1. 2014 “Towards an HIV Cure” symposium Melbourne
Induction and Clearance of Latent HIV Infection:
an Ex-Vivo Assessment of Immune Effectors using Cells from ART-treated Patients
DM Margolis1, C Garrido1, JM Sung1, S Lam2, R Bateson1, B Allard1, N Dahl1, CR Cruz2, P Castillo-Caro3, MT Ngo3, J Kuruc1, A Crooks1, CM Rooney3, CM Bollard2, and NM Archin1
1University of North Carolina Chapel Hill, NC; 2Children’s National Medical Center, Washington, DC; 3Baylor College of Medicine, Houston, Texas

2. Model systems to assess clearance
Primary cell systems
Cytotoxic T cells
NK cells
ADCC antibodies
Immunotoxins
Selective apoptosis inducers

3. Why Ex-vivo Expanded CTLs
Bypasses impaired immune response
Precisely controlled quantity & timing of administration
CTLs can persist
Safe and effective for treatment of viral infections in oncology patients
As complex as the immune system is and given how profound the depth and breadth of immune exhaustion in chronic HIV infection is, there are many avenues to approaching the problem of enhancing the immune system in order to clear latent HIV infection;
This work focuses on ex-vivo expanded CTLs for proof of concept work as this approach bypasses many of the obstacles to a robust immune response that occur in the face of chronic HIV infection, including dysfunctional and impaired DC function, CD4 help and CD8 proliferation; additionally, we can precisely control the timing of administration-which will be an important consideration if combining with a latency reactivating agent, which may have its own impact (transient or otherwise) on the immune system, as well as control over the number and type of CTLs that are ultimately infused
AM Leen, et al. Nat Med. 2006
Bollard, et al. Blood. 2007

4. Ex vivo Expansion of HIV-specific T cells
ART to prevent
In vitro spread ++
HIV-CTLs
T cell
Immature DC
PBMC
freeze
HXTCs
IL IL IL-2
IL-12
IL-15
Here you can see the method for generating the ex-vivo expanded HIV-CTLs; T cells derived from patient PBMCs undergo stimulation with matured autologous DC and then irradiated PHAblasts that have been loaded with multiple overlapping consensus clade b peptides for gag, pol, and nef in order to minimize the impact of CTL escape variants; This is followed by a third and final round of stimulation with irradiated loaded K562 cells that have been modified to overepress costimulatory molecules to really amp up the numbers; The use of the modified, irradiated K562 cells has been found to be safe for clinical use;
Using this method, CTls were expanded by anywhere from 37 to 300 fold
PHAblast
CD80/86
4-1BBL
CD32
+ K562
Mature DC
+ gag/ pol/ nef
Cath Bollard, DC Children’s
Clio Rooney, Baylor and PACT

5. HXTCs are a Mixture of Phenotypes
82% CD8+ T cells
19% CD4+ T cells
80% EM T cells
13% CM T cells
IFNγ Release
to Cognate Peptides

6. HXTCs Inhibit Productively Infected Cells
CD8 deplete patient PBMCs
Activate 2-3 days
Superinfect w/ virus via spinoculation
+ autologous unexpanded CD8 or expanded HIV-CTLs (or no effector control)
Co-culture x 7 days
Media change q3-4 days
Supernatant harvested for p24 ELISA
wash
Plate in triplicate
HIV-CTLs
To measure the antiviral activity of the HIV-CTLs, we modified a viral inhibition assay well described in the literature; HIV-CTLs, or as a comparator unexpanded autologous CD8s, or as a control, media only are added to autologous targets that had been superinfected with JR-CSF;
Yang, et al. JID 2012
Freel, et al. J Virol. 2012

7. HXTCs Inhibit Autologous Reservoir Virus
HIV p24 (% of no effector at day 7)
E:T
E:T
E:T
E:T

8. Ex-Vivo Latency Clearance Assay: A modified quantitative viral outgrowth Assay
CTL Expansion
CD8 negative selection
PBMCs
CD8
HXTCs
No Effectors or or
Resting CD4
Negative selection
CD8, CD14, CD16, CD19,
CD56, glycophorin A
Cx with ARVs
24H
Induction
PHA/IL2 or
VOR
Add CTLs
Co Cx
24H
Add Allo
Feeders
CoCx x2
Measure p24 at
Day 15
To do this, we modified the standard quantitative outgrowth assay-outlined below-in which resting CD4 cells are isolated and activated with PHA/IL-2- to include the addition of effector cells-either the ex=vivo expanded HIV-CTLs, or autologous unexpanded CD8s as a comparator, or as a control, no effectors/just media are added and allowed to co-culture with the reactivated CD4 cells for 24 hours prior to addition of allogeneic CD8 depleted PBMCs to amplify any residual infection;
wash
wash
Media changes q3-4 days
Plate: 0.5-1x106/well, 12 wells group

