Background Limited information exists on nursing home (NH) residents regarding BNT162b2/Pfizer vaccine efficacy in preventing SARS-CoV-2 and severe Covid-19, and its association with post-vaccine humoral response. Methods 396 residents from seven NHs suffering a SARS-CoV-2 B.1.1.7 (VOC-α) outbreak at least 14 days after a vaccine campaign were repeatedly tested using SARS-CoV-2 real-time reverse-transcriptase polymerase chain reaction on nasopharyngeal swab test (RT-PCR). SARS-CoV-2 Receptor-Binding Domain (RBD) of the S1 subunit (RBD-IgG) was measured in all residents. Nucleocapsid antigenemia (N-Ag) was measured in RT-PCR-positive residents, and serum neutralizing antibodies in vaccinated residents from one NH. Results The incidence of positive RT-PCR was lower in residents vaccinated by two doses (22.7%) vs one dose (32.3%) or non-vaccinated residents (43.7%)(p<0.01). Covid-19-induced deaths were observed in 10.4% of the non-vaccinated residents, in 6.4% of those who had received one dose, and in 0.9% with two doses (p=0.0007). Severe symptoms were more common in infected non-vaccinated (21.0%) vs vaccinated residents (47.6%, p=0.002). Higher levels of RBD-IgG (n=325) were associated with a lower SARS-CoV-2 incidence. No in vitro serum neutralization activity was found for RBD-IgG levels below 1,050 AU/mL. RBD-IgG levels were inversely associated with N-Ag levels, found as a risk factor of severe Covid-19. Conclusions Two BNT162b2/Pfizer doses are associated with a 48% reduction of SARS-CoV-2 incidence and a 91.3% reduction of death risk in residents from NHs facing a VOC-α outbreak. BNT162b2/Pfizer efficacy was partly predicted by post-vaccine RBD-IgG levels.

Needle-free Epicutaneous For t 2 DNA Vaccine is Effective for Preventing and Treating Biting Midge (Forcipomyia taiwana) allergy in a murine modelTo the Editor,Allergen-specific immunotherapy (ASIT) remains the only treatment capable of inducing immune tolerance to the corresponding allergen and potentially treating the root cause of the allergic disease.1 As the treatment course of protein-based vaccines for ASIT is time-consuming, an easily administered epicutaneous anti-allergic DNA-based vaccine is an attractive method, especially in light of the COVID-19 pandemic.2The biting midge, Forcipomyia taiwana , is the most prevalent cause of biting insect allergy in Taiwan. It is a tiny hematophagous midge that attacks en masse. As many as 60% of exposed individuals develop allergic reactions to the bites.3 The midge is widely distributed throughout Taiwan and southern China. For t 2 is the most predominant,with 75% of midge-allergic patients showing specific IgE to For t 2.4 Allergic reactions to midge bites are not limited to humans but also seen in livestock, such as horses, cattle, sheep, and donkeys, causing significant veterinarian problems.E.coli -expressed For t 2 recombinant protein (rFor t 2) was used as an allergen to sensitize and challenge the mice.5For t 2-encoding fragment (GenBank accession EU678971) was amplified by PCR. The PCR products were subcloned into pVAX1 (Life Technologies, Carlsbad, CA) . The experiments were designed using two approaches: therapeutic and prophylactic (Fig 1). The therapeutic approach is to imitate ASIT in human with established allergy while the prophylactic approach to non-allergics. Twenty-five μg For t 2 DNA was determined as the optimal dose after dose-finding experiments (Supplementary Fig S1). For each treatment, the hair of the abdominal area of the mice was removed using a depilatory,tape-stripped,then patched with 25 μg For t 2 DNA vaccine for one hour and removed.A total of three treatments were given spaced one week apart (Fig 1 and Fig S2). For t 2 proteins were detected in the patched skin and the immune organ spleen at 24 hoursand had significantly increased at 48 hours after last treatment (Fig S3). Scratch bouts after rFor t 2 challenge were used as a clinical surrogate of itch. We measured For t 2-specific IgE, IgG1 and IgG2a in the sera as well as mRNA and proteins of IL-13, interferon-gamma, IL-10, and FOXP3 in the culture supernatants of splenocytes after stimulation with various doses of rFor t 2 at 37℃ for 3-5 days by ELISA and real-time quantitative PCR. Histopathology of the challenged skins was examined.After epicutaneous DNA vaccination, the allergen-induced itchin both groups significantly improved, and For t 2-specific IgE and IgG1/IgG2a ratio decreased significantly at week 6 or week 8 (Fig 2). Levels of mRNA and protein of IL-13 decreased significantly, but IFN-gamma and IL-10 remained unchanged. Expression of FOXP3 mRNA increased (Fig 2, protein data not shown). Eosinophils infiltration in the challenged skin significantly decreased (Fig S4).This is the first study to demonstrate an epicutaneous needless anti-allergic DNA vaccine that effectively treats an established allergic condition and prevents the development of an allergic disease using biting midge allergy as a model. After epicutaneous DNA vaccination, in addition to allergen-induced itch, the changes of multiple biomarkers suggest that immune tolerance was induced after the epicutaneous DNA vaccine.Our data show that though the molecular weight of the For t 2 DNA vaccine is as high as 4000 base pairs, it can penetrate the dermal barrier and translate the corresponding protein in the targeted skin and the spleen of the vaccinated mice. It is possible that the DNA vaccine passes the epidermis via the hair follicles as the skin is tape-stripped before epicutaneous vaccination.6The mode of this anti-allergic epicutaneous DNA vaccine may potentially be used in other specific immunotherapies for other allergens.Mey- Fann Lee1Chi-Sheng Wu2 Shyh-Jye Lin3Yi-Hsing Chen2,4*1Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan2Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan3School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan4School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan

