1. HIV Cures and Immunotherapy: New Horizons
Daniel C. Douek, MD, MRCP, PhD
Bethesda, Maryland
EDITED: 03/29/16
Atlanta, Georgia: April 8, 2016

2. After attending this presentation, participants will be able to:
Learning Objectives
After attending this presentation, participants will be able to:
Describe the primary objectives of cure approaches
Contrast the benefits and drawbacks of different cure approaches
Describe how different approaches may achieve a cure goal

3. Yes No Only in some studies
Latency reversing agents (eg: vorinostat, romidepsin) reduce the size of the HIV reservoir in clinical studies:
Yes No
Only in some studies 1

4. To cure an HIV-infected person the latent HIV-reservoir needs to be reduced by at least:
103 times
105 times
109 times 1

5. HIV-specific monoclonal antibodies administered to HIV-infected people in clinical studies to date
Reduce plasma virus load
Reduce the size of the reservoir
Select for resistant virus strains
1 and 3 1

6. The Cascade This is one reason we need a cure
Number and percentage of HIV-infected persons engaged in selected stages of the continuum of HIV care in the United States
2x105
100%
80%
62%
41%
36%
28%
4x105
6x105
8x105
10x105
12x105
HIV infected
HIV diagnosed
Linked to HIV care
Retained in HIV care
Prescribed ARVs
Suppressed virus load
≤200c/ml
Number of Individuals
Cohen 2012; Rowan 1012; Hall 2012; MMWR 2011; Gardner CID 2011; Burns CID 2010
This is one reason we need a cure
Even with drugs that are 100% effective at suppressing virus we are unable to achieve this in 100% of infected people

7. Three Histories of HIV-infected Cells In Vivo
ART
Virus
Transmission
Cellular
Self-renewal
Cellular
Latency
This depicts different behavior of cells. Longevity and proliferation.
May correspond to behavior of different subsets.
Different viruses may be associated with these different cells.
Multiple mechanisms govern these events

8. Ongoing HIV Viremia During cART
cART reduces plasma HIV RNA > 6 to 7 log10
But viremia persists indefinitely (0.1-3 copies RNA/mL)
Source of viremia unknown 8

9. How To Cure HIV-Infected People
Multiple mechanisms account for HIV persistence
Unifying theme: find and diminish size of the HIV reservoir
Reduce seeding of latent pool with early/more ART
Reverse latency (shock and kill)
Increase HIV-specific immune function (vaccines)
Reduce immune activation
Gene therapy targeting the virus and the host
Allogeneic stem cell transplantation
Combination therapy may be necessary

10. How To Cure HIV-Infected People
Hematopoietic transplantation with cells resistant to infection
Doing well off ARV therapy > 5 years
No replication competent HIV
No PBMC DNA, intermittent very low plasma HIV RNA and rectal DNA
Waning HIV antibodies and no HIV-specific T cells
Even though this procedure works, it is highly unlikely that it will ever translate into an accessible approach

11. Boston Patients: Virus Recrudescence
Hematopoietic transplantation with cells susceptible to infection
Despite 1000 – 10,000 fold reductions in reservoir size, virus rebounded
Modeling: latent reservoir will have to be depleted > 105 fold (Hill, PNAS ‘14)
A single virus accounts for recrudescence

12. Shock and Kill Activate the infected T cell “Shock” Virus is produced
Immune system kills the cell
ART stops new infections

13. Current Status of LRA Clinical Trials
Numerous LRAs identified in studies with cell lines and primary T cells
Relative to T cell activation, few LRAs work well ex vivo with cells from patients
In clinical trials, evidence for increase in cell-associated and plasma HIV RNA
In clinical trials, no reduction in the reservoir yet demonstrated
Single LRA
+ Bryostatin
+ Disulfiram
% max T cell stim
Romidepsin
Søgaard PLoS P 2015
We have explored these issues using our primary cells model. When latency is reversed through T cell activation, the infected cells die quickly. But when latency is reversed with a histone deacytlase inhibitor that does not induce T cell activation, the cells don’t die. They just sit there and produce virus.
Laird JCI 2015

