Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.
Journal: 2002/October - Antimicrobial Agents and Chemotherapy
ISSN: 0066-4804
PUBMED: 12019106
Abstract:
The synthetic peptide T-20 (enfuvirtide) represents the first of a new class of antiretroviral compounds to demonstrate in vivo potency by targeting a step in viral entry. T-20 inhibits a conformational change in the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein (gp41) that is required for fusion between HIV-1 and target cell membranes. The initial phase I clinical trial of T-20 treatment for HIV-infected patients thus provided a unique opportunity to evaluate the emergence of resistant virus in vivo to this novel class of antiretroviral agents. All four patients who received an intermediate dose of T-20 (30 mg twice daily) had an initial decline in plasma viral load over the first 10 days but a rising trend by day 14, suggestive of selection for resistant virus. Plasma virus derived from patients enrolled in all dosage groups of the phase I T-20 trial was analyzed by population sequencing before and after treatment. While no mutations were found within a highly conserved 3-amino-acid sequence (GIV) known to be critical for fusion at baseline, after 14 days of therapy, virus from one patient in the 30-mg dose group (30-1) developed a mutation in this motif, specifically an aspartic acid (D) substitution for glycine (G) at position 36. Multiple env clones were derived from the plasma virus of all four patients in the 30-mg dosage group. Sequence analysis of 49 clones derived from the plasma of patient 30-1 on day 14 revealed that 25 clones contained the G36D mutation, while 8 contained the V38A mutation. Dual mutations involving G36D and other residues within the HR1 domain were also identified. In 5 of the 49 env clones, other mutations involving residues 32 (Q32R or Q32H) and 39 (Q39R) were found in combination with G36D. Cloned env sequences derived from the plasma virus of subject 30-3 also had single mutations in the GIV sequence (V38M and I37V) detectable following therapy with T-20. The plasma virus from subjects 30-2 and 30-4 did not contain changes within the GIV sequence. To analyze the biological resistance properties of these mutations, we developed a novel single-cycle HIV-1 entry assay using JC53BL cells which express beta-galactosidase and luciferase under control of the HIV-1 long terminal repeat. Full-length env clones were derived from the plasma virus of patients 30-1 and 30-3 and used to generate pseudotyped virus stocks. The mean 50% inhibition concentrations (IC(50)s) for mutants G36D and V38A (patient 30-1) were 2.3 microg/ml and 11.2 microg/ml, respectively, statistically significant increases of 9.1- and 45-fold, respectively, compared with those of wild-type Env. The IC(50) for the V38 M mutation (patient 30-3) was 7.6 microg/ml, an 8-fold increase compared with that of the wild type. The I37V mutation resulted in an IC(50) 3.2-fold greater than that of the wild type. Envs with double mutations (Q32R plus G36D and Q32H plus G36D) exhibited a level of resistance similar to that of G36D alone. These findings provide the first evidence for the rapid emergence of clinical resistance to a novel class of HIV-1 entry inhibitors and may be relevant to future treatment strategies involving these agents.
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Antimicrob Agents Chemother 46(6): 1896-1905

Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy

Howard Hughes Medical Institute, Departments of, Medicine, Microbiology, Biostatistics, University of Alabama at Birmingham, Birmingham, Alabama 35294, Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, Alabama 352335
Corresponding author. Mailing address: University of Alabama at Birmingham, Department of Medicine, LHRB 613, 701 19th St. South, Birmingham, AL 35294. Phone: (205) 934-0051. Fax: (205) 975-7300. E-mail: ude.bau@cjseppak.
Received 2001 Sep 7; Revised 2001 Dec 5; Accepted 2002 Mar 21.

