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Curr HIV/AIDS Rep. Author manuscript; available in PMC 2023 August 22.
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Published in final edited form as:

Curr HIV/AIDS Rep. 2022 June ; 19(3): 194–206. doi:10.1007/s11904-022-00604-2.

HIV-1 Reservoir Persistence and Decay: Implications for Cure

Strategies
Edward F. Kreider1, Katharine J. Bar2
1Perelman School of Medicine, University of Pennsylvania, Stemmler Hall Room 130-150, 3450

Hamilton Walk, Philadelphia, PA 19104-6073, USA

2Perelman School of Medicine, University of Pennsylvania, 502D Johnson Pavilion, 3610
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Hamilton Walk, Philadelphia, PA 19104-0673, USA

Abstract
Purpose of Review—Despite suppressive antiretroviral therapy (ART), a viral reservoir
persists in individuals living with HIV that can reignite systemic replication should treatment
be interrupted. Understanding how HIV-1 persists through effective ART is essential to develop
cure strategies to induce ART-free virus remission.

Recent Findings—The HIV-1 reservoir resides in a pool of CD4-expressing cells as a range of

viral species, a subset of which is genetically intact. Recent studies suggest that the reservoir on
ART is highly dynamic, with expansion and contraction of virus-infected cells over time. Overall,
the intact proviral reservoir declines faster than defective viruses, suggesting enhanced immune
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clearance or cellular turnover. Upon treatment interruption, rebound viruses demonstrate escape
from adaptive and innate immune responses, implicating these selective pressures in restriction
of virus reactivation. Cure strategies employing immunotherapy are poised to test whether host
immune pressure can be augmented to enhance reservoir suppression or clearance. Alternatively,
genomic engineering approaches are being applied to directly eliminate intact viruses and shrink
the replication-competent virus pool.

Summary—New evidence suggests host immunity exerts selective pressure on reservoir viruses
and clears HIV-1 infected cells over years on ART. Efforts to build on the detectable, but
insufficient, reservoir clearance via empiric testing in clinical trials will inform our understanding
of mechanisms of viral persistence and the direction of future cure strategies.

Keywords
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HIV-1 reservoirs; Persistence; Viral rebound; Cure

Introduction
The management of people living with HIV (PLWH) has evolved over the course of the
pandemic, transforming a near certain death sentence into a manageable chronic illness

Katharine J. Bar bark@pennmedicine.upenn.edu.

Conflict of Interest The authors declare no competing interests.
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with appropriate treatment [1, 2]. Despite medical advances that have markedly improved
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disease outcomes, discontinuation of antiretroviral therapy (ART) reliably results in viral

recrudescence [3, 4] and, thus, PLWH must remain on therapy for the duration of their lives.
Lifelong treatment can be complicated by drug toxicities [5, 6], medication interactions
[7, 8], adherence issues [9], stigma [10], and ongoing costs to individuals and healthcare
systems [11]. Thus, interventions to induce ART-free virus remission are a priority.

The major barrier to such a cure is the viral reservoir—a collection of viral species that
persist through ART and is capable of productively infecting new cells to reestablish viremia
upon antiretroviral cessation. Accurate characterization of this viral pool is challenging,
but of vital importance to determining mechanisms of virus persistence and to evaluate
cure strategies. Here, we will provide an overview of our current understanding of
HIV-1 persistence, highlighting newly realized reservoir features that pose obstacles and
opportunities for cure interventions. Recent work by several groups reinforces the theme
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that viral reservoirs are highly dynamic, but cells harboring rebound-competent viruses
appear to decrease in size slowly on suppressive ART. We will then highlight several cure
interventions that seek to enhance this viral clearance through immunomodulation or gene
therapy.

Persistent HIV-1 Reservoirs

Viral and Cellular Reservoirs—Primate immunodeficiency viruses, including HIV-1,
mediate both active and latent infection and are capable of infecting multiple CD4-
expressing cell types. Further complicating reservoir characterization, HIV-infected cells
reside within lymphoid tissues, blood, viscera, and immune-privileged sites [12]. Reservoir
viruses can endure long-term ART as integrated DNA proviruses in cells, or intact virions
within tissue networks, that are long-lived or self-renewing. At treatment interruption,
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a subset of the reservoir that is both replication- and rebound-competent evades host
restriction and rekindles systemic virus replication. Years of study in both PLWH and
animal models of persistence have shed light on the heterogeneous cellular and viral species
responsible for reservoir maintenance.

