Prophylactic human papillomavirus (HPV) vaccines are commercially available for prevention of infection with cancerogenic HPV genotypes but are not able to combat pre-existing HPV-associated disease. In this study, we designed a nanomaterial-based therapeutic HPV vaccine, comprising manganese (Mn4+)-doped silica nanoparticles (Mn4+-SNPs) and the viral neoantigen peptide GF001 derived from the HPV16 E7 oncoprotein. We show in mice that Mn4+-SNPs act as self-adjuvants by activating the inflammatory signaling pathway via generation of reactive oxygen species, resulting in immune cell recruitment to the immunization site and dendritic cell maturation. Mn4+-SNPs further serve as Ag carriers by facilitating endo/lysosomal escape via depletion of protons in acidic endocytic compartments and subsequent Ag delivery to the cytosol for cross-presentation. The Mn4+-SNPs+GF001 nanovaccine induced strong E7-specific CD8+ T cell responses, leading to remission of established murine HPV16 E7-expressing solid TC-1 tumors and E7-expressing transgenic skin grafts. This vaccine construct offers a simple and general strategy for therapeutic HPV and potentially other cancer vaccines.

Persistent infection with high-risk human papillomavirus (HPV) genotypes HPV16 and 18 is the major cause of cervical and other anogenital and oropharyngeal cancers (1). Commercially available HPV vaccines prevent HPV infection by inducing neutralizing Abs against structural viral capsid proteins and consequently prevent development of HPV-associated disease (2). These vaccines, however, are not effective to treat HPV-induced malignancies because of the absence of viral capsid protein expression by HPV-transformed cells. HPV-transformed cells express regulatory viral proteins, dominantly the viral oncoproteins E6 and E7, which act as neoantigens and therewith provide an opportunity to develop Ag-specific targeted therapy. Nevertheless, because of complex mechanisms of immune suppression and tumor escape strategies established in the tumor microenvironment, the development of efficient HPV-specific immunotherapy remains a challenge (3).

Most preclinical studies evaluating HPV-targeted immunotherapy have been performed in a transplantable tumor model derived from immortalized lung cells (tissue culture-1 [TC-1]), which express HPV16-E6 and E7 oncogenes (4). Alternatively, a skin graft model has been developed to more rigorously evaluate therapeutic HPV vaccines (5). In this model, the skin from transgenic mice expressing the HPV16 E7 oncoprotein driven by the keratin 14 promoter (K14E7) is grafted onto recipient mice. K14E7 mice display epidermal hyperplasia and local immune suppression comparable to cervical intraepithelial neoplasia in humans (6) and are therefore a suitable model to study vaccine efficacies preclinically. K14E7 skin grafts are not rejected by wild type recipients or recipients harboring a high frequency of E7-specific T cells (5, 7). Therapeutic HPV vaccines can be evaluated by their ability to promote rejection of K14E7 skin grafts, therewith providing supportive preclinical evidence for further clinical studies.

Cancer immunotherapy, and particularly Ag-specific therapeutic immunization, has received recognition for its potential in cancer treatment through activation of the adaptive immune system (8). Despite the success of tumor-specific immunotherapy in animal studies, translation to clinical effectiveness is often limited. Generally, exogenous Ags are trafficked through the endocytosis pathway in APCs, resulting in preferential Ag presentation on MHC class II (MHC II) to activate CD4+ helper T cells (9). However, cross-presentation, in which exogenous Ags are presented by MHC class I (MHC I) in APCs, is critical to induce cytotoxic CD8+ T cell responses, which are key mediators of anti-tumor immunity. The newly emerging field of synthetic nanomaterial– or biomaterial scaffold–based vaccines offers a wide potential for cancer immunotherapy (1012), mainly focusing on engineering materials with codelivered adjuvants (for example, immune-stimulatory CpG) and Ags to induce augmented Ag-specific CD8+ T cell responses (13, 14). Recently, a few studies have reported immunogenicity of nanomaterial-based vaccines (nanovaccines) as cancer immunotherapy with simple incorporation of nanomaterial-based adjuvants (nanoadjuvants) and Ags and have demonstrated that nanoadjuvants can efficiently facilitate Ag transfer from acidic endocytic organelles to the cytosol for cross-presentation (15) or specifically target cross-presenting dendritic cells (DCs) (16). These polymers, emulsions, or lipid-based nanovaccines have been evaluated in a preclinical HPV-expressing solid TC-1 tumor model, showing great therapeutic efficacy but focusing on early stage disease at which solid tumors are not yet established (1517). Thus, there is still a need for highly immunogenic therapeutic nanovaccines that demonstrate efficacy in more rigorous preclinical HPV disease models to predict successful treatment of established HPV-induced malignant disease in humans.

In the attempt to develop a potent adjuvant suited for immunotherapy of HPV-induced malignant disease, we rationally designed a formulation based on a silica-based nanomaterial together with the immune-stimulatory metal manganese (Mn). The Mn4+-doped silica nanoparticles (Mn4+-SNPs) are characterized by a hollow raspberry-like morphology containing nanospikes, and these features have previously been demonstrated to enhance adjuvancy (18, 19). Furthermore, the doped Mn contained a high oxidative state (Mn4+), which we predicted will facilitate proton depletion in acidic endosomal compartments, thereby facilitating endosome escape of codelivered HPV-specific Ag for cross-presentation by APCs. We incorporated the immune-dominant peptide GF001 of the HPV16 E7 oncoprotein into the Mn4+-SNPs nanoadjuvant and tested the formulation for immunogenicity and efficacy in murine HPV tumor and skin grafting models.

The Mn4+-SNPs nanoadjuvant as a pH-regulating system showed enhanced Ag delivery into the cytosolic MHC I pathway. Mn4+-SNPs further induced reactive oxygen species (ROS), thereby activating the inflammatory signaling pathway via the NLRP3 inflammasome, resulting in infiltration of innate immune cells at the immunization site and DC maturation in draining lymph nodes (LNs) (dLNs). We generated a therapeutic HPV vaccine comprising the Mn4+-SNP nanovaccine and HPV16 E7 peptide, which induced potent CD8+ T cell responses, eradicated established E7-expressing TC-1 tumors, and promoted remission of E7-expressing transgenic skin grafts.

Mice were housed at Biological Research Facility of the Translational Research Institute under specific pathogen-free conditions. C57BL/6J mice were ordered from the Animal Resources Centre (Perth, Australia). Transgenic E7TCR269 mice were bred as previously described (20). Transgenic K14E7 mice expressing the HPV16E7 oncoprotein under the K14 promotor were originally provided by P. Lambert (University of Wisconsin-Madison) (21). CASP-1/ mice were kindly provided from K. Stacey (The University of Queensland, Australia). Female mice at 6–12 wk were used in experiments. All animal experiments were approved by The University of Queensland Animal Ethics Committee (UQDI/452/16 and UQDI/405/19).

