Intra-cheek immunization as a novel vaccination route for therapeutic vaccines of head and neck squamous cell carcinomas using plasmo virus-like particles

Abstract

Despite current therapy, head and neck squamous cell carcinomas (HNSCCs) arising from various mucosal sites of the upper aero-digestive tract frequently relapse in a loco-regional manner and have a poor prognosis. Our objective was to validate an innovative mucosal route of vaccination using plasmo virus-like particles (pVLPs) in a pre-clinical orthotopic model of HNSCCs. For this purpose, we used pVLP-E7, that are plasmid DNA encoding retroviral virus-like particles carrying a truncated E7 oncoprotein from HPV-16 as antigen model, to vaccinate mice bearing pre-established TC-1 tumors implanted into the buccal mucosa. pVLP-E7 were combined with clinical grade TLR agonists (Imiquimod and CpG-ODN). In this pre-clinical orthotopic model, whose tumor microenvironment resembles to those of human HNSCCs, different mucosal vaccination routes were tested for their ability to elicit efficient immune and antitumoral responses. Results showed that mucosal intra-cheek (IC) vaccinations using pVLP-E7, comparatively to intradermic vaccinations (ID), gave rise to higher mobilization of mucosal (CD49a(+)) CD8(+) specific effector T cells in both tumor draining lymph nodes (TdLNs) and tumor microenvironment resulting in better antitumor effects and in a long-term protection against tumor rechallenge. In vivo CD8(+) depletion demonstrated that antitumoral effects were fully dependent upon the presence of CD8(+) T cells. Validation of IC mucosal vaccinations with pVLPs combined with adjuvants using a pre-clinical orthotopic model of HNSCC provides valuable pre-clinical data to rapidly envision the use of such therapeutic vaccines in patients with HNSCCs, inasmuch as vaccinal components and adjuvants can be easily obtained as clinical grade reagents.

Keywords: Head and neck squamous cell-carcinomas;; intra-cheek route; mucosal immunization; plasmo virus-like particles; pre-clinical orthotopic model; therapeutic vaccines; tumor microenvironment.

Figure 1.

Human Oral Squamous Cell Cancers are inflammatory neoplasms. Single cell suspensions were obtained from human OSCC samples (n = 7) and healthy gingiva (n = 7), and analyzed by flow cytometry. (A) Gating strategy: after dead cells and doublets exclusion, nine subpopulations were identified within CD45⁺ cells: (a) CD15⁺CD11b⁺ (granulocytes), (b) CD14⁺CD11b⁺ (macrophages), (c) CD19⁺CD3⁻ (B cells), (d) CD56⁺CD3⁻ (Natural Killer cells), (e) CD3⁺CD4⁺ (CD4⁺ T cells), (f) CD3⁺CD8⁺ (CD8⁺ T cells), (g) CD3⁺CD4⁺CD25⁺FoxP3⁺CD127⁻ (Treg), (h) Lin-1neg (CD3⁻CD19⁻CD56⁻) HLA-DR⁺CD11c⁻ (pDC) and (i) Lin-1negCD-11c⁺HLA-DR⁺CD14⁻ (mDC). (B) Number of CD45⁺ cells per gram (g) of tissue (left panel), cell number of indicated subset per g of tissue (upper right panel) and percent of indicated subset within CD45⁺ cells (lower right panel) are presented. NS, non-statistical difference = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2.

Orthotopic tumor models for OSCCs. C57BL/6 mice (5 mice per group) were injected with TC-1-Luc cells using subcutaneous (SC), intra-cheek (CH) or intra-lingual (IL) routes. (A) A representative bioluminescence imaging is shown one-week after TC-1-Luc injection (left panel). Kaplan–Meier curves show tumor-specific survival rates (right panel). (B) Flow cytometry analysis of cell suspensions from tumors obtained two weeks after TC-1-Luc cell injection in C57BL/6 mice (5 mice per group). For gating strategy, see Fig. S1. Number of CD45⁺ cells per mm³ of tumor (left panel), cell number of indicated subset per mm³ of tumor (upper right panel) and percent of indicated subset within CD45⁺ cells (lower right panel) are presented. (C) Flow cytometry analysis of cell suspensions from tumors obtained two weeks after injection of NR-S1 cells using SC and IC routes in C3H mice (5 mice per group). Number of CD45⁺ cells per mm³ of tumor (left panel), cell number of indicated subset per mm³ of tumor (upper right panel) and percent of indicated subset within CD45⁺ cells (lower right panel) are showed. NS, non-statistical difference = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3.

