNα1-S27 contributes to the low affinity for IFNAR2, we created an IFNα1 mutein, IFNα1-S27F, and compared it to wild-type IFNα1 and IFNα2. Substitution of phenylalanine for serine increased affinity for IFNAR2 ∼4-fold and commensurately enhanced activation of STAT1, STAT3, and STAT5, transcription of a subset of interferon stimulated genes, and restriction of vesicular stomatitis virus infection in vitro. Structural modeling suggests that S27 of IFNα1 disrupts the IFNα:IFNAR2 hydrophobic interface that is otherwise stabilized by F27 and that replacing S27 with phenylalanine partially restores the hydrophobic surface. Disruption of the hydrophobic IFNα:IFNAR2 interface by the unique S27 of IFN α1 contributes to its low affinity and weak antiviral activity. [ABSTRACT FROM AUTHOR]

Intestinal epithelial cells (IECs) are constantly exposed to commensal flora and pathogen challenges. How IECs regulate their innate immune response to maintain gut homeostasis remains unclear. Interferons (IFNs) are cytokines produced during infections. While type I IFN receptors are ubiquitously expressed, type III IFN receptors are expressed only on epithelial cells. This epithelium specificity strongly suggests exclusive functions at epithelial surfaces, but the relative roles of type I and III IFNs in the establishment of an antiviral innate immune response in human IECs are not clearly defined. Here, we used mini-gut organoids to define the functions of types I and III IFNs to protect the human gut against viral infection. We show that primary non-transformed human IECs, upon viral challenge, upregulate the expression of both type I and type III IFNs at the transcriptional level but only secrete type III IFN in the supernatant. However, human IECs respond to both type I and type III IFNs by producing IFN-stimulated genes that in turn induce an antiviral state. Using genetic ablation of either type I or type III IFN receptors, we show that either IFN can independently restrict virus infection in human IECs. Importantly, we report, for the first time, differences in the mechanisms by which each IFN establishes the antiviral state. Contrary to type I IFN, the antiviral activity induced by type III IFN is strongly dependent on the mitogen-activated protein kinases signaling pathway, suggesting a pathway used by type III IFNs that non-redundantly contributes to the antiviral state. In conclusion, we demonstrate that human intestinal epithelial cells specifically regulate their innate immune response favoring type III IFN-mediated signaling, which allows for efficient protection against pathogens without producing excessive inflammation. Our results strongly suggest that type III IFN constitutes the frontline of antiviral response in the human gut. We propose that mucosal surfaces, particularly the gastrointestinal tract, have evolved to favor type III IFN-mediated response to pathogen infections as it allows for spatial segregation of signaling and moderate production of inflammatory signals which we propose are key to maintain gut homeostasis. [ABSTRACT FROM AUTHOR]

After viral infection, type I and III interferons (IFNs) are coexpressed by respiratory epithelial cells (RECs) and activate the ISGF3 transcription factor (TF) complex to induce expression of a cell-specific set of interferon-stimulated genes (ISGs). Type I and III IFNs share a canonical signaling pathway, suggesting that they are redundant. Animal and in vitro models, however, have shown that they are not redundant. Because TFs dictate cellular phenotype and function, we hypothesized that focusing on TF-ISG will reveal critical combinatorial and nonredundant functions of type I or III IFN. We treated BEAS-2B human RECs with increasing doses of IFNβ or IFNλ1 and measured expression of TF-ISG. ISGs were expressed in a dose-dependent manner with a nonlinear jump at intermediate doses. At subsaturating combinations of IFNβ and IFNλ1, many ISGs were expressed in a pattern that we modeled with a cubic equation that mathematically defines this threshold effect. Uniquely, IFNβ alone induced early and transient IRF1 transcript and protein expression, while IFNλ1 alone induced IRF1 protein expression at low levels that were sustained through 24 h. In combination, saturating doses of these 2 IFNs together enhanced and sustained IRF1 expression. We conclude that the cubic model quantitates combinatorial effects of IFNβ and IFNλ1 and that IRF1 may mediate nonredundancy of type I or III IFN in RECs. [ABSTRACT FROM AUTHOR]