Mice were s.c. injected with particles or vaccines in 100 μl of PBS at the tail base. Unimmunized mice were used as a control. The detailed amount and timeline were described in the figure captions.

Cetyltrimethylammonium chloride (25% by weight in water), tetraethoxysilane, disodium maleate, FITC-conjugated OVA conjugate (FITC-OVA), 2′,7′-dichlorofluorescin diacetate (DCF-DA), monochlorobimane, glutathione (GSH), and NaAc·3H2O were purchased from Sigma-Aldrich. MnSO4·H2O was purchased from ChemSupply. HPV16 E7 peptide (49–57) RAHYNIVTF (GF001, >95% purity) was purchased from Auspep. LysoTracker Red DND-99 was purchased from Thermo Fisher Scientific. Inhibitors (Ebselen, Z-YVAD-FMK) were purchased from Abcam. PE-RAHYNIVTF-MHC I dextramer and negative control PE dextramer were purchased from Immudex. TC-1 cells were provided by T.C. Wu (Johns Hopkins University). RPMI 1640 (Life Technologies) complete medium were supplemented with 10% FBS (Life Technologies) and 1% of penicillin–streptomycin–glutamine (Life Technologies). All cell culture was conducted in a humidified incubator at 37°C supplied with 5% CO2. Abs used for flow cytometry are listed in Supplemental Table I.

SNPs were synthesized as described previously (22). Briefly, NaAc·3H2O (0.3 g) and cetyltrimethylammonium chloride (6.24 g) were dissolved in water (54 g) under stirring at 60°C. Tetraethoxysilane (4.35 ml) was added to the aqueous solution. After stirring for 24 h, the solid particles were harvested after centrifugation at 20,000 rpm for 10 min and washed with water. The as-synthesized particles were air dried at 50°C. SNPs were obtained after calcination at 650°C for 5 h.

Mn4+-SNPs were synthesized using a modified hydrothermal treatment method (23). The as-synthesized SNPs without calcination process were used as hard templates. Fifty milligrams of as-synthesized SNPs were suspended in 10 ml of distilled water under bath sonication. MnSO4·H2O (0.8 mmol) and disodium maleate (1.6 mmol) were dissolved in 10 ml of distilled water, which was then mixed with the SNP suspension. The mixed solution was hydrothermally treated at 180°C for 12 h in an autoclave. The particles were obtained by centrifugation at 20,000 rpm for 10 min. Finally, Mn4+-SNPs were calcinated at 550°C for 5 h to remove the surfactant in the as-synthesized hard templates and convert Mn2+ into a Mn4+ state.

Nanoparticle samples suspended in ethanol were dropped on a carbon-covered Cu grid (Beijing XXBR Technology) for transmission electron microscopy (TEM) analysis using a FEI Tecnai F30 (300 kV) or a JEOL 1010 (100 kV). Scanning TEM (STEM) images were obtained on a Hitachi HF5000 TEM operated at 200 kV. Energy-dispersive x-ray mapping analysis was performed on a JEOL 2100 at 200 kV. The nanoparticles were degassed under vacuum at 453K for 14 h, and nitrogen sorption analysis was conducted at 77K using a Micromeritics ASAP Tristar II 3020 system. The Brunauer–Emmett–Teller method was used to calculate the surface area. The total pore volume was calculated from the absorbed nitrogen amount at the highest relative pressure P/P0 = 0.99. X-ray photoelectron spectroscopy (XPS) analysis was conducted on a Kratos Axis ULTRA X-ray photoelectron spectrometer. Casa XPS version 2.3.14 software was used for peak fitting and atomic percentage calculations. The ζ potential of nanoparticles was measured by a Malvern Nano ZS Zetasizer in PBS at room temperature.

One milligram of SNPs or Mn4+-SNPs were suspended in 1 ml of PBS pH 7.2 (neutral), 5.0 (acidic), or GSH (0, 2, 10 mM) PBS pH 7.2, followed by shaking at 150 rpm and 37°C. After 24 h, the dissolved Mn2+ and silicon amounts in PBS superannuants were measured by inductively coupled plasma optical emission spectrometry (ICP-OES).

Bone marrow macrophages (BMMs) were cultured as previously described (24). Bone marrow cells were harvested from femur and tibia of naive C57BL/6J mice and seeded in a petri dish with BMM culture medium (RPMI complete medium in the presence of 100 ng/ml GM-CSF [PeproTech]). On day 3 and 6, the cells were washed and supplied with fresh BMM culture medium. On day 7, BMMs were harvested for further treatment. Bone marrow–derived DCs (BMDCs) were cultured as described previously (25). Briefly, the harvested bone marrow cells were cultured in BMDC culture medium (RPMI complete medium supplemented with 10 ng/ml IL-4 and 20 ng/ml GM-CSF). The medium was replaced with fresh BMDC culture medium on day 3 and 5. On day 6, BMDCs were harvested for further experiments.

BMMs were seeded in a 24-well plate with 2.5 × 105 cells/well in 500 μl BMM culture medium and then treated with nanoparticles (30 μg/ml) for 4 h. After washing, BMMs were incubated with 40 μM DCF-DA in 500 μl of fresh BMM culture medium for 20 min. Hoechst (1:500) dilution was used to stain BMMs at room temperature for 10 min. After washing, the fluorescence images of DCF converted by intracellular ROS in BMMs were taken by confocal microscopy (FV1200, Olympus, Tokyo, Japan) under an excitation of 485 nm and emission of 530 nm. In another experiment, BMMs were seeded in a 96-well black/clear flat-bottom plate for nanoparticle and DCF-DA treatment, and then the fluorescence intensity dichlorofluorescin was detected by plate reader (PHERAstar). To quantify the intracellular ROS level per cell, 5 × 105 BMMs in 1 ml of BMM culture medium were seeded in a sterile polystyrene flow cytometry tube with snap cap (Corning). After treatment with particles (30 μg/ml) for 4 h, BMMs were incubated with 40 μM DCF-DA for 20 min. BMMs were washed with cold PBS and resuspended in 250 μl of FACS buffer (PBS containing 2% FBS and 1 mM EDTA) for flow cytometry analysis (BD LSRFortessa ×20). Cells without treatment of nanoparticles were used as control.

First, 5 × 105 BMMs in 1 ml of BMM culture medium were seeded in flow cytometry tubes, followed by the addition of nanoparticles (30 μg/ml). After 4 h, the cells were incubated with 40 μM monochlorobimate at room temperature for 30 min. The cells were washed and resuspended in FACS buffer for flow cytometry analysis (excitation at 360 ± 40 nm and emission at 460 ± 40 nm). Propidium iodide was added at a 1:10,000 dilution for dead cell discrimination just before flow cytometry analysis. Cells without the addition of nanoparticles were used as control.

BMMs (1 × 106) were seeded in a six-well plate, followed by treatment with free FITC-OVA, Mn4+-SNPs+FITC-OVA, or SNPs+FITC-OVA (final concentration of 30 μg/ml SNP and 6 μg/ml FITC-OVA). After 2- or 6-h incubation, the cells were washed and stained with 75 nM LysoTracker Red DND-99 in fresh medium for 30 min. After washing, the cells were stained with Hoechst (1:500). The images of cells were acquired by fluorescence microscopy (FV1200; Olympus).

