Single-Molecule Diagnostics to Support Curative Interventions for Tuberculosis and HIV

Abstract

Tuberculosis (TB) and the human immunodeficiency virus (HIV) are two of the leading causes of death worldwide. Tuberculosis is curable, but because of the difficulties of diagnosing it, many people with TB—and the majority of those killed by it—never begin treatment. HIV can be treated with lifelong medication. But if drug resistance develops or treatment is interrupted, the virus resurges. This could be prevented by an HIV cure that either clears HIV from the body or keeps the virus suppressed without continued therapy. Next-generation diagnostics will play a central role in supporting access to existing TB cures and future HIV cures. In this thesis, I describe the advancement of digital enzyme-linked immunosorbent assay (ELISA) protein detection methods in service of curing these two deadly infectious diseases.

Existing TB diagnostics rely heavily on sputum, which is highly infectious, leading to increased TB cases among health care workers and limiting access to places with appropriate biosafety precautions. We developed a multiplexed Single Molecule Array (Simoa) digital ELISA that can diagnose TB from biomarkers in urine. Our assay is highly sensitive, as demonstrated in diverse cohorts totaling approximately 600 individuals.

Simoa is a robust and widely used platform, but its accessibility is limited because it relies heavily on advanced microwell and imaging technology. We developed a new digital ELISA platform, called Molecular On-bead Signal Amplification for Individual Counting (MOSAIC), that performs the final readout step with a flow cytometer, bringing digital ELISA within reach of many hospitals and other health care centers. In addition to reducing instrumentation and cost, MOSAIC also allows for greater sensitivity and higher-order multiplexing than Simoa. It is, to our knowledge, the most sensitive protein measurement technique ever developed, with attomolar limits of detection.

Finally, I describe the application of MOSAIC toward the development of HIV cures and longer-acting antiretroviral medications. These depend on a deeper understanding of the biology of HIV, and when they are ready for clinical trials, will also need highly sensitive tests to characterize the virus-host interactions and determine whether they are working. We developed ultrasensitive Simoa and MOSAIC assays for 20 circulating host and viral proteins and measured them in a cohort of 17 individuals with HIV whose treatment was interrupted, to evaluate which biomarkers could predict when the virus would rebound. Baseline levels of these biomarkers did not predict viral rebound, but changes over time did, highlighting the need for scalable personalized approaches.

HIV and TB are two of the great diseases in the world. The next generation of diagnostic technologies, a urine test conducted on expensive instrumentation, and newly identified circulating biomarkers will not in themselves solve these problems. But these more sensitive assays are one step closer to the true biology of these diseases, and these advances in accessibility bring this this ultrasensitive monitoring one step closer to the clinic.