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3/31/2017
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Robert F. Siliciano, MD, PhDProfessor of Medicine
The Johns Hopkins UniversityBaltimore, Maryland
New Technologies Applied to an HIV Cure
FORMATTED: 03/20/17
Atlanta, Georgia: March 30, 2017
Slide 2 of 54
New Technologies Applied to an HIV Cure
Robert F. Siliciano MDPhDJohns Hopkins University
School of MedicineHoward Hughes Medical Institute
Disclosures: None
Slide 3 of 54
Financial Relationships With Commercial Entities
Dr Siliciano has no relevant financial affiliations to disclose. (Updated 03/20/17/)
Atlanta, Georgia: March 30, 2017
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Slide 4 of 54
Learning Objectives
After attending this presentation, learners will be able to:
Describe the barriers to curing HIV infection
Describe the current approaches to measuring and eliminating the latent HIV reservoir
Slide 5 of 54
Time Post Infection
(weeks) (years)
1,000,000
100,000
10,000
1000
100Pla
sma
HIV
RN
A (
cop
ies/
ml)
Preventing viral rebound is the goal!
ART
Slide 6 of 54
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Time (months)
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
HIV replication dynamics
Limit ofdetection
R0 = 8 -10
Setpoint
ART
t1/2 = 1d
t1/2 = 14d
Intensify
Wei et al. Nature 1995Ho et al, Nature 1995Perelson et al, Nature 1997Finzi et al, Nature Med 1999Dornadula et al, JAMA 1999Dinoso et al, PNAS, 2009Robb and Ananworanich, COHA, 2016
Residual viremia
†
v +
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Slide 7 of 54
Establishment of the latentreservoir in resting CD4+ T cells
Naive
Mem
ory
Ag
††††
†
†
Slide 8 of 54
Establishment of the latentreservoir in resting CD4+ T cells
Naive
Mem
ory
Ag
††††
†
†
HIV
Ag
†
Slide 9 of 54
Quantitative viral outgrowthassay
PHA + irradiatedallogeneic PBMC
p24Ag
180-200ml blood
Purified restingCD4+ T cells
d2: add CD4+
lymphoblastsfrom HIV-donors
d7: add CD4+
lymphoblastsfrom HIV-donors
Finzi et al., Science, 1997
1/1,000,000
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Slide 10 of 54
10000
Slow decay of latently infectedCD4+ T cells
-
Time to eradication> 73.4 years
0.0001
0.001
0.01
0.1
1
10
100
1000
0 1 2 3 4 5 6 7
Time on ART (years)
Fre
quen
cy(p
er 1
06ce
lls)
0.00001
Finzi et al., Nature Med., 1999Siliciano et al., Nature Med., 2003
Slide 11 of 54
Slow decay of the reservoir
Finzi et al, Nature Med 1999; Siliciano et al., Nature Med., 2003
Crook et al, JID2015
t1/2 = 43 months
t1/2 = 44 months
Slide 12 of 54
Time on ART (months)
Lorenzo‐Redondo et al, Nature 2016
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Slide 13 of 54
Labile infected cells populations dominate early
Blankson et al, JID 1999Rosenbloom et al., submitted
Slide 14 of 54
-3 0 3 6
0.72
0.99 0.98
1
0.61
0.87
0.99
0.95
0.9
B
Months on ART: 0–3 3 6
Early sampling creates misleadingappearance of clocklike evolution
Rosenbloom et al., submitted
Slide 15 of 54
0
20
40
60
80
100
0.01 0.1 1 10 100
Concentration/IC50
m =1
m =1 .5
m =2
m =3
m =5
fa /fu = (D/IC50 )m
fa = fn affectedfu = fn unaffectedD = dosem = slope parameter
Infe
ctio
n (%
of c
ontr
ol)
(Hill coefficient)
Shen et al, Nat Med 2008Shen et al, Sci Trans Med 2011
Sampah et al, PNAS 2011Jilek et al, Nat Med 2012
Rosenbloom et al, Nat Med 2012Rabi et al, JCI 2012
Efficacy of HIV Drugs
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CmaxCmin
0
20
40
60
80
100
0.01 0.1 1 10 100
m =1
m =1 .5
m =2
m =3
m =5
Linear-Log Dose Response Curve
Concentration/IC50
Infe
ctio
n (%
of c
ontr
ol)
Shen et al, Nat Med 2008Shen et al, Sci Trans Med 2011
Sampah et al, PNAS 2011Jilek et al, Nat Med 2012
Rosenbloom et al, Nat Med 2012Rabi et al, JCI 2012
Slide 17 of 54
CmaxCmin
0.00001
0.0001
0.001
0.01
0.1
1
10
100
0.01 0.1 1 10 100
m =1
m =1 .5
m =2
m =3
m =5
Log-Log Dose Response Curve
Concentration/IC50
Infe
ctio
n (%
of c
ontr
ol)
IC50
Shen et al, Nat Med 2008Shen et al, Sci Trans Med 2011
Sampah et al, PNAS 2011Jilek et al, Nat Med 2012
Rosenbloom et al, Nat Med 2012Rabi et al, JCI 2012
Slide 18 of 54
Chronic Hepatitis C infection
• Continuous, high level viremia
• Rapid viral evolution
• Drug resistance with suboptimal treatment
Feld et al., NEJM, 2015