BMMs (2.5 × 105 cells) in 500 μl of BMM culture medium were seeded in a 24-well plate. BMMs were then incubated for 14 h in the presence (or absence) of LPS (10 ng/ml). The (primed) cells were treated with 15 μg/ml nanoparticles for 48 h. The supernatants of the activated cells were collected after centrifugation at 350 × g for 5 min to perform the IL-1β ELISA, following the manufacturer’s procedures. In another version of the experiment, BMMs were treated with 50 μM Ebselen (antioxidants) or 10 μM ZYVAD-fmk (an inhibitor of caspase-1) for 30 min prior to the addition of nanoparticles. The cells without the addition of nanoparticles were used as control.

BMDCs (5 × 105) in 1 ml of BMDC culture medium were seeded in a sterile polystyrene flow cytometry tube with snap cap (Corning), followed by the addition of SNPs or Mn4+-SNPs (15 μg/ml). After 24- or 48-h incubation, BMDCs were washed with cold PBS for flow cytometry analysis.

The loading capacity of Mn4+-SNPs with GF001 was estimated by direct detect spectrometer analysis. GF001 (10 μl, 4 μg/μl) were mixed with Mn4+-SNPs (10 μl, 2 mg/ml). In a control group, PBS without Mn4+-SNPs was mixed with GF001 solution. The solutions were incubated on ice for 30 min. After that, the supernatants were collected by centrifugation at 15,000 rpm for 15 min at 4°C. Then, 2 μl of supernatants or GF001 standard solutions (0–4 mg/ml) was added to the sample position of an assay-free card placed on the spotting tray. The card was then inserted into a direct detect spectrometer (Merck) for sample measurements.

Mice were sacrificed by CO2 asphyxiation, and skin, LNs, and spleen samples were harvested for single-cell processing and analysis. Skin samples (∼1 cm2) at the injection site and LNs (inguinal, cervical, and brachial/axillary) were chopped finely with scissors in 0.5 ml of digestion buffer, containing 0.2 mg/ml DNase I (Thermo Fisher Scientific) and 1 mg/ml collagenase D (Roche) in PBS. Chopped skin was further topped up with digestion buffer to 3 ml and shaken at 37°C for 1 h before passing through a 70-μm cell strainer (BD Falcon) with 10 ml of PBS. The samples were centrifuged at 4°C and 350 × g for 5 min, and the cell pellets were resuspended in FACS buffer or complete RPMI medium. To obtain splenocytes, spleens were mechanically disrupted and meshed using a syringe plunger through a 70-μm cell strainer and washed with 10 ml FACS buffer. Erythrocytes were removed by ammonium–chloride–potassium lysing buffer. Splenocytes were resuspended in FACS buffer or complete RPMI medium after washing.

Single-cell suspensions were incubated with Fc blocking Ab (anti-CD16/32) and LIVE/DEAD stain (Aqua, Thermo Fisher Scientific) on ice. The cells from skin samples were stained with a master mix of anti-mouse mAbs (Supplemental Table I) to F4/80 (FITC), TCRβ (Pacific Blue), CD45 (Percp-Cy5.5), CD8a (allophycocyanin), CD11b (PB), EpCAM (allophycocyanin), MHC II (allophycocyanin-Cy7), CD103 (PE), CD11c (PE-Cy7), Ly6G (Alexa Fluor 700), and Ly6C (Brilliant Violet 711). The cells from LNs were stained with a master mix of Abs specific to CD11c (PE-Cy7), MHC II (allophycocyanin-Cy7), CD80 (FITC), CD86 (PB), and EpCAM (PE). BMDCs were stained with a master mix of Abs specific to CD11c (PE-Cy7), MHC II (allophycocyanin-Cy7), CD80 (FITC), CD86 (PB), and EpCAM (allophycocyanin). For dextramer staining, splenocytes were incubated with RAHYNIVTF-MHC I dextramer or negative control dextramer before Fc blocking and DEAD/LIVE cell staining. After washing, cells were incubated with a master mix of Abs specific to TCR-β (Pacific Blue), CD8a (PerCP-Cy5.5), and CD4 (Alexa Fluor 700) for flow cytometry analysis. For intracellular cytokine staining, cells were restimulated with 10 μg/ml of GF001 in RPMI complete medium overnight, and GolgiPlug (BD GolgiPlug, containing brefeldin A) was added into each well at a 1:1000 dilution for an additional 4 h. After washing, Fc blocking, and DEAD/LIVE staining, the cells were stained with a master mix of anti-mouse Abs specific to TCR-β (Pacific Blue), CD8a (allophycocyanin), and CD4 (PE-Cy7). After fixation and permeabilization (BD Cytofix/Cytoperm), the cells were intracellularly stained with Abs specific to IFN-γ (FITC) or isotype Ab. After washing, the stained cells were resuspended in FACS buffer and acquired using a BD LSRFortessa ×20 flow cytometer.

Skin samples harvested from the immunization site were fixed in 10% neutral buffered formalin overnight and then embedded in paraffin for H&E staining. The sections were observed under an Olympus BX63 upright microscope. For immunofluorescence staining, the skin samples were embedded in optimal cutting temperature compound (Sakura). Tissue sections were cryosectioned using the Leica Cryostat CM1950 at 4-μm thickness, followed by fixation treatment in acetone at −20°C for 10 min and permeabilization with 0.1% Triton-X for 15 min. Then, 10% goat serum was used to block sections for 1 h. Sections were treated with primary Ab rat anti-mouse Ly6G (1:200) or rat anti-mouse MHC II (1:200) in PBS solution in the dark for 1 h, followed by incubation with a secondary goat anti-rat Ab (Alexa Fluor 555) (Invitrogen) in PBS solution for 1 h. All sections were stained with DAPI (1:500) for 20 min and then mounted using ProLong Gold Antifade mounting medium (Thermo Fisher Scientific). All washes were performed using 0.025% Triton-X PBS. Images were obtained using a FV1200 Olympus microscope.

Briefly, 2.5 × 105 lymphocytes in 100 μl of RPMI complete medium were seeded in a MultiScreen-IP filter 96-well plate (Merck) coated with capture Ab against IFN-γ (AN-18; eBioscience) and incubated with or without 10 μg/ml E7 peptide GF001. After 18-h incubation, cells were removed, and plates were incubated with a biotinylated Ab against IFN-γ (R4-6A2; eBioscience). HRP-conjugated streptavidin (Sigma-Aldrich) was incubated with the plate. After washing, urea hydrogen peroxide and 3, 3′-diaminobenzidine tablets (Sigma-Aldrich) were dissolved in PBS and added to the plate for detection. Spots were counted by an AID ELISPOT reader system (Autoimmun Diagnostika).

