Human immunodeficiency virus (HIV) is a serious global health issue with 36.7 million people infected worldwide and over 70% of these people living in low- to middle-income countries. Despite increased access to antiretroviral therapy (ART) to manage HIV and recent declines in HIV-related deaths throughout these countries, the emergence of drug-resistant (DR) strains of HIV threatens the continued efficacy of available ART. Drug resistance testing can be used to inform proper ART regimens, improve patient outcomes, and prevent further transmission. However, the gold-standard method (Sanger sequencing) is costly, requires sophisticated laboratory resources, and is unable to detect low amounts of DR HIV that can lead to treatment failure. To address this technology gap, the Frenkel Lab at Seattle Children’s Research Institute developed a sensitive, inexpensive, laboratory-based Oligonucleotide Ligation Assay (OLA). The OLA identifies single nucleotide polymorphisms (SNPs) known to confer resistance to ART via highly specific ligation of DNA probes targeting a SNP of interest. In order to expand application of the OLA to lesser-equipped settings, the Frenkel Lab is collaborating with the Lai and Lutz Labs (UW Bioengineering) to re-engineer the OLA into an integrated, paper-based device. As part of this effort, the present project aims to simplify the critical ligation process into a solid-phase enzymatic reaction system that can be integrated with a previously developed paper-based detection device. Compared to the laboratory assay, the solid-phase format provides a less intensive user protocol, protection from contaminants, and seamless transfer of analyte to downstream detection. Several affinity-based and bioconjugation-based enzyme immobilization methods will be investigated to create the solid-phase system. Quantitative analysis of enzymatic activity and immobilization efficiency will then be used to identify the best-performing method(s). Ultimately, simplification of the ligation process is expected to move the OLA closer to implementation in resource-limited areas impacted by HIV.

The Oligonucleotide Ligation Assay (OLA) specifically detects point mutations in the HIV polymerase gene conferring drug resistance. Screening patients for drug-resistant HIV is a necessary step in determining the proper treatment. However, the traditional OLA is complex and lengthy, making it non-ideal for low-resource settings. We are exploiting a novel approach to adapt the OLA into a point-of-care, paper-based format. To enable downstream isothermal ligation and paper-based detection steps, the amplification step of the OLA must be designed to preferably generate many copies of single-stranded HIV DNA (ssDNA). Typically, this can be achieved using a Linear-After-The-Exponential Polymerase Chain Reaction (LATE-PCR), which uses an uneven ratio of forward and reverse primers. Though the LATE-PCR is a useful tool in producing large amounts of ssDNA, it involves a plethora of parameters whose contributions to the reaction output are not well understood. Thus, the LATE-PCR is often optimized via trial and error and may not yield the most desirable outcome. In order to minimize wasted resources from unnecessary trials and achieve a high-efficiency amplification, we aim to understand the behavior, predict the outcome, and develop a platform for LATE-PCR optimization. To accomplish this, we have observed the behavior of LATE-PCR reactions by measuring ssDNA output when altering key reaction parameters such as the number of cycles run, primer concentration ratios, enzyme concentrations, enzyme rates, salt concentrations, and primer sequences. Analysis of these results will allow us to understand the effects of these parameters on the thermodynamics and kinetics of the LATE-PCR. This will form the basis of a computational model which will accurately predict reaction behavior, and provide specific LATE-PCR reactions given user-defined reaction parameters and desired ssDNA output. Future research will focus on expanding the LATE-PCR model to other instrument-free isothermal amplification approaches for a simplified point-of-care OLA.

