Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Abstract

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Delta and R47Delta, have been constructed by deleting the alpha47 gene and the promoter region of US11 from gamma34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the alpha47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Delta-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early alpha47 promoter, which suppresses the reduced growth properties of gamma34.5-deficient mutants. G47Delta and R47Delta showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Delta was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 x 10(6) plaque forming units, G47Delta was as safe as G207. These results suggest that G47Delta may have enhanced antitumor activity in humans.

Figure 1

Structure of G47Δ. (a) Schematic of the HSV-1 genome showing the regions modified in G47Δ. The HSV-1 genome consists of long and short unique regions (Ul and Us) each bounded by terminal (T) and internal (I) repeat regions (Rl and Rs). The parental virus G207 was engineered from wild-type HSV-1 strain F by deleting 1 kb within both copies of the γ34.5 gene and inserting the E. coli lacZ gene into the ICP6 coding region. G47Δ was derived from G207 by deleting 312 bp from the ICP47 locus, as indicated. (b) Map of the ICP47 locus showing locations of the overlapping 3′ coterminal transcripts (US10, US11, and ICP47), ORFs (thick arrow), and ICP47 splice junctions (∧). (c) Map of plasmid pIE12Δ used to generate deletion by homologous recombination with the indicated flanking sequences. Whereas US11 is regulated as a true late gene in wild-type HSV-1, deletion between the indicated BstEII and EcoNI sites places US11 under control of the ICP47 immediate-early promoter. Restriction site abbreviations: B, BamHI; Bs, BstEII; EN, EcoNI; Nr, NruI; E, EcoRI.

Figure 2

Virus yields of replication-competent HSV-1 mutants in various cell lines. Cells were seeded on 6-well plates at 5 × 10⁵ cells per well. Triplicate wells were infected with R3616, R47Δ, G207, G47Δ, or strain F at an moi of 0.01. At 24 h after infection, cells were scraped into the medium and progeny virus was titered on Vero cells. In all cell lines tested, G47Δ showed a significantly higher replication capability than G207. Results represent the mean of triplicates ± SD.

Figure 3

Cytopathic effect of G47Δ in vitro. Cells were plated into 6-well plates at 2 × 10⁵ cells per well. After a 24-h incubation, cells were infected with G207 or G47Δ at an moi of 0.01 or 0.1, or without virus (Control). The number of surviving cells was counted daily and expressed as a percentage of mock-infected controls. G47Δ exhibited a significantly stronger cytopathic effect than did G207 in all three human tumor cell lines (U87MG, melanoma 624, and melanoma 888) at an moi of 0.01, and also in Neuro2a murine neuroblastoma cells at an moi of 0.1. The results are the mean of triplicates ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; G207 versus G47Δ, unpaired t test.

Figure 4

G47Δ precludes down-regulation of MHC class I expression in infected host cells. (a) Flow cytometric analyses of MHC class I expression in Detroit 551 human fibroblast cells 48 h after infection with HSV-1 (moi = 3). Whereas all HSVs with an intact α47 gene (wild-type strain F and G207) significantly down-regulated MHC class I expression, G47Δ completely precluded the down-regulation. (b) Time course of MHC class I down-regulation in Detroit 551 cells infected with HSV-1. For each virus, the peak value of MHC class I expression at 6, 24, or 48 h after infection, as analyzed by flow cytometry, was expressed as a percentage of the peak value of mock-infected cells (Control) at each time point. MHC class I down-regulation by G207 and R3616 occurred in a time-dependent fashion. Dissociation of MHC class I expression between α47-deleted mutants (G47Δ and R47Δ) and α47-intact viruses became apparent at 24–48 h after infection. (c) Flow cytometric analyses of MHC class I expression in human melanoma cell lines 24 h after infection with G207 and G47Δ. G47Δ caused a partial preclusion of MHC class I down-regulation in melanomas 1102 and 938, resulting in greater MHC class I expression than G207.

Figure 5

G47Δ-infected tumor cells stimulate T cells to a greater extent than G207-infected tumor cells. Human melanoma cells were mock infected (no virus) or infected with G207 or G47Δ at an moi of 3, and after 3–6 h, were cocultured with an equal number of responding human T cells for 18 h. T cell stimulation was assessed by an increase in IFN-γ release into conditioned media. G47Δ-infected melanoma 1102 cells caused a significantly greater stimulation of TIL888 cells compared with G207-infected 1102 cells (P < 0.01, unpaired t test). G47Δ-infected 938 melanoma cells also stimulated TIL1413 cells, although the improvement was not statistically significant compared with G207-infected 938 cells (P = 0.1, unpaired t test). Neither G207- nor G47Δ-infected melanoma 888 cells caused a significant stimulation of TIL888 cells.

Figure 6

G47Δ exhibits greater antitumor efficacy than G207 in vivo. S.c. tumors of U87MG human glioma (Left) or Neuro2a murine neuroblastoma (Right) were generated in 6-week-old female athymic mice or 6-week-old female A/J mice, respectively. Established tumors of ≈6-mm diameter were inoculated with G207 or G47Δ (1 × 10⁶ pfu) or Mock (PBS with 10% glycerol) on days 0 and 3. G47Δ treatment was significantly more efficacious than G207 in both tumor models, resulting in smaller average tumor volumes (P < 0.05 for U87MG on day 24 and P < 0.001 for Neuro2a on day 15; G207 versus G47Δ, unpaired t test).