Actinohivin, a broadly neutralizing prokaryotic lectin, inhibits HIV-1 infection by specifically targeting high-mannose-type glycans on the gp120 envelope

Abstract

The lectin actinohivin (AH) is a monomeric carbohydrate-binding agent (CBA) with three carbohydrate-binding sites. AH strongly interacts with gp120 derived from different X4 and R5 human immunodeficiency virus (HIV) strains, simian immunodeficiency virus (SIV) gp130, and HIV type 1 (HIV-1) gp41 with affinity constants (KD) in the lower nM range. The gp120 and gp41 binding of AH is selectively reversed by (alpha1,2-mannose)3 oligosaccharide but not by alpha1,3/alpha1,6-mannose- or GlcNAc-based oligosaccharides. AH binding to gp120 prevents binding of alpha1,2-mannose-specific monoclonal antibody 2G12, and AH covers a broader epitope on gp120 than 2G12. Prolonged exposure of HIV-1-infected CEM T-cell cultures with escalating AH concentrations selects for mutant virus strains containing N-glycosylation site deletions (predominantly affecting high-mannose-type glycans) in gp120. In contrast to 2G12, AH has a high genetic barrier, since several concomitant N-glycosylation site deletions in gp120 are required to afford significant phenotypic drug resistance. AH is endowed with broadly neutralizing activity against laboratory-adapted HIV strains and a variety of X4 and/or R5 HIV-1 clinical clade isolates and blocks viral entry within a narrow concentration window of variation (approximately 5-fold). In contrast, the neutralizing activity of 2G12 varied up to 1,000-fold, depending on the virus strain. Since AH efficiently prevents syncytium formation in cocultures of persistently HIV-1-infected HuT-78 cells and uninfected CD4+ T lymphocytes, inhibits dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated capture of HIV-1 and subsequent virus transmission to CD4+ T lymphocytes, does not upregulate cellular activation markers, lacks mitogenic activity, and does not induce cytokines/chemokines in peripheral blood mononuclear cell cultures, it should be considered a potential candidate drug for microbicidal use.

FIG. 1.

Kinetic analysis of AH interaction with III_B gp120 isolated from CHO cell cultures (A) and from the baculovirus system (C) and with HxB2 gp41 isolated from P. pastoris (E) using the SPR technology. Serial 2-fold analyte dilutions (covering a concentration range of from 2.5 to 80 nM) were injected over the surface of the gp120-bound (A to D) or gp41-bound (E and F) sensor chip. The experimental data (colored curves) were fit using the 1:1 binding model (black lines) to determine the kinetic parameters. The data are representative examples of the results of three independent experiments. The biosensor chip densities were 822 RU (or 6.9 fmol) for gp120 from CHO (A and B), 725 RU (or 6.0 fmol) for gp120 from baculovirus (C and D), and 637 RU (or 15.5 fmol) for gp41 (E and F). For the titration experiments, AH at a fixed concentration (20 nM) was incubated for 30 min with increasing concentrations of (α1,2-man)₃ (curve 1, 200 nM; curve 2, 2 μM; curve 3, 10 μM; curve 4, 20 μM; and curve 5, 200 μM) and injected over the three surfaces. The amplitudes at 3 min after injection were used to calculate the IC₅₀s.

FIG. 2.

The CBAs AH and HHA (0.5 μM) were subjected for binding to III_B gp120 (origin, CHO cells; chip density, 3,840 RU [∼32 fmol]) under both acidic (pH 4.0; red and blue) and neutral (pH 7.4; green and magenta) conditions. The association phase of AH and HHA was monitored for 3 min.

FIG. 3.

Kinetic analysis of the MAb 2G12 interaction with the III_B gp120 isolated from baculovirus (A) and CHO cell cultures (B) and with gp41 (HxB2) isolated from P. pastoris (C) using the SPR technology. Serial 2-fold analyte dilutions (covering concentration ranges of from 4 to 125 nM 2G12 for the gp120 interaction [A and B] and 4 nM to 4 μM for the gp41 interaction [C]) were injected over the surface. The experimental data (colored curves) were fit using the 1:1 binding model (black lines) to determine the kinetics. The same biosensor chip used for Fig. 1 was used in the experiment whose results are shown here.

FIG. 4.

(A) Competition between different CBAs (10 μM HHA [magenta], 10 μM UDA [green], 2 μM anti-gp120 G212 MAb [light blue]) with 0.5 μM AH (red) for binding to the III_B gp120 (CHO origin; chip density, 360 RU [∼3 fmol]). AH (10 μM) was injected (time point 1), followed after 2 min by injection of 10 μM AH in the presence of another CBA (time point 2). (B) The test compounds were injected over the surface at a high concentration (>100-fold the K_D).

FIG. 5.

Inhibition of the binding of anti-gp120 MAb 2G12 of HIV-1(NL4.3)-infected MT-4 cells in the presence of serial dilutions of AH: 1.6 μM (A), 0.32 μM (B), and 0.064 μM (C). Gray, blue, and red histograms represent the background fluorescence, MAb 2G12 binding when it was preincubated with AH, and MAb 2G12 binding, respectively. MFI, mean fluorescence intensity.

FIG. 6.

Schedule of selection of AH resistance development in HIV-1(III_B)-infected CEM cell cultures exposed to increasing AH concentrations. Arrows indicate the time points when virus isolates were taken for further characterization. AH_1 and AH_2 represent two independent subcultivation schedules in which for each passage suspensions of AH-exposed CEM cell cultures were transferred. AH_3 represents the series of subcultivations that were started from the last passage of AH_2 and for which the supernatants of the AH-exposed CEM cell cultures were transferred at each passage. AH_4 was split from the AH_1 selection series after the 46th subcultivation and independently subcultured for an additional 12 passages.

FIG. 7.

Mapping of the mutated glycosylation sites (deleted [red balls], unchanged [green balls], or created [blue ball]) in gp120 of HIV-1(III_B) strains isolated upon AH drug pressure in CEM cell cultures.

FIG. 8.

Alignment of the envelope protein sequences of a variety of laboratory HIV-1 strains, clinical HIV-1 isolates, HIV-2(ROD), SIV(mac251), and SIV(sm239). The sequences are derived from the NCBI database, and the alignment was performed using the MUSCLE program (http://www.ebi.ac.uk). The N-glycosylation motifs are indicated in yellow. The glycosylation sites that were found to be mutated in AH-exposed HIV-1(III_B) isolates are indicated in red.