HHS SBIR PA-14-101

Awareness of the mechanisms of HIV persistence and latent reservoirs has greatly increased over the last decade.  The scientific community now agrees that the time has come to pursue research towards a cure for HIV.  In addition, eradication efforts are facilitated by the presence of a clear target, the integrated, latent HIV provirus in resting CD4+ T-cells. A few years ago, HIV cure research moved into the spotlight following a report in the New England Journal of Medicine that an HIV-infected patient had no detectable viral load and declining HIV antibody titers following CCR5 delta32/delta32 stem-cell transplantation (the “Berlin” patient).  This case study demonstrated that a cure from HIV infection was technically achievable. The development of highly active antiretroviral therapy (HAART) has resulted in long-term suppression of HIV replication in infected individuals to levels below the limit of detection using standard diagnostic assays. Curing HIV infection will require the elimination of long-lived reservoir cells that persist despite antiretroviral therapy.

For this FOA, NIH is especially interested in Small Business Innovation Research (SBIR) grant applications from Small Business Concerns (SBCs) to develop novel semi-automated, medium-throughput assays that correlate with replication-competent, re-activatable virus from latently infected cells. Infected resting CD4+ T-cells and monocytes/macrophages have been shown to contribute to the persistent HIV reservoir. It has been estimated that in HIV-positive individuals on HAART, approximately one per million resting CD4+ T cells in the peripheral blood contain latent proviruses capable of producing replication-competent virus. Quantifying cells harboring latent provirus that can be re-activated under a changing environment is critical for evaluating HIV elimination strategies, but the low frequency of these cells poses a logistical challenge. The current gold-standard assay for quantifying replication competent HIV is a limiting dilution, quantitative viral outgrowth assay (QVOA) that is labor-intensive, requires a high number of cells, and has low sensitivity.  Other drawbacks of the QVOA assay are residual intracellular HAART pools that may suppress virus replication in vitro and the persistence of epigenetic silencing in the absence of latency-reversing agents. Therefore, the QVOA assay may not accurately reflect the total number of replication-competent viruses, creating a need for the development of more efficient and sensitive replacement assays.

The second goal of this FOA focuses on novel, sensitive and super-resolution imaging techniques to visualize the HIV reservoir before and after therapeutic interventions. Technology developed in the past 10 years has dramatically increased the ability of scientists to directly visualize viral proteins and nucleic acids in situ.

This FOA will support applications in the following areas:

(1) Sensitive in vitro assays that measure changes in the residual pool of HIV-infected cells and integrated proviruses (that persist despite effective antiretroviral therapy) before and after cure interventions.

Examples of research that would fit within the scope of this FOA include, but are not limited to those listed below:

• Modifications of the current viral outgrowth assay to improve throughput, cost, sensitivity, and reproducibility;
• Development of an indicator cell line assay that has the same or a higher sensitivity as the current viral outgrowth assay;
• Use of advanced detection technologies to quantify latent HIV;
• Development of a high-throughput molecular assay that could serve as a surrogate for the viral outgrowth assay in quantifying replication-competent latent HIV reservoirs;
• Assays based on detecting specific mRNA species associated with reactivation of latent HIV and virus production;
• Sensitive assays that detect HIV production after reactivation from latency; and
• Determination of the cellular source of the persistent virus using cellular proteins incorporated into the virion, such as tetraspanins from macrophages.

Applications proposing research in the following areas will NOT be supported under in vitro assay development:

• Approaches that are not focused on latent or persistent HIV reservoirs;
• Point-of-care HIV assays not in support of HIV cure research;
• Assays solely based on unspliced mRNA; and
• Approaches proposing clinical trials.

(2) Sensitive non-toxic in vivo or in situ molecular imaging techniques that measure the residual viral reservoir in tissues or monitor the experimental treatment of fully HAART-suppressed HIV-infected humanized mice, SIV/SHIV-infected monkeys, or HIV-infected individuals.

Examples include:

• Non-antibody based radiolabelled probes;
• Super-resolution methods, e.g., fluorescein arsenical helix binder (FlAsH-PALM);
• Fluorescent in situ hybridization methods;
• RNA trans-splicing methods;
• Fluorescent HIV/SIV reporter viruses; and
• Biosensors that distinguish physical differences between HIV-infected and uninfected cells.

It is important to emphasize that the topics listed above are only meant to be illustrative, and not a comprehensive list of appropriate topics, or exclusive of other appropriate topics. Applications may propose projects that are highly innovative or that are enhancements of current approaches. In either case, studies must significantly advance the current state of the art and have commercial potential.