The most thoroughly characterized component of the reservoir are transcriptionally silent
DNA proviruses integrated within memory CD4+ T cells [13, 14]. Ex vivo stimulation
of latently infected memory T cells yields infectious virus, implicating this pool as a
substantial contributor to the reservoir [13, 15]. Interrogations of this long-lived proviral
reservoir indicate that it may be enriched in certain cellular subsets, e.g., central and
stem cell memory CD4 + T cells, which persist through low-level cycling or exhibit self-
renewal without activation [16, 17]. While less numerous, effector memory CD4+ T cells
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have higher frequencies of intact and inducible provirus [18–20]; thus, they may serve as
important sources of reactivating virus. Furthermore, CD4+ T cells expressing exhaustion
markers, including the immune checkpoint receptors programmed death protein 1 (PD-1),
cytotoxic lymphocyte-associated protein 4 (CTLA-4), and others, are key reservoirs as they
are enriched for HIV and/or SIV infection [21, 22]. Determining the key features and
relative contributions of the CD4 + T cell reservoir based on tissue location, function, and
differentiation status remains an active area of study (reviewed in [23]). Apart from T

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cells, latently infected tissue macrophages have also been proposed as components of the
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viral reservoir, as these cells can be productively infected and can durably persist [24, 25].
Replication-competent virus has been retrieved from tissue macrophages in the male genital
tract [26], and from the brain and circulation in specific animal models [27–29]; however,
the scarcity of tissue macrophages within largely inaccessible tissues challenges verification
of these findings.

In addition to quiescent HIV-1 proviruses, cells with active viral transcription and translation
have been identified in PLWH on suppressive ART and implicated in both viral persistence
and immunopathogenesis. Cell-associated viral RNA (CA-RNA), which reflects proviral
transcription, extrachromosomal circular viral DNA, which is inherently nonself-renewing,
and HIV-1 proteins, reflecting ongoing viral translation, remain detectable in PLWH during
periods of suppressed viremia [30–32]. In theory, expression of viral RNA and protein
offer opportunities for immune recognition and clearance of infected cells in the absence
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of virus replication. Indeed, comprehensive single-cell characterization approaches suggest

that actively transcribing and translating reservoirs are better targeted for clearance by host
immune pressures [33••]. Some expanded HIV + T cell clones, however, persist for years
despite being transcriptionally active [33••], suggesting cellular persistence and proliferation
outcompete immune clearance in the majority of PLWH. These HIV-1 viral products within
the blood and lymphoid tissues reflect that activity of the reservoir and correlate with
time to viral rebound [4, 34]. The relative contributions of transcriptionally silent DNA
proviruses, actively transcribing infected cells, or virions in other forms (e.g., trapped within
the follicular dendritic cell network) to the pool of rebound-competent viruses remain an
important topic of study [35•].

Characterization of the other extreme of the viral reservoir, deeply latent proviruses, has
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also informed our understanding of virus persistence. Studies of the proviral landscape
of elite controllers (ECs), a rare subset of PLWH who maintain viral suppression in the
absence of ART [36], demonstrate that ECs in general have less frequent, but detectable,
replication-competent proviruses [37, 38]. Importantly, the proviruses in ECs are enriched in
regions of heterochromatin and exhibit long distances to the nearest transcription start site
[39••], suggesting a lower likelihood of reactivation in vivo. These deeply latent viruses may
also be enriched in some PLWH on long-term suppressive ART and appear less responsive
to activation and latency reversal [33••]. The enrichment of deeply latent viruses in ECs and
after long-term ART supports the hypothesis that effective anti-HIV-1 immune responses can
clear more transcriptionally active proviruses [40••, 41] and has spurred efforts to augment
host immune responses to enhance reservoir clearance [36, 42]
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The Challenge of Measuring a Highly Defective Proviral Reservoir

Attempts to accurately measure heterogeneous cellular reservoirs are further hampered by
the extent to which provirus is defective. Proviral DNA genomes are rendered replication-
incompetent by large deletions, incomplete viral coding sequences, and apolipoprotein
B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G) hypermutation [3,
4, 43–45, 46••, 47]. Though unable to reactivate, some of these defective genomes are
capable of viral protein expression, and thus serve as potential sources of pathogenic

