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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

Complement and isotype dependence of anti-A33 MAb neutralization of VACV EEV.VACV EEV neutralization activity of purified anti-A33 MAbs in the absence (MAbs) or presence (MAbs+10% C´) of complement. Rabbit anti-A33 polyclonal Abs (N628) were used as positive control. Anti-B5 MAbs B126 (IgG2a), and B96 (IgG1) were used as positive and negative neutralization controls, respectively. Human anti-ditrophenol (DNP, IgG1) and VACV EEV (EEV) were used as negative controls. Error bars indicate SEM in each condition. Dashed line indicates the 50% of plaques number of VACV EV in panels A and B. The data are representative of two experiments. Three more experiments were done and they show comparable results.
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ppat.1005148.g001: Complement and isotype dependence of anti-A33 MAb neutralization of VACV EEV.VACV EEV neutralization activity of purified anti-A33 MAbs in the absence (MAbs) or presence (MAbs+10% C´) of complement. Rabbit anti-A33 polyclonal Abs (N628) were used as positive control. Anti-B5 MAbs B126 (IgG2a), and B96 (IgG1) were used as positive and negative neutralization controls, respectively. Human anti-ditrophenol (DNP, IgG1) and VACV EEV (EEV) were used as negative controls. Error bars indicate SEM in each condition. Dashed line indicates the 50% of plaques number of VACV EV in panels A and B. The data are representative of two experiments. Three more experiments were done and they show comparable results.

Mentions: All anti-A33 MAbs were then tested for their ability to neutralize EEV in vitro by complement dependent and independent processes (Fig 1). None of the antibodies was able to neutralize EEV in the absence of complement (Fig 1A). IgG1 anti-A33 mAbs A17D7 and A25F2 at 10 μg/ml exhibited no complement mediated EEV neutralization, consistent with the inability of recruiting complement by IgG1 MAbs in general (Fig 1B). However, IgG2a (A26C7, A25D11, A27D7, and A20G2) and IgG2b (A2C7) anti-A33 MAbs at 10 μg/ml exhibited strong complement-mediated EEV neutralization in the presence of complement (Fig 1B). Anti-B5 MAbs B126 (IgG2a) at the same concentration show comparable neutralization activity as anti-A33 mAbs of appropriate isotypes. Irrelevant IgG1 anti-DNP MAbs (DNP) and anti-B5 MAbs B96 (IgG1) at the same concentration showed no effect (Fig 1B). These data support a model where anti-A33 MAbs can neutralize EEV in the presence of complement via opsonization of the EEV particle surface.


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)

Complement and isotype dependence of anti-A33 MAb neutralization of VACV EEV.VACV EEV neutralization activity of purified anti-A33 MAbs in the absence (MAbs) or presence (MAbs+10% C´) of complement. Rabbit anti-A33 polyclonal Abs (N628) were used as positive control. Anti-B5 MAbs B126 (IgG2a), and B96 (IgG1) were used as positive and negative neutralization controls, respectively. Human anti-ditrophenol (DNP, IgG1) and VACV EEV (EEV) were used as negative controls. Error bars indicate SEM in each condition. Dashed line indicates the 50% of plaques number of VACV EV in panels A and B. The data are representative of two experiments. Three more experiments were done and they show comparable results.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005148.g001: Complement and isotype dependence of anti-A33 MAb neutralization of VACV EEV.VACV EEV neutralization activity of purified anti-A33 MAbs in the absence (MAbs) or presence (MAbs+10% C´) of complement. Rabbit anti-A33 polyclonal Abs (N628) were used as positive control. Anti-B5 MAbs B126 (IgG2a), and B96 (IgG1) were used as positive and negative neutralization controls, respectively. Human anti-ditrophenol (DNP, IgG1) and VACV EEV (EEV) were used as negative controls. Error bars indicate SEM in each condition. Dashed line indicates the 50% of plaques number of VACV EV in panels A and B. The data are representative of two experiments. Three more experiments were done and they show comparable results.
Mentions: All anti-A33 MAbs were then tested for their ability to neutralize EEV in vitro by complement dependent and independent processes (Fig 1). None of the antibodies was able to neutralize EEV in the absence of complement (Fig 1A). IgG1 anti-A33 mAbs A17D7 and A25F2 at 10 μg/ml exhibited no complement mediated EEV neutralization, consistent with the inability of recruiting complement by IgG1 MAbs in general (Fig 1B). However, IgG2a (A26C7, A25D11, A27D7, and A20G2) and IgG2b (A2C7) anti-A33 MAbs at 10 μg/ml exhibited strong complement-mediated EEV neutralization in the presence of complement (Fig 1B). Anti-B5 MAbs B126 (IgG2a) at the same concentration show comparable neutralization activity as anti-A33 mAbs of appropriate isotypes. Irrelevant IgG1 anti-DNP MAbs (DNP) and anti-B5 MAbs B96 (IgG1) at the same concentration showed no effect (Fig 1B). These data support a model where anti-A33 MAbs can neutralize EEV in the presence of complement via opsonization of the EEV particle surface.

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