Immune targeting of HIV-1 reservoir cells: a path to elimination strategies and cure

Abstract

Successful approaches for eradication or cure of HIV-1 infection are likely to include immunological mechanisms, but remarkably little is known about how human immune responses can recognize and interact with the few HIV-1-infected cells that harbour genome-intact viral DNA, persist long term despite antiretroviral therapy and represent the main barrier to a cure. For a long time regarded as being completely shielded from host immune responses due to viral latency, these cells do, on closer examination with single-cell analytic techniques, display discrete footprints of immune selection, implying that human immune responses may be able to effectively engage and target at least some of these cells. The failure to eliminate rebound-competent virally infected cells in the majority of persons likely reflects the evolution of a highly selected pool of reservoir cells that are effectively camouflaged from immune recognition or rely on sophisticated approaches for resisting immune-mediated killing. Understanding the fine-tuned interplay between host immune responses and viral reservoir cells will help to design improved interventions that exploit the immunological vulnerabilities of HIV-1 reservoir cells.

Fig. 1 |. Multidimensional analysis of HIV-1 reservoir cells.

Integrative approaches for analysing HIV-1 reservoir cells, focusing on a hierarchical pattern involving individual tissues, cells, proviral chromosomal locations, proviral sequences and viral RNA transcripts. Proposed assays for analysis at each step are listed. Due to the rarity of HIV-1 reservoir cells, investigations frequently involve large numbers of lymphocytes collected by leukapheresis or by autopsy procedures; biopsy samples can also be used, but may yield more limited cell counts. The phenotype and transcriptional programme of viral reservoir cells can be assessed with various novel single-cell next-generation sequencing technologies, including assays for single-cell sorting (focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq)), single-cell proteogenomic profiling (phenotypic and proviral sequencing (PheP-seq)), and single-cell transcriptional/epigenetic profiling assays (expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq), ASAP-seq, DOGMA-seq). Moreover, matched integration site and proviral sequencing (MIP-seq) and PRIP-seq (parallel HIV-1 RNA, integration site and proviral sequencing) can be used to evaluate the chromosomal location of intact HIV-1 proviruses using integration site loop amplification (ISLA) or ligation-mediated PCR (LM-PCR), and their positioning relative to genome-wide epigenetic and structural chromatin features determined by ATAC-seq, chromatin immunoprecipitation followed by sequencing (ChIP–seq) and high-throughput chromosome conformation capture (Hi-C) sequencing. Proviral sequences can be characterized at the single-genome level using near full-length proviral sequencing (FLIP-seq/FLIPS assays) or the Q4 PCR assay; the intact proviral DNA assay (IPDA) can be more sensitive for quantification of intact proviruses, but less specific due to possible misclassification of defective proviruses as intact.

Fig. 2 |. Selection and evolution of the HIV-1 reservoir cell pool.

a, Kinetic evolution of the frequency of intact and defective proviruses during antiretroviral therapy, based on quantitative analysis using the intact proviral DNA assay (IPDA) and near full-length proviral sequencing assays. Intact proviruses display a more complex, multi-phasic decline; more profound reductions within the first months after antiretroviral suppression treatment (ART) initiation are succeeded by phases of slower decline. During prolonged ART over two decades, intact proviruses can either continue to decay, remain stable or increase^,,. Defective proviruses display a monophasic decay after treatment initiation but then remain mostly stable or decrease very slowly. b, Longitudinal immune selection of HIV-1 reservoir cells revealed by chromosomal integration site profiling as qualitative markers of HIV reservoir cells. At ART initiation, intact HIV-1 proviruses are typically integrated in accessible genomic locations in euchromatin (yellow); intact proviruses in heterochromatin locations (blue, green and red) are rarely detected at this stage. HIV-1 reservoir cells undergo a profound transformation during long-term ART, with clonal expansion of reservoir cells containing intact proviruses in heterochromatin locations, and de-selection of cells containing intact proviruses in euchromatin. c, Genomic, phenotypic and transcriptional immune selection features of viral reservoir cells that survive during long-term ART. HIV-1 reservoir cells at this stage frequently harbour intact proviruses at chromosomal locations associated with proviral transcriptional repression (such as centromeric satellite DNA, zinc finger (ZNF) genes, gene deserts or lamina-associated domains) and display phenotypic properties associated with enhanced resistance to immune-mediated killing, such as upregulation of immune checkpoint markers (TIGIT, BTLA, KLRG1, PD1), markers associated with resistance to immune-mediated killing (PVR, HLA-E, PDL1, HVEM) and survival markers (CD127, CD44, CD28, CD49d). Transcriptional signatures of large clones harbouring intact HIV-1 typically reveal effector memory cell signatures with upregulation of cytotoxicity markers, and/or transcriptional signatures associated with cell survival and proviral transcriptional repression.

Fig. 3 |. Immune strategies for targeting HIV-1 reservoir cells.

a, Possible immunological strategies for eliminating HIV-1 reservoir cells. Current experimental approaches for HIV-1 eradication involve manipulation of innate and adaptive immune effector cells, administration of broadly neutralizing antibodies (bNAbs) or pharmacological targeting of immune checkpoint markers, cell-intrinsic immune recognition pathways, or cell death or cell survival cascades. Viral latency-reversing agents (LRAs) may increase the immunological vulnerability of viral reservoir cells to these interventions. b, A proposed staggered approach for targeting HIV-1 reservoir cells. An initial induction phase would aim for reducing the pool of rebound-competent viral reservoir cells, primarily through pharmacological LRAs and innate immune modulators; such therapies would preferentially eliminate cells with proviruses integrated in accessible euchromatin locations, whereas proviruses with integration site features of ‘deep latency’ would persist. During a subsequent immune control phase, viral breakthroughs from rare residual reservoir cells would be prevented by functional HIV-specific memory T cell or B cell responses that could be induced by, for example, therapeutic vaccines. CAR T cells, chimeric antigen receptor T cells; H3K27ac, histone H3 acetylated at K27; H3K27me3, H3 trimethylated at K27; IFNα, interferon-α; TLR, Toll-like receptor.