1. 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.

2. 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-to- bedside”)

3. HIV life cycle
From Han et al, Nat Rev, 2007

4. However, the virus rebounds after cessation of therapy
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
START
STOP
HAART
Circulating virus
Limit of detection
Time
HIV hides in reservoir that are not sensitive to current therapies

5. HIV persists during ART
Several studies in the late 1990s identified a reservoir of HIV in long-lived cells in the body that persists despite ART.

6. 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.
The long-lived nature of memory CD4 T cells explains the ability of latent HIV to persist despite HAART. Memory CD4 T cells can copy themselves in a process known as “homeostatic proliferation,” and if the CD4 T cell is latently infected the HIV DNA is copied along with the genome of the cell.

7. Where is the HIV reservoir?
Stem cells give rise to the multiple lineages of cells that make up the immune system, The primary target of HIV is the T-helper (Th) or CD4 T cell.
From

8. HIV latency From Siliciano et al. Cold Spring Harb Perspect Med 2011
HIV latency is thought to result (at least in part) when the virus infects an activated CD4 T cell that is transitioning into a long-lived memory state. The memory CD4 T cell can persist with HIV integrated into the cell’s genome, and the virus only becomes active again if the CD4 T cell is re-activated. For example, if the memory CD4 T cell developed in response to activation by influenza antigens, that memory CD4 T cell will remain in a “resting” state until it encounters influenza again. A subsequent re-encounter with influenza—even if many years later—will activate that memory CD4 T cell and, if it contains HIV, also trigger virus production.
From Siliciano et al. Cold Spring Harb Perspect Med 2011

9. Terminally differentiated
CD4 T cells
Naïve
Central
memory
Transitional
Effector
Terminally differentiated Ag
“Stem cell” memory
IFN-g
IL-2
Apoptosis
CD4 T cells exist in a variety of states, all derived from the most youthful population called naïve CD4 T cells. Naïve CD4 T cells are generated in the bone marrow, then undergo a selection process in the thymus gland (located behind the breastbone) before entering the circulation to patrol for antigens that they recognize via a structure on the surface called a T-cell receptor (TCR). Naïve CD4 T cell production is maximal at birth and declines dramatically in adulthood, dwindling to a trickle in old age. When a naïve CD4 T cell encounters an antigen it recognizes, it becomes activated, divides many times and generates a population of “memory” CD4 T cells (specific for the same antigen) that can differentiate further into different memory types. The most long-lived memory CD4 T cells are stem cell memory and central memory cells.
Survival
Activation
Self renewal
Sallusto et al. Nature 1999 ; Riou et al. J Exp Med ; Ahmed et al. Nat. Rev Immunol 2009 ;
Gattinoni et al. Nat Med 2011 ; Farber et al. Nat. Rev Immunol 2014

10. Contribution of CD4 T cells to the HIV reservoir
HIV persists in central, transitional and effector memory CD4 T cells
Chomont et al. Nat Med 2009

11. 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
HIV-infected cells can persist at multiple sites in the body, research continues to investigate the contribution of different anatomical sites to HIV persistence on HAART.

12. « Size » of the HIV reservoir
The « real reservoir » ?
Ho et al. Cell 2013

13. Estimated HIV reservoir size
The frequency of cells harboring HIV integrated DNA is per 106 CD4 T cells
% 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)
=> % 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
Extrapolating from published studies, the total number of CD4 T cells containing latent, replication-competent HIV is likely to be in the range of 10 thousand to 10 million.
The total number of « reservoir CD4 T cells » in suppressed individuals may be 100, ,000,000

14. HIV persistence Minimal decay of the HIV reservoir
Studies have shown that the size of the HIV reservoir is very stable over time.
Minimal decay of the HIV reservoir
Siliciano et al. Nat Med 2003

15. Half life of the HIV reservoir
Although there is some evidence for a slow decline in the level of the HIV reservoir in people on HAART, it is estimated that it would take at least a lifetime for all latently infected cells to die off without additional intervention.
Siliciano et al. Nat Med 2003

16. Reservoir established rapidly after infection
A study in macaques infected with SIV, HIV’s simian equivalent, has found that even if HAART is started 3 days after infection, the establishment of a persistent reservoir of virus is not prevented. The graphs show different groups of animals that received HAART starting at day 3, day 7, day 10 or day 14 after SIV infection. In all cases, SIV viral load rebounded after HAART was interrupted.

