barriers to hiv cure - ias-usa · 5/2/2016 1 janet m. siliciano, phd associate professor of...
TRANSCRIPT
5/2/2016
1
Janet M. Siliciano, PhDAssociate Professor of Medicine
Johns Hopkins University School of Medicine
Baltimore, Maryland
Barriers to HIV Cure
FINAL: 04-08-16
Washington, DC: April 15, 2016
Slide 2 of 54
Financial Relationships With Commercial Entities
Dr Siliciano has no relevant financial affiliations
to disclose. (Updated 04/14/16)
Slide 3 of 54
Learning Objectives
After attending this presentation, participants will
be able to:
Describe how the latent reservoir for HIV arises
List current approaches to curing HIV infection
Describe how these approaches will be evaluated
clinically
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Slide 4 of 54
How does early ART affect likelihood of cure?
13%
18%
69% 1. Smaller latent reservoir
2. More rapid reservoir decay
3. No effect
Slide 5 of 54
How is the latent reservoir best measured?
3%
18%
27%
52% 1. Viral outgrowth assay
2. DNA PCR assays
3. Plasma HIV RNA
4. Western blot
Slide 6 of 54
0.001
0.01
0.1
1
10
100
1000
10000
100000
1000000
0 100 200 300
Time on ART (d)
Viral dynamics in patients on ART
Start
ART
†
v +
Limit of Detection
(50 copies/ml)
t1/2 = 1 day
t1/2 = 14 days
Activated CD4+ T cells
Eradication in
2-3 years
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Slide 7 of 54
Physiology of resting andactivated CD4+ T cells
Slide 8 of 54
Establishment of immunologic memory
Ag
††††
†
†
Ag
††††
†
†
Slide 9 of 54
†
HIV infection of activated and resting CD4+ T cells
Ag
†
HIV
AgHIV
HIV
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Slide 10 of 54
Establishment of the latentreservoir in resting CD4+ T cells
Ag
††††
†
†
HIV
Slide 11 of 54
U 3 R U 5
Modulatory region
Enhancer
Core
Cell DNA
AP1 NFAT1 USF1 Ets1 LEF NFBNFAT
Sp1 TBP LBP1
NFκB sites in the HIV LTR
Nabel G, et al. Nature. 1987;326:711-713.Tong-Starksen SE, et al. PNAS. 1987;84:6845:6849.Bohnlein E, et al. Cell. 1988;53:827-836.Duh EJ, et al. PNAS. 1989;86:5974-5978.
Slide 12 of 54
Reactivation of latent HIV
Ag
††††
†
†
HIV
Ag
†
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Slide 13 of 54
An assay for latently infected cells
PHA + irradiatedallogeneic PBMC
p24
Ag
180-200ml blood
Purified restingCD4+ T cells
d2: add CD4+
lymphoblasts from HIV-donors
d7: add CD4+
lymphoblasts from HIV-donors
Chun et al., Nature, 1997Finzi et al., Science, 1997
1/1,000,000
Slide 14 of 54
10000
Slow decay of latently infected
CD4+ 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)
Freq
uen
cy(p
er 1
06
cells
)
0.00001
Finzi et al., Nature Med., 1999Siliciano et al., Nature Med., 2003
Slide 15 of 54
Slow decay of the reservoir
Siliciano et al., Nature Med., 2003
Crook et al, JID 2015
t1/2 = 43 months
t1/2 = 44 months
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Slide 16 of 54
Ag
Deng et al., submitted
Latency results from infection of
memory precursor cells
Slide 17 of 54
0.001
0.01
0.1
1
10
100
1000
10000
100000
1000000
0 100 200
Time on HAART (days/years)
Limit of
Detection
(50 copies/ml)
Start Therapy
years200
Hermankova et al, JAMA, 2001
Persaud et al, J Virol, 2003
Kieffer et al, J Infect Dis, 2004
Nettles et al, JAMA, 2005
Bailey et al, J Virol, 2006
Brennan et al, J Virol, 2009
•Sensitive to current regimen
•Archival
•Non-evolving
Residual viremia
Ag
HAART
1 copy/ml
Slide 18 of 54
0.001
0.01
0.1
1
10
100
1000
10000
100000
1000000
0 100 200
Time on HAART (days/years)
Limit of
Detection
(50 copies/ml)
Start Therapy
years200
Residual viremia
Ag
HAART
1 copy/ml
Dinoso et al, PNAS, 2009
Many later raltegravir
intensification studies
