Subcutaneous immunotherapy for bee venom allergy induces epitope spreading and immunophenotypic changes in allergen-specific memory B cells.

BACKGROUND: Allergen immunotherapy (AIT) is the only disease-modifying treatment for allergic disorders. We have recently discovered that allergen-specific memory B-cells (Bmem) are phenotypically altered after 4 months sublingual AIT for ryegrass pollen allergy. Whether these effects are shared with subcutaneous AIT (SCIT) and affect the epitope-specificity of Bmem remain unknown. OBJECTIVE: To evaluate the phenotype and antigen-receptor sequences of Bmem specific to the major bee venom (BV) allergen Api m 1 before and after ultra-rush SCIT for BV allergy. METHODS: Recombinant Api m 1 protein tetramers were generated to evaluate basophil activation in a cohort of BV allergic individuals before and after BV SCIT. Comprehensive flow cytometry was performed to evaluate and purify Api m 1-specific Bmem. Ig genes from single Api m 1-specific Bmem were sequenced and structurally modeled onto Api m 1. RESULTS: SCIT promoted class-switching of Api m 1-specific Bmem to IgG2 and IgG4 with increased expression of CD23 and CD29. Furthermore, modeling of Api m 1-specific Ig from Bmem identified a suite of possible new and diverse allergen epitopes on Api m 1 and highlights epitopes that may preferentially be bound by Ig after SCIT. CONCLUSION: AIT induces shifting of epitope specificity and phenotypic changes in allergen-specific Bmem.

Fourth dose bivalent COVID-19 vaccines outperform monovalent boosters in eliciting cross-reactive memory B cells to Omicron subvariants.

Bivalent COVID-19 vaccines comprising ancestral Wuhan-Hu-1 (WH1) and the Omicron BA.1 or BA.5 subvariant elicit enhanced serum antibody responses to emerging Omicron subvariants. Here, we characterized the RBD-specific memory B cell (Bmem) response following a fourth dose with a BA.1 or BA.5 bivalent vaccine, in direct comparison with a WH1 monovalent fourth dose. Healthcare workers previously immunized with mRNA or adenoviral vector monovalent vaccines were sampled before and one month after a fourth dose with a monovalent or a BA.1 or BA.5 bivalent vaccine. Serum neutralizing antibodies (NAb) were quantified, as well as RBD-specific Bmem with an in-depth spectral flow cytometry panel including recombinant RBD proteins of the WH1, BA.1, BA.5, BQ.1.1, and XBB.1.5 variants. Both bivalent vaccines elicited higher NAb titers against Omicron subvariants compared to the monovalent vaccine. Following either vaccine type, recipients had slightly increased WH1 RBD-specific Bmem numbers. Both bivalent vaccines significantly increased WH1 RBD-specific Bmem binding of all Omicron subvariants tested by flow cytometry, while recognition of Omicron subvariants was not enhanced following monovalent vaccination. IgG1+ Bmem dominated the response, with substantial IgG4+ Bmem only detected in recipients of an mRNA vaccine for their primary dose. Thus, Omicron-based bivalent vaccines can significantly boost NAb and Bmem specific for ancestral WH1 and Omicron variants and improve recognition of descendent subvariants by pre-existing, WH1-specific Bmem beyond that of a monovalent vaccine. This provides new insights into the capacity of variant-based mRNA booster vaccines to improve immune memory against emerging SARS-CoV-2 variants and potentially protect against severe disease. ONE-SENTENCE SUMMARY: Omicron BA.1 and BA.5 bivalent COVID-19 boosters, used as a fourth dose, increase RBD-specific Bmem cross-recognition of Omicron subvariants, both those encoded by the vaccines and antigenically distinct subvariants, further than a monovalent booster.

Homologous but not heterologous COVID-19 vaccine booster elicits IgG4+ B-cells and enhanced Omicron subvariant binding.