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inflammation [48, 49]. In many cohorts, defective proviruses comprise more than 95%
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of all integrated viral genomes [40••, 50], complicating measurements of total versus
functional virus. Indeed, for years, the field lacked reservoir measurement assays that
were both feasible and accurate. Long considered the gold standard, the quantitative viral
outgrowth assay (QVOA) is now understood to provide a minimum estimate of replication-
competent virus using ex vivo stimulation of longlived CD4+ T cells [51, 52], as repetitive
stimulations yield increasing numbers of virus-producing cells [13, 53••]. Furthermore,
QVOA is resource-intensive and requires many input cells. Thus, many investigators have
relied on assays measuring total viral DNA, which overestimate the replication-competent
reservoir and do not consistently correlate with QVOA measures, but are high-throughput,
inexpensive, efficient across virus subtypes, and feasible with fewer cells [54•, 55•, 56,
57]. Assays measuring inducible provirus, like the Tat/Rev-Induced Limited Dilution Assay
(TILDA), which uses limiting dilution to measure the frequency of cells producing multiply-
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spliced HIV RNA after stimulation, and Envelope Detection by Induced Transcription-based
Sequencing (EDITS), which uses sequencing to detect cells producing both mature env
RNA and functional HIV proteins after stimulation, provide a middle ground for stringency
and feasibility, but often correlate poorly with QVOA [52, 58–61]. Thus, methodologic
limitations have hindered accurate assessment of reservoir size in humans, particularly in the
restricted samples available in clinical trials.

Aiming for a more feasible measurement of functional viruses, two groups independently
developed novel approaches to estimate the number of proviruses with intact sequences. The
intact proviral DNA assay (IPDA) [53••] and quadruplex quantitative PCR (Q4PCR) [62]
employ three and four quantitative PCR probes at two and four genomic loci, respectively,
for a single template quantitative PCR reaction to distinguish provirus with select defects
versus those without (of note, Q4PCR adds a subsequent near-full-length sequencing step).
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Notably, IPDA and Q4PCR values correlate with replication-competent measurements using
QVOA [50, 53••, 63]. Modeling of longitudinal reservoir decay kinetics by IPDA across
cohorts suggests intact sequences wane more rapidly than the minimal decline seen in
defective sequences [40••, 46••]. This pattern of intact proviral decay across cohorts further
supports evidence that cells harboring intact sequences are preferentially lost, through
immune clearance mechanisms or apoptosis of the infected cell. The high-throughput
nature and reduced input cell requirements of these quantitative approaches also make
them attractive assays for research and clinical trials [55•], while issues of efficiency and
validation across diverse HIV-1 subtypes remain to be addressed [40••, 64, 65]. Moving
forward, these estimates of the intact provirus pool are being widely employed as endpoints
in clinical trials and applied across clinical cohorts [66].
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The Dynamics of Reservoir Persistence

Following ART initiation, viral and cellular reservoirs are not stagnant. While the intact
reservoir on ART declines slowly, viral persistence is characterized by co-occurring
expansion and contraction of infected CD4+ T cell populations. Building on the
identification of expanded T cell clones bearing identical proviruses at shared integration
sites [67, 68], we now infer that many proviruses sampled by QVOA and total or integrated
HIV-1 DNA assays are derived from identical T cell clones, which can undergo expansion

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either prior to or after HIV-1 infection [69], be maintained independent of viral production,
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and drive the low-level viremia observed on suppressive ART [70•]. Informed by improved
sequencing and novel single-cell analysis techniques, we now understand that the relative
frequencies of the infected T cell clones shift substantially over time [3, 43, 44, 47, 53••,
67, 71–74] via several processes, including antigen-specific T cell proliferation [69, 75],
homeostatic T cell maintenance [16, 76, 77], and proto-oncogene integration effects [67, 72].
Thus, HIV-1 persistence in CD4+ T cells is a complex process driven by mechanisms closely
linked to intrinsic T cell immunity, which poses challenges to the development of safe and
parsimonious interventions to reduce clonal reservoirs.

