Oncolytic herpes simplex virus 1 encoding 15-prostaglandin dehydrogenase mitigates immune suppression and reduces ectopic primary and metastatic breast cancer in mice

Abstract

Oncolytic herpes simplex virus 1 (HSV-1) viruses armed with immunomodulatory transgenes have shown potential for enhanced antitumor therapy by overcoming tumor-based immune suppression and promoting antitumor effector cell development. Previously, we reported that the new oncolytic HSV-1 virus, OncSyn (OS), engineered to fuse tumor cells, prevented tumor growth and metastasis to distal organs in the 4T1/BALB/c immunocompetent breast cancer mouse model, suggesting the elicitation of antitumor immune responses (Israyelyan et al., Hum. Gen. Ther. 18:5, 2007, and Israyelyan et al., Virol. J. 5:68, 2008). The OSV virus was constructed by deleting the OS viral host shutoff gene (vhs; UL41) to further attenuate the virus and permit dendritic cell activation and antigen presentation. Subsequently, the OSVP virus was constructed by inserting into the OSV viral genome a murine 15-prostaglandin dehydrogenase (15-PGDH) expression cassette, designed to constitutively express 15-PGDH upon infection. 15-PGDH is a tumor suppressor protein and the primary enzyme responsible for the degradation of prostaglandin E2 (PGE2), which is known to promote tumor development. OSVP, OSV, and OS treatment of 4T1 tumors in BALB/c mice effectively reduced primary tumor growth and inhibited metastatic development of secondary tumors. OSVP was able to significantly inhibit the development and accumulation of 4T1 metastatic tumor cells in the lungs of treated mice. Ex vivo analysis of immune cells following treatment showed increased inflammatory cytokine production and the presence of mature dendritic cells for the OSVP, OSV, and OS viruses. A statistically significant decrease in splenic myeloid-derived suppressor cells (MDSC) was observed only for OSVP-treated mice. These results show that intratumoral oncolytic herpes is highly immunogenic and suggest that 15-PGDH expression by OSVP enhanced the antitumor immune response initiated by viral infection of primary tumor cells, leading to reduced development of pulmonary metastases. The availability of the OSVP genome as a bacterial artificial chromosome allows for the rapid insertion of additional immunomodulatory genes that could further assist in the induction of potent antitumor immune responses against primary and metastatic tumors.

Fig. 1.

Schematic representation of the genomic structure of oncolytic recombinant viruses OSV and OSVP. (A) Representation of the prototypic arrangement of the HSV-1 OncSyn (OS) genome with the unique long (UL) and unique short (US) regions flanked by the terminal repeat (TR) regions. The Δ denotes the approximate location of the OS genomic deletion between the UL and US regions (26), and the position of the gB syncytial mutation is indicated. The expanded area below the depicted genome shows the genomic region from 84531 to 96068 nucleotides encompassing the UL38-UL43 genes. (B) Genomic organization of the OSV recombinant virus showing deletion of the UL41 gene. (C) Genomic organization of the OSVP recombinant virus showing insertion of the 15-PGDH gene cassette in place of the deleted UL41 sequences. SV40, simian virus 40.

Fig. 2.

Plaque morphology and growth characteristics of OS OSV and OSVP. (A) Nearly confluent Vero cell monolayers were infected with wild-type HSV-1(F), OS, OSV, or OSVP viruses at MOI of 0.001. Individual viral plaques were visualized 48 h postinfection by immunohistochemistry and photographed at the same magnification with a phase-contrast microscope. (B) A minimum of 30 plaques for each virus (prepared as described in the legend to panel A) were measured, and individual plaque sizes are indicated on the x axis, with the horizontal bar indicating the means for each group. Error bars indicate standard error of the means. All pairwise comparisons of the means using two-tailed t tests indicated significance (P < 0.0185) except for the comparison between OSV and OSVP (P = 0.1473). (C) Nearly confluent 4T1 cell monolayers were infected with the above-mentioned viruses at an MOI of 5. Triplicate culture supernatants were collected at 48 hpi, and viral titers were determined (PFU/ml).