•Residual viremia cannot be reduced
by treatment intensification
Add 4th drug
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Slide 19 of 54
The first cure•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 20 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
Matched
allogeneic
HSCT
“Boston Patient B”
Stop
ART
Below limitof detection
Slide 21 of 54
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
AZT3TC
LPV/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
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Slide 22 of 54
Approaches to HIV cure
Gene Rx
Prevent reactivation
†
Induce elite control
TCR pathway agonists
LRAs(HDACi) †
†
†
Shock and kill
Slide 23 of 54
Other approaches to HIV cure
Gene Editing Strategies used in Cure Research: target integrated provirus with engineered
nucleases (ZFN,TALENS,) or CRISPR/Cas9
Problems
• No way to deliver enzymes into every infected cell in vivo
• Off target effects
xx
Slide 24 of 54
Other approaches to HIV cure
Gene Rx – ZFN targeting CCR5 gene in patient CD4+ T cells or HSC. Reinfuse
engineered, HIV-resistant cells back into patients
Problems
HIV can still replicate in non-engineered cells. (In Berlin patient, CCR5+ host cells
eliminated by conditioning regimen and graft vs. host effects)
xx
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Slide 25 of 54
Fundamental approach to HIV cure
TCR pathway agonists
LRAs(HDACi) †
†
†
•How do we measure the
reservoir in eradication trials?
•How do we identify latency
reversing agents?
•Will cells be eliminated following
reversal of latency?
Slide 26 of 54
Current status of LRA trials
TCR pathway agonists
LRAs(HDACi)
†
†
†
• Numerous LRAs identified in studies
with transformed cell lines and
primary T cell model systems
• Few shown to work ex vivo with cells
from patients
• In clinical trials, no reduction in the
reservoir yet demonstrated
• Some evidence for slight transient
increases in plasma HIV RNA after
LRA treatment (romidepsin,
panobinostat, TLR7 agonist)
• In clinical trials, evidence for
increases in cell-associated HIV RNA
(Archin et al.)
Slide 27 of 54
Assay for reversal of latency using patient resting CD4+ T cells
500 x106 restingCD4+ T cells
+Test compound
Bullen et al., Nature Med, 2014
Measure intracellular HIV RNAand virion release
5 x 106 cells/wellShan et al, J Virol, 2013Laird et al, PLOS Pathogens, 2013
LRA
TCRagonist
Positive control
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10
Slide 28 of 54
Induction of HIV RNAs by LRAsDM
SO C
ontro
lVo
rinos
tat
Rom
idep
sinPa
nobi
nostat
Disu
lfira
m JQ1
Bryo
stat
in-1
PMA
+ Iono
myc
in
•Total RNA
•polyA primers
•18 hrs
Bullen et al, Nat Med 2014
Slide 29 of 54
Induction of HIV RNAs by combinations of LRAs
% o
f P
MA
/io
no
myc
in
+ Bryostatin-1 + Disulfiram
Disram
JQ1
Pano
bino
stat
Rom
idep
sin
Vorin
ostat
Bryo
stat
in-1
Disram
JQ1
Pano
bino
stat
Rom
idep
sin
Vorin
osta
t
Pano
bino
stat
Romid
epsin
Vorin
osta
t
Pros
tratin
Rom
idep
sin
0
20
40
60
80
100
Perc
ent P
MA
+ io
nom
ycin
Single LRA + Bryostatin-1 + Prostratin
Disram
JQ1
Pano
bino
stat
Rom
idep
sin
Vorin
ostat
Bryo
stat
in-1
Disram
JQ1
Pano
bino
stat
Rom
idep
sin
Vorin
osta
t
Pano
bino
stat
Romid
epsin
Vorin
osta
t
Pros
tratin
Rom
idep
sin
0
20
40
60
80
100
Perc
ent P
MA
+ io
nom
ycin
Single LRA + Bryostatin-1 + Prostratin
Single LRA
Laird et al, in preparation
Slide 30 of 54
Fundamental approach to HIV cure
TCR pathway agonists
LRAs(HDACi) †
†
†
•How do we measure the
reservoir in eradication trials?
•How do we identify latency
reversing agents?
•Will cells be eliminated
following reversal of latency?
