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Sep 11, 1992 - ... KoJIMA*, TAKASHI SHIMADAt, SAMUEL BRODER§, AND HIROAKI MITSUYA* ...... Clark, P., Hizi, A. & Hughes, S. H. (1992) Nature (London).
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 562-566, January 1993 Medical Sciences

Changes in drug sensitivity of human immunodeficiency virus type 1 during therapy with azidothymidine, dideoxycytidine, and dideoxyinosine: An in vitro comparative study TAKUMA SHIRASAKA*, ROBERT YARCHOANt, MARY C. O'BRIEN*, ROBERT N. HUSSON*, BARRY D. ANDERSON*, Eui KoJIMA*, TAKASHI SHIMADAt, SAMUEL BRODER§, AND HIROAKI MITSUYA*¶ *Experimental Retrovirology Section, tRetrovirology Diseases Section, Medicine Branch, and Clinical Hematology Branch, National Heart, Lung and Blood Institute, National Cancer Institute, Bethesda, MD 20892

Communicated by Howard M. Temin, September 11, 1992 (received for review April 22, 1992)

Human immunodeficiency virus type 1 ABSTRACT (1IV-1) strains were Isolated from nine patients before and after prolonged therapy with either an alternating regimen of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dldeoxycytldin (ddC) (AZT/ddC) or 2',3'-deoxyinosine (ddl) alone. AN strains obtained from four patients who received AZT/ddC for up to 41 mo were highly insensitive to AZT in vitro. Only one strain obtained after AZT/ddC therapy showed reduced susceptibility to ddC in addition to AZT and had previously unreported amino acid substitutions in the viral polymeraseending pol region, whereas three other strains had one or more of the five previously reported AZT-related mutations. In flve HIV-1 strains from patents who received ddI for up to 29 mo, no appreciable decrease in sensitivity to ddl was detect. t amino Two strains Isolated after ddI therapy had no acid mutations, although three strains had a mutation reportedly acited with ddI administration. These data suggest that HIV-1 develops reduced susceptibility to AZT more readily than to ddC and ddI and/or that the reduced susceptibility to ddC and ddI is modest in degree. Moreover, the present data suggest that an alternating regimen of AZT and . ddC does not block the emergence of AZT- ensitve va It should be noted, however, that the current results do not provide a basds for concluding that AZT/ddC or ddI is inferior, equivalent, or superior to AZT as therapy of AIDS.

MATERIALS AND METHODS Nucleosides. ddC and ddl for in vitro use were provided by the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, whereas AZT was purchased from Sigma. of Clinical HIV-1 Strains. HIV-1 strains were isolated from four patients with AIDS or AIDS-related complex (ARC) (patients 100, 101, 103, and 104) who received an alternating regimen of AZT (7 days) and ddC (7 days) (AZT/ddC) for 15, 22, 41, and 32 mo, respectively. Patient 103 received ddI for the last 3 mo in addition to AZT/ddC therapy. HIV-1 strains were also isolated from five patients with AIDS or ARC (patients 200, 201, 203, 204, and 205), who received ddl for 25, 20, 29, 18, and 20 mo, respectively. Patients 203, 204, and 205 had received AZT before ddl therapy for 12, 3, and 12 mo, respectively. To isolate primary HIV-1 strains, peripheral blood mononuclear cells (PBMCs) (1-2 x 106) from each patient were cocultured with phytohemagglutinin (PHA)-activated PBMCs (PHA-PBMCs, 1-2 x 106) from HIV-1-seronegative volunteers in 24-well microtiter culture plates in 2 ml of culture medium [RPMI 1640 medium/15% heat-inactivated fetal calf serum/recombinant interleukin 2 at 10 units per ml (Amgen Biologicals)/4 mM L-glutamine/penicillin at 50 units per ml/streptomycin at 50 ug/ml]. When the level of p24 Gag protein in the culture supernatant reached .10 ng/ml as assessed by RIA (Dupont), the supernatant was collected as

The ability to provide effective long-term antiretroviral therapy using single agents for human immunodeficiency virus type 1 (HIV-1) infection became a complex issue when HIV-1 strains that were less susceptible to 3'-azido-3'-deoxythymidine (AZT or zidovudine) in vitro were isolated from patients with AIDS who received long-term AZT therapy (1-3). Two

a source of infectious virions.