Reservoir evaluations at key timepoints of infection, including immediately following

transmission and at ART initiation, have further shed light on reservoir dynamics. Studies
of early ART initiation in human and nonhuman primate (NHP) models have conclusively
shown that a longlived reservoir can be established within days of HIV-1 or SIV acquisition
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[78–80]. Reservoir size can be markedly, but not totally, reduced with early ART initiation
[41, 81], and these reservoirs decay slowly as they become increasingly clonal over years
of suppressive ART [14, 15, 43, 82]. Recent reports from several groups have shown that
the replication-competent reservoir has a marked enrichment for viruses that are genetically
and phenotypically similar to those circulating at the time of ART initiation [83••, 84••,
85]. The bias towards later viruses suggests that antiretrovirals may be a stabilizing force
on HIV-1-infected cell survival and persistence, which is supported by studies modeling
NHP infection showing slower turnover of cells infected at ART initiation [86]. If much
of the persistent reservoir is seeded at the time of ART initiation, this juncture serves as
a window of opportunity to attempt reservoir reduction—an approach that is now being
tested in several ongoing and in-development clinical trials. Alternatively, the dearth of
early circulating proviruses may indicate preferential immune clearance of archival wild-
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type viruses, resulting in the persistence of proviral escape variants [87]. Reassuringly,
ART-suppressed PLWH have been shown to retain effective HIV-specific CD8+ T cell
responses without widespread escape in sampled proviruses [87, 88]. The exact contribution
of cytolytic T cells to reservoir reduction has been challenging to measure, but recent
evaluation of acutely infected individuals suggests that greater magnitude and quality of
the cellular immune response correlates with smaller reservoir size [89]. The potential
for immune surveillance to drive preferential loss of archival viruses supports the use of
immunotherapy to enhance the potency and breadth of adaptive responses as eradication
strategies.

The Rebound-Competent Reservoir

Phenotypic studies of rebound viruses have identified selective pressures that shape
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the rebound-competent reservoir and highlight processes that can be exploited for cure
interventions. Analytic treatment interruption (ATI) results in viral rebound in nearly 95%
of ART-suppressed PLWH at 12 weeks post-ART cessation [4] with multiple genetically
distinct virus populations reactivating nearly simultaneously [3, 84••, 90–92]. Notably,
these rebound viruses are both genetically and phenotypically distinct from the replication-
competent viruses sampled from the same participants’ blood on ART [3, 90, 91, 93]. One
salient distinction between rebound and reservoir viruses is their relative sensitivity to type

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I interferons [84••]. The unique resistance to interferons seen in rebound viruses suggests
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that innate immunity restricts which reservoir variants can reignite systemic replication.
Accordingly, intense sampling in the period between treatment interruption and detection
of rebound viremia has revealed that plasmacytoid dendritic cell frequency and soluble
inflammatory markers both spike prior to peripheral viral RNA detection [94]. Together,
these results highlight the innate pressure within the tissues as viruses reactivate locally.
Similarly, adaptive immune responses restrict rebound, with breakthrough viruses showing
resistance to contemporaneously circulating autologous neutralizing antibodies [3, 95].
Because replicating HIV-1 is a sensitive rheostat of the cumulative selective pressures it
is experiencing, further comparison of rebound virus phenotype to that of the remainder of
the reservoir may elucidate immune responses that can be leveraged into cure strategies.

Interventions to Clear or Suppress HIV-1 Reservoirs

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Broadly Neutralizing Antibody Immunotherapy—Over the last decade, the field has
collectively isolated numerous broadly neutralizing antibodies (bnAbs) capable of inhibiting
genetically diverse HIV-1 strains, a subset of which have been tested in human trials. Passive
immunization with bnAbs can protect from transmission of neutralization-sensitive viruses
[96, 97]; thus, their elicitation has been a major focus in HIV prevention efforts [98]. In
PLWH, bnAbs have been shown to decrease viremia and maintain viral suppression in
those with sensitive viral reservoirs [90, 91, 99–105], demonstrating bnAbs’ ability to act as
suppressive ART. The promise of bnAbs for HIV cure efforts, however, lies in their potential
for effector function–mediated clearance of infected cells and immunomodulation, activities
that have not yet been conclusively demonstrated in human trials.