Human type I interferons ( IFNs) include IFN-β and 12 subtypes of IFN-α. During viral infection, infiltrating memory CD4 + T cells are exposed to IFNs, but their impact on memory T-cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN-α8 and -α10 most potently enhanced expression of IFN-γ, IL-2, and IL-4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL-2 and IL-4 correlated with the time of preincubation with type I IFN, IFN-γ production was enhanced best when IFN-α was added immediately preceding or simultaneously with T-cell stimulation. Comparison of T-cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN-α best enhanced cytokine expression when CD4 + T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T-cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4 + T-cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation. [ABSTRACT FROM AUTHOR]

Type-I interferon (IFN-I) has been increasingly implicated in HIV-1 pathogenesis. Various studies have shown elevated IFN-I and an IFN-I-induced gene and protein expression signature in HIV-1 infection, yet the elevated IFN-I species has not been conclusively identified, its source remains obscure and its role in driving HIV-1 pathogenesis is controversial. We assessed IFN-I species in plasma by ELISAs and bioassay, and we investigated potential sources of IFN-I in blood and lymph node tissue by qRT-PCR. Furthermore, we measured the effect of therapeutic administration of IFNα in HCV-infected subjects to model the effect of IFNα on chronic immune activation. IFN-I bioactivity was significantly increased in plasma of untreated HIV-1-infected subjects relative to uninfected subjects (p = 0.012), and IFNα was the predominant IFN-I subtype correlating with IFN-I bioactivity (r = 0.658, p<0.001). IFNα was not detectable in plasma of subjects receiving anti-retroviral therapy. Elevated expression of IFNα mRNA was limited to lymph node tissue cells, suggesting that peripheral blood leukocytes are not a major source of IFNα in untreated chronic HIV-1 infection. Plasma IFN-I levels correlated inversely with CD4 T cell count (p = 0.003) and positively with levels of plasma HIV-1 RNA and CD38 expression on CD8 T cells (p = 0.009). In hepatitis C virus-infected subjects, treatment with IFN-I and ribavirin increased expression of CD38 on CD8 T cells (p = 0.003). These studies identify IFNα derived from lymph nodes, rather than blood leukocytes, as a possible source of the IFN-I signature that contributes to immune activation in HIV-1 infection. [ABSTRACT FROM AUTHOR]

Human respiratory syncytial virus (HRSV) and, to a lesser extent, human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), can re-infect symptomatically throughout life without significant antigenic change, suggestive of incomplete or short-lived immunity. In contrast, re-infection by influenza A virus (IAV) largely depends on antigenic change, suggestive of more complete immunity. Antigen presentation by dendritic cells (DC) is critical in initiating the adaptive immune response. Antigen uptake by DC induces maturational changes that include decreased expression of the chemokine receptors CCR1, CCR2, and CCR5 that maintain DC residence in peripheral tissues, and increased expression of CCR7 that mediates the migration of antigen-bearing DC to lymphatic tissue. We stimulated human monocyte-derived DC (MDDC) with virus and found that, in contrast to HPIV3 and IAV, HMPV and HRSV did not efficiently decrease CCR1, 2, and 5 expression, and did not efficiently increase CCR7 expression. Consistent with the differences in CCR7 mRNA and protein expression, MDDC stimulated with HRSV or HMPV migrated less efficiently to the CCR7 ligand CCL19 than did IAV- stimulated MDDC. Using GFP-expressing recombinant virus, we showed that the subpopulation of MDDC that was robustly infected with HRSV was particularly inefficient in chemokine receptor modulation. HMPV- or HRSV-stimulated MDDC responded to secondary stimulation with bacterial lipopolysaccharide or with a cocktail of proinflammatory cytokines by increasing CCR7 and decreasing CCR1, 2 and 5 expression, and by more efficient migration to CCL19, suggesting that HMPV and HRSV suboptimally stimulate rather than irreversibly inhibit MDDC migration. This also suggests that the low concentration of proinflammatory cytokines released from HRSV- and HMPV-stimulated MDDC is partly responsible for the low CCR7-mediated migration. We propose that inefficient migration of HRSV- and HMPV-stimulated DC to lymphatic tissue contributes to reduced adaptive responses to these viruses. [ABSTRACT FROM AUTHOR]