To deplete macrophages or neutrophils, mice were injected i.p. with 200 μl of clodronate liposomes (Clodrosome, 18.4 mM; Encapsula NanoSciences) or 250 μg anti-Ly6G (clone 1A8, no. BP0075-1; Bio X Cell), respectively. Control mice received equal amounts of control liposomes without clodronate (Encapsome; Encapsula NanoSciences) or isotype Ab (rat IgG2a, no. BP0089; Bio X Cell). Depletion was validated from blood 1 d after treatment (26) or spleen 4 d after treatment (27) using flow cytometry.

TC-1 cells were thawed and cultured for 6 d prior to injection (7). TC-1 cells were s.c. injected at the back of mice. In therapeutic vaccination regimen, tumor-bearing mice were mixed and randomly allocated to different groups before immunization. Tumor sizes were measured every 2 or 3 d using a digital caliper. The tumor volume was calculated using the following equation: tumor volume = (length × width2)/2. Animals were euthanized when the tumor size reached 1.0 cm3 or developed ulceration. The dose of TC-1 cells and the timeline were described in the figure captions.

Snap-frozen TC-1 tumors were homogenized in 1 ml TRIzol using a homogenizer (T10 basic ULTRA-TURRAX; IKA). After centrifugation, the clear aqueous layer of tumor lysate mixture was collected for RNA extraction using the QIAGEN RNeasy Mini Kit, following the manufacturer’s procedures. Purified RNA samples were used to synthesize cDNA together with Superscript III Reverse Transcriptase (Invitrogen) and random hexametric primers. The one-step reverse transcription settings were set as follows: 5 min at 65°C, 1 min at 4°C, 5 min at 25°C, 60 min at 50°C, 15 min at 70°C, and hold at 4°C. cDNA products were stored at −20°C until used for RT-PCR.

Quantitative real-time PCR analysis was performed on the QuantStudio 7 Real-Time PCR system (Applied Biosystems) using SYBR Green Master Mix (Invitrogen) and Mus musculus primer sequences designed for SYBR assays, according to the manufacturer’s procedures. The designed primers (Table I) were purchased from Integrated DNA Technologies. Relative gene expression was calculated using 2-ΔCt where ΔCt values were calculated from the difference between the Ct values of target genes and average Ct values of two housekeeping genes.

Skin grafting was carried out as previously described (5). Briefly, the ear skins of C57BL/6J and K14E7 donor mice were split into dorsal and ventral halves using forceps. The recipient E7TCR269 mice were anesthetized and received double dorsal ear skins to the flank region. Grafts were protected by covering with Bactigras (Smith+Nephew) and sensitive dressing lengths (Elastoplast). Recipient mice were wrapped with bandages of Flex-wrap (Lyppard) and micropore tape. At day 7 postgrafting, the bandages were removed. Skin grafts were recorded weekly by taking photographs together with a ruler. Graft sizes were determined using Fiji Imaging software.

Statistical analysis was performed using GraphPad Prism 8.0. The detailed statistical methods are described in each figure legend.

The data generated or analyzed in this paper are available from the corresponding authors upon request.

Using a modified hydrothermal treatment method (23), we produced a potent nanoadjuvant by doping SNPs with Mn4+ (Mn4+-SNPs). TEM images of Mn4+-SNPs showed uniform hollow spheres of ∼70 nm in diameter (Fig. 1A), which were decorated with ultrasmall “bubbles” with a diameter of ∼6 nm and a wall thickness of 1–2 nm on the surface (Fig. 1B). The raspberry-like morphology and hollow outer cavities were further visualized by STEM (Fig. 1C). STEM imaging (Fig. 1D) and elemental mapping of silicon (Fig. 1E) and manganese (Fig. 1F) of Mn4+-SNPs indicated uniform distribution of Mn in the framework of SNPs. Energy-dispersive spectroscopy of Mn4+-SNPs showed an atomic percentage of Mn of 8.4% (Supplemental Fig. 1A). Nitrogen analysis was used to evaluate the porosity. Mn4+-SNPs demonstrated a type IV isotherm with a major capillary condensation at high relative pressures, indicative of large pore sizes (Fig. 1G). The Barrett–Joyner–Halenda pore size distribution curve derived from the adsorption branch suggested dual pore sizes of 3.8 and 65.8 nm (Fig. 1H), corresponding to the outer and inner hollow cavity, respectively, which was consistent with the TEM observation. The surface area and total pore volume of Mn4+-SNPs was 419 m2g−1 and 0.62 cm3g−1, respectively.

FIGURE 1.

Mn4+-SNPs display a hollow raspberry-like structure with uniform doping of oxidative Mn4+ in the silica framework. TEM images (A and B), STEM images (C and D), elemental mapping (E and F), nitrogen isotherm curve (G), and Barrett–Joyner–Halenda pore size distribution curve (H) derived from adsorption branch, XPS spectrum survey (I), XPS spectra of Mn 2p3/2 (J), and Si 2p3/2 (K) of Mn4+-SNPs.

FIGURE 1.

Mn4+-SNPs display a hollow raspberry-like structure with uniform doping of oxidative Mn4+ in the silica framework. TEM images (A and B), STEM images (C and D), elemental mapping (E and F), nitrogen isotherm curve (G), and Barrett–Joyner–Halenda pore size distribution curve (H) derived from adsorption branch, XPS spectrum survey (I), XPS spectra of Mn 2p3/2 (J), and Si 2p3/2 (K) of Mn4+-SNPs.

Close modal

XPS was used to evaluate the chemical valence state of Mn and the interaction nature between the doped Mn and SiO2 framework. The XPS survey of Mn4+-SNPs (Fig. 1I) showed an atomic percentage of Mn of 9.6% and Si of 19.2%, similar to the energy-dispersive spectroscoy results. XPS spectra of Mn 2p3/2 exhibited a characteristic strong peak of Mn4+ at 642.3 eV and satellite peaks at higher binding energies of 643.9 and 646.8 eV (Fig. 1J) (28). The XPS spectra of Si 2p3/2 displayed two types of Si peaks: Si-O-Mn at 102.9 and 103.5 eV and Si-O-Si at 103.6 and 104.2 eV (Fig. 1K). The atomic percentage of Si in Si-O-Mn and Si-O-Si was 10.4 and 9.8%, respectively. The percentage of Si in Si-O-Mn was in accordance with the percentage of Mn, indicating a high doping content of Mn (∼50% of Si-O-Mn) in the silica framework. To investigate the significance of Mn4+ doped in SNPs, bare SNPs (Supplemental Fig. 1B–D) with a similar particle size (∼70 nm) and mesopore size (∼2.8 nm) were synthesized for comparison, according to our previous report (22). Both Mn4+-SNPs and SNPs displayed a similar surface charge of ∼−20 mV (Supplemental Fig. 1E).

ROS initiate inflammation and innate immune cell recruitment and can control invading pathogens (29). We therefore designed Mn4+-SNPs with the aim to enhance ROS production and an associated adjuvant effect. To investigate the ROS induction capacity of Mn4+-SNPs, mouse BMMs were incubated with Mn4+-SNPs or SNPs and an intracellular ROS probe 2’,7’-dichlorodihydrofluorescein diacetate. Both Mn4+-SNPs and SNPs induced enhanced green fluorescence in BMMs under confocal microscopy (Fig. 2A), indicating elevated intracellular ROS generation. We quantified the intensity of green fluorescence by flow cytometry (Fig. 2B) and plate reader (Fig. 2C) and found that Mn4+-SNPs induced significantly higher levels of ROS compared with SNPs. The calcination process on SNPs promotes the generation of three membered siloxane rings, which produces hydroxyl radicals inside cells (30). Intracellular antioxidant enzymes (e.g., GSH) tend to consume ROS generated by SNPs. In contrast, doped Mn4+ oxidizes GSH in the cytosol to GSH disulfide (31), thus promoting ROS production when compared with SNPs. To verify the reaction between Mn4+-SNPs and GSH, we incubated Mn4+-SNPs with different concentrations of GSH and detected the dissolved ions by ICP-OES analysis. The released Mn2+ and Si from degraded Si-O-Mn in Mn4+-SNPs was significantly promoted by increased levels of GSH (Fig. 2D). To further confirm this reaction intracellularly, an intracellular GSH probe monochlorobimane was used to detect changes in GSH levels in BMMs after particle treatment. Stimulation with Mn4+-SNPs resulted in significantly higher reduction of intracellular GSH than SNPs (Fig. 2E), which is consistent with increased ROS generation.

FIGURE 2.

Mn4+-SNPs induce ROS generation, inflammasome activation, innate immune cell recruitment, and DC maturation. (AC) BMMs were incubated with or without (control) the addition of particles, followed by incubation with ROS detection probe H2DCFDA. (A) ROS-induced fluorescence was detected by confocal microscopy images, (B) change in median fluorescence intensity (MFI) (n = 4) was analyzed by flow cytometry, and (C) fluorescence intensity change (n = 4) was determined by plate reader. (D) Particles were incubated in pH = 7.2 PBS solutions with different GSH concentrations at 37°C in a shaker for 24 h. Soluble Mn2+ and Si ions released from particles in the supernatants were measured by ICP-OES analysis (n = 3). (E) BMMs were treated with particles, followed by incubation with GSH probe monochlorobimane. Intracellular GSH content change was detected by flow cytometry in BMMs (n = 3). (F) BMMs pretreated with LPS (10 ng/ml) were incubated with particles (15 μg/ml) for 48 h. The IL-1β levels in the supernatants were determined by ELISA (n = 3). (GI) Mice were s.c. immunized with particles for 48 h. Innate immune cells at the skin injection site were analyzed by flow cytometry (G, n = 6) and immunofluorescence staining (with anti–MHC II or anti-Ly6G in red and DAPI in blue, H). (I) Histogram overlays and MFI (n = 3) of CD80 and CD86 on CD11c+MHC II+ DCs derived from inguinal dLNs was analyzed by flow cytometry. Data are presented as means ± SD (B–F) or means ± SEM (G and I). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, analyzed by unpaired two-tailed t test (B, C, and E), one-way (G and I) or two-way ANOVA followed by Sidak multiple comparisons test (D and F). ns, not significant.

FIGURE 2.

Mn4+-SNPs induce ROS generation, inflammasome activation, innate immune cell recruitment, and DC maturation. (AC) BMMs were incubated with or without (control) the addition of particles, followed by incubation with ROS detection probe H2DCFDA. (A) ROS-induced fluorescence was detected by confocal microscopy images, (B) change in median fluorescence intensity (MFI) (n = 4) was analyzed by flow cytometry, and (C) fluorescence intensity change (n = 4) was determined by plate reader. (D) Particles were incubated in pH = 7.2 PBS solutions with different GSH concentrations at 37°C in a shaker for 24 h. Soluble Mn2+ and Si ions released from particles in the supernatants were measured by ICP-OES analysis (n = 3). (E) BMMs were treated with particles, followed by incubation with GSH probe monochlorobimane. Intracellular GSH content change was detected by flow cytometry in BMMs (n = 3). (F) BMMs pretreated with LPS (10 ng/ml) were incubated with particles (15 μg/ml) for 48 h. The IL-1β levels in the supernatants were determined by ELISA (n = 3). (GI) Mice were s.c. immunized with particles for 48 h. Innate immune cells at the skin injection site were analyzed by flow cytometry (G, n = 6) and immunofluorescence staining (with anti–MHC II or anti-Ly6G in red and DAPI in blue, H). (I) Histogram overlays and MFI (n = 3) of CD80 and CD86 on CD11c+MHC II+ DCs derived from inguinal dLNs was analyzed by flow cytometry. Data are presented as means ± SD (B–F) or means ± SEM (G and I). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, analyzed by unpaired two-tailed t test (B, C, and E), one-way (G and I) or two-way ANOVA followed by Sidak multiple comparisons test (D and F). ns, not significant.

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To determine whether elevated ROS was associated with inflammasome activation, we analyzed IL-1β secretion by BMMs. Mn4+-SNPs but not SNPs significantly enhanced IL-1β secretion in LPS-primed BMMs (Fig. 2F). Treatment of BMM with antioxidant (Ebselen) and caspase-1 inhibitor (ZYVAD-fmk) prior to the addition of Mn4+-SNPs significantly decreased the secretion of IL-1β, suggesting that the NLRP3 inflammasome pathway was involved. We used CASP-1−/− mice to test the role of this inflammasome pathway in vivo. C57BL/6J mice s.c. immunized with Mn4+-SNPs but not SNPs led to significantly enhanced infiltration of Ly-6G+ neutrophils, F4/80+ macrophages, and monocytes and a slight increase of CD11b+ DCs to the skin (Fig. 2G, Supplemental Fig. 2A). In contrast, CASP-1−/− mice immunized with Mn4+-SNPs showed a significant decrease in recruitment of these innate immune cells, particularly neutrophils. Further, H&E staining demonstrated that Mn4+-SNPs but not SNPs induced cell infiltration into the injection site (Supplemental Fig. 2B), among which Ly-6G+ neutrophils and MHC II+ APCs were identified (Fig. 2H). Combined, these results demonstrate that Mn4+-SNPs induce ROS and lead to inflammasome activation, which drives immune cell recruitment to the injection site.

To determine the adjuvant effect of Mn4+-SNPs on APCs, we examined the expression levels of costimulatory molecules (CD80/CD86) on DCs. Costimulation is critical to naive T cell activation (32). Treatment of BMDCs with Mn4+-SNPs but not SNPs led to significantly elevated expression of MHC II, CD80, and CD86 (Supplemental Fig. 2C). Administration of Mn4+-SNPs s.c. induced significant upregulation of CD80 and CD86 in CD11c+MHC II+ DCs of draining inguinal LNs (Fig. 2I). These data suggest that the Mn4+-SNP nanomaterial is a potent adjuvant.