Booster vaccinations are recommended to improve protection against severe disease from SARS-CoV-2 infection. With primary vaccinations involving various adenoviral vector and mRNA-based formulations, it remains unclear if these differentially affect the immune response to booster doses. We examined the effects of homologous (mRNA/mRNA) and heterologous (adenoviral vector/mRNA) vaccination on antibody and memory B cell (Bmem) responses against ancestral and Omicron subvariants. Healthy adults who received primary BNT162b2 (mRNA) or ChAdOx1 (vector) vaccination were sampled 1-month and 6-months after their 2nd and 3rd dose (homologous or heterologous) vaccination. Recombinant spike receptor-binding domain (RBD) proteins from ancestral, Omicron BA.2 and BA.5 variants were produced for ELISA-based serology, and tetramerized for immunophenotyping of RBD-specific Bmem. Dose 3 boosters significantly increased ancestral RBD-specific plasma IgG and Bmem in both cohorts. Up to 80% of ancestral RBD-specific Bmem expressed IgG1+. IgG4+ Bmem were detectable after primary mRNA vaccination, and expanded significantly to 5-20% after dose 3, whereas heterologous boosting did not elicit IgG4+ Bmem. Recognition of Omicron BA.2 and BA.5 by ancestral RBD-specific plasma IgG increased from 20% to 60% after the 3rd dose in both cohorts. Reactivity of ancestral RBD-specific Bmem to Omicron BA.2 and BA.5 increased following a homologous booster from 40% to 60%, but not after a heterologous booster. A 3rd mRNA dose generates similarly robust serological and Bmem responses in homologous and heterologous vaccination groups. The expansion of IgG4+ Bmem after mRNA priming might result from the unique vaccine formulation or dosing schedule affecting the Bmem response duration and antibody maturation.

IgE in allergy: It takes two.

A type 2 memory B cell subset is poised to differentiate into IgE-producing plasma cells in individuals with allergies (Ota et al. and Koenig et al.).

Accurate determination of house dust mite sensitization in asthma and allergic rhinitis through cytometric detection of Der p 1 and Der p 2 binding on basophils (CytoBas).

BACKGROUND: House dust mite (HDM) is the most common allergen trigger globally for allergic rhinitis and atopic asthma. OBJECTIVES: To expedite accurate confirmation of allergen sensitization, we designed fluorescent allergen tetramers to directly stain specific IgE on basophils to detect specific allergen sensitization using the flow cytometric CytoBas assay. METHODS: Recombinant proteins of major HDM allergens (component), Der f 1, Der p 1, and Der p 2 were biotinylated and conjugated with fluorochrome streptavidins as tetramers. Blood samples from 64 patients who are HDM-allergic and 26 controls that are non-HDM-sensitized were incubated with allergen tetramers for evaluation of basophil binding (CytoBas) and activation (BAT) with flow cytometry. RESULTS: The tetramers effectively bound and activated basophils from patients who are allergic but not from controls who are nonsensitized. CytoBas with Der p 1 as a single allergen had comparable sensitivity and specificity (92% and 100%) to BAT (91% and 100%) in detecting allergen sensitization, as did CytoBas with Der p 2 (95% and 96%) to BAT (95% and 87%). A positive staining for Der p 1 and/or Der p 2 in CytoBas was 100% sensitive and 96% specific for HDM allergy. CONCLUSIONS: CytoBas has diagnostic accuracy for group 1 and group 2 HDM allergens that is comparable to BAT, but with additional advantages of multiple allergen components in a single tube and no requirement for in vitro basophil activation. These findings endorse a single, multiplex CytoBas assay for accurate and component-resolved diagnosis of aeroallergen sensitization in patients with allergic asthma and/or rhinitis.

Phase 1 trial supports safety and mechanism of action of peptide immunotherapy for peanut allergy.

