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Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer.

Matho MH, Schlossman A, Meng X, Benhnia MR, Kaever T, Buller M, Doronin K, Parker S, Peters B, Crotty S, Xiang Y, Zajonc DM - PLoS Pathog. (2015)

Bottom Line: To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33.While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously.A27D7 binding is resistant to single alanine substitutions within the A33 epitope.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, United States of America.

ABSTRACT
Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.

No MeSH data available.


Related in: MedlinePlus

Structures of A33/A2C7, A20G2, and A27D7-Fab complexes.Structures of (A) A33/A2C7, (B) A20G2, and (C) A27D7-Fab complexes. Structures are shown as cartoon representations, with light chains in green and heavy chains in orange (top), while the antibody footprints on the molecular surface of A33 are shown below. In (C), A33 subunits (SUs) are shown in different colors because the epitope spreads asymmetrically over both SUs (SU A in aquamarine, SU B in grey). Footprints on A33 is colored according to contacting CDR loops (L1, brown; L3, yellow; H1, green; H2, cyan; H3, blue). Stripe patterns highlight A33 residues that elicit contacts with more than 1 CDR. The continuous line and the asterisk indicate A33 dimer axis. Letters indicate when this axis constitutes a crystallographic axis (X) along with the A33 dimerization axis (P). A33 residues that are part of the orthopox variation profile are labeled in magenta.
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ppat.1005148.g004: Structures of A33/A2C7, A20G2, and A27D7-Fab complexes.Structures of (A) A33/A2C7, (B) A20G2, and (C) A27D7-Fab complexes. Structures are shown as cartoon representations, with light chains in green and heavy chains in orange (top), while the antibody footprints on the molecular surface of A33 are shown below. In (C), A33 subunits (SUs) are shown in different colors because the epitope spreads asymmetrically over both SUs (SU A in aquamarine, SU B in grey). Footprints on A33 is colored according to contacting CDR loops (L1, brown; L3, yellow; H1, green; H2, cyan; H3, blue). Stripe patterns highlight A33 residues that elicit contacts with more than 1 CDR. The continuous line and the asterisk indicate A33 dimer axis. Letters indicate when this axis constitutes a crystallographic axis (X) along with the A33 dimerization axis (P). A33 residues that are part of the orthopox variation profile are labeled in magenta.

Mentions: To analyze the binding mode of each Fab to A33 we set out to determine the crystal structure of one representative of each of the three different neutralizing MAbs bound to A33. To increase our chances of success we started out with all five neutralizing antibodies and finally determined the crystal structures of the complexes of A33/A2C7, A33/A20G2, and A33/A27D7 to 2.3 Å, 2.9 Å, and 1.6 Å resolution, respectively (Table 1 and Figs 4 and 5). To assess the impact of A33 epitope residues in antibody binding, we have further generated A33 variants with selected mutations in the epitope of each of the three crystallized antibodies and determined the antibody binding affinity using BLI (S2 Table and Fig 6 and S3 Fig)


Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer.

Matho MH, Schlossman A, Meng X, Benhnia MR, Kaever T, Buller M, Doronin K, Parker S, Peters B, Crotty S, Xiang Y, Zajonc DM - PLoS Pathog. (2015)

Structures of A33/A2C7, A20G2, and A27D7-Fab complexes.Structures of (A) A33/A2C7, (B) A20G2, and (C) A27D7-Fab complexes. Structures are shown as cartoon representations, with light chains in green and heavy chains in orange (top), while the antibody footprints on the molecular surface of A33 are shown below. In (C), A33 subunits (SUs) are shown in different colors because the epitope spreads asymmetrically over both SUs (SU A in aquamarine, SU B in grey). Footprints on A33 is colored according to contacting CDR loops (L1, brown; L3, yellow; H1, green; H2, cyan; H3, blue). Stripe patterns highlight A33 residues that elicit contacts with more than 1 CDR. The continuous line and the asterisk indicate A33 dimer axis. Letters indicate when this axis constitutes a crystallographic axis (X) along with the A33 dimerization axis (P). A33 residues that are part of the orthopox variation profile are labeled in magenta.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556652&req=5

ppat.1005148.g004: Structures of A33/A2C7, A20G2, and A27D7-Fab complexes.Structures of (A) A33/A2C7, (B) A20G2, and (C) A27D7-Fab complexes. Structures are shown as cartoon representations, with light chains in green and heavy chains in orange (top), while the antibody footprints on the molecular surface of A33 are shown below. In (C), A33 subunits (SUs) are shown in different colors because the epitope spreads asymmetrically over both SUs (SU A in aquamarine, SU B in grey). Footprints on A33 is colored according to contacting CDR loops (L1, brown; L3, yellow; H1, green; H2, cyan; H3, blue). Stripe patterns highlight A33 residues that elicit contacts with more than 1 CDR. The continuous line and the asterisk indicate A33 dimer axis. Letters indicate when this axis constitutes a crystallographic axis (X) along with the A33 dimerization axis (P). A33 residues that are part of the orthopox variation profile are labeled in magenta.
Mentions: To analyze the binding mode of each Fab to A33 we set out to determine the crystal structure of one representative of each of the three different neutralizing MAbs bound to A33. To increase our chances of success we started out with all five neutralizing antibodies and finally determined the crystal structures of the complexes of A33/A2C7, A33/A20G2, and A33/A27D7 to 2.3 Å, 2.9 Å, and 1.6 Å resolution, respectively (Table 1 and Figs 4 and 5). To assess the impact of A33 epitope residues in antibody binding, we have further generated A33 variants with selected mutations in the epitope of each of the three crystallized antibodies and determined the antibody binding affinity using BLI (S2 Table and Fig 6 and S3 Fig)

Bottom Line: To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33.While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously.A27D7 binding is resistant to single alanine substitutions within the A33 epitope.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, United States of America.

ABSTRACT
Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.

No MeSH data available.


Related in: MedlinePlus