Abstract

The synthetic peptide T-20 (enfuvirtide) represents the first of a new class of antiretroviral compounds to demonstrate in vivo potency by targeting a step in viral entry. T-20 inhibits a conformational change in the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein (gp41) that is required for fusion between HIV-1 and target cell membranes. The initial phase I clinical trial of T-20 treatment for HIV-infected patients thus provided a unique opportunity to evaluate the emergence of resistant virus in vivo to this novel class of antiretroviral agents. All four patients who received an intermediate dose of T-20 (30 mg twice daily) had an initial decline in plasma viral load over the first 10 days but a rising trend by day 14, suggestive of selection for resistant virus. Plasma virus derived from patients enrolled in all dosage groups of the phase I T-20 trial was analyzed by population sequencing before and after treatment. While no mutations were found within a highly conserved 3-amino-acid sequence (GIV) known to be critical for fusion at baseline, after 14 days of therapy, virus from one patient in the 30-mg dose group (30-1) developed a mutation in this motif, specifically an aspartic acid (D) substitution for glycine (G) at position 36. Multiple env clones were derived from the plasma virus of all four patients in the 30-mg dosage group. Sequence analysis of 49 clones derived from the plasma of patient 30-1 on day 14 revealed that 25 clones contained the G36D mutation, while 8 contained the V38A mutation. Dual mutations involving G36D and other residues within the HR1 domain were also identified. In 5 of the 49 env clones, other mutations involving residues 32 (Q32R or Q32H) and 39 (Q39R) were found in combination with G36D. Cloned env sequences derived from the plasma virus of subject 30-3 also had single mutations in the GIV sequence (V38M and I37V) detectable following therapy with T-20. The plasma virus from subjects 30-2 and 30-4 did not contain changes within the GIV sequence. To analyze the biological resistance properties of these mutations, we developed a novel single-cycle HIV-1 entry assay using JC53BL cells which express β-galactosidase and luciferase under control of the HIV-1 long terminal repeat. Full-length env clones were derived from the plasma virus of patients 30-1 and 30-3 and used to generate pseudotyped virus stocks. The mean 50% inhibition concentrations (IC50s) for mutants G36D and V38A (patient 30-1) were 2.3 μg/ml and 11.2 μg/ml, respectively, statistically significant increases of 9.1- and 45-fold, respectively, compared with those of wild-type Env. The IC50 for the V38 M mutation (patient 30-3) was 7.6 μg/ml, an 8-fold increase compared with that of the wild type. The I37V mutation resulted in an IC50 3.2-fold greater than that of the wild type. Envs with double mutations (Q32R plus G36D and Q32H plus G36D) exhibited a level of resistance similar to that of G36D alone. These findings provide the first evidence for the rapid emergence of clinical resistance to a novel class of HIV-1 entry inhibitors and may be relevant to future treatment strategies involving these agents.

Abstract

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex controls key processes of viral entry. This complex determines viral tropism and mediates membrane fusion and thus enables virus entry and infection of the host cell. The HIV-1 envelope protein is initially synthesized as a 160-kDa polyprotein precursor (gp160) that is extensively glycosylated. Proteolytic cleavage of gp160 produces the gp120 surface subunit and the gp41 transmembrane subunit, which form oligomers and associate with each other through noncovalent interactions on the surface of the virion. On the surface of target cells, the gp120 surface subunit binds to its receptor and coreceptors and the gp41 transmembrane subunit mediates the fusion of the viral and cellular membranes (for a review, see reference 8). Subsequent to receptor binding, conformational changes occur in gp41 that lead to membrane fusion (7, 13, 18, 19, 21). To attain a fusion-active conformation, specific regions of the gp41 ectodomain must interact. The ectodomain contains a hydrophobic fusion peptide (FP) sequence at the amino terminus, followed by two leucine zipper-like motifs (heptad repeat 1 [HR1] and HR2). HR1 and HR2 consist of a 4,3 hydrophobic repeat that is predictive of an alpha-helical secondary structure and characteristic of coiled coils. Ultimately, through coiled coil interactions a trimer of antiparallel dimers of HR1 and HR2 is predicted to form (12). The formation of this six-stranded helical bundle induces a hairpin structure that brings the viral and cell membranes into proximity for fusion (2, 23).

Previous studies reported that synthetic peptides based on the HR2 sequence effectively block HIV-induced membrane fusion and infection by cell-free virus (9, 12, 24-26). T-20, previously known as DP178 and now as enfuvirtide, is a 36-amino-acid synthetic peptide homologous to the last 36 amino acids of HR2 (3, 24). By competitively binding HR1, T-20 blocks formation of the hairpin structure necessary for fusion (3, 26). Studies in vitro have shown that T-20 inhibits cell-free HIV-1 infection and virus-mediated cell-cell fusion (24, 26). After in vitro passage for 6 weeks in the presence of increasing concentrations of T-20, resistant variants of HIV-1 evolved (16). Sequence analysis indicated that resistant virus contained mutations within a sequence of three amino acid residues (GIV, positions 36 to 38) that are highly conserved in the HR1 domain (16). Mutations at both position 36 (G to D or S) and 38 (V to M) caused a marked decrease in susceptibility to T-20 inhibition (16). An intermediate level of sensitivity was observed with single mutations at position 36 (G36S).

