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Kazuhisa YOSHIMURA Center for AIDS Research Kumamoto Univ., Japan

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

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Kazuhisa YOSHIMURA Center for AIDS Research Kumamoto Univ., Japan

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  1. 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. 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. Two mechanisms of resistance to CCR5 inhibitors HIV Competitive Resistance IC50 shift Unbounded Coreceptors used more efficiently 100 gp41 CD4i Drug-sensitive gp120 CD4bs % Inhibition V3 loop CCR5 inhibitor 50 CD4 Drug-resistant 0 Drug Concentration CCR5 ? Non-competitive Resistance HIV Plateau down gp41 CD4i 100 Inhibitor-bound Coreceptors used Drug-sensitive gp120 CD4bs V3 loop % Inhibition 50 CCR5 inhibitor Drug-resistant CD4 0 Drug Concentration CCR5

  4. 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. PM1/ CCR5 PM1 PM1/CCR5 and PM1 cells Primary isolate (Sub B, R5) CCR5 CXCR4 CCR5 CXCR4 Infection Infection CD4 CXCR4 CCR5 R5 primary virus 48 passages with MVC Low CCR5 passage 48 passages with no drug

  6. In vitro selection of MVC resistant variant using subtype B primary isolate（HIV-1Y1） 10000 1000 MVC selection (in PM1/CCR5) 100 MVC conc. (nM) 10 1 Control passage (in PM1/CCR5) Low CCR5 passage (in PM1) 0 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

  7. Susceptibility of MVC-resistant variant to CCR5 inhibitors 100 MVC 90 Control passage 80 70 60 Low CCR5 passage (in PM1 cells) % inhibition 50 40 30 20 MVC resistant 10 0 0.1 1 10 100 1000 10000 Concentration (nM) 100 APL 100 SCH-C 90 90 80 80 70 % inhibition 70 % inhibition 60 60 50 50 40 40 30 30 20 20 10 10 0 0 1 10 100 1000 10000 10 100 1000 10000 Concentration (nM) Concentration (nM)

  8. Low CCR5 passage gp120 in PM1 cells K8R(SP) C11W(SP) V84I(C1) A436T(C4) I464T(V5) D141N(V1) E321D(V3) F317W(V3) Comparison of gp120 sequences between Low-CCR5 passaged and MVC resistant variants MVC-resistant gp120 in PM1/CCR5 cells V65K(C1) K8R(SP) C11W(SP) I333L(C3) Gp120 Gp120 T297I(V3) I464T(V5) K305R(V3) D141N(V1) M309I(V3) V3 V200I (C2) V3 E137K(V1) K148Q(V1) D187G(V2) M434I (C4) E321D(V3) F317L(V3) 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)

  9. T297I (V3) M434I (C4) V200I (C2) K305R (V3) T297I (V3) M434I (C4) V200I (C2) T297I (V3) M434I (C4) T297I (V3) IC50 values of the passaged viruses 104 103 MVC resistant (PM1/CCR5) IC50 (MVC, nM) 102 Low CCR5 passage (PM1) 101 Passage Control (PM1/CCR5) 41 48 40 33 32 31 30 29 28 27 26 25 24 21 16 14 13 12 Passage number 10 9 7 5 0 10000 10000 10000 7000 5000 3000 5000 4000 2000 3000 2500 2000 2000 1000 250 170 150 120 80 60 MVC conc (nM) 40 20 0

  10. B I333L(C3) T297I(V3) V3 M309I(V3) M434I(C4) 21P(0.8µM) IC50 :3.1µM E321D(V3) F317L (V3) D C I333L(C3) I333L(C3) T297I(V3) T297I(V3) K305R(V3) V3 V3 M309I(V3) V200I(C2) V200I(C2) M309I(V3) M434I(C4) M434I(C4) 34P(8µM) IC50 :>10µM 41P(10µM) IC50 :>10µM E321D(V3) E321D(V3) F317L(V3) F317L(V3) The sites of mutations appeared during in vitro selection by MVC A I333L(C3) T297I(V3) V3 M309I(V3) 17P (0.3µM) E321D(V3) IC50 :0.2µM F317L (V3)

  11. Two-step mechanism of resistance to MVC 1-14 passages 1st STEP Low-CCR5 (Low conc MVC) adaptation 100 Drug-sensitive 90 HIV 80 IC50 shift Low-CCR5 passage 70 gp41 % Inhibition 60 gp120 50 40 CCR5 inhibitor (MVC) V3 loop 30 MVC-resistant 20 CD4 10 0 1 10 100 1000 CCR5 Drug Concentration 33~48 passages 2nd STEP MVC-bound Coreceptors used 100 Drug-sensitive 90 HIV 80 gp41 IC50 shift 70 Low-CCR5 passage gp120 60 % Inhibition 50 Plateau shift 40 V3 loop CCR5 inhibitor (MVC) 30 20 CD4 MVC-resistant 10 0 CCR5 1 10 100 1000 10000 Drug Concentration

  12. Susceptibility of the passaged variants to anti-Env MAbs Control passage (in PM1/CCR5) MVC-Resistant (in PM1/CCR5) 100 100 V3 90 90 CD4bs V3 MAb(KD-247) 80 80 CD4i MAb(4E9C) 70 70 60 60 CD4bs MAb(b12) 50 50 40 40 30 30 CD4bs 20 20 V3 10 10 CD4i 0 0 CD4i -10 -10 0.01 0.1 1 10 100 0.01 0.1 1 10 100 % Inhibition MVC-Resistant (in PM1/CCR5) +MVC(10000nM) Low CCR5 passage (in PM1) 90 90 CD4i V3 80 80 CD4bs CD4bs 70 70 CD4i 60 60 50 50 40 40 30 30 20 20 V3 10 10 0 0 -10 -10 0.01 0.1 1 10 100 0.01 0.1 1 10 100 Concentration (µg/ml)

  13. Susceptibility of the each passaged variant to autologous plasma IgG 80 Control passage (in PM1/CCR5) 70 Control passage (in PM1) Low CCR5 passage (in PM1) MVC resistant 60 MVC resistant +MVC(10000nM) 50 MVC-Resistant + MVC(10000nM) 40 % Inhibition 30 20 10 MVC-Resistant 0 Control passage -10 1 10 100 1000 Concentration (µg/ml)

  14. Conclusion 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. 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. 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. MVC resistant variants became sensitive to anti-V3 MAb and autologous plasma IgG under high concentrations of MVC.

  15. Center for AIDS Research Kumamoto University Collaborators Shuzo Matsushita (Kumamoto-U) Yosuke Maeda (Kumamoto-U) Hiroaki Mitsuya (NCI, NIH) Sachi Fukunishi (ViiV) Dennis Burton (The Scripps Research Institute) • CAIDS • Shigeyoshi Harada • Aki Hamaji • Akiko Shibata • Noriko Shirai 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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