eCD4-Ig Limits HIV-1 Escape More Effectively than CD4-Ig or a Broadly Neutralizing Antibody

Abstract

The engineered antibody-like entry inhibitor eCD4-Ig neutralizes every human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus isolate it has been tested against. The exceptional breadth of eCD4-Ig derives from its ability to closely and simultaneously emulate the HIV-1 receptor CD4 and coreceptors, either CCR5 or CXCR4. Here we investigated whether viral escape from eCD4-Ig is more difficult than that from CD4-Ig or the CD4-binding site antibody NIH45-46. We observed that a viral swarm selected with high concentrations of eCD4-Ig was increasingly resistant to but did not fully escape from eCD4-Ig. In contrast, viruses selected under the same conditions with CD4-Ig or NIH45-46 fully escaped from those inhibitors. eCD4-Ig-resistant viruses acquired unique changes in the V2 apex, V3, V4, and CD4-binding regions of the HIV-1 envelope glycoprotein (Env). Most of the alterations did not directly affect neutralization by eCD4-Ig or neutralizing antibodies. However, alteration of Q428 to an arginine or lysine resulted in markedly greater resistance to eCD4-Ig and CD4-Ig, with correspondingly dramatic losses in infectivity and greater sensitivity to a V3 antibody and to serum from an infected individual. Compensatory mutations in the V3 loop (N301D) and in the V2 apex (K171E) partially restored viral fitness without affecting serum or eCD4-Ig sensitivity. Collectively, these data suggest that multiple mutations will be necessary to fully escape eCD4-Ig without loss of viral fitness.IMPORTANCE HIV-1 broadly neutralizing antibodies (bNAbs) and engineered antibody-like inhibitors have been compared for their breadths, potencies, and in vivo half-lives. However, a key limitation in the use of antibodies to treat an established HIV-1 infection is the rapid emergence of fully resistant viruses. Entry inhibitors of similar breadths and potencies can differ in the ease with which viral escape variants arise. Here we show that HIV-1 escape from the potent and exceptionally broad entry inhibitor eCD4-Ig is more difficult than that from CD4-Ig or the bNAb NIH45-46. Indeed, full escape was not observed under conditions under which escape from CD4-Ig or NIH45-46 was readily detected. Moreover, viruses that were partially resistant to eCD4-Ig were markedly less infective and more sensitive to antibodies in the serum of an infected person. These data suggest that eCD4-Ig will be more difficult to escape and that even partial escape will likely extract a high fitness cost.

Keywords: CCR5; CD4; HIV-1; eCD4-Ig; viral entry.

FIG 1

Neutralization of SHIV-SF162P3 swarms after extensive passage in the presence of NIH45-46, CD4-Ig, or eCD4-Ig. SHIV-SF162P3 was amplified for three passages on GHOST CCR5⁺ cells in the absence of inhibitor and then was passaged for 60 rounds of selection in the presence of NIH45-46, CD4-Ig, or eCD4-Ig or in the absence of any inhibitor, as indicated. Inhibitor concentrations were determined from the estimated IC₉₀ values for the swarm at a given passage, up to 100 μg/ml. Passages were conducted in 3 (no inhibitor control), 4 (CD4-Ig), or 5 (NIH45-46 and eCD4-Ig) independent wells. Neutralization of each selected swarm was measured for each inhibitor, as indicated at the top of each panel. Dashed lines indicate neutralization curves for individual wells, and solid lines indicate averages among wells selected with the same inhibitor. Note that swarms selected with NIH45-46 (top right, green) and CD4-Ig (bottom left, red) were fully resistant to the respective inhibitors, whereas virus selected with eCD4-Ig remained partially sensitive to this inhibitor (bottom right, blue) but was fully resistant to CD4-Ig (bottom right, red). Error bars represent standard errors of the means (SEM).

FIG 2

Env substitutions found in SHIV-162P3 swarms selected by eCD4-Ig. A plot of Env amino acid sequences from viruses passaged in the absence of inhibitor or in the presence of eCD4-Ig was generated with the Highlighter utility. A single SHIV-SF162P3 isolate (GenBank accession no. KF042063.1) was used as a reference sequence. Bars indicate changes from this reference sequence to the amino acids indicated. The corresponding regions of Env are indicated at the bottom of the figure. C1 to C5, gp120 constant regions 1 to 5; V1 to V5, gp120 variable regions 1 to 5; SP, signal peptide; FP, fusion peptide; TM, transmembrane region.