In addition to aiding the expression of costimulatory signals, cytosolic delivery of Ags for cross-presentation is critical to elicit cytotoxic CD8+ T cell immune responses (23). One strategy to achieve cytosolic Ag delivery is to deplete protons in the acidic endocytic organelles, which results in endo/lysosome escape and enables transfer of Ags to the cytosol to promote cross-presentation (15). The chemistry of doped Mn4+ determines that Mn4+-SNPs can efficiently consume H+, as shown in the inset of Fig. 3A. The ICP-OES data verified this reaction, in which Mn4+-SNPs showed a significant release of Mn2+ and Si from degraded Si-O-Mn in Mn4+-SNPs under acidic conditions (Fig. 3A). A similar level of degraded Si at pH 7.2 was derived from Si in Si-O-Si in both particles. We investigated the effect of Mn4+-SNPs on cytosolic Ag delivery. We incubated BMMs with Mn4+-SNPs loaded with the FITC-labeled model Ag OVA (FITC-OVA). Mn4+-SNP+FITC-OVA colocalized with endo/lysosomes at 2 h but achieved endo/lysosome escape at 6 h (Fig. 3B). In contrast, free FITC-OVA and FITC-OVA delivered by SNPs mainly remained colocalized with endo/lysosomes. These data demonstrate that Mn4+-SNPs can facilitate Ag endosome escape and therewith target Ag to the cytosol, where the Ag can access the MHC I presentation pathway.

FIGURE 3.

Mn4+-SNPs deplete protons and facilitate Ag delivery to the cytosol of APCs by endo/lysosome escape, resulting in robust Ag-specific CD8+ T cell response, independent of macrophages and neutrophils. (A) SNPs and Mn4+-SNPs were incubated in PBS solutions at different pH values at 37°C in a shaker for 24 h. Soluble Mn2+ or Si ions released from particles in the supernatant were detected by ICP-OES analysis (n = 3). The inset depicts the reaction between H+/GSH and Mn4+-SNPs. (B) BMMs were incubated with FITC-OVA, Mn4+-SNPs+FITC-OVA, or SNPs+FITC-OVA for 2 or 6 h, followed by staining with Hoechst (blue) and LysoTracker (red). Cells were analyzed using confocal microscopy. (CE) C57BL/6J mice were immunized s.c. with the HPV nanovaccine Mn4+-SNPs +GF001 twice 2 wk apart. One week after the last immunization, IFN-γ secretion by E7-specific CD8+ T cells in spleen and inguinal dLNs was measured by ELISPOT (C, n = 6), and E7–MHC I Dextramer positive CD8+ T cells in spleen were analyzed by flow cytometry (D and E, n = 6). (F) C57BL/6J mice were i.p. treated with a macrophage-depleting clodronate liposome (Clodrosome) or control liposome (Encapsome) or treated with a neutrophil-depleting anti-Ly6G Ab or isotype control. Two days later, mice were immunized s.c. with Mn4+-SNPs+GF001 nanovaccine. One week later, E7-specific CD8+ T cell priming was measured by ELISPOT (F, n = 6). Data are presented as means ± SD (A) or means ± SEM (C, E, and F). ****p < 0.0001, analyzed by one-way (E) or two-way ANOVA (A, C, and F) followed by Sidak multiple comparisons test.

FIGURE 3.

Mn4+-SNPs deplete protons and facilitate Ag delivery to the cytosol of APCs by endo/lysosome escape, resulting in robust Ag-specific CD8+ T cell response, independent of macrophages and neutrophils. (A) SNPs and Mn4+-SNPs were incubated in PBS solutions at different pH values at 37°C in a shaker for 24 h. Soluble Mn2+ or Si ions released from particles in the supernatant were detected by ICP-OES analysis (n = 3). The inset depicts the reaction between H+/GSH and Mn4+-SNPs. (B) BMMs were incubated with FITC-OVA, Mn4+-SNPs+FITC-OVA, or SNPs+FITC-OVA for 2 or 6 h, followed by staining with Hoechst (blue) and LysoTracker (red). Cells were analyzed using confocal microscopy. (CE) C57BL/6J mice were immunized s.c. with the HPV nanovaccine Mn4+-SNPs +GF001 twice 2 wk apart. One week after the last immunization, IFN-γ secretion by E7-specific CD8+ T cells in spleen and inguinal dLNs was measured by ELISPOT (C, n = 6), and E7–MHC I Dextramer positive CD8+ T cells in spleen were analyzed by flow cytometry (D and E, n = 6). (F) C57BL/6J mice were i.p. treated with a macrophage-depleting clodronate liposome (Clodrosome) or control liposome (Encapsome) or treated with a neutrophil-depleting anti-Ly6G Ab or isotype control. Two days later, mice were immunized s.c. with Mn4+-SNPs+GF001 nanovaccine. One week later, E7-specific CD8+ T cell priming was measured by ELISPOT (F, n = 6). Data are presented as means ± SD (A) or means ± SEM (C, E, and F). ****p < 0.0001, analyzed by one-way (E) or two-way ANOVA (A, C, and F) followed by Sidak multiple comparisons test.

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To test the utility of Mn4+-SNPs as a therapeutic cancer vaccine, we generated a HPV nanovaccine by combining Mn4+-SNPs with the MHC I–restricted immune-dominant HPV16 E7 peptide GF001 (RAHYNIVTF). GF001 was loaded onto Mn4+-SNPs via electrostatic interaction between positively charged GF001 (isoelectric point = 9.95) and negatively charged Mn4+-SNPs (Supplemental Fig. 1E), with a high loading capacity of 479 ± 108 μg/mg determined by direct detect spectrometer. We immunized mice with Mn4+-SNP+GF001 and examined the E7-specific CD8+ T cell response, which is a key mediator of HPV+ solid tumor rejection (33). Mn4+-SNPs+GF001 induced potent E7-specific IFN-γ secretion in CD8+ T cells in both spleen and inguinal dLNs (Fig. 3C). We further observed a significantly higher proportion of E7–MHC I Dextramer+CD8+ T cells in mice immunized with Mn4+-SNPs+GF001 compared with unimmunized mice (Fig. 3D, 3E). To determine tolerability of Mn4+-SNPs+GF001, livers of treated animals were analyzed by Masson trichrome staining (Supplemental Fig. 3A) and revealed no obvious abnormality, suggesting low systemic vaccine toxicity.

We observed that immunization of Mn4+-SNPs induced significant recruitment of innate immune cells at the injection site, particularly macrophages and neutrophils (Fig. 2G); thus, we next sought to investigate the role of macrophages or neutrophils on CD8+ T cell priming after immunization with Mn4+-SNPs+GF001. Administration of clodronate liposome (Clodrosome) or anti-Ly6G mAb resulted in significant depletion of macrophages or neutrophils, respectively (Supplemental Fig. 4). The extent of E7-specific CD8+ T cell immune response induced by Mn4+-SNPs+GF001 vaccine remained unaffected either by depleting macrophages or neutrophils compared with the control-treated mice (Fig. 3F), suggesting that macrophages or neutrophils are not essential for CD8+ T cell priming with this nanovaccine immunization strategy.

