Research ArticleCancer

Repurposing rotavirus vaccines for intratumoral immunotherapy can overcome resistance to immune checkpoint blockade

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Science Translational Medicine  23 Oct 2019:
Vol. 11, Issue 515, eaat5025
DOI: 10.1126/scitranslmed.aat5025
  • Fig. 1 Proinflammatory features of vaccines.

    NF-κB activation in transgenic HEK-293 cell lines expressing specific Toll-like receptors (TLRs) and NF-κB–Luc reporter system by different dilutions of (A) M. tuberculosis vaccine (BCG Pasteur), (B) the typhoid fever vaccine (Tyavax), (C and D) rotavirus vaccines (Rotarix and Rotateq, respectively). Peptidoglycan (PG), lipopolysaccharide (LPS), and tumor necrosis factor–α (TNF-α) were used as positive controls for stimulation of TLR2, TLR4, and NF-κB, respectively. Data are represented as RLU (relative luminescence unit) bioluminescence intensity. Red lines represent the activity of the natural ligand for the TLR, blue lines represent the activity of the vaccine, and the black line is the negative control (medium). Each data point is means ± SEM; n = 3 wells per dilution. The 293T parental cell line does not express any TLR but contains NF-κB reporter system. Other TLRs tested are shown in fig. S1. (E) Heatmap summarizing the NF-κB stimulatory properties across 14 vaccines at optimal dilution (i.e., dilution providing the highest bioluminescence intensity) using the 293T transgenic reporter cell lines expressing specific TLRs and NF-κB–Luc reporter system. Normalization was performed with the corresponding natural TLR ligand at optimal dilution as a positive control (100%). TLR9 and TLR6 cell lines have not been depicted because of the lack of positive control activation of the corresponding NF-κB cell lines by synthetic ligands. (F) Viability of 293T parental cell lines at different dilutions of Rotarix by SRB assay. Each data point is means ± SEM; n = 3 wells per dilution.

  • Fig. 2 Rotavirus vaccines have in vivo antitumor activity against pediatric tumor models resistant to anti–CTLA-4 and anti–PD-(L)1 and in vitro oncolytic properties against cancer cell lines.

    (A) Effects on the tumor growth of intraperitoneal (IP) injections of 200 μg of anti–CTLA-4 (αCTLA-4) or anti–PD-(L)1 (αPD-1/αPD-L1) antibodies, either alone or in combination, in the murine Neuro2a neuroblastoma model (syngeneic to A/J mouse strain background). Briefly, mice were injected subcutaneously with 5 × 106 of the Neuro2a murine tumor cell line. The therapy started at day 7 (black arrow) for a total of four injections with 3-day intervals. Tumors sizes were estimated as the product of their perpendicular diameters (in square millimeters). Every data point is the mean value of the tumor sizes from the mice belonging to the same treatment group at a given time point ± SEM, n = 5 to 10 mice per group. (B) Intratumoral (IT) injections of anti-infectious disease vaccines (BCG, Tyavax, Rotarix, and Rotateq) in the Neuro2a syngeneic mouse tumor model. Three consecutive IT injections of 100 μl of vaccines were performed every 3 days and starting around day 7 after tumor cells inoculation. n = 10 mice per group, each group was compared to IT PBS (unpaired one-tailed t test); ns, not significant; *P < 0.05 and **P < 0.005. Cytotoxic effects were evaluated by SRB assay on the neuroblastoma human cell line (SH-SY5Y) after 5 days of incubation with (C) dilutions of the Rotateq excipient, (D) dilutions of the five pure rotavirus strains contained in the Rotateq vaccine (rotavirus concentration, 10 to 100× higher than in the vaccine), (E) dilutions of the Rotateq vaccine (made of a mixture of ~5.5 × 106 pfu/ml of five different rotavirus strains) versus pure rotavirus strains (mixture of five different rotavirus strains at 5.5 × 107 to 5.5 × 108 pfu/ml). Means ± SEM, n = 3 wells per dilution. Unpaired one-tailed t test, *P < 0.05, ***P < 0.001, and ****P < 0.0001. (F) IC50 of rotavirus strains determined by cytotoxicity SRB assay on a variety of human cancer cell lines and (G) murine tumor cell lines. (H) IC50 of rotavirus strains determined by cytotoxicity SRB assay on normal cells (fibroblast and HMECs) compared to human triple-negative breast cancer (MDA-MB-231) and human neuroblastoma (SH-SY5Y) cancer cell lines. (I) IC50 of live versus inactivated rotavirus strains determined by cytotoxicity SRB assay on murine tumor cell lines. Serial dilution (1:10) was performed from 1:20 to 1:2,000,000. Incubation time of 3 to 5 days depending on the cell line. (F to I) Means ± SEM, n = 3 wells per dilution.