Background: Human respiratory syncytial virus (HRSV), and to a lesser extent human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), re-infect symptomatically throughout life without antigenic change, suggestive of incomplete immunity. One causative factor is thought to be viral interference with dendritic cell (DC)-mediated stimulation of CD4+ T cells. Methodology, Principal Findings: We infected human monocyte-derived DC with purified HRSV, HMPV, HPIV3, or influenza A virus (IAV) and compared their ability to induce activation and proliferation of autologous CD4+ T cells in vitro. IAV was included because symptomatic re-infection without antigenic change is less frequent, suggesting that immune protection is more complete and durable. We examined virus-specific memory responses and superantigen-induced responses by multiparameter flow cytometry. Live virus was more stimulatory than inactivated virus in inducing DC-mediated proliferation of virus-specific memory CD4+ T cells, suggesting a lack of strong suppression by live virus. There were trends of increasing proliferation in the order: HMPV

The pathways for differentiation of human CD4+ T cells into functionally distinct subsets of memory cells in vivo are unknown. The identification of these subsets and pathways has clear implications for the design of vaccines and immune-targeted therapies. Here, we show that populations of apparently naive CD4+ T cells express the chemokine receptors CXCR3 or CCR4 and demonstrate patterns of gene expression and functional responses characteristic of memory cells. The proliferation history and T cell receptor repertoire of these chemokine-receptor' cells suggest that they are very early memory CD4+ T cells that have "rested down" before acquiring the phenotypes described for "central" or "effector" memory T cells. In addition, the chemokine-receptor' "naïve" populations contain Th1 and Th2 cells, respectively, demonstrating that Thl/Th2 differentiation can occur very early in vivo in the absence of markers conventionally associated with memory cells. We localized ligands for CXCR3 and CCR4 to separate foci in T cell zones of tonsil, suggesting that the chemokine-receptor' subsets may be recruited and contribute to segregated, polarized micro- environments within lymphoid organs. Importantly, our data suggest that CD4+ T cells do not differentiate according to a simple schema from naïve → CD45RO+ noneffector/central memory → effector/effector memory cells. Rather, developmental pathways branch early on to yield effector/memory populations that are highly heterogeneous and multifunctional and have the potential to become stable resting cells. [ABSTRACT FROM AUTHOR]

Bruton's tyrosine kinase (Btk) is required for human and mouse B cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice. Unlike Src proteins, Btk lacks a negative regulatory domain at the COOH terminus and may rely on cytoplasmic Btk-binding proteins to regulates its kinase activity by trans-inhibitor mechanisms. Consistent with this possibility, IBtk, which we identified as an inhibitor of Btk, bound to the PH domain of Btk. IBtk downregulated Btk kinase activity, Btk-mediated calcium mobilization and nuclear factor-κB?driven transcription. These results define a potential mechanism for the regulation of Btk function in B cells. [ABSTRACT FROM AUTHOR]