Advantage of intra-cheek vaccinations for inducing local and loco-regional antigen-specific CD8⁺ T-cell responses in tumor-bearing mice. (A) IFNγ ELISpot assay performed at day 10 using cell suspensions from draining lymph nodes (dLNs), spleen and non-draining lymph nodes (ndLNs), C57BL/6 mice being immunized at days 0–2–4 with pVLP-E7 using intra-dermal (ID), intra-cheek (IC) or intranasal (IN) routes of vaccination. Data were obtained from three separate experiments using 4–6 mice per group. (B) IFNγ ELISpot assay performed at day 10 using cell suspensions from dLNs, C57BL/6 mice (4–5 mice) being immunized at days 0–2–4 with pVLP-E7 or E7 polypeptide (+ CpG-ODN) using the ID or IC route. (C) and (D) C57BL/6 mice were injected in the cheek with TC-1-Luc cells, and then ID or IC immunized with pVLP-E7 at days 7–9–11 following tumor challenge. (C) Detection of E749-57-specific CD8⁺ T cells: cell suspensions from tumors were pooled from either 5 mice (Experiment 1) or 6 mice (Experiment 2) and stained with E7-tetramers at day 18. Data obtained from Experiment 1 (5 mice pooled) and Experiment 2 (6 mice pooled). (D) IFNγ ELISpot assay performed using cell suspensions from tumor draining lymph nodes (TdLNs) cells at day 18. Presented data are pooled from two separate experiments (5 and 6 mice per group). (E) Kaplan–Meier curves showing tumor-free survival rates. For ELISpot assays, cells were loaded and stimulated with E749-57 peptide. Background (always ≤ 100 spots per 10⁶ cells) obtained with not pulsed cells was subtracted due to some variability between the different tissues tested. NS, non-statistical difference = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4.

Adjuvants enhanced the therapeutic antitumor effects of intra-cheek vaccinations. C57BL/6 mice bearing intra-cheek TC-1-Luc tumors were ID or IC immunized at days 7–9–11 (arrow) with pVLP-E7 in the presence of adjuvants: CpG-ODN + Imiquimod (CpG/IMQ). As controls, one group of mice received CpG/IMQ alone and another received PBS. (A) Monitoring of tumor volume measured every 2–4 d; tumor-free rates are indicated in italic letters. (B) Kaplan–Meier curves showing tumor-free survival rates. Presented data are pooled from three separate experiments using 3–4 mice per group. *p < 0.05; ***p < 0.001; ****p < 0.0001.

Figure 5.

The therapeutic effect of intra-cheek vaccinations correlates with high specific CD8⁺ T-cell responses. C57BL/6 mice bearing intra-cheek TC-1-Luc tumors were ID or IC immunized at days 7–9–11 with pVLP-E7 in the presence of adjuvants: CpG-ODN + Imiquimod (CpG/IMQ). As controls, one group of mice received CpG/IMQ alone and another received PBS. (A) Detection by flow cytometry of E7-tetramer⁺ cells in single cell suspensions obtained from TdLNs and tumors pooled from 5 mice (Experiment 1) and 6 mice (Experiment 2) at day 18. Numbers of E749-57-tetramer⁺, CD8⁺ and CD49a⁺ expressing cells/mm³ (tumors) and per ×10⁶ cells (TdLNs) are presented. Presented data were obtained from two separate experiments using 5 and 6 mice, respectively. (B) E7-specific IFNγ ELISpot assay: cells from TdLNs were loaded and stimulated with E749-57 peptide and spot numbers are expressed by 10⁶ cells. The background (always ≤ 100 spots per 10⁶ cells) obtained with not pulsed cells has been subtracted. Presented data are pooled from two separate experiments using 5 and 6 mice per group, respectively. (C) Number of CD4⁺ T cells per mm³ of tumor (upper left panel), number of Treg per mm³ of tumor (upper right panel), percent of Treg in CD4⁺ cells in tumor (lower left panel) and ratio Treg/T effector cells (CD4⁺ and CD8⁺ T cells) in tumor (lower right panel) are showed. (D) Effect of the in vivo CD8⁺ depletion in C57BL/6 mice bearing intra-cheek TC-1-Luc tumors when IC immunized with pVLP-E7 combined with CpG/IMQ at days 7–9–11 (arrow). One week before the first vaccination and then once a week, mice received anti-CD8⁺ mAb (100 mg, intraperitoneally) or isotype-matched control mAb. Kaplan–Meier curves showing tumor-free survival rates. NS, non-statistical difference = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6.

Long-term protection induced by intra-cheek vaccinations. Immunized C57BL/6 mice showing complete tumor regression (3–7 mice per group), were rechallenged with TC-1 cells at day 200. (A) Monitoring of tumor volume measured every 2–4 d. (B) Blood detection by flow cytometry of E749-57-tetramer expressing cells in CD8⁺ T cells at day 250. A representative analysis is shown. (C) Percentages of CD62L, CD44 and CD49a cells in CD8⁺ T cells. Data was obtained from three separate experiments. NS, non-statistical difference = p > 0.05; *p < 0.05; **p < 0.01.