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A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
Evolution of a functionally intact but antigenically distinct DENV fusion loop.
Rita M Meganck1, Deanna Zhu2, Stephanie Dong2
1Department of Molecular Microbiology and Immunology, Saint Louis University, Saint Louis, United States.
Researchers engineered a mutated dengue virus (DENV) variant to evade antibody-dependent enhancement, a major challenge for DENV vaccines. This modified DENV may lead to safer vaccines for individuals without prior DENV exposure.
Area of Science:
- Virology
- Immunology
- Vaccinology
Background:
- Antibody-dependent enhancement (ADE) complicates dengue virus (DENV) vaccine development and is linked to severe secondary DENV infections.
- ADE is associated with antibodies targeting the conserved fusion loop (FL) motif on the DENV envelope protein, crucial for viral entry.
Purpose of the Study:
- To engineer a DENV variant with a mutated FL that evades FL-targeting antibodies.
- To assess the neutralization sensitivity of engineered DENV variants to different antibody types.
Main Methods:
- Utilized saturation mutagenesis and directed evolution to create a DENV variant with a mutated FL (D2-FL).
- Combined FL mutations with a modified prM cleavage site to generate a mature DENV variant (D2-FLM).
- Evaluated neutralization titers using serum from DENV-infected non-human primates (NHP).
Main Results:
- Engineered DENV variants (D2-FL and D2-FLM) were not neutralized by FL-targeting antibodies.
- D2-FLM evaded both prM- and FL-targeting antibodies but remained sensitive to other antibodies.
- Heterotypic (DENV4) NHP serum showed reduced neutralization of D2-FL and D2-FLM compared to wildtype DENV2, while homotypic (DENV2) NHP serum showed similar neutralization.
Conclusions:
- D2-FL and D2-FLM are valuable tools for characterizing cross-reactive (CR) antibody subtypes.
- These engineered DENV variants represent a promising platform for developing safer live-attenuated DENV vaccines, particularly for naive individuals and children.
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