Aui, P. M. | Search bioRxiv

bioRxiv2023

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)

BackgroundHouse dust mite (HDM) is the commonest allergen trigger globally for allergic rhinitis and atopic asthma. To expedite accurate confirmation of allergen sensitization, we designed fluorescent allergen tetramers to directly stain specific IgE on basophils to detect allergen sensitization using the flow cytometric CytoBas assay. MethodsRecombinant 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 HDM-allergic patients and 26 non-HDM-sensitized controls were incubated with allergen tetramers for evaluation of basophil binding (CytoBas) and activation (BAT) with flow cytometry. ResultsThe tetramers effectively bound and activated basophils from allergic patients but not non-sensitized controls. CytoBas with Der p 1 as a single allergen had comparable sensitivity and specificity (92% and 100%) to BAT (91% and 100%), similarly for CytoBas with a single Der p 2 (95% and 96%) to BAT (95% and 87%) in detecting allergen sensitization. A positive staining for Der p 1 and/or Der p 2 was 100% sensitive and 96% specific for HDM allergy. ConclusionsCytoBas has diagnostic accuracy for group 1 and group 2 HDM allergens that is comparable to a BAT assay, 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. CAPSULE SUMMARYA single flow cytometry stain of basophils (CytoBas) with both Der p 1 and Der p 2 provides >95% specificity and sensitivity for detection of functional HDM allergen sensitization. HighlightsO_LIFluorescent tetramers of recombinant Der f 1, Der p 1 and Der p 2 can be used to detect functional IgE sensitization to house dust mite (HDM) by flowcytometric detection on basophils (CytoBas). C_LIO_LIA single CytoBas assay with inclusion of both Der p 1 and Der p 2 can detect HDM sensitization with >95% sensitivity and specificity. C_LI

immunology↗

bioRxiv2022

Double-dose mRNA vaccination to SARS-CoV-2 progressively increases recognition of variants-of-concern by Spike RBD-specific memory B cells

BackgroundSARS-CoV-2 vaccination with BNT162b2 (Pfizer BioNTech) has been shown to be 95% effective.1 Double-dose vaccination generates high levels of spike-specific antibodies, memory B cells (Bmem) and T cells. However, variants of concern (VoC) with mutations in the spike Receptor Binding Domain (RBD) can evade antibody responses. Booster vaccinations improve antibody recognition of VoC, but it is unclear if this is due to higher total antibodies or their capacity to bind VoC. We here addressed the capacity of surface Ig on single Wuhan-specific Bmem after first and second dose BNT162b2 vaccination to recognize variant RBD. MethodsSamples were collected from 30 healthy COVID-19 naive individuals pre-BNT162b2 vaccination, 3 weeks post-dose 1 and 4-weeks post-dose 2. Plasma antibodies and Bmem were evaluated using recombinant RBD proteins of the Wuhan, Gamma and Delta strains. ResultsAll individuals generated a robust antibody response to BNT162b2 vaccination with all participants producing neutralizing antibodies following dose 2. IgM+ and IgG+ RBD-specific Bmem were generated after one vaccine dose, and those expressing IgG1 increased in absolute number after dose 2. The majority of RBD-specific Bmem bound the Gamma and/or Delta variants, and this proportion significantly increased after the second dose. ConclusionThe second dose of BNT162b2 increases the number of circulating Ig-class switched RBD-specific Bmem. Importantly, the second dose of vaccination is required for a high frequency of RBD-specific Bmem to recognize Gamma and Delta variants. This suggests that dose 2 not only increases the number of RBD-specific Bmem but also the affinity of the Bmem to overcome the point mutations in VoC.

immunology↗