Enhanced HIV-1 neutralization by antibody heteroligation

Abstract

Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti-HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation.

Fig. 1.

Design and production of HIV-gp120/41 BiAbs. (A) Schematic diagram shows the gp120/41 BiAbs made as scFv₂-Fc IgG-like molecules bearing one antibody binding site to gp120 and the other to gp41. scFv, single-chain variable fragment; V_H, heavy-chain variable domain; V_L, light-chain variable domain; H, hinge; L, (G₄S)₃ linker; C_H, heavy-chain constant domain; h-IgG1, human IgG1. (B) Silver staining and Western blot analyses of gp120/41 BiAbs and BiAb controls (anti-gp120/mGO53 heterodimers). (C) Binding analyses of 10-188 IgG antibody and scFv₂-Fc control to gp120 and gp140 antigens measured by ELISA. The x axis shows the antibody concentration (nM) required to obtain the ELISA values (OD_{405 nm}) indicated on the y axis. The dotted lines show mGO53 negative control (61). (D) Neutralizing activity of 10-188 IgG and scFv₂-Fc control, measured by TZM-bl assay. The x axis shows the antibody concentration (nM) required to achieve IC₅₀ for each virus indicated on the y axis. The MNR values comparing the IC₅₀ concentrations of 10-188 scFv₂-Fc and 10-188 IgG are given for each tested virus. All experiments were performed at least in duplicate. Error bars indicate SEM. *Not neutralized; **negative control.

Fig. 2.

Dual antigen binding of HIV-gp120/41 BiAbs. (A) SPR analyses of the interaction of the anti-gp120/41 BiAbs, IgG, and BiAb controls (BiAb ctr.) with the gp140, gp120, and gp41 ligands immobilized at low density on the sensor chips (100 RU). Graphs show SPR sensorgrams over time for the binding of the selected antibodies. (B) Graph shows SPR analysis of the interaction of the anti-gp120/41 BiAbs monovalently bound to gp120 immobilized on the low-density chip (100 RU) (binding 1) with in solution-injected gp41 (binding 2), as illustrated by the schematic diagram (Left). (C) SPR analyses show no interaction of the BiAb controls monovalently bound to gp120 immobilized on the low-density chip (100 RU) (binding 1) with in solution-injected gp41, as illustrated by the schematic diagram (Left). All experiments were performed at least in duplicate. Representative data are shown.

Fig. 3.

Neutralizing activity of HIV-gp120/41 BiAbs. (A) Graph shows neutralization curves of SS1196.1 pseudovirus for 10-188 BiAb and controls. The x axis shows the antibody concentration (nM) required to achieve 50% neutralization (IC₅₀), indicated by the dashed line. (B) Bar graph shows the MNR values comparing the IC₅₀ concentrations of the anti-gp120/41 BiAbs and parental anti-gp120 IgGs for each tested virus. (C) Pie charts show the frequency of virus variants showing enhanced neutralization with the anti-gp120/41 BiAbs compared with parental anti-gp120 IgG antibodies. (D) Dot graph shows the IC₅₀ concentrations of anti-gp120/41 BiAbs compared with controls for the neutralization of the selected HIV viruses. Median IC₅₀ values are indicated for each group by horizontal lines. (E) Bar graph shows the MNR values comparing the IC₅₀ concentrations of the parental anti-gp120 IgGs mixed with 5-25 anti-gp41 IgG and anti-gp120 IgG mAbs alone for the selected viruses. (F) Bar graph shows the fold decreases for the neutralization activity of the anti-gp120/41 BiAbs in presence of gp41^ID peptide against the selected pseudoviruses.