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Inhibition of HCV replication by direct acting antiviral drugs
Koizumi et al, PNAS 2017
• HCV antivirals also have cooperative dose response curves and sygnergies that produce very high IIP
• HCV infection is readily curable
• HCV has no latentform
Slide 20 of 54
ART is completely suppressive but not curative due to latent reservoir
• Host immune system, including latently infected cells, largely eliminated by condition regimen (chemo + irradiation and by graft vs host disease.
• Donor cells protected from HIV infection due to absence of CCR5
Slide 21 of 54
TDFFTCRAL
1
10
100
1000
10,000
100,000
1,000000
Time after Rx interruption (months)
Henrich et al, JID, 2013
0 2 4 6 8
10,000,000
‐30‐42
Matchedallogeneic
HSCT
“Boston Patient B”
StopART
Below limitof detection
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The Mississippi baby
Persaud D et al., NEJM 2013
10
100
1000
10,000
100,000
1,000000
Months after Birth
0 3010 20 40 50
AZT3TCLPV/r
ART discontinued
Below limitof detection
>2 years
These delayed rebound cases prove that HIV can persist in a latent form for years and then begin to replicate
Slide 23 of 54
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Time (months)
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
HIV replication dynamics
Limit ofdetection
R0 = 8 -10
Setpoint
ART
t1/2 = 1d
t1/2 = 14d R0 =1.4
StopART
14d
Slide 24 of 54
Limit ofdetection
R0 = 8 -10
Setpoint
ART
t1/2 = 1d
t1/2 = 14dR0 = 1.4
StopART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Time (months)
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
HIV replication dynamics
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Slide 25 of 54
ART
StopART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
Davey et al, PNAS 1999
Rebound dynamics
1
Time after interruption of ART (months)
0 21 43 5 6 7-1
Slide 26 of 54
ART
StopART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
Rebound dynamics
Rothenberger et al, PNAS 2014Hill et al, PNAS 2014
1
Time after interruption of ART (months)
0 21 43 5 6 7-1
• Variation in rebound time << variation in reservoir size
• Multiple cells reactivate per day
• Exponential• Rebound in ~2 wks
• Long delays when <1 cell reactivates per day
Slide 27 of 54
ART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
Drugwashout
Appearance ofproductively infected cells
Exponentialgrowth
Rebound dynamics
1
Time after interruption of ART (months)
0 21 43 5 6 7-1
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Slide 28 of 54
Bonsignori et al, Imm Rev 2017
Broadly neutralizing antibodies
• Block infection and target infected cells
• Neutralize diverse HIV isolates
• Arise slowly generally after escape
• Unusual structures make them difficult to induce with vaccines
• Can be administered passively as Ab infusion or with AAV vectors
• Role in HIV cure
Slide 29 of 54
ART
StopART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
Slight delay with bNAb infusionVRC01
1
Time after interruption of ART (months)
0 21 43 5 6 7-1
Bar et al, NEJM 2016Scheid et al Nature 2016
Slide 30 of 54
StopART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Pla
sma
SIV
RN
A (
copi
es/m
l)
Borducchi et al, Nature 2016
Time after interruption of ART (months)
0 21 43 5 6 7
Reservoir reduction vs immune control
-1
Ad26/MVA + TLR7a
ShamAd26/MVA
TLR7a
Reservoirreduction
Immunecontrol
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Slide 31 of 54
ART
StopART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Byrareddy et al, Science 2016
α4β7 antibody
α4β7 antibody
Control antibody
Pla
sma
SIV
RN
A (
copi
es/m
l)
1
Time after interruption of ART (months)
0 21 43 5 6 7-1
Reservoir reduction vs immune control
Slide 32 of 54
ART
StopART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
Rothenberger et al, PNAS 2014
Time after interruption of ART (months)
0 21 43 5 6 7-1
Reservoir reduction vs immune control
Slide 33 of 54
ART
StopART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
Boston patients
Mississippibaby
0 2412
Patientson ART
Henrich et al, AIM 2014Persaud et al, NEJM 2015
Time after interruption of ART (months)
Reservoir reduction vs immune control
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ART
StopART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
?
