Abstract
BackgroundNon-typeable Haemophilus influenzae (NTHi) is a Gram negative microorganism residing in the human nasopharyngeal mucosa and occasionally causing infections of both middle ear and lower respiratory airways. A broadly protective vaccine against NTHi has been a long-unmet medical need, as the high genetic variability of this bacterium has posed great challenges.ResultsIn this study, we developed a robust serum bactericidal assay (SBA) to optimize the selection of protective antigens against NTHi. SBA takes advantage of the complement-mediated lysis of bacterial cells and is a key in vitro method for measuring the functional activity of antibodies. As a proof of concept, we assessed the bactericidal activity of antibodies directed against antigens known to elicit a protective response, including protein D used as carrier protein in the Synflorix pneumococcal polysaccharide conjugate vaccine. Prior to SBA screening, the accessibility of antigens to antibodies and the capacity of the latter to induce C3 complement deposition was verified by flow cytometry. Using baby rabbit serum as a source of complement, the proposed assay not only confirmed the bactericidal activity of the antibodies against the selected vaccine candidates, but also showed a significant reproducibility.ConclusionsConsidering the rapidity and cost-effectiveness of this novel SBA protocol, we conclude that it is likely to become an important tool to prove the capability of antibodies directed against recombinant antigens to induce NTHi in vitro killing and to both select new protective vaccine candidates, and predict vaccine efficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0420-x) contains supplementary material, which is available to authorized users.
Highlights
Non-typeable Haemophilus influenzae (NTHi) is a Gram negative microorganism residing in the human nasopharyngeal mucosa and occasionally causing infections of both middle ear and lower respiratory airways
NTHi is a commensal in the human nasopharyngeal mucosa and may occasionally act as a pathogen initiating infections of both the upper and lower respiratory airways, causing conditions such as otitis media (OM), chronic obstructive pulmonary disease (COPD), and invasive diseases such as meningitis and sepsis, especially in neonates and the elderly [1,2,3]
Selection of the antigens to develop a Serum Bactericidal Assay for NTHi The aim of this study was to set up a robust assay to screen the antigens capable of raising functional antibodies to kill NTHi
Summary
Non-typeable Haemophilus influenzae (NTHi) is a Gram negative microorganism residing in the human nasopharyngeal mucosa and occasionally causing infections of both middle ear and lower respiratory airways. A broadly protective vaccine against NTHi has been a long-unmet medical need, as the high genetic variability of this bacterium has posed great challenges. Non-typeable Haemophilus influenzae (NTHi) is a Gram negative microorganism that differs from the other Hi serotypes for the lack of a polysaccharide capsule. A broadly protective vaccine against NTHi is a current medical need. One of the major hurdles to NTHi vaccine development is the high genetic diversity among strains, leading to the necessity to identify antigens carrying functional epitopes able to induce cross-protective antibodies [4]. Major (P1, P2, P4, P5) and minor (P6, D15, Ercoli et al BMC Microbiology (2015) 15:87
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