17th International HIV Drug Resistance Workshop 10-14 June 2008, Sitges, Spain

RESISTANCE TO CCR5mAb RoAb3952 IS ASSOCIATED WITH A SHIFT IN BINDING FROM THE EXTRACELLULAR DOMAIN 2 TO THE N TERMINUS OF CCR5

Antivir Ther. 2008; 13(Suppl. 3):A4 (abstract no. 2)

A Jekle¹, M Chhabra¹, E Chow¹, S Meier¹, S Sankuratri¹, M Brandt², N Cammack¹ and G Heilek^1
1Roche Palo Alto LLC, Palo Alto, CA, USA ²Roche Diagnostics GmbH, Penzberg, Germany

BACKGROUND: RoAb3952 is a de-immunized version of the previously published mouse anti-CCR5 monoclonal antibody (mAb) ROAb14. RoAb3952, which binds to the extracellular loop 2 (ECL2) of CCR5, has potent antiviral activity in vitro. To determine the pattern of viral resistance against RoAb3952, we passaged two HIV-1 isolates in human peripheral blood mononuclear cells (PBMCs) in the presence of RoAb3952.

METHODS: The CCR5-tropic HIV-1 isolates Bal and CC1/85 were passaged in vitro in the presence of increasing RoAb3952 concentrations. CD8-depleted PBMC and genetically diverse, high titre viruses were used to facilitate fast resistance development. The sensitivity of resistant and no drug control (NDC) viruses to RoAb3952 and other CCR5mAbs was measured in PBMCs and single- cycle assays. The env genes of these viruses were sequenced to identify mutations associated with resistance to RoAb3952.

RESULTS: Passaging of Bal and CC1/85 in the presence of increasing concentrations of RoAb3952 resulted in the selection of two highly RoAb3952-resistant viruses. The NDC viruses remained sensitive to RoAb3952. In the presence of another CCR5mAb (RoAb13) recognizing the N-terminal of CCR5, the phenotype was reversed; both the Bal_3952res and CC1/85_3952res virus were more sensitive to RoAb13 compared with their respective NDC virus, suggesting a shift in the binding ability of these Envs. Sequence analysis revealed that Bal_3952res differed from Bal_NDC in only two amino acid positions (K163N and S531A). K163N is located in the V2 region, whereas S531A is located in gp41. S531A was also detected in CC1/85_3952res, which had additional mutations throughout gp120. Furthermore, pseudotyped virus containing Envs of CC1/85_3952res and Bal_3952res, but not of Bal_NDC and CC1/85_NDC, were also resistant to RoAb3952 and the CCR5mAb 2D7.

CONCLUSIONS: Using high titred, genetically diverse viruses and CD8-depleted human PBMCs, we were able to select virus strains resistant to the CCR5mAb RoAb3952. Resistance to RoAb3952 was associated with a change in the binding properties of the virus; RoAb3952-sensitive virus still required binding to ECL2 of CCR5 for viral entry, whereas RoAb3952-resistant virus was capable of efficiently using the N-terminal loop of CCR5 for viral entry in the presence of an antibody that binds to CCR5 ECL2.

2008-06-10
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