Affinity maturation of an anti-V antigen IgG expressed in situ through adenovirus gene delivery confers enhanced protection against Yersinia pestis challenge

T. J. Van Blarcom, C. Sofer-Podesta, J. Ang, J. L. Boyer, Ronald Crystal, G. Georgiou

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Genetic transfer of neutralizing antibodies (Abs) has been shown to confer strong and persistent protection against bacterial and viral infectious agents. Although it is well established that for many exogenous neutralizing Abs increased antigen affinity correlates with protection, the effect of antigen affinity on Abs produced in situ after adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal Ab, 2C12.4, recognizes the Yersinia pestis type III secretion apparatus protein, LcrV (V antigen), and confers protection in mice when administered as an IgG intraperitoneally or after genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad). The 2C12.4 Ab was expressed as a single-chain variable fragment (scFv) in Escherichia coli and was shown to display an equilibrium dissociation constant (K D)3.5 nM by surface plasmon resonance analysis. The 2C12.4 scFv was subjected to random mutagenesis, and variants with increased affinity were isolated by flow cytometry using the anchored periplasmic expression bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower K D values (H8, K D 100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV, giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen Abs 3 days after immunization, with 10 9, 10 10 or 10 11 particle units (pu). After intranasal challenge with 363 LD 50 (lethal dose, 50%) of Y. pestis CO92, 54% of the mice immunized with 10 10 pu of AdαV.H8 survived through the 14 day end point compared with only 15% survivors for the group immunized with AdαV expressing the lower-affinity 2C12.4 (P0.04; AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing Ab delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but also possibly for other pathogens.

Original languageEnglish
Pages (from-to)913-921
Number of pages9
JournalGene Therapy
Volume17
Issue number7
DOIs
Publication statusPublished - 1 Jul 2010
Externally publishedYes

Fingerprint

Yersinia pestis
Adenoviridae
Immunoglobulin G
Antigens
Neutralizing Antibodies
Mutagenesis
Genes
Immunization
Human Adenoviruses
Single-Chain Antibodies
Antibody Affinity
Surface Plasmon Resonance
Lethal Dose 50
Flow Cytometry
Escherichia coli
Antibodies
Proteins

Keywords

  • adenovirus gene transfer
  • affinity maturation
  • antibody
  • protection
  • Yersinia pestis

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Affinity maturation of an anti-V antigen IgG expressed in situ through adenovirus gene delivery confers enhanced protection against Yersinia pestis challenge. / Van Blarcom, T. J.; Sofer-Podesta, C.; Ang, J.; Boyer, J. L.; Crystal, Ronald; Georgiou, G.

In: Gene Therapy, Vol. 17, No. 7, 01.07.2010, p. 913-921.

Research output: Contribution to journalArticle

@article{ed77e249a39543568dd25058acc55dc5,
title = "Affinity maturation of an anti-V antigen IgG expressed in situ through adenovirus gene delivery confers enhanced protection against Yersinia pestis challenge",
abstract = "Genetic transfer of neutralizing antibodies (Abs) has been shown to confer strong and persistent protection against bacterial and viral infectious agents. Although it is well established that for many exogenous neutralizing Abs increased antigen affinity correlates with protection, the effect of antigen affinity on Abs produced in situ after adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal Ab, 2C12.4, recognizes the Yersinia pestis type III secretion apparatus protein, LcrV (V antigen), and confers protection in mice when administered as an IgG intraperitoneally or after genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad). The 2C12.4 Ab was expressed as a single-chain variable fragment (scFv) in Escherichia coli and was shown to display an equilibrium dissociation constant (K D)3.5 nM by surface plasmon resonance analysis. The 2C12.4 scFv was subjected to random mutagenesis, and variants with increased affinity were isolated by flow cytometry using the anchored periplasmic expression bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower K D values (H8, K D 100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV, giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen Abs 3 days after immunization, with 10 9, 10 10 or 10 11 particle units (pu). After intranasal challenge with 363 LD 50 (lethal dose, 50{\%}) of Y. pestis CO92, 54{\%} of the mice immunized with 10 10 pu of AdαV.H8 survived through the 14 day end point compared with only 15{\%} survivors for the group immunized with AdαV expressing the lower-affinity 2C12.4 (P0.04; AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing Ab delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but also possibly for other pathogens.",
keywords = "adenovirus gene transfer, affinity maturation, antibody, protection, Yersinia pestis",
author = "{Van Blarcom}, {T. J.} and C. Sofer-Podesta and J. Ang and Boyer, {J. L.} and Ronald Crystal and G. Georgiou",
year = "2010",
month = "7",
day = "1",
doi = "10.1038/gt.2010.42",
language = "English",
volume = "17",
pages = "913--921",
journal = "Gene Therapy",
issn = "0969-7128",
publisher = "Nature Publishing Group",
number = "7",

