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. 2000 Feb 29;97(5):2208-13.
doi: 10.1073/pnas.040557897.

Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors

J N Parker  1 , G Y Gillespie, C E Love, S Randall, R J Whitley, J M Markert
Affiliations

Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors

J N Parker et al. Proc Natl Acad Sci U S A. .

Abstract

Genetically engineered, neuroattenuated herpes simplex viruses (HSVs) expressing various cytokines can improve survival when used in the treatment of experimental brain tumors. These attenuated viruses have both copies of gamma(1)34.5 deleted. Recently, we demonstrated increased survival of C57BL/6 mice bearing syngeneic GL-261 gliomas when treated with an engineered HSV expressing IL-4, as compared with treatment with the parent construct (gamma(1)34. 5(-)) alone or with a virus expressing IL-10. Herein, we report construction of a conditionally replication-competent mutant expressing both subunits of mIL-12 (M002) and its evaluation in a syngeneic neuroblastoma murine model. IL-12 induces a helper T cell subset type 1 response, which may induce more durable antitumor effects. In vitro studies showed that, when infected with M002, both Vero cells and murine Neuro-2a neuroblastoma cells produced physiologically relevant levels of IL-12 heterodimers, as determined by ELISA. M002 was cytotoxic for Neuro-2a cells and human glioma cell lines U251MG and D54MG. Neurotoxicity studies, as defined by plaque-forming units/LD(50), performed in HSV-1-sensitive A/J strain mice found that M002 was not toxic even at high doses. When evaluated in an intracranial syngeneic neuroblastoma murine model, median survival of M002-treated animals was significantly longer than the median survival of animals treated with R3659, the parent gamma(1)34.5(-) mutant lacking any cytokine gene insert. Immunohistochemical analysis of M002-treated tumors identified a pronounced influx of CD4(+) T cells and macrophages as well as CD8(+) cells when compared with an analysis of R3659-treated tumors. We conclude that M002 produced a survival benefit via oncolytic effects combined with immunologic effects meditated by helper T cells of subset type 1.

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Figures

Figure 1
Figure 1
Schematic representation of mIL-12-expressing HSV (M002). Line 1 illustrates the HSV-1 (F) Δ305 genome, which contains a 501-bp deletion within the tk gene, as indicated by the Δ symbol. UL and US represent the unique long and unique short sequences, respectively. The inverted repeat sequences are indicated by a, b, and c, with subscripts n and m representing variable numbers of a sequences. a1 and as represent the a sequences flanking the UL and US terminal repeats. Line 2 shows the sequence arrangement of the recombinant HSV R3659. The BstEII–StuI fragment within the γ134.5 gene was replaced by the chimeric α27-tk gene in the inverted sequences ab (shown above) and b′a′ (not shown) flanking the UL sequence. Line 3 shows the sequence arrangements of the relevant regions in the recombinant mIL-12-expressing HSV M001 (tk−) or M002 (tk+). NcoI restriction sites are indicated.
Figure 2
Figure 2
Southern blot hybridization confirming the presence of mIL-12 in M002. Viral DNAs were isolated, digested with NcoI, and electrophoretically separated, and Southern blot hybridization was performed as described in Materials and Methods. The predicted fragment sizes for each viral DNA (1.76 kb for HSV-1 (F), 0.7 kb for R3659, and 2.2 kb for M002) are indicated by arrows. The 1.6-kb, 0.52-kb, and all smaller bands of the 1-kb DNA ladder (Life Technologies, Grand Island, NY) will also hybridize to the probe, which has the same vector backbone as the ladder.
Figure 3
Figure 3
In vitro replication of M001 in human glioma cells. Replicating monolayers of U251MG (Upper) or D54MG (Lower) human malignant glioma cell lines were infected at 1 pfu per cell with HSV-1 (F) (closed diamonds), R3659 (closed circles), or M001 (open circles). Replicate cultures were harvested at 12, 24, 48, and 72 h after infection, and virus titers were determined on Vero cell monolayers.
Figure 4
Figure 4
Survival of A/J strain mice with intracerebral Neuro-2a neuroblastomas treated with M002. Neuro-2a cells (1 × 104/5 μl) were injected intracerebrally in A/J mice. After 5 days, intracerebral tumors were injected with 10 μl of saline or 2 × 107 pfu of HSV R3659 or HSV M002, and mouse survival was monitored. Median survival for saline-treated mice was 19.8 days versus 50.5 days for M002-treated mice (P = 0.0002) and 19.5 days for HSV R3659-treated mice. Histologic examination of the brains of survivors killed at 59 days identified no persistent tumor.
Figure 5
Figure 5
Immunohistologic identification of inflammatory cell infiltrates. A/J female mice were injected intracerebrally with Neuro-2a cells (1 × 105/5 μl) and 5 days later were injected intratumorally with 1 × 107 pfu of HSV R3659 (A, C, E, and G) or HSV M002 (B, D, F, and H). After 6 days, the mice were killed, and their brains were removed intact and embedded in OCT for preparation of frozen sections. Serial 10-μm-thick sections were reacted with rat monoclonal antibodies to CD4+ (A and B) or CD8+ T (C and D) cells or macrophages (E and F); antibody binding was detected by using horseradish peroxidase-labeled anti-rat antibody, and sections were counterstained with Mayer's hematoxylin. Hematoxylin-eosin stained adjacent sections are also shown (G and H).
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