Available online at www.sciencedirect.
com
ScienceDirect
Which therapeutic strategy will achieve a cure for HIV-1?
Anthony R Cillo and John W Mellors
Strategies to achieve a cure for HIV-1 infection can be
broadly classified into three categories: eradication cure
(elimination of all viral reservoirs), functional cure (immune
control without reservoir eradication), or a hybrid cure
(reservoir reduction with improved immune control). The
many HIV-1 cure strategies being investigated include
modification of host cells to resist HIV-1, engineered T cells
to eliminate HIV-infected cells, broadly HIV-1 neutralizing
monoclonal antibodies, and therapeutic vaccination, but the
‘kick and kill’ strategy to expose latent HIV-1 with latency
reversing agents (LRAs) and kill the exposed cells through
immune effector functions is currently the most actively
pursued. It is unknown, however, whether LRAs can deplete
viral reservoirs in vivo or whether current LRAs are
sufficiently safe for clinical use.
Address
Division of Infectious Diseases, School of Medicine, University of
Pittsburgh, Pittsburgh, PA, USA
Corresponding author: Mellors, John W (jwm1@pitt.edu)
Current Opinion in Virology 2016, 18:14–19
This review comes from a themed issue on Antiviral strategies
Edited by Raymond F Schinazi and Erik de Clercq
For a complete overview see the Issue and the Editorial
Available online 15th March 2016
http://dx.doi.org/10.1016/j.coviro.2016.02.001
1879-6257/Published by Elsevier B.V.
Introduction
Despite the success of antiretroviral therapy (ART) in
fully suppressing viral replication, HIV-1 remains an
incurable viral infection. Recently, significant resources
have been invested in developing and evaluating poten-
tially curative strategies for HIV-1 infection. A cure for
HIV-1, defined as control of viral replication in the
absence of ART, could theoretically be achieved through
several varied approaches and goals: an eradication cure
(elimination of all viral reservoirs); a functional cure
(control of viral replication without reservoir eradication),
or a hybrid cure (reduction of the reservoir and boosting of
immune responses) (Figure 1). Here, we discuss specific
therapeutic approaches aimed at achieving a cure of
HIV-1 (summarized in Table 1), with a focus on the
‘kick and kill’ strategy.
Current Opinion in Virology 2016, 18:14–19
Main body
Specific strategies to achieve a cure for HIV-1
Eradication cure
The solitary example of an eradication cure to date is
Timothy Ray Brown, the Berlin patient [1], whose HIV-
infected immune system was replaced, through allogene-
ic hematopoietic stem cell transplantation, with one that
was resistant to HIV-1 as a result of a homozygous
CCR5D32 gene mutation of the donor’s cells. Even in
the case of near complete replacement of the recipient’s
immune system, viral eradication is not guaranteed; the
Boston patients who underwent allogeneic hematopoietic
stem cell transplant with wild-type CCR5 donors experi-
enced viral rebound despite persistence of <0.001% of
host cells in peripheral blood [2,3]. In addition, alloge-
neic transplantation has high morbidity and mortality, and
is thus not suitable for those without life-threatening
hematologic malignancies. As a consequence, most strat-
egies to achieve a cure of HIV-1 infection have focused on
achieving either a functional cure or a hybrid cure, as
illustrated below.
Functional cure
One strategy to achieve a functional cure is the editing of
the human genome in either autologous CD4+ T cells [4]
or autologous hematopoietic stem cells [5,6] by homing
endonucleases to knock out the gene for CCR5, thereby
blocking the ability of HIV-1 to enter via the main
coreceptor. Tebas et al. have advanced this concept to
clinical trials, using cyclophosphamide conditioning to
achieve successful engraftment of zinc-finger modified
autologous CD4+ T cells [4]. Questions still remain
regarding the efficacy of the CCR5 knockout approach,
namely the proportion of cells that need to be modified to
prevent viral rebound and whether CXCR4-tropic virus
emerges over time. A recent alternative approach has
been to modify autologous cells to express CRISPR/
Cas9 to disrupt the provirus rather than a host gene
[7], although this approach is complicated by proviral
sequence diversity. Others have attempted to prevent
the spread of infection in a macaque model by adding an
expression cassette for a fusion inhibitor (mC46) in au-
tologous cells, preventing virus entry [8], but it is un-
known whether enough target cells can be modified and
whether HIV-1 can evolve resistance to this inhibitor over
time.
Another strategy to achieve a functional cure is the
introduction of designer immune responses, one of which
is chimeric antigen receptor (CAR) expressing effector
cytotoxic T lymphocytes targeting HIV-1 epitopes on
MHC-I or cell-surface HIV-1 envelope. CAR T cells
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 Curative strategies for HIV-1 Cillo and Mellors 15