FIG 3

Repeated substitutions in key regions of Env unique to eCD4-Ig-selected swarms. Env mutations present in several individual isolates, not found in control swarms, are indicated. Sequences from the SHIV-SF162P3 reference sequence found in the apex region (residues 168 to 171), the base of V3 (residues 296 to 306), V4 (residues 382 to 390), and the CD4-binding site (residues 425 to 432) are shown. Changes from this reference sequence are shown, along with the number of independent wells in which a given pattern was observed. Note that K171E and Q428K emerged independently in several wells in combination with a mutation in the V3 base.

FIG 4

Neutralization of Env variants bearing eCD4-Ig-selected substitutions. HIV-1 pseudotyped with Env of the SHIV-SF162P3 sequence or the same Env modified to include the indicated eCD4-Ig-selected substitutions was used to infect TZM-bl cells in the presence of the indicated concentrations of CD4-Ig or eCD4-Ig. Luciferase activity, normalized to that for each variant in the absence of inhibitor, is shown. Error bars represent SEM. The experiment is representative of two or three with similar results. Note that only variants bearing a substitution at Q428 were more resistant to these inhibitors.

FIG 5

Lower infectivity of partially eCD4-Ig-resistant Env variants. A luciferase-encoding HIV-1 pseudovirus pseudotyped with Env of the SHIV-SF162P3 reference sequence or the same Env modified to include the indicated eCD4-Ig-selected substitutions was incubated at the indicated p24 amounts with either HOS.CCR5 or Cf2Th-CCR5 cells stably expressing human CD4. Luciferase activity was measured 36 to 48 h postinfection. Error bars represent SEM, and the results are representative of two independent experiments. Note that variants bearing the Q428K substitution were markedly less infective than the reference SHIV-SF162p3 Env sequence or other Env variants.

FIG 6

Characterization of eCD4-Ig-induced residue changes with monoclonal and polyclonal antibodies. TZM-bl cells were incubated with HIV-1 pseudoviruses pseudotyped with Env of the SHIV-SF162P3 sequence or variants including eCD4-Ig-selected substitutions, in the presence of the indicated concentrations of the neutralizing antibodies N6 (CD4-binding site), 10-1074 (V3 glycan), 447-52D (V3 loop), or E51 (CD4-induced) or with serum from an HIV-1-positive person. Results are representative of two independent experiments. Error bars represent SEM. Note that V3 loop substitutions increased sensitivity to 447-52D and E51 and variants with the Q428K substitution were more susceptible to serum neutralization.

FIG 7

Modeling of Env trimers, highlighting some of the eCD4-Ig selected mutated residues. (A) Model of apex V2 glycan bNAb PG9 in complex with the BG505 SOSIP.664 trimer (PDB accession no. 5VJ6) (67). gp120 is depicted in tan, gp41 in pink, and PG9 in gray. (B) Detailed view of the structure in panel A. Apex residue K171, which is altered to glutamic acid in some eCD4-Ig-selected Env variants, is highlighted in magenta. (C) Model of CD4-induced bNAb 412d heavy chain (412d_HC) in complex with an Env trimer in a partially open conformation, created by combining the crystal structure of BG505 SOSIP gp140 with that of 412d complexed with HIV-1 YU2 gp120 (PDB accession no. 4NCO and accession no. 2QAD) (68, 69). 412d_HC is depicted in green. (D) Detailed view of the structure in panel C. Residue N301, which is altered to aspartic acid in some eCD4-Ig-selected Env variants, is shown in light blue. (E) Model of the BG505 DS-SOSIP trimer in complex with CD4 (PDB accession no. 5U1F) (70). CD4 is depicted in dark blue. (F) Detailed view of the structure in panel E. CD4-binding site residue Q428, which is altered to lysine or arginine in some eCD4-Ig-selected Env variants, is highlighted in green.