BACKGROUND: Food allergy is a leading cause of anaphylaxis worldwide. Allergen-specific immunotherapy is the only treatment shown to modify the natural history of allergic disease, but application to food allergy has been hindered by risk of severe allergic reactions and short-lived efficacy. Allergen-derived peptides could provide a solution. PVX108 comprises seven short peptides representing immunodominant T-cell epitopes of major peanut allergens for treatment of peanut allergy. METHODS: Pre-clinical safety of PVX108 was assessed using ex vivo basophil activation tests (n = 185). Clinical safety and tolerability of single and repeat PVX108 doses were evaluated in a first-in-human, randomized, double-blind, placebo-controlled trial in peanut-allergic adults (46 active, 21 placebo). The repeat-dose cohort received six doses over 16 weeks with safety monitored to 21 weeks. Exploratory immunological analyses were performed at pre-dose, Week 21 and Month 18 after treatment. RESULTS: PVX108 induced negligible activation of peanut-sensitised basophils. PVX108 was safe and well tolerated in peanut-allergic adults. There were no treatment-related hypersensitivity events or AEs of clinical concern. The only events occurring more frequently in active than placebo were mild injection site reactions. Exploratory immunological analyses revealed a decrease in the ratio of ST2+ Th2A:CCR6+ Th17-like cells within the peanut-reactive Th pool which strengthened following treatment. CONCLUSION: This study supports the concept that PVX108 could provide a safe alternative to whole peanut immunotherapies and provides evidence of durable peanut-specific T-cell modulation. Translation of these findings to clinical efficacy in ongoing Phase 2 trials would provide important proof-of-concept for using peptides to treat food allergy.

COVID-19 Adenoviral Vector Vaccination Elicits a Robust Memory B Cell Response with the Capacity to Recognize Omicron BA.2 and BA.5 Variants.

Following the COVID-19 pandemic, novel vaccines have successfully reduced severe disease and death. Despite eliciting lower antibody responses, adenoviral vector vaccines are nearly as effective as mRNA vaccines. Therefore, protection against severe disease may be mediated by immune memory cells. We here evaluated plasma antibody and memory B cells (Bmem) targeting the SARS-CoV-2 Spike receptor-binding domain (RBD) elicited by the adenoviral vector vaccine ChAdOx1 (AstraZeneca), their capacity to bind Omicron subvariants, and compared this to the response to mRNA BNT162b2 (Pfizer-BioNTech) vaccination. Whole blood was sampled from 31 healthy adults pre-vaccination and 4 weeks after dose one and dose two of ChAdOx1. Neutralizing antibodies (NAb) against SARS-CoV-2 were quantified at each time point. Recombinant RBDs of the Wuhan-Hu-1 (WH1), Delta, BA.2, and BA.5 variants were produced for ELISA-based quantification of plasma IgG and incorporated separately into fluorescent tetramers for flow cytometric identification of RBD-specific Bmem. NAb and RBD-specific IgG levels were over eight times lower following ChAdOx1 vaccination than BNT162b2. In ChAdOx1-vaccinated individuals, median plasma IgG recognition of BA.2 and BA.5 as a proportion of WH1-specific IgG was 26% and 17%, respectively. All donors generated resting RBD-specific Bmem, which were boosted after the second dose of ChAdOx1 and were similar in number to those produced by BNT162b2. The second dose of ChAdOx1 boosted Bmem that recognized VoC, and 37% and 39% of WH1-specific Bmem recognized BA.2 and BA.5, respectively. These data uncover mechanisms by which ChAdOx1 elicits immune memory to confer effective protection against severe COVID-19.

Combined immunodeficiency and impaired PI3K signaling in a patient with biallelic LCP2 variants.