  • Fig. 3 In vitro oncolytic properties of rotavirus strains translate into in vivo immune activation and antitumor immunity.

    (A) Therapeutic activity of pure and diluted IT rotavirus strains against A20 B cell lymphoma tumors. Briefly, 5 × 106 tumor cells were injected subcutaneously on day 0, three IT injections of 100 μl of pure rotavirus, or 1:10 and 1:100 dilutions were performed every 3 days on days 7, 10, and 13. Tumor sizes (length × width) were evaluated twice a week. n = 10 mice per group; unpaired one-tailed t test, *P < 0.05; log-rank test for survival, **P = 0.001. Each group was compared to PBS-injected group. The therapeutic activity of 1:100 diluted IT rotavirus was also tested against two murine syngeneic neuroblastoma model NXS2 (B) (n = 8 or 9 mice per group; 5 × 106 tumor cells injected per mice) and Neuro2a (C) (n = 10 mice per group; 5 × 106 tumor cells injected per mice). Unpaired one-tailed t tests; log-rank test for survival rate, *P < 0.05 and ***P = 0.0001. (D) Tumor-infiltrating immune cells were analyzed by flow cytometry 24 hours after the first IT Rotavirus injection in the Neuro2a tumor model. Results are presented as a percentage of cells from the tumor-infiltrative CD45+ population, and Tregs were gated as Foxp3+ cell in the IT CD4+ population. Rotavirus-injected tumors were compared to PBS-injected tumor. Data as means ± SEM, n = 5 mice per group; unpaired one-tailed t tests, *P < 0.05, **P < 0.005, and ***P < 0.0005. (E) Flow cytometry analysis of CD86, MHC-II, and PD-L1 expression on human PBMC-derived M0 macrophages after 96 hours of coculture with live and inactivated rotavirus (dilution, 1:10). Data as means ± SEM, n = 2; unpaired one-tailed t test, *P < 0.05 and **P < 0.005.

  • Fig. 4 Rotavirus induces the type I IFN pathway and release of ATP by tumor cells.

    (A) List of the 23 shared genes commonly up-regulated by A20 and EMT6 cancer cell lines upon in vitro rotavirus exposure with at least 1.5 log2 fold change in expression and a P < 0.005 (mean values of triplicates). For the two log2 fold change columns, a gradient from white to red allows identification of the genes with the weakest (white) to the strongest (red) up-regulations. Genes highlighted in gray are in common with the list of genes up-regulated in A20 upon exposure with inactivated rotavirus (see Fig. 7D). (B) Pictures of immunofluorescence staining (magnification, ×20) with anti-MxA–specific monoclonal antibodies and secondary detection on A20 tumors harvested from Balb/c mice after either IT rotavirus or IT PBS treatment. Staining was performed on frozen tumor sections. An isotype control was used as a negative control for MxA detection (in green) after DAPI (4′,6-diamidino-2-phenylindole) counterstaining (in blue). The means (±SEM) of immunofluorescence intensity as measured by the ImageJ software are presented in the right panel. Data from n = 2 mice in each group with three tumor sections. Unpaired one-tailed t test, **P = 0.005. (C) ATP release in the cell culture supernatant upon several dilutions of rotavirus exposure on human (SH-SY5Y neuroblastoma) or murine (Neuro2a neuroblastoma and A20 B-cell lymphoma) tumor cell lines. Measurement performed by luminescence assay using CellTiter-Glo Luminescent Cell Viability Assay kit 48 hours after incubation with different dilutions of rotavirus. Results are reported as RLU intensity corresponding to relative ATP release intensity. Data as means ± SEM, n = 3 wells per dilution; one-way ANOVA test, ****P < 0.0001, ***P < 0.0005, and **P < 0.005.

