A herpes simplex virus type 1 mutant deleted for γ34.5 and LAT kills glioma cells in vitro and is inhibited for in vivo reactivation

Abstract

To create an oncolytic herpes simplex virus type 1 ( HSV -1 ) that is inhibited for reactivation, we constructed a novel herpes recombinant virus with deletions in the 34.5 and LAT genes. The LAT gene was replaced by the gene for green fluorescent protein, thereby allowing viral infection to be followed. This virus, designated DM33, is effective in killing primary and established human glioma cell lines in culture. DM33 is considerably less virulent following intracerebral inoculation of HSV -susceptible BALB / c mice than the wild -type HSV -1 strain McKrae. The safety of this virus is further supported by the retention of its sensitivity to ganciclovir and its relatively limited toxicity against cultured human neuronal cells, astrocytes, and endothelial cells. The ability of DM33 to spontaneously reactivate was tested in a rabbit ocular infection model that accurately depicts human herpes infection and reactivation. Following ocular infection of rabbits, spontaneous reactivation was detected in 83% ( 15 / 18 ) of the eyes infected with wild -type McKrae. In contrast, none of the eyes infected with DM33 had detectable reactivation. The efficacy of this virus in cultured human glioma cell lines, its safety, confirmed by its inability to reactivate, and its attenuated neurovirulence make DM33 a promising oncolytic agent for tumor therapy.

Recombinant Herpes Simplex Virus Type 1 (HSV-1) Codelivering Interleukin-12p35 as a Molecular Adjuvant Enhances the Protective Immune Response against Ocular HSV-1 Challenge

“…1B), and a LAT-␥34.5-null mutant expressing IL-4 (vIL-4) (45). Ocular challenge of mice with these recombinant viruses showed that, similar to the parental virus (49) and other recombinant HSV-1 lacking the ␥34.5 gene (8), these recombinant viruses are also attenuated (46). CL7 and spleen cells were grown in RPMI 1640 supplemented with 10% fetal calf serum.…”

“…Briefly, pLAT-IL12p35 or pLAT-IL12p40 were cotransfected individually with infectious HSV-1 double mutant (LAT-␥34.5-null mutant) DNA by using the calcium phosphate method. This virus is a mutant of HSV-1 strain McKrae in which 1.8 kb of LAT and 0.9 kb of ␥34.5 have been deleted (49). Viruses from the cotransfection were plated, and the isolated plaques were picked and then screened for insertion of IL-12p35 or IL-12p40 gene by using restriction digestion and Southern blot analysis.…”

“…The arrow at Ϫ28 indicates the LAT TATA box. (B) LAT-␥34.5-null mutant (dbl-p) virus is a mutant of HSV-1 strain McKrae in which 1.8 kb of LAT (nucleotides Ϫ161 to ϩ1667) and 0.9 kb of ␥34.5 (nt ϩ6309 to ϩ7103) have been deleted (49). The deleted regions are indicated by "XXXXXX" and "XXX."…”

Comparison of Adjuvant Efficacy of Herpes Simplex Virus Type 1 Recombinant Viruses Expressing TH1 and TH2 Cytokine Genes

“…Briefly, pLAT-IL-2, pLAT-IL-4, or pLAT-IFN␥ was cotransfected with infectious HSV-1 double mutant (DM-33) DNA by the calcium phosphate method. The DM-33 (LAT-␥34.5-null mutant) virus is a mutant of HSV-1 strain McKrae in which 1.8 kb of LAT and 0.9 kb of ␥34.5 have been deleted (42). Viruses from the cotransfection were plated, and the isolated plaques were picked and then screened for the presence of the IL-2, IL-4, or IFN-␥ gene using restriction digestion and Southern blot analysis.…”

Advance in herpes simplex viruses for cancer therapy

“…In another study, DM33 demonstrated dose-dependent antitumor activity in glioma cell lines. The safety of DM33 was supported by its attenuated toxicity against neuronal cells, astrocytes, and endothelial cells [26]. Based on its efficacy and safety, DM33 shows considerable promise as an oncolytic virus for cancer treatment.…”