[Construction of a new oncolytic virus oHSV2hGM-CSF and its anti-tumor effects].
OBJECTIVE
The aim of this study was to construct a new oncolytic virus oHSV2hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV1hGM-CSF.


METHODS
oHSV2hGM-CSF was a replication-competent, attenuated HSV2 based on the HG52 virus (an HSV2 strain). It was engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and insertion of the gene encoding hGM-CSF. To measure the in vitro killing effect of the virus, 15 human tumor cell lines (HeLa, Eca-109, PG, HepG2, SK/FU, CNE-2Z, PC-3, SK-OV3, A-549, 786-0, MCF-7, Hep-2, HT-29, SK-Mel-28, U87-MG) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at MOI = 1 (multiplicity of infection, MOI), or left uninfected. The cells were harvested 24 and 48 hours post infection, and observed under the microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3 x 10(6) PFU (plaque forming unit, PFU) for three times when the tumor volume reached 7-8 mm3. The tumor volume was measured at 3-day intervals and animal survival was recorded.


RESULTS
Both oHSV2hCM-CSFand oHSV1hGM-CSF induced widespread cytopathic effects at 24 h after infection. OHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSV1hGM-CSF. In the in vitro killing experiments for the cell lines HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSV1hGM-CSF under the same conditions. For the mouse experiments, it was observed that oHSV2hGM-CSF significantly inhibited the tumor growth. At 15 days after B16R tumor cells inoculation, the tumor volumes of the PBS, oHSV1hGCM-CSF and oHSV2hGM-CSF groups were (374.7 +/- 128.24) mm3, (128.23 +/- 45.32) mm3 (P < 0.05, vs. PBS group) or (10.06 +/- 5.1) mm3 (P < 0.01, vs. PBS group), respectively (mean +/- error). The long term therapeutic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days after tumor cells inoculation whereas all the mice in the PBS group died by day 22 (P < 0.01). The anti-tumor mechanism of the newly constructed oHSV2hGM-CSF against B16R cell tumor appeared to include the directly oncolytic activity and the induction of anti-tumor immunity to some degree.


CONCLUSIONS
The findings of our study demonstrate that the newly constructed oHSV2hGM-CSF has potent anti-tumor activity in vitro to many tumor cell lines and in vive to the transplanted B16R tumor models.
Department of Immunology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
Gui-Lan
GL
Shi
Xiu-Fen
XF
Zhuang
Xiang-Ping
XP
Han
Jie
J
Li
Yu
Y
Zhang
Shu-Ren
SR
Zhang
Bin-Lei
BL
Liu
D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D017353:Gene Deletion; D005818:Genetic Engineering; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D018258:Herpesvirus 2, Human / Q000235:genetics* / Q000276:immunology; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D008546:Melanoma, Experimental / Q000473:pathology / Q000628:therapy* / Q000821:virology; D051379:Mice; D008810:Mice, Inbred C57BL; D050130:Oncolytic Virotherapy / Q000379:methods*; D050504:Oncolytic Viruses / Q000235:genetics* / Q000502:physiology; D011897:Random Allocation; D047368:Tumor Burden; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
C540071:ICP34.5 protein, human herpesvirus-2; C105843:ICP47 protein, Herpes simplex virus; D017874:Immediate-Early Proteins; D014764:Viral Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor