Characterization of human class-switched polymeric (immunoglobulin M [IgM] and IgA) anti-human immunodeficiency virus type 1 antibodies 2F5 and 2G12

Abstract

We have previously generated human monoclonal anti-human immunodeficiency virus type 1 (anti-HIV-1) antibodies 2F5IgG and 2G12IgG with an exceptional cross-clade neutralizing potential. 2F5IgG and 2G12IgG passively administrated to macaques were able to confer complete protection from both intravenous and mucosal challenge with pathogenic HIV-simian immunodeficiency virus chimeric strains and have shown beneficial effects in a phase-1 clinical trial. We now class-switched 2F5 and 2G12 to the immunoglobulin M (IgM) or IgA isotype, to enforce features like avidity, complement activation, or the potential to neutralize mucosal transmission. For this purpose we expressed functional polymeric 2F5 and 2G12 antibodies in CHO cells and evaluated their anti-HIV-1 activity in vitro. The class switch had a strong impact on the protective potential of 2F5 and 2G12. 2G12IgM inhibited HIV-1 infection of peripheral blood mononuclear cell cultures up to 28-fold-more efficiently than the corresponding IgG and neutralized all of the primary isolates tested. The 2F5 and 2G12 antibodies of all isotypes were able to interact with active human serum to inhibit viral infection. Furthermore, we demonstrated that polymeric 2F5 and 2G12 antibodies but not the corresponding IgGs could interfere with HIV-1 entry across a mucosal epithelial layer in vitro. Although polymeric 2F5 antibodies had only limited potential in the standard neutralization assay, the results from the mucosal assay suggest that 2F5 and 2G12 antibodies may have a high potential to prevent natural HIV-1 transmission in vivo.

FIG. 1.

Molecular distribution of the polymeric anti-HIV-1 antibodies. Western blots of denaturating nonreducing Tris-3 to 8% acetate (for IgM) and Tris-7% acetate (for IgA) gels were stained with goat anti-human κ light chain. The blots compare supernatants and concentrates containing 2F5IgM (lanes 2 and 3), 2G12IgM (lanes 6 and 7), 2F5IgA (lanes 8 and 9), 2F5IgG (lane 4), and human IgM from serum (lanes 1 and 5). The arrows indicate the positions of pentameric and monomeric IgM (M) and dimeric and monomeric IgA (A), respectively. The position of the 200- and 116-kDa molecular mass marker proteins are indicated.

FIG. 2.

Specificity ELISA. Comparison of 2F5IgA, 2F5IgM, and 2G12IgM CHO supernatants with 2F5IgG and 2G12IgG. Antibody concentrations (in nanograms/milliliter) are plotted against optical density (OD) values. For the specificity ELISA, we used the HIV-1 envelope antigens ELDKWA (amino acids 662 to 667) (A), gp160 (B), and gp120 (C) and a mouse anti-2G12 idiotype antibody (D). The samples were detected by anti-human κ light chain AP conjugate.

FIG. 3.

Neutralization curves for 2F5 and 2G12 antibodies against primary isolates. The dose-dependent inhibition of primary isolates was assessed by measuring p24 concentrations in HIV-infected PBMC cultures. The antibody dilutions (in micrograms/milliliter) are plotted against the p24 levels relative to p24 in the blank (without addition of antibody) (V_n/V₀). The horizontal lines on the y axes indicate the points of 50, 90, and 99% virus reduction. The primary isolates used were P7/366 (A), S2/04 (B), P3/366 (C), and P2/71 (D). MAb, monoclonal antibody.

FIG. 4.

Immunofluorescence analysis for transcytosis of 2F5 and 2G12 antibodies. A polarized monolayer of Me-180 cells grown on transwells was incubated with 2F5IgA, human sIgA, 2G12IgM, 2F5IgM, or 2F5IgG from the basal side. After 2 h, the cells were fixed and stained for the human κ light chain. The membrane cell layer was scanned from the basal to the apical side. Pictures A (basal) to E (apical) represent spaced confocal pictures of 2F5IgA-loaded cells. Pictures F and G represent pictures of the basal and the apical side of a cell layer loaded with human sIgA. Pictures H (basal) and I (intra-cellular) show cells loaded with 2G12IgM, pictures J (basal) and K (intracellular) show cells loaded with 2F5IgM, and picture L shows the basal side of cells loaded with 2F5IgG. Picture M shows the negative control (basal) without the addition of antibodies. The left side shows the micrograph, the right side shows the fluorescent signal.

FIG. 5.

Transwell assay of in vitro transepithelial neutralization. An epithelial monolayer was preincubated with the antibodies from the basal side before HIV-infected PBMCs were added from the apical side. After 3 h of coincubation, p24 levels on both sides of the transwell were determined by ELISA. The reduction is presented as the percentage of p24 found in the basal medium when incubated with the antibodies compared to p24 levels in the basal medium of the isotype control, representing uninhibited transcytosis of the virus.