In both human and NHP studies, antibody effector function has been associated with
improved prevention, decreased viral load, and limited immunopathogenesis (reviewed in
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[106]), but bnAbs’ capacity to recognize and enhance clearance of HIV-infected cells is less
well demonstrated. Modeling studies of bnAb administration in viremic hosts demonstrate
accelerated decline in viremia as compared to small molecule ART, suggesting that bnAbs
mediate additional activities beyond cell-free virus neutralization [107]. NHP studies of
bnAbs with or without latency reversal agents have also suggested clearance of infected
cells, but these studies may not represent PLWH as they rely on extremely early ART
initiation following infection with viruses prone to spontaneous control [108, 109].

A “vaccinal effect,” or enhancement of adaptive immune responses that persist after

antibody levels wane, is well characterized in oncology [110, 111], and is implicated, but
less well substantiated with anti-HIV bnAbs [103, 112–114]. Preclinical studies have shown
possible immunomodulation from bnAbs administered with early ART. In SHIV-infected
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NHPs, passive immunization can result in improved T cell functionality, decreased cellular
exhaustion, and more potent autologous nAb responses [109, 115, 116]. Perhaps most
intriguingly, early bnAb immunotherapy in SHIV-infected NHPs has been associated with
durable virus control [109, 117, 118]. Prolonged virus suppression has also been seen post-
bnAb administration in small human studies [99], but the bnAb-specific contribution and
mechanism of action in human and NHP post-intervention control is yet to be determined.

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Despite excitement, concerns surrounding bnAb use remain. Most daunting, viral escape
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variants have been described for every class of antibody and there exists no reliable way
to screen the entire viral archive for resistance. The field continues to develop more broad
and potent agents and combination therapy to attempt to overcome these challenges. Thus,
despite these caveats, bnAb immunotherapy holds promise and is being tested in multiple
clinical trials of combination cure strategies.

Latency Reversal and Immune Enhancement

Multiple strategies seek to enhance host immune responses to induce more effective
clearance and suppression of the HIV-1 reservoir in a broad range of PLWH. Given the
heterogeneity of the reservoir, combination strategies are likely needed, including latent
virus reactivation, suppression of virus replication, and improved cellular clearance. Many
promising agents have demonstrated efficacy in oncology, where the life-threatening nature
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of cancer allows tolerance of drug-related toxicities. Paramount to the repurposing of these

immunomodulatory agents to HIV cure is demonstrating safety in otherwise healthy PLWH.

A long-standing goal of the cure field is the development of safe and effective latency
reversal agents, or LRAs, to specifically induce latently infected cells to reactivate, thus
exposing them to immune clearance. The first-generation LRAs to reach clinical trials were
largely histone deacetylase inhibitors (HDACi). These agents demonstrated safety and in
vitro activity but failed to induce consistent on-ART viremia or reductions in reservoir
measures [119–122]. Recent clinical trials of HDACi in combination with therapeutic
vaccines [123] and bnAbs [124], failed to meet their primary outcomes of decreases
in reservoir measures or time to rebound, respectively. Newer LRAs, including second
mitochondrial inhibitor of caspase (SMAC) mimetics, which target noncanonical NF-kB
signaling, and antibodies that transiently deplete CD8+ T cells [125–127], have shown
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more robust and reliable latency reversal across animal models [128, 129], and are
poised for human studies. Other agents aim to induce both latency reversal and enhanced
immune function; furthest in clinical development are IL-15 superagonists, Toll-like receptor
agonists, and immune checkpoint inhibitors.

The IL-15 superagonist N-803 is a fusion protein of mutant IL-15 and a dimeric IL-15
receptor αSu/Fc developed for enhanced pharmacokinetics and tissue distribution [130,
131]. In vitro use has shown latency reversal within primary cell models of HIV-1 latency
[132]. NHP data suggest enhanced cellular immune activity and migration to key tissue
sites [127, 133], but limited on-ART viremia when used independently. Notably, when
coupled with depletion of CD8+ T cells, LRA activity was markedly increased [126,
127]. N-803 has shown promise in several phase 1 cancer trials [134–136], and the first-
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in-PLWH study demonstrated a reasonable safety profile, with injection site reactions and
lymphadenopathy as the primary toxicities [137]. In exploratory analyses, investigators
observed early increases in HIV + CD4 T cells as measured by IPDA and EDITS, followed
by decreases in the size of the inducible reservoir as measured by EDITS. Building on
this demonstration of tolerability and possible biologic activity, multiple phase 1 studies are
currently testing N-803 in combination with early ART initiation, bnAbs, and therapeutic
vaccination.

