Refined methods for propagating vesicular stomatitis virus vectors that are defective for G protein expression.
Propagation-defective vesicular stomatitis virus (VSV) vectors that encode a truncated G protein (VSV-Gstem) or lack the G gene entirely (VSV-DeltaG) are attractive vaccine vectors because they are immunogenic, cannot replicate and spread after vaccination, and do not express many of the epitopes that elicit neutralizing anti-VSV immunity. To consider advancing non-propagating VSV vectors towards clinical assessment, scalable technology that is compliant with human vaccine manufacturing must be developed to produce clinical trial material. Accordingly, two propagation methods were developed for VSV-Gstem and VSV-DeltaG vectors encoding HIV gag that have the potential to support large-scale production. One method is based on transient expression of G protein after electroporating plasmid DNA into Vero cells and the second is based on a stable Vero cell line that contains a G gene controlled by a heat shock-inducible transcription unit. Both methods reproducibly supported production of 1 x 10(7) to 1 x 10(8) infectious units (I.U.s) of vaccine vector per milliliter. Results from these studies also showed that optimization of the G gene is necessary for abundant G protein expression from electroporated plasmid DNA or from DNA integrated in the genome of a stable cell line, and that the titers of VSV-Gstem vectors generally exceeded VSV-DeltaG.
Witko
Susan E
SE
Pfizer Vaccine Research, 401 North Middletown Road, Pearl River, NY 10965, United States.
Johnson
J Erik
JE
Kalyan
Narender K
NK
Felber
Barbara K
BK
Pavlakis
George N
GN
Sidhu
Maninder K
MK
Hendry
R Michael
RM
Udem
Stephen A
SA
Parks
Christopher L
CL
D000818:Animals; D002522:Chlorocebus aethiops; D005822:Genetic Vectors; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000172:deficiency*; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA; D014709:Vero Cells; D018116:Vesiculovirus / Q000235:genetics* / Q000254:growth & development*; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000172:deficiency*; D014776:Virus Cultivation / Q000379:methods; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus