Synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector.
A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6.6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected insect cells made poliovirus proteins that included the structural proteins VP0, VP1 and VP3. Poliovirus particles were recovered from extracts of the infected cells and demonstrated to be free from detectable levels of RNA and to be non-infectious in tissue culture. After particle purification by CsCl gradient centrifugation and immunization of outbred mice, antibodies to the structural proteins, including neutralizing antibodies, were obtained. Other recombinant baculoviruses, containing the majority of the capsid coding region of P3/Leon/37 (e.g. AcCAP21, nucleotide residues 742 to 3318), made an unprocessed precursor to the poliovirus structural proteins. These data suggested that processing of the poliovirus gene product by the AcLeon construct was catalysed by the poliovirus-encoded proteases. The results demonstrated that antigenic and immunogenic poliovirus proteins and empty particles can be made in insect cells by recombinant baculoviruses.
Urakawa
T
T
NERC Institute of Virology, Oxford, U.K.
Ferguson
M
M
Minor
P D
PD
Cooper
J
J
Sullivan
M
M
Almond
J W
JW
Bishop
D H
DH
D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004279:DNA, Viral / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta / Q000382:microbiology; D051379:Mice; D010957:Plasmids; D017955:Poliovirus / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015678:Viral Structural Proteins; D014771:Virion / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity
D000914:Antibodies, Viral; D004279:DNA, Viral; D014764:Viral Proteins; D015678:Viral Structural Proteins