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Slide 31 of 54
Fate of infected CD4 cells after
latency reversal in vivo is unknown
HDACi
αCD3+αCD28
64 7Days after reactivation
32 5
120
100
60
80
40
20
00
Res
idu
al G
FP+
cells
(%)
TCR pathway agonists
HDACi
†
Shan et al, Immunity, 2012
Slide 32 of 54
0
20
40
60
80
100
Surv
ivin
g in
fecte
d c
ells
(%
)
Elite suppressor 1
Elite suppressor 2
Elite suppressor 3
Time of coculture (days)
0 2 4 6 8
Shan et al, Immunity, 2012
CTL killing of latently infected
cells treated with SAHANormal donor 1
Normal donor 2
Normal donor 3
E:T = 1:1
Slide 33 of 54
0
20
40
60
80
100
Surv
ivin
g in
fecte
d c
ells
(%
)
Elite suppressor 1
Elite suppressor 2
Elite suppressor 3
Time of coculture (days)
0 2 4 6 8
Shan et al, Immunity, 2012
CTL killing of latently infected
cells treated with SAHANormal donor 1
Normal donor 2
Normal donor 3
HAART patient 1
HAART patient 2
HAART patient 3
HAART patient 4
HAART patient 5
HAART patient 6
E:T = 1:1
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Slide 34 of 54
0
50
100
WF9 SL9 TV9 TL9 HA9 PY9 VI9 FK10
0
50
100
GK9 EV9SL9 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
en
cy
of
vari
an
ts (
%)
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 MutationType Not Determined
CTL escape variants dominate in the
latent reservoir of chronic patients
Deng et al, Nature, 2015
Slide 35 of 54
Fundamental approach to HIV cure
TCR pathway agonists
LRAs(HDACi) †
†
†
•How do we measure the
reservoir in eradication trials?
•How do we identify latency
reversing agents?
•Will cells be eliminated following
reversal of latency?
Slide 36 of 54
An assay for latently infected cells
PHA + irradiatedallogeneic PBMC
p24
Ag
180-200ml blood
Purified restingCD4+ T cells
d2: add CD4+
lymphoblasts from HIV-donors
d7: add CD4+
lymphoblasts from HIV-donors
Chun et al., Nature, 1997Finzi et al., Science, 1997
1/1,000,000
5/2/2016
13
Slide 37 of 54
Assays for latent HIV
TCR
agonist•Viral outgrowth
assay (VOA)
•DNA PCR PCR for proviral DNA
Measure intracellular
HIV RNA•Induction of HIV
RNA
TCR
agonist
Measure virion release•Induction of virion
production
TCR
agonist
Slide 38 of 54
Infe
cte
d c
ell
frequency
(per
10
6)
Viral
outgrowth
10
100
1,000
0.1
Resting CD4 PBMC Resting CD4Cell/tissue
Assay
PBMC 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
RN
A (co
pies/m
l)
Viral outgrowth vs PCR assays
Integrated HIV DNA
Total HIV
DNA
Residual
viremiaPBMC
2 LTR
circles
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 39 of 54
Non-induced proviruses
PHA + irradiated
allogeneic PBMC
Non-induced
proviruses
Are they inducible?
full length, single genome analysis
Ho et al, Cell, 2013
Resting
CD4+ T cells
p24
Ag
d2: add CD4+
lymphoblasts
from HIV-
donors
d7: add CD4+
lymphoblasts
from HIV-
donors
5/2/2016
14
Slide 40 of 54
Non-induced proviral clones (n=213)
N
NH
O
O
N
N
NH2
O
TGG TAG
Trp Stop
Hypermutated 32.4%
Ho et al, Cell, 2013
Slide 41 of 54
10 20 30 40 50 60 70 80 90 100....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEA
9CC3_31E5_gag_hypermut I.....I..E.....*.................L...G................A....................K..F....V........DV......
9CC3_31E11_gag_hypermut IS....I..R.....*...Q...K.........L.*.GK.........S.....A......R........R....K..F....V..Y...K.DV......
20CB4_36D12_gag_hypermut I..........Q...*.R.......N.R.........................AG......E....A.K......K..F...........K.DV......
20TB1_33C3_gag_hypermut I..........Q...*.R.......N.R.........................AG......E....A.K......K..F...........K.DV......
20TB1_33C9_gag_hypermut I..........Q...*.R.......N.R.........................AG......E....A.K......K..F...........K.DV......
20TB3_33G10_gag_hypermut I........R.Q...*.......E.N.R..........K..............AG......E....A.K......K..F...........K.DV......
