RESUMO
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.
Assuntos
Antígenos de Dermatophagoides , Proteínas de Artrópodes , Asma , Basófilos , Cisteína Endopeptidases , Citometria de Fluxo , Pyroglyphidae , Rinite Alérgica , Humanos , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Basófilos/imunologia , Cisteína Endopeptidases/imunologia , Animais , Rinite Alérgica/imunologia , Rinite Alérgica/diagnóstico , Asma/imunologia , Asma/diagnóstico , Feminino , Adulto , Citometria de Fluxo/métodos , Masculino , Pyroglyphidae/imunologia , Pessoa de Meia-Idade , Adolescente , Adulto Jovem , Imunoglobulina E/imunologia , Imunoglobulina E/sangue , Alérgenos/imunologia , Sensibilidade e Especificidade , CriançaRESUMO
BACKGROUND: The RTS,S malaria vaccine is currently recommended for children aged 5-6 months in regions with moderate-to-high Plasmodium falciparum transmission. However, vaccination only confers 55% efficacy over 12 months and wanes within 18 months. The immunological mechanisms of RTS,S-mediated immunity are poorly understood; therefore, we aimed to identify antibody response types associated with protection against malaria in children vaccinated with RTS,S. METHODS: In this post-hoc analysis, we evaluated antibody responses in 737 children aged 1-4 years vaccinated with RTS,S in a phase 2b clinical trial conducted in Mozambique in 2003. We evaluated all available samples collected from children 30 days after the three-dose vaccination schedule at study month 3 (M3; n=737 available of 803 children allocated to receive RTS,S). For comparison, we tested a subset of samples collected before vaccination at study month 0 (M0; n=50) and from children in the control vaccine group (M0 n=25; M3 n=99). We quantified the induction of antibodies to different regions of the vaccine antigen that function by fixing serum complement proteins and binding to Fcγ receptors (FcγRs; FcγRI, FcγRIIa, and FcγRIII) expressed on immune cells as potential mechanisms of immunity. FINDINGS: Functional antibody responses to the C-terminal region of the vaccine antigen, circumsporozoite protein (CSP), were associated with a reduced risk of malaria (C1q p=0·0060, FcγRIIa p=0·014, and FcγRIII p=0·019). These associations remained significant in male participants when the analyses were stratified by sex (C1q p=0·012, FcγRI p=0·023, FcγRIIa p=0·0070, and FcγRIII p=0·0080). IgA to the central repeat (p=0·0010) and C-terminal (p=0·0040) regions of CSP were also associated with protection. We show that IgA can bind FcαRI and mediate opsonic phagocytosis using a serum pool and monoclonal antibodies. Multiparameter analysis using machine-learning methods suggest that IgA, complement fixation, and FcγRI binding were most predictive of protection against malaria (hazard ratio <1) and suggested that associations differed between male and female participants. INTERPRETATION: We provide evidence that functional antibody responses mediated by IgG and IgA are associated with protection against malaria in young children vaccinated with RTS,S, and suggest potential differences in the correlates of immunity between males and females. These findings reveal new avenues that could be used to achieve malaria vaccines with higher efficacy. FUNDING: National Health and Medical Research Council, Australia, and Thrasher Research Fund.