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Resistance Profile of a Novel Broadly Neutralizing Anti-HIV Monoclonal Antibody, KD-247, that Has Favorable Synergism with Anti-CCR5 Inhibitors in vitro
Kazuhisa Yoshimura*1, J Shibata1, A Honda1, T Murakami2, H Mitsuya3, A Koito1, and S Matsushita1
1Ctr for AIDS Res, Kumamoto Univ, Japan; 2Chemo-Sero-Therapeutic Res Inst, Kumamoto, Japan; and 3Ctr for AIDS Res, Kumamoto Univ, Japan
Background: KD-247,
a broadly neutralizing monoclonal
antibody, was generated by sequential immunization with V3 peptides derived
from HIV-1 subtype B primary isolates. In
this study, we induced HIV-1 variants escape from KD-247 in vitro using R5 virus, JR-FL, to increasing concentrations of
KD-247, and defined the virological properties of pseudotyped HIV-1 clone carrying the escape-associated env gene mutation. We also evaluated
the anti-HIV-1 interactions between KD-247 and various CCR5 inhibitors in vitro.
Methods: To
select HIV-1 variants resistant to KD-247 in
vitro, we exposed PM1-CCR5 cell,
which had high expression of CCR5 to JR-FL and
the virus was serially passaged in the presence of increasing concentrations
of KD-247. The monoclonal antibody concentration was increased to 1 mg/mL.
We determined the amino acid sequence of the gp120-encoding region
of the JR-FL
escape mutant from KD-247. To confirm that the mutation was
associated with the reduced sensitivity of the escape variant, we constructed a
pseudotyped virus containing the mutation. The multiple-drug-effect analysis of
Chou et al. was used to analyze the effects of combinations of KD-247 with CCR5
inhibitors.
Results: At passage 8 in the culture where JR-FL was
propagating in the presence of KD-247 (1000 mg/mL), 1 amino acid substitution, Gly to Glu at position 314
(G314E), in the V3-tip of gp120 was identified. To determine whether this
substitution was responsible for KD-247 resistance, a single-round replication assay was
performed. The pseudotype virus with G314E was
completely resistant to KD-247. Unexpectedly, this mutant virus was sensitive
to CCR5 inhibitors, RANTES, rsCD4, and anti-CCR5 monoclonal
antibody, but resistant to
anti-CD4 monoclonal antibody compared with wild type virus. We
also evaluated the anti-HIV-1 interactions between KD-247 and various CCR5 inhibitors in vitro. Combinations of
KD-247 with the CCR5 inhibitors showed highly synergistic interactions at
the 50, 75, and 90% inhibitory concentrations.
Conclusions: The
present data suggest that KD-247 has at least 3 advantages: the viral acquisition of resistance is
substantially needed a very high concentration of KD-247 in vitro; the escaped variant becomes more sensitive to CCR5
inhibitors and rsCD4, and less dependent on CD4 binding for entry; and
combinations of KD-247 with the CCR5 inhibitors show highly synergistic
interactions.
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