Background: Although FoxP3 + regulatory T (Treg) cells constitute a highly heterogeneous population, with different regulatory potential depending on the disease context, distinct subsets or phenotypes remain poorly defined. This hampers the development of immunotherapy for allergic and autoimmune disorders. Objective: This study aimed at characterizing distinct FoxP3 + Treg subpopulations involved in the suppression of Th2-mediated allergic inflammation in the lung. Methods: We used an established mouse model of allergic airway disease based on ovalbumin sensitization and challenge to analyze FoxP3 + Tregs during the induction and resolution of inflammation, and identify markers that distinguish their most suppressive phenotypes. We also developed a new knock-in mouse model ( Foxp3creCd103dtr) enabling the specific ablation of CD103 +FoxP3 + Tregs for functional studies. Results: We found that during resolution of allergic airway inflammation in mice >50% of FoxP3 + Treg cells expressed the integrin CD103 which marks FoxP3 + Treg cells of high IL-10 production, increased expression of immunoregulatory molecules such as KLRG1, ICOS and CD127, and enhanced suppressive capacity for Th2-mediated inflammatory responses. CD103 +FoxP3 + Tregs were essential for keeping allergic inflammation under control as their specific depletion in Foxp3creCd103dtr mice lead to severe alveocapillary damage, eosinophilic pneumonia, and markedly reduced lifespan of the animals. Conversely, adoptive transfer of CD103 +FoxP3 + Tregs effectively treated disease, attenuating Th2 responses and allergic inflammation in an IL-10-dependent manner. Conclusion: Our study identifies a novel regulatory T cell population, defined by CD103 expression, programmed to prevent exuberant type 2 inflammation and keep homeostasis in the respiratory tract under control. This has important therapeutic implications.