BACKGROUND: Inborn errors affecting components of the T-cell receptor signaling cascade cause combined immunodeficiency with various degrees of severity. Recently, homozygous variants in LCP2 were reported to cause pediatric onset of severe combined immunodeficiency with neutrophil, platelet, and T- and B-cell defects. OBJECTIVE: We sought to unravel the genetic cause of combined immunodeficiency and early-onset immune dysregulation in a 26-year-old man who presented with specific antibody deficiency, autoimmunity, and inflammatory bowel disease since early childhood. METHODS: The patient was subjected to whole-exome sequencing of genomic DNA and examination of blood neutrophils, platelets, and T and B cells. Expression levels of the Src homology domain 2-containing leukocyte protein of 76 kDa (SLP76) and tonic and ligand-induced PI3K signaling were evaluated by flow-cytometric detection of phosphorylated ribosomal protein S6 in B and T cells. RESULTS: Compound heterozygous missense variants were identified in LCP2, affecting the proline-rich repeat domain of SLP76 (p.P190R and p.R204W). The patient's total B- and T-cell numbers were within the normal range, as was platelet function. However, neutrophil function, numbers of unswitched and class-switched memory B cells, and serum IgA were decreased. Moreover, intracellular SLP76 protein levels were reduced in the patient's B cells, CD4+ and CD8+ T cells, and natural killer cells. Tonic and ligand-induced levels of phosphorylated ribosomal protein S6 and ligand-induced phosphorylated PLCγ1 were decreased in the patient's B cells and CD4+ and CD8+ T cells. CONCLUSIONS: Biallelic variants in LCP2 impair neutrophil function and T-cell and B-cell antigen-receptor signaling and can cause combined immunodeficiency with early-onset immune dysregulation, even in the absence of platelet defects.

Trait profiles in difficult-to-treat asthma: Clinical impact and response to systematic assessment.

BACKGROUND: Multidisciplinary systematic assessment improves outcomes in difficult-to-treat asthma, but without clear response predictors. Using a treatable-traits framework, we stratified patients by trait profile, examining clinical impact and treatment responsiveness to systematic assessment. METHODS: We performed latent class analysis using 12 traits on difficult-to-treat asthma patients undergoing systematic assessment at our institution. We examined Asthma Control Questionnaire (ACQ-6) and Asthma Quality of Life Questionnaire (AQLQ) scores, FEV1 , exacerbation frequency, and maintenance oral corticosteroid (mOCS) dose, at baseline and following systematic assessment. RESULTS: Among 241 patients, two airway-centric profiles were characterized by early-onset with allergic rhinitis (n = 46) and adult onset with eosinophilia/chronic rhinosinusitis (n = 60), respectively, with minimal comorbid or psychosocial traits; three non-airway-centric profiles exhibited either comorbid (obesity, vocal cord dysfunction, dysfunctional breathing) dominance (n = 51), psychosocial (anxiety, depression, smoking, unemployment) dominance (n = 72), or multi-domain impairment (n = 12). Compared to airway-centric profiles, non-airway-centric profiles had worse baseline ACQ-6 (2.7 vs. 2.2, p < .001) and AQLQ (3.8 vs. 4.5, p < .001) scores. Following systematic assessment, the cohort showed overall improvements across all outcomes. However, airway-centric profiles had more FEV1 improvement (5.6% vs. 2.2% predicted, p < .05) while non-airway-centric profiles trended to greater exacerbation reduction (1.7 vs. 1.0, p = .07); mOCS dose reduction was similar (3.1 mg vs. 3.5 mg, p = .782). CONCLUSION: Distinct trait profiles in difficult-to-treat asthma are associated with different clinical outcomes and treatment responsiveness to systematic assessment. These findings yield clinical and mechanistic insights into difficult-to-treat asthma, offer a conceptual framework to address disease heterogeneity, and highlight areas responsive to targeted intervention.

Current advances in house dust mite allergen immunotherapy (AIT): Routes of administration, biomarkers and molecular allergen profiling.

Allergy to house dust mites (HDM) is a perennial respiratory disease that affect more than half a billion people worldwide. Dermatophagoides pteronyssinus and D. farinae, two HDM species, are major sources of indoor allergens triggering allergic inflammation. Although symptomatic drugs are widely used to block the allergic reaction, allergen immunotherapy is the only curative treatment of IgE-mediated type I respiratory allergies. In this article, we review recent advances in various routes of allergen immunotherapy. We particularly focus on subcutaneous (SCIT) and sublingual (SLIT) immunotherapy, used as a reference therapy since they have transformed allergic treatments by improving symptoms (asthma and rhinitis) as well as the quality of life of patients. We also highlight recent data in more exploratory routes (i.e., oral, intralymphatic, epicutaneous and intradermal) and discuss respective advantages of various route, as well as their foreseen modes of action. Finally, we provide an update on biomarkers as well as on the relevance of the molecular profiling of allergic individuals related to treatment efficacy or asthma prediction.

