Establishment of a modified opsonophagocytic killing assay for anti-pneumococcal surface protein A antibody

Eisuke Kuroda; Yuka Koizumi; Zhenyu Piao; Hiroki Nakayama; Kazunori Tomono; Kazunori Oishi; Shigeto Hamaguchi; Yukihiro Akeda

doi:10.1016/j.mimet.2023.106804

Establishment of a modified opsonophagocytic killing assay for anti-pneumococcal surface protein A antibody

J Microbiol Methods. 2023 Sep:212:106804. doi: 10.1016/j.mimet.2023.106804. Epub 2023 Aug 4.

Authors

Eisuke Kuroda¹, Yuka Koizumi², Zhenyu Piao³, Hiroki Nakayama², Kazunori Tomono⁴, Kazunori Oishi⁵, Shigeto Hamaguchi⁶, Yukihiro Akeda⁷

Affiliations

¹ Department of Infection Control and Prevention, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan; Division of Infection Control and Prevention, Osaka University Hospital, Osaka University, Suita, Osaka, Japan; Department of Transformative Infection Control Development Studies, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; Division of Fostering Required Medical Human Resources, Center for Infectious Disease Education and Research (CiDER), Osaka University, Suita, Osaka, Japan.
² Discovery Research Department, Innovative Vaccine Research and Development Division, The Research Foundation for Microbial Diseases of Osaka University, Osaka, Japan.
³ Biotechnology Section, Biomedical Science Center, The Research Foundation for Microbial Diseases of Osaka University, Osaka, Japan.
⁴ Department of Infection Control and Prevention, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan; Division of Infection Control and Prevention, Osaka University Hospital, Osaka University, Suita, Osaka, Japan.
⁵ Toyama Institute of Health, Imizu, Toyama, Japan.
⁶ Department of Infection Control and Prevention, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan; Division of Infection Control and Prevention, Osaka University Hospital, Osaka University, Suita, Osaka, Japan; Division of Fostering Required Medical Human Resources, Center for Infectious Disease Education and Research (CiDER), Osaka University, Suita, Osaka, Japan; Department of Transformative Analysis for Human Specimen, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Electronic address: hamaguchi@hp-infect.med.osaka-u.ac.jp.
⁷ Division of Infection Control and Prevention, Osaka University Hospital, Osaka University, Suita, Osaka, Japan; Thailand-Japan Research Collaboration Centre on Emerging and Re-emerging Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

PMID: 37543109
DOI: 10.1016/j.mimet.2023.106804

Abstract

Streptococcus pneumoniae (pneumococcus) is a pathogenic gram-positive bacterium that causes pneumonia, meningitis, and sepsis. Pneumococcal surface protein A (PspA) induces antibodies that protect against lethal infections by pneumococci. PspA is a choline-binding protein present on the cell surface of almost all pneumococcal strains and is a non-capsular polysaccharide vaccine candidate. For research and development of PspA-based vaccines, an in-vitro test system to measure the activity of functional antibodies capable of killing pneumococci is essential. The opsonophagocytic killing (OPK) assay is used to evaluate the opsonic activity of functional antibodies induced by capsular polysaccharide (CPS)-based vaccines (standard OPK assay). Despite the potential of anti-PspA antibodies to protect against lethal infections in mice, the standard OPK assay fails to evaluate anti-PspA antibodies. Using a pneumococcal surface protein C-deficient strain and extending the incubation time of opsonized bacteria, complement, and HL-60 cells reportedly results in enhanced bactericidal activity (modified OPK assay). We aimed to measure the bactericidal activity of anti-PspA antibodies in intact pneumococcal strains. We optimized the pneumococcal culture method used in the OPK assay to increase the efficiency of anti-PspA antibody-mediated phagocytosis of HL-60 cells. As thick capsules hinder phagocytosis, we attempted to obtain pneumococci with thin capsules through an improved culture method. As pneumococci attached to cells exhibit thin capsules, pneumococci cultured in Todd Hewitt yeast extract (THY) broth were spread on blood agar plates and incubated for 4 h. cpsA mRNA transcript levels in pneumococci cultured on blood agar were lower than those in pneumococci cultured in THY broth. OPK activity against pneumococci expressing PspA of clades 1-5 was reasonably well detected using pneumococci cultured on blood agar in the modified OPK assay. The modified OPK assay for anti-PspA antibody using pneumococci cultured on blood agar represents a useful assay to determine the killing activity of functional anti-PspA antibodies against pneumococci.

Keywords: Anti-PspA antibody; C3 complement deposition; Opsonophagocytic killing assay; Pneumococcal surface protein A; Streptococcus pneumoniae.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Agar
Animals
Antibodies, Bacterial
Bacterial Proteins / metabolism
Capsules
Membrane Proteins
Mice
Pneumococcal Infections*
Pneumococcal Vaccines
Polysaccharides
Streptococcus pneumoniae*

Substances

Membrane Proteins
Agar
Capsules
Antibodies, Bacterial
Polysaccharides
Bacterial Proteins
Pneumococcal Vaccines