Table 2.
Summary of isotypic distribution, MPER reactivities, and HIV-1 neutralization profiles of B cell hybridomas derived from peripheral immune tissues of immunized 2F5 complete KI mice
| A. Primary screens of hybridoma
clonesa | |||||
|---|---|---|---|---|---|
| Fusion name (method) | Mouse genotype/background | Tissue source of B-cells | Neutralizing clones/MPER±clones assayed b,c,d | ||
| IgM | IgG | Total (IgM+G) | |||
| V5 (electrofusion) | 2F5 VH+/+ × VL+/+/WT (B6) | Spleen | 342/344 | 149/153 | 491/497 |
| LN | 54/54 | 15/16 | 69/70 | ||
| Total % that neutralize | 396/398 (99.5%) | 164/169 (97.0%) | 560/567 (98.8%) | ||
| V6 (PEGe) | 2F5 VH+/+ × VL+/+/Eμ-bcl2 tg (B6) | Spleen | 39/45 | 46/48 | 85/93 |
| V6 (electrofusion) | Spleen | 263/271 | 907/926 | 1170/1197 | |
| LN | 14/15 | 37/38 | 51/53 | ||
| Total % that neutralize | 316/354 (89.3%) | 990/1012 (97.8%) | 1306/1343 (97.2%) | ||
| B. Neutralization profiles of
purified IgG+ mAbs selected from cloned hybridoma
linesf | |||||
|---|---|---|---|---|---|
| Clone ID | IC50 concentration (μg/ml) for neutralization of HIV-1 | ||||
| A.92UG037.1 | B.MN.3 | B.SF162.LS | B.6535.3 | B.BG1168.1 | |
| V4-SE 2 CL1-1 | 0.12 | <0.01 | 0.34 | 7.61 | 0.78 |
| V4-SE 6 CL1-1 | 0.12 | <0.01 | 0.45 | 4.81 | 1.04 |
| V4-SE 8 CL1-1 | 0.14 | 0.01 | 0.54 | 5.6 | 1.1 |
| V4-SE 361 CL1-1 | 0.21 | 0.03 | 3.04 | 2.75 | 2.37 |
| V4-SP 1B5 CL1-1 | 0.32 | 0.06 | 1.18 | 17.3 | 1.53 |
| V4-SE 18 CL1-2 | 0.06 | <0.01 | 0.23 | 2.81 | 0.64 |
| V4-SE 372 CL1-1 | 0.14 | <0.01 | 0.38 | 7.19 | 1.04 |
| V4-SP 1G3 CL1-1 | 0.14 | 0.02 | 0.55 | 7.66 | 0.98 |
| V5-SP 3H6 CL1-1 | 0.24 | 0.03 | 2.42 | 2.37 | 2.55 |
| V5-SE 4 CL1-1 | 0.13 | <0.01 | 0.38 | 6.48 | 1.04 |
| V5-SE 10 CL1-1 | 0.16 | 0.01 | 0.5 | 9.88 | 0.8 |
| V5-SP 3B11G | 0.13 | <0.01 | 0.38 | 7.23 | 0.97 |
| h2F5g | 0.28 | 0.03 | 1.49 | 6.93 | 1.93 |
Fusions were performed using NSO myeloma fusion partner lines using previously described methods (12) and represent separate experiments, each using individual immunized mice sacrificed 4 d after either fifth or sixth boosts. Data shown represent clones identified in primary screens of fusions in which total splenocytes or LN cells (lympholyte-M separated) were plated at limiting dilutions (1000 and 10000 cells/well for electrofusions and PEG fusions, respectively) in 96-well plates.
Non-clonal wells i.e. with>1 isotype, either as identified in ELISAs or which were subcloned for further analysis and had conflicting isotype identity (based on DNA sequence analysis) were excluded from this summary.
Secreting wells were identified by ELISA, using a general anti-mouse Ig H chain (G+M+A) reagent, and isotypes of secreting clones were subsequently determined by using anti-mouse H chain γ, μ, and α-specific reagents in ELISAs. MPER reactivity assays of culture supernatants were performed as previously described (4,11,12,26) using a plate-bound peptide SP62, which encodes the nominal 2F5-specific MPER epitope.
Neutralization in culture supernatants was determined using the TZM-bl HIV-1 Env pseudorvirus infectivity assay (Seaman et al., 2010), using the HIV-1 B.MN3 isolate as the test strain for primary screens and using >50% neutralization inhibition scores as the cutoff for positive neutralization activity, as previously described (12,31). The majority of non-neutralizing well supernatants were found not to neutralize due to low supernatant Ig concentration.
PEG=polyethylene glycol-mediated hybridoma fusion.
Shown are 50% neutralization concentrations (IC50) of affinity-purified mAbs required to neutralize each HIV-1 isolate listed in the TZM-bl cell assay.
Affinity-purified h2F5 (recombinant human 2F5) was used as a positive control; values shown are averages of three experiments.