We tested the efficacy of the Mn4+-SNPs+GF001 nanovaccine in the E7-expressing TC-1 solid tumor model (4). Using a preventive immunization regimen (Fig. 4A), mice immunized with Mn4+-SNPs+GF001 or Quil-A (a well-defined saponin-based cellular response adjuvant) +GF001 were completely protected from tumor growth (Fig. 4B) with 100% survival (Fig. 4C) compared with unimmunized or Mn4+-SNPs immunized controls. Using a therapeutic vaccination regimen (Supplemental Fig. 3B), mice immunized with Mn4+-SNPs+GF001 or Quil-A+GF001 showed significant tumor suppression compared with control groups (Supplemental Fig. 3C, 3E). The survival rate of Mn4+-SNPs+GF001 and Quil-A+GF001 immunized mice was 80% by day 60 (Supplemental Fig. 3D). Notably, one mouse immunized with Mn4+-SNPs+GF001 showed an obvious tumor remission from day 12 to 21 (from ∼200 to 80 mm3, Supplemental Fig. 3E); however, it later progressed, suggesting tumor escape.

FIGURE 4.

The HPV nanovaccine Mn4+-SNPs+GF001 prevents tumor growth and eradicates established HPV16 E7-expressing solid tumors. (AC) Preventive immunization regimen (A): C57BL/6 mice were immunized with a 5:1 ratio of Mn4+-SNPs (250 μg) and GF001 (50 μg) s.c. twice 3 wk apart. 1 × 106 TC-1 cells were inoculated 1 wk after the last immunization. Tumor growth was assessed for 50 d. (B) Tumor growth curves showing mean values of tumor sizes of each experimental group (n = 5) with SEM and (C) Kaplan–Meier survival curves (n = 5 or 10). (DG) Therapeutic immunization regimen (D): C57BL/6 mice were inoculated with 2 × 105 TC-1 cells and subsequently immunized s.c. thrice in weekly intervals with different Mn4+-SNPs:GF001 ratios (5:1–250 μg:50 μg; 3:1–150 μg:50 μg; 1:1–50 μg:50 μg) starting at day 7 post–tumor inoculation. Tumor growth was monitored until day 60. (E) Survival curves of C57BL/6 mice (n = 13 and 18) and (F) individual tumor growth curves of therapeutic immunization model. (G) mRNA expression of E7 and MHC I from TC-1 tumors at the end point of therapeutic immunization experiment (n = 5 and 6). Data (D–F) were pooled from two to three independent experiments (combined n = 13–18/experimental group). Data are presented as means ± SEM (B and G). Survival analyses used the log-rank (Mantel–Cox) test (C and E). *p < 0.05, **p < 0.01, ****p < 0.0001, analyzed by one-way (G) or two-way ANOVA (B) followed by Sidak multiple comparisons test. ns, not significant.

FIGURE 4.

The HPV nanovaccine Mn4+-SNPs+GF001 prevents tumor growth and eradicates established HPV16 E7-expressing solid tumors. (AC) Preventive immunization regimen (A): C57BL/6 mice were immunized with a 5:1 ratio of Mn4+-SNPs (250 μg) and GF001 (50 μg) s.c. twice 3 wk apart. 1 × 106 TC-1 cells were inoculated 1 wk after the last immunization. Tumor growth was assessed for 50 d. (B) Tumor growth curves showing mean values of tumor sizes of each experimental group (n = 5) with SEM and (C) Kaplan–Meier survival curves (n = 5 or 10). (DG) Therapeutic immunization regimen (D): C57BL/6 mice were inoculated with 2 × 105 TC-1 cells and subsequently immunized s.c. thrice in weekly intervals with different Mn4+-SNPs:GF001 ratios (5:1–250 μg:50 μg; 3:1–150 μg:50 μg; 1:1–50 μg:50 μg) starting at day 7 post–tumor inoculation. Tumor growth was monitored until day 60. (E) Survival curves of C57BL/6 mice (n = 13 and 18) and (F) individual tumor growth curves of therapeutic immunization model. (G) mRNA expression of E7 and MHC I from TC-1 tumors at the end point of therapeutic immunization experiment (n = 5 and 6). Data (D–F) were pooled from two to three independent experiments (combined n = 13–18/experimental group). Data are presented as means ± SEM (B and G). Survival analyses used the log-rank (Mantel–Cox) test (C and E). *p < 0.05, **p < 0.01, ****p < 0.0001, analyzed by one-way (G) or two-way ANOVA (B) followed by Sidak multiple comparisons test. ns, not significant.

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To further optimize the vaccination regimen and formulation, we included an additional booster injection and compared different ratios of Mn4+-SNPs to GF001 peptide while keeping the Ag amount stable and increased the TC-1 dose 2-fold to test the vaccine more rigorously (Fig. 4D). On day 7 post–tumor inoculation, mice displayed established solid tumors with a size of 50–200 mm3. A 5:1 ratio of Mn4+-SNPs:GF001 (as used in Fig. 3, Supplemental Fig. 3) including the additional booster injection failed to suppress tumor growth (Fig. 4F), resulting in only ∼20% survival (Fig. 4E). In contrast, mice immunized with a 3:1 ratio of Mn4+-SNPs:GF001 showed significantly improved tumor eradication (9 out of 18 mice tumor free) compared with mice receiving a 5:1 or 1:1 ratio (Fig. 4F). This was associated with significantly prolonged survival (3:1 [50%] versus 5:1 [20%] and 1:1 [0%]; Fig. 4E). These results indicate that the optimized 3:1 formulation induced enhanced anti-tumor responses. Based on the loading capacity (479 ± 108 μg/mg), the minimum amount of Mn4+-SNPs to load 50 μg of E7 Ag is ∼100 μg; thus, the 1:1 ratio formulation (50 μg Mn4+-SNPs: 50 μg E7 peptide) is expected to contain free Ag, which may result in the induction of tolerance if picked up by APCs without nanoadjuvant (34), thus leading to decreased anti-tumor immune responses. At a 5:1 or 3:1 ratio, the Ag was expected to be fully loaded to Mn4+-SNPs, and no free Ag should be present in these formulations. With increasing adjuvant dose while keeping the Ag dose stable, the carried Ag molecules in each nanoparticle will relatively decrease with an increase in adjuvant. We hypothesize that the optimal ratio (3:1) is determined by an optimal balance of the immunogenic dose of adjuvant and the ratio between adjuvant and Ag.

