Background: The high-affinity humanized monoclonal antibody (MAb) KD-247 reacts with a tip region in gp120-V3 and cross-neutralizes primary isolates with a matching neutralization sequence motif.
Methods: We induced an HIV-1 variant that was resistant to KD-247 by exposing the JR-FL virus to increasing concentrations of KD-247 in PM1/CCR5 cells, which expressed high levels of CCR5 in vitro. We determined the amino acid sequence of the gp120-encoding region of the JR-FL escape mutant from KD-247. To confirm that this substitution was responsible for the KD-247-resistance, a single-round replication assay was performed. We further evaluated the anti-HIV-1 interactions between KD-247 and various CCR5 inhibitors in vitro.
Results: At passage 8 of the culture in the presence of 1000 μg/ml KD-247, one amino acid substitution, Gly to Glu at position 314 (G314E), was identified in the V3-tip of gp120. A pseudotyped virus with the G314E mutation was highly resistant to KD-247. Unexpectedly, this mutant virus was sensitive to CCR5 inhibitors, RANTES, recombinant human soluble CD4 (rsCD4) and an anti-CCR5 MAb, but resistant to an anti-CD4 MAb, compared with the wild-type virus. We also found that combinations of KD-247 and CCR5 inhibitors were highly synergistic.
Conclusions: The present data suggest that KD-247 has certain advantages for possible passive immunotherapy. They are: high concentrations of KD-247 are needed for viral acquisition of KD-247 resistance; the escape variants are more sensitive to CCR5 inhibitors and rsCD4; and there are high levels of synergism between KD-247 and CCR5 inhibitors at all concentrations tested.
From the aDivision of Clinical Retrovirology and Infectious Diseases, Center for AIDS Research, Graduate School of Medical Sciences, Kumamoto, University, Kumamoto, Japan
bDepartment of Medical Virology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
cThe Chemo-Sero-Therapeutic Research Institute, Kyokushi, Kikuchi, Kumamoto, Japan
dDepartment of Internal Medicine II, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.
Received 21 February, 2006
Accepted 27 July, 2006
Correspondence to S. Matsushita, Division of Clinical Retrovirology and Infectious Diseases, Center for AIDS Research, Kumamoto University, Kumamoto 860-0811, Japan. Tel: +81 96 373 6536; fax: +81 96 373 6537; e-mail: firstname.lastname@example.org
These data were presented at the Thirteenth Conference on Retroviruses and Opportunistic Infections. Denver, CO, February 2006 [abstract 506].
Note: K. Yoshimura and J. Shibata contributed equally to this work.