Development of a serological assay to predict antibody bactericidal activity against non-typeable Haemophilus influenzae.
BACKGROUND
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.


RESULTS
In 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.


CONCLUSIONS
Considering 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.
University of Leicester, Department of Genetics, Adrian building, Leicester, UK. ge39@leicester.ac.uk.
Giuseppe
G
Ercoli
Novartis Vaccines & Diagnostics s.r.l. (a GSK company), Via Fiorentina 1, 53100, Siena, Italy. buki.baddal@gmail.com.
Buket
B
Baddal
Novartis Vaccines & Diagnostics s.r.l. (a GSK company), Via Fiorentina 1, 53100, Siena, Italy. alessandra.greco@novartis.com.
Greco
G
Alessandra
Novartis Vaccines & Diagnostics s.r.l. (a GSK company), Via Fiorentina 1, 53100, Siena, Italy. sara.x.marchi@gks.com.
Sara
S
Marchi
Novartis Vaccines & Diagnostics s.r.l. (a GSK company), Via Fiorentina 1, 53100, Siena, Italy. roberto.x.petracca@gsk.com.
Roberto
R
Petracca
Novartis Vaccines & Diagnostics s.r.l. (a GSK company), Via Fiorentina 1, 53100, Siena, Italy. maria.x.arico@gsk.com.
Beatrice
B
Aricò
Novartis Vaccines & Diagnostics s.r.l. (a GSK company), Via Fiorentina 1, 53100, Siena, Italy. mariagrazia.x.pizza@gsk.com.
Mariagrazia
M
Pizza
Novartis Vaccines & Diagnostics s.r.l. (a GSK company), Via Fiorentina 1, 53100, Siena, Italy. marco.x.soriani@gsk.com.
Marco
M
Soriani
Novartis Vaccines & Diagnostics s.r.l. (a GSK company), Via Fiorentina 1, 53100, Siena, Italy. silvia.x.rossi-paccani@gsk.com.
Silvia
S
Rossi-Paccani
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000942:Antigens, Bacterial / Q000276:immunology*; D001770:Blood Bactericidal Activity; D006168:Guinea Pigs; D006193:Haemophilus influenzae / Q000187:drug effects / Q000276:immunology* / Q000502:physiology*; D007118:Immunoassay / Q000379:methods; D051379:Mice; D050296:Microbial Viability / Q000187:drug effects*; D011817:Rabbits
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial