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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

Treatment with antibody A27D7 protects mice against a lethal ECTV challenge.Mice were treated with A2C7, A20G2 or A27D7 at T = -1 or T = +1 relative to a 1000 PFU ECTV challenge. (A) Mice were monitored for morbidity, as measured by weight-change. Mice treated with A27D7 at T = +1 were fully protected against morbidity. N = 5 mice/group. Data presented is from 1 of 2 studies—both with similar results. (B) Mice were monitored for mortality until T = +21. Only mice treated with A27D7 were significantly protected against challenge. P = 0.0004 for T = +1 and P = 0.006 for T = -1 (90% protected). N = 10 animals/group. Data is pooled from 2 independent studies of 5 animals/group. Controls include no ECTV challenge (PBS/NT), challenge without treatment (NT), treatment with vehicle only (Veh) or CDV treatment.
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ppat.1005148.g007: Treatment with antibody A27D7 protects mice against a lethal ECTV challenge.Mice were treated with A2C7, A20G2 or A27D7 at T = -1 or T = +1 relative to a 1000 PFU ECTV challenge. (A) Mice were monitored for morbidity, as measured by weight-change. Mice treated with A27D7 at T = +1 were fully protected against morbidity. N = 5 mice/group. Data presented is from 1 of 2 studies—both with similar results. (B) Mice were monitored for mortality until T = +21. Only mice treated with A27D7 were significantly protected against challenge. P = 0.0004 for T = +1 and P = 0.006 for T = -1 (90% protected). N = 10 animals/group. Data is pooled from 2 independent studies of 5 animals/group. Controls include no ECTV challenge (PBS/NT), challenge without treatment (NT), treatment with vehicle only (Veh) or CDV treatment.

Mentions: In order to demonstrate the in vivo protective capabilities of antibodies A2C7, A27D7, and A20G2 we evaluated their efficacy following a lethal challenge with ECTV. Mice were treated with a single 100 μg IP injection of the antibodies at either T = -1 or T = +1 relative to challenge with 1000 PFU of ECTV. An additional group of mice was treated with the potent antiviral cidofivir (CDV), which is known to be protective against this dose of ECTV. Following challenge, we observed that mice treated with antibodies A2C7 and A20G2 were not able to statistically protect mice against challenge when compared to mice treated with vehicle (Fig 7A). Conversely, mice treated with the A27D7 antibody were fully protected against challenge when the antibody was administered at T = +1 (P = 0.0004) and were 90% protected (P = 0.006) when the antibody was administered at T = -1 (Fig 7A). Although weight-loss was significant following the T = -1 administration, we found negligible levels of weight-loss when the antibody was administered at T = +1 (Fig 7A). Indeed, at T = +1 we found that A27D7 protected against mortality and morbidity with the equivalency of the CDV-treated controls (Fig 7B). The slightly reduced efficacy of the antibodies when administered at T = -1 is likely a reflection of the antibody half-life, which we have not addressed in this assay. At T = +21 days post-challenge, we re-challenged the mice with a 10,000 PFU inoculum of ECTV—all previously challenged mice were protected against subsequent mortality and morbidity, indicating that the antibodies did not impede the generation of memory and protective immunity.


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)

Treatment with antibody A27D7 protects mice against a lethal ECTV challenge.Mice were treated with A2C7, A20G2 or A27D7 at T = -1 or T = +1 relative to a 1000 PFU ECTV challenge. (A) Mice were monitored for morbidity, as measured by weight-change. Mice treated with A27D7 at T = +1 were fully protected against morbidity. N = 5 mice/group. Data presented is from 1 of 2 studies—both with similar results. (B) Mice were monitored for mortality until T = +21. Only mice treated with A27D7 were significantly protected against challenge. P = 0.0004 for T = +1 and P = 0.006 for T = -1 (90% protected). N = 10 animals/group. Data is pooled from 2 independent studies of 5 animals/group. Controls include no ECTV challenge (PBS/NT), challenge without treatment (NT), treatment with vehicle only (Veh) or CDV treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556652&req=5

ppat.1005148.g007: Treatment with antibody A27D7 protects mice against a lethal ECTV challenge.Mice were treated with A2C7, A20G2 or A27D7 at T = -1 or T = +1 relative to a 1000 PFU ECTV challenge. (A) Mice were monitored for morbidity, as measured by weight-change. Mice treated with A27D7 at T = +1 were fully protected against morbidity. N = 5 mice/group. Data presented is from 1 of 2 studies—both with similar results. (B) Mice were monitored for mortality until T = +21. Only mice treated with A27D7 were significantly protected against challenge. P = 0.0004 for T = +1 and P = 0.006 for T = -1 (90% protected). N = 10 animals/group. Data is pooled from 2 independent studies of 5 animals/group. Controls include no ECTV challenge (PBS/NT), challenge without treatment (NT), treatment with vehicle only (Veh) or CDV treatment.
Mentions: In order to demonstrate the in vivo protective capabilities of antibodies A2C7, A27D7, and A20G2 we evaluated their efficacy following a lethal challenge with ECTV. Mice were treated with a single 100 μg IP injection of the antibodies at either T = -1 or T = +1 relative to challenge with 1000 PFU of ECTV. An additional group of mice was treated with the potent antiviral cidofivir (CDV), which is known to be protective against this dose of ECTV. Following challenge, we observed that mice treated with antibodies A2C7 and A20G2 were not able to statistically protect mice against challenge when compared to mice treated with vehicle (Fig 7A). Conversely, mice treated with the A27D7 antibody were fully protected against challenge when the antibody was administered at T = +1 (P = 0.0004) and were 90% protected (P = 0.006) when the antibody was administered at T = -1 (Fig 7A). Although weight-loss was significant following the T = -1 administration, we found negligible levels of weight-loss when the antibody was administered at T = +1 (Fig 7A). Indeed, at T = +1 we found that A27D7 protected against mortality and morbidity with the equivalency of the CDV-treated controls (Fig 7B). The slightly reduced efficacy of the antibodies when administered at T = -1 is likely a reflection of the antibody half-life, which we have not addressed in this assay. At T = +21 days post-challenge, we re-challenged the mice with a 10,000 PFU inoculum of ECTV—all previously challenged mice were protected against subsequent mortality and morbidity, indicating that the antibodies did not impede the generation of memory and protective immunity.

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