Effect of Preexisting Anti-Herpes Immunity on the Efficacy of Herpes Simplex Viral Therapy in a Murine Intraperitoneal Tumor Model
2000; Elsevier BV; Volume: 2; Issue: 4 Linguagem: Inglês
10.1006/mthe.2000.0133
ISSN1525-0024
AutoresEric S. Lambright, Eugene H. Kang, Seth Force, Michael Lanuti, David Caparrelli, Larry R. Kaiser, Steven M. Albelda, Katherine L. Molnar-Kimber,
Tópico(s)CAR-T cell therapy research
ResumoHSV-1716, a replicating nonneurovirulent herpes simplex virus type 1, has shown efficacy in treating multiple types of human tumors in immunodeficient mice. Since the majority of the human population has been previously exposed to herpes simplex virus, the efficacy of HSV-based oncolytic therapy was investigated in an immunocompetent animal tumor model. EJ-6-2-Bam-6a, a tumor cell line derived from h-ras-transformed murine fibroblast, exhibit a diffuse growth pattern in the peritoneal cavity of BALB/c mice and replicate HSV-1716 to titers observed in human tumors. An established intraperitoneal (ip) tumor model of EJ-6-2-Bam-6a in naive and HSV-immunized mice was used to evaluate the efficacy of single or multiple ip administrations of HSV-1716 (4 × 106 pfu/treatment) or of carrier cells, which are irradiated, ex vivo virally infected EJ-6-2-Bam-6a cells that can amplify the viral load in situ. All treated groups significantly prolonged survival versus media control with an approximately 40% long-term survival rate (cure) in the multiply treated, HSV-naive animals. Prior immunization of the mice with HSV did not significantly decrease the median survival of the single or multiply treated HSV-1716 or the carrier cell-treated groups. These studies support the development of replication-selective herpes virus mutants for use in localized intraperitoneal malignancies. HSV-1716, a replicating nonneurovirulent herpes simplex virus type 1, has shown efficacy in treating multiple types of human tumors in immunodeficient mice. Since the majority of the human population has been previously exposed to herpes simplex virus, the efficacy of HSV-based oncolytic therapy was investigated in an immunocompetent animal tumor model. EJ-6-2-Bam-6a, a tumor cell line derived from h-ras-transformed murine fibroblast, exhibit a diffuse growth pattern in the peritoneal cavity of BALB/c mice and replicate HSV-1716 to titers observed in human tumors. An established intraperitoneal (ip) tumor model of EJ-6-2-Bam-6a in naive and HSV-immunized mice was used to evaluate the efficacy of single or multiple ip administrations of HSV-1716 (4 × 106 pfu/treatment) or of carrier cells, which are irradiated, ex vivo virally infected EJ-6-2-Bam-6a cells that can amplify the viral load in situ. All treated groups significantly prolonged survival versus media control with an approximately 40% long-term survival rate (cure) in the multiply treated, HSV-naive animals. Prior immunization of the mice with HSV did not significantly decrease the median survival of the single or multiply treated HSV-1716 or the carrier cell-treated groups. These studies support the development of replication-selective herpes virus mutants for use in localized intraperitoneal malignancies. IntroductionReplication-selective herpes simplex viruses that have deletions in the RL1 gene (HSV-1716 and HSV-R3616), the ribonuclease reductase (ICP6) gene (hrR3), or both genes (G207, MGH-1) were first shown to exhibit efficacy against various brain tumors (1Chambers R. et al.Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a SCID mouse model of human malignant glioma.Proc. Natl. Acad. Sci. USA. 1995; 92: 1411-1415Crossref PubMed Scopus (181) Google Scholar, 2Kesari S. et al.Therapy of experimental human brain tumors using a neuroattenuated herpes simplex virus mutant.Lab. Invest. 1995; 73: 636-648PubMed Google Scholar, 3Mineta T. Rabkin S.D. Yazaki T. Hunter W.D. Martuza R.L. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas.Nat. Med. 1995; 1: 938-943Crossref PubMed Scopus (684) Google Scholar). HSV-1716, R3616, hrR3, and G207 have also shown efficacy in animal models against a wide variety of other human tumors, including melanoma (4Randazzo B. et al.Treatment of experimental intracranial murine melanoma with a neuroattenuated herpes simplex virus 1 mutant.Virology. 