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Agents targeting the Toll-like receptors (TLRs), which are pathogen pattern recognition
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receptors central to innate antiviral immunity, have also demonstrated different degrees
of LRA activity in preclinical studies. In S(H)IV-infected rhesus macaques (RM), TLR-7
agonists have demonstrated transient on-ART viremia and delays in rebound in some, but
not all studies [138, 139]. Similar variability was shown across studies of early ART-treated
S(H)IV-infected RM, where TLR-7 agonists, in combination with vaccination or bnAbs,
failed to induce on-ART viremia, but led to substantial delays in time to rebound [108, 140].
Extension of this strategy to a more robust infection model showed more modest effects
[141]. In a recent phase I trial in PLWH who controlled viremia to low levels prior to ART, a
TLR-7 agonist showed enhanced interferon responses and modest decreases in the reservoir
as measured by IPDA [142]. As with IL-15 superagonists, TLR agonists in combination
with other agents are currently being tested in humans, trials that will shed light on whether
this form of innate immune engagement can enhance reservoir clearance.
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Another key focus of immunotherapy are checkpoint inhibitors (CPI), because HIV
reservoirs are known to be enriched in cells expressing immune checkpoint receptors and
CPIs have shown remarkable immunomodulation and improved outcomes in oncology [143,
144]. In NHP models of HIV-1 persistence, several CPIs have shown efficacy. Studies
testing anti-PD-1 and anti-CTLA-4 in SIV-infected RM demonstrate marked increases in
cellular cycling and differentiation and consistent LRA activity [145]. These CPIs have
been tested in pilot studies of people living with HIV and cancer, showing promising
reservoir reductions in these unique patient populations [146••]. In healthy PLWH, however,
phase 1 trials of ant-PD-1 or anti-PDL1 have been stopped due to immune-mediated
toxicities, highlighting concerns that the therapeutic index for many CPI approaches may
not be suitable for otherwise healthy PLWH. Thus, the potential of CPIs may depend on
development of safer interventions that balance immune activity with toxicities.
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Cellular and Gene Therapy

Novel approaches in cellular and gene therapy have advanced in multiple fields to the
point of clinical testing or clinical efficacy. One promising approach adopted from oncology
is engineered chimeric antigen receptor T (CAR T) cells, which have shown remarkable
success in the treatment of hematologic malignancies, including cancer remission over 10
years post-treatment [147]. CAR T cells have long been studied as retroviral eradication
strategies given their ability to recognize and kill infected cells, but HIV-1 poses distinct
challenges compared to antigen-rich hematologic malignancies. Lentiviral-specific CAR T
cells potently kill cells infected with diverse HIV-1 isolates [148•, 149, 150], with newer
generations capable of peripheral viral clearance in humanized mouse models of infection
[149, 151]. Unfortunately, the same efficacy has been more challenging in NHP models.
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While issues of engraftment may be overcome by vaccination with the CAR’s antigen
[152] or direct modification of hematopoietic stem cells to induce CAR expression in all
progeny [153, 154], anti-CAR humoral responses [155] and inability to mediate SHIV/SIV
containment in NHPs [156] remain major hurdles. Following technological improvements
in strain cross-reactivity and co-stimulatory signals, HIV-1 CARs have recently reentered
human clinical testing [157]. While multiple constructs are currently being evaluated [158],
a recent trial employing a bnAb-based CAR reported the preliminary result of declines in

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CA-RNA and intact proviral DNA coincident with CAR T cell infusion, as well as modestly
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delayed time to rebound compared to matched historical controls [159].