36.7 million people are living with HIV worldwide, with most in low- to middle-income countries. Among many clinical interventions for controlling HIV, Antiretroviral Therapy (ART) has shown success in reducing HIV transmission. However, the effectiveness of these ART is undermined by rising drug-resistant HIV. The lab-based Oligonucleotide Ligation Assay (OLA) can specifically detect HIV drug resistance as an alternative to expensive complete-genome sequencing, but its reliance on moderately expensive lab equipment prevents its universal access. Our lab, in collaboration with the Frenkel Lab (Seattle Children’s Research Institute) and the Lai Lab (UW Bioengineering), is developing a simplified OLA-based HIV drug resistance test. In the OLA, PCR amplification of the HIV Pol region is necessary before subsequent detection of Single Nucleotide Polymorphisms (SNPs) conferring HIV drug resistance. Current amplification relies on a thermal cycler, a >$2.5k instrument not widely available in low resource settings. Isothermal Helicase Dependent Amplification (HDA) eliminates need for a thermal cycler. HDA has been used to amplify the conserved Gag and Env regions of HIV, however it has never been used for amplification of the Pol region, a non-conserved region containing mutations that interfere with DNA annealing in isothermal amplification. We have designed and validated three sets of primers that successfully amplified three regions in the HIV Pol gene covering six common SNPs that confer HIV drug resistance against 1st-line ART. These primer sets each could detect down to 10 copies of HIV Pol DNA per reaction.  We are currently working on a multiplexed HDA assay that simultaneously amplifies three regions in a single tube to reduce cost and consolidate workflow. Implementation of HDA will allow OLA to be further simplified for use in low resource settings by allowing accurate diagnoses of HIV-infected individuals, controlling transmission rates, and reducing the evolution of drug-resistant HIV within entire populations.

Anti-retroviral therapies (ART) are an effective strategy to treat HIV patients by suppressing viral replication and reducing transmission. However, error-prone HIV reverse transcriptase (RT) can cause single nucleotide polymorphisms (SNPs) associated with drug-resistant HIV. To ensure effective treatment, HIV patients must be tested for drug-resistant SNPs prior to initiating ART. The Oligonucleotide Ligation Assay (OLA) is a lab-based sensitive method for detecting drug-resistant SNPs in HIV, developed and validated by the Frenkel lab at Seattle Children’s Research Institute (SCRI)- however complex workflow has limited implementation in low-resource labs. To address the need for an HIV drug resistance test in these regions, the UW Bioengineering Lutz lab, in collaboration with the Frenkel lab and the UW Bioengineering Lai lab, are attempting to re-engineer the lab-based traditional OLA into an equipment-free, point-of-care format. We are currently exploiting innovative chemistry and autonomous wicking properties of paper-based microfluidics to integrate the OLA into a credit-card-sized cartridge device. Multichannel capillary-based sequential delivery platform is used to supply the reagents required in all main steps of the OLA: nucleic acid extraction, amplification, ligation, and detection, to the different regions of the paper-based device. To enable incubation during amplification and ligation a pull-tab technology is used to physically disconnect the circuit. After these reactions are completed, the tab is pulled to re-activate the next sequential delivery steps. Currently, a proof-of-concept integrated device has been designed. To initially test the microfluidic physics, five fluid sources were colored to permit visualization of the flow. A successful controlled sequential delivery to mimic all steps in the OLA has been demonstrated. Next, the chemistry of each step will be incorporated into this cartridge device. The success of this work will enable non-instrumented, sensitive detection of HIV drug resistance and viral load that requires only two user steps: addition of a rehydration buffer and activation of pull-tabs.

Nucleic acid (NA) extraction is a critical step in molecular diagnostic assays, since it can impact the sensitivity and specificity of the subsequent amplification step such as the Polymerase Chain Reaction (PCR). Most commercially-available NA extraction approaches capture both disease target NA and other non-target NAs. However, these non-target NAs compete with the target NA for resources and as a result can reduce the efficiency of PCR to detect a disease target of interest. Here, we present a strategy to specifically capture NA sequence of HIV from lysed infected human CD4 cells. After cell lysis, restriction enzymes are used to excise the HIV pol sequence from proviral DNA integrated in human genome. After a heat-denaturing step, biotinylated DNA probes hybridize to the HIV pol sequence. This solution containing the duplexes of pol gene/biotinylated probes is transferred to a microcentrifuge filter tube containing streptavidin agarose bead resin. Subsequent centrifugation retains the duplexes of biotinylated probes and HIV pol while removing other undesirable non-target NAs and possible contaminants in cell lysate. Finally, the target sequence is eluted from the biotinylated probes and with sodium hydroxide and subsequently neutralized prior to addition to PCR. For initial optimization of this capture system, synthetic HIV pol at a concentration ranged from of 50 – 100,000 copies were used as mock specimens. The method presented achieved 60% ~ 100% HIV DNA target recovery. In the next stage of this work, the effectiveness of capture will be tested with the upstream sample preparation procedures. We are currently investigating capture efficiency and restriction enzyme activity in different sample solute conditions in an effort to streamline the two steps. The successful development of this sequence-specific capture will potentially improve the sensitivity of the PCR assay and allow for early HIV diagnostics.