Regression of established human papillomavirus type 16 (HPV-16) immortalized tumors in vivo by vaccinia viruses expressing different forms of HPV-16 E7 correlates with enhanced CD8(+) T-cell responses that home to the tumor site

Abstract

Using vaccinia virus as a live vector, we show that the expression of human papillomavirus type 16 (HPV-16) E7 fused to a nonhemolytic portion of the Listeria monocytogenes virulence factor, listeriolysin O (LLO), induces an immune response that causes the regression of established HPV-16 immortalized tumors in C57BL/6 mice. The vaccinia virus construct expressing LLO fused to E7 (VacLLOE7) was compared with two previously described vaccinia virus constructs: one that expresses unmodified E7 (VacE7) and another that expresses E7 in a form designed to direct it to intracellular lysosomal compartments and improve major histocompatibility complex class II-restricted responses (VacSigE7LAMP-1). C57BL/6 mice bearing established HPV-16 immortalized tumors of 5 or 8 mm were treated with each of these vaccines. Fifty percent of the mice treated with VacLLOE7 remained tumor free 2 months after tumor inoculation, whereas 12 to 25% of the mice were tumor free after treatment with VacSigE7LAMP-1 (depending on the size of the tumor). No mice were tumor free in the group given VacE7. Compared to VacE7, VacSigE7LAMP-1 and VacLLOE7 resulted in increased numbers of H2-D(b)-specific tetramer-positive CD8(+) T cells in mouse spleens that produced gamma interferon and tumor necrosis factor alpha upon stimulation with RAHYNIVTF peptide. In addition, the highest frequency of tetramer-positive T cells was seen in the tumor sites of mice treated with VacLLOE7. An increased efficiency of E7-specific lysis by splenocytes from mice immunized with VacLLOE7 was also observed. These results indicate that the fusion of E7 with LLO not only enhances antitumor therapy by improving the tumoricidal function of E7-specific CD8(+) T cells but may also increase the number of antigen-specific CD8(+) T cells in the tumor, the principle site of antigen expression.

FIG. 1

Schematic of the different forms of E7 expressed by each vaccinia virus recombinant. In each form, the pSC11 vaccinia virus vector was used to insert the gene of interest into the thymidine kinase (TK) gene of the WR host strain of vaccinia virus.

FIG. 2

VacLLOE7 causes long-term regression of tumors established from 2 × 10⁵ TC-1 cells injected s.c. into C57BL/6 mice. Mice were injected 11 and 18 days after tumor challenge with 10⁷ PFU of VacLLOE7, VacSigE7LAMP-1, or VacE7/mouse i.p. or were left untreated (naive). Eight mice per treatment group were used, and the cross section for each tumor (average of two measurements) in each mouse is shown for the indicated days after tumor inoculation.

FIG. 3

CTL responses to E7 induced by VacLLOE7 surpass those seen with VacSigE7LAMP-1 and VacE7. Mice were injected twice i.p. 7 days apart with 10⁷ PFU. Thirteen days after the final injection, spleen cells from two mice per treatment group were harvested and pooled as described in Materials and Methods. EL4 cells, EL4 cells pulsed with a peptide from HPV-16 E7 (RAHYNIVTF) (EL4 + E7 peptide), or EL4 cells stably transfected with full-length HPV-16 E7 (EL4E7) were compared with the parental EL4 cell line as targets for lysis. CTL activity is expressed as the average percent specific lysis for measurements at each E:T ratio as described in Materials and Methods. Error bars indicate standard deviations for measurements in triplicate or quadruplicate. Results shown are representative of three independent experiments.

FIG. 4

Inflammatory cytokine production in response to RAHYNIVTF by CD8⁺ T cells is enhanced in the spleens of mice vaccinated with VacLLOE7. Following two i.p. injections 7 days apart with 10⁷ PFU of VacGag, VacE7, VacSigE7LAMP-1, or VacLLOE7, spleen cells from two mice per treatment group were harvested 13 days after the second injection. Whole splenocytes were incubated with 1 μM HPV-16 E7 peptide RAHYNIVTF for 5 h in the presence of a Golgi transport inhibitor, and the levels of intracellular IFN-γ (A) and TNF-α (B) were determined by flow cytometry as described in Materials and Methods. Values shown are percentages of activated CD8⁺ T lymphocytes (gated on CD62L) that are IFN-γ positive. IFN-γ production in the presence of an irrelevant peptide (AMQMLKETI) from HIV Gag was no more than 10% the value shown for each vaccinia virus recombinant expressing E7. Responses shown are representative of three experiments.

FIG. 5

VacLLOE7 and VacSigE7LAMP-1 induce similar numbers of tetramer-positive T cells that are specific for E7–H2-D^b and that secrete IFN-γ in response to the HPV-16 peptide RAHYNIVTF. Spleen cells from mice vaccinated twice i.p. with each vaccinia virus construct were harvested 7 days after the last vaccination, incubated with 1 μM HPV-16 E7 (A) or HIV Gag (B) peptide, and stained for surface markers as described in Materials and Methods. Cells were gated on CD8⁺ CD62L^lo, and IFN-γ production by E7–H2-D^b tetramer-positive cells was determined. IFN-γ production in the presence of an irrelevant peptide (AMQMLKETI) from HIV Gag was less than 10% the value shown for each vaccinia virus recombinant expressing E7. Responses shown are representative of two independent experiments.

FIG. 6

In vitro proliferative responses to exogenous E7 protein are significantly reduced in the spleens of mice vaccinated with VacLLOE7. Mice were given two i.p. injections of 10⁷ PFU of VacGAG, VacE7, VacSigE7LAMP-1, or VacLLOE7. Ten days after the last injection, spleen cells from two mice per vaccination group were harvested and enriched for T cells over nylon wool columns. T cells from each vaccination group were incubated with equal numbers of syngeneic γ-irradiated APCs as described in Materials and Methods. T-cell proliferation is shown as the mean of triplicate measurements of [³H]thymidine incorporation at each concentration of E7. Error bars are standard deviations of the mean at each dose. Results shown are representative of two experiments.

FIG. 7

Ex vivo frequencies of E7–H2-D^b-specific tetramer-positive CD8⁺ T cells in the tumors of mice treated with VacLLOE7 are enhanced over those seen with VacSigE7LAMP-1 and VacE7. Tumors were homogenized in the presence of collagenase and DNase to aid in the isolation of T lymphocytes. Cells were gated for live lymphocytes based on their forward scatter and side scatter to exclude cellular debris, and CD8⁺ T cells reactive for E7–H2-D^b were identified without in vivo stimulation or gating for activated T cells.

FIG. 8

IFN-γ secretion by E7–H2-D^b-specific tetramer-positive CD8⁺ cells in the spleens of tumor-bearing mice is indistinguishable between mice receiving different vaccinia virus constructs expressing E7. Spleen cells from vaccinated mice were harvested and stimulated as described in the legend to Fig. 5. Plots are of percentages of activated (CD62L^lo) CD8⁺ cells that secreted IFN-γ in response to the HIV Gag control peptide (A) or RAHYNIVF (B).