TY - JOUR

T1 - Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping

AU - Khlusevich, Yana

AU - Matveev, Andrey

AU - Baykov, Ivan

AU - Bulychev, Leonid

AU - Bormotov, Nikolai

AU - Ilyichev, Ivan

AU - Shevelev, Georgiy

AU - Morozova, Vera

AU - Pyshnyi, Dmitrii

AU - Tikunova, Nina

N1 - Publisher Copyright: © 2018

PY - 2018/4/1

Y1 - 2018/4/1

N2 - In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15–19 aa and 232–237 aa, are involved in binding with neutralizing anti-p35 antibodies.

AB - In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15–19 aa and 232–237 aa, are involved in binding with neutralizing anti-p35 antibodies.

KW - Antibodies, Neutralizing/genetics

KW - Antibodies, Viral/genetics

KW - Ectromelia virus/genetics

KW - Epitope Mapping

KW - Humans

KW - Neutralization Tests

KW - Peptide Library

KW - Single-Chain Antibodies/genetics

KW - Smallpox/immunology

KW - Variola virus/chemistry

KW - Viral Envelope Proteins/chemistry

UR - http://www.scopus.com/inward/record.url?scp=85041508838&partnerID=8YFLogxK

U2 - 10.1016/j.antiviral.2018.02.006

DO - 10.1016/j.antiviral.2018.02.006

M3 - Article

C2 - 29427674

AN - SCOPUS:85041508838

VL - 152

SP - 18

EP - 25

JO - Antiviral Research

JF - Antiviral Research

SN - 0166-3542

ER -