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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

Fab/A33 binding interactions.(A) Determination of A33/Fab binding stoichiometry by size exclusion chromatography (SEC). Fab/A33 complex formation of five different MAbs (A2C7, A26C7, A25D11, A27D7, and A20G2) is illustrated. Two major peaks corresponding in size to 1 Fab: 1 A33 dimer (red and blue curve) and 2 Fabs: 1 A33 dimer (brown, cyan and grey) complexes are visible. Presence of both Fab and A33 in each peak are confirmed by SDS-PAGE (bottom left corner). Molecular weight markers with sizes in kDa are shown as a reference (thin grey curve). (B) Real-time A33 binding curves to immobilized MAbs as assessed by BLI. Note that A33 dissociates much slower from MAbs A2C7 and A20G2, compared to A27D7, likely to its ability to simultaneous bind to two Fabs.
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ppat.1005148.g003: Fab/A33 binding interactions.(A) Determination of A33/Fab binding stoichiometry by size exclusion chromatography (SEC). Fab/A33 complex formation of five different MAbs (A2C7, A26C7, A25D11, A27D7, and A20G2) is illustrated. Two major peaks corresponding in size to 1 Fab: 1 A33 dimer (red and blue curve) and 2 Fabs: 1 A33 dimer (brown, cyan and grey) complexes are visible. Presence of both Fab and A33 in each peak are confirmed by SDS-PAGE (bottom left corner). Molecular weight markers with sizes in kDa are shown as a reference (thin grey curve). (B) Real-time A33 binding curves to immobilized MAbs as assessed by BLI. Note that A33 dissociates much slower from MAbs A2C7 and A20G2, compared to A27D7, likely to its ability to simultaneous bind to two Fabs.

Mentions: To assess the interaction between the antibody and A33 we prepared the Fab portion of all five neutralizing antibodies (A2C7, A26C7, A25D11, A27D7, and A20G2) and assessed Fab binding to recombinant A33 using size exclusion chromatography (SEC) (Fig 3A). We observed two major peaks of different molecular weight (MW), indicating that the Fab’s bound to A33 with different stoichiometries. Both Fab’s A26C7 and A27D7 eluted with a lower MW when bound to A33, while the Fab’s of A2C7, A25D11, and A20G2 formed a higher MW complex with A33. The SEC data indicated that a single Fab of A26C7 and A27D7 bound to one A33 dimer, while A2C7, A25D11, and A20G2 each bound with one Fab to one A33 subunit (2 Fab’s per A33 dimer). We next assessed the real-time binding kinetics with the three representative antibodies A2C7, A27D7, and A20G2. Intact antibodies were immobilized on biosensors and binding to A33 in solution was measured by Biolayer interferometry (BLI) (S2 Table and Fig 3B). A33 bound with very high affinity to the antibodies A2C7 and A20G2 (KD ~65 pM), consistent with the SEC data, where the A33 dimer can simultaneously bind to two separate Fab’s. This binding mode is characterized by a very slow dissociation rate. A33 also bound with high affinity to the antibody A27D7 (KD~14nM), yet roughly 400-fold weaker compared to A2C7 and A20G2. Since the A33 dissociation rates were very slow for both A2C7 and A20G2, which made calculation of the binding kinetics less robust, we repeated the binding assay but immobilized A33 instead of the MAbs (S2 Table). Both A2C7 and A20G2 showed similar binding to chip bound A33 in this reversed orientation with a ~2 to 4-fold difference in KD each (S2 Table). However, MAb A27D7 showed very slow dissociation from A33, consistent with a model in which each Fab can bind to two separate A33 dimers on the sensor tip. As a result, the apparent binding affinity increased from 14 nM (MAb immobilized) to beyond 1pM (A33 immobilized), as no dissociation could be reliably measured. This led to A27D7 being the antibody with the highest affinity in this setting. All subsequent kinetic binding studies using A33 mutants were performed using A33 immobilized on the sensor tip. This binding orientation also mimics the antibody binding during infection, where A33 is embedded in the EEV membrane.