Reservoir reduction vs immune control
• In patients with starting treatment after the acute phase, control may be much more difficult to achieve due to:
• Immune exhaustion
• Post-Rx control has been observed with early ART
• High viral diversity
• Escape mutations
0 2412
Time after interruption of ART (months)
Slide 35 of 54
0
50
100
WF9 SL9 TV9 TL9 HA9 PY9 VI9 FK10
0
50
100
GK9 EV9 SL9 TV9 EI8GLY9 DL9 FK10
0
50
100
KK9 RK9 SV9 TL9 HA9 GL9
0
50
100
KK9 RK9 LY9 SV9 TL9 HA9 GL9
0
50
100
RY11 VL8 TW10 YL9 QW9
0
50
100
LY9 IW9 KF11 TW10 QW9
Fre
qu
ency
of
vari
ants
(%
)
CTL epitopes in HIV-1 Gag
Acute Pt10A*02:01
Chronic Pt 18A*02:01
Acute Pt 12A*03:01
Chronic Pt 39A*03:01
Acute Pt07B*58:01
Chronic Pt12B*57:01
Documented Escape Diminished Response Mutation Type Not Determined
CTL escape variants dominate in the latent reservoir of chronic patients
Deng et al, Nature, 2015
Slide 36 of 54
ART
StopART
1
10
100
1000
10,000
100,000
1,000,000
10,000,000
Pla
sma
HI V
-1 R
NA
(cop
es/m
l)
Time after interruption of ART (years)0 21
Reservoir reduction vs immune control
Immunecontrol
Reservoirreduction
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Slide 37 of 54
Time to rebound
Hill et al, PNAS 2014
Time to rebound
Log
redu
ctio
n in
late
nt r
eser
voir
1 wk 1 mo 10 yr1 yr3 mo Lifetime
6
3
1
2
5
4
Berlin pt.
Bostonpt. B
Bostonpt. A
Chun et al.
Miss.baby
Slide 38 of 54
Adaptive probability of cure
Hill et al. PLoS Pathogens 2016
Slide 39 of 54
0.001
0.01
0.1
1
10
100
1000
10000
100000
1000000
0 100 200 300
Time on ART (days)
Assay for plasma HIV RNA sped development of ART
Start ART
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Slide 40 of 54
Quantitative viral outgrowthassay
PHA + irradiatedallogeneic PBMC
p24Ag
180-200ml blood
Purified restingCD4+ T cells
d2: add CD4+
lymphoblastsfrom HIV-donors
d7: add CD4+
lymphoblastsfrom HIV-donors
Finzi et al., Science, 1997
1/1,000,000
Slide 41 of 54
Infe
cted
cel
l fre
que
ncy
(per
106 )
Viraloutgrowth
10
100
1,000
0.1
Resting CD4 PBMC Resting CD4Cell/tissue
AssayPBMC Resting CD4 Rectal CD4
Chronic AcuteChronic Chronic AcuteAcuteChronic AcuteCohort Chronic AcuteAcute
Plasma
Chronic
1
10,000
0.1
10
100
1,000
1
10,000
Plasm
as HIV RNA (co
pies/m
l)Viral outgrowth vs PCR assays
Integrated HIV DNATotal HIV
DNAResidualviremia
PBMC
2 LTRcircles
Chronic Acute Chronic Acute
Total HIV DNA
r = 0.38p = 0.28
r = 0.70p < 0.01
r = 0.41p = 0.13
r = 0.05p = 0.86
rho = 0.19p = 0.31
rho = 0.07p = 0.71300x
Eriksson et al, PLOS Pathogens, 2013
Slide 42 of 54
Non-induced proviruses
PHA + irradiatedallogeneic PBMC Are they inducible?
