Description:
Fast Track Proposals will be accepted.
Direct to Phase II will not be accepted.
Number of anticipated awards: 1-2
Budget (total costs):
Phase I: $300,000 for up to 1 year;
Phase II: $2,000,000 for up to 3 years.
Background
Despite effective antiretroviral therapy (ART), HIV-1 persists in all infected individuals as proviral DNA within long-lived memory CD4+ T cells. Early studies on PBMC from HIV-infected donors on suppressive ART demonstrated that a subset of proviruses could be induced to replicate in tissue culture. This replication-competent HIV reservoir constitutes the primary barrier for curing HIV infection. In this context, the number of full-length (intact) HIV proviruses represents the upper limit of the replication-competent HIV reservoir whereas infectious units per million (IUPM) measured by the Quantitative Viral Outgrowth Assay constitute the lower limit of this reservoir of interest.
To measure success, the design of HIV cure strategies should be accompanied by the development of fast and reliable assays that accurately measure changes in the replication-competent HIV reservoir. In addition, for practical reasons, HIV reservoir assays should only require small sample sizes, either a few million cells or a tissue biopsy. Recently, several assays have been developed to replace the time-consuming Quantitative Viral Outgrowth Assay, but validation for clinical applications and commercial purposes is lagging behind.
Project Goal
The overall goal of this project is to develop and commercialize sequence-based HIV reservoir assays for clinical HIV cure interventions. Specifically, the assay should be designed as an analytical tool to monitor the size of the replication-competent HIV reservoir in clinical research and if successful, in prospective clinical trials. Essential characteristics for commercially applicable HIV reservoir assays are reproducibility, low labor intensity, medium-to-high throughput performance, and correlation with the replication-competent HIV reservoir. When designing the requested assays, it needs to be also taken into consideration that besides internal sequence deletions, lethal mutagenesis, such as G-A hypermutations, stop codons within the HIV open reading frames and nonfunctional LTR promoters could also present blockades in the HIV replication cycle.
An additional goal of this project is to develop secondary assays that are not tissue culture-based and discriminate between actively transcribed and latent full-length proviruses. Applicants also need to provide a plan for evaluation how their assay correlates with the Quantitative Viral Outgrowth Assay and other recently developed HIV reservoir assays, such as the Tat/Rev Induced Limiting Dilution Assay (TILDA), and why their assay is superior to similar reservoir assays. The ultimate goal of this project is to develop an assay that accurately measures the size of the HIV reservoir defined as the “barrier” to the HIV cure, which needs to be eliminated to prevent viral rebound.
Phase I activities may include, but are not limited to:
• Developing medium-to-high throughput sequence-based assays that accurately reflect the size of the replication-competent reservoir.
• Developing standardized controls for the sequence-based assays.
• Confirming that the sequences detected correspond to full-length HIV proviruses.
• Determining the following assay parameters:
o Specificity: Will the assay only detect full-length proviruses?
o Sensitivity: Will the assay detect small levels of full-length proviruses? What is the dynamic range and is it adequate?
o Interference: will components in the assay sample interfere with the assay (for example, blood anticoagulants, such as heparin)?
o Robustness: Can the assay cope with small changes in the assay sample/equipment/operator?
o Accuracy: Is the assay capable of accurately determining the absolute number of full-length proviruses?
Phase II activities may include, but are not limited to:
• Determining the utility of the assay for clinical samples.
• Testing clinical samples from diverse cohorts of HIV+ individuals with varying levels of residual viral reservoirs.
• Validating the developed assays under CLIA and ICH harmonized Good Clinical Practices.
• Determining that the assays qualify for FDA regulatory submissions.
• Determining assay performance for different HIV subtypes and drug-resistant strains.
• Determining assay performance in tissues versus blood.
• Demonstrating that the assay can measure changes in the size of the latent HIV reservoir in response to an intervention.
