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Accurate, High-Throughput Quantification of HIV Reservoirs Using Optical Scanning

HIV viral protein expression and functional virus reservoir size are rapidly and accurately quantified using optical scanning

Problem:

HIV can incorporate itself into the DNA of a host cell without producing circulating virus. When lying dormant in these reservoirs, HIV cannot be detected and eradicated by the immune system of the host.

As most of the HIV DNA in these reservoirs is hypermutated and incapable of producing infectious virions, a major hurdle in efforts to cure HIV, and to manage care of patients on antiretroviral therapy, is accurately quantifying the size of infection-competent viral reservoirs without interference from defective HIV DNA.

Current methods to quantify viral load are labor-intensive and prone to systematic errors.

Solution:

This invention identifies replication-competent HIV reservoirs by screening for the presence of one protein (Gag) and downregulation of another (CD4) on the surface of cells. These features of cells infected with replication-competent HIV are encoded at disparate ends of the viral DNA; consequently, one of these features without the other is unlikely to occur in a cell that contains a replication competent provirus (the form of HIV integrated into the human chromosome).

Fiber-optic assay scanning technology couples high-throughput optical scanning methods with an assay that recognizes target proteins via binding of dye-bearing antibodies to selectively identify cells infected with functional HIV. This method has the potential to quantify HIV reservoirs with faster results, improved accuracy, less labor, less technical expertise, and smaller required sample sizes.

Advantages:

Improved accuracy of HIV reservoir quantification
Distinguishes between replication competent and defective proviruses
Rapid, high-throughput quantification
Faster turn-around time
Smaller samples tolerated

Applications:

Monitoring HIV patients who are receiving antiretroviral therapy to determine baseline and induced reservoir expression
Clinical research to determine the effects of new drugs such as latency reversal agents on HIV

Fiber-optic assay scanning technology (FAST) images show HIV Gag (red) and internalized CD4 (green) on the surface of cells. These images can be merged (right) to identify and quantify cells infected with replication-competent HIV that is expressed. A nuclear stain (blue) is used to show cells.