Human Anti-HIV-1 gp120 V3 Recombinant Antibody (clone 2424)

Titration curves of the six V3 MAbs against JRFL. Neutralization assay was performed with JRFL pseudovirus and TZM.bl cells. MAbs were titrated and incubated with virus at 37°C for 1 h. TZM.bl cells were then added to the virus-MAb mixtures in DMEM containing 10% fetal bovine serum and DEAE-dextran. Neutralization activity was assessed 48 h later using the luciferase detection system. The parvovirus-specific MAb 1418 was included as a negative control.

Kumar, R., Pan, R., Upadhyay, C., Mayr, L., Cohen, S., Wang, X. H.,... & Gorny, M. K. (2015). Functional and structural characterization of human V3-specific monoclonal antibody 2424 with neutralizing activity against HIV-1 JRFL.Journal of virology, JVI-01280.

Neutralization of JRFL pseudovirus by V3 MAbs 2424, 447-52D, 3074, and 3869 was evaluated after treatment with or without sCD4. Virus was pretreated with 2.5 μg/ml of sCD4 for 30 min at 37°C or left untreated and then incubated with serially diluted MAb for 1 h at 37°C. TZM.bl cells were added to virus-MAb mixtures in DMEM containing 10% fetal bovine serum and DEAE-dextran. After 48 h, virus infection was measured based on β-galactosidase activity.

Kumar, R., Pan, R., Upadhyay, C., Mayr, L., Cohen, S., Wang, X. H.,... & Gorny, M. K. (2015). Functional and structural characterization of human V3-specific monoclonal antibody 2424 with neutralizing activity against HIV-1 JRFL.Journal of virology, JVI-01280.

HIV-1 JRFL pseudoviruses bearing wild-type or mutated Envs were produced in transfected 293T cells and incubated by titrated V3 MAbs (2424, 447-52D, 2219, 2557, 3074, and 3869) for 1 h at 37°C. TZM.bl cells were then added to the virus-MAb mixtures in complete DMEM containing DEAE-dextran. Neutralization activity was assessed after 48 h.

Kumar, R., Pan, R., Upadhyay, C., Mayr, L., Cohen, S., Wang, X. H.,... & Gorny, M. K. (2015). Functional and structural characterization of human V3-specific monoclonal antibody 2424 with neutralizing activity against HIV-1 JRFL.Journal of virology, JVI-01280.

The binding of MAbs (1 μg/ml) to gp120 used at 1 μg/ml to coat the well surface was detected by ELISA. An irrelevant control MAb, MAb 1418, was also tested in parallel and displayed no binding to any gp120 in the panel (OD405 ≤ 0.2; data not shown).

Kumar, R., Pan, R., Upadhyay, C., Mayr, L., Cohen, S., Wang, X. H.,... & Gorny, M. K. (2015). Functional and structural characterization of human V3-specific monoclonal antibody 2424 with neutralizing activity against HIV-1 JRFL.Journal of virology, JVI-01280.

(A) HIV-1 JRFL pseudoviruses were produced in transfected 293T cells in the absence or presence of 25 μM kifunensine (JRFLKif) and tested for neutralization by V3 MAbs (2424, 447-52D, 2219, 2557, 3869, and 3074), MAb 2G12, CD4bs MAb b12, or an irrelevant MAb, MAb 1418. (B) HIV-1 JRFL pseudoviruses were produced in 293T cells with or without 20 μM swainsonine (JRFLSwain) and tested for neutralization by V3 MAbs 2424 and 447-52D. Serially diluted MAbs were incubated with virus at 37°C for 1 h. TZM.bl cells were then added to the virus-MAb mixtures in complete DMEM containing DEAE-dextran. Neutralization activity was assessed after 48 h. Results with 2424 and 447-52D were averages from duplicate wells, and data from one representative experiment are shown. (C) V3 MAb reactivity with gp120 JRFL was assessed in ELISA. Recombinant gp120 protein (1 μg/ml) was coated on 96-well plates and reacted with serially diluted V3 MAbs.

Kumar, R., Pan, R., Upadhyay, C., Mayr, L., Cohen, S., Wang, X. H.,... & Gorny, M. K. (2015). Functional and structural characterization of human V3-specific monoclonal antibody 2424 with neutralizing activity against HIV-1 JRFL.Journal of virology, JVI-01280.