Background: Serological tests are a powerful tool in the monitoring of infectious diseases and the detection of host immunity. However, manufacturers often provide diagnostic accuracy data generated through biased studies and the performance in clinical practice is essentially unclear.  Objectives: We aimed to determine the diagnostic accuracy of various serological testing strategies for (a) identification of patients with previous coronavirus disease-2019 (COVID-19) and (b) prediction of neutralizing antibodies against SARS-CoV-2 in real-life clinical settings.  Methods: We prospectively included 2’573 consecutive health-care workers and 1’085 inpatients with suspected or possible previous COVID-19 at a Swiss University Hospital. Various serological immunoassays based on different analytical techniques (enzyme-linked immunosorbent assays, ELISA; chemiluminescence immunoassay, CLIA; electrochemiluminescence immunoassay, ECLIA; lateral-flow immunoassay, LFI), epitopes of SARS-CoV-2 (nucleocapsid, N; receptor-binding domain, RBD; extended RBD, RBD+; S1 or S2 domain of the spike [S] protein, S1/S2), and antibody subtypes (IgG, pan-Ig) were conducted. A positive real-time PCR test from a nasopharyngeal swab was defined as previous COVID-19. Neutralization assays with live SARS-CoV-2 were performed in a subgroup of patients to assess neutralization activity (n=201).  Results: The sensitivity to detect patients with previous COVID-19 was ≥85% in anti-N ECLIA (86.8%) and anti-S1 ELISA (86.2%). Sensitivity was 84.7% in anti-S1/S2 CLIA, 84.0% in anti-RBD+ LFI, 81.0% in anti-N CLIA, 79.2% in anti-RBD ELISA, and 65.6% in anti-N ELISA. The specificity was 98.4% in anti-N ECLIA, 98.3% in anti-N CLIA, 98.2% in anti-S1 ELISA, 97.7% in anti-N ELISA, 97.6% in anti-S1/S2 CLIA, 97.2% in anti-RBD ELISA, and 96.1% in anti-RBD+ LFI. The sensitivity to detect neutralizing antibodies was ≥85% in anti-S1 ELISA (92.7%), anti-N ECLIA (91.7%), anti-S1/S2 CLIA (90.3%), anti-RBD+ LFI (87.9%), and anti-RBD ELISA (85.8%). Sensitivity was 84.1% in anti-N CLIA, and 66.2% in anti-N ELISA. The specificity was ≥97% in anti-N CLIA (100%), anti-S1/S2 CLIA (97.7%), and anti-RBD+ LFI (97.9%). Specificity was 95.9% in anti-RBD ELISA, 93.0% in anti-N ECLIA, 92% in anti-S1 ELISA, and 65.3% in anti-N ELISA. Diagnostic accuracy measures were consistent among subgroups.  Conclusions: The diagnostic accuracy of serological tests for SARS-CoV-2 antibodies varied remarkably in clinical practice, and the sensitivity to identify patients with previous COVID-19 deviated substantially from the manufacturer’s specifications. The data presented here should be considered when using such tests to estimate the infection burden within a specific population and determine the likelihood of protection against re-infection.

Allergen immunotherapy (AIT) has gained a permanent place in the therapeutic arsenal for the patient with allergy. Particularly, substantial evidence has been established for the efficacy of AIT in allergic rhinitis. A hallmark of AIT is it disease modifying effect resulting in persistent benefit after the treatment has been terminated. Both the subcutaneous and sublingual mode of administration appear to be safe. It is, however, a matter of debate whether AIT can be implemented for patients with asthma. EAACI and GINA guidelines recommend sublingual AIT in house dust mite driven asthma. The question however remains whether the different available forms of AIT should be used for allergic asthma in general.

COVID-19 Vaccines Tolerated in Patients with Paclitaxel and Docetaxel Allergy

Mass cytometry-based identification of a unique T-cell signature in childhood allergic asthma Short title: T-cell signature in paediatric AA

Background: The administration of L-glutamine (Gln) suppresses allergic airway inflammation via the rapid upregulation of MAPK phosphatase (MKP)-1, which functions as a negative regulator of inflammation by deactivating p38 and JNK mitogen-activated protein kinases (MAPKs). However, the role of endogenous Gln remains to be elucidated. Therefore, we investigated the mechanism by which endogenous Gln regulates MKP-1 induction and allergic airway inflammation in an ovalbumin-based murine asthma model. Methods: We depleted endogenous Gln levels using l-γ-glutamyl- p-nitroanilide (GPNA), an inhibitor of the Gln transporter ASCT2, and glutamine synthetase small interfering (si)RNA. Lentivirus expressing MKP-1 was injected to achieve overexpression of MKP-1. Asthmatic phenotypes were assessed using our previously developed ovalbumin-based murine model, which is suitable for examining sequential asthmatic events, including neutrophil infiltration. Gln levels were analyzed using a Gln assay kit. Results: GPNA or glutamine synthetase siRNA successfully depleted endogenous Gln levels. Importantly, homeostatic MKP-1 induction did not occur at all, which resulted in prolonged p38 MAPK and cytosolic phospholipase A 2 (cPLA 2) phosphorylation in Gln-deficient mice. Gln deficiency augmented all examined asthmatic reactions, but it exhibited a strong bias toward increasing the neutrophil count, which was not observed in MKP-1-overexpressing lungs. This neutrophilia was inhibited by a cPLA 2 inhibitor and a leukotriene B4 inhibitor, but not by dexamethasone. Conclusion: Gln deficiency leads to the impairment of MKP-1 induction and activation of p38 MAPK and cPLA 2, resulting in the augmentation of neutrophilic, more so than eosinophilic, airway inflammation.