Fig. 3.

PGE2 degradation by OSVP. Nearly confluent monolayers of 4T1 cells were infected with the indicated viruses at an MOI of 5. After 2 days culture supernatants were harvested and assayed for PGE2 by ELISA. P denotes transient expression with a 15-PGDH mammalian expression vector, while “No Tx” denotes mock-treated cells. PGE2 assays were performed in triplicates, and error bars represent the 95% confidence interval (CI) of the mean.

Fig. 4.

Intratumor treatment with OS, OSV, or OSVP viruses. A total of 1 × 10⁵ viable 4T1 cells were implanted subcutaneously in the interscapular area of BALB/c mice. Tumors were measured using a digital caliper at defined time intervals prior to and after treatment (x axis). Median tumor volumes over time are shown on the y axis. Tumors were injected with each virus (1 × 10⁷ PFU/ml in PBS buffer), or PBS alone when tumors reached approximately 80 to 90 mm³ in volume. Tumor volumes were measured prior to (negative values on the x axis) and after the injections. Tumors were treated with each virus at days 1, 3, and 6, as indicated by arrows. The asterisk indicates statistical significance by nonparametric analysis (31). The results shown are from one of three independent experiments that produced similar results.

Fig. 5.

Quantification of pulmonary metastases. Virus-treated mice bearing 4T1 tumors were sacrificed 30 days post-tumor inoculation, and both lungs were harvested from each treated mouse and homogenized for cell culture. Clonogenic metastatic foci derived from total lung homogenates were enumerated by limited dilution culture in the presence of 6-thioguanine. Individual resistant colonies were counted and used to determine the number of CFU per animal (x axis). Shown are the calculated CFU of each mouse sorted by treatment. Means for each treatment group are represented by horizontal bars with error bars delineating 95% CI. The mean number of metastatic colonies recovered from the OSVP-treated group was significantly less than that of the PBS control group (Mann-Whitney test; P = 0.0474), but other pairwise comparisons did not prove statistically significant. The results shown are from one of three independent experiments that produced similar results. Not all samples yielded colonies, and the ability to recover colonies was used to determine pulmonary metastasis incidence rates for each group (shown below the chart).

Fig. 6.

Immunophenotypes of draining lymph node lymphocytes and splenocytes. 4T1 tumor-bearing mice with similarly sized and well-developed tumors were given a single intratumoral injection of the indicated viruses. After 2 days, cells from draining lymph nodes (B and D) or spleens (A and C) were isolated and analyzed for MDSC markers (Gr1⁺, CD11b⁺) (A), CD4+CD25+FoxP3+ regulatory T cells (B), and CD83 (C and D). Indicated phenotypes are shown as the mean percentage of total live cells interrogated by flow cytometry (error bars represent 95% CI of the means) (n = 3 unless otherwise noted). Asterisks with brackets indicate pairwise statistical significance. Asterisks alone indicate statistical significance over all pairwise comparisons among the treatments (two-tailed t test; P < 0.05).

Fig. 7.

Cytokine immunoprofiles of lymphocytes derived from treated mice. Mice with similarly sized 4T1 tumors (200 to 300 mm²) were treated twice with a 3-day interval. Two days following treatments with OS, OSV, OSVP, or PBS (control), lymphocytes from draining lymph nodes were isolated and cultured for 24 h alone (A) or with immobilized anti-CD3 stimulatory antibody (B) at a concentration of 100,000 cells per ml. TNF-α, tumor necrosis factor alpha. Supernatants were assayed for TH1/TH2 cytokine production by Bioplex (Bio-Rad). Shown are the mean quantities of the indicated cytokines in pg/ml with error bars representing 95% CI (n = 3). Shown are results from one of two experiments with similar results.