full length, single genome analysis
Ho et al, Cell, 2013Bruner et al, Nature Med 2016
RestingCD4+ T cells
p24Ag
d2: add CD4+
lymphoblastsfrom HIV-donors
d7: add CD4+
lymphoblastsfrom HIV-donors
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Slide 43 of 54
LTR gag
pol
vif
vpr
vpu nef
tat
rev
LTR
gag env
Bruner et al, Nature Med 2016
Landscape of HIV proviruses
Slide 44 of 54
Landscape of HIV proviruses
Packaging signal deletion
Intact
Hypermutated
Deletion at 3’ endof genome
Very large internal deletion
Deletion at 5’ endof genome
Hypermutatedand deleted
Key:
ART during chronic infection2% 5%
21%
36%
21%
7% 8%
ART during acute infection
5%2%
22%
26%8%
19%
18%
Bruner et al, Nature Med 2016
Slide 45 of 54
Intact
QVOA, intact, and total proviruses
•We need a scalable assay for intact proviruses to guide clinical trials of cure strategies
QVOA ddgag Total
•The number of intact proviruses provides a much more accurate upper limit on reservoir size than standard DNA PCR assays
Infe
cted
cel
l fre
quen
cy(p
er 1
06)
Ho et al Cell, 2013Bruner et al, Nat Med, 2016Hosmane et al, submitted
Defective
•Each round of stimulation induces additional proviruses
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Slide 46 of 54
Clonal expansion detected by integration site analysis
Maldarelli et al, Science, 2014 Wagner et al, Science, 2014
Slide 47 of 54
HXB2
0.02
P04
HXB2
0.02
P03
HXB20.02
P02
HXB20.02
P01
P06
HXB20.02
0.02
HXB2
P07
P08
0.02
HXB2
0.02HXB2
P09
P10
0.02HXB2
0.02
P11
HXB2
P12
0.02
HXB2
0.02
HXB2
P05
Independent isolates of replication-competent HIV with identical sequence
Stimulation1234
Slide 48 of 54
HXB2
0.02
P04
HXB2
0.02
P03
HXB20.02
P02
HXB20.02
P01
P06
HXB20.02
0.02
HXB2
P07
P08
0.02
HXB2
0.02HXB2
P09
P10
0.02HXB2
0.02
P11
HXB2
P12
0.02
HXB2
0.02
HXB2
P05
Expanded cellular clones accountfor the majority of the reservoir
57%
• Antigen• Cytokines• Integration site effects
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Slide 49 of 54
10000
The reservoir does not increaseover time in patients on ART
-
Half‐life: 44 monthsTime to eradication: > 73.4 years
0.0001
0.001
0.01
0.1
1
10
100
1000
0 1 2 3 4 5 6 7Time on ART (years)
Fre
quen
cy(p
er 1
06ce
lls)
0.00001
Finzi et al., Nature Med., 1999Siliciano et al., Nature Med., 2003
Slide 50 of 54
Jennifer KwonKatie Bruner
Thanks
Ya‐Chi Ho
Nina Hosmane
Janet SilicianoJanet Siliciano
Alex MurrayAlison Hill and Daniel Rosenbloom
Slide 51 of 54
CollaboratorsSteve DeeksJohn MellorsDoug RichmanDan BarouchBrad JonesRichard FlavellDave MargolisJoel GallantJoe Cofrancesco
Matt StrainSarah PalmerUna O’DohertyJoe WongSteve Yukl
FundingNIH: Martin Delaney CollaboratoriesHoward Hughes Medical InstituteJohns Hopkins Center for AIDSResearch
Bill and Melinda Gates FoundationGilead
Thanks
Atlanta, Georgia: March 30, 2017
3/31/2017
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Robert F. Siliciano, MD, PhDProfessor of Medicine
The Johns Hopkins UniversityBaltimore, Maryland
New Technologies Applied to an HIV Cure
FORMATTED: 03/20/17
Atlanta, Georgia: March 30, 2017
Atlanta, Georgia: March 30, 2017
