Local HPV Recombinant Vaccinia Boost Following Priming with an HPV DNA Vaccine Enhances Local HPV-Specific CD8+ T-cell-Mediated Tumor Control in the Genital Tract

Abstract

Purpose: Two viral oncoproteins, E6 and E7, are expressed in all human papillomavirus (HPV)-infected cells, from initial infection in the genital tract to metastatic cervical cancer. Intramuscular vaccination of women with high-grade cervical intraepithelial neoplasia (CIN2/3) twice with a naked DNA vaccine, pNGVL4a-sig/E7(detox)/HSP70, and a single boost with HPVE6/E7 recombinant vaccinia vaccine (TA-HPV) elicited systemic HPV-specific CD8 T-cell responses that could traffic to the lesion and was associated with regression in some patients (NCT00788164).

Experimental design: Here, we examine whether alteration of this vaccination regimen by administration of TA-HPV vaccination in the cervicovaginal tract, rather than intramuscular (IM) delivery, can more effectively recruit antigen-specific T cells in an orthotopic syngeneic mouse model of HPV16(+) cervical cancer (TC-1 luc).

Results: We found that pNGVL4a-sig/E7(detox)/HSP70 vaccination followed by cervicovaginal vaccination with TA-HPV increased accumulation of total and E7-specific CD8(+) T cells in the cervicovaginal tract and better controlled E7-expressing cervicovaginal TC-1 luc tumor than IM administration of TA-HPV. Furthermore, the E7-specific CD8(+) T cells in the cervicovaginal tract generated through the cervicovaginal route of vaccination expressed the α4β7 integrin and CCR9, which are necessary for the homing of the E7-specific CD8(+) T cells to the cervicovaginal tract. Finally, we show that cervicovaginal vaccination with TA-HPV can induce potent local HPV-16 E7 antigen-specific CD8(+) T-cell immune responses regardless of whether an HPV DNA vaccine priming vaccination was administered IM or within the cervicovaginal tract.

Conclusions: Our results support future clinical translation using cervicovaginal TA-HPV vaccination.

Conflict of interest statement

Conflict of Interest: T.-C. Wu and Richard B.S. Roden are co-founders of and have an equity ownership interest in Papivax LLC. Also, they own Papivax Biotech Inc. stock options and are members of Papivax Biotech Inc.'s Scientific Advisory Board. Additionally, under a licensing agreement between Papivax Biotech Inc. and the Johns Hopkins University, Dr. Wu and Dr. Roden are entitled to royalties on an invention described in this article. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies

©2015 American Association for Cancer Research.

Figures

Figure 1. Characterization of the E7-specific CD8+ T cell immune response using intracellular IFN-γ cytokine staining followed by flow cytometry analysis

C57BL/6 mice (5 per group) were intramuscularly vaccinated with pNGVL4a-sig/E7(detox)/HSP70 DNA (50μg per mouse) prime followed six days later by TA-HPV (1x107 per mouse) boost, DNA prime followed by DNA boost, TA-HPV only or received no vaccination. One week after the last immunization, splenocytes were analyzed by flow cytometry. A. Data shown are from a representative flow cytometry analysis. The number in the right upper corner indicates the number of CD8+ IFN-γ+ E7-specific T cells in 105 total splenocytes. B. Bar graph. Values are shown as mean ± SD, *p<0.05, **p<0.01, ns, not significant.

Figure 2. Characterization of E7-specific CD8+ T cells in splenocytes and cervicovaginal tract using E7 peptide-loaded H-2Db tetramer and intracellular IFN-γ staining

C57BL/6 mice (5 per group) were vaccinated with pNGVL4a-sig/E7(detox)/HSP70 (50μg per mouse) prime followed 7 days later by TA-HPV (1x107 per mouse) boost with the regimen administered either within cervicovaginal tissues or intramuscularly. One week after the last immunization, splenocytes and cervicovaginal cells were prepared for the detection of total or HPV16 E7-specific CD8+ T cells by either tetramer or IFN-γ intracellular staining. Representative flow cytometry analysis by HPV16 E7 tetramer staining of splenocytes (A) and cervicovaginal cells (D). Summary of flow cytometry analysis of E7 tetramer+ CD8+ T cells in splenocytes (B), total CD8+ T cells (C) and E7 tetramer+ CD8+ T cells (E) in cervicovaginal tissues. Representative flow cytometry analysis by IFN-γ intracellular staining of splenocytes (F) and cervicovaginal cells (H). Summary of flow cytometry analysis of E7-specific IFN-γ –producing CD8+ T cells in splenocytes (G) and in cervicovaginal tissues (I). Values are shown as mean ± SD, *p<0.05, **p<0.01, ns, not significant.