1995; 211: 94-101Crossref PubMed Scopus (99) Google Scholar, 5Randazzo B.P. Bhat M.G. Kesari S. Fraser N.W. Brown S.M. Treatment of experimental subcutaneous human melanoma with a replication-restricted herpes simplex virus mutant.J. Invest. Dermatol. 1997; 108: 933-937Abstract Full Text PDF PubMed Scopus (54) Google Scholar), malignant mesothelioma (6Kucharczuk J.C. et al.Use of a replication-restricted recombinantherpes virus to treat localized human malignancy.Cancer Res. 1997; 57: 466-471PubMed Google Scholar), colon cancer (7Carroll N.M. Chiocca E.A. Takahashi K. Tanabe K.K. Enhancement of gene therapy specificity for diffuse colon carcinoma liver metastases with recombinant herpes simplex virus.Ann. Surg. 1996; 224: 323-329Crossref PubMed Scopus (85) Google Scholar, 8Yoon S.S. Carroll N.M. Chiocca E.A. Tanabe K.K. Cancer gene therapy using a replication-competent herpes simplex virus type 1 vector.Ann. Surg. 2000; 228: 366-374Crossref Scopus (68) Google Scholar, 9Yoon S.S. Nakamura H. Carroll N.M. Bode B.P. Chiocca E.A. Tanabe K.K. An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma.FASEB J. 2000; 14: 301-311Crossref PubMed Scopus (105) Google Scholar), breast cancer (10Toda M. Rabkin S.D. Martuza R.L. Treatment of human breast cancer in a brain metastatic model by G207 areplication competent multimutated herpes simplex virus 1.Hum. Gene Ther. 2000; 9: 2173-2185Google Scholar), ovarian cancer (11Coukos G. et al.Use of carrier cells to deliver a replication-selective herpes simplex virus-1 mutant for the intraperitoneal therapy of epithelial ovarian cancer.Clin. Cancer Res. 1999; 5: 1523-1537PubMed Google Scholar, 12Coukos G. et al.Multi-attenuated herpes simplex virus-1 mutant G207 exerts cytotoxicity against epithelial ovarian cancer but not normal mesothelium and is suitable for intraperitoneal oncolytic therapy.Cancer Gene Ther. 2000; 7: 275-283Crossref PubMed Scopus (63) Google Scholar), head and neck tumors (13Carew J.F. Kooby D.A. Halterman M.W. Federoff H.J. Fong Y. Selective infection and cytolysis of human head and neck squamous cell carcinoma with sparing of normal mucosa by a cytotoxic herpes simplex virus type 1 (G207).Hum. Gene Ther. 1999; 10: 1599-1606Crossref PubMed Scopus (78) Google Scholar), prostate cancers (14Advani S.J. et al.Replication-competent nonneuroinvasivegenetically engineered herpes virus is highly effective in the treatment of therapy-resistant experimental human tumors.Cancer Res. 1999; 59: 2055-2058PubMed Google Scholar), and lung cancer (15Lambright E.S. et al.Oncolytic therapy using a mutant type 1 herpes simplex virus and the role of the immune response.Ann. Thoracic Surg. 1999; 68: 1756-1762Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 16Toyoizumi T. Mick R. Abbas A.E. Kang E.H. Kaiser L.R. Molnar-Kimber K.L. Combined therapy with chemotherapeutic agents and herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) in human non-small cell lung cancer.Hum. Gene Ther. 1999; 10: 3013-3029Crossref PubMed Scopus (95) Google Scholar). Viruses with mutations in ICP6, the RL1 region, or both show 103, 105, or greater reduced neurovirulence, respectively (17Chou J. Kern E. Whitley R. Roizman B. Mapping of herpes simplex virus-1 neurovirulence to g1 34.5 agene nonessential for growth in culture.Science. 1990; 250: 1262-1266Crossref PubMed Scopus (631) Google Scholar, 18Hunter W.D. et al.Attenuated, replication-competent herpes simplex virus type 1 mutant G207: Safety evaluation of intracerebral injection in nonhuman primates.J. Virol. 1999; 73: 6319-6326Crossref PubMed Google Scholar, 19Valyi-Nagy T. et al.The herpes simplex virus type 1 strain 17+ gamma 34.5 deletion mutant 1716 is avirulent in SCID mice.J. Gen. Virol. 1994; 75: 2059-2063Crossref PubMed Scopus (62) Google Scholar) and do not produce encephalitis when inoculated peripherally. Additionally, these mutants replicate as well as their wild-type parental strain in a variety of dividing cells lines but replicate poorly in cells not undergoing mitosis (12Coukos G. et al.Multi-attenuated herpes simplex virus-1 mutant G207 exerts cytotoxicity against epithelial ovarian cancer but not normal mesothelium and is suitable for intraperitoneal oncolytic therapy.Cancer Gene Ther. 2000; 7: 275-283Crossref PubMed Scopus (63) Google Scholar, 20Brown S.M. Harland J. MacLean A.R. Podlech J. Clements J.B. Cell type and cell state determine differential in vitro growth of non-neurovirulent ICP34.5-negative herpes simplex virus types 1 and 2.J. Gen. Virol. 1994; 75: 2367-2377Crossref PubMed Scopus (84) Google Scholar).Our group has focused on the use of replication-selective HSV-1 viruses in the treatment of localized peripheral tumors including malignant mesothelioma (6Kucharczuk J.C. et al.Use of a replication-restricted recombinantherpes virus to treat localized human malignancy.Cancer Res. 1997; 57: 466-471PubMed Google Scholar) and ovarian carcinoma (11Coukos G. et al.Use of carrier cells to deliver a replication-selective herpes simplex virus-1 mutant for the intraperitoneal therapy of epithelial ovarian cancer.Clin. Cancer Res. 1999; 5: 1523-1537PubMed Google Scholar, 12Coukos G. et al.Multi-attenuated herpes simplex virus-1 mutant G207 exerts cytotoxicity against epithelial ovarian cancer but not normal mesothelium and is suitable for intraperitoneal oncolytic therapy.Cancer Gene Ther. 2000; 7: 275-283Crossref PubMed Scopus (63) Google Scholar). Intraperitoneal injection of HSV-1716 or irradiated "carrier" cells infected with HSV-1716 into immunodeficient animals bearing established human malignant mesothelioma and ovarian carcinoma cells resulted in significant survival increases in treated animals with minimal systemic toxicity.Although herpes viral gene therapy for malignant disease has shown promise in immunodeficient animal models, a major question is the influence of an intact immune system. Preexisting anti-herpes immunity is known to decrease lesion size with subsequent HSV-1 infection in both humans and mice (21Halford W.P. Veress L.A. Gebhardt B.M. Carr D.J. Innate and acquired immunity to herpes simplex virus type 1.Virology. 1997; 236: 328-337Crossref PubMed Scopus (54) Google Scholar, 22Mester J.C. Rouse B.T. The mouse model and understanding immunity to herpes simplex virus.Rev. Infect. Dis. 1991; 13(Suppl. 11): S935-S945Crossref PubMed Scopus (51) Google Scholar, 23Whitley R.J. Kimberlin D.W. Roizman B. Herpes simplex viruses.Clin. Infect. Dis. 2000; 26: 541-555Crossref Scopus (440) Google Scholar). The effect of previously established immunity on the efficacy of herpes-mediated oncolytic therapy and the ability to administer repeated doses of virus remains an unresolved issue.Herrlinger et al. reported that preexisting HSV immunity markedly decreased the initial HSV-hrR3 uptake in an experimental brain tumor model (24Herrlinger U. et al.Pre-existing herpes simplex virus 1 (HSV-1) immunity decreases butdoes not abolish genetransfer to experimental brain tumors by a HSV-1 vector.Gene Ther. 2000; 5: 809-819Crossref Scopus (70) Google Scholar), but Chahlavi et al. observed that anti-herpes immunity did not seriously impair efficacy in subcutaneous or brain tumor nodules in their mouse tumor model (25Chahlavi A. Rabkin S. Todo T. Sundaresan P. Martuza R. Effect of prior exposure to herpes simplex virus 1 on viral vector-mediated tumor therapy in immunocompetent mice.Gene Ther. 1999; 6: 1751-1758Crossref PubMed Scopus (86) Google Scholar). The role of anti-herpes immunity has been a difficult question to study because oncolytic herpes viruses such as G207, hrR3, and HSV-1716 do not replicate as well in murine tissue and cells lines as human cell lines.With our interest in mesothelioma and ovarian carcinoma, the first aim of this study was to develop an immunocompetent animal model of localized intraperitoneal malignancy in which we could study the potential therapeutic effects of a replication-selective HSV-1 vector. This was accomplished using an h-ras-transformed fibroblast that grows in a diffuse pattern on the various surfaces of the peritoneal cavity in BALB/c mice. The second aim was to evaluate the efficacy of administration of the replication-selective HSV-1 mutant, HSV-1716, in this immunocompetent model to determine the effects of treating a localized intraperitoneal murine tumor. The ability of single or repeated viral treatments to prolong survival was compared in naive and HSV-immunized animals. Our final aim was to evaluate the effect of anti-herpes