Herpes Simplex Virus Glycoprotein D Targets a Specific Dendritic Cell Subset and Improves the Performance of Vaccines to Human Papillomavirus-Associated Tumors

Immunization with gDE7 admixed with poly (I:C) induces activation of multifunctional and effector memory CD8⁺ T cells. A–E, Immunizations with gDE7 admixed with poly (I:C) induce the differentiation of multifunctional CD8⁺ T cells. Mice were immunized with two subcutaneous doses of gDE7 or E7 admixed or not with poly (I:C) on days 1 and 8 after challenge. Spleen cells were analyzed by flow cytometry 14 days after the last immunization. A, Multiparametric flow cytometry of multifunctional CD8⁺ T cells. The frequencies of IFNγ (B), TNFα (C), or IL2 (D) single producers CD8⁺ T cells were depicted. *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, After gating on CD8⁺ T cells, Boolean combinations were created using the FlowJo software to determine the frequency of all possible combinations of cytokine production. *, P < 0.05 versus all other experimental groups in IL2-producing CD8⁺ T cells; **, P < 0.01 versus all other experimental groups in IFNγ -and TNFα-producing CD8⁺ T cells. Data represent the mean ± SD of 4–5 mice per group from 1 of 2 independent experiments with similar results. F, Administration of gDE7 admixed with poly (I:C) induces the differentiation of effector memory E7-specific CD8⁺ T cells. Spleen cells from challenged and immunized mice were labeled with an E7 H-2Db–specific dextramer and subjected to phenotypic analysis using CD44 and CD62L T-cell markers by flow cytometry. Effector memory (CD44⁺CD62L^low) and central memory (CD44⁺CD62L^high) CD8⁺ T-cell ratio in mice challenged with TC-1 cells and subsequently immunized with two doses (days 1 and 8 post-challenge) of tested vaccine formulations. ***, P < 0.001; •, P < 0.05 versus all other experimental groups in T_EM. Data represent the mean ± SD of 4–5 mice per group from 1 of 2 independent experiments with similar results. G and H, Immunization with gDE7 admixed with poly (I:C) induces high frequency of tumor-infiltrating E7-specific CD8⁺ T cells. Mice were challenged with 7.5 × 10⁴ TC-1 cells suspended in Matrigel and were immunized with two subcutaneous doses (days 1 and 8 after challenge) of tested vaccine formulations. Four days after the last dose, mice were euthanized, and tumors/Matrigel were removed for tumor microenvironment analysis by flow cytometry. G, Frequencies of tumor-infiltrating CD8⁺ T cells induced by the tested vaccine formulations. H, Cells recovered from tumors/Matrigel of E7 admixed with poly (I:C), gDE7, and gDE7 admixed with poly (I:C) immunized mice were labeled with an E7 H-2Db–specific dextramer, and the frequencies of tumor-infiltrating E7-specific CD8⁺ T cells were determined. ***, P < 0.001 versus all other experimental groups. Data represent the mean ± SD of 4 mice per group from 1 of 2 independent experiments with similar results.

Control of tumor development mediated by gDE7 admixed with poly (I:C) vaccine formulation prevents expansion of splenic immunosuppressive cells. For immunosuppressive cells analysis, mice were challenged with 7.5 × 10⁴ TC-1 cells and immunized with two subcutaneous doses (days 1 and 8 after challenge) of gDE7 or E7 admixed or not with poly (I:C). A and B, Spleen Treg cells do not expand in mice immunized with gDE7-based antitumor vaccines. For Treg analysis, spleen cells were incubated with 10 μg/mL of E7 protein for 5 days. After the incubation time, spleen cells were stained with anti-CD4, anti-CD25, and anti-Foxp3, and the frequency of Treg cells was determined. Numbers inside plots represent the frequency of CD25⁺Foxp3⁺ cells over all CD4⁺ T cells of a representative mouse. **, P < 0.01. Data represent the mean ± SD of 5 mice per group from 1 of 2 independent experiments with similar results. C and D, Spleen MDSCs do not expand in mice challenged with TC-1 cells and immunized with gDE7-based antitumor vaccines. Spleen cells from challenged and immunized mice were stained with anti-CD45, anti-CD11b, and anti-Gr-1 antibodies, and the frequency of MDSCs is depicted. Numbers inside plots represent the frequency of CD11b⁺ Gr-1⁺ over all CD45⁺ cells of a representative mouse. **, P < 0.01. Data represent the mean ± SD of 5 mice per group of 1 of 2 independent experiments with similar results.

Targeting of gDE7 to mouse CD11c⁺ CD8α⁺ DC leads to DC activation. gD and gDE7 recombinant proteins bind with higher affinity to mouse DC subset specialized in antigen cross-presentation. A, Gating strategy for analysis of gD, gDE7, or E7 binding and activation of isolated mouse DCs. Doublets and CD3⁺/CD19⁺/CD49b⁺ cells were excluded from analysis. CD11c⁺MHC-II⁺ cells were gated and subsequently separated by the expression of CD8α⁺. B, Isolated mouse DCs (4 × 10⁵ cells/well) were incubated with gD, gDE7, or E7 proteins at equimolar amounts. Protein binding was detected in 10⁵ cells using mouse anti-His tag and anti-mouse IgG-PE monoclonal antibodies. C, MFI of protein binding on CD11c⁺ CD8α⁺ and CD11c⁺ CD8α⁻ DCs. ***, P < 0.001; •, P < 0.05; and •••, P < 0.001 versus E7 on CD11c⁺ CD8α⁺ DCs; ###, P < 0.001 versus E7 on CD11c⁺ CD8α⁻ DCs. Data represent the mean of triplicate assays ± SD of 1 of 2 independent experiments with similar results. gD and gDE7 recombinant proteins promote activation of mouse DCs specialized in antigen cross-presentation. DC activation was determined by upregulation of surface molecules and intracellular cytokine production. MFI of CD40 (D) and CD86 (E) and IL12 production (F) on CD11c⁺ CD8α⁺ and CD11c⁺ CD8α⁻ DCs. ***, P < 0.001; ••, P < 0.01; and •••, P < 0.001 versus E7 on CD11c⁺ CD8α⁺ DCs; #, P < 0.05 versus E7 on CD11c⁺ CD8α⁻ DCs. ★, P < 0.05 versus iDC. Data represent the mean of duplicate assays ± SD of 1 of 2 independent experiments with similar results. iDC, cells incubated with apyrogenic saline.