  • Fig. 5 Intratumoral rotavirus synergizes with and overcomes the resistance to immune checkpoint–targeted therapies.

    Tumor growth and survival rates of mice treated with three IT injections of 100 μl of rotavirus strains (1:100 dilution) and/or four IP injections of 200 μg of αCTLA-4 with 3-day intervals in (A) the Neuro2a neuroblastoma and (B) the A20 B cell lymphoma tumor models. n = 10 mice per group and each group compared to PBS; unpaired one-tailed t test comparing the mean tumor sizes of each group, *P < 0.05 and **P < 0.005; survival rate (log-rank test), ****P < 0.0001. (C) Survival of mice treated with systemic anti–CTLA-4 together with either IT or systemic rotavirus in the A20 B cell lymphoma model. n = 8 to 9 mice per group; survival rate (log-rank test), **P = 0.001. (D) Tumor growth and survival rates in the EMT6 murine breast mammary carcinoma and (E) the CT26 murine colorectal tumor models. Same protocol as described in (A) and (B); means ± SEM, n = 9 to 10 mice per group. Combinations of IT rotavirus vaccine and/or IP αCTLA-4 in the Neuro2a neuroblastoma (F) and the A20 B cell lymphoma models (G). Same protocol as above; means ± SEM, n = 9 to 10 mice per group; unpaired one-tailed t test, *P < 0.05. Combination group (rotavirus vaccine + αCTLA-4) is compared to PBS group. (H) Tumor growth and survival rates of IT rotavirus and/or IP αPD-L1 in the A20 B cell lymphoma model. Same protocol as above; means ± SEM, n = 7 to 8 mice per group; unpaired one-tailed t test, *P < 0.05. Combination group (rotavirus + αPD-L1) is compared to PBS group.

  • Fig. 6 Intratumoral rotavirus therapy in combination with anti–CTLA-4 generates a tumor-specific CD8+ T cell immune response.

    (A) In vivo antitumor effects of three injections of 1:100 rotavirus strain into Neuro2a tumors growing in NSG immunodeficient mice. Tumor growth (left) and mice survival rates (right). n = 9 to 10 mice per group; unpaired one-tailed t test. A20 tumor growth after CD8, CD4, or NK cell depletions in mice treated with IT rotavirus alone (B) and IT rotavirus + IP αCTLA-4 (C). Same therapeutic protocol as described before; means ± SEM, n = 10 mice per group; unpaired one-tailed t test, *P < 0.05. Each group is compared to control group. (D) Survival rates of mice cured from A20 lymphoma (left) and Neuro2a neuroblastoma (right) after rechallenge with the same tumor. n = 5 mice per group; log-rank test, **P < 0.005. (E) Tumor growth in mice cured from A20 lymphoma and rechallenged with both A20 lymphoma and 4T1 mammary carcinoma cells. Means ± SEM, n = 3 mice per group; unpaired one-tailed t test, *P < 0.05. Intracellular IFN-γ and membrane activation markers on CD8+ T cells from cured versus control mice cocultured in vitro with A20 lymphoma cells (F) or Neuro2a neuroblastoma cells (G). Briefly, splenocytes from mice cured of an A20 lymphoma or Neuro2a tumor by IT rotavirus + IP αCTLA-4 and splenocytes from control mice bearing A20 lymphoma tumors or Neuro2a tumors but treated with IT PBS were cocultured with the relevant A20/Neuro2a tumor cell line for 4 days. Then, CD8 T cells were stained for intracellular IFN-γ and surface activation markers. Means ± SEM, n = 3 mice per group; unpaired one-tailed t test, *P < 0.05.

  • Fig. 7 Intrinsic rotavirus components are driving the proinflammatory features necessary for the synergy with anti–CTLA-4.

