Unique allergen-specific human IgE monoclonal antibodies derived from patients with allergic disease.

Introduction: Allergic reactions are mediated by human IgE antibodies that bind to and cross-link allergen molecules. The sites on allergens that are recognized by IgE antibodies have been difficult to investigate because of the paucity of IgE antibodies in a human serum. Here, we report the production of unique human IgE monoclonal antibodies to major inhaled allergens and food allergens that can be produced at scale in perpetuity. Materials and methods: The IgE antibodies were derived from peripheral blood mononuclear cells of symptomatic allergic patients, mostly children aged 3-18 years, using hybridoma fusion technology. Total IgE and allergen-specific IgE was measured by ImmunoCAP. Their specificity was confirmed through ELISA and immunoblotting. Allergenic potency measurements were determined by ImmunoCAP inhibition. Biological activity was determined in vitro by comparing ß-hexosaminidase release from a humanized rat basophilic cell line. Results: Human IgE monoclonal antibodies (n = 33) were derived from 17 allergic patients with symptoms of allergic rhinitis, asthma, atopic dermatitis, food allergy, eosinophilic esophagitis, or red meat allergy. The antibodies were specific for five inhaled allergens, nine food allergens, and alpha-gal and had high levels of IgE (53,450-1,702,500 kU/L) with ratios of specific IgE to total IgE ranging from <0.01 to 1.39. Sigmoidal allergen binding curves were obtained through ELISA, with low limits of detection (<1 kU/L). Allergen specificity was confirmed through immunoblotting. Pairs of IgE monoclonal antibodies to Ara h 6 were identified that cross-linked after allergen stimulation and induced release of significant levels of ß-hexosaminidase (35%-80%) from a humanized rat basophilic cell line. Conclusions: Human IgE monoclonal antibodies are unique antibody molecules with potential applications in allergy diagnosis, allergen standardization, and identification of allergenic epitopes for the development of allergy therapeutics. The IgE antibody probes will enable the unequivocal localization and validation of allergenic epitopes.

Differential cellular and humoral immune responses in immunocompromised individuals following multiple SARS-CoV-2 vaccinations.

Introduction: The heterogeneity of the immunocompromised population means some individuals may exhibit variable, weak or reduced vaccine-induced immune responses, leaving them poorly protected from COVID-19 disease despite receiving multiple SARS-CoV-2 vaccinations. There is conflicting data on the immunogenicity elicited by multiple vaccinations in immunocompromised groups. The aim of this study was to measure both humoral and cellular vaccine-induced immunity in several immunocompromised cohorts and to compare them to immunocompetent controls. Methods: Cytokine release in peptide-stimulated whole blood, and neutralising antibody and baseline SARS-CoV-2 spike-specific IgG levels in plasma were measured in rheumatology patients (n=29), renal transplant recipients (n=46), people living with HIV (PLWH) (n=27) and immunocompetent participants (n=64) post third or fourth vaccination from just one blood sample. Cytokines were measured by ELISA and multiplex array. Neutralising antibody levels in plasma were determined by a 50% neutralising antibody titre assay and SARS-CoV-2 spike specific IgG levels were quantified by ELISA. Results: In infection negative donors, IFN-γ, IL-2 and neutralising antibody levels were significantly reduced in rheumatology patients (p=0.0014, p=0.0415, p=0.0319, respectively) and renal transplant recipients (p<0.0001, p=0.0005, p<0.0001, respectively) compared to immunocompetent controls, with IgG antibody responses similarly affected. Conversely, cellular and humoral immune responses were not impaired in PLWH, or between individuals from all groups with previous SARS-CoV-2 infections. Discussion: These results suggest that specific subgroups within immunocompromised cohorts could benefit from distinct, personalised immunisation or treatment strategies. Identification of vaccine non-responders could be critical to protect those most at risk.

Longitudinal T Cell Responses against Ancestral, Delta, and Omicron SARS-CoV-2 Variants Determined by Rapid Cytokine Release Assay in Whole Blood.

T cell immunity to natural SARS-CoV-2 infection may be more robust and longer lived than Ab responses. Accurate assessment of T cell responses is critical for understanding the magnitude and longevity of immunity across patient cohorts, and against emerging variants. By establishing a simple, accurate, and rapid whole blood test, natural and vaccine-induced SARS-CoV-2 immunity was determined. Cytokine release in whole blood stimulated with peptides specific for SARS-CoV-2 was measured in donors with previous PCR-confirmed infection, suspected infection, or with no exposure history (n = 128), as well as in donors before and after vaccination (n = 32). Longitudinal assessment of T cell responses following initial vaccination and booster vaccination was also conducted (n = 50 and n = 62, respectively). Cytokines were measured by ELISA and multiplex array. IL-2 and IFN-γ were highly elevated in PCR-confirmed donors compared with history-negative controls, with median levels ∼33-fold and ∼48-fold higher, respectively. Receiver operating curves showed IL-2 as the superior biomarker (area under the curve = 0.9950). Following vaccination, all donors demonstrated a positive IL-2 response. Median IL-2 levels increased ∼32-fold from prevaccination to postvaccination in uninfected individuals. Longitudinal assessment revealed that T cell responses were stable up to 6 mo postvaccination. No significant differences in cytokine production were observed between stimulations with Wuhan, Delta, or Omicron peptides. This rapid, whole blood-based test can be used to make comparable longitudinal assessments of vaccine-induced T cell immunity across multiple cohorts and against variants of concern, thus aiding decisions on public health policies.

Simultaneous quantification of specific food allergen proteins using a fluorescent multiplex array.

The aim was to develop a fluorescent multiplex array for simultaneously measuring regulated food allergens using specific allergen protein molecules from peanut, tree nut, cow's milk, egg, soy, fish, shellfish, sesame, mustard and celery. Microspheres coupled to specific monoclonal antibodies were used for allergen detection, with purified allergens as reference standards.Standard curves for 17 allergens covered a 5-log dynamic range. Intra- and inter-assay acceptance criteria were within 70-130% recovery and a CV of ≤15%. Food reference materials contained high levels of their respective major allergens (2000-175,000 µg/g), Similar high allergen levels were found in 10 selected foods analysed using a 9-plex array. Egg, milk, peanut, hazelnut and walnut allergens were detectable in chocolate bars with incurred allergens at 3, 10, 30, and 100 ppm. The multiplex array is an efficient tool for measuring specific food allergens, with applications for risk assessment and standardization of therapeutic products for food allergy.