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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.

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Presentation on theme: "Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 Maraviroc-resistant subtype B primary HIV-1 induced in vitro selection became highly."— Presentation transcript:

1 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

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

3 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 100 50 0 % Inhibition 100 50 0 % Inhibition Drug Concentration HIV gp41 gp120 V3 loop CD4bs CD4i Non-competitive Resistance 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-resistant IC 50 shift Plateau down ? Two mechanisms of resistance to CCR5 inhibitors

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

5 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 Primary isolate (Sub B, R5) PM1 CCR5CXCR4 CCR5CXCR4 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

6 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 In vitro selection of MVC resistant variant using subtype B primary isolate （ HIV-1 Y1 ） 0 1 10 100 1000 10000 024681012141618202224262830323436384042444648 Passage number MVC conc. (nM) Low CCR5 passage (in PM1) Control passage (in PM1/CCR5) MVC selection (in PM1/CCR5)

7 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 Susceptibility of MVC-resistant variant to CCR5 inhibitors Concentration (nM) % inhibition 0 10 20 30 40 50 60 70 80 90 100 10100100010000 APL Concentration (nM) % inhibition 0 10 20 30 40 50 60 70 80 90 100 110100100010000 SCH-C Concentration (nM) % inhibition 0 10 20 30 40 50 60 70 80 90 100 0.1110100100010000 Control passage MVC resistant MVC Low CCR5 passage (in PM1 cells)

8 Antiviral Immunity and Transmission June 19, 2011 Abst. #: 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 passage gp120 in PM1 cells K8R(SP) C11W(SP) Gp120 V3

9 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 IC 50 (MVC, nM) 10 1 10 2 10 3 10 4 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 2500 3000 2000 4000 5000 3000 5000 7000 10000 41 10000 48 10000 IC 50 values of the passaged viruses T297I (V3) M434I (C4) V200I (C2) T297I (V3) M434I (C4) V200I (C2) K305R (V3) T297I (V3) M434I (C4)

10 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 F317L (V3) I333L(C3) 17P (0.3µM) A V3 IC 50 :0.2µM M309I(V3) E321D(V3) T297I(V3) 21P(0.8µM) IC 50 :3.1µM M434I(C4) B F317L (V3) I333L(C3) V3 E321D(V3) T297I(V3) M309I(V3) 34P(8µM) IC 50 :>10µM F317L(V3) I333L(C3) M434I(C4) V200I(C2) C V3 E321D(V3) T297I(V3) M309I(V3) 41P(10µM) IC 50 :>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

11 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 % Inhibition Drug Concentration Drug-sensitive MVC-resistant IC 50 shift Plateau shift Two-step mechanism of resistance to MVC 0 10 20 30 40 50 60 70 80 90 100 1101001000 1-14 passages 0 10 20 30 40 50 60 70 80 90 100 110100100010000 33~48 passages HIV gp41 gp120 V3 loop MVC-bound Coreceptors used CCR5 CD4 CCR5 inhibitor (MVC) HIV gp41 gp120 V3 loop CCR5 CD4 CCR5 inhibitor (MVC) Low-CCR5 (Low conc MVC) adaptation MVC-resistant IC 50 shift Low-CCR5 passage Low-CCR5 passage 1st STEP 2nd STEP

12 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 % Inhibition Concentration (µg/ml) Susceptibility of the passaged variants to anti-Env MAbs -10 0 10 20 30 40 50 60 70 80 90 0.010.1110100 -10 0 10 20 30 40 50 60 70 80 90 100 0.010.1110100 -10 0 10 20 30 40 50 60 70 80 90 100 0.010.1110100 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) V3 CD4bs CD4i -10 0 10 20 30 40 50 60 70 80 90 0.010.1110100 MVC-Resistant (in PM1/CCR5) +MVC(10000nM) V3 CD4bs CD4i V3 CD4bs CD4i V3 CD4bs CD4i

13 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 % Inhibition Concentration (µg/ml) Susceptibility of the each passaged variant to autologous plasma IgG -10 0 10 20 30 40 50 60 70 80 1101001000 Control passage (in PM1/CCR5) MVC resistant +MVC(10000nM) Control passage Low CCR5 passage (in PM1) MVC-Resistant + MVC(10000nM) Control passage (in PM1) MVC resistant

14 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 Conclusion 1.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 (IC 50 : 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.

15 Antiviral Immunity and Transmission June 19, 2011 Abst. #: TUAA0105 CAIDS Shigeyoshi Harada Aki Hamaji Akiko Shibata Noriko Shirai 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

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