Background: Nonimmediate (delayed) allergic reactions to penicillins are common and some of them can be life-threatening. The genetic factors influencing these reactions are unknown/poorly known/poorly understood. We assessed the genetic predictors of a delayed penicillin allergy that cover the HLA loci. Methods: Using next-generation sequencing (NGS), we genotyped the MHC region in 24 patients with delayed hypersensitivity compared with 20 patients with documented immediate hypersensitivity to penicillins recruited in Italy. Subsequently, we analyzed in silico Illumina Immunochip genotyping data that covered the HLA loci in 98 Spanish patients with delayed hypersensitivity and 315 with immediate hypersensitivity compared to 1,308 controls. Results: The two alleles DRB3*02:02:01:02 and DRB3*02:02:01:01 were reported in twenty cases with delayed reactions (83%) and ten cases with immediate reactions (50%), but not in the Allele Frequency Net Database. Bearing at least one of the two alleles increased the risk of delayed reactions compared to immediate reactions, with an OR of 8.88 (95% CI, 3.37–23.32; P <0.0001). The haplotype (ACAA) from rs9268835, rs6923504, rs6903608, and rs9268838 genetic variants of the HLA-DRB3 genomic region was significantly associated with an increased risk of delayed hypersensitivity to penicillins (OR, 1.7; 95% CI: 1.06–1.92; P=0.001), but not immediate hypersensitivity. Conclusion: We showed that the HLA-DRB3 locus is strongly associated with an increased risk of delayed penicillin hypersensitivity, at least in Southwestern Europe. The determination of HLA-DRB3*02:02 alleles in the risk management of severe delayed hypersensitivity to penicillins should be evaluated further in larger population samples of different origins.

LETTER TO THE EDITORNo apparent impact of incremental dosing on eliciting dose at double-blind, placebo-controlled peanut challengeOlaya Álvarez García,1,2 Joan Bartra,1,3 Monica Ruiz-Garcia,1Isabel J. Skypala,1,4 Stephen R. Durham,1,4 Robert J. Boyle,1 E.N. Clare Mills,5 Paul J. Turner1

SARS-CoV-2 caused one of the most devastating pandemics in the recent history of mankind. Due to various countermeasures, including lock-downs, wearing masks and increased hygiene, the virus has been controlled in some parts of the world. More recently, the availability of vaccines, based on RNA or Adenoviruses, have greatly added to our ability to keep the virus at bay, again in some parts of the world only. While available vaccines are effective, it would be desirable to also have more classical vaccines at hand for the future. Key feature of vaccines for long-term control of SARS-CoV-2 would be inexpensive production at large scale, ability to make multiple booster injections and long-term stability at +4 oC. Here we describe such a vaccine candidate, consisting of the SARS-CoV-2 receptor binding motif grafted genetically onto the surface of the immunologically optimized cucumber mosaic virus, called CuMV TT-RBM. Using bacterial fermenter production and continuous flow centrifugation, the productivity of the production process is estimated to be >2.5 million doses per 1000 liter fermenter run and the vaccine candidate is stable for at least 14 months at 4°C. We further demonstrate that the candidate vaccine is highly immunogenic in mice and rabbits and induces more high avidity antibodies compared to convalescent human sera and antibodies induced are more cross-reactive to mutant RBDs for variants of concern (VoC). Furthermore, antibody responses are neutralizing and long-lived. This, the here presented VLP-based vaccine may be a good candidate for use as conventional vaccine in the long-term.

Access to the manuscript published in Allergy  https://doi.org/10.1111/all.15037

Increased circulating CRTH2+Tregs are associated with asthma control and exacerbation Short running title:Increased circulating CRTH2+Tregs among patients with asthmaTeerapol Chantveerawonga, Sasipa Sangkangjanavanicha,b, Chirawat Chiewchalermsria,c, Panitan Pradubpongsaa, Wat Mitthamsiria, Sarawut Jindaratd, Ming Wange, Mübeccel Akdisf, Milena Sokolowskaf, Cezmi A. Akdisf, Atik Sangasapaviliyaa, Tadech Boonpiyathada,faDivision of Allergy and Clinical Immunology, Department of Medicine, Phramongkutklao Hospital, Bangkok, Thailandb Division of Allergy, Immunology and Rheumatology, Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, ThailandcDepartment of Medicine, Panyananthaphikkhu Chonprathan Medical Center, Srinakharinwirot University, Nonthaburi, ThailanddDepartment of Pharmacology, Phramongkutklao College of Medicine, Bangkok, ThailandeDepartment of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, and the Beijing Key Laboratory of Nasal Diseases, Beijing Institute of Otolaryngology, Beijing, ChinafSwiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, and the Christine Kühne-Center for Allergy Research and Education (CK-CARE), Davos, Switzerland

