CONCEPTS IN BASIC
SCIENCE AND
TRANSLATIONAL
RESEARCH
HIV Cure Research Training Curriculum
Nicolas Chomont, University of Montreal
Richard Jefferys, Treatment Action Group
The HIV CURE training curriculum is a collaborative project aimed at making HIV
cure research science accessible to the community and the HIV research field.
Definitions
•
Basic science: Laboratory studies that aim to further
understanding of the mechanisms involved in
phenomena e.g. the mechanisms of HIV persistence
•
Translational research: Research that aims to
translate knowledge gained from basic science into
the clinic (sometimes referred to as “bench-tobedside”)
HIV life cycle
From Han et al, Nat Rev, 2007
Current anti-HIV drugs do not eradicate HIV
HIV infection is characterized by
high levels of circulating viruses
in the blood
Antiretroviral drugs (HAART) are capable
of suppressing HIV, even to undetectable
levels
However, the virus
rebounds after
cessation of therapy
STOP
START
Circulating virus
HAART
Limit of detection
Time
HIV hides in reservoir that are not sensitive to current therapies
HIV persists during ART
How does HIV persist during ART?
Active reservoir
Ongoing
viral replication
Latent reservoir
• T cell survival: Reservoir cells are memory T cells. These cells, which are generated
after infection or vaccination, keep the memory of the immune system for decades.
• Proliferation: Reservoir cells, like other memory T cells, divide very slowly to
maintain the memory of the immune system.
Where is the HIV reservoir?
From http://textbookofbacteriology.net
HIV latency
From Siliciano et al. Cold Spring Harb Perspect Med 2011
CD4 T cells
“Stem cell”
memory
Naïve
Central
memory
Transitional
memory
Effector
memory
Terminally
differentiated
Ag
IFN-g
IL-2
Survival
Self renewal
Apoptosis
Activation
Sallusto et al. Nature 1999 ; Riou et al. J Exp Med. 2007 ; Ahmed et al. Nat. Rev Immunol 2009 ;
Gattinoni et al. Nat Med 2011 ; Farber et al. Nat. Rev Immunol 2014
Contribution of CD4 T cells to the HIV reservoir
Contribution to the HIV
reservoir size (%)
100
80
60
40
20
0
TN
TCM
T TM
T EM
T TD
HIV persists in central, transitional and effector memory CD4 T cells
Chomont et al. Nat Med 2009
Where does HIV persist during ART?
• At the anatomical level: Potential “hiding places”
• Brain
• Lymph nodes (inc. B cell follicles)
• Peripheral blood
• Gut
• Bone marrow
Adapted from A. Fauci
« Size » of the HIV reservoir
The « real reservoir » ?
Ho et al. Cell 2013
Estimated HIV reservoir size
The frequency of cells harboring HIV integrated DNA is 10-1000 per 106 CD4 T cells
 0.1-0.001% of CD4 T cells contain HIV integrated DNA
Among these cells, 0.1-1% are able to produce infectious viral particles upon
stimulation (Finzi, Siliciano Nat. Med. 1999)
=> 0.001-0.000001% of CD4 T cells harbor infectious HIV
The total number of CD4 T cells in humans is estimated to 200X109
(Gasunov and De Boer, Trends in Immunology, 2007)
This calculation does not include additional reservoirs such as tissue macrophages
The total number of « reservoir CD4 T cells » in suppressed individuals
may be 100,000 -10,000,000
HIV persistence
Minimal decay of the HIV reservoir
Siliciano et al. Nat Med 2003
Half life of the HIV reservoir
Siliciano et al. Nat Med 2003
Reservoir established rapidly after infection
Early ART restricts the size of the HIV reservoir
(RV254)
Integrated HIV DNA
(copies/106 PBMCs)
10000
1000
100
FI
FIII
Chron
10
1
0.1
0
20
40
60
80
100
Time (weeks)
Very early ART (<2 weeks after infection) dramatically reduces the
size of the HIV reservoir
Higher CD4 count, smaller reservoir
Absolute CD4 count
 = -0.38
p = 0.03
10000
Integrated HIV DNA copies
per 106 CD4 T cells
CD4/CD8 ratio
1000
1000
100
100
10
10
1
200
p < 0.0001
10000
1
700
1200
CD4 count (cells/µl)
<1
>1
CD4/CD8 ratio
Translating basic science into interventions
Two strategies to eliminate the reservoir:
• Reactivation of HIV replication from its latent reservoir
Cytokines, chemical
compounds…
Uninfected cells
HIV-induced cell death
• Interfering with the immunological mechanisms that contribute to HIV persistence
T cell survival
Antibodies, cytokines,
gene therapy,
chemotherapy
Proliferation
Targeting molecular mechanisms of HIV latency
•
Histones
• Cellular proteins that encase genes and prevent their
transcription
• HIV genes can be freed form histone entrapment by
drugs called histone deacetylase (HDAC) inhibitors
• HDAC inhibitors promote production of HIV RNA (and
maybe proteins) by latently infected cells
• Vorinostat, panobinostat and romidepsin being evaluated
in clinical trials
Targeting molecular mechanisms of HIV latency
Targeting molecular mechanisms of HIV latency
Targeting molecular mechanisms of HIV latency
Gamma-c Cytokines:
IL-7
IL-15