Determination of Sensitivity of Clinical HIV-1 Strains Agant Drus. In the current study, each pair of pre- and posttherapy isolates to be compared was simultaneously titrated for infectivity and assessed for drug sensitivity. To determine changes in drug sensitivity, PHA-PBMCs were first exposed to various concentrations of drug for 2 hr, then exposed to a 20x tissue culture 50% infectious dose (TCID50) of each virus isolate. To confirm that the viral dose used was the desired one, the inoculum was retitrated for TCID50 in the same assay. The drug concentrations used were 0.0005, 0.005, 0.05, 0.5, 5, 50 ,uM for AZT; 0.005, 0.05, 0.5, 2, 5 ,uM for ddC; and 0.1, 1, 10, 50, 100 juM for ddM. The sensitivity of a given HIV-1 strain against a drug was defined as the drug concentration that yielded 50% p24 Gag protein-negative

2',3'-dideoxynucleosides, 2',3'-dideoxyinosine (ddN or didanosine) and 2',3'-dideoxycytidine (ddC), have been shown to have clinical activity in patients with HIV-1 infection (4-8). St. Clair et al. (9) have recently reported that patients who received long-term AZT therapy and then ddl therapy developed reduced susceptibility to ddI and that the emergence of ddI insensitivity was associated with a reversion to a more AZT-sensitive phenotype. However, the behavior of HIV-1 at the genetic and phenotypic level upon exposure to multiple antiretroviral agents is as yet poorly understood. In the current study, we specifically asked how easily HIV-1 develops reduced sensitivity to three drugs: AZT, ddC, and ddI, when given as single drugs or in combination. We also asked whether the addition of ddC to AZT therapy in an alternating regimen could block the emergence of AZT-insensitive HIV-1 variants.

wells (CN5o) on days 8-10 in culture. When concentrations of p24 Gag protein were 15% clones sequenced. Dashes indicate amino acid residues conserved with respect to the reference sequence. In strain ERS100pre K -- E substitution at codon 32 was seen in four of four [designated as K32E(4/4)], E44K(4/4), and i135T(4/4). The following other substitutions were found: in strain ERS100post, D67N(8/8), K7°R(8/8), i'35T(8/8), R199K(8/8), T215Y(6/8), and K219Q(8/8); in strain ERS101pr,, i135T(4/4), P157L(1/4), K201I(1/4), and 1202V(3/4); in strain ERS101pos, D67N(8/8), T*9D(8/8), K70R(8/8), V9°I(8/8), i135T(8/8), K"54E(2/8), I202V(8/8), T215F(8/8), and K219Q(8/8); in strain ERS103pre, K46R(1/4) and K166R(4/4); in strain ERS103psto, E44G(1/4), D 21N(1/4), D123N(1/4), Y146H(1/4), K'66R(4/4), G'90E(1/4), and Q222P(1/4); in strain ERSl03post-lmo, A62V(8/8), V75I(8/8), F77L(8/8), F'16Y(8/8), V118I(2/8), Q151M(8/8), K166R(7/7), and I202V(7/7); in strain ERS104pr,, K22R(1/4), E40K(1/4), and D67G(1/4); and in strain ERS104post, V106I(8/8), T215Y(6/8), and T215D(2/8, shown by *) were identified. Single-letter amino acid code is used. Amino acids shown in lowercase letters represent variable amino acid residues (19).

had received AZT therapy before ddI therapy and then received ddI monotherapy. Patients 200 and 201, however, had received no prior AZT therapy. Therefore, these data indicate that patients who received only ddI monotherapy developed the Leu-74 -+ Val substitution. Restoration of Sensitivity to AZT of AZT-Insensitive HIV-1 Strain after Chaning to ddI Therapy. Three patients, 203, 204, and 205, had received AZT prior to ddI therapy. Before ddI, each of these three patients' HIV-1 isolates showed a substantial'reduced susceptibility to AZT (Fig. 2A at time 0). Interestingly, these strains resumed relative sensitivity to AZT after switching to ddM. HIV-12o4pre had the Lys-70 Arg(3/4) substitution that should be related to the prior AZT therapy; however, after 18 mo of ddI therapy followed by 3 mo of AZT therapy, this patient's posttherapy strain lost the Lys-70 -* Arg substitution, reverting to the wild-type amino acid Lys-70, while it acquired the Thr-215 -* Tyr substitution that is thought to correlate with insensitivity to AZT (2). In fact, the CN50 of HIV-1204pot (16 1.M) was still relatively high, indicating that HIV-12wpost was yet substantially insensitive to AZT (Fig. 2A).

HIV-1205' also had the AZT-related Lys-70 -* Arg(1/4) substitution at entry that was presumably from prior AZT therapy. However, this substitution was not seen after 20 mo of ddI therapy. Thus, the HIV-1 strain from patient 205 may have regained sensitivity to AZT by losing this substitution after ddI therapy. Patient 203, who had previously received AZT for 12 mo, had a fall in his CD4 count after 26 mo of ddI therapy (Fig. 2A), and he expressed a desire to try AZT. During the 26 mo of ddI therapy, his HIV-1 had become relatively sensitive to AZT. Genetically the virus maintained the Thr-215 -* Tyr substitution, while acquiring a new Leu-74 -* Val substitution (Table 1). This result suggests that the acquisition of Leu-74 -- Val rendered the AZT-insensitive variant somewhat sensitive to AZT, as St. Clair et al. (9) reported. When this patient then received AZT for 2 mo, insensitivity to AZT increased rapidly (Fig. 2 and Table 1). This increase was accompanied by a genotypic reversal to wild-type Leu-74 in nearly 80% of the clones, whereas all the clones retained the Thr-215 -* Tyr substitution (Table 1). This rapid loss of the Leu-74 -+ Val substitution within 2 mo of ddI discontinuation

Medical Sciences: Shirasaka et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

VIRUS STRAIN

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AMINO ACID SEQUENCE 30 40 50 60 70 80 90 VKQWPLTEEK IKaLvEICTE MEKEGKISKi GPENPYNTPV FAIKKKDSTK WRKLVDFREL NkrTQDFWEV

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110 120 130 140 100 150 160 QLGIPHPAGL KkKKSVTVLD VGDAYFSVPL DkdFRKYTAF TIPSiNNETP gIRYQYNVLP QGWKGSPAIF e

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FIG. 4. Deduced amino acid sequences of RT in HIV strains isolated from patients receiving ddl. Four and eight clones were sequenced for pretherapy strains and posttherapy strains, respectively. The following substitutions were found: in strain ERS200p,,, V211(3/4), G45R(1/4), G51R(1/4), G"R(1/4), k102R(1/4), 1142T(1/4), and q2O7H(4/4); in strain ERS2OOp.,t, T69A(2/8), L74V(8/8), and q2°7H(7/8); in strain ERS201pe, 1142T(4/4) and T165I(4/4); in strain ERS201POSt, L74V(8/8), P142T(8/8), T165I(8/8), and Y181C(7/8); in strain ERS203pre, M41L(4/4), K43E(4/4), I94M(1/4), A"S(4/4), L210W(4/4), and T21 Y(4/4); in strain ERS203p.t, T39A(6/8), M41L(8/8), K43E(3/8), L74V(8/8), A98S(8/8), V1181(4/8), P142V(4/8), M184V(5/8), L21OW(6/8), T215Y(7/8), and LmH(7/8); in strain ERS204pFC, P25S(1/4), D67N(1/4), K70R(3/4), and E194T(4/4); in strain ERS204post, T39I(2/8), F17L(3/8), E'94T(8/8), and T215Y(8/8); in strain ERS205.r, 147V(1/4), S48T(4/4), V60I(1/4), K70R(1/4), W"R(1/4), L149F(1/4), I'67N(1/4), E16D(4/4), I178L(2/4), and Q197R(1/4); and in strain ERS205post, E169D(8/8) and I178L(8/8).

contrasts strikingly with the persistence of the Thr-215 -- Tyr substitution in nearly 90% of clones despite 26 mo of ddI therapy (Table 1).