Kilby et al. reported results from the first phase I clinical trial of T-20, which provided proof of concept for potent, dose-related virologic suppression by inhibiting a step in viral entry and/or fusion (10). All four of the subjects who received a 100-mg dose twice daily experienced a marked decline (mean, −1.96 log10) in plasma HIV-1 RNA (vRNA), while those in the 3- and 10-mg BID dose groups had minimal or no changes in viral load after the 14-day course of therapy. Subjects treated with the 30-mg BID dose were particularly intriguing from the standpoint of this investigation, because incomplete suppression of virus replication (median decline of −0.62 log10 among these four patients) suggested the possibility of selection for T-20-resistant HIV-1. Here we report, for the first time, the in vivo emergence of HIV-1 resistance to a viral entry inhibitor.

Acknowledgments

This research was supported by National Institute of Health grants CA73470, R41 AI46112, and AI35467; and facilities of the central AIDS virus, biostatistical, genetic sequencing, and flow cytometry cores of the Birmingham Center for AIDS Research (P30-AI-27767).

We thank Trimeris, Inc., Durham, N.C., for providing T-20 and for helpful comments regarding the manuscript. We also thank David Kabat for JC53 cells and Tranzyme Inc. for use of the JC53BL-13 reporter cell line.

Acknowledgments

REFERENCES

REFERENCES

References

  • 1. Adachi, A., H. E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, and M. A. Martin. 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol.59:284-291.
  • 2. Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of gp41 from the HIV envelope glycoprotein. Cell89:263-273. [[PubMed]
  • 3. Chen, C.-H., T. J. Matthews, C. B. McDanal, D. P. Bolognesi, and M. L. Greenberg. 1995. A molecular clasp in the human immunodeficiency virus (HIV) type 1 TM protein determines the anti-HIV activity of gp41 derivatives: implications for viral fusion. J. Virol.69:3771-3777.
  • 4. Condra, JH., et al. 1995. In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature374:569-571. [[PubMed][Google Scholar]
  • 5. Derdeyn, C. A., J. M. Decker, J. N. Sfakianos, Z. Zhang, W. A. O'Brien, L. Ratner, G. M. Shaw, and E. Hunter. 2001. Sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor. J. Virol.75:8605-8614.
  • 6. Derdeyn, C. A., J. M. Decker, J. N. Sfakianos, X. Wu, W. A. O'Brien, L. Ratner, J. C. Kappes, G. M. Shaw, and E. Hunter. 2000. Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120. J. Virol.74:8358-8367.
  • 7. Hart, T. K., R. Kirsh, H. Helleos, R. W. Street, D. M. Lambert, S. R. Petteway, J. Leary, and P. Bugelski. 1991. CD4 HIV-1 interactions: binding of soluble CD4 (sT4) to HIV-1 and HIV-1 infected cells induces shedding of envelope gp120. Proc. Natl. Acad. Sci. USA88:2189-2193.
  • 8. Hunter, E. 1997. Viral entry and receptors, p. 71-120. In J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • 9. Jiang, S., K. Lin, N. Strick, and A. R. Neurath. 1993. HIV-1 inhibition by a peptide. Nature365:113.. [[PubMed]
  • 10. Kilby, J. M., S. Hopkins, T. M. Venetta, B. DiMassimo, G. A. Cloud, J. Y. Lee, L. Alldredge, E. Hunter, D. Lambert, D. Bolognesi, T. Matthews, M. R. Johnson, M. A. Nowak, G. M. Shaw, and M. S. Saag. 1998. Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nat. Med.4:1302-1307. [[PubMed]
  • 11. Kilby, J. M., J. P. Lalezari, J. J. Eron, M. Carlson, C. Cohen, R. C. Arduino, J. C. Goodgame, J. E. Gallant, P. Volberding, R. L. Murphy, F. Valentine, M. S. Saag, E. L. Nelson, P. R. Sista, and A. Dusek. The safety, plasma pharmacokinetics, and antiviral activity of subcutaneous enfuvirtide (T-20), a peptide inhibitor of gp41-mediated virus fusion, in HIV-infected adults. AIDS Res. Hum. Retrovir., in press. [[PubMed]