immunity on the efficacy of treatment of tumors using HSV-1716-infected "carrier cells"—that is, cells that were irradiated, infected with the mutant herpes virus, and injected into the peritoneal cavity (11Coukos G. et al.Use of carrier cells to deliver a replication-selective herpes simplex virus-1 mutant for the intraperitoneal therapy of epithelial ovarian cancer.Clin. Cancer Res. 1999; 5: 1523-1537PubMed Google Scholar). We were interested in this delivery system because administering these carrier cells could theoretically protect the viral vector from host neutralizing antibodies.Materials and MethodsHerpes simplex vectorsHSV-1716, which was originally isolated in the laboratory of S. M. Brown (Glasgow, Scotland) (26MacLean A.R. Ul-Fareed M. Roberson L. Harland J. Brown S.M. Herpes simplex virus type 1 deletion variant 1714 and 1716 pinpoint neurovirulence-related sequences in Glasgow strain 17+ between immediate early gene 1 and the 'a' sequence.J. Gen. Virol. 1991; 72: 631-639Crossref PubMed Scopus (220) Google Scholar), and KOS1.1 (27Sandstrom I.K. Foster C.S. Wells P.A. Knipe D. Caron L. Greene M.I. Previous immunization of mice with herpes simplex virus type-1 strain MP protects against secondary corneal infection.Clin. Immunol. Immunopathol. 1986; 40: 326-334Crossref PubMed Scopus (20) Google Scholar) were passaged for use as previously described (16Toyoizumi T. Mick R. Abbas A.E. Kang E.H. Kaiser L.R. Molnar-Kimber K.L. Combined therapy with chemotherapeutic agents and herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) in human non-small cell lung cancer.Hum. Gene Ther. 1999; 10: 3013-3029Crossref PubMed Scopus (95) Google Scholar). HSV-1716 contains a 759-bp deletion in each copy of the RL1 region of the HSV I strain 17+ genome (26MacLean A.R. Ul-Fareed M. Roberson L. Harland J. Brown S.M. Herpes simplex virus type 1 deletion variant 1714 and 1716 pinpoint neurovirulence-related sequences in Glasgow strain 17+ between immediate early gene 1 and the 'a' sequence.J. Gen. Virol. 1991; 72: 631-639Crossref PubMed Scopus (220) Google Scholar). The RL1 region encodes the ICP 34.5 and thus no ICP 34.5 protein expression occurs in HSV-1716-infected cells. KOS1.1 was used to induce anti-HSV immunity in mice, as previously described (28Brenner G. Nicholas C. Moynihan J. Similar immune response to non-lethal infection with herpes simplex virus-1 in sensitive (BALB/c) and resistant (C57BL/6) strains of mice.Cell. Immunol. 1994; 157: 510-524Crossref PubMed Scopus (39) Google Scholar).Cell lines and cultureEJ-6-2-Bam-6a is a VIH3T3 mouse fibroblast cell line transformed with the h-ras oncogene that was derived from a human bladder carcinoma (29Bernstein S. Weinberg R. Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA..Proc. Natl. Acad. Sci. USA. 1985; 82: 1726-1730Crossref PubMed Scopus (88) Google Scholar) (American Type Culture Collection, Manassas, VA). It was maintained in DMEM with 10% fetal calf serum (FCS, Atlanta Biologicals, Norcross, GA), 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM glutamine (Mediatch, Washington, DC). Cells were harvested for tumor experiments during exponential growth of the cell culture.In vitro cell viability assaysEJ-6-2-Bam-6a cells were infected with HSV-1716 (diluted in serum-free DMEM) at multiplicity of infection (m.o.i.) values of 0.01. 0.1, 1, and 10 for 4 h. After 4 h, medium containing 10% heat-inactivated FCS was added and plates were incubated at 37°C for 1 h. The cells were trypsinized and transferred to 96-well plates at a density of 2000 cells/well (n = 6 per condition). Viable cell number was assessed by colorimetric assay (CellTiter 96 aqueous nonradioactive MTS cell proliferation assay; Promega, Madison, WI) which measures viable cell dehydrogenase activity by absorbency at 490 nm. Error bars represent standard errors of the mean.One-step viral growth curvesEJ-6-2-Bam-6a cells (106) were plated in T25 flasks plates and infected 24 h later with HSV-1716 at a m.o.i. of 0.01. A sample was harvested at time points of 0, 6, 19, 24, and 48 h by cell scraping and collection of the cell lysate, as previously described (16Toyoizumi T. Mick R. Abbas A.E. Kang E.H. Kaiser L.R. Molnar-Kimber K.L. Combined therapy with chemotherapeutic agents and herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) in human non-small cell lung cancer.Hum. Gene Ther. 1999; 10: 3013-3029Crossref PubMed Scopus (95) Google Scholar). The samples were frozen and stored at –80°C. The cell lysates were then thawed and titered by plaque assay on baby hamster kidney cell monolayers, as previously described (16Toyoizumi T. Mick R. Abbas A.E. Kang E.H. Kaiser L.R. Molnar-Kimber K.L. Combined therapy with chemotherapeutic agents and herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) in human non-small cell lung cancer.Hum. Gene Ther. 1999; 10: 3013-3029Crossref PubMed Scopus (95) Google Scholar). Burst size was defined by the calculation of fold increase in titer from t = 0 to t = 24 h.Carrier cellsEJ-6-2-Bam-6a cells were plated in T75 flasks and grown until the cells reached 80% confluency (approximately 1 × 107 cells/flask). After cells were irradiated (3000 rads) in the flask, they were infected with HSV-1716 at a m.o.i. of 2 for 1 h (5 ml serum-free DMEM with rocking), and the medium was replaced with DMEM with 10% FCS. After a 3-h incubation, the cells were harvested with trypsin, counted, and injected intraperitoneally as described below.Neutralizing antibody assayMouse serum was heat inactivated at 56°C for 1 h and then serially diluted (1:25–1:5000) in serum-free DMEM. The diluted serum was added in equal volumes to HSV-1716 (400 pfu/ml) and incubated at 4°C for 20 min. The mixture (100 μl) was then applied to 5 × 104 BHK cells (at 80% confluency) growing in 96-well plates. Viral plaques were measured as described (16Toyoizumi T. Mick R. Abbas A.E. Kang E.H. Kaiser L.R. Molnar-Kimber K.L. Combined therapy with chemotherapeutic agents and herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) in human non-small cell lung cancer.Hum. Gene Ther. 1999; 10: 3013-3029Crossref PubMed Scopus (95) Google Scholar) and the neutralizing antibody titer was defined as the highest dilution that yielded a 50% reduction in plaques.Animals and immunizationFemale BALB/c mice (6 weeks old) were obtained from Taconic Laboratory (Germantown, NY) and maintained in the animal facility at the Wistar Institute (Philadelphia, PA). Mice were at least 8 weeks of age before use. In the indicated groups, mice were immunized with a herpes simplex virus type 1, KOS (5 × 105 pfu injected intraperitoneally), approximately 6 weeks prior to tumor injection. The Animal Use Committees of the Wistar Institute and the University of Pennsylvania in compliance with the Guide for the Care and Use of Laboratory Animals (NIH No. 85-23, revised 1985) approved all animal protocols.Intraperitoneal tumor modelIntraperitoneal tumor was established in BALB/c mice by intraperitoneal (ip) injection of 1 × 105 EJ-6-2-Bam-6a cells in 0.5 ml of serum-free DMEM. At 1 week, when macroscopic tumor was identified on the bowel mesentery in four of four sacrificed mice, the mice were randomly divided into groups (n = 10/group) and injected ip with one of the following treatments: medium (500 μl) alone once or three times (every third day), HSV-1716 (4 × 106 pfu/500 μl medium) once or HSV-1716 three times (every third day), HSV-1716-infected carrier cells (4 × 106 cells/500 μl DMEM) once or three times (every third day), or uninfected, irradiated EJ-6-2-Bam-6a cells (4 × 106 cells/500 μl DMEM) given three times (every third day). For the experiments in immunized mice, the presence of neutralizing antibodies (1:2000 ± 200) was confirmed, and mice were then injected with tumor. The presence of tumor was subsequently confirmed as described above and the mice were then randomized for treatment. The naive groups were age-matched. Survival curves are plotted using Kaplan–Meier analysis.Statistical analysisAll in vitro cell viability data are expressed as means ± standard error of the mean. Survival studies were analyzed using the Mantel–Cox log-rank test.ResultsHSV-1716 Replicates in EJ-6-2-Bam-6a CellsTo study the effect of an intact immune system and anti-herpes immunity on the efficacy of HSV-1716, a murine tumor cell line capable of growth within the peritoneal cavity and with the ability to support viral replication to the same extent as in human cell lines was sought. Human cell lines such as the mesothelioma cell line, REN, supports HSV-1716 viral replication with a burst size of 6600 (6Kucharczuk J.C. et al.Use of a replication-restricted recombinantherpes virus to treat localized human malignancy.Cancer Res. 1997; 57: 466-471PubMed Google Scholar). Some murine cell lines such as CT-26 have a burst size of 2–4; others such as Lewis lung exhibit a burst size of 20 (15Lambright E.S. et al.Oncolytic therapy using a mutant type 1 herpes simplex virus and the role of the immune response.Ann. Thoracic Surg. 1999; 68: 1756-1762Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Although the NIH3T3 fibroblast line was susceptible to HSV, but not tumorigenic, the EJ-6-2-Bam-6a cell line, an h-ras-transformed fibroblast line of the NIH3T3 line, is indeed tumorigenic (29Bernstein S. Weinberg R. Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA..Proc. Natl. Acad. Sci. USA. 1985; 82: 1726-1730Crossref PubMed Scopus (88) Google Scholar). A one-step growth curve was performed to determine the extent of HSV-1716 replication in both nonirradiated and irradiated EJ-6-2-Bam-6a cells (Fig. 1), since carrier cells must be rendered incapable of tumor formation (e.g., by radiation) for use in the clinical setting. It should be noted that inoculation of EJ-6-2-bam-6a cells (105) that had received 3000 rads did not induce tumors in five of five mice after intraperitoneal injection nor in five of five mice after subcutaneous bilateral flank injection (n = 10 flanks). The mice were euthanized 8 weeks after the initial tumor cell inoculum and necropsy revealed no evidence of tumors. The burst sizes for EJ-6-2-Bam-6a and for irradiated EJ-6-2-Bam-6a cells were 2210 ± 120 and 350 ± 20. Thus, these data indicate that EJ-6-2-Bam-6a can support HSV-1716 replication to a similar extent as many human cell lines.HSV-1716 Is Oncolytic in the EJ-6-2-Bam-6a Cell LineThe in vitro oncolytic effect of HSV-1716 on EJ-6-2-Bam-6a was assessed at various m.o.i. values ranging from 0.01 to 10. Cell viability was determined after 6 days using a cell proliferation assay. As demonstrated in Fig. 2, EJ-6-2-Bam-6a cells were quite sensitive to the lytic effects of the virus. Cells exposed to m.o.i. values as low as 0.01 showed a greater than 75% cell death versus control. Cells infected at m.o.i. values of 1 or higher had a greater than 90% cell death versus control.FIG. 2In vitro oncolytic effect of HSV-1716 on EJ-62-Bam-6a cells. Subconfluent monolayers of cells were infected at varying m.o.i. values from 0.01 to 10 on day 0. Cell survival on day 6 was accessed via cell viability assay (see Materials and Methods). Error bars represent standard errors of the mean of three independenView Large Image Figure ViewerDownload Hi-res image Download (PPT)Efficacy of HSV-1716 Therapy in Mice with Established Intraperitoneal TumorThe antitumor effects of HSV-1716 therapy were evaluated in survival studies in an immunocompetent, intraperitoneal tumor model. Injection of 1 × 105 EJ-6-2-Bam-6a cells into mice led to reproducible, diffuse intraperitoneal tumor nodules located on the small bowel mesentery, hepatic hilum, and surface of the diaphragm. Lesions less than 1 mm in size were identified 7 days post tumor inoculation. As the tumor burden increases, the abdomen becomes distended in the absence of ascites. Necropsy reveals multiple (>100) nodules in the abdomen measuring from 1 to 5 mm in size. These nodules are observed throughout the abdomen growing diffusely on the small bowel mesentery, omentum, and hepatic hilum with the greatest tumor burden in the small bowel mesentery (data not shown). Death of the animals was often due to intestinal obstruction secondary to tumor burden. Median survival was 4 weeks with virtually all animals dying by 50 days (Fig. 3). Since multiple administrations of HSV-based oncolytic viruses have improved efficacy in several models (14; Blank et al. and Abbas et al., unpublished data), the efficacy of three treatments was also assessed.FIG. 3Kaplan–Meier survival analysis of efficacy of HSV1716 treatment in an intraperitoneal tumor model in both naive (A) and immunized (B) BALB/c mice. Intraperitoneal tumor was established in an immunocompetent (BALB/c) mouse by intraperitoneal (ip) injection of 1 × 105 