Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection

Abstract

Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.

Keywords: Eastern equine; aerosol; encephalitis; human antibodies; monoclonal; neutralizing; prophylaxis; therapy.

Figure 1.. Human anti-EEEV mAbs isolated from an EEEV survivor potently neutralize Sindbis (SINV)/EEEV and WT EEEV.

(A–B) Representative neutralization curves of neutralizing human anti-EEEV mAbs (A) or Fabs (B) against SINV/EEEV. Neutralization curves of potent (left) or moderate (right) neutralizing human anti-EEEV mAbs (open squares; A) or Fab molecules (open circles; B) against SINV/EEEV with mAb (A) or Fab (B) concentration (nM) on the x-axis and % relative infectivity on the y-axis. A positive control mouse mAb, EEEV-86 (dark purple) (Kim et al., 2019), and a negative control mAb, rDENV-2D22 (black), were included. (C) Neutralization curves of human anti-EEEV mAbs against EEEV. Neutralization curves of potent (left) or moderate (right) neutralizing human anti-EEEV mAbs (open diamonds) against EEEV with mAb concentration (nM) on the x-axis and % relative infectivity on the y-axis. A positive control mouse anti-EEEV ascites fluid, ATCC (+)ve, and a negative control mAb (black), were included. (D) Half-maximal inhibitory concentration (IC₅₀) values (pM) for human anti-EEEV mAbs or Fabs against SINV/EEEV and mAbs against EEEV strain FL93–939. IC₅₀ values (pM) for neutralizing human anti-EEEV mAbs or Fabs against SINV/EEEV or mAbs against EEEV are indicated in the table. Neutralizing human anti-EEEV mAbs are listed in order of increasing IC₅₀ value against SINV/EEEV. IC₅₀ value in pM is indicated by the orange heat map (<33 [dark orange], 33.01 to 333 [medium orange], 333.01 to 3,333 [light orange], <10,000 [lightest orange]). Isotype is indicated as heavy chain (IgG1 or IgA1), light chain (κ or λ) as determined by antibody gene sequencing. Data in A–B represent mean ± SD of technical triplicates and are representative of three independent focus reduction neutralization test (FRNT) experiments. Data in C represent mean ± SD of technical triplicates of a plaque reduction neutralization test (PRNT) experiment.

Figure 2.. Neutralizing human anti-EEEV mAbs bind to SINV/EEEV particles and/or recombinant EEEV E2 glycoprotein.

(A) Binding ratio of neutralizing human anti-EEEV mAbs to SINV/EEEV particles versus recombinant monomeric EEEV E2 glycoprotein. A dotted line indicates 32 pM EC₅₀ values for binding, revealing distinct binding patterns of human anti-EEEV mAbs to SINV/EEEV particles and EEEV E2 glycoprotein. Neutralizing human anti-EEEV mAbs are labeled with the anti-EEEV mAb name and are colored according to binding group (Group 1 [red] = virus>protein binding; Group 2 [green] = strong (SINV/EEEV EC₅₀ = <32 pM) virus » protein binding; Group 3 [purple] = weak (SINV/EEEV EC₅₀ = >32 pM) virus » protein binding; Group 4 [orange] = protein>virus binding). (B) EC₅₀ values (pM) for binding of neutralizing human anti-EEEV mAbs to SINV/EEEV particles or EEEV E2 glycoprotein. Neutralizing human anti-EEEV mAbs are listed in order of binding group and increasing EC₅₀ value for binding to SINV/EEEV particles. EC₅₀ value in pM is indicated by the blue heat map (<32 [dark blue], 32.01 to 100 [medium blue], 100.01 to 320 [light blue], <10,000 [lightest blue]). Ratio of binding to SINV/EEEV particles versus EEEV E2 glycoprotein is indicated as the ratio of EC₅₀ values, corresponding to Figure 2A. Increasing depth of green color indicates lower ratios (<0.1 [dark green], 0.1 to 1.0 [medium green], 1.01 to 2.0 [light green], >2.0 [lightest green]), suggesting recognition of a quaternary epitope on virion particles. (C) Representative binding curves of neutralizing human anti-EEEV mAbs to four different antigens. Binding curves of neutralizing human anti-EEEV mAbs to SINV/EEEV particles (green) and EEEV E2 glycoprotein (blue), with mAb concentration (nM) on the x-axis and optical density at 405 nm on the y-axis. Binding to EEEV E1 (purple) or CHIKV E1 (pink) glycoproteins was not detected. Data in A–C represent mean ± SD of technical triplicates and are representative of three independent experiments. See also Figure S1 for recombinant IgG1, IgA1, or Fab binding reactivity to SINV/EEEV particles or EEEV E2 glycoprotein.

Figure 3.. Human anti-EEEV mAbs recognize three neutralizing antigenic determinants on the EEEV E2 glycoprotein.

