Fowlpox-based survivin vaccination for malignant mesothelioma therapy

Abstract

Survivin protein is an attractive candidate for cancer immunotherapy since it is abundantly expressed in most common human cancers and mostly absent in normal adult tissues. Malignant mesothelioma (MM) is a deadly cancer associated with asbestos or erionite exposure for which no successful therapies are currently available. In this study, we evaluated the therapeutic efficacy of a novel survivin-based vaccine by subcutaneous or intraperitoneum injection of BALB/c mice with murine fiber-induced MM tumor cells followed by vaccination with recombinant Fowlpox virus replicons encoding survivin. Vaccination generated significant immune responses in both models, leading to delayed tumor growth and improved animal survival. Flow cytometry and immunofluorescence analyses of tumors from vaccinated mice showed CD8(+) T-cell infiltration, and real-time PCR demonstrated increased mRNA and protein levels of immunostimulatory cytokines. Analyses of survivin peptide-pulsed spleen and lymph node cells from vaccinated mice using ELISPOT and intracellular cytokine staining confirmed antigen-specific, interferon-γ-producing CD8(+) T-cell responses. In addition pentamer-based flow cytometry showed that vaccination generated survivin-specific CD8(+) T cells. Importantly, vaccination did not affect fertility or induce autoimmune abnormalities in mice. Our results demonstrate that vaccination with recombinant Fowlpox expressing survivin improves T-cell responses against aggressive MM tumors and may form the basis for promising clinical applications.

Figure 1

Survivin and MHC-I expression in human MM. (A) Immunoblotting with survivin and MHC-I antibodies on human mesothelial cells (HM) and 7 human MM cell lines. HM served as a normal tissue negative control and positive controls included HeLa cells known to express both survivin and MHC-I. (B) Immunohistochemical staining for survivin and MHC-I in different histological subtypes of human MM. Original magnification: 200x. Representative sections of epithelioid, biphasic and sarcomatoid MM tumors stained with H/E, survivin and MHC-I.

Figure 2

Survivin and MHC-I expression in MM tumors induced in mice by i.p. injection of carcinogenic fibers. BALB/c mice were i.p. injected with PBS (control) or 4 mg of fibers: crocidolite, Turkish erionite or U.S. erionite. (A) Immunohistochemical staining for survivin in MM tumors from six different mice i.p. injected with crocidolite (CRH5, CRH11), Turkish erionite (EKKH3, EKKH5) or US erionite (EOH8, EOH9). Original magnification: 200x. H/E staining was performed to localize malignant cells, and sections from the same specimens were stained using survivin antibodies. (B) Immunoblotting with survivin antibodies of MM cells isolated from peritoneal fluid of fiber-injected mice. MM cells were isolated as described in Materials and Methods, placed in culture for 5 passages and then lysed. Positive and negative controls included AB12 mouse MM cell line and P815 mouse mastocytoma cell line, respectively, with α-tubulin used as loading control. (C) Frequency (as percentage of cell population) of H-2K^d expressing MM cells isolated from fiber-injected mice determined by flow cytometric analysis.

Figure 3

Survivin vaccination with Fowlpox vectors delays tumor growth and improves survival in s.c. mouse models of MM. BALB/c mice bearing palpable tumors (n = 6 for each group) were vaccinated with two i.m. injections of FP-surv or FP-ctrl (one week apart). Tumor volume (A) and animal survival (B) are shown for injection with three different cell lines: AB12 (Top), CRH5 (Middle) and EKKH5 (Bottom). Tumors were measured weekly and a Student's t test was used to compare means of each group. For survival, mice were followed until tumors reached volumes of 1,500 mm³ (AB12 and CRH5) or 100 mm³ (EKKH5) w. (C) Tumors from survivin-vaccinated mice exhibited intra-tumor necrosis with the presence of lymphoid cells. AB12 tumors from FP-ctrl and FP-surv vaccinated mice displayed different macroscopic appearance after 17 days from the first immunization (Top). Tumors were excised, paraffin embedded and stained with H/E. Representative AB12 tumor tissues at 20x (Middle) and 200x (Bottom) magnification. (D) Tumors were collected and enzyme-digested as described in the Materials and Methods section and stained with anti-CD8-APC. Live cells were distinguished from dead cells and debris using LIVE/DEAD® cell viability dye. Top: Flow cytometry analysis of representative data from AB12 tumors in survivin-vaccinated and control mice. Bottom: CD8⁺ T cells were counted in AB12 subcutaneous tumors. Results represent mean ± S.E. and statistical significance was determined by Student's t test (N = 5 mice/group).

Figure 4

Induction of IFN-γ secreting CD8⁺ T lymphocytes by survivin vaccination. BALB/c mice were vaccinated with two i.m. injections of FP-surv or FP-ctrl (one week apart). Five days after the last vaccination spleens and inguinal lymph node cells were isolated and enumerated for assays. (A) Secretion of IFN-γ was evaluated by ELISPOT assay. T cells were activated with survivin_46–54 peptide in the presence of 5 IU/mL IL-2 for 24 h or incubated with no peptide (NP). The results are presented as spot-forming cells per 10⁶ cells and results represent the mean ± S.E. (N = 5 mice/group). (B) Intracellular IFN-γ in CD8⁺ T cells was evaluated by flow cytometric ICC analyses of spleen/lymph ± node cells cultured with or without survivin_46–54 peptide. Left Panel: Representative data from flow cytometric analysis of survivin-vaccinated and control mice. Lymphocytes were either not stimulated (NP) or stimulated with survivin_46–54 peptide. CD8⁺ T cells were distinguished using a marker gate in the CD3 vs. CD8 dot plot. Right panel: Percentage of IFN-γ expressing CD8⁺ T cells was evaluated with data are presented as mean ± S.E. (N = 5 mice/group). Statistical significance was determined by Student's t test in both ELISPOT and intracellular IFNγ staining assays.

Figure 5

Fowlpox vectors expressing survivin induce CD8⁺ T cell tumor infiltration which improve survival in i.p. mouse models of MM. (A) BALB/c mice were injected i.p. with AB12 (Left) or CRH5 (Right) MM cells (day 0). After 7 days, mice (n = 10 mice/group) were vaccinated with 1 × 10⁷ PFU of FP-surv and boosted with the same vaccine 7 days later. FP-ctrl was used as control. Mice were followed for signs of distress and then sacrificed when morbidity was evident. Log-rank analysis was used to determine significance. (B) Tumors from survivin-vaccinated and control mice were collected and enzyme-digested and stained with APC-anti-mouse CD8 and FITC-anti-CD4 as described in Materials and Method. Live cells were distinguished from debris using LIVE/DEAD® cell viability dye. Left panel: Representative data from flow cytometric analysis of AB12 tumors in survivin-vaccinated and control mice. Right panel: Percentage of CD8⁺ T cells in AB12 and CRH5 tumors from survivin-vaccinated and control mice. Results represent mean ± S.E. (N = 5 mice/group) with means of each group compared using a Student's t test. (C) Frozen sections of tumors obtained from survivin-vaccinated and control mice were stained with H&E or used for immunofluorescence. Anti-CD8 in immunofluorescence analysis was detected with Alexa Fluor 594 (red) and nuclei detected with DAPI (blue). Both H&E and fluorescence images represent AB12 tumor tissues (400×). Right panel: Percentage CD8⁺ T cells were counted on 10 fields with at least 100 cells in the same slide. Values are expressed as the mean ± S.E. percentage of positive cells per total counted and statistical significance was determined by Student's t test.