110 120 130 140 150 160 170 180 190 200....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 LDKIEEEQNKSKKKAQQAAADTGH--SNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAM
9CC3_31E5_gag_hypermut ............N..........NSS.S...................SL...........I................................R......
9CC3_31E11_gag_hypermut ....K.......N.........RNSS.S...............I...SL..K....*...I.K................K.............R......
20CB4_36D12_gag_hypermut .K.....................N--.S............................*...........................................
20TB1_33C3_gag_hypermut .K.....................N--.S............................*...........................................
20TB1_33C9_gag_hypermut .K.....................N--.S............................*...........................................
20TB3_33G10_gag_hypermut .E.....................N--.S............................*.....................................R.....
210 220 230 240 250 260 270 280 290 300....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 QMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVD
9CC3_31E5_gag_hypermut .............*..L....................................H...V........*.........K....V.....K.R..........
9CC3_31E11_gag_hypermut .............*..L.....R.......K...K....R.........R*..H...V..RK....*..............V.....K.K..K.....IN
20CB4_36D12_gag_hypermut .............*....................................*.........................K.......................
20TB1_33C3_gag_hypermut .............*....................................*.........................K.......................
20TB1_33C9_gag_hypermut .............*....................................*.........................K.......................
20TB3_33G10_gag_hypermut .............*..L.............K...................*.........R.........R................K.......K....
310 320 330 340 350 360 370 380 390 400....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 RFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKE
9CC3_31E5_gag_hypermut ..........CT.D...*.........S.........................R.R.............C.M............P.....T.........
9CC3_31E11_gag_hypermut Q.........CT.D...*.........S..N........R.............K.RR.....K......C.M.........K..PK..K.T.........
20CB4_36D12_gag_hypermut Q................*.........S...................................I...........NA.............PI........
20TB1_33C3_gag_hypermut Q................*.........S...................................I...........NA.............PI........
20TB1_33C9_gag_hypermut Q................*.........S...................................I...........NA.............PI........
20TB3_33G10_gag_hypermut .................*.........S...........R.............R.RR......I..........AN.........K....P.........
410 420 430 440 450 460 470 480 490 500....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 GHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPPEESFRSGVET--------TTPPQKQEPI----DKELYPL
9CC3_31E5_gag_hypermut ..I.........R..*..R..........K......R.....H....E.............A....F.E..--------.........V----...M...
9CC3_31E11_gag_hypermut R.I....K..K..S.*..R.KR..I....K......K.....H....E.............A....FKE..--------.........V----...M...
20CB4_36D12_gag_hypermut E...K.......R..*..R.......N.........R.....H....E.............A....F.E..TTPSQKQEP..S....L.DLDK.......
20TB1_33C3_gag_hypermut E...K.......R..*..R.......N.........R.....H....E.............A....F.E..TTPSQKQEP..S....L.DLDK.......
20TB1_33C9_gag_hypermut E...K.......R..*..R.......N.........R.....H....E.............A....F.E..TTPSQKQEP..S....L.DLDK..GQ---
20TB3_33G10_gag_hypermut R.V..H.K..K.R..*..R.......N..K......K.....H....K..................FKE..--------...S....L.DLDK.......
510....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 TSLRSLFGNDPSSQ*
9CC3_31E5_gag_hypermut A..K..........*
9CC3_31E11_gag_hypermut A..K...S......*
20CB4_36D12_gag_hypermut A............K*
20TB1_33C3_gag_hypermut A............K*
20TB1_33C9_gag_hypermut ---..........K*
20TB3_33G10_gag_hypermut A............K*
32.4% of non-induced proviruses
have lethal GA hypermutation10 20 30 40 50 60 70 80 90 100....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATA
9CC3_31E5_gag_hypermut ..A...............A.........A.............A...A................C.......A..................C........T
9CC3_31E11_gag_hypermut ..AA..............A........A.A............A...AA..........A....C.....AAA..................C........T
20CB4_36D12_gag_hypermut ..A..............................C........A...A.....G..................A.....C....GG................
20TB1_33C3_gag_hypermut ..A..............................C........A...A.....G..................A.....C....GG................
20TB1_33C9_gag_hypermut ..A..............................C........A...A.....G..................A.....C....GG................
20TB3_33G10_gag_hypermut ..A........................A.A...C........A...A.......................AA.....C....GG................
110 120 130 140 150 160 170 180 190 200....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 TAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATC
9CC3_31E5_gag_hypermut ...........G.......A...........T.................A.............CT.................A..G........G.....