Genomic engineering is also being employed to directly inactivate proviruses, bypassing

the need for host immune effectors. One promising approach is the expression of HIV-1-
specific recombinases. Because the proviral sequence is flanked by identical long terminal
repeats, or LTRs, recombinases can mediate near-full-length provirus excision. Although
early iterations of designer recombinases recognized a small subset of isolates [160],
subsequent efforts yielded Brec1, a Cre recombinase that recognizes a 34-nucleotide span
around the polyA stem loop present in 90% of subtype A, B, and C isolates [161]. While
Brec1 demonstrates in vitro recombination activity and mediates clearance of viral RNA in
a humanized mouse model [161, 162], some concerns about nonspecific Brec1 substrate
activity have been reported [163]. This approach, while elegant, is less flexible than
others given the difficulty in adjusting the recombinase recognition sequence and would
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be rendered useless by anti-drug adaptive immune responses.

Alternatively, proviral inactivation has been achieved using the readily adaptable clustered
regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/
cas9) nuclease system for lentiviral genomic editing [164]. CRISPR/cas9 approaches have
mediated viral restriction in vitro, ex vivo, and in NHP models [165, 166•, 167], by targeting
cas9 endonuclease activity with guide RNAs specific to conserved HIV sequence motifs,
thereby damaging or excising HIV proviral genes. Rapid viral mutational escape has been
seen with the use of single guide RNAs [168–172]; however, viral escape can be overcome
by using multiple guides, resulting in greater in vitro editing efficiency [173, 174]. More
than half of published strategies target the LTR, which offers benefit over coding sequence
targeting; as LTR function depends on nucleic acid sequence, the LTR is highly conserved,
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and each provirus is flanked by two LTRs, offering double the targets and possible proviral
excision. Despite theoretical benefits, indels are more frequent within LTRs as compared
to coding sequences (unpublished observation, E. Kreider, W. Fischer, B. Korber, and
G. Shaw). Unlike other engineered nucleases, panels of guide RNAs targeting conserved
regions can be rapidly generated that offer broad inter-subtype cross-reactivity [175–178].
These findings support the notion that in vivo editing of the proviral pool is a tenable
approach to reservoir clearance. Whether CRISPR/cas9 delivery, efficiency, and specificity
can overcome the obstacles of proviral DNA reservoir eradication in humans remains to be
seen.

Conclusions
The rebound-competent HIV-1 reservoir that persists through suppressive ART remains the
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ultimate barrier to drug-free virus remission. Recent work has highlighted the multiple
processes that shape viral persistence, including the spectrum of cell types, locations,
and activation states comprising the reservoir; the highly defective nature of the proviral
population; and the expansion and contraction of host cells that themselves play important
immunologic roles. These findings reinforce the consistent theme that intact proviruses
decline over time, which supports ongoing development of cure strategies that build on
these reservoir clearing processes. In the coming years, deep interrogation of preclinical

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animal studies [179–181] and human clinical trials that seek to accelerate reservoir decay
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or eradicate DNA proviruses will help identify promising cure interventions and further our
understanding of reservoir persistence (Fig. 1).

Acknowledgments
Funding EFK is supported by T32 AI118684–05 and BEAT-HIV Delaney Collaboratory UM1 AI164570; KJB is
supported by the Penn CFAR P30 AI 045008; P01 AI131338; R01 AI62646; R01 MH128155; UM1 AI068636; and
BEAT-HIV Delaney Collaboratory UM1 AI164570.

Human and Animal Rights and Informed Consent All reported studies/ experiments with human or animal
subjects performed by the authors have been previously published and complied with all applicable ethical
standards (including the Helsinki declaration and its amendments, institutional/national research committee
standard, and international/ national/institutional guidelines).

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Fig. 1.
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Dynamics of the HIV-1 reservoir following ART initiation. A Schematization of three

measures of the proviral reservoir size plotted over time. “Total proviral DNA” corresponds
to quantitative measurements of any proviral genomes, which are generally stable overtime.
Methods that can differentiate defective from intact proviruses like IPDA and Q4PCR have
shown relative stability of rthe defective pool, but a gradual decline in the intact proviral
reservoir (red line). B Multiple forces that impact the size of the intact (filled color circles)
and defective (white circles) proviral reservoir have been identified and/or postulated. Forces

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that expand population size are listed on top and forces thought to cause decay of the intact
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reservoir shown on bottom. C With time on suppressive ART, cells harboring intact proviral
populations decrease in total number and become more clonal, as indicated by the expansion
of certain clones (e.g., red clone) and loss of others (e.g., yellow clone). Figure created with
BioRender
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