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Development of aptamer based HIV-1
entry inhibitor prophylactic drugs
Grace LondonCSIR, Biosciences
Emerging Health Technologies Platform
Pretoria, South AfricaPretoria, South Africa
2nd International Conference and Exhibition on Pathology
Properties of Aptamers
� Aptamers areantibodies
nucleic acids with properties of
� simple in vitro process called SELEXGenerated by
� High affinity and specificity� High affinity and specificity
� Small in size and fold in 3 –D structure (e.g RNAaptamers)
� Resistant to nucleases and chemically stableJoubert et al ., 2010
� Low toxicity and non(immunogenic
Applications of aptamers
Marro et al ., 2005 Rotherham et al ., 2012
Rusconi et al ., 2004
Green et al ., 2001
Appl. Microbiol. Biotechnol., 2005, Nov 11, 1-8
Anti-gp120 aptamers as HIV-1 entry inhibitors
Apt 2Apt 1
Apt 2 bind recombinant gp120Apt 1 bind gp120 trimer
Apt = aptamerKhati et al ., 2003; Cohen et al., 2008
HIV-1 entry and inhibitors
HIVHIV
HIV
gp120Co-receptor binding
gp41gp120- CD4 receptor binding
gp41gp120- CD4 receptorbinding
gp120
CD4
CCR5CXCR4 gp41 Mediated
Fusion
Host Cell
Pierson and Doms ., Current Top Microbiol Immunol (2003) 281 pp 1-27
Outline of the study
1. Evaluate efficacy of anti-gp120aptamer
subtype C
against
HIV-1
2. Testtoxicitytoxicity
3. Map “aptatope’s ”on
HIV-1gp120
4. Test
synergy with other entryinhibitors
AptamersC
inhibit entry of HIV-1 subtype
Env pseudoviruses
�
�
�
50 nM = No inhibitionNT = Not titred
* viruses using CXCR4 coreceptor
Aptamer IC50
Env clone Stages of disease Geographic location Apt 1 Apt 2
CAP45.2.00.G3 Acute/early S.Africa 2.6 0.3
ZM233M.PB6 Acute/early Zambia 4.9 0.1
ZM249M.PL1 Acute/early Zambia >50 0.6
ZM53M.PB12 Acute/early Zambia 20.1 0.8
ZM109F.PB4 Acute/early Zambia 14.1 >50
ZM197M.PB7 Acute/early Zambia 19.2 0.4
CAP210.200.E8 Acute/early S.Africa 2.2 0.1
ZM135M.PL10a Acute/early Zambia 2.8 NT
ZM214M.PL5 Acute/early Zambia 17.6 0.2
DU172.17 Acute/early S.Africa 12.1 0.6
DU156.12 Acute/early S.Africa 3.3 0.6
DU422.1 Acute/early S.Africa >50 0.3
CAP08.2.00.F6 Acute/early S.Africa 1.2 1
� Apt 1 = inhibited 84 % viruses� Mean IC50 6.6 ± 8.1 nM
� Apt 2 = inhibited 79 % viruses� Mean IC50 0.4 ± 0.3 nM
CAP08.2.00.F6 Acute/early S.Africa 1.2 1
CAP61.2.00.F10 Acute/early S.Africa 0.3 0.4
CAP63.2.00.A9J Acute/early S.Africa 1.7 0.1
CAP84.2.00.32J Acute/early S.Africa 29.1 >50
CAP85.2.00.09J Acute/early S.Africa 16.1 0.1
CAP239.2.00.G3J Acute/early S.Africa 7.1 0.2
RP1.12* Acute/early S.Africa 4.7 0.6
RP4.3 Acute/early S.Africa >50 0.5
COT6.15 Chronic S.Africa 0.9 >50
COT9.6 Chronic S.Africa 6.1 >50
DU151.2 Acute/early S.Africa >50 >50
DU123.6 Acute/early S.Africa >50 >50
Conc Acute/early S.Africa 5 0.1
CAP288.2.00.5 Acute/early S.Africa 9.5 0.3
CAP206.2.00.E8 Acute/early S.Africa 3.8 0.7
CAP244.2.00.D3 Acute/early S.Africa 2.3 0.4
RP6.6 Acute/early S.Africa 3.7 0.4
CAP88.2.00.B6J Acute/early S.Africa 1.6 1
IN8362.25 Acute/early India 18.2 NT
IN0013095.211 Acute/early India 0.2 NT
% viruses
neutralized 84% 79%
Mean IC50 (nM) 6.6 ± 8.1 0.4 ± 0.3
Aptamers inhibit entry ofPBMC
HIV-1 subtype C
Virus controlApt 2
Aptamer[nM] Aptamer][nM]
Mean IC80 in PMBC = 80 ± 11.8 nM
Aptamer[nM] Aptamer [nM]
UCLA1 (nM)
Virus Control
M
.5
.0
OT= 2
)lm
Aptamerssubtype
Du422IC80 = 6
nM
inhibit entry of HIV-1C in Macrophages
COT9IC80 = 29
nMDu422
COT97.5 2
UCLA1(nM)
VirusControl
UCLA1 (nM)
7.5
5.0
2
C 9
*** p <0.0019 nMVir*ups
<C0o.n0t5rolNS: notsignificant
*** p < 0.001* p <0.05NS: not
significant
NS IC801
5.02.5
Virus controlApt 21
2.50.0 *** p < 0.001
* p < 0.05NS: not significant
03.6 11 33� 100
Control
1 UCLA1 (nM)
3.6 11 33 100
Apt UCLA1 (nM)
CoNnStrol
Aptamer [nM]amer [nM]
0.0
2.5 03.6 11 3.6 11 33ADA100 Contr �
P24
(ng P
m 24l)
(ng
ml)
P24
(ng
ml)
p24
(ng
/ml) p24
(ng
/ml)
p24
(ng
/
IC80 = 29 nM
C
IC80
2�
IC80 = 6 nM
Du422IC80 = 6 n
7
5
NS
0 3.6 11
SW3314 100
IUCC8L0A=12(
n6Mn) M
3.6 11 33ADA100
Control
UCICL8A01=(n3M0) nM
0
Control
�
0.03 4033 100 ControlSW11
4ADA3.