Figure 1

Eradication Cure Eradication

Timothy Ray Cure
Brown (Berlin No functional HIV-1
Patient) remaining in the body

Hybrid Cure Functional Cure

Reduce Hybrid Functional Elite
reservoir size Cure Cure controllers
and diversity Reduced functional Control of HIV VISCONTI
with “kick” reservoirs & improve without ART or Post-ART
Enhance immune control deleterious controllers
immune without ART immunologic effects
Host cell
responses modification
with “kill”

Current Opinion in Virology

Broad definitions of a cure for HIV-1, with specific examples. An eradication cure would be one in which no functional HIV-1 remains in the body,
and is represented by allogeneic stem cell transplantation and complete replacement of the recipient’s HIV-infected immune system. Functional
cure, illustrated by elite controllers and post-treatment controllers (and also possibly successful host cell modification), is defined as control of
HIV-1 replication without ART, immune dysfunction (i.e. depletion of CD4+ T cells or persistent immune activation) or reservoir eradication. A
hybrid cure could be achieved by reduction in the size and diversity of the latent reservoir (as in an eradication cure), combined with enhanced
immune control of HIV-1.

have proven efficacious for B cell lymphoma by targeting
CD19 [9], but at the cost of healthy B cells. Off-target
specificity of engineered T cells may be a significant
safety barrier in this approach, as fatal cardiac toxicity
occurred in myeloma and melanoma trials using affinity
purified T cells [10,11]. Whether sufficient numbers
of CAR modified T cells could be targeted to invariant
HIV-1 epitopes and maintained in vivo are important
challenges for this approach.
Considerable effort has gone into the discovery and
characterization of bNAbs for HIV-1 envelope, but elicit-
ing bNAb-like antibodies through therapeutic vaccina-
tion remains difficult because of the extensive affinity
maturation required to achieve the often structurally
abnormal bNAbs (reviewed by [12]). However, passive
infusion of a bNAb or a cocktail of bNAbs could prove to
be a successful approach to an ART-free functional cure.
The AIDS Clinical Trials Group (ACTG) study A5342 is

Table 1

Specific HIV-1 curative strategies under investigation

Overarching strategy Specific strategy Application of specific strategies

Eradication cure
Functional cure
Hybrid cure
Allogeneic stem cell transplantation
Genome editing
Designer immune responses
Control of viral replication
Kick and kill
 Timothy Ray Brown (Berlin patient) [1]
 Boston patients [2,3]
 Knockout of CCR5 in CD4+ T cells or hematopoietic stem cells [4–6]
 Knockout of HIV-1 proviruses [7]
 Expression of a viral fusion inhibitor [8]
 Chimeric antigen receptors on CD8+ T cells [9]
 Broadly neutralizing monoclonal antibodies [12]
 Highly effective CD8+ T cell response to control viral replication,
similar to elite controllers [13–17]
 Post-treatment control of viral replication, similar to the VISCONTI
cohort controllers [18]
 LRAs disrupt proviral latency, followed by death of infected cells
 Reduced reservoir size and diversity permits suppression of viral
replication with boosted immune responses