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)

Fab/A33 binding interactions.(A) Determination of A33/Fab binding stoichiometry by size exclusion chromatography (SEC). Fab/A33 complex formation of five different MAbs (A2C7, A26C7, A25D11, A27D7, and A20G2) is illustrated. Two major peaks corresponding in size to 1 Fab: 1 A33 dimer (red and blue curve) and 2 Fabs: 1 A33 dimer (brown, cyan and grey) complexes are visible. Presence of both Fab and A33 in each peak are confirmed by SDS-PAGE (bottom left corner). Molecular weight markers with sizes in kDa are shown as a reference (thin grey curve). (B) Real-time A33 binding curves to immobilized MAbs as assessed by BLI. Note that A33 dissociates much slower from MAbs A2C7 and A20G2, compared to A27D7, likely to its ability to simultaneous bind to two Fabs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556652&req=5

ppat.1005148.g003: Fab/A33 binding interactions.(A) Determination of A33/Fab binding stoichiometry by size exclusion chromatography (SEC). Fab/A33 complex formation of five different MAbs (A2C7, A26C7, A25D11, A27D7, and A20G2) is illustrated. Two major peaks corresponding in size to 1 Fab: 1 A33 dimer (red and blue curve) and 2 Fabs: 1 A33 dimer (brown, cyan and grey) complexes are visible. Presence of both Fab and A33 in each peak are confirmed by SDS-PAGE (bottom left corner). Molecular weight markers with sizes in kDa are shown as a reference (thin grey curve). (B) Real-time A33 binding curves to immobilized MAbs as assessed by BLI. Note that A33 dissociates much slower from MAbs A2C7 and A20G2, compared to A27D7, likely to its ability to simultaneous bind to two Fabs.
Mentions: To assess the interaction between the antibody and A33 we prepared the Fab portion of all five neutralizing antibodies (A2C7, A26C7, A25D11, A27D7, and A20G2) and assessed Fab binding to recombinant A33 using size exclusion chromatography (SEC) (Fig 3A). We observed two major peaks of different molecular weight (MW), indicating that the Fab’s bound to A33 with different stoichiometries. Both Fab’s A26C7 and A27D7 eluted with a lower MW when bound to A33, while the Fab’s of A2C7, A25D11, and A20G2 formed a higher MW complex with A33. The SEC data indicated that a single Fab of A26C7 and A27D7 bound to one A33 dimer, while A2C7, A25D11, and A20G2 each bound with one Fab to one A33 subunit (2 Fab’s per A33 dimer). We next assessed the real-time binding kinetics with the three representative antibodies A2C7, A27D7, and A20G2. Intact antibodies were immobilized on biosensors and binding to A33 in solution was measured by Biolayer interferometry (BLI) (S2 Table and Fig 3B). A33 bound with very high affinity to the antibodies A2C7 and A20G2 (KD ~65 pM), consistent with the SEC data, where the A33 dimer can simultaneously bind to two separate Fab’s. This binding mode is characterized by a very slow dissociation rate. A33 also bound with high affinity to the antibody A27D7 (KD~14nM), yet roughly 400-fold weaker compared to A2C7 and A20G2. Since the A33 dissociation rates were very slow for both A2C7 and A20G2, which made calculation of the binding kinetics less robust, we repeated the binding assay but immobilized A33 instead of the MAbs (S2 Table). Both A2C7 and A20G2 showed similar binding to chip bound A33 in this reversed orientation with a ~2 to 4-fold difference in KD each (S2 Table). However, MAb A27D7 showed very slow dissociation from A33, consistent with a model in which each Fab can bind to two separate A33 dimers on the sensor tip. As a result, the apparent binding affinity increased from 14 nM (MAb immobilized) to beyond 1pM (A33 immobilized), as no dissociation could be reliably measured. This led to A27D7 being the antibody with the highest affinity in this setting. All subsequent kinetic binding studies using A33 mutants were performed using A33 immobilized on the sensor tip. This binding orientation also mimics the antibody binding during infection, where A33 is embedded in the EEV membrane.

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