The recent approval of Palforzia for treatment of peanut allergy by the US Food and Drug Administration (USFDA) predicts that additional products for oral immunotherapy (OIT) are on the horizon. In this article, the authors review the legal framework by which the USFDA regulates products for OIT of food allergy and the clinical data that demonstrated that the safety and effectiveness profile of Palforzia supported approval and conclude with a discussion of oral food challenge (OFC) as a clinical endpoint to demonstrate safety and effectiveness of OIT products. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) infects and causes disease in infants and reinfects with reduced disease throughout life without significant antigenic change. In contrast, reinfection by influenza A virus (IAV) largely requires antigenic change. The adaptive immune response depends on antigen presentation by dendritic cells (DC), which may be too immature in young infants to induce a fully protective immune response against RSV reinfections. We therefore compared the ability of RSV and IAV to activate primary human cord blood (CB) and adult blood (AB) myeloid DC (mDC). While RSV and IAV infected with similar efficiencies, RSV poorly induced maturation and cytokine production in CB and AB mDC. This difference between RSV and IAV was more profound in CB mDC. While IAV activated CB mDC to some extent, RSV did not induce CB mDC to increase the maturation markers CD38 and CD86 or CCR7, which directs DC migration to lymphatic tissue. Low CCR7 surface expression was associated with high expression of CCR5, which keeps DC in inflamed peripheral tissues. To evaluate a possible inhibition by RSV, we subjected RSV-inoculated AB mDC to secondary IAV inoculation. While RSV-inoculated AB mDC responded to secondary IAV inoculation by efficiently upregulating activation markers and cytokine production, IAV-induced CCR5 downregulation was slightly inhibited in cells exhibiting robust RSV infection. Thus, suboptimal stimulation and weak and mostly reversible inhibition seem to be responsible for inefficient mDC activation by RSV. The inefficient mDC stimulation and immunological immaturity in young infants may contribute to reduced immune responses and incomplete protection against RSV reinfection. [ABSTRACT FROM AUTHOR]

Host-influenza virus interplay at the transcript level has been extensively characterized in epithelial cells. Yet, there are no studies that simultaneously characterize human host and influenza A virus (IAV) genomes. We infected human bronchial epithelial BEAS-2B cells with two seasonal IAV/H3N2 strains, Brisbane/10/07 and Perth/16/09 (reference strains for past vaccine seasons) and the well-characterized laboratory strain Udorn/307/72. Strand-specific RNA sequencing (RNA-seq) of the infected BEAS-2B cells allowed for simultaneous analysis of host and viral transcriptomes, in addition to pathogen genomes, to reveal changes in mRNA expression and alternative splicing (AS). In general, patterns of global and immune gene expression induced by the three IAVs were mostly shared. However, AS of host transcripts and small nuclear RNAs differed between the seasonal and laboratory strains. Analysis of viral transcriptomes showed deletions of the polymerase components (defective interfering-like RNAs) within the genome. Surprisingly, we found that the neuraminidase gene undergoes AS and that the splicing event differs between seasonal and laboratory strains. Our findings reveal novel elements of the host-virus interaction and highlight the importance of RNA-seq in identifying molecular changes at the genome level that may contribute to shaping RNA-based innate immunity. Importance The use of massively parallel RNA sequencing (RNA-seq) has revealed insights into human and pathogen genomes and their evolution. Dual RNA-seq allows simultaneous dissection of host and pathogen genomes and strand-specific RNA-seq provides information about the polarity of the RNA. This is important in the case of negative-strand RNA viruses like influenza virus, which generate positive (complementary and mRNA) and negative-strand RNAs (genome) that differ in their potential to trigger innate immunity. Here, we characterize interactions between human bronchial epithelial cells and three influenza A/H3N2 strains using strand-specific dual RNA-seq. We focused on this subtype because of its epidemiological importance in causing significant morbidity and mortality during influenza epidemics. We report novel elements that differ between seasonal and laboratory strains highlighting the complexity of the host-virus interplay at the RNA level. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) infects small foci of respiratory epithelial cells via infected droplets. Infection induces expression of type I and III interferons (IFNs) and proinflammatory cytokines, the balance of which may restrict viral replication and affect disease severity. We explored this balance by infecting two respiratory epithelial cell lines with low doses of recombinant RSV expressing green fluorescent protein (rgRSV). A549 cells were highly permissive, whereas BEAS-2B cells restricted infection to individual cells or small foci. After infection, A549 cells expressed higher levels of IFN-β-, IFN-γ-, and NF-λB-inducible proinflammatory cytokines. In contrast, BEAS-2B cells expressed higher levels of antiviral interferon-stimulated genes, pattern recognition receptors, and other signaling intermediaries constitutively and after infection. Transcriptome analysis revealed that constitutive expression of antiviral and proinflammatory genes predicted responses by each cell line. These two cell lines provide a model for elucidating critical mediators of local control of viral infection in respiratory epithelial cells. IMPORTANCE Airway epithelium is both the primary target of and the first defense against respiratory syncytial virus (RSV). Whether RSV replicates and spreads to adjacent epithelial cells depends on the quality of their innate immune responses. A549 and BEAS-2B are alveolar and bronchial epithelial cell lines, respectively, that are often used to study RSV infection. We show that A549 cells are permissive to RSV infection and express genes characteristic of a proinflammatory response. In contrast, BEAS-2B cells restrict infection and express genes characteristic of an antiviral response associated with expression of type I and III interferons. Transcriptome analysis of constitutive gene expression revealed patterns that may predict the response of each cell line to infection. This study suggests that restrictive and permissive cell lines may provide a model for identifying critical mediators of local control of infection and stresses the importance of the constitutive antiviral state for the response to viral challenge. [ABSTRACT FROM AUTHOR]

