Cloning, expression and functional activities of a single chain antibody fragment directed to fusion protein of respiratory syncytial virus.
BACKGROUND
HNK20 is a murine IgA which is currently being investigated in clinical trials against respiratory syncytial virus (RSV) infections in infants and young children.


OBJECTIVE
To produce a single chain antibody fragment (scFv) from HNK20 hybridoma cells and assess its functional activities in vitro and in vivo (mouse model).


METHODS
The V regions of heavy and light chains were cloned and linked by a sequence encoding for (Gly4 Ser)3 and expressed in Escherichia coli.


RESULTS
Over 100 mg/l of the HNK20-scFv was produced in shake flasks after induction with isopropyl (beta-D-thiogalactopyranoside (IPTG). ScFv was purified under native conditions on a Ni2+ affinity column and migrated as a single band of 34 kDa on sodium dodecyl sulfate (SDS)-gels. ScFv demonstrated similar affinity as its parent IgA molecule, neutralized RSV in vitro and significantly reduced RSV titers in lungs of mice when administered intranasally shortly before or a day after RSV challenge.


CONCLUSIONS
It is possible that this scFv or its derivatives, when applied by intranasal or pulmonary route, will be useful for treatment of RSV infections in infants and young children.
Guirakhoo
F
F
OraVax, Cambridge, MA 02139, USA. fguirakh@oravax.com
Catalan
J
J
Monath
T
T
Weltzin
R
R
D000818:Animals; D003001:Cloning, Molecular; D005260:Female; D015870:Gene Expression; D015707:HN Protein; D007070:Immunoglobulin A / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000276:immunology; D013696:Temperature; D014170:Transformation, Genetic; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins / Q000235:genetics / Q000276:immunology*; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology*
D015707:HN Protein; D007070:Immunoglobulin A; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins; C116204:attachment protein G