DISCUSSION In this study, three of four post-AZT/ddC strains showed no significant decrease in sensitivity to ddC, although all strains had developed high levels of AZT insensitivity. In a separate pediatric study, we have isolated an additional seven pairs of pre- and post-AZT/ddC strains and have seen the development of a high level ofAZT insensitivity in four such patients, despite the addition of ddC to the therapy (R.N.H., T.S., P. A. Pizzo, and H.M., unpublished observations). These results suggest that although the alternating regimen of AZT and ddC may reduce the side effects of each drug and conceivably have clinical advantages over monotherapy, the combined AZT/ddC therapy does not prevent the development of HIV-1 variants less susceptible to AZT. One AZT/ddC-insensitive strain, HIV-11o3post41mo, had no previously reported AZT-related mutations, but seven dis-

tinctive amino acid substitutions were found in this strain. These results suggest either that the amino acid substitutions in HIV-1l13post41mo provided a replicative advantage in the presence of AZT that exceeded that provided by combinations of the previously reported AZT-related substitutions or that the mutations observed in HIV-1lO3post-lmo caused conformational changes incompatible with the previously reported AZT-related substitutions. The most remarkable substitution identified in HIV-11o3pst is the substitution for glutamine at position 151 with methionine [Gln-151 -* Met(8/ 8)]. This residue is part of a highly conserved amino acid sequence (Leu-Pro-Gln-Gly, amino acids 149-152 in Fig. 3), termed motif B, which is seen in each of 26 different retroviruses (13, 14) including HIV-1, HIV-2, simian immunodeficiency virus, human T-cell leukemia virus, and a number of animal retroviruses. Most recent x-ray crystallography of the HIV-1 RT has suggested that Gln-151 lies in (or adjacent to) the 3-strand 8, which is located close to the putative polymerase active site (15). Thus, the Gln-151 Met substitution could alter the interaction between the RT and dNTP or change the interaction between the enzyme and the template.

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Table 1. Phenotypic and genotypic changes of HIV-1 strains sequentially isolated from a patient receiving AZT or ddI Drug sensitivity Clones Clones Residue (CNmo), ALM examined no Antiviral (%) 74 215 no. AZT ddC ddI therapy WT M 4(100) 4 1 17 32 12-mo AZT Additional M M 7 (88) 8 26-mo ddI 2.3 3.2 22 M WT 1(12) Additional WT M 7 (78) 9 3.2 22 2-mo AZT 32 M M 2 (22) Residue 74 represents leucine and valine for wild-type (WT) and mutant (M) amino acids, respectively; whereas residue 215 represents threonine and tyrosine for wild-type and mutant amino acids, respectively.

In the current study, no appreciable decrease in sensitivity to ddI was detected in five different post-ddl HIV-1 strains, although the decreased sensitivity could have been so modest in degree that the difference was not readily detected. St. Clair and coworkers (9) have reported that HIV-1 strains isolated after ddI therapy developed decreased sensitivity (up to 26-fold) to ddI as compared with pretherapy isolates. It is possible that their use of RT activity and different PBMC batches rather than the use of biologically more stable p24 Gag protein and a single PBMC batch for comparison could have resulted in greater variability. The acquisition of the Leu-74 -- Val substitution in addition to the Thr-215 -* Tyr substitution appears to provide HIV-1 with a replication advantage in the presence of ddI; however, in the absence of ddI, the Leu-74 -- Val substitution may cause a significant replication disadvantage. This result may explain why HIV-1 mutants with the Leu-74 -+ Val substitution were rapidly eliminated from the virus population when therapy no longer included ddI (Table 1), although elucidation of the conformational and functional basis for the decreased susceptibility to AZT and the Leu-74 -* Val substitution-related restoration of AZT sensitivity will depend on continued analysis of RT by x-ray crystallography and related technologies (15, 16). HIV-1 apparently undergoes selection pressure to develop reduced susceptibility to AZT. RT of AZT-insensitive variants could, in theory, discriminate between AZT triphosphate and dTTP by the presence or absence of the tubular and bulky 3'-azido group (17). In this regard, the close structural resemblance of ddATP and ddCTP (the putative active moieties of ddI and ddC, respectively) to their natural corresponding nucleotides (dATP and dCTP, respectively) may make it more difficult for the virus to mutate to efficiently exclude ddATP or ddCTP, while preserving the function of RT. Indeed, molar-refraction data (18) indicate that an azido group is about three times larger than a hydroxyl group, whereas a hydrogen molecule is 2-fold smaller than a hydroxyl group. It is possible that the mutations affect the

