Evaluation of infection parameters in the production of replication-defective HSV-1 viral vectors.
Herpes simplex virus type-1 (HSV-1) is a neurotrophic human pathogen that establishes life-long latency in the nervous system. Our laboratory has extensively engineered this virus to retain the ability to persist in neurons without expression of lytic genes or disease phenotype. Highly defective, replication-incompetent HSV mutants are thus potentially ideal for transfer of therapeutic transgenes to human nerves where long-term therapy of nervous system disease may be provided. A prerequisite for using recombinant HSV vectors for therapeutic gene delivery to humans is the development of methods for large-scale manufacture of HSV vectors. Here we report studies to identify infection parameters that result in high-yield production of immediate early gene deletion mutant HSV vectors in complementing cells that supply the deleted essential viral functions in trans. Virus yield was correlated with various culture media conditions that included pH, glucose metabolism, and serum levels. The results demonstrated that systematic media exchange to remove lactate derived from high-level glucose consumption, maintenance of tissue culture pH at 6.8, and the use of 5% fetal bovine serum gave the highest yield of infectious virus. The data indicate that these are important parameters to consider for high-yield, large-scale virus production.
Ozuer
Ali
A
Department of Chemical Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Wechuck
James B
JB
Russell
Brian
B
Wolfe
Darren
D
Goins
William F
WF
Glorioso
Joseph C
JC
Ataai
Mohammad M
MM
D000818:Animals; D002522:Chlorocebus aethiops; D003673:Defective Viruses / Q000235:genetics* / Q000254:growth & development; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D014709:Vero Cells; D010948:Viral Plaque Assay