    (A) Tumor growth of A20 tumors in mice preimmunized or not with oral rotavirus vaccine and treated with the combination of IT rotavirus and IP αCTLA-4. Same therapeutic protocol as described before; means ± SEM, n = 10 mice per group. (B) Tumor growth of A20 tumors upon treatment with IT live or inactivated rotavirus, alone or in combination with αCTLA-4. Same therapeutic protocol as described before; means ± SEM, n = 9 to 10 mice per group; unpaired one-tailed t test, *P < 0.05. Each group is compared to the PBS group. (C) Heatmap representing the list of genes commonly differentially expressed (i.e., up-regulated or down-regulated) by A20 tumor cells upon incubation with active (live) or inactivated (“Inact”) virus or PBS (“Ctrl”); genes selected on the basis of |log2 fold change| of ≥1 and P ≤ 0.005; experiment performed was in triplicate; each triplicate data are depicted. (D) List of the six genes commonly up-regulated by A20 tumor cells exposed to active (live) or inactivated rotavirus and by EMT6 tumor cells exposed to live rotavirus as compared to media. For each gene, the log2 fold change value and the P value are provided. A gradient of colors from white to red has been attributed to the log2 fold change values from lowest to highest values, respectively, under all conditions.

  • Fig. 8 IT rotavirus with systemic anti–CTLA-4 generates a systemic antitumor immunity, which eradicates distant, noninjected established tumors.

    (A) Picture illustrating the two-tumor mouse model. Briefly, mice were injected with either A20 or Neuro2a tumor cells on both flanks. IT therapy was performed in only one of the two tumors and started when both tumors were established (~day 7). Growth of injected (left) or uninjected (“distant”; middle) tumors and survival (right) of mice treated with IT rotavirus in one tumor (“injected”) and IP anti–CTLA-4 in the A20 (B) and Neuro2a (C) models. n = 10 mice per group; survival rate (log-rank test), ****P < 0.0001 and ***P = 0.0004. (D and E) Proportion and phenotype of tumor-infiltrating immune cells from injected and noninjected (distant) tumors upon IT rotavirus in one tumor (injected) and IP anti–CTLA-4. Flow cytometry analysis; tumors were excised 24 hours after the second IT injection of rotavirus. CD11b and CD8 are gated from CD45+ cells for OX40 and CD86 marker analysis; means ± SEM, n = 3 mice per group; unpaired one-tailed t test, *P < 0.05 and **P < 0.005. Each group is compared to PBS.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/515/eaat5025/DC1

    Fig. S1. Proinflammatory features of anti-infectious disease vaccines.

    Fig. S2. Immune gene expression of A20 tumor cells upon in vitro active and inactivated rotavirus exposure.

    Fig. S3. Number of genes differentially expressed upon exposure of A20 cells with active or inactivated rotavirus in vitro.

    Fig. S4. Immune gene expression of A20 tumors upon in vivo active and inactivated rotavirus exposure.

    Fig. S5. Impact of in vitro rotavirus exposure on human myeloid cells.

    Table S1. Contingency table of the number of genes differentially expressed (steady, up-regulated, or down-regulated) between EMT6 and A20 cancer cell lines upon rotavirus exposure.

    Table S2. Number of genes commonly regulated between A20 and EMT6 cancer cell lines upon rotavirus exposure.

    Table S3. Number of genes commonly regulated upon exposure of A20 cancer cells with active rotavirus or inactivated rotavirus in comparison with control media.

    Table S4. Genes differentially expressed by A20 tumors in vivo upon active versus inactivated IT rotavirus therapy.

    Data file S1. Primary data.

    Data file S2. Gene expression data.

  • The PDF file includes:

    • Fig. S1. Proinflammatory features of anti-infectious disease vaccines.
    • Fig. S2. Immune gene expression of A20 tumor cells upon in vitro active and inactivated rotavirus exposure.
    • Fig. S3. Number of genes differentially expressed upon exposure of A20 cells with active or inactivated rotavirus in vitro.
    • Fig. S4. Immune gene expression of A20 tumors upon in vivo active and inactivated rotavirus exposure.
    • Fig. S5. Impact of in vitro rotavirus exposure on human myeloid cells.
    • Table S1. Contingency table of the number of genes differentially expressed (steady, up-regulated, or down-regulated) between EMT6 and A20 cancer cell lines upon rotavirus exposure.
    • Table S2. Number of genes commonly regulated between A20 and EMT6 cancer cell lines upon rotavirus exposure.
    • Table S3. Number of genes commonly regulated upon exposure of A20 cancer cells with active rotavirus or inactivated rotavirus in comparison with control media.
    • Table S4. Genes differentially expressed by A20 tumors in vivo upon active versus inactivated IT rotavirus therapy.

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    Other Supplementary Material for this manuscript includes the following:

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