Proc. Nad. Acad Sci. USA 90 (1993)

template-binding site in RT and change the ability of the enzyme to discriminate between natural dNTP and ddNTP, as proposed by Kohlstaedt et al. (15). However, the relationship between the structural resemblance of the nucleotides and development of drug resistance is as yet highly hypothetical and requires future assessment. We thank Robert Wittes, Marvin Reitz, John Driscoll, Samuel Wilson, and John Erickson for helpful discussions and the medical and nursing staffs of the Medicine Branch of the National Cancer Institute for their help. 1. Larder, B. A., Darby, G. & Richman, D. D. (1989) Science 243, 1731-1734. 2. Larder, B. A. & Kemp, S. D. (1989) Science 246, 1155-1158. 3. Kellam, P., Boucher, C. A. B. & Larder, B. A. (1992) Proc. Nati. Acad. Sci. USA 89, 1934-1938. 4. Mitsuya, H. & Broder, S. (1986) Proc. Nati. Acad. Sci. USA 83, 1911-1915. 5. Mitsuya, H., Yarchoan, R. & Broder, S. (1990) Science 249, 1533-1544. 6. Yarchoan, R., Perno, C.-F., Thomas, R. V., Klecker, R. W., Allain, J.-P., Wills, R. J., McAtee, N., Fischl, M. A., Dubinsky, R., McNeely, M. C., Mitsuya, H., Pluda, J. M., Lawley, T. J., Leuther, M., Safai, B., Collins, J. M., Myers, C. E. & Broder, S. (1988) Lancet 1, 76-81. 7. Yarchoan, R., Mitsuya, H., Thomas, R. V., Pluda, J. M., Hartman, N. R., Perno, C.-F., Marczyk, K. S., Allain, J.-P., Johns, D. G. & Broder, S. (1989) Science 245, 412-415. 8. Lambert, J. S., Seidlin, M., Reichman, R. C., Plank, C. S., Laverty, M., Morse, G. D., Knupp, C., McLaren, C., Pettinelli, C., Valentine, F. T. & Dolin, R. (1990) N. Engl. J. Med. 322, 1333-1340. 9. St. Clair, M. H., Martin, J. L., Tudor-Williams, G., Bach, M. C., Vavro, C. L., King, D. M., Kellam, P., Kemp, S. D. & Larder, B. A. (1991) Science 253, 1557-1559. 10. McConway, M. G., Chapman, R. S., Beastall, G. H., Brown, E., Tillman, J., Bonar, J. A., Hutchinson, A., Allison, T., Finayson, J., Weston, R., Beckett, G. J., Carter, G. D., Carlyle, E., Herbertson, R., Blundell, G., Edwards, W., Glen, A. C. A. & Reid, A. (1989) Clin. Chem. 35, 289-291. 11. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A. & Arnheim, N. (1985) Science 230, 13501354. 12. Fitzgibbon, J. E., Howell, R. E., Harberzettl, C. A., Sperber, S. J., Gocke, D. J. & Dubin, D. T. (1992) Antimicrob. Agents Chemother. 36, 153-157. 13. Xiong, Y. & Eickbush, T. H. (1990) EMBO J. 9, 3353-3362. 14. Poch, O., Sauvaget, I., Delarue, M. & Tordo, N. (1989) EMBO J. 8, 3867-3874. 15. Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783-1790. 16. Arnold, E., Jacob-Monila, A., Nanni, R. G., Williams, R. L., Lu, X., Ding, J., Clark, A. D., Jr., Zhang, A., Ferris, A. L., Clark, P., Hizi, A. & Hughes, S. H. (1992) Nature (London) 357, 85-89. 17. Cameron, A., Mastropaolo, D. & Cameron, N. (1987) Proc. Nati. Acad. Sci. USA 84, 8239-8242. 18. Hansch, C., Leo, A., Unger, S. H., Kim, K. H., Nikaitani, D. & Lien, E. J. (1973) J. Med. Chem. 16, 1207-1216. 19. Myers, G., Berzofsky, J. A., Korber, B. & Smith, R. F. (1991) Human Retroviruses and AIDS (Los Alamos National Lab., Los Alamos, NM).