Chapter 33
Testing Anti-Pneumococcal Antibody Function Using
Bacteria and Primary Neutrophils
Manmeet Bhalla, Shaunna R. Simmons, Essi Y. I. Tchalla,
and Elsa N. Bou Ghanem
Abstract
Antibodies against Streptococcus pneumoniae (pneumococcus) following vaccination are crucial for host
protection against invasive pneumococcal infections. The antibodies induced by pneumococcal vaccines act
as opsonins to mediate bacterial uptake and killing by host phagocytic cells, especially polymorphonuclear
leukocytes (PMNs) also called neutrophils. Therefore, it is important to measure not only the levels of
antibodies induced by a pneumococcal vaccine candidate but their actual functional capacity in mediating
bacterial opsonization and killing by PMNs. Here, we describe a protocol to demonstrate effective deposition of vaccine-induced antibodies on the surface of S. pneumoniae by flow cytometry and subsequent
opsonophagocytic killing (OPH) by murine bone-marrow derived PMNs.
Key words Streptococcus pneumoniae, Opsonophagocytic killing, Antibodies, Neutrophils, Flow
cytometry
1 Introduction
Streptococcus pneumoniae (pneumococcus) are Gram-positive bacteria with >90 serotypes classified based on the composition of the
capsular polysaccharides [1]. These bacteria typically reside asymptomatically in the nasopharynx [1]. However, in certain susceptible
individuals, S. pneumoniae can cause invasive pneumococcal diseases (IPD) including meningitis, pneumonia and bacteremia
[2]. Increased mortality rates associated with IPD are often seen
in high-risk populations such as young children, the elderly, and
immunocompromised patients [2].
Two vaccines covering common disease causing bacterial serotypes that rely on production of anti-capsular antibodies (Ab) are
available [3]. The pneumococcal polysaccharide vaccine (PPSV or
Pneumovax
® ) covers 23 serotypes and consists of repeating polysaccharides that can directly cross-link B cell receptors leading to
antibody production independent of T-cells [4]. The
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_33, © Springer Science+Business Media, LLC, part of Springer Nature 2021
559
Testing Anti-Pneumococcal Antibody Function Using
Bacteria and Primary Neutrophils
Manmeet Bhalla, Shaunna R. Simmons, Essi Y. I. Tchalla,
and Elsa N. Bou Ghanem
Abstract
Antibodies against Streptococcus pneumoniae (pneumococcus) following vaccination are crucial for host
protection against invasive pneumococcal infections. The antibodies induced by pneumococcal vaccines act
as opsonins to mediate bacterial uptake and killing by host phagocytic cells, especially polymorphonuclear
leukocytes (PMNs) also called neutrophils. Therefore, it is important to measure not only the levels of
antibodies induced by a pneumococcal vaccine candidate but their actual functional capacity in mediating
bacterial opsonization and killing by PMNs. Here, we describe a protocol to demonstrate effective deposition of vaccine-induced antibodies on the surface of S. pneumoniae by flow cytometry and subsequent
opsonophagocytic killing (OPH) by murine bone-marrow derived PMNs.
Key words Streptococcus pneumoniae, Opsonophagocytic killing, Antibodies, Neutrophils, Flow
cytometry
1 Introduction
Streptococcus pneumoniae (pneumococcus) are Gram-positive bacteria with >90 serotypes classified based on the composition of the
capsular polysaccharides [1]. These bacteria typically reside asymptomatically in the nasopharynx [1]. However, in certain susceptible
individuals, S. pneumoniae can cause invasive pneumococcal diseases (IPD) including meningitis, pneumonia and bacteremia
[2]. Increased mortality rates associated with IPD are often seen
in high-risk populations such as young children, the elderly, and
immunocompromised patients [2].
Two vaccines covering common disease causing bacterial serotypes that rely on production of anti-capsular antibodies (Ab) are
available [3]. The pneumococcal polysaccharide vaccine (PPSV or
Pneumovax
® ) covers 23 serotypes and consists of repeating polysaccharides that can directly cross-link B cell receptors leading to
antibody production independent of T-cells [4]. The
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_33, © Springer Science+Business Media, LLC, part of Springer Nature 2021
559