COVID-19 vaccination with BNT162b2 and ChAdOx1 vaccines induces nasal neutralizing antibodiesDeclercq Jozefien(1,2,5)*, Tobback Els(3)*, Vanhee Stijn(2,5), Natalie De Ruyck(1), Gerlo Sarah(4), Gevaert Philippe(1)**, Vandekerckhove Linos(3,4)***shared co-first authorship** shared last authorshipUpper Airways Research Lab URL, Department of Otorhinolaryngology, Ghent University, Ghent, BelgiumLaboratory of immunoregulation, VIB Center for Inflammation Research, Ghent, BelgiumDepartment of General Internal Medicine, Ghent University Hospital, Ghent, BelgiumHIV Cure Research Centre, Department of Internal Medicine and Pediatrics, Ghent University, Ghent, BelgiumDepartment of Internal Medicine and Pediatrics, Ghent University, Ghent, BelgiumCorresponding author:Prof. Dr. Philippe GevaertUpper Airways Research Lab URL, Department of Otorhinolaryngology, Ghent University,C Heymanslaan 10, 1P1Ghent, Belgium+3293324922Philippe.gevaert@ugent.beFinancial support: no fundingWord count: 591

Background Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the 4 structural proteins of SARS-CoV-2. Methods VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography and characterized by tunable resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. Results Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1 biased T cell responses, reduced virus load and prevented lung pathology upon live virus challenge in vaccinated animals. Conclusion These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

Background: Early exposure to allergens through a defect skin barrier has been proposed as a mechanism for inducing sensitization and development of allergic diseases. We hypothesized that early-onset, severe atopic dermatitis (AD) is associated with development of aeroallergen sensitization and allergic rhinitis. Methods: We included 368 children from the Copenhagen Prospective Studies on Asthma in Childhood 2000 (COPSAC 2000) at-risk mother-child cohort. AD was diagnosed prospectively based on Hanifin&Rajka’s criteria and severity assessed using the Scoring Atopic Dermatitis (SCORAD) index. Early-onset AD was defined as debut ≤1 year, late-onset as debut from 1-6 years. Aeroallergen sensitization and allergic rhinitis were diagnosed at ages 6-7 and 12 years. Associations between early-onset and late-onset AD and allergy endpoints were calculated using general estimating equations (GEE) models to compute the overall odds ratios (OR) for both time points. Results: Early-onset AD (yes/no) and severity (SCORAD) were associated with development of aeroallergen sensitization during childhood; GEE OR=1.68 [1.08; 2.62], p=0.02 and 1.08 [1.03; 1.12], p<0.001, whereas late-onset was not; GEE OR=1.65 [0.92; 2.94], p=0.08 and 1.01 [0.97; 1.06], p=0.55. The same trend was seen for allergic rhinitis with significant association between early-onset AD and allergic rhinitis; GEE OR=1.56 [1.01; 2.41], p=0.04 and severity; GEE OR=1.09 [1.05; 1.13], p<0.001, whereas late-onset AD showed no association. The effects on sensitization and rhinitis of early-onset vs. late-onset AD severity were significantly different: p-interaction sensitization=0.03 and p-interaction rhinitis<0.01. Conclusion: Increasing severity of early-onset AD, but not late-onset AD, associates with aeroallergen sensitization and allergic rhinitis later in childhood.

NK cells and Lipoxin A4 promote resolution of eosinophilic inflammation after nasal allergen challenge

Role of activation of the coagulation system in the pathogenesis of urticariaTaro Yasuma,1 Corina N. D’Alessandro-Gabazza,1 Tetsu Kobayashi,2 John Morser,3 Esteban C Gabazza.1*1Department of Immunology,2Department of Pulmonary and Critical Care Medicine, Mie University Faculty and Graduate School of Medicine, and Mie University Hospital, Edobashi 2-174, Tsu, Mie 514-8507, Japan.3Division of Hematology, Stanford University School of Medicine, 291 Campus Drive, Stanford, CA 94305, United States*Correspondence: Esteban C Gabazza, MD, PhD, Department of Immunology, Mie University Graduate School of Medicine, Edobashi 2-174, Postal Code 514-8507, Tsu-city, Mie, Japan. Tel.: +81 59 231 5017; fax: +81 59 231 5225.E-mail:gabazza@doc.medic.mie-u.ac.jpWord count: 590