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16 Antiviral strategies
currently being undertaken to test whether a bNAb can
reduce the size of the reservoir in vivo. Complicating
factors with non-autologous bNAbs are the generation of
anti-bNAb antibodies, and the pre-existence of HIV-1
envelope with escaped epitopes to the bNAbs, which
could limit long-term efficacy.
Modulation of T cell mediated immunity could also
control viral replication in the absence of ART. Rare
elite controllers are capable of controlling viral replication
in the absence of ART, often due to favorable HLA
alleles and highly effective CD8+ T cell responses
[13–15]. The vast majority of people infected with
HIV-1 do not control viral replication, and would require
therapeutic vaccination to elicit the proper cytotoxic
T cell (CTL) responses for control of viral replication.
This may be possible in the context of a highly effective,
patrolling CTL response capable of preventing systemic
viral spread. Hansen et al. demonstrated that a rhesus
CMV-vectored vaccine could control viremia following
challenge and eliminate virally-infected cells [16,17].
Though promising, the utility of this approach in the
context of an established latent infection in humans is
currently uncertain. Another example of immune control
is exhibited by a limited number of individuals in the
VISCONTI cohort, who have shown durable control of
viremia following early initiation of ART in the absence
of favorable elite controller alleles [18]. Immunologic
correlates of control in this cohort have yet to be defined.
Hybrid cure
A strategy that aims to both reduce the viral reservoir and
enhance immune control of the residual reservoir is
referred to as the ‘kick and kill’ or ‘shock and kill’
strategy. In this approach, reservoir reduction is achieved
by pharmacologically reactivating latent proviruses (kick)
with latency reversing agents (LRAs), followed by sub-
sequent death of the infected cell through either immune
clearance or cytopathic effects of HIV-1 protein expres-
sion. The kick component has been tested with numerous
candidate LRAs in proof of principle clinical trials. As
such, the remainder of this review will focus on the results
of clinical trials of LRAs to date, the preclinical assess-
ment of new LRAs, and perspectives on the future of this
approach.
Clinical trials of HIV-1 latency reversing agents
The best-studied putative LRAs to date have been
histone deacetylase inhibitors (HDACi), with vorinostat,
panobinostat and romidepsin having been investigated
clinically (Table 2). Trials of vorinostat have shown
upregulation of cellular HIV-1 RNA expression
[19,20], though not consistently after repeated dosing
[21], and without concomitant increases in plasma viremia
[19,20]. HDACi may also have longer term effects on
gene expression in vivo than previously thought, as gene
profiling revealed protracted gene dysregulation follow-
Current Opinion in Virology 2016, 18:14–19
Box 1 Key points
 HIV-1 curative strategies can be broadly classified into eradication
cure, functional cure, or hybrid cure.
 An eradication cure is least likely to be achieved because of the
persistence of long-lived, CD4+ T cells with intact proviruses
throughout the human body.
 Control of HIV-1 in the absence of reservoir reduction (functional
cure) will be more difficult to achieve compared with strategies that
both reduce the reservoir and enhance HIV-1 specific immune
effector functions (hybrid cure).
 Many specific strategies are under investigation for their ability to
achieve a cure of HIV-1, with kick and kill being the most
intensively pursued approach to date.
 Clinical trials of latency reversing agents have generally shown
increases in cellular HIV-1 RNA from baseline, but their effect on
HIV-1 protein expression, virion production and the latent inducible
reservoir are undefined.
 Reduction in the size and diversity of the viral reservoir, in
conjunction with modulation of immune responses is an under-
developed but vital area of functional HIV-1 cure research.
ing HDACi treatment [20]. This gene dysregulation may
contribute to the impaired CTL function observed ex vivo
following treatment with HDACi [22]. Trials of the more
potent HDACi panobinostat and romidepsin have also
been performed, and have shown upregulation of cellular
HIV-1 RNA [23,24]. In contrast to studies of vorinostat,
modest increases in plasma viremia were detected by
either transcription-mediated amplification (TMA) or the
Roche COBAS AmpliPrep/TaqMan platform following
dosing with panobinostat or romidepsin. Though not an
HDACi, disulfiram has also been shown to modestly
increase plasma viremia and cellular unspliced HIV-1
RNA levels [25,26].
Trials of LRAs to date should be interpreted with caution,
in part because assays used to measure virion HIV-1 RNA
in plasma (TMA and AmpliPrep/TaqMan platforms)
cannot readily distinguish virion-associated viral RNA
from HIV-1 DNA in plasma resulting from cellular cyto-
toxicity from the HDACi. Another important caveat is
that cellular HIV-1 RNA expression following HDACi
treatment could be a result of readthrough transcripts,
which can be mistaken for increases in functional
unspliced HIV-1 RNA [27]. Finally, it will be important
in future studies to assess whether increases in cellular
HIV-1 transcription are related to increases in translation
of HIV-1 proteins, rendering reactivated cells visible to
the immune system.
Ex vivo assessments of new latency reversing agents
Results from clinical trials have generated optimism that
the kick and kill strategy can be used to reverse latency.
However, HDACi only antagonize one known mecha-
nism of proviral latency, namely nucleosome structure.
Mechanisms of proviral latency are complex and multi-
factorial, and have been reviewed elsewhere (reviewed in
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 Curative strategies for HIV-1 Cillo and Mellors 17