(A) Competition-binding groups of neutralizing human anti-EEEV mAbs to recombinant EEEV E2 monomeric glycoprotein as determined through biolayer interferometry. Mouse domain B (magenta) and human mAbs were incubated with EEEV E2 glycoprotein to identify the number of antigenic determinants recognized by these mAbs. The first mAb incubated with E2 is shown in the left-hand column and the second mAb is shown in the top column. Black boxes indicate competition, or reduction in maximum signal for binding of the second mAb to <33%. Grey boxes indicate intermediate competition, or reduction in maximum signal for binding of the second mAb to between 33 to 67%. White boxes indicate no competition, or little to no reduction in maximum signal for binding of the second mAb to >67%. Each mAb is colored based on binding group as defined in Figure 2. IC₅₀ (pM) values for neutralization activity against SINV/EEEV are indicated in parentheses (Figure 1D). (B) Heat map of critical residues for neutralizing human anti-EEEV mAbs as determined through alanine-scanning mutagenesis library analysis. The average percent binding of each neutralizing human anti-EEEV mAbs is indicated for the critical residues identified (<25% binding of mAb in which at least two mAbs exhibited >70% binding to control for expression; D1-L267) and for the previously characterized murine anti-EEEV mAbs (Kim et al., 2019) and the VEEV-specific human mAb, F5 (Hunt et al., 2010; Porta et al., 2014). The heat map displays average % binding relative to WT EEEV E2 glycoprotein with dark blue (>70%), light blue (25–70%), and light green (<25%). Residues are colored based on E2 domain (N-link - purple, Domain A - red, Arch 1 - magenta, Domain B - cyan, and Arch 2 - orange). Each mAb is colored based on binding group as defined in Figure 2 and ordered to correspond with the competition-binding groups as defined in Figure 3A. Data represents mean of at least two independent experiments. (C) Epitope mapping of critical alanine and arginine residues previously identified for neutralizing murine anti-EEEV mAbs binding to the E2 glycoprotein. Critical residues for binding of murine anti-EEEV mAbs as previously determined through alanine and arginine mutagenesis analyses were mapped onto the 4.2 Å cryo-EM reconstruction of EEEV VLP (EMD-22276; PBD ID: 6XO4) for comparison to the critical alanine residues identified for human anti-EEEV mAbs (see Figure 3D). A trimeric top view of the E2 (green) and E1 (red) glycoproteins is shown with critical residues (spheres) for murine anti-EEEV mAbs that recognize the E2 domains A, B, and A/B. Residues are colored based on E2 domain (Domain A - red and Domain B - cyan). (D) Epitope mapping of critical alanine residues identified for neutralizing human anti-EEEV mAbs binding to the E2 glycoprotein. Critical residues for binding of human anti-EEEV mAbs as identified through alanine-scanning mutagenesis library analyses (Figure 3B) were mapped as described in Figure 3C. Residues are colored based on E2 domain (N-link - purple, Domain A - red, Arch 1 - magenta, Domain B - cyan, and Arch 2 - orange). Yellow spheres indicate the previously identified SINV/EEEV neutralization escape mutants (M68T, G192R, and L227R) (Kim et al., 2019). Each mAb is presented with its respective E2 domain and is colored based on binding group as defined in Figure 2. See Figure S2 for a bar graph representation of the percent binding of each mAb to the alanine residues described in Figure 3B. See Figure S3 for neutralization activity of mAbs against the SINV/EEEV escape mutants (M68T, G192R, and L227R). See Table S1 for critical alanine residues identified for each mAb.

Figure 4.. EEEV-33 recognizes a critical domain A epitope on SINV/EEEV particles for inhibition of viral entry or fusion.

