Recombinant adeno-associated virus expressing human papillomavirus type 16 E7 peptide DNA fused with heat shock protein DNA as a potential vaccine for cervical cancer

Abstract

In this study, we explore a potential vaccine for human papillomavirus (HPV)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus (AAV) as a gene delivery vector. The tumor vaccine was devised by constructing a chimeric gene which contained HPV type 16 E7 cytotoxic T-lymphocyte (CTL) epitope DNA (M. C. Feltkamp, H. L. Smits, M. P. Vierboom, R. P. Minnaar, B. M. de Jongh, J. W. Drijfhout, J. ter Schegget, C. J. Melief, and W. M. Kast, Eur. J. Immunol. 23:2242-2249, 1993) fused with the heat shock protein gene as a tumor vaccine delivered via AAV. Our results demonstrate that this vaccine can eliminate tumor cells in syngeneic animals and induce CD4- and CD8-dependent CTL activity in vitro. Moreover, studies with knockout mice with distinct T-cell deficiencies confirm that CTL-induced tumor protection is CD4 and CD8 dependent. Taken together, the evidence indicates that this chimeric gene delivered by AAV has potential as a cervical cancer vaccine.

FIG. 1

Northern blot analysis. RNA (20 μg) was fractionated by agarose gel electrophoresis, blotted, and hybridized with ³²P-labeled E7CTL-hsp DNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA separately. A GAPDH probe was used to ensure equal RNA loading. Lane 1, 293 cells infected with rAAV lacZ; lane 2, 293 cells infected with rAAV E7CTL-hsp.

FIG. 2

X-Gal staining of the cryostat sections of the muscle injected with rAAV lacZ. (A) Five-week-old mice were injected with rAAV lacZ (3 × 10¹⁰ viral particles) in the tibialis anterior muscle. After 14 days, the cryostat sections were stained with X-Gal and eosin. (B) Control mice without rAAV lacZ injection.

FIG. 3

In vivo tumor elimination assay. Groups of 10 mice were injected subcutaneously with 5 × 10⁴ TC-1 or B16F1 tumor cells and, after 1 week, were immunized with 5 × 10¹⁰ viral particles of rAAV E7CTL-hsp or rAAV lacZ or phosphate-buffered saline (PBS) (mock). The tumor volume was monitored once a week. The data are represented as means and standard errors of each group.

FIG. 4

HPV-16 E7 49 to 57 specific CTL responses induced by immunization with rAAV E7CTL-hsp. C57BL/6 mice were i.m. immunized with rAAV E7CTL-hsp. Four weeks after the vaccination, the spleens were collected and analyzed for in vitro CTL assay. (A) TC-1 cells; (B) B16F1 cells; (C) B16F1 cells pulsed with E7 peptide 49 to 57, (D) B16F1 cells pulsed with HPV-16 E5 peptide 6 to 13. The data were the averages of five vaccinated mice. E/T, effector/target. PBS, phosphate-buffered saline.

FIG. 5

Proliferation of rAAV E7CTL-hsp-vaccinated lymphocytes in response to E7 peptide 44 to 62. C57BL/6 mice were i.m. immunized with rAAV E7CTL-hsp, rAAV lacZ, or mock. Four weeks after the vaccination, the spleens were collected and analyzed for T-cell proliferation measured by incorporation of [³H]thymidine. The data were the averages of five vaccinated mice of each group. The error bars represent standard errors. Stimulation index was defined as the ratio of mean counts per minute of incorporated [³H]thymidine for cells with antigen (E7 peptide 44 to 62) to mean counts per minute for cells without any added antigen. PBS, phosphate-buffered saline.

FIG. 6

CD4- and CD8-dependent lymphocytes in tumor eradication by rAAV E7CTL-hsp vaccination. KOI (CD8-deficient) (A) and KOII (CD4-deficient) (B) mice were subcutaneously injected with 5 × 10⁴ TC-1 tumor cells. One week later, they were divided into two groups for treatments with 5 × 10¹⁰ viral particles of rAAV lacZ or rAAV E7CTL-hsp. Each group consists of six mice. Tumor volume was checked once a week.