Development of flow cytometric opsonophagocytosis and antibody-mediated complement deposition assays for non-typeable Haemophilus influenzae

• Published in: BMC microbiology Vol. 18; no. 1; p. 167
• Main Authors: Thomas, Stephen R, Leung, Stephanie, Knox, Katy, Wilkinson, Tom M A, Staples, Karl J, Lestrate, Pascal, Wauters, Dominique, Gorringe, Andrew, Taylor, Stephen C
• Format: Journal Article
• Language: English
• Published: England BioMed Central 29.10.2018; BMC
• Subjects: Antibodies - immunology; Antibody; Assaying; Chronic obstructive pulmonary disease; Clinical trials; Coefficient of variation; Colonization; Complement; Complement component C3b; Complement System Proteins - immunology; Deposition; Ear diseases; Epidemiology; Flow Cytometry; Haemophilus Infections - immunology; Haemophilus influenzae; HL-60 Cells; Human populations; Humans; Immune response; Immunity; Immunologic Techniques; Infants; Laboratories; Lung diseases; Medical research; Methodology; Nasopharynx; Nasopharynx - microbiology; Neutrophils; Non-typeable Haemophilus influenzae; Obstructive lung disease; Opsonin Proteins - immunology; Opsonophagocytosis; Otitis media; Phagocytosis; Proteins; Reproducibility of Results; Respiratory Burst - immunology; Sensitivity and Specificity; Streptococcus infections; Vaccine; Vaccines; Flow cytometry; Complement; Vaccine; Antibody; Non-typeable Haemophilus influenzae; Opsonophagocytosis
• ISSN: 1471-2180; 1471-2180
• DOI: 10.1186/s12866-018-1314-5

Haemophilus influenzae is found in the nasopharynx of 80% of the human population. While colonisation with non-typeable Haemophilus influenzae (NTHi) is usually asymptomatic, it is capable of causing acute and chronic otitis media (OM) in infants, invasive disease in susceptible groups and is the leading cause of exacerbations of patients with chronic obstructive pulmonary disease (COPD). Current methods for assessing functional antibody immunity to NTHi are limited and labour intensive. Flow cytometric assays could provide an attractive alternative to evaluate immune responses to candidate vaccines in clinical trials. We have developed a duplexed flow-cytometric uptake and oxidative burst opsonophagocytosis assay (fOPA). We have also developed a duplexed antibody-mediated complement C3b/iC3b and C5b-9 deposition assay (CDA). Antibody-mediated C3b/iC3b deposition correlated with opsonophagocytic uptake (r = 0.65) and with opsonophagocytic oxidative burst (r = 0.69). Both fOPA and CDA were reproducible, with the majority of samples giving a coefficient of variation (CV) of < 20% and overall assay CVs of 14% and 16% respectively. The high-throughput flow cytometric assays developed here were successfully optimised for use with NTHi. Assays proved to be sensitive and highly reproducible for the measurement of bacterial uptake and oxidative burst opsonophagocytosis and antibody-mediated deposition of C3b/iC3b and C5b-9. These assays are useful tools for use in large scale epidemiological studies and to assist in the assessment of functional antibody induced by NTHi candidate vaccines.