Whether respiratory syncytial virus (RSV) induces severe infantile pulmonary disease may depend on viral strain and expression of types I and III interferons (IFNs). These IFNs impact disease severity by inducing expression of many anti-viral IFN-stimulated genes (ISGs). To investigate the impact of RSV strain on IFN and ISG expression, we stimulated human monocyte-derived DCs (MDDCs) with either RSV A2 or Line 19 and measured expression of types I and III IFNs and ISGs. At 24 h, A2 elicited higher ISG expression than Line 19. Both strains induced MDDCs to express genes for IFN-β, IFN-α1, IFN-α8, and IFN-λ1-3, but only A2 induced IFN-α2, -α14 and -α21. We then show that IFN-α8 and IFN-α14 most potently induced MDDCs and bronchial epithelial cells (BECs) to express ISGs. Our findings demonstrate that RSV strain may impact patterns of types I and III IFN expression and the magnitude of the ISG response by DCs and BECs. [ABSTRACT FROM AUTHOR]

Innate interferons (IFN) are comprised of multiple Type I and III subtypes. The in vivo kinetics of subtype responses during human immunodeficiency virus (HIV) infection is not well defined. Using the acute simian immunodeficiency virus (SIV) infection model, we show that plasma IFNα levels peak at day 10 post-infection (pi) after which they rapidly declined. The mRNA expression of Type I and III IFN subtypes were significantly elevated in the lymph nodes (LN) at day 10 pi. Though the expression levels of all subtypes declined by day 14–31 pi, numerous subtypes remained elevated suggesting that ongoing viral replication in LN continues to drive induction of these subtypes. Interestingly, treatment with reverse transcriptase (RT) inhibitors at day 7 pi significantly suppressed plasma IFNα responses by day 10 pi that significantly correlated with cell-associated SIV DNA loads suggesting that RT byproducts such as viral DNA likely plays a role in driving IFN responses during acute SIV infection. Quantification of Type I and III subtype transcripts in sorted subsets of LN CD4+ and CD8+ T cells, CD14+/CD14− monocytes/macrophages, and total CD11c/CD123+ dendritic cells (DC) at day 10 pi showed that DC expressed ∼3–4 log more subtype transcripts as compared to the other subsets. Taken together, our results provide new insights into the kinetics of innate interferon responses during early stages of infection, and provide evidence that DC’s are a major in vivo source of innate IFN during acute SIV infection. [ABSTRACT FROM AUTHOR]