Bromodomain inhibitors
JQ1
I-BET
HDAC inhibitors
Saha (vorinostat)
Panobinostat
Romidepsin
PKC agonists:
Prostratin
Bryostatin
Richman et al. Science 2009
Targeting molecular mechanisms of HIV latency
T-cell activation
PKC
Cytokines
HDACi
othe
r
Spina et al., Plos Pathogens 2013 Dec;9(12):e1003834
Targeting molecular mechanisms of HIV latency
• CD4 T cells respond to signals from their environment via
receptors on the cell surface
• The receptors expressed on a CD4 T cell also fluctuate in
response to signaling from the environment
• HIV latency in CD4 T cells is associated with the
expression of receptors that are involved in maintaining
the CD4 T cell in a resting state
• These receptors are referred to as “negative regulators”
or “immune checkpoints” as they are also involved in
preventing immune reactions to self (autoimmunity)
PD-1
•
•
•
Negatively regulates T cell responses (Freeman J Exp Med 2000, Wei PNAS 2013)
Two known ligands: PD-L1 and PD-L2, mostly expressed by
myeloid cells (Freeman J Exp Med 2000, Latchman Nat Immunol 2001)
Blocking PD-1 interaction with its ligands restores HIV specific
T cell functions (Day Nature 2006, Trautmann Nat Med 2006, Porichis Blood 2011)
PD-1 and the HIV reservoir
Integrated HIV DNA
(copies/106 CD4+ T cells)
10000
1000
100
10
r = 0.61
p < 0.0001
1
1
10
100
PD-1+ CD4 T cells (%)
The frequency of cells harboring integrated HIV DNA correlates
with PD-1 expression
Hatano et al. JID 2013
PD-1 and more
REV I EW S
b Co-inhibition of T cells following interaction with
Co-stimulation of T cells following interaction
with counter-receptors on APCs
PC
counter-receptors on APCs
T cell
MHC
TCR
B7-H2
ICOS
TReg cell
activation
MHC
class II
IDO
B7-1
–
TCR
LAG3
?
B7-2
CTLA4
B7-1
B7-2
CD28
CD70
CD27
B7-DC
LIGHT
HVEM
– B7-H1
HVEM
LIGHT
CD40L
CD40
B7-H1
PD1
B7-1
CD160
HVEM
4-1BB
4-1BBL
OX40L
OX40
TL1A
DR3
GITRL
GITR
CD30L
CD30
Unknown
IM1 ligand
Proliferation
Cytokine
production
Cytotoxic
function
Memory
formation
Survival
CD48
SLAM
Unknown
PD1H
receptor
Tolerance
Exhaustion
Apoptosis
Collagen
LAIR1
TIM4
CD2
+ CD48
CD155
CD112
CD113
CD155
CD226
CD112
TReg–TCon co-signalling interactions
cell
PD1H
?
– Galectin 9
CD58
Reg
PD1H
TIM1
TIM4
SLAM
?
Cell cycle
inhibition
Inhibition of
TIM1
?
?
BTLA
Unknown
PD1H
receptor
Unknown
TIM1
receptor
TIM3
?
Unknown
TIM4
receptor
2B4
TIGIT
d Co-signalling interactions through multiple interfaces
TCon cell
Chen L, Nat Rev Imm. 2013
Expression of multiple negative regulators
Fold over frequency
of infected CD4 T cells
12
CD4
9
Triple Negative
Single Positive
6
Double Positive
Triple Positive
3
0
0
CD4
1
2
3
Number of negative regulators expressed
Memory CD4
CD4 T cells expressing multiple negative regulators are highly
enriched for integrated HIV DNA
R. Fromentin, Means +/-SD from 5 ART subjects
Reactivation of the latent HIV reservoir
P = 0.017
P = 0.041
100000
Viral production
(HIV RNA copies)
10000
1000
100
10
1
NS
Mock
MK3475
isotype
+SEA/SEB (0.3 ng/mL)
Blocking PD-1 in vitro induces a modest but significant increase in
viral production in latently infected CD4 T cells
R. Fromentin
Targeting immunological mechanisms of HIV
latency
• The negative regulators PD-1, LAG-3 and TIGIT identify
CD4 T cells harboring integrated HIV DNA
• Blocking these receptors may revert HIV latency and
possibly also enhance HIV-specific T cell responses
• A clinical trial of an antibody to PD-L1 is ongoing (ACTG
A5326)
• PD-1 blockade may also be studied
One reason that could explain the reduced toxicity could be that the PD1/PD-L1 checkpoint interaction
takes place peripherally, i.e., at the tumor site, whereas the CTLA4/B7 interaction occurs mostly
centrally, i.e., in the lymphoid organs [78]. Most of the toxicity associated with anti-PD-1/PD-L1
was immune related, as well as with anti-CTLA-4 therapy [10,11]. The most frequent adverse
events recorded, regardless of causality, were fatigue, decreased appetite, diarrhea, nausea, dyspnea,
constipation, vomiting, rash, pyrexia and headache [10]. The Grade 3/4 adverse event rate was 14% in
patients receiving nivolumab. Interestingly, one unique and potentially life-threatening toxicity for
these agents is pneumonitis, which occurred in 3% of patients, but only 1%–3% developed a Grade 3
or 4 pneumonitis [10,51,52]. No clear relationship was reported between the incidence of this side
effect and tumor type, dose level or the number of doses received. In the majority of cases, it was
reversible with treatment discontinuation and/or glucocorticoid administration, but three patients died
despite the use of infliximab and mycophenolate [10]. Mild infusion reactions were observed in
patients receiving anti-PD-L1 treatment, whereas severe adverse effects were infrequently noted [11].