9. HXTCs Clear Infected Cells Emerging from Latency
PHA Stimulation VOR Induction
425
532
250 20 40 60 80
100
120
% viral recovery
No Effectors
CD8
HXTC 1 2 3 4 5 6 7 8
#wells positive
(out of 12 total)
Et 1:10
Et 1:10
Black dot is pt 492 with all 3
Some with only Nef response suppressed
p <0.03 Wilcoxon signed rank
p <0.02 t test

10. VOR does not Impair CD8 Antiviral Activity at Physiogically Relevant Exposures
Viral inhibition
assay at E:T
ratio 1:1 B.
Control 24 48 72 20 40 60 80
100
No CD8 Control
335nM
500nM
1000nM
No VOR
HIV p24 (ng/ml)
hours
Patient 532

11. Why NKs to target residual HIV
Crucial innate immune effectors against viral infections
Do not recognize specific antigens nor require prior antigen sensitization
Function is balanced by inhibitory and activating receptors
Kill cells by release of granzymes and perforins, which causes apoptosis
NK cells may help control HIV-1 infection
Memory NK cells may also provide a more effective antiviral response
NK function may be augmented by clinically applicable cytokines

12. VIRAL INHIBITION ASSAY
Unstimulated NKs
PBMCs
Negative isolation
NK cells
IL-2 /IL-15 stimulated NKs
No effectors
Negative isolation
≠ E:T ratios
Super-infection (autologous reservoir virus)
CD4+T cells
HIV-CD4+ cells +/- effectors
PHA 24h
7 days
HIV-p24

13. VIRAL INHIBITION ASSAY† † † † † †
CD4+
Targets
only
1: :10 1:100
1: :10 1:100
1: :10 1:100
NK cells
IL2-stimulated NK cells
IL15-stimulated NK cells
†: p <0.01

14. LATENCY CLEARANCE ASSAY
PBMCs
Negative isolation
NK cells
Resting CD4+T cells
ARV 24h
Stimulation
CD4+T cells +/- effectors
Add feeders
Unstimulated NKs
IL-2 stimulated NKs
Measure number of positive wells for p24 at day 15
24h
No effectors
Compare number of positive wells with/without effectors

15. LATENCY CLEARANCE ASSAY
PHA reactivation
VOR reactivation
VOR
Positive trends but more assays needed

16. IMPACT OF VOR ON NK FUNCTION
NKs were treated with or without 335 nM VOR overnight
For degranulation cells 4 hours in the presence of K562 cell targets
VOR
Viral inhbition assays
Degranulation
Accepted 270 registrations, approximately 270 people attended over the two days.
20 oral abstracts accepted, of which 8 were LB’s. 52 poster abstracts were accepted = total of 72 abstracts
51 full and partial scholarships were offered.
VOR did not impair NK cytotoxicity or antiviral activity

17. Summary
Polyclonal HXTCs with activity against multiple peptides can be generated in clinically relevant numbers
HXTCs and cytokine-treated NK cells show enhanced antiviral activity
Using both lab strain JR-CSF virus and autologous reservoir virus
NK cells and HXTCs reduce viral recovery from resting CD4 cells reactivated with PHA/IL-2, demonstrating a potential to clear latent HIV infection ex-vivo
Preliminary results also show reduction in viral recovery from resting CD4 cells reactivated with VOR, and no adverse effect on function at relevant VOR exposures

18. Children’s National Medical Center
Juila Sung MD
Carolina Garrido Pavon, PhD
Natalia Soriano, PhD
Nancie Archin, PhD
Noelle Dahl
Rosalie Bateson
Brigette Allard
Joann Kuruc, MSN RN
Amanda Crooks
Cynthia Gay, MD, MPH
Children’s National Medical Center
Catherine Bollard, MD
Sharon Lam
Special thanks to the
HIV+ volunteers