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A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion
Published on: August 14, 2018
Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition
Enas Sheik-Khalil1, Mark-Anthony Bray, Gülsen Özkaya Şahin
1Department of Laboratory Medicine, Lund University, Lund, Sweden. enas.sheik-khalil@med.lu.se.
Insights
A new automated assay can simultaneously measure HIV neutralization and cell-cell fusion inhibition. This high-throughput method aids in developing an effective HIV vaccine by assessing antibody responses.
Area of Science:
- Immunology
- Virology
- Biotechnology
Background:
- Standardized assays are crucial for detecting HIV-neutralizing antibody responses.
- Developing an effective HIV vaccine relies on robust methods to evaluate immune responses.
Purpose of the Study:
- To present a novel, automated, high-throughput assay for evaluating HIV neutralization and cell-cell fusion inhibition.
- To enable simultaneous assessment of two key antiviral mechanisms within a single assay.
Main Methods:
- Developed a high-throughput, high-content automated plaque reduction (APR) assay using automated microscopy and image analysis.
- Quantified HIV neutralization by measuring the reduction in fluorescent plaque numbers.
- Assessed cell-cell fusion inhibition by measuring the reduction in plaque area.
Main Results:
- Demonstrated that neutralization strength correlates with the virus's ability to form syncytia.
- Introduced the inhibitory concentration of plaque area reduction (ICpar) as a metric for fusion inhibition.
- Showed that inhibiting cell-to-cell fusion requires higher reagent concentrations than inhibiting virus neutralization.
Conclusions:
- An automated, image-based, high-throughput HIV plaque reduction assay was developed.
- This assay allows for the first time simultaneous evaluation of viral neutralization and cell-cell fusion inhibition.
- The findings indicate distinct concentration requirements for inhibiting viral neutralization versus cell-to-cell fusion.
Background:
Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine.
Methods:
Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area.
Results:
We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition.
Conclusions:
We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.
