Kazuhisa YOSHIMURA Center for AIDS Research Kumamoto Univ., Japan
description
Transcript of Kazuhisa YOSHIMURA Center for AIDS Research Kumamoto Univ., Japan
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
Maraviroc-resistant subtype B primary HIV-1 induced in vitro selection became highly sensitive to anti-gp120 neutralizing antibodies and autologous plasma IgG under high concentrations of the CCR5 inhibitor
Kazuhisa YOSHIMURACenter for AIDS Research Kumamoto Univ., Japan
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
Background
Resistance to entry inhibitors has been a particularly difficult problem since these drugs target the env gene, which is the most variable of all the HIV genes. For CCR5 inhibitors such as maraviroc (MVC), several possible mechanisms of resistance can be expected.
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
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Drug Concentration
Drug Concentration
HIV
gp41
gp120
V3 loop
CD4bs
CD4i
Non-competitiveResistance
Inhibitor-bound Coreceptors used
CCR5
CD4
CCR5 inhibitor
HIV
gp41
gp120
V3 loop
CD4bs
CD4i
CCR5
CD4
CCR5 inhibitor
Competitive Resistance Unbounded
Coreceptors used more efficiently
Drug-sensitive
Drug-sensitive
Drug-resistant
Drug-resistant
IC50 shift
Plateau down
?
Two mechanisms of resistance to CCR5 inhibitors
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
Objective
The aim of this study was to generate maraviroc-resistant viruses by serial passage with a CCR5-tropic subtype B primary HIV-1 in high CCR5 expressing cell line PM1/CCR5 and to characterize the resultant viruses in order to better understand the mechanisms of resistance to MVC in vitro.
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
Primary isolate (Sub B, R5)
PM1
CCR5 CXCR4CCR5 CXCR4
CD4
CXCR4
CCR5
R5 primary virus
PM1/CCR5
Infection
PM1/CCR5 and PM1 cells
48 passages with MVC
48 passages with no drug
Infection
Low CCR5 passage
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
In vitro selection of MVC resistant variant using subtype B primary isolate ( HIV-1Y1 )
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MV
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onc.
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Low CCR5 passage (in PM1)
Control passage (in PM1/CCR5)
MVC selection (in PM1/CCR5)
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
Susceptibility of MVC-resistant variant to CCR5 inhibitors
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APL
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SCH-C
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Control passage
MVC resistant
MVC
Low CCR5 passage (in PM1 cells)
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
F317L(V3)
T297I(V3)
I333L(C3)
M434I(C4)
V200I(C2)
K305R(V3)
E321D(V3)
I464T(V5)
M309I(V3)D141N(V1)E137K(V1) K148Q(V1) D187G(V2)
MVC-resistant gp120 in PM1/CCR5 cells K8R(SP)
C11W(SP)V65K(C1)
1) PM1 passaged variants (same as MVC selection, in green) → K8R(SP), C11W(SP), D141N(V1), E321D(V3), I464T(V5)2) PM1 passaged variants (different from MVC selection, in pink) → V84I(C1), E189A(V2), F317W(V3), A436T(C4) 3) MVC escape variants (different from PM1 passaged, in yellow) → V65K(C1), E137K(V1), K148Q(V1), D187G(V2), V200I(C2), T297I(V3),
K305R(V3), M309I(V3), F317L(V3), I333L(C3), 402insT(V4), M434I(C4)
Comparison of gp120 sequences between Low-CCR5 passaged and MVC resistant variants
F317W(V3)E321D(V3)
I464T(V5)
V84I(C1)
A436T(C4)
D141N(V1)
Low CCR5 passagegp120 in PM1 cells K8R(SP)
C11W(SP)
Gp120 Gp120
V3 V3
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
IC50
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Passage Control (PM1/CCR5)
Low CCR5 passage (PM1)
MVC resistant (PM1/CCR5)
Passage number
MVC conc (nM)
