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Antiviral Immunity and Transmission June 19, 2011 Abst. #: 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 YOSHIMURA Center for AIDS Research Kumamoto Univ., Japan

Kazuhisa YOSHIMURA Center for AIDS Research Kumamoto Univ., Japan

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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 YOSHIMURA Center for AIDS Research Kumamoto Univ., Japan. Background - PowerPoint PPT Presentation

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Page 1: 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

Page 2: Kazuhisa YOSHIMURA Center 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.

Page 3: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105

100

50

0

% In

hibi

tion

100

50

0

% In

hibi

tion

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

Page 4: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

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.

Page 5: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

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

Page 6: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105

In vitro selection of MVC resistant variant using subtype B primary isolate ( HIV-1Y1 )

0

1

10

100

1000

10000

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

Passage number

MV

C c

onc.

(nM

)

Low CCR5 passage (in PM1)

Control passage (in PM1/CCR5)

MVC selection (in PM1/CCR5)

Page 7: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105

Susceptibility of MVC-resistant variant to CCR5 inhibitors

Concentration (nM)

% in

hibi

tion

0

10

20

30

40

50

60

70

80

90

100

10 100 1000 10000

APL

Concentration (nM)

% in

hibi

tion

010

20

30

40

50

60

70

80

90

100

1 10 100 1000 10000

SCH-C

Concentration (nM)

% in

hibi

tion

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000 10000

Control passage

MVC resistant

MVC

Low CCR5 passage (in PM1 cells)

Page 8: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

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

Page 9: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105

IC50

(MV

C, n

M)

101

102

103

104

Passage Control (PM1/CCR5)

Low CCR5 passage (PM1)

MVC resistant (PM1/CCR5)

Passage number

MVC conc (nM)

0 5 7 9 10 12 13 14 16 21 24 25 26 27 28 29 30 31 32 33 40

0 20 40 60 80 120 150 170 250 1000 2000 2000 2500 3000 2000 4000 5000 3000 5000 7000 10000

41

10000

48

10000

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)

Page 10: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

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)

C

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

Page 11: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105

% In

hibi

tion

% In

hibi

tion

Drug Concentration

Drug Concentration

Drug-sensitive

Drug-sensitive

MVC-resistant

IC50 shift

Plateau shift

Two-step mechanism of resistance to MVC

0102030405060708090100

1 10 100 1000

1-14 passages

0

10

20

30

40

50

60

7080

90

100

1 10 100 1000 10000

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

Page 12: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105

% In

hibi

tion

Concentration (µg/ml)

Susceptibility of the passaged variants to anti-Env MAbs

-10

0

10

20

30

40

50

60

70

80

90

0.01 0.1 1 10 100

-100

102030405060708090

100

0.01 0.1 1 10 100-100

1020

3040

50607080

90100

0.01 0.1 1 10 100

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

-10

0

10

20

30

40

50

60

70

80

90

0.01 0.1 1 10 100

MVC-Resistant (in PM1/CCR5)+MVC(10000nM)

V3CD4bs

CD4i

V3

CD4bsCD4i

V3CD4bs

CD4i

Page 13: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

Antiviral Immunity and Transmission June 19, 2011Abst. #: TUAA0105

% In

hibi

tion

Concentration (µg/ml)

Susceptibility of the each passaged variant to autologous plasma IgG

-10

0

10

20

30

40

50

60

70

80

1 10 100 1000

Control passage (in PM1/CCR5)

MVC resistant +MVC(10000nM)

Control passage

Low CCR5 passage (in PM1)

MVC-Resistant

MVC-Resistant + MVC(10000nM)

Control passage (in PM1)

MVC resistant

Page 14: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

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.

Page 15: Kazuhisa YOSHIMURA Center for AIDS Research  Kumamoto Univ., Japan

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