}

TY - JOUR

T1 - Affinity maturation of an anti-V antigen IgG expressed in situ through adenovirus gene delivery confers enhanced protection against Yersinia pestis challenge

AU - Van Blarcom, T. J.

AU - Sofer-Podesta, C.

AU - Ang, J.

AU - Boyer, J. L.

AU - Crystal, Ronald

AU - Georgiou, G.

PY - 2010/7/1

Y1 - 2010/7/1

N2 - Genetic transfer of neutralizing antibodies (Abs) has been shown to confer strong and persistent protection against bacterial and viral infectious agents. Although it is well established that for many exogenous neutralizing Abs increased antigen affinity correlates with protection, the effect of antigen affinity on Abs produced in situ after adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal Ab, 2C12.4, recognizes the Yersinia pestis type III secretion apparatus protein, LcrV (V antigen), and confers protection in mice when administered as an IgG intraperitoneally or after genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad). The 2C12.4 Ab was expressed as a single-chain variable fragment (scFv) in Escherichia coli and was shown to display an equilibrium dissociation constant (K D)3.5 nM by surface plasmon resonance analysis. The 2C12.4 scFv was subjected to random mutagenesis, and variants with increased affinity were isolated by flow cytometry using the anchored periplasmic expression bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower K D values (H8, K D 100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV, giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen Abs 3 days after immunization, with 10 9, 10 10 or 10 11 particle units (pu). After intranasal challenge with 363 LD 50 (lethal dose, 50%) of Y. pestis CO92, 54% of the mice immunized with 10 10 pu of AdαV.H8 survived through the 14 day end point compared with only 15% survivors for the group immunized with AdαV expressing the lower-affinity 2C12.4 (P0.04; AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing Ab delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but also possibly for other pathogens.

AB - Genetic transfer of neutralizing antibodies (Abs) has been shown to confer strong and persistent protection against bacterial and viral infectious agents. Although it is well established that for many exogenous neutralizing Abs increased antigen affinity correlates with protection, the effect of antigen affinity on Abs produced in situ after adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal Ab, 2C12.4, recognizes the Yersinia pestis type III secretion apparatus protein, LcrV (V antigen), and confers protection in mice when administered as an IgG intraperitoneally or after genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad). The 2C12.4 Ab was expressed as a single-chain variable fragment (scFv) in Escherichia coli and was shown to display an equilibrium dissociation constant (K D)3.5 nM by surface plasmon resonance analysis. The 2C12.4 scFv was subjected to random mutagenesis, and variants with increased affinity were isolated by flow cytometry using the anchored periplasmic expression bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower K D values (H8, K D 100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV, giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen Abs 3 days after immunization, with 10 9, 10 10 or 10 11 particle units (pu). After intranasal challenge with 363 LD 50 (lethal dose, 50%) of Y. pestis CO92, 54% of the mice immunized with 10 10 pu of AdαV.H8 survived through the 14 day end point compared with only 15% survivors for the group immunized with AdαV expressing the lower-affinity 2C12.4 (P0.04; AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing Ab delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but also possibly for other pathogens.

KW - adenovirus gene transfer

KW - affinity maturation

KW - antibody

KW - protection

KW - Yersinia pestis

UR - http://www.scopus.com/inward/record.url?scp=77954537924&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77954537924&partnerID=8YFLogxK

U2 - 10.1038/gt.2010.42

DO - 10.1038/gt.2010.42

M3 - Article

VL - 17

SP - 913

EP - 921

JO - Gene Therapy

JF - Gene Therapy

SN - 0969-7128

IS - 7

ER -