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IC50 values of the passaged virusesT297I (V3)M434I (C4) V200I (C2)
T297I (V3)M434I (C4) V200I (C2) K305R (V3)
T297I (V3)
T297I (V3)M434I (C4)
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
F317L (V3)
I333L(C3)
17P (0.3µM)
A
V3
IC50 :0.2µM
M309I(V3)
E321D(V3)
T297I(V3)
21P(0.8µM)IC50 :3.1µM
M434I(C4)
B
F317L (V3)
I333L(C3)
V3
E321D(V3)
T297I(V3)
M309I(V3)
34P(8µM)IC50 :>10µM
F317L(V3)
I333L(C3)
M434I(C4)
V200I(C2)
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V3
E321D(V3)
T297I(V3)
M309I(V3)
41P(10µM)IC50 :>10µM
K305R(V3)
D
F317L(V3)
I333L(C3)
V3
E321D(V3)
T297I(V3)
M434I(C4)
V200I(C2) M309I(V3)
The sites of mutations appeared during in vitro selection by MVC
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
% In
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% In
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Drug Concentration
Drug Concentration
Drug-sensitive
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MVC-resistant
IC50 shift
Plateau shift
Two-step mechanism of resistance to MVC
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1-14 passages
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33~48 passages
HIV
gp41gp120
V3 loop
MVC-bound Coreceptors used
CCR5
CD4
CCR5 inhibitor(MVC)
HIV
gp41gp120
V3 loop
CCR5
CD4
CCR5 inhibitor(MVC)
Low-CCR5 (Low conc MVC) adaptation
MVC-resistant
IC50 shift
Low-CCR5 passage
Low-CCR5 passage
1st STEP
2nd STEP
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
% In
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Concentration (µg/ml)
Susceptibility of the passaged variants to anti-Env MAbs
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Control passage (in PM1/CCR5)
Low CCR5 passage (in PM1)
MVC-Resistant (in PM1/CCR5)
CD4bs MAb(b12)
CD4i MAb(4E9C)
V3 MAb(KD-247)V3CD4bs
CD4i
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V3CD4bs
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CD4bsCD4i
V3CD4bs
CD4i
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
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Susceptibility of the each passaged variant to autologous plasma IgG
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Control passage
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MVC-Resistant
MVC-Resistant + MVC(10000nM)
Control passage (in PM1)
MVC resistant
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
Conclusion1. The substitutions in the SP (K8R and C11W), V1 (D141N), V3
(E321D), V5 (I464T) induced at low concentrations of MVC or by passaging in PM1 cells play a key role in adaptation for low density of CCR5 molecule on the target cells.
2. In addition to these substitutions, four mutations in the C2 (V200I), V3 (T297I and K305R), C4 (M434I) were acquired to replicate under high concentrations of MVC.
3. Low-CCR5 passage variant became moderate resistant to MVC (IC50: around 200nM), and also became sensitive to anti-CD4bs MAb, CD4i MAb and the plasma IgG, but not anti-V3 MAb.
4. MVC resistant variants became sensitive to anti-V3 MAb and autologous plasma IgG under high concentrations of MVC.
Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105
CAIDS
Shigeyoshi Harada
Aki Hamaji
Akiko Shibata
Noriko Shirai
Center for AIDS ResearchKumamoto University
Collaborators
Shuzo Matsushita (Kumamoto-U)
Yosuke Maeda (Kumamoto-U)
Hiroaki Mitsuya (NCI, NIH)
Sachi Fukunishi (ViiV)
Dennis Burton (The Scripps Research Institute)
Financially support• Grants from the Ministry of Health, Labour and Welfare, Japan (H23-AIDS-001; H22-“Research on Publicly
Essential Drugs and Medical Devices”-general-007)
• The Program of Founding Research Centers for Emerging and Re-emerging Infectious Diseases
• The Global COE Program Global Education and Research Center Aiming at the Control of AIDS