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The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity.

Hunt AR, Frederickson S, Maruyama T, Roehrig JT, Blair CD - PLoS Negl Trop Dis (2010)

Bottom Line: We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119.The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically.Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.

View Article: PubMed Central - PubMed

Affiliation: Microbiology, Immunology and Pathology Department, Colorado State University, Fort Collins, Colorado, United States of America. Ann.Hunt@colostate.edu

ABSTRACT

Background: Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2(c,d,e,f,g,h)) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.

Methods: We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.

Findings: Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 A resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.

Conclusions: The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.

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Mapping the E2 ectodomain.A. A top view of the E2 density of one spike looking down the 3‐fold axis (shown as the large purple spot corresponding to three merged carbohydrate moieties at position 46). B. A side view of one E2 molecule with the approximate location of Venezuelan equine encephalitis virus (VEEV) E2 peptide 13 (amino acids 241–265) shown in black. A and B: The markers on the E2 glycoprotein that correspond to carbohydrate moieties at positions 46, 160, 196, 200, 216, 262, and 318 are shown in purple, red, blue, orange, green, pink, and light blue, respectively. Position 216 (green) was also identified with a cryoEM map of a Fab‐Ross River virus complex. Approximate locations of the VEEV murine MAb anti‐mE2c binding site and critical neutralization site are shown by a dotted circle; the proposed binding site of the VEEV human MAb anti‐hE2c is shown in cyan.
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pntd-0000739-g003: Mapping the E2 ectodomain.A. A top view of the E2 density of one spike looking down the 3‐fold axis (shown as the large purple spot corresponding to three merged carbohydrate moieties at position 46). B. A side view of one E2 molecule with the approximate location of Venezuelan equine encephalitis virus (VEEV) E2 peptide 13 (amino acids 241–265) shown in black. A and B: The markers on the E2 glycoprotein that correspond to carbohydrate moieties at positions 46, 160, 196, 200, 216, 262, and 318 are shown in purple, red, blue, orange, green, pink, and light blue, respectively. Position 216 (green) was also identified with a cryoEM map of a Fab‐Ross River virus complex. Approximate locations of the VEEV murine MAb anti‐mE2c binding site and critical neutralization site are shown by a dotted circle; the proposed binding site of the VEEV human MAb anti‐hE2c is shown in cyan.

Mentions: Although the crystal structure of the alphavirus E2 glycoprotein has not been solved, cryoEM reconstructions of E2 have been reported [25], [30], [72]. The 9Å resolution cryoEM map of the SV E2 presented by Mukhopadhyay et al. [72] was annotated with markers representing locations of glycosylation sites, the protein N-terminus, and a neutralizing Fab binding site. We have adapted their figure to show the probable surface-accessible location of the hMAb F5 nIgG binding site (E2 aa115–119) and its relationship with other markers (Figure 3A,B). Mapping of this epitope to a unique E2 neutralization site was based on the data presented in this study: (1) epitope binding by hMAb F5 nIgG was more sensitive to 0.3% BPL treatment than epitopes recognized by neutralizing mMAbs, (2) hMAb F5 was able to neutralize all anti-VEEV mMAb neutralization escape variant viruses and therefore did not bind to E2 residues 182–207, defined as the “critical” neutralization domain, and (3) hMAb F5 neutralization escape variant viruses vF5-3 and vF5-5 defined a neutralization epitope involving E2 aa115–119. Results from studies in mice using VEEV E2 synthetic peptides as vaccines have been included in the proposed map of the E2 ectodomain to complement the placement of the hE2c epitope (Figure 3B). Previously, we identified two peptide vaccines, VE2pep01 (E2 aa1–25) and VE2pep13 (E2 aa241–265) that protected mice from virulent VEEV challenge [73]–[75]. We also isolated an anti-peptide MAb, 1A2B-10, specific for E2 aa1–19, which passively protected mice challenged with VEEV varieties 1AB, 1C, and 1D [76]. None of the anti-peptide antibodies, either polyclonal or monoclonal, had virus-neutralizing activity, indicating that their cognate peptides were not likely to be surface-accessible or lacked the appropriate conformation. The proposed configuration of the E2 molecule shown in Fig. 3B places the hE2c epitope (E2 aa115–119) on the surface of the spike above the more cryptic locations of the E2 N-terminus (VE2pep01) and aa 241–265 (VE2pep13). Such an arrangement would be in agreement with the current knowledge of the structure of the E2 glycoprotein, the location of specific markers, and functional attributes of specified epitopes. We are now in collaboration to obtain structural data on Fab-virion complexes to determine actual binding sites of F5 nIgG and other human and murine MAbs.


The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity.

