RESUMEN
Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/química , Ensayo de Inmunoadsorción Enzimática/métodos , Glicoproteínas Hemaglutininas del Virus de la Influenza/sangre , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Concentración de Iones de Hidrógeno , Subtipo H3N2 del Virus de la Influenza A/química , Gripe Humana/sangre , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunologíaRESUMEN
BACKGROUND: Detections of influenza A subtype-specific antibody responses are often complicated by the presence of cross-reactive antibodies. We developed two novel multiplex platforms for antibody detection. The multiplexed magnetic fluorescence microsphere immunoassay (MAGPIX) is a high-throughput laboratory-based assay. Chembio Dual Path Platform (DPP) is a portable and rapid test that could be used in the field. METHODS: Twelve recombinant globular head domain hemagglutinin (GH HA1) antigens from A(H1N1)pdm09 (pH1N1), A(H2N2), A(H3N2), A(H5N1), A(H7N9), A(H9N2), A(H13N9), B/Victoria lineage, B/Yamagata lineage viruses, and protein A control were used. Human sera from U.S. residents either vaccinated (with H5N1 or pH1N1) or infected with pH1N1 influenza viruses and sera from live bird market workers in Bangladesh (BDPW) were evaluated. GH HA1 antigens and serum adsorption using full ectodomain recombinant hemagglutinins from A(pH1N1) and A(H3N2) were introduced into the platforms to reduce cross-reactivity. RESULTS: Serum adsorption reduced cross-reactivity to novel subtype HAs. Compared to traditional hemagglutination inhibition or microneutralization assays, when serum adsorption and the highest fold rise in signals were used to determine positivity, the correct subtype-specific responses were identified in 86%-100% of U.S. residents exposed to influenza antigens through vaccination or infection (N=49). For detection of H5N1-specific antibodies in sera collected from BDPW, H5 sensitivity was 100% (six of six) for MAGPIX, 83% (five of six) for DPP, H5 specificity was 100% (15/15), and cross-reactivity against other subtype was 0% (zero of six) for both platforms. CONCLUSION: MAGPIX and DPP platforms can be utilized for high-throughput and in-field detection of novel influenza virus infections.
