Strategies to eradicate HIV from infected patients: elimination of latent provirus reservoirs

Abstract

35 years since identification of HIV as the causative agent of AIDS, and 35 million deaths associated with this disease, significant effort is now directed towards the development of potential cures. Current anti-retroviral (ART) therapies for HIV/AIDS can suppress virus replication to undetectable levels, and infected individuals can live symptom free so long as treatment is maintained. However, removal of therapy allows rapid re-emergence of virus from a highly stable reservoir of latently infected cells that exist as a barrier to elimination of the infection with current ART. Prospects of a cure for HIV infection are significantly encouraged by two serendipitous cases where individuals have entered remission following stem cell transplantation from compatible HIV-resistant donors. However, development of a routine cure that could become available to millions of infected individuals will require a means of specifically purging cells harboring latent HIV, preventing replication of latent provirus, or destruction of provirus genomes by gene editing. Elimination of latently infected cells will require a means of exposing this population, which may involve identification of a natural specific biomarker or therapeutic intervention to force their exposure by reactivation of virus expression. Accordingly, the proposed "Shock and Kill" strategy involves treatment with latency-reversing agents (LRA) to induce HIV provirus expression thus exposing these cells to killing by cellular immunity or apoptosis. Current efforts to enable this strategy are directed at developing improved combinations of LRA to produce broad and robust induction of HIV provirus and enhancing the elimination of cells where replication has been reactivated by targeted immune modulation. Alternative strategies may involve preventing re-emergence virus from latently infected cells by "Lock and Block" intervention, where transcription of provirus is inhibited to prevent virus spread or disruption of the HIV provirus genome by genome editing.

Keywords: AIDS; Anti-retroviral therapy; Cure; Genome editing; HIV; Immune modulation; Latency-reversing agents; Lock and block; Shock and kill; Transcriptional regulation; Virus latency.

Fig. 1

Transcription factors controlling activation and repression of the HIV-1 LTR. a Transcription factors mediating the activation of HIV-1 transcription. The enhancer region of the 5′ HIV-1 LTR binds multiple transcriptional activator proteins [AP1, NF-κB, SP1, NFAT, GABP/Ets, USF1/2/TFII-I (RBF-2)] that recruit general transcription factors and co-activator complexes to stimulate transcription by RNA Polymerase II (Pol II). Transcription of the HIV-1 5′ mRNA region produces the TAR (TAT-Responsive) RNA stem-loop structure that binds the viral TAT protein, which recruits elongation factor pTEFB to inhibit negative regulators of pausing, DSIF and NELF, and promote elongation by RNA Pol II. The 5′ LTR is associated with two strongly positioned nucleosomes, designated nuc-0 and nuc-1; transcriptional activation from the LTR is causes dissociation of nuc-1 near the core promoter. b Factors causing repression of HIV-1 transcription in unstimulated cells. In unstimulated cells, activator proteins are replaced by transcriptional repressors (NF-κB p50, CBF-1) that recruit histone deacetylase and histone methyltransferase complexes. Several LTR-bound factors are converted from activators to repressors (SP1/3 (RBF-2)/TFII-I) that recruit HDAC enzymes (HDAC1/2/3). The multifunctional factor YY1 (Yin Yang 1) is associated with the latent provirus 5′ LTR and also recruits HDACs. Several factors, including CTIP-2, recruit histone methyltransferases (Suv39H1) that promote transcriptional silencing and spreading of repressive chromatin

Fig. 2

Mechanisms for the establishment of HIV-1 provirus latency. a Productive infection; in productively infected cells, expression from integrated HIV-1 provirus is stimulated by signal-responsive (cell signaling) transcriptional activators (A) bound to the 5′HIV-LTR enhancer. The viral transactivator Tat produces strong positive feedback activation of LTR-directed transcriptional elongation by RNA Polymerase II (Pol II) to maintain productive infection. b Epigenetic establishment of latency; in cells where signaling is down-regulated (T cells that revert to latency) signal-responsive transcriptional activators are replaced with repressor proteins (R) that recruit histone modifying factors which cause the formation of repressive nucleosomes (RN) through histone deacetylation and histone methylation. Repressive chromatin inhibits transcriptional initiation and elongation from the 5′ LTR promoter. c Immediate latency; approximately 50% of newly infected cells produce integrated HIV provirus where LTR transcription is repressed within 24 h. Immediate latency is associated with low levels of cell signaling, and requires the interaction of YY1 with the 5′ LTR, but the mechanism(s) producing this mode of latency have yet to be determined (?). d Silenced Provirus; repressor proteins (R) bound to the transcriptionally repressed 5′ HIV LTR recruit silencing complexes which promote spreading of silenced (S) heterochromatin onto adjacent viral and cellular chromosomal DNA

Fig. 3

Potential strategies to eliminate cells latently infected with HIV-1. Combined Shock and enhanced Kill. Cells latently infected with HIV-1 provirus may produce sporadic transcripts (A), which mostly terminate shortly upon initiation because of RNA Polymerase II pausing factors (DSIF, NELF). Occasional transcripts may produce viral gene products, including the viral transactivator TAT. Combinations of latency-reversing agents (LRA) may be employed to produce broad and robust reactivation of provirus mRNA expression and production of viral gene products (B) (g, gag; e, env; t, tat; v, vif; n, nef). Killing of cells with reactivated provirus could be encouraged by additional intervention, including: (i) enhanced cellular apoptosis induced by viral proteins; (ii) enhanced cellular and/or humoral immune responses towards HIV-infected cells; (iii) production of designer chimeric antigen receptor (CAR)-expressing CD8 T cells that target HIV env or gag gene products expressed on the surface of infected cells; (iv) antibody drug conjugates (ADC) using env- or gag-specific antibodies coupled to toxic effectors; (v) recombinant Dual Affinity Retargeting Antibodies (DART) may promote specific elimination of specific subsets of HIV-1 infected cells following reactivation of provirus through recognition of a cell surface marker (CD3) in combination with viral gene products (env). Lock and block. Therapeutic intervention that prevents the expression of HIV gene products could “lock down” sporadic expression in latently infected cells, to prevent re-emergence of virus when anti-retroviral therapy is removed. Such strategies may involve inhibiting factors that activate transcription from the HIV LTR (C), prevent elongation of RNA transcripts from the viral LTR promoter (D) or enhancing effects of repressor proteins bound to the LTR regulatory region (E). Genome Editing of HIV provirus. Recombinant TALEN or CRISPR/Cas9 gene editing molecules delivered to latently infected cells designed to produce double-stranded cleavage at highly conserved regions of the provirus genome (F). Double-stranded breaks repaired by non-homologous end joining results in deletions of the HIV genome that prevents further replication (G)