14. The Kill?
Reactivate the virus with LRA and then clear the infected cells
Will the virus kill the infected cells?
– “After reversal of latency in an in vitro model, infected resting CD4 T cells survived despite viral cytopathic effects” (Shan, Immunity 2012)
Can the immune system help?
– Most of the virus has mutated to escape the immune response and escape variants dominate in the latent reservoir of chronic subjects (Deng, Nature 2015)
Therapeutic vaccination?
– Transient expansion of T cells that do not recognize escaped HIV epitopes (Koup, JID 2014)
Testing of killing of cells infected with autologous virus was only done in 2 subjects. Rapid outgrowth of autologous virus from PBMC favored very fit viruses, therefore selected against escape mutants. No virus sequencing was done.

15. Gene Therapy Deliver a therapeutic agent to a cell using a gene
Provide something to inhibit or kill HIV
Anti-HIV antisense RNA, fusion inhibitor C46, transdominant Rev
Remove something that HIV needs: CCR5
Using antisense RNA, intrabodies, targeted DNA nucleases
Real lesson we learned from the Berlin and Boston patients:
You need to remove the virus and the target cells
Remove the target cell. This is what we learned from the Berlin and Boston patients. Taking away the virus isn’t enough, because it’s too difficult. You need to remove the traget cells too.

16. Summary of Findings
Infusion of CCR5-specific zinc-finger nuclease-treated ex vivo expanded autologous T cells is generally safe and well tolerated
Durable increases seen in both CD4 and total T cell counts
CCR5 modified T cells persist long-term in vivo and persist longer than unmodified cells during treatment interruption
Change in HIV DNA
All subjects in a cohort of chronically infected INR saw long-term CD4 T cell reconstitution and HIV reservoir decay
(Sekaly, Lalezari, Blick CROI 2016)

17. Antibodies Used In Virus Infections
Pathogen
Product Description
Indication
Measles
Concentrated human gamma globulin
Prevention
Polio
CMV
Cytomegalovirus Immune Globulin
Hepatitis A
Immune serum globulin (ISG)
Prevention (travel)
Hepatitis B
Hepatitis B Immune Globulin
Post Exposure
Rabies
Rabies Immune Globulin
RSV
mAb (palivizumab) for prophylaxis of high risk infants
Prevention in high risk Infants
VZIG
Varicella Zoster Immune Globulin
Current clinical use
NHP studies going back to 1999 show that anti-HIV mAb can completely protect against acquisition of infection

18. Potential Targets for Neutralization
V1V2 Glycan:
PG9, PG16 PGT CAP256-VRC26.25
PGDM1400
HIV Envelope
N332 Glycan Supersite:
PGT128, DH542,
PGT121,
CD4 Binding Site:
PG04, CH31, 12A12, VRC13,
VRC01, VRC01LS,
VRC07-523LS, 3BNC117, N6
V1V2-glycan
Glycan-V3
CD4bs
gp41 MPER:
2F5, 4E10, 10e8
gp120/41 interface
8ANC195 PGT
gp120/41 interface
MPER

19. Antibody Potency/Breadth
Breadth: % of Envs neutralized
Broad and potent 87 80 96 97
Less broad but 500x more potent 54 62 73 48 98
Very broad, very potent 98
Panel of 206 HIV Env pseudoviruses from all major clades
More potent
Virus IC80 titer mg/ml

20. Clinical Use of HIV-Specific Antibodies
Prevention and treatment with antibodies are different
Prevent acquisition of infection
Prevention
Block transmission
Have to deal with greater viral diversity
Different mechanism of action?
mAbs complementary to ARV
Potential to reduce HIV reservoir (Cure)
Treatment
Block virus entry
Cell killing
CD4 T-cell
NK cell killing of infected cells

21. Clinical Use of HIV-Specific Antibodies
During acute HIV infection, with ARVs, to rapidly reduce viremia and limit seeding the virus reservoir
To maintain long-term viral suppression induced by ARV eg: LA-ARV + mAb given once every 2-3 months
Reduce cell-associated virus reservoir by cell-mediated killing – functions distinct from ARVs; combine with LRAs?
Latently infected cell
Natural killer
cell lysis
Macrophage
phagocytosis
Stimulate viral expression (LRA) 21