Table 2

Recent clinical trials of latency reversing agents

Clinical trial, NCT identifier
Single-dose vorinostat,
NCT01319383
Pilot study of disulfiram,
NCT01286259
Short-course vorinostat,
NCT01319383
Short-course vorinostat,
NCT01365065
Short-course panobinostat,
NCT01680094
Multi-dose romidepsin,
NCT02092116
Short-course disulfiram,
NCT01944371
First author, location(s) of trial
Archin et al. [19], University of North
Carolina Chapel Hill
Spivak, Andrade et al. [25],
University of Utah/Johns Hopkins
University
Archin et al. [21], University of North
Carolina Chapel Hill
Elliott et al. [20], Alfred Hospital and
Monash University/Burnet Institute/
University of Melbourne
Rasmussen et al. [23], Aarhus
University
Søgaard et al. [24], Aarhus
University
Elliott et al. [26], Monash University
and Alfred Hospital/University of
California San Francisco
Primary endpoint
Changes in unspliced cellular HIV-1
RNA in resting CD4+ T cells
Changes in plasma HIV-1 RNA
levels
Changes in unspliced cellular HIV-1
RNA in resting CD4+ T cells
Changes in unspliced cellular HIV-1
RNA in total CD4+ T cells
Changes in unspliced cellular HIV-1
RNA in total CD4+ T cells
Changes in unspliced cellular HIV-1
RNA in total CD4+ T cells
Changes in unspliced cellular HIV-1
RNA in total CD4+ T cells
Results of the trial
Mean 4.8-fold increase in cellular
HIV-1 RNA over pre-vorinostat
No significant change in viremia
during disulfiram, but 1.88-fold
increase observed post-treatment
No significant increase in cellular
HIV-1 RNA over time
Median peak increase of 7.4-fold
cellular HIV-1 RNA from pre-
vorinostat; protracted gene
dysregulation observed
Median maximum increase
of 3.5-fold from pre-panobinostat;
no change in time to rebound off
ART
2.4–5.0-fold increase in cellular
HIV-1 RNA from pre-romidepsin
Increases in unspliced HIV-1 RNA
observed in all groups during and
post disulfiram treatment, ranging
from 1.6- to 2.5-fold

