Related Experiment Video
Updated: Jun 9, 2026
Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens
Published on: April 5, 2019
The efficacy of pneumococcal capsular polysaccharide-specific antibodies to serotype 3 Streptococcus pneumoniae
Kevin Fabrizio1, Catherine Manix, Haijun Tian
1Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Abstract:
The efficacy of antibody immunity against Streptococcus pneumoniae stems from the ability of opsonic, serotype (ST)-specific antibodies to pneumococcal capsular polysaccharide (PPS) to facilitate killing of the homologous ST by host phagocytes. However, PPS-specific antibodies have been identified that are protective in mice, but do not promote opsonic killing in vitro, raising the question of how they mediate protection in vivo. To probe this question, we investigated the dependence of antibody efficacy against lethal systemic (intraperitoneal, i.p.) infection with Streptococcus pneumoniae serotype 3 (ST3) on macrophages and neutrophils for the following PPS3-specific monoclonal antibodies (MAbs) in survival experiments in mice using a non-opsonic human IgM (A7), a non-opsonic mouse IgG1 (1E2) and an opsonic mouse IgG1 (5F6). The survival of A7- and PPS3-specific and isotype control MAb-treated neutrophil-depleted and neutrophil-sufficient and macrophage-depleted and macrophage-sufficient mice were determined after i.p. challenge with ST3 strains 6303 and WU2. Neutrophils were dispensable for A7 and the mouse MAbs to mediate protection in this model, but macrophages were required for the efficacy of A7 and optimal mouse MAb-mediated protection. For A7-treated mice, macrophage-depleted mice had higher blood CFU, cytokines and peripheral neutrophil levels than macrophage-sufficient mice, and macrophage-sufficient mice had lower tissue bacterial burdens than control MAb-treated mice. These findings demonstrate that macrophages contribute to opsonic and non-opsonic PPS3-specific MAb-mediated protection against ST3 infection by enhancing bacterial clearance and suggest that neutrophils do not compensate for the absence of macrophages in the model used in this study.
Insights
Macrophages are crucial for antibody protection against Streptococcus pneumoniae infections, even when antibodies do not promote bacterial killing. Neutrophils play a limited role in this protective mechanism.
Area of Science:
- Immunology
- Microbiology
- Infectious Diseases
Background:
- Antibody immunity against Streptococcus pneumoniae relies on opsonic antibodies targeting pneumococcal capsular polysaccharide (PPS) for phagocyte-mediated killing.
- Some PPS-specific antibodies protect mice without promoting in vitro opsonic killing, raising questions about their in vivo mechanisms.
Purpose of the Study:
- To investigate the roles of macrophages and neutrophils in mediating protection by different types of PPS3-specific monoclonal antibodies (MAbs) against Streptococcus pneumoniae serotype 3 (ST3) infection in mice.
- To differentiate the contributions of opsonic and non-opsonic antibody mechanisms in vivo.
Main Methods:
- Survival experiments in mice challenged with ST3 strains after treatment with non-opsonic (A7, 1E2) and opsonic (5F6) PPS3-specific MAbs.
- Depletion of neutrophils and/or macrophages to assess their necessity for MAb-mediated protection.
- Quantification of blood colony-forming units (CFU), cytokine levels, and peripheral neutrophil counts.
Main Results:
- Neutrophils were dispensable for the protective efficacy of both non-opsonic and opsonic MAbs.
- Macrophages were essential for the efficacy of the non-opsonic IgM MAb (A7) and for optimal protection by the mouse IgG1 MAbs.
- In the absence of macrophages, A7-treated mice showed increased bacterial load and inflammation, while macrophage-sufficient mice had reduced bacterial burden in tissues.
Conclusions:
- Macrophages are critical for both opsonic and non-opsonic antibody-mediated protection against ST3 infection.
- Macrophages enhance bacterial clearance and contribute significantly to host defense, independent of neutrophil function in this model.
- Neutrophils do not compensate for macrophage absence in mediating protection against ST3 infection with these MAbs.
Related Concept Videos
01:31Defense Against Bacterial Pathogens
Phagocytes
Phagocytes are the frontline soldiers of the immune system. They include neutrophils and macrophages. Neutrophils are the most abundant type of white blood cell and are quickly mobilized to the site of infection. Macrophages are larger cells that patrol...
01:25Immune Surveillance by NK Cells and Phagocytes
Natural Killer Cells: The Fast Responders
NK cells are large granular lymphocytes found in the blood and lymphatic system. These...