22. Phase I Trial of VRC01: Single Infusion
# 20
# 21
# 22
# 23
# 24
# 25
# 26
# 27
Δ log10 virus load (copies/ml)
Days post VRC01-infusion
3 Patterns:
Profound and maintained suppression
Transient suppression
No suppression
Lynch, Science Trans. Med. 2015

23. Pre-Infusion HIV Sensitivity to VRC01
Non-responders
VRC01 IC80 (μg/ml) 20 21 22 23 24 25 26
VRC01 27
IC80 > 20 ug/ml
More sensitive
median
The two low-responder participants had most resistant virus quasi-species before VRC01 infusion
Lynch, Science Trans. Med. 2015

24. Profile of a 2nd Generation mAb Product
Cost comparable to ARV drugs
Cover 98-99% of virus diversity
10-fold more potent than current mAbs
Given by SQ injection once every 3-4 months (vs IV infusion every 2 months) 24

25. Combined mAbs: Improved Potency and Breadth
2 mAbs > 98% coverage
Broader
Kong, JV 2015
More potent
Combination of 2+ mAbs improves potency and breadth

26. Mutations to Increase Half-Life
Motavizumab-YTE
VRC01-LS
Mota-YTE
>20 mg/ml for >6 months
Mota
Robbie, AAChem 2013
At least fold increase in half-life in healthy adults
Decreases dose needed by 5-10 fold
Potentially extends dosing interval to every 6 months
Means (± standard deviations) of mota-YTE and motavizumab serum concentrations after a single dose. d, days.

27. Bifunctional Antibodies
Bispecific T cell engager (BITE)
Dual-affinity re-targeting protein (DART)
Infected CD4 T cell
anti-Env
anti-CD3
CD8 T cell
Potential to mediate cell killing of infected CD4 T cell
Products exist and have entered clinical trials for cancer
Little in vivo data for treatment of HIV, even in animal models

28. Activation and Lysis of Human CD4 Cells Latently Infected with HIV-1.
Bifunctional Antibodies: Proof of Concept
Volume 125 September 28, 2015
October 20, 2015
Activation and Lysis of Human CD4 Cells Latently Infected with HIV-1.
A. Pegu, M. Asokan, L. Wu, K. Wang, J. Hataye, J. P. Casazza, X. Guo, W. Shi, I. Georgiev, T. Zhou, X. Chen, S. O’Dell, J. Todd, P. D. Kwong, S. S. Rao, Z. Yang, R. A. Koup, J. R. Mascola & G. J. Nabel.
November 5, 2015
Dual-Affinity Re-Targeting Proteins Direct T Cell-Mediated Cytolysis of Latently HIV-Infected Cells.
Sung JA, Pickeral J, Liu L, Stanfield-Oakley SA, Lam CK, Garrido C, Pollara J, LaBranche C, Bonsignori M, Moody MA, Yang Y, Parks R, Archin N, Allard B, Kirchherr J, Kuruc JD, Gay CL, Cohen MS, Ochsenbauer C, Soderberg K, Liao HX, Montefiori D, Moore P, Johnson S, Koenig S, Haynes BF, Nordstrom JL, Margolis DM, Ferrari G.
Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells.
Sloan DD, Lam C-YK, Irrinki A, Liu L, Tsai A, Pace CS, Kaur J, Murry JP, Balakrishnan M, Moore PA, Johnson S, Nordstrom JL, Cihlar T, Koenig S.
BITEs and DARTs mediate killing of HIV-infected cells in vitro

29. Conclusions for an HIV Cure
LRAs show poor reactivation and no reduction in reservoir size
Gene therapy may be used to target HIV and/or CCR5
Clinical studies are ongoing and some show reservoir reduction
Env-specific mAbs are in promising proof of concept studies
More potent mAbs and combinations of mAbs are on the horizon
New approaches to cell-mediated killing of infected T cells
Combinations of approaches may be used:
LA-ARVs + mAbs, LRAs + BITEs, gene therapy + mAbs + LRAs