Structural basis for differential neutralization of ebolaviruses

Abstract

There are five antigenically distinct ebolaviruses that cause hemorrhagic fever in humans or non-human primates (Ebola virus, Sudan virus, Reston virus, Taï Forest virus, and Bundibugyo virus). The small handful of antibodies known to neutralize the ebolaviruses bind to the surface glycoprotein termed GP₁,₂. Curiously, some antibodies against them are known to neutralize in vitro but not protect in vivo, whereas other antibodies are known to protect animal models in vivo, but not neutralize in vitro. A detailed understanding of what constitutes a neutralizing and/or protective antibody response is critical for development of novel therapeutic strategies. Here, we show that paradoxically, a lower affinity antibody with restricted access to its epitope confers better neutralization than a higher affinity antibody against a similar epitope, suggesting that either subtle differences in epitope, or different characteristics of the GP₁,₂ molecules themselves, confer differential neutralization susceptibility. Here, we also report the crystal structure of trimeric, prefusion GP₁,₂ from the original 1976 Boniface variant of Sudan virus complexed with 16F6, the first antibody known to neutralize Sudan virus, and compare the structure to that of Sudan virus, variant Gulu. We discuss new structural details of the GP₁-GP₂ clamp, thermal motion of various regions in GP₁,₂ across the two viruses visualized, details of differential interaction of the crystallized neutralizing antibodies, and their relevance for virus neutralization.

Keywords: Ebola; Filovirus; Sudan virus; antibodies; ebolavirus; neutralization: glycoprotein; structure.

Figure 1

Cartoon representation of trimer of SUDV-Bon GP_1,2 bound to 16F6. The GP₁ chains are colored in various shades of blue and GP₂ chains are colored in white. The light chain of 16F6 is shown in pale yellow and the heavy chain of 16F6 is colored bright orange. The sugar residues on the glycoprotein are shown in ball and colored cyan. Lysine residues critical for receptor binding are shown in ball and stick and colored orange.

Figure 2

Secondary structure interactions between SUDV-Bon GP₁ (colored blue and purple) and GP₂ (colored light grey). (A) Close up of the continuous seven-stranded twisted β sheet formed by five β strands of the GP₁ base (β2, β1, β13, β3, and β6 in blue) and two antiparallel β strands of the GP₂ internal fusion loop (β19 and β20 in grey). (B) The first heptad repeat of GP₂ (which contains the grey coils labeled HR1A and HR1B and the loop afterwards) is clamped by a horseshoe surface formed by five β strands of GP₁ base (β12, β5, β6, β3, β13), a second small β sheet of the GP₁ base (β1 and β2) and a glycan (pink ball-and-stick). To orient the reader, key residues are labeled.

Figure 3

Interaction of residues in the chain reversal region in (A) the prefusion SUDV-Bon GP_1,2 and (B) the post fusion EBOV-May GP₂. Prefusion SUDV-Bon is used here as it is better ordered than prefusion EBOV-May. Interacting residues are shown in ball and stick. In (A), different monomers of GP₁ are colored blue and purple and the three copies of GP₂ are all colored light grey. In (B), the three copies of GP₂ are colored different shades of grey. Equivalent residues in the 3-fold related protomers are labeled with ´ and ´´ respectively. Hydrogen bonds are shown as red dashed lines. The residue R609* in the postfusion form is an engineered mutation to replace the cysteine residue (C609) in the native protein that is involved in a disulfide bond with C53 of GP₁.

Figure 4

Residues at the interface of SUDV-Bon GP_1,2 and 16F6 (cutoff distance of 3.5 Å). GP₁ is colored purple, GP₂ is colored white, the 16F6 heavy chain is colored orange and the light chain is colored pale yellow. Hydrogen bonds are shown as red dashed lines.

Figure 5

Comparison of thermal factors (B-factors) of various regions of SUDV GP_1,2 (Panels A and B). Regions of low thermal mobility are deep blue (deep blue color set at ~ 80 Ų), whereas regions of high thermal mobility are red (deep red color set at ~ 220 Ų). The glycan cap and C-terminal “stem” regions of SUDV GP_1,2 have higher B-values than the core region, indicating higher motion in those regions.

Figure 6

Plots of number of deuterons vs. time. These compare the deuteration levels of key peptides of EBOV GP_1,2 (shown in blue) to their counterparts in SUDV-Gul GP_1,2 (shown in magenta), measured over a time scale of 10-1000 sec using deuterium exchange mass spectrometry [17].

Figure 7

The N-terminal peptide of GP₂ in (A) EBOV-May GP_1,2 and (B) SUDV-Bon GP_1,2. In both panels, GP₂ is shown in ball-and-stick and colored grey. GP₁ is colored purple and the heavy and light chains of the antibody are colored bright orange and pale yellow, respectively. Note that the first eight residues of GP₂ are disordered in SUDV-Bon/-Gul GP₂ and are shown as black dots. Hydrogen bonds in (A) are shown as dashed red lines. There are no hydrogen bonds observed in (B) as the N-terminal peptide is disordered. The glycan residue connected to N563 is shown in ball and stick and colored light pink.

Figure 8

Cartoon illustration of the different modes of antibody binding. (A) KZ52 binds perpendicular to the central axis of GP_1,2 (parallel to the viral membrane), while 16F6 recognizes the overlapping epitope from an acute angle. The central axes of the two Fabs make a 50° angle with each other, and the constant portion of the 16F6 Fab is 50 Å lower than that of KZ52. Fab heavy chains are colored orange, while light chains are colored yellow. Approximate location of the viral membrane is indicated by a dotted line. C terminal portions of GP₂ that anchor the visualized trimer to the viral membrane are disordered and not drawn here. (B) Model of a 16F6 IgG binding SUDV GP_1,2 (purple) based on the 16F6 Fab-SUDV-Bon GP_1,2 complex (PDB: 3VE0). (C) Model of a KZ52 IgG binding EBOV-May GP_1,2 (blue) based on the KZ52 Fab-EBOV-May GP_1,2 complex structure (PDB: 3CSY). The IgG and GP_1,2 fragments are drawn to scale in the models.

Figure 9

The ability of 16F6 and KZ52 IgGs to neutralize SUDV GP_1,2-bearing VSIV or EBOV GP_1,2-bearing VSIV, plotted in a log (A) and linear (B) scales. VSIV particles were pre-incubated with the indicated concentrations of antibody for one hour at room temperature, and then exposed to Vero cells at 37° C. Viral infectivity was scored at 16 hours post-infection. The starting titers were 9×10⁷ IU/ml for EBOV and 3×10⁷ IU/ml for SUDV on Vero cells [16].