[28]). Activation of NF-kB through agonism of protein
kinase C (PKC) with bryostatin-1 or ingenol derivatives,
which permits binding of this transcription factor to the
HIV-1 long terminal repeat [29], has recently been tested
in ex vivo systems by several groups. The PKC agonist
bryostatin-1 has been investigated in vivo for cancer
[30,31], and is currently in a Phase II trial for Alzheimer’s
disease (NCT02431468). A trial in Spain in which bryos-
tatin was given to HIV-1 patients is currently closed, and
results are pending (BRYOLAT, NCT02269605).
Another aspect of proviral latency that is amenable to
therapeutic intervention is the availability of P-TEFb
(consisting of Cyclin T1 and CDK9, reviewed by [32]) to
interact with the viral protein trans-activator of transcrip-
tion (Tat) [33]. Following release of P-TEFb from the
suppressive 7SK snRNA complex [34], Cyclin T1 and
CDK9 can interact with bromodomain and extra terminal
domain 4 (BRD4) as part of the super-elongation com-
plex; however, this limits availability for P-TEFb for
interaction with Tat [35]. Bromodomain inhibitors
function by preventing the interaction of P-TEFb with
BRD4, allowing Tat to bind P-TEFb and facilitating
HIV-1 transcriptional elongation [36]. BRD4 inhibitors
have been developed for the treatment of cancer [37,38],
and are in early phase trials for hematologic malignancies
[39].
Recent ex vivo studies have focused on combinations of
PKC inhibitors, or BRD4 inhibitors, with HDACi. These
studies have revealed that PKC agonists alone are gener-
ally the most potent LRAs [27,40,41,42], likely due to
partial T cell activation through the PKC pathway [43].
HDACi in combination with bryostatin-1 or ingenol
appears to increase cellular HIV-1 mRNA transcription
at or near the level of synergy [40]. Several studies have
also shown that PKC agonists in conjunction with BRD4
inhibitors increase cellular HIV-1 mRNA transcription in
at least a supra-additive manner [40,41,42]. These
studies suggest that HIV-1 transcription can be modulat-
ed by LRAs targeting HDAC, PKC or BRD4. However,
the relative potency of LRAs compared with full T cell
activation at the level of individual proviruses [44] should
be investigated for promising LRAs. An important unan-
swered question is whether these compounds alone or in
combination will be safe enough for use in HIV-positive
individuals who are healthy on suppressive ART.
Compounds with narrow therapeutic indices in preclini-
cal animal studies should not be pursued in human
studies.
One current knowledge gap that exists in the study of
LRAs is whether they have caused a decrease in the size
of the inducible reservoir ex vivo. To achieve a hybrid
cure, it will likely be necessary to reduce both the size and
diversity of the reservoir with LRAs; assays will be
required to determine if LRAs in preclinical development
can reduce the size of the reservoir, and the degree of
additional immunologic control bNAbs or CTL responses
that will be required to prevent viral escape after cessa-
tion of ART. An approach to a functional cure consisting
of a reduction in the size and diversity of the reservoir,
followed by boosting of immune responses to conserved
viral epitopes could be thought of as a ‘kick, kill and
contain’ strategy. Careful studies of the change in reser-
voir size and investigation of control by immunologic
mechanisms will inform whether a kick, kill and contain
strategy can be used to achieve a cure of HIV-1.

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18 Antiviral strategies
Conclusion
Many strategies to achieve an HIV-1 cure are being
studied (Box 1). Early proof of principle clinical trials
of LRAs have shown increased levels of HIV-1 RNA
transcription, and ex vivo assessments of new LRAs tar-
geting different aspect of proviral latency are underway.
Open questions are whether reservoir reduction is
achieved with LRAs and whether LRAs or LRA combi-
nations have favorable toxicity profiles. Improving the kill
aspect of the kick and kill strategy by enhancing immune
effectors is a critical component of the ‘kick, kill and
contain’ strategy. Many promising strategies for an HIV-1
cure are in their infancy, but it is unknown whether any
strategy can achieve an ART-free remission of HIV-1.
Nevertheless, such a goal should be pursued rigorously
with vigilance for potential toxicities of the approaches
taken.
Conflict of interest statement
ARC has no conflicts to disclose. JWM is a consultant to
Gilead Sciences, has received grant funding from Gilead
Sciences, and owns shares of Co-Crystal, Inc.
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