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. 2006 Aug;80(15):7308-15.
doi: 10.1128/JVI.00725-06.

Serial passage through human glioma xenografts selects for a Deltagamma134.5 herpes simplex virus type 1 mutant that exhibits decreased neurotoxicity and prolongs survival of mice with experimental brain tumors

Amish C Shah  1 , Kathleen H Price, Jacqueline N Parker, Sharon L Samuel, Sreelatha Meleth, Kevin A Cassady, G Yancey Gillespie, Richard J Whitley, James M Markert
Affiliations

Serial passage through human glioma xenografts selects for a Deltagamma134.5 herpes simplex virus type 1 mutant that exhibits decreased neurotoxicity and prolongs survival of mice with experimental brain tumors

Amish C Shah et al. J Virol. 2006 Aug.

Abstract

Previous studies have described in vitro serial passage of a Deltagamma(1)34.5 herpes simplex virus type 1 (HSV-1) strain in SK-N-SH neuroblastoma cells and selection of mutants that have acquired the ability to infect and replicate in this previously nonpermissive cell line. Here we describe the selection of a mutant HSV-1 strain by in vivo serial passage, which prolongs survival in two separate experimental murine brain tumor models. Two conditionally replication-competent Deltagamma(1)34.5 viruses, M002, which expresses murine interleukin-12, and its parent virus, R3659, were serially passaged within human malignant glioma D54-MG cell lines in vitro or flank tumor xenografts in vivo. The major findings are (i) viruses passaged in vivo demonstrate decreased neurovirulence, whereas those passaged in vitro demonstrate a partial recovery of the neurovirulence associated with HSV-1; and (ii) vvD54-M002, the virus selected after in vivo serial passage of M002 in D54-MG tumors, improves survival in two independent murine brain tumor models compared to the parent (unpassaged) M002. Additionally, in vitro-passaged, but not in vivo-passaged, M002 displayed changes in the protein synthesis profile in previously nonpermissive cell lines, as well as early U(S)11 transcription. Thus, a mutant HSV-1 strain expressing a foreign gene can be selected for enhanced antitumor efficacy via in vivo serial passage within flank D54-MG tumor xenografts. The enhanced antitumor efficacy of vvD54-M002 is not due to restoration of protein synthesis or early U(S)11 expression. This finding emphasizes the contribution of the in vivo tumor environment for selecting novel oncolytic HSV specifically adapted for tumor cell destruction in vivo.

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Figures

FIG. 1.
FIG. 1.
Schematic of selection of novel Δγ134.5 HSV-1 by serial passage. (A) Selection by in vivo serial passage. (B) Selection by in vitro serial passage.
FIG. 2.
FIG. 2.
In vitro and in vivo replication of parent and passaged viruses. (A) D54-MG cells were cultured in 24-well plates in vitro, and replicate wells were infected (MOI = 0.1) with either HSV-1 (F), parent viruses (R3659 or M002), or a serially passaged virus. Samples were harvested 12, 24, 36, 48, and 72 hpi. (B) Replication time course after intratumoral injection of viruses in D54-MG brain tumors established intracranially in SCID mice.
FIG. 3.
FIG. 3.
Recombinant IL-12 production after M002 and vvD54-M002 infection. IL-12 production after infection with viruses was determined using an ELISA kit (R&D Systems) specific for murine IL-12 p70 heterodimer. Shown are the levels of IL-12 detected after vvD54-M002 or M002 infection of (A) D54-MG cells (72 hpi, P = 0.0124; 84 hpi, P = 0.0093) and (B) Neuro-2a cells (48 hpi, P = 0.0152; 72 hpi, P = 0.0008).
FIG. 4.
FIG. 4.
Protein synthesis profiles from infected D54-MG human glioma cells and SK-N-SH human neuroblastoma cells. An autoradiographic image of protein production from cells pulse-labeled with 35S-labeled l-methionine 12 hpi (MOI = 50) with either HSV-1 (F), parent viruses (M002 or R3659), or viruses serially passaged in vivo or in vitro in (A) SK-N-SH cells or (B) D54-MG cells.
FIG. 5.
FIG. 5.
Expression kinetics of US11 from infected Vero cells. Shown is US11 expression 12 hpi from Vero cells in the absence (−) or presence (+) of 300 μg/ml PAA infected. Cells were infected with R5104 (early US11 expression), HSV-1 (F), M002, or the in vivo- and in vitro-passaged M002 viruses. The arrow indicates US11.
FIG. 6.
FIG. 6.
Survival of mice bearing human D54-MG brain tumors after vvD54-M002 treatment. Immunocompromised SCID mice bearing D54-MG brain tumors were treated with either saline (closed circles) as a control or 1 × 107 PFU of either R3659 (closed diamonds), M002 (closed squares), or vvD54-M002 (open squares) after 7 days. The survival graph shows two separate studies combined. Cohorts were compared using Kaplan-Meier statistical analysis.
FIG. 7.
FIG. 7.
Survival of mice bearing Neuro-2a brain tumors after vvD54-M002 treatment. Immunocompetent A/J mice bearing Neuro-2a brain tumors were treated with either saline (closed circles) as a control or 1 × 107 PFU of either R3659 (closed diamonds), M002 (closed squares), or vvD54-M002 (open squares) after 5 days. The survival graph shows two separate studies combined. Cohorts were compared using Kaplan-Meier statistical analysis.
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