Measuring the Success of HIV-1 Cure Strategies

Abstract

HIV-1 eradication strategies aim to achieve viral remission in the absence of antiretroviral therapy (ART). The development of an HIV-1 cure remains challenging due to the latent reservoir (LR): long-lived CD4 T cells that harbor transcriptionally silent HIV-1 provirus. The LR is stable despite years of suppressive ART and is the source of rebound viremia following therapy interruption. Cure strategies such as "shock and kill" aim to eliminate or reduce the LR by reversing latency, exposing the infected cells to clearance via the immune response or the viral cytopathic effect. Alternative strategies include therapeutic vaccination, which aims to prime the immune response to facilitate control of the virus in the absence of ART. Despite promising advances, these strategies have been unable to significantly reduce the LR or increase the time to viral rebound but have provided invaluable insight in the field of HIV-1 eradication. The development and assessment of an HIV-1 cure requires robust assays that can measure the LR with sufficient sensitivity to detect changes that may occur following treatment. The viral outgrowth assay (VOA) is considered the gold standard method for LR quantification due to its ability to distinguish intact and defective provirus. However, the VOA is time consuming and resource intensive, therefore several alternative assays have been developed to bridge the gap between practicality and accuracy. Whilst a cure for HIV-1 infection remains elusive, recent advances in our understanding of the LR and methods for its eradication have offered renewed hope regarding achieving ART free viral remission.

Keywords: DNA/RNA quantification; HIV-1; latency; latent reservoir; persistence.

Figure 1

Different strategies for HIV-1 cure. From top to bottom. Shock and kill relies on reversal of latency using a range of different compounds including TLR agonists and HDACis, followed by CTL mediated cell clearance, whilst ART blocks new infections caused by virus release. Lock in and apoptosis utilizes latency reversal agents, as well as a Pr55Gag inhibitor to block virus budding from the cell. The build-up of viral RNA and proteins leads to apoptosis of the infected cell. Block and lock approaches aim to reinforce latency mechanisms by using siRNAs or Tat inhibitors to disrupt cellular epigenetic regulators or viral replication, respectively (red cells represent HIV-1 latently infected cells).

Figure 2

Comparison of assays that measure replication competent provirus specifically or all provirus. Cells for analysis come from either peripheral blood or from anatomical compartments. From left to right: following DNA extraction, multiple HIV-1 DNA forms can be assayed by PCR based on the primer position. For integrated HIV-1 DNA assays, a primer targeting repeated Alu sequences within the human genome are paired with a HIV-1 specific primer. Total HIV-1 DNA can be measured by primers specific for regions within the viral genome, this is most commonly performed with primers targeting conserved regions within the LTR. Non-integrated HIV-1 DNA forms such as 2-LTR and 1-LTR circular DNA can be measured by primers specific that will amplify junctions that are only present in these DNA forms. The intact proviral DNA assay (IPDA) uses primers within the packaging signal (Ψ) and env to determine replication competence. This assay also uses primers targeting regions within the human genome to measure cell numbers and correct for DNA shearing. Replication competence is determined when both sequences are present from ddPCR. The quadruplex PCR (Q4PCR) uses primers within Ψ, env, gag, and pol to quantify provirus in limiting dilutions, and NGS is uses to confirm replication competence in reactions with 2/4 of the sequences present. Cell based assays use purified cell samples to measure virus or RNA production following stimulation. The viral outgrowth assay (VOA) uses limiting dilutions of CD4 T cells that are stimulated with PMA and irradiated PBMCs to induce viral gene expression; viral outgrowth is supported by incubation with HIV-1 negative donor cells and measured by p24 ELISA, viral RNA or reverse transcriptase activity. Cell associated (CA) RNA or tat/rev induced limiting dilution assays measure viral RNAs following HIV-1 activation, reducing time to read out when compared to the VOA. Assays in blue shaded area are not specific for cells infected with replication competent provirus because viral DNA is measured indiscriminately. Assays in shaded orange area are more specific for replication competent provirus, or in the case of the VOA, only measure replication competent provirus.