Indeed, irAEs were observed in 39% of patients and included rash, hypothyroidism, hepatitis and, less
frequently, sarcoidosis, diabetes mellitus and myasthenia gravis. These adverse events were
predominantly of Grade 1 or 2 and were managed with treatment interruption or discontinuation. The
Grade 3/4 adverse event rate was 9% in patients receiving BMS-936559 [49] and was managed with
glucocorticoids. Table 2 summarizes the main serious adverse effects of checkpoint inhibitors.
Risk consideration of immune checkpoint
blockers
• Manipulating the tight regulation of the immune system has to be
carefully evaluated (autoimmunity?)
• So far, clinical studies with ICBs have been performed in patients with
cancer
Table 2. Grade 3–4 serious adverse events of immune checkpoints inhibitors.
Serious Adverse Events
(Grade 3 and 4)
Rash and/or pruritus
Diarrhea
Nausea or vomiting
Colitis
Hypophysitis
Hypothyroidism
Hypopituitarism
Adrenal insufficiency
Increase in alanine
aminotransferase
Increase in aspartate
aminotransferase
Hepatitis
Fatigue
Pneumonitis
Ipilimumab
[9*,42,72,74,76]
Tremelimumab
[44–46]
Dermatologic
3.2%–4%
2.5%–18%
Gastrointestinal
4%–5.3%
5%–21%
<5%
8%–13%
2%–21%
2.1%–18%
Endocrine
0.8
2%
0
1%
0.8
1%
1.5
1%
Hepatic
Anti-PD1
(Nivolumab,
Lambrolizumab)
[3,51 **,52]
Anti-PD-L1
(BMS-936559)
[4]
1%–4%
<1%
1%–3%
0
2%
<1%
<1%
1%
1%
Non reported
0
0
0
0
<1%
1%–7%
0
1.5%–22%
Not reported
0.8%–18%
Not reported
1%–6%
<1%
<3%
6%–10%
Not reported
1%
2%–13%
1%
Not reported
2%
1%–3%
<5%
3%
Not reported
Gelao et al. Toxins 2014
Notes: * In this study, ipilimumab is in combination with dacarbazine; ** in this study, nivolumab is in
combination with ipilimumab.
• Possible additional side effects in HIV-infected individuals?
Targeting immunological mechanisms of HIV
latency
• Other approaches with the potential to
interfere with the proliferation and/or survival
of latently infected CD4 T cells also being
explored (e.g. mTOR inhibitors, auranofin)
Translational research
• Basic research findings on molecular and
immunological mechanisms of HIV persistence
are being translated into clinical trials of
possible interventions
• These are many other examples of translational
research in the HIV cure field, trials of gene
therapies, therapeutic vaccines and immunebased therapies also based on basic research
discoveries
• Additional CUREiculum modules provide more
information on all these approaches:
http://www.avac.org/cureiculum
Acknowledgments
CRCHUM
Rémi Fromentin
VGTI Florida
Claire Vandergeeten
Francesco Procopio
Mariam Lawani
Wendy Bakeman
Amanda McNulty
Jessica Brehm
Deanna Kulpa
Rafick-Pierre Sékaly
MHRP
Jintanat Ananworanich
Jerome Kim
Merlin Robb
Nelson Michael
Institut Pasteur
Asier-Saez-Cirion
Merck
Daria Hazuda
Mike Miller
Richard Barnard
UCSF
Hiroyu Hatano
Ma Somsouk
Peter Hunt
Elisabeth Sinclair
Rick Hecht
Rebecca Hoh
Lorrie Epling
Mike McCune
Steven Deeks
Westmead Institute
Sarah Palmer
Eunok Lee
McGill
Jean-Pierre Routy
VRC
Danny Douek
Eli Boritz
UNC
Karine Dubé
AVAC
Jessica Handibode
Collaborators
The study participants!