(A) Entry blockade of SINV/EEEV by EEEV-33. An entry blockade assay (open squares) was performed by extensive washing of EEEV-33 (red) or the control mAb (black), from the medium following internalization of SINV/EEEV into Vero cells. Representative neutralization curves are shown for EEEV-33 and rDENV-2D22 as determined through FRNT (closed squares; see Figure 1A) or the entry blockade assay with mAb concentration (nM) on the x-axis and percent relative infectivity on the y-axis. (B) Post-attachment neutralization of SINV/EEEV. A post-attachment neutralization assay (starred circles) was performed by incubation of Vero cells with SINV/EEEV at 4°C for 1 hour followed by addition of EEEV-33 (red) or rDENV-2D22 (black) at 4°C for 1 hour. Cells were incubated at 37°C for 15 min prior to addition of an overlay and incubation at 37°C for 18 h. Representative neutralization curves are shown for EEEV-33 and rDENV-2D22 as determined through FRNT (closed squares; see Figure 1A) or the post-attachment neutralization assay with mAb concentration (nM) on the x-axis and percent relative infectivity on the y-axis. (C–D) Cryo-EM reconstruction of SINV/EEEV in complex with EEEV-33 Fab. Cryo-EM structure of EEEV-33 Fab complex (~7.2 Å) showing radially colored surface representation of full (C) and cross section (D) of the map. (E) EEEV-33 Fab binding footprint to E2 trimeric spikes on SINV/EEEV particles. View of map surface to illustrate binding of EEEV-33 Fab (red) to the q3 and i3 spikes along the icosahedral 2-fold axis. (F) EEEV-33 Fab constant domain contact interactions. Close-up view of EEEV-33 Fab binding to the i3 spike (black circle in E), in which overlapping Fab constant domain density is observed. (G) Cryo-EM E2 trimeric view of EEEV-33 Fab binding with critical alanine residues. Critical alanine residues identified for EEEV-33 are indicated with spheres to illustrate the epitope of EEEV-33 corresponds with the SINV/EEEV (PDB ID: 6MX4) and EEEV-143 Fab (mutated sequence of PDB: 6MWX) docked and rigid body refined cryo-EM model of rEEEV-33 Fab in complex with SINV/EEEV. Sphere color corresponds to E2 domain (N-link - purple, Domain A - red, Arch 1 - magenta, Domain B - cyan, and Arch 2 - orange) as described in Figure 3D. Data in A–B represent mean ± SD of technical triplicates and are representative of two independent experiments. See Figure S4 for additional views of EEEV-33 Fab binding to the E2 trimer on SINV/EEEV particles.

Figure 5.. EEEV-143 recognizes a critical domain B epitope on SINV/EEEV particles for inhibition of viral entry or fusion into cells.

(A) Entry blockade of SINV/EEEV by EEEV-143. An entry blockade assay (open squares) was performed by extensive washing of EEEV-143 (orange) or the control mAb rDENV-2D22 (black), from the medium following internalization of SINV/EEEV. Representative neutralization curves are shown for EEEV-143 and rDENV-2D22 as determined through FRNT (closed squares; see Figure 1A) or the entry blockade assay with mAb concentration (nM) on the x-axis and percent relative infectivity on the y-axis. (B) Post-attachment neutralization of SINV/EEEV by EEEV-143. A post-attachment neutralization assay (starred circles) was performed by incubation of Vero cells with SINV/EEEV at 4°C for 1 h followed by addition of EEEV-143 (orange) or rDENV-2D22 (black) at 4°C for 1 h. Cells then were incubated at 37°C for 15 min prior to addition of an overlay and incubation at 37°C for 18 h. Representative neutralization curves are shown for EEEV-143 and rDENV-2D22 as determined through FRNT (closed squares; see Figure 1A) or the post-attachment neutralization assay with mAb concentration (nM) on the x-axis and percent relative infectivity on the y-axis. (C–D) Cryo-EM reconstruction of SINV/EEEV in complex with EEEV-143 Fab. Cryo-EM structure of EEEV-143 Fab complex (~8.3 Å) showing radially colored surface representation of full (C) and cross section (D) of the map. (E) EEEV-143 Fab binding footprint to E2 trimeric spikes on EEEV virus-like particles (VLPs). View of map surface to illustrate binding of EEEV-143 Fab (in orange) to the q3 and i3 spikes along the icosahedral 2-fold axis. (F) EEEV-143 Fab constant domain contact interactions. Close-up view of EEEV-143 Fab binding to the q3 and i3 spikes (black circles in E), in which overlapping Fab constant domain density is observed around the 2-fold axis. Fabs bound to the q3 and i3 spikes across the 3-fold axis are ~11 Å apart, in which the flexibility of the Fab may allow for contacts to occur. (G) Cryo-EM E2 trimeric view of EEEV-143 Fab binding with critical alanine residues. Critical alanine residues identified for EEEV-143 are indicated with spheres to illustrate the epitope of EEEV-143 corresponds with the EEEV VLP (EMD-22276; PDB ID: 6XO4) and EEEV-143 Fab (mutated sequence of PDB: 6MWX) docked and rigid body refined cryo-EM model. Sphere color corresponds to E2 domain (N-link - purple, Domain A - red, Arch 1 - magenta, Domain B - cyan, and Arch 2 - orange) as described in Figure 3D. Data in A–B represent mean ± SD of technical triplicates and are representative of two independent experiments. See Figure S4 for additional views of EEEV-143 Fab binding to the E2 trimer on SINV/EEEV particles.

Figure 6.. Prophylactic administration of mice with EEEV-33 EEEV-143 protects in an aerosol challenge model.