9CC3_31E11_gag_hypermut ......A....G...AA..A...........T............A....A.............CT.................AA.G........G.....
20CB4_36D12_gag_hypermut .................................................A.........G...G....................A.............G.
20TB1_33C3_gag_hypermut .................................................A.........G...G....................A.............G.
20TB1_33C9_gag_hypermut .................................................A.........G...G....................A.............G.
20TB3_33G10_gag_hypermut ...............A.................................A.........G...G..................A.A.............G.
210 220 230 240 250 260 270 280 290 300....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 CCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCT
9CC3_31E5_gag_hypermut ...A......................A........T.............GT...........................TG.............A......
9CC3_31E11_gag_hypermut ...A......A...............A........T.............GT........A...........A......TG.............A......
20CB4_36D12_gag_hypermut ....A.....................A........T...........................A.......A......TG.G...........A..G...
20TB1_33C3_gag_hypermut ....A.....................A........T...........................A.......A......TG.G...........A..G...
20TB1_33C9_gag_hypermut ....A.....................A........T...........................A.......A......TG.G...........A..G...
20TB3_33G10_gag_hypermut ....A.....................A........T...........................A.......A......TG.G...........A......
310 320 330 340 350 360 370 380 390 400....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 TTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACAC------AGCAATCAGGTCAGCCAAAATT
9CC3_31E5_gag_hypermut ..............A.......................C.....T........G...............A..AGCAGT....GC..A.............
9CC3_31E11_gag_hypermut ............A.........................C.....T........G............A..A..AGCAGT....GC..A.............
20CB4_36D12_gag_hypermut ...A.G...............................................................A..------....GC................
20TB1_33C3_gag_hypermut ...A.G...............................................................A..------....GC................
20TB1_33C9_gag_hypermut ...A.G...............................................................A..------....GC................
20TB3_33G10_gag_hypermut .....G...............................................................A..------....GC................
410 420 430 440 450 460 470 480 490 500....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 ACCCTATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAG
9CC3_31E5_gag_hypermut .........................A..G............T..T...................................A...................
9CC3_31E11_gag_hypermut ......................A..A..G..A.........T..T.........A..............A..........A.....A.............
20CB4_36D12_gag_hypermut ........................................A............................A..................A...........
20TB1_33C3_gag_hypermut ........................................A............................A..................A...........
20TB1_33C9_gag_hypermut ........................................A............................A..................A...........
20TB3_33G10_gag_hypermut ........................................A............................A..................A...........
510 520 530 540 550 560 570 580 590 600....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 CCCAGAAGTGATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATG
9CC3_31E5_gag_hypermut .........A..............................................................T.....AA.A..............T...
9CC3_31E11_gag_hypermut .........A...........................AA.................................T.....AA.A..............T...
20CB4_36D12_gag_hypermut .........A.......................................................................A..................
20TB1_33C3_gag_hypermut .........A.......................................................................A..................
20TB1_33C9_gag_hypermut .........A.......................................................................A..................
20TB3_33G10_gag_hypermut .........A....................................................................A..AA.................
610 620 630 640 650 660 670 680 690 700
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 CAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAA
9CC3_31E5_gag_hypermut ........................................AA......T...................................................
9CC3_31E11_gag_hypermut ..........................A.............AA......T.................A........................A........
20CB4_36D12_gag_hypermut ..G.....................................AA..........................................................
20TB1_33C3_gag_hypermut ..G.....................................AA..........................................................
20TB1_33C9_gag_hypermut ..G.....................................AA..........................................................
20TB3_33G10_gag_hypermut ..G.......................A.............A.......T..........................................A........
710 720 730 740 750 760 770 780 790 800
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
B.FR.83.HXB2_LAI_IIIB_BRU_K034 GGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAAATTTATAAAAGATG
9CC3_31E5_gag_hypermut .A...................................A.....................C...........G.............G..C..........A
9CC3_31E11_gag_hypermut .AAA.............A...................A.........A...AA......C...........G........A..A.G..C..........A
20CB4_36D12_gag_hypermut .A.................................................A....................................C...........
20TB1_33C3_gag_hypermut .A.................................................A....................................C...........
20TB1_33C9_gag_hypermut .A.................................................A....................................C...........
20TB3_33G10_gag_hypermut .A...................................A.............A............................A.......C...........
