Table I. Neutralizing activity of mAbs derived from rescued 2F5 complete (VH+/+ × VL+/+) hybridoma lines
Neutralization of B.MN and other HIV-1 isolates by purified mAbsa
IC50 for Neutralization of HIV-1 (μg/ml)
HIV-1 IsolateV3-1.4V3-4.1V3-6.2V3-10.1AID 3G11
Neutralization activity of V3-1.4 mAb: comparison of pentameric and monomeric IgMb
IC50 for Neutralization of HIV-1 (μg/ml)
HIV-1 Isolate2F5V3-1.4 PentamerV3-1.4 Monomer
  • Initial screens involved assaying supernatants from all 800 original wells plated and then rescreening all 58 secreting clones (i.e., with Ab production >0.05 μg/ml) from the 77 subcloned hybridoma lines using culture supernatants that were concentrated/normalized at >2 μg/ml for assessing neutralization of the HIV-1 B.MN isolate. Neutralization in culture supernatants was determined using the TZM-bl HIV-1 Env pseudorvirus infectivity assay as previously described (22) and using the MN clade B HIV-1 isolate (a strain particularly sensitive to neutralization by IgM bnAbs) as the initial test indicator line. Four hybridoma supernatants from these initial screens—V3-1.4, V3-4.1, V3-6.2, and V3-10.1—had 99.0, 91.2, 93.8, and 92.4% neutralization inhibition scores, respectively, whereas the other 54 hybridomas had ≤35%.

  • a Shown are 50% neutralization titers (IC50) concentrations of the affinity-purified IgM+ mAbs V3-1.4, V3-4.1, V3-6.2, and V3-10.1 that were required to neutralize each HIV-1 isolate shown in the TZM-bl assay. The purified IgM+ mAb of the hybridoma line AID 3G11, which is negative for MPER binding and poly-/self-reactivity (Fig. 4), was used as a negative control in the TZM-bl neutralization assay.

  • b Prior to use in neutralization assays, the original (pentameric) form of the purified V3-4.1 mAb or its reduced (monomeric) form was verified by Western blot and/or FPLC analysis. As a further comparison, neutralization activity was also measured for the original human 2F5 IgG mAb.