Hunt AR, Frederickson S, Maruyama T, Roehrig JT, Blair CD - PLoS Negl Trop Dis (2010)

Mapping the E2 ectodomain.A. A top view of the E2 density of one spike looking down the 3‐fold axis (shown as the large purple spot corresponding to three merged carbohydrate moieties at position 46). B. A side view of one E2 molecule with the approximate location of Venezuelan equine encephalitis virus (VEEV) E2 peptide 13 (amino acids 241–265) shown in black. A and B: The markers on the E2 glycoprotein that correspond to carbohydrate moieties at positions 46, 160, 196, 200, 216, 262, and 318 are shown in purple, red, blue, orange, green, pink, and light blue, respectively. Position 216 (green) was also identified with a cryoEM map of a Fab‐Ross River virus complex. Approximate locations of the VEEV murine MAb anti‐mE2c binding site and critical neutralization site are shown by a dotted circle; the proposed binding site of the VEEV human MAb anti‐hE2c is shown in cyan.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903468&req=5

pntd-0000739-g003: Mapping the E2 ectodomain.A. A top view of the E2 density of one spike looking down the 3‐fold axis (shown as the large purple spot corresponding to three merged carbohydrate moieties at position 46). B. A side view of one E2 molecule with the approximate location of Venezuelan equine encephalitis virus (VEEV) E2 peptide 13 (amino acids 241–265) shown in black. A and B: The markers on the E2 glycoprotein that correspond to carbohydrate moieties at positions 46, 160, 196, 200, 216, 262, and 318 are shown in purple, red, blue, orange, green, pink, and light blue, respectively. Position 216 (green) was also identified with a cryoEM map of a Fab‐Ross River virus complex. Approximate locations of the VEEV murine MAb anti‐mE2c binding site and critical neutralization site are shown by a dotted circle; the proposed binding site of the VEEV human MAb anti‐hE2c is shown in cyan.
Mentions: Although the crystal structure of the alphavirus E2 glycoprotein has not been solved, cryoEM reconstructions of E2 have been reported [25], [30], [72]. The 9Å resolution cryoEM map of the SV E2 presented by Mukhopadhyay et al. [72] was annotated with markers representing locations of glycosylation sites, the protein N-terminus, and a neutralizing Fab binding site. We have adapted their figure to show the probable surface-accessible location of the hMAb F5 nIgG binding site (E2 aa115–119) and its relationship with other markers (Figure 3A,B). Mapping of this epitope to a unique E2 neutralization site was based on the data presented in this study: (1) epitope binding by hMAb F5 nIgG was more sensitive to 0.3% BPL treatment than epitopes recognized by neutralizing mMAbs, (2) hMAb F5 was able to neutralize all anti-VEEV mMAb neutralization escape variant viruses and therefore did not bind to E2 residues 182–207, defined as the “critical” neutralization domain, and (3) hMAb F5 neutralization escape variant viruses vF5-3 and vF5-5 defined a neutralization epitope involving E2 aa115–119. Results from studies in mice using VEEV E2 synthetic peptides as vaccines have been included in the proposed map of the E2 ectodomain to complement the placement of the hE2c epitope (Figure 3B). Previously, we identified two peptide vaccines, VE2pep01 (E2 aa1–25) and VE2pep13 (E2 aa241–265) that protected mice from virulent VEEV challenge [73]–[75]. We also isolated an anti-peptide MAb, 1A2B-10, specific for E2 aa1–19, which passively protected mice challenged with VEEV varieties 1AB, 1C, and 1D [76]. None of the anti-peptide antibodies, either polyclonal or monoclonal, had virus-neutralizing activity, indicating that their cognate peptides were not likely to be surface-accessible or lacked the appropriate conformation. The proposed configuration of the E2 molecule shown in Fig. 3B places the hE2c epitope (E2 aa115–119) on the surface of the spike above the more cryptic locations of the E2 N-terminus (VE2pep01) and aa 241–265 (VE2pep13). Such an arrangement would be in agreement with the current knowledge of the structure of the E2 glycoprotein, the location of specific markers, and functional attributes of specified epitopes. We are now in collaboration to obtain structural data on Fab-virion complexes to determine actual binding sites of F5 nIgG and other human and murine MAbs.

Bottom Line: We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119.The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically.Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.

View Article: PubMed Central - PubMed

Affiliation: Microbiology, Immunology and Pathology Department, Colorado State University, Fort Collins, Colorado, United States of America. Ann.Hunt@colostate.edu

ABSTRACT

Background: Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2(c,d,e,f,g,h)) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.

Methods: We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.

Findings: Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 A resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.

Conclusions: The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.

Show MeSH
Related in: MedlinePlus