ATG ATA
MI
start codon mutation
TGG TAA, TAG, TGA
Tryptophan stop codon
nonsense mutation
Ho et al, Cell, 2013
HXB2
Pt 09 clone 31E05
Pt 09 clone 31E11
Pt 20 clone 36D12
Pt 20 clone 33C03
Pt 20 clone 33C09
Pt 20 clone 33G10
HXB2
Pt 09 clone 31E05
Pt 09 clone 31E11
Pt 20 clone 36D12
Pt 20 clone 33C03
Pt 20 clone 33C09
Pt 20 clone 33G10
Slide 42 of 54
Ho et al, Cell, 2013
45.5% of non-induced proviruses
have large internal deletions
N
NH
O
O
N
N
NH2
O
TGG TAG
Trp Stop
Hypermutated 32.4%
Large internaldeletion45.5%
5/2/2016
15
Slide 43 of 54
LTR gag
pol
vif
vpr
vpu nef
tat
rev
LTR
gag env
Pt. KB7
Pt. KB6
Pt. KB5
Pt. KB3
Pt. KB8
Deletions and hypermutation
Bruner et al, submitted
Slide 44 of 54
Non-induced proviral clones (n=213)11.7% Intact
genome
Deletion in ψ/MSD site 6.5%
Nonsense mutations/INDELS 3.8%
Hypermutated 32.4%
Large internaldeletion45.5%
Ho et al, Cell, 2013
N
NH
O
O
N
N
NH2
O
TGG TAG
Trp Stop
Non-induced proviruses
Slide 45 of 54
Replication of intact
non-induced viruses
Patient 16
0.01
0.1
1
10
100
1000
0 3 5
Patient 17
1 70.01
0.1
1
10
100
1000
Patient 10
0.01
0.1
1
10
100
1000
0 3 5
Time post infection (days)
Patient 20
1 70.01
0.1
1
10
100
1000
p2
4 (
ng
/ml) NL4-3
Rep-Comp
Intact non-induced
Replication capacity of intact non-
induced proviruses
5/2/2016
16
Slide 46 of 54
Size of latent reservoir
VOA
Intact
HIV DNA
Scale=100/106
62 fold
Ho et al, Cell, 2013
Slide 47 of 54
180-200ml blood
Purified restingCD4+ T cells
Can intact non-induced proviruses
be induced?
PHA + irradiated
allogeneic PBMC
Recover cells from
negative wells
p24
Ag
d2: add CD4+
lymphoblasts from HIV-donors
d7: add CD4+
lymphoblasts from HIV-donors
Ho et al, Cell, 2013
Slide 48 of 54
Can intact non-induced proviruses
be induced?
Ho et al, Cell, 2013
Nina Hosmane
47% 53%
PHA+ allo PBMC+ -
39% 61%
PHA+ allo PBMC+ -
39% 61%
PHA+ allo PBMC+ -
Resting CD4+ T cells
5/2/2016
17
Slide 49 of 54
Take home points
• DNA PCR assays widely used for reservoir analysis
mainly defect grossly defective proviruses
• The quantitative viral outgrowth assay remains the
best available assay for the latent reservoir, but better
assays are urgently needed.
• There is no clinical assay for the latent reservoir
• Other approaches: transient blips following LRA
administration, time to rebound after ART
interruption
Slide 50 of 54
Predicting time to rebound after reservoir reductions
Hill et al, PNAS 2014
Slide 51 of 54
Time to rebound
Hill et al, PNAS 2014
Time to rebound
Log
red
uct
ion
inla
ten
t re
serv
oir
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
5/2/2016
18
Slide 52 of 54
Time Post Infection
(weeks) (years)
1,000,000
100,000
10,000
1000
100Pla
sm
a H
IV R
NA
(c
op
ies
/ml)
cART
Therapeutic vaccination
cLRAs
What will cure look like?
Slide 53 of 54
Ya-Chi Ho Korin Bullen
Greg LairdLiang Shan Kai Deng
Robert Siliciano
Slide 54 of 54
CollaboratorsSteve DeeksRichard FlavellDave MargolisJoel GallantJoe CofrancescoDoug RichmanMartin Nowak
Matt StrainSarah PalmerUna O’DohertySteve YuklJohn Mellors
FundingNIH: Martin Delaney Collaboratories
CARE and DAREHoward Hughes Medical InstituteFoundation for AIDS Research
(amfAR): ARCHEJohns Hopkins Center for AIDS
ResearchBill and Melinda Gates Foundation
Thanks