6
3.6 11 33 100
UCLA1 (nM)
Control
Mean ICIC80 = 26UnCMLA1 (nM)
SW14
IC80 = 30
nMin MDM = 23 ± 10.4 nM30
8023 ADA40
IC80 = 30 nM20
1
230
10�
200 2
3.6 11 33 100 Control
3.6 11 33 100 ntrol1
��
�UCLA1(nM)
10�
�
0 � 0�
3.6 11 33 100 Contr�ol
3.6 11 33 100 Contro�l��
AptamU0CLA1 (nM)
���AptaUmCeLrA[n1M(n]M)
3.6 11 33 100
UCLA1 (nM)
er[nM]0
Control 3.6 11 33 100
UCLA1 (nM)
Control
p24
(ng/m
l) p24
(ng
/ml)
p24
(ng
/ml)
p24
(ng
/ml)
p24
(ng
/ml)
p24
(ng
/ml)
�4�0�
���
30 ���
0
IC80
Co
20
���
10 �����
IC80 = 26 nM
3
�
�
1
UCLA1 (nM)�
�
Aptamers exhibit no cytotoxicity
TZM-bl
MTS based-assayPBMC
MTS based-assay120 120
100 100
80 80
60 60
Ce
llV
iab
ility
Ce
llV
iab
ility
40 40
2020
00
%C
%C
Aptamers interact with conserved
residues on gp120
Aptamers bind to amino acids within the coreceptor (CoRbs) CCR5 binding site
Mufhandu, H et al., J. Virol. (2012,)86(9), pp. 4989
Synergy of aptamers withinhibitors
HIV-1 entry
100100UACpLtA21+Tb21
02UACpLtA21
TT2200
7575
T 20
5010010050
UCLA1+T20
UCLA1
T207525 25
5050 0 0
0.01 0.1 1 10 100 0.01 0.1 1 10 100
µM Lo2g5 Synergism : CI = 0.3 – 0.925
%in
hib
itio
n
ZM249
75
Du156 T 20
ZM249 T20Du156 T20
UCLA1+b12
UCLA1
b12
µM Lo2g5 Synergism : CI = 0.3 – 0.925
Antagonism : CI = >1Du172 b121
SW7 b120.1
00.01
100
0
0.010.1 10 100
100
1 10 100
UCLA1+b12
UCLA1
b1275 75
100 10050 Apt 2 + b12
Apt 2T20
50
75 7525 25
50 5000.01
00.1 1 10 100 0.01
2525
µM (Log)0 00.01 0.1 1 10 100 0.01 0.1 1 10 100
µM (Log)
%%
inIh n
ibh
i it %bio
in tIi n
ohn
i
bit
ion 0.1 1 10 100
µM (
SW7 b12Du172 b12
Summary
� Anti-gp120
RNA
aptamers
areefficacious
against
HIV-1subtype C isolates ( concentration in
nanomolar)
� They interact with conserved residues on gp120, delay virusresistance
� Not toxic in different cell types
� Synergy with other entry inhibitors, combination therapy withother drugs
� Anti-gp120 aptamers can be developed as entry inhibitordrugs
Acknowledgements
CollaboratorsFunding
•
•
•
•
NICD (Lynn Morris)
UKZN (Alexander Pym)
UCT & GSH (B. Mayosi)
University of Oxford, UK (WilliamJames)James)
• The Scripps Research Institute, USA (Dennis Burton)
Reagents
• Los Alamos National Lab, USA (Basil ISwanson)
•
•
IAVI
NIH AIDS Reagents
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