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Updated: Aug 23, 2025
Optimization of a Quantitative Micro-neutralization Assay
Published on: December 14, 2016
Relationship between neutralizing and opsonizing monoclonal antibodies against foot-and-mouth disease virus
Artur Summerfield1,2, Heidi Gerber1, Rebeka Schmitt1
1Institute of Virology and Immunology, Köniz, Switzerland.
Abstract:
Previous studies demonstrated that polyclonal antibodies against foot-and-mouth disease virus (FMDV) generated by vaccination can mediate immune functions not only through virus neutralization but also through promoting virus uptake by macrophages and dendritic cells that are otherwise resistant to FMDV infection. This causes abortive infections resulting in activation, enhanced antigen presentation but also cell death. Here we report the use of RAW264.7 cells representing a murine macrophage cells line to characterize opsonizing functions of a collection of monoclonal antibodies (mAbs) against FMDV O and A serotypes. We demonstrate that all neutralizing immunoglobulin G isotype mAbs are able to opsonize FMDV resulting in increased cell death of RAW264.7 cells. In contrast, neutralizing IgM antibodies did not possess this activity. Opsonization was observed with broader reactivity within the serotype when compared to neutralization. Importantly, the anti-O serotype D9 mAb reacting with the continuous epitope within the G-H loop of VP1 that contains the RGD binding site of FMDV, opsonized several FMDV serotypes despite its restricted neutralizing activity within the O serotype. Furthermore, by generating RAW264.7 cells expressing bovine CD32, an easy-to-use cell-based assay system to test for bovine antibody-dependent enhanced infection of FMDV was generated and tested with a collection of sera. The data indicate that opsonizing titers correlated better with vaccine dose when compared to neutralizing titers. On the other hand, neutralization and opsonization titers were similar predictive of protection. We conclude that low avidity interactions are sufficient to mediate Fcγ receptor-mediated immune functions that could contribute to protective immune responses against FMDV.
Insights
Antibodies against foot-and-mouth disease virus (FMDV) can enhance virus uptake by immune cells, leading to cell death and improved antigen presentation. Opsonizing antibodies, particularly IgG, show broader reactivity and correlate better with vaccine response than neutralizing antibodies.
Area of Science:
- Immunology
- Virology
- Veterinary Medicine
Background:
- Polyclonal antibodies against foot-and-mouth disease virus (FMDV) mediate immune functions beyond neutralization, including enhanced virus uptake by macrophages and dendritic cells.
- This uptake can lead to abortive infections, resulting in immune cell activation, enhanced antigen presentation, and cell death.
Purpose of the Study:
- To characterize the opsonizing functions of monoclonal antibodies (mAbs) against FMDV serotypes O and A using a murine macrophage cell line (RAW264.7).
- To develop and test a cell-based assay for bovine antibody-dependent enhanced infection of FMDV.
- To compare the correlation of opsonizing and neutralizing antibody titers with vaccine dose and protection.
Main Methods:
- Utilized RAW264.7 cells to assess the opsonizing capacity of various monoclonal antibodies (mAbs) against FMDV.
- Generated RAW264.7 cells expressing bovine CD32 to create an assay for bovine antibody-dependent enhanced infection.
- Tested a collection of sera using the developed cell-based assay.
Main Results:
- All neutralizing immunoglobulin G (IgG) isotype mAbs opsonized FMDV, increasing RAW264.7 cell death, while neutralizing IgM antibodies did not.
- Opsonization demonstrated broader reactivity within serotypes compared to neutralization.
- A specific anti-O serotype mAb (D9) opsonized multiple FMDV serotypes despite limited neutralization activity.
- Opsonizing titers correlated better with vaccine dose than neutralizing titers, and both opsonization and neutralization titers were similar predictors of protection.
Conclusions:
- Low avidity interactions are sufficient for Fcγ receptor-mediated immune functions against FMDV.
- Opsonization is a significant immune mechanism contributing to protective responses against FMDV.
- The developed cell-based assay provides a valuable tool for evaluating FMDV antibody responses in cattle.
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