RNA sequencing of single allergen-specific memory B cells after grass pollen immunotherapy: Two unique cell fates and CD29 as a biomarker for treatment effect.

BACKGROUND: Sublingual immunotherapy (SLIT) for grass pollen allergy can modify the natural history of allergic rhinitis and is associated with increased allergen-specific IgG4 . IgG4 competitively inhibits functional IgE on the surface of effector cells, such as mast cells and basophils, from binding to allergens. To further understand the important role memory B-cell (Bmem) responses play in mediating the beneficial effects of SLIT, we assessed changes in allergen-specific Bmem subsets induced by SLIT for grass pollen allergy. METHODS: Blood samples were collected twice outside the pollen season from twenty-seven patients with sensitization to ryegrass pollen (RGP; Lolium perenne) and seasonal rhinoconjunctivitis. Thirteen received 4-month pre-seasonal SLIT for grass pollen allergy, and 14 received standard pharmacotherapy only. Single-cell RNA sequencing was performed on FACS-purified Lol p 1-specific Bmem before and after SLIT from four patients, and significant genes were validated by flow cytometry on the total cohort. RESULTS: Four months of SLIT increased RGP-specific IgE and IgG4 in serum and induced two Lol p 1-specific Bmem subsets with unique transcriptional profiles. Both subsets had upregulated expression of beta 1 integrin ITGB1 (CD29), whereas IGHE (IgE), IGHG4 (IgG4 ), FCER2 (CD23), and IL13RA1 were upregulated in one subset. There was an increase in the proportion of Lol p 1+ Bmem expressing surface IgG4 , CD23, and CD29 after SLIT. CONCLUSIONS: A clinically successful 4 months course of SLIT for grass pollen allergy induces two transcriptionally unique Bmem fates. Associated changes in surface-expressed proteins on these Bmem subsets can be used as early biomarkers for treatment effects.

Diagnostic value of a medical algorithm for investigation of perioperative hypersensitivity reactions.

BACKGROUND: Evaluation of perioperative hypersensitivity (POH) is challenging, and accurate screening tools are needed to optimize the diagnostic process. We aimed to assess and validate the diagnostic value of a published algorithm (using tryptase and clinical presentation) to identify appropriate individuals for further testing for IgE-mediated POH. METHODS: We analysed the clinical presentation (tryptase elevation, cardiovascular, respiratory, skin involvement) of patients proceeding to testing for possible IgE-mediated POH at a single tertiary referral centre, relative to subsequent skin testing and specific IgE results. Clinical presentations by drug class were also determined. RESULTS: In 293 consecutive patients, the use of a published algorithm based on one or more of; (i) defined increase in serum tryptase, (ii) involvement of at least two-organ systems, or (iii) presentation with new urticaria and/or angioedema; was highly sensitive [98.8% (CI95: 95.7-99.9%)] but less specific [34.6% (CI95: 25.7-44.4%)] in identifying patients testing positive on skin testing and/or specific IgE. Presentation with cardiovascular symptoms was also sensitive [89.8%(CI95: 84.2-94.0%)], while the combination of respiratory symptoms and increased tryptase was most specific [85.9%(CI95:76.6-92.5%)]. Respiratory involvement was more common in neuromuscular blocking agent allergy, while urticaria/angioedema was more common in antibiotic allergy. CONCLUSION: The published algorithm (of tryptase rise, two-organ involvement or new urticaria/angioedema) is highly sensitive, and appropriate as a screening tool to identify patients suitable for testing for IgE-mediated POH.

Phenotypic Distinctions Between Omega-5-Gliadin Allergy and Peanut Allergy: Clinical Profile, Reaction Rates and Triggers, and Quality of Life.