EJ-6-2-Bam-6a. Macroscopic tumor was identified in all mice observed (n = 4) and experimental mice were randomly divided into groups (n = 10/group) and injected intraperitoneally with medium alone once or medium alone three times (every third day) and HSV-1716 (4 × 106 pfu/500 μl medium) once or HSV-1716 three times (every third day). (B) In addition, a group of mice were immunized with a herpes simplex type 1 virus, KOS (5 × 105 pfu injected intraperitoneally), 6 weeks prior to tumor injection. After confirmation of the presence of neutralizing antibodies (1:2000 ± 200), mice were injected with tumor cells, tumor nodules were established, and then mice were randomized for treatment as in the naive experiments. Mice were followed daily for survival, and the data were analyzed using the Mantel–Cox log-ranView Large Image Figure ViewerDownload Hi-res image Download (PPT)After macroscopic tumor nodules were established and observed in four of four animals, groups of animals (n = 10) were injected ip with either vehicle or one dose of virus (4 × 106 pfu) or three doses of virus (4 × 106 pfu) at 3-day intervals. As shown in Fig. 3A, a single HSV-1716 administration significantly improved survival versus control (P = 0.0002, log-rank), although no long-term survival was observed. Three doses of HSV-1716 significantly prolonged survival over control (P < 0.0001, log-rank) and over a single injection (P = 0.0179, log-rank). Importantly, there was a 50% long-term survival rate (cure) in the multiply treated animals.Impact of Preexisting HSV ImmunityPreexisting anti-herpes immunity is prevalent in the human population and may influence the efficiency of infection of our viral therapy (24Herrlinger U. et al.Pre-existing herpes simplex virus 1 (HSV-1) immunity decreases butdoes not abolish genetransfer to experimental brain tumors by a HSV-1 vector.Gene Ther. 2000; 5: 809-819Crossref Scopus (70) Google Scholar). Thus, the efficacy of HSV-1716 therapy was also determined in mice that had been previously immunized with the wild-type herpes simplex virus 1 strain, KOS (5 × 105 pfu), intraperitoneally. Anti-HSV immunity was assessed by the induction of neutralizing antibody (NAb) as a function of time. As expected, the serum of control mice (n = 4) that had not been inoculated did not neutralize HSV-1716 at any dilution. Anti-HSV NAbs were detectable in all mice (n = 5) at a dilution of 1:2000 ± 200 by day 35 postimmunization. Intraperitoneal tumor was established in these immunized animals and age-matched naive mice. The mice were randomized and groups of animals (n = 10) were injected ip with vehicle (medium), one dose of HSV-1716 (4 × 106 pfu), or three doses of HSV-1716 (4 × 106 pfu) at 3-day intervals. The immunized, tumor-bearing mice treated with medium had virtually identical median survival times compared to the naive mice (Fig. 3B vs Fig. 3A). Median survival was 32 days with all control animals dead by 50 days.A single HSV-1716 treatment of tumor-bearing, immunized mice significantly improved survival versus control (P = 0.0021, log-rank) with no long-term survival. There was no significant difference in the efficacy of a single HSV-1716 treatment of established ip tumor between naive and immunized mice (P = 0.1). Multiple HSV-1716 treatments significantly prolonged survival over control (P < 0.0001, log-rank) and over a single treatment (P = 0.0228, log-rank). Again, a significant number of animals (30%) in the multiply treated group were long-term survivors compared to none of the singly treated animals. The efficacy of three administrations of HSV-1716 was not significantly different in naive and immunized mice (P = 0.2).These studies indicate that both single and multiple injections of HSV-1716 have significant antitumor activity in an immunocompetent mouse model of peritoneal tumor, even after immunization against HSV. Multiple injections of virus, even in immunized animals, showed significantly greater efficacy and led to long-term survival in 30–50% of animals.Viral Delivery via Carrier CellsHSV infection can spread via release of virus and subsequent uptake of virus in adjacent cells and it can also spread via cell-to-cell contact (30Weeks B.S. Sund
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