17. Early ART restricts the size of the HIV reservoir (RV254)
Very early ART (<2 weeks after infection) dramatically reduces the size of the HIV reservoir
Studies in humans have sown that longer duration of untreated HIV infection is associated with a larger reservoir size.

18. Integrated HIV DNA copies per 106 CD4 T cells
Higher CD4 count, smaller reservoir
Absolute CD4 count
CD4/CD8 ratio
> 1
< 1
p < 1 10
100
1000
10000
10000
 = -0.38
p = 0.03
1000
Integrated HIV DNA copies per 106 CD4 T cells
100 10
Lower CD4 T cell counts and lower CD4/CD8 ratios are also associated with larger reservoir size. 1
200
700
1200
CD4 count (cells/µl)

19. HAART HAART HAART HAART HAART HAART
Translating basic science into interventions
Two strategies to eliminate the reservoir:
Reactivation of HIV replication from its latent reservoir
HAART
Cytokines, chemical compounds…
HAART
HAART
Uninfected cells
HAART
HAART
HAART
HIV-induced cell death
Interfering with the immunological mechanisms that contribute to HIV persistence
Scientific knowledge about the latent HIV reservoir is facilitating the development of interventions that may be able to disrupt latency, in hopes of ultimately being able to achieve a cure.
Antibodies, cytokines, gene therapy, chemotherapy
T cell survival
Proliferation

20. 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
Laboratory studies have identified many cellular proteins that are involved in maintaining HIV in a latent state in resting CD4 T cells. Histones are a key example. The effect of histones can be counteracted with drugs called histone deacetylase (HDAC) inhibitors, and several HDAC inhibitors are already approved as treatments for cancer. Several clinical trials have now reported that HDAC inhibitors can activate latent HIV. Drugs with the capacity to reverse HIV latency are now commonly referred to as latency-reversing agents (LRAs).

21. Targeting molecular mechanisms of HIV latency
This diagram shows how HDAC inhibitors can liberate HIV from histones, allowing the viral genes to transcribe messenger RNA which in turn generates virus proteins and, ultimately, new virions capable of infecting other cells.

22. Targeting molecular mechanisms of HIV latency

23. 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

24. Targeting molecular mechanisms of HIV latency
This slide shows different latency reversing agents working in different cell types
Protein kinase C (PKC) – being targeted with PKC agonists e.g. Bryostatin
Bromodomain proteins – possible targets with inhibitors (e.g. JQ1 in vitro)
JAK/STAT pathway – modulated by cytokines (e.g. IL-15)
P-ETFb
TLR (TLR-7 agonist)
HIV Tat protein may be master regulator of latency
T-cell activation
PKC
Cytokines
HDACi
other
Spina et al., Plos Pathogens 2013 Dec;9(12):e

25. 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)

26. 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)
The PD-1 receptor was one of the first negative regulators identified. The molecules PD-1 interacts with (ligands) are PD-L1 and PD-L2. Studies of HIV-positive individuals have found that expression of PD-1 by HIV-specific CD4 and CD8 T cells is associated with the dysfunction of these cells. In laboratory experiments, blockade of PD-1 restores the function of HIV-specific CD4 and CD8 T cells. Antibodies that block PD-1 have shown success as cancer treatments and are under investigation in HIV,

27. PD-1 and the HIV reservoir
The frequency of cells harboring integrated HIV DNA correlates with PD-1 expression
Hatano et al. JID 2013

28. PD-1 and more
Chen L, Nat Rev Imm. 2013

29. Expression of multiple negative regulators1 2 3
Number of negative regulators expressed
CD4
Memory CD4
CD4 T cells expressing multiple negative regulators are highly enriched for integrated HIV DNA
R. Fromentin, Means +/-SD from 5 ART subjects

30. Reactivation of the latent HIV reservoir
A PD-1 blocking antibody activates HIV production by latently infected CD4 T cells isolated from HIV-positive individuals.
Blocking PD-1 in vitro induces a modest but significant increase in viral production in latently infected CD4 T cells
R. Fromentin

31. 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

32. 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
Possible additional side effects in HIV-infected individuals?
Gelao et al. Toxins 2014

33. 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)

34. 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 immune- based therapies also based on basic research discoveries
Additional CUREiculum modules provide more information on all these approaches:

35. Acknowledgments UNC Karine Dubé AVAC Jessica Handibode 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

36. The study participants!
Collaborators
The study participants!