Background: Different phenotypes of food allergy may exist, exhibiting distinct clinical features, and driven by different pathogenic mechanisms. We compared omega-5-gliadin (O5G) allergy to peanut allergy, focusing on clinical features, reaction rates and triggers, and quality of life (QOL). Methods: We surveyed adults with O5G allergy and peanut allergy regarding their diagnosis, co-morbidities, allergic reactions, and QOL measured by the FAQLQ-AF. Results: We received responses from 43/80 (54%) individuals with O5G allergy and 43/130 (33%) with peanut allergy. Compared to peanut allergic individuals, those with O5G allergy were older at age of onset (37.2 vs 2.5 years, p < 0.001), had fewer additional atopic conditions (0.88 vs 2.93, p < 0.001) or food allergies (0.15 vs 1.86, p < 0.001), and more frequent reactions before diagnosis (1.085 vs 0.29 per month, p < 0.05) Reaction rates improved in both groups following diagnosis. Reactions to peanut were more often triggered by accidental exposure (84% vs 26%, p < 0.001) and being away from home (65% vs 28%, p < 0.001), while reactions to O5G were more often due to deliberate ingestion (30% vs 9%, p < 0.05) or unexpected exercise (35% vs 2%, p < 0.001). Overall QOL score was similar between groups (4.2 in O5G allergy, 4.7 in peanut allergy, p = 0.12), but worse among women and those with additional food allergies. Conclusion: Phenotypic differences between O5G and peanut allergy support the development of different clinical approaches and the possibility of targeting distinct pathogenic mechanisms for prevention and treatment. Quality of life was impaired to a similar degree between groups.

Humoral immunity and B-cell memory in response to SARS-CoV-2 infection and vaccination.

Natural infection with SARS-CoV-2 induces a robust circulating memory B cell (Bmem) population, which remains stable in number at least 8 months post-infection despite the contraction of antibody levels after 1 month. Multiple vaccines have been developed to combat the virus. These include two new formulations, mRNA and adenoviral vector vaccines, which have varying efficacy rates, potentially related to their distinct capacities to induce humoral immune responses. The mRNA vaccines BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) elicit significantly higher serum IgG and neutralizing antibody levels than the adenoviral vector ChAdOx1 (AstraZeneca) and Ad26.COV2.S (Janssen) vaccines. However, all vaccines induce Spike- and RBD-specific Bmem, which are vital in providing long-lasting protection in the form of rapid recall responses to subsequent infections. Past and current SARS-CoV-2 variants of concern (VoC) have shown the capacity to escape antibody neutralization to varying degrees. A booster dose with an mRNA vaccine following primary vaccination restores antibody levels and improves the capacity of these antibodies and Bmem to bind viral variants, including the current VoC Omicron. Future experimental research will be essential to evaluate the durability of protection against VoC provided by each vaccine and to identify immune markers of protection to enable prognostication of people who are at risk of severe complications from COVID-19.

New insights into human immune memory from SARS-CoV-2 infection and vaccination.

Since early 2020, the world has been embroiled in an ongoing viral pandemic with SARS-CoV-2 and emerging variants resulting in mass morbidity and an estimated 6 million deaths globally. The scientific community pivoted rapidly, providing unique and innovative means to identify infected individuals, technologies to evaluate immune responses to infection and vaccination, and new therapeutic strategies to treat infected individuals. Never before has immunology been so critically at the forefront of combatting a global pandemic. It has now become evident that not just antibody responses, but formation and durability of immune memory cells following vaccination are associated with protection against severe disease from SARS-CoV-2 infection. Furthermore, the emergence of variants of concern (VoC) highlight the need for immunological markers to quantify the protective capacity of Wuhan-based vaccines. Thus, harnessing and modulating the immune response is key to successful vaccination and treatment of disease. We here review the latest knowledge about immune memory generation and durability following natural infection and vaccination, and provide insights into the attributes of immune memory that may protect from emerging variants.