  • 12. Li, Y., J. C. Kappes, J. A. Conway, R. W. Price, G. M. Shaw, and B. H. Hahn. 1991. Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: identification of replication-competent and -defective viral genomes. J. Virol.65:3973-3985.
  • 13. Lu, M., S. C. Blacklow, and P. S. Kim. 1995. A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat. Struct. Biol.2:1075-1082. [[PubMed]
  • 14. Moore, J. P., R. A. M. Keating, R. A. Weiss, and Q. J. Sattentau. 1990. Dissociation of gp120 from HIV-1 virions induced by soluble CD4. Science250:1139.. [[PubMed]
  • 15. Naldini, L., U. Blomer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. A. Verma, and D. Trono. 1996. In vivo gene delivery and stable gene transduction of nondividing cells by a lentivirus vector. Science272:263-267. [[PubMed]
  • 16. Platt, E. J., K. Wehrly, S. E. Kuhmann, B. Chesebro, and D. Kabat. 1998. Effects of CCR5 and CD4 cell surface concentrations on infection by macrophagetropic isolates of human immunodeficiency virus type 1. J. Virol.72:2855-2864.
  • 17. Rimsky, L. T., D. C. Shugars, and T. J. Matthews. 1998. Determinants of human immunodeficiency virus type 1 resistance to gp41-derived inhibitory peptides. J. Virol.72:986-993.
  • 18. Saag, M. S., E. A. Emini, O. L. Laskin, J. Douglas, W. I. Lapidus, W. A. Schleif, R. J. Whitley, C. Hildebrand, V. W. Byrnes, J. C. Kappes, et al. 1993. A short-term clinical evaluation of L-697,661, a non-nucleoside inhibitor of HIV-1 reverse transcriptase. New Eng. J. Med.329:1065-1072. [[PubMed]
  • 19. Sattentau, Q. J., and J. P. Moore. 1991. Conformational changes induced in the human immunodeficiency virus envelope glycoprotein by soluble CD4 binding. J. Exp. Med.174:407-415.
  • 20. Sattentau, Q. J., and J. P. Moore. 1993. The role of CD4 in HIV binding and entry. Philos. Trans. R. Soc. Lond. B Biol. Sci.342:59-66. [[PubMed]
  • 21. Schuurman, R., M. Nijhuis, R. van Leeuwen, P. Schipper, D. de Jong, P. Collis, S. A. Danner, J. Mulder, C. Loveday, C. Christopherson, et al. 1995. Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug-resistant virus populations in persons treated with lamivudine (3TC). J. Infect. Dis.171:1411-1419. [[PubMed]
  • 22. Sullivan, N., Y. Sun, J. Li, L. Hoffmann, and J. Sodroski. 1995. Replicative function and neutralization sensitivity of envelope glycoproteins from primary and T-cell line-passaged human immunodeficiency virus type 1 isolates. J. Virol.69:4413-4422.
  • 23. Wei, X., S. K. Ghosh, M. E. Taylor, V. A. Johnson, E. A. Emini, P. Deutsch, J. D. Lifson, S. Bonhoeffer, M. A. Nowak, B. H. Hahn, et al. 1995. Viral dynamics in human immunodeficiency virus type 1 invection. Nature373:117-122. [[PubMed]
  • 24. Weissenhorn, W., A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley. 1997. Atomic structure of the extodomain from HIV-1 gp41. Nature387:426-430. [[PubMed]
  • 25. Wild, C., T. Greenwell, and T. Matthews. 1993. A synthetic peptide from HIV-1 gp41 is a potent inhibitor of virus-mediated cell-cell fusion. AIDS Res. Hum. Retrovir.9:1051-1053. [[PubMed]
  • 26. Wild, C. T., T. Oas, C. B. McDanal, D. Bolognesi, and T. J. Matthews. 1992. A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition. Proc. Natl. Acad. Sci. USA89:10537-10541.
  • 27. Wild, C. T., D. C. Shugars, T. K. Greenwell, C. B. McDanal, and T. J. Matthews. 1994. Peptides corresponding to a predictive alpha-helical domain of HIV-1 gp41 are potent inhibitors of virus infection Proc. Natl. Acad. Sci. USA91:9770-9774.
  • 28. Wu, X., H. Liu, H. Xiao, J. A. Conway, E. Hunter, and J. C. Kappes. 1997. Functional RT and IN incorporated into HIV-1 particles independently of the Gag-Pol precursor protein. EMBO J.16:5113-5122.
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