(A) Anti-EEEV mAbs protect against EEEV lethality. EEEV-33 (red; n=11) and EEEV-143 (orange; n=5) were administered prophylactically (24 h prior to virus challenge) at 100 μg via the IP route to CD-1 female mice (4–6-weeks old). EEEV-33 or EEEV-143 protected mice with 91 or 100% survival, respectively, against EEEV (FL93–939) aerosol challenge (1,631 to 1,825 PFU/mouse) compared to the control mAb rDENV-2D22 (black; n=10) (Fibriansah et al., 2015). (B) EEEV-143 protects against a higher inoculation dose of EEEV. EEEV-143 (orange; n=5) was administered as described in Figure 6A. EEEV-143 exhibited 100% prophylactic survival against EEEV (FL93–939) aerosol challenge (2,739 PFU/mouse). rDENV-2D22 (black; n=5) served as a negative control. (C) In vivo imaging system (IVIS) images of CD-1 mice for EEEV-33, EEEV-143, and rDENV-2D22 prophylactically treated groups at days 4–5 after EEEV aerosol challenge. IVIS images for EEEV-33 (red), EEEV-143 (orange), and rDENV-2D22 (black). One of the mice in the EEEV-33 group and three of the mice in the rDENV-2D22 negative control group died prior to IVIS imaging on day 5 after virus challenge. (D) Luminescence intensity of IVIS images. Total flux (photons/second) for the corresponding IVIS images in Figure 6C of the EEEV-33, EEEV-143, and rDENV-2D22 groups is indicated. ~1 × 10⁵ total flux is the background for uninfected mice. One-way ANOVA with Dunnett’s multiple comparisons correction was used to compare luminescence of the images to the rDENV-2D22 control group. *p < 0.01. Data A, C, D represent combined in vivo data for EEEV-33 (n=6, n=5) or rDENV-2D22 (n=5, n=5) in two independent experiments (1,631 to 1,825 PFU/mouse). Data in A represent in vivo data for EEEV-143 (n=5) in one independent experiment (1,631 to 1,825 PFU/mouse). Data in B–D represent in vivo data for EEEV-143 (n=5) in one independent experiment (2,739 PFU/mouse). Data in A–B, the survival curves were compared using the log-rank test with Bonferroni multiple comparison correction. *p < 0.05, **p < 0.01, ns = not significant.

Figure 7.. Post-exposure therapy with EEEV-33 and EEEV-143 partially protects mice in an aerosol challenge model.

(A) Anti-EEEV mAbs protect against EEEV lethality as post-exposure therapy. EEEV-33 (red; n=11) and EEEV-143 (orange; n=5) were administered therapeutically (24 h post virus challenge) at 100 μg via the IP route to CD-1 female mice (4–6-weeks old). EEEV-33 or EEEV-143 exhibited 27% or 80% therapeutic survival, respectively, against EEEV (FL93–939) aerosol challenge (1,631–1,825 PFU/mouse) compared to the control mAb (black; n=5). (B) EEEV-143 administration at a higher inoculation dose of EEEV. EEEV-143 (orange; n=5) was administered as described in Figure 7A. EEEV-143 exhibited 20% therapeutic survival against EEEV (FL93–939) aerosol challenge (2,739 PFU/mouse). (C) EEEV-143 administration at 200-μg dose after EEEV exposure. EEEV-143 (orange; n=5) was administered as described in Figure 7A except at a 200 μg dose. EEEV-143 conferred 20% therapeutic survival against EEEV (strain FL93–939) aerosol challenge (1,897 PFU/mouse) compared to the control mAb (black; n=5; 200 μg). (D) In vivo imaging system (IVIS) images of CD-1 mice for EEEV-33 and EEEV-143 therapeutically treated group at days 4–5 after EEEV aerosol challenge. IVIS images for EEEV-33 (red) or EEEV-143 (orange). Four of the mice in the EEEV-33 group died prior to IVIS imaging on day 5 after virus challenge. (E) Luminescence intensity of IVIS images. Total flux (photons/second) for the corresponding IVIS images in Figure 7D of the EEEV-33 and EEEV-143 groups are indicated. ~1 × 10⁵ total flux is the background for uninfected mice. Data A, B, D, E represent combined in vivo data for EEEV-33 (n=6, n=5) in two independent experiments (1,631 to 1,825 PFU/mouse). Data in A represent in vivo data for EEEV-143 (n=5) and rDENV-2D22 (n=5) in one independent experiment (1,631 to 1,825 PFU/mouse). Data in B, D, E represent in vivo data for EEEV-143 (n=5) in one independent experiment (2,739 PFU/mouse). Data in C represent in vivo data for EEEV-143 (n=5) and rDENV-2D22 (n=5) in one independent experiment (1,897 PFU/mouse). Data in A-C, the survival curves were compared using the log-rank test with Bonferroni multiple comparison correction. *p < 0.05, **p < 0.01, ns = not significant.