Figure 3. Characterization of in vivo therapeutic antitumor effects generated by pNGVL4a-sig/E7(detox)/HSP70 prime and TA-HPV boost via ICV or IM vaccination

C57BL/6 mice (5 per group) were challenged with luciferase-expressing TC-1 cells (2x104 per mouse) in the submucosa of the vagina. One day later, mice were immunized with pNGVL4a-sig/E7(detox)/HSP70 and 7 days later, mice were immunized with TA-HPV via ICV or IM. The signal in the vagina was monitored by luminescence on day 3 and day 17 after injection of TC-1 luciferase-expressing cells. A. Luminescence images of representative mice challenged with luciferase-expressing TC-1 tumor and treated according to the various treatment regimens. B. Bar graph showing luminescence intensity, values are shown as mean ± SD, *p<0.05, **p<0.01, ns, not significant. C. Kaplan-Meier survival analysis of mice in various treatment groups.

Figure 4. Characterization of α4β7 and CCR9 expression by E7-specific CD8+T cells in cervicovaginal tissue and iliac lymph node

C57BL/6 mice (5 per group) were vaccinated with pNGVL4a-sig/E7(detox)/HSP70 (50μg per mouse) prime followed six days later by TA-HPV (1x107 per mouse) boost either within cervicovaginal tissues or intramuscularly. One week after the second immunization, cervicovaginal and iliac lymph node cells were analyzed by flow cytometry using E7 peptide-loaded tetramer, antibody against α4β7 and CCR9. Representative flow cytometry analysis of α4β7 (A), CCR9 (C) in the cervicovaginal cells and α4β7 in the iliac lymph node cells (E) by E7-specific CD8+ T cells. Summary of flow cytometry analysis of α4β7 expression by E7-specific CD8+ T cells in cervicovaginal tissues (B), iliac lymph node (F) and CCR9 expression in cervicovaginal tissues (D). Values are shown as mean ± SD, *p<0.05, **p<0.01, ns, not significant.

Figure 5. Integrin β7 is required for E7-specific CD8+ T cell homing to the cervicovaginal tissue and enhanced therapeutic antitumor effect

Wild type ( WT) and Itgb7−/ − mice were vaccinated ICV with pNGVL4a-sig/E7(detox)/HSP70 (50μg per mouse) prime followed 7 days later by TA-HPV (1x107 per mouse) boost. One week after the last immunization, cervicovaginal tissues and splenocytes were analyzed by flow cytometry. To test the role of integrin β7 in the antitumor effect generated by vaccination, WT or Itgb7−/ − C57BL/6 mice (5 per group) were challenged with luciferase-expressing TC-1 cells (2x104 per mouse) in the submucosa of the vagina. One day later, mice were immunized with pNGVL4a-Sig/E7(detox)/HSP70 and 7 days later, mice were immunized with TA-HPV. The signal in the vagina was monitored by luminescence on day 3 and day 17 after injection of TC-1 luciferase-expressing cells. Representative flow cytometry analysis of splenocytes (A) and cervicovaginal cells (C). Summary of flow cytometry analysis of E7-specific CD8+ T cells in splenocytes (B) and in cervicovaginal tissues (D). Luminescence images of representative mice challenged with luciferase-expressing TC-1 tumor and treated according to the various treatment regimens (E). Summary of the tumor luminescence intensity (F). Kaplan-Meier survival analysis of mice in various treatment groups (G). Values are shown as mean ± SD, *p<0.05, **p<0.01, ns, not significant.

Figure 6. Induction of integrin α4β7 by vaginal DC and is mediated by retinoic acid

CD11c+ dendritic cells were isolated from either the spleen or the cervicovaginal tract. These cells were then co-cultured with CFSE-labeled OT-1 cells at a ratio of 1:5 at the presence of OVA protein and IL-2 for 4 days. To inhibit retinoic acid receptor (RAR),a RAR-β inhibitor, LE540 was added into the cell culture. The cells were then stained with anti-mouse CD8 and α4β7. Representative flow cytometry analysis of α4β7 by OT-1 cells after stimulation with OVA protein pulsed dendritic cells from either spleen or vagina (A) or at the presence of RAR-β inhibitor, LE540 (C). Summary of the flow cytometry data of α4β7 expression by OT-1 cells after stimulation with OVA protein pulsed dendritic cells from either spleen or vagina (B) or at the presence of RAR-β inhibitor, LE540 (D).