After an initial regression of TC-1 tumor growth, we eventually observed outgrowth of TC-1 tumors in multiple animals (Fig. 4F). In cervical cancer patients, tumor escape can be mediated through downregulation of neoantigen and MHC I (35). To understand the mechanisms underlying TC-1 tumor escape, we compared the expression of E7 and MHC I between TC-1 tumors from unimmunized mice and late escaped tumors from immunized mice and found that escaped tumors expressed significantly less E7 and MHC I (Fig. 4G, Table I), likely explaining why these outgrowing TC-1 tumors were no longer responsive to vaccine-induced immune responses.

Table I.
List of primers and the sequences used in RT-qPCR
PrimerPrimer Sequence
E7  
 Forward 5′-TGACTCTACGCTTCGGTTGT-3′ 
 Reverse 5′-AGAACAGATGGGGCACACAAT-3′ 
H-2kb  
 Forward 5′-CGCGACGCTGCTGCGCACAG-3′ 
 Reverse 5′-TACAATCTGGGAGAGACAGA-3′ 
Rpl5  
 Forward 5′-GTACATCGGAAGCACATCATGG-3′ 
 Reverse 5′-CTCCATCATGTCTGGAGTTACG-3′ 
Hprt  
 Forward 5′-AGCGTCGTGATTAGCGATGA-3′ 
 Reverse 5′-CTCGAGCAAGTCTTTCAGTCCT-3′ 
PrimerPrimer Sequence
E7  
 Forward 5′-TGACTCTACGCTTCGGTTGT-3′ 
 Reverse 5′-AGAACAGATGGGGCACACAAT-3′ 
H-2kb  
 Forward 5′-CGCGACGCTGCTGCGCACAG-3′ 
 Reverse 5′-TACAATCTGGGAGAGACAGA-3′ 
Rpl5  
 Forward 5′-GTACATCGGAAGCACATCATGG-3′ 
 Reverse 5′-CTCCATCATGTCTGGAGTTACG-3′ 
Hprt  
 Forward 5′-AGCGTCGTGATTAGCGATGA-3′ 
 Reverse 5′-CTCGAGCAAGTCTTTCAGTCCT-3′ 

We next tested the therapeutic efficacy of Mn4+-SNPs+GF001 in the K14E7 skin grafting model that resembles human HPV-associated epithelial neoplasia more closely (6). Transgenic K14E7 skin displaying hyperproliferative epithelium and associated immune tolerance was grafted to transgenic E7TCR269 mice, which express an E7-specific MHC I–restricted TCR (20). K14E7 skin grafts are not spontaneously rejected, and conventional immunization with E7 protein and adjuvants fails to promote skin graft rejection (36). E7TCR269 mice received K14E7 and C57BL/6J skin grafts, followed by nanovaccine treatment at day 21 and 28 postgrafting (Fig. 5A). K14E7 grafts of mice immunized with Mn4+-SNPs+GF001 showed a significant reduction in size (∼30%) compared with unimmunized mice (∼0%), whereas K14E7 grafts of mice immunized with Mn4+-SNPs alone displayed negligible reduction (∼5%) (Fig. 5B, 5C). We further observed significantly enhanced E7-specific IFN-γ production by CD8+ T cells from spleens of E7TCR269 recipient mice immunized with Mn4+-SNPs+GF001 compared with unimmunized mice or mice immunized with Mn4+-SNPs alone. The magnitude of E7-specific CD8+ T cell response strongly correlated with the degree of K14E7 graft reduction in mice immunized with Mn4+-SNPs+GF001 (Supplemental Fig. 3G).

FIGURE 5.

The HPV nanovaccine Mn4+-SNPs+GF001 promotes reduction of well-healed HPV E7-expressing skin grafts. (A) Experiment timeline: E7TCR269 recipients received skin grafts from K14.E7 and C57BL/6J mice on day 0 and were immunized with a 3:1 ratio of 100 μg Mn4+-SNPs and 33.3 μg GF001 (determined optimal in Supplemental Fig. 3F) s.c. on days 21 and 28. Grafts were monitored up to day 63. (B) Representative photos of skin grafts on day 21 and 63. (C) Graft sizes on day 21 were depicted as baseline and the percentage of skin graft reduction over time was calculated from photographs and imaging software (n = 11 or 12). Data represents means ± SEM of two pooled independent experiments. (D) At end point, IFN-γ secretion by E7-specific CD8+ T cells in splenocytes after restimulation with E7 peptide GF001 was measured using ELISPOT (n = 5 and 6). Each data point represents individual animals with mean of experimental group and SEM indicated. ****p < 0.0001, **p < 0.01, ns (p > 0.05), analyzed by two-way ANOVA followed by Sidak multiple comparisons test (C and D).

FIGURE 5.

The HPV nanovaccine Mn4+-SNPs+GF001 promotes reduction of well-healed HPV E7-expressing skin grafts. (A) Experiment timeline: E7TCR269 recipients received skin grafts from K14.E7 and C57BL/6J mice on day 0 and were immunized with a 3:1 ratio of 100 μg Mn4+-SNPs and 33.3 μg GF001 (determined optimal in Supplemental Fig. 3F) s.c. on days 21 and 28. Grafts were monitored up to day 63. (B) Representative photos of skin grafts on day 21 and 63. (C) Graft sizes on day 21 were depicted as baseline and the percentage of skin graft reduction over time was calculated from photographs and imaging software (n = 11 or 12). Data represents means ± SEM of two pooled independent experiments. (D) At end point, IFN-γ secretion by E7-specific CD8+ T cells in splenocytes after restimulation with E7 peptide GF001 was measured using ELISPOT (n = 5 and 6). Each data point represents individual animals with mean of experimental group and SEM indicated. ****p < 0.0001, **p < 0.01, ns (p > 0.05), analyzed by two-way ANOVA followed by Sidak multiple comparisons test (C and D).

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Together, these results indicate that the novel therapeutic HPV nanovaccine Mn4+-SNPs+GF001 is highly immunogenic and promotes anti-tumor immunity against HPV-associated cancers in preclinical animal studies.

In summary, we have developed a safe and potent nanoadjuvant Mn4+-SNP, which activated the inflammatory signaling pathway via ROS generation and NLRP3 inflammasome activation, resulting in immune cell recruitment to the injection site and DC maturation in dLNs in the absence of systemic adverse effects. Mn4+-SNPs facilitated delivery of Ags to the cytosol for cross-presentation by depleting protons in endo/lysosomes. The therapeutic HPV nanovaccine was formed by combining the Mn4+-SNPs with the MHC I–dominant HPV16 E7 peptide GF001, which induced robust E7-specific CD8+ T cell immune responses against HPV16 E7-expressing solid tumors and skin grafts. This nanotechnology represents a promising strategy for cancer immunotherapy to treat HPV-associated and potentially other types of invasive cancers for which tumor neoantigens are known.

Progress in synthetic design of biodegradable silica materials has increased the feasibility of fabricating sophisticated nanotechnology with controllable architecture. Thus, silica materials are widely used as promising vaccine delivery platforms (14, 3739). However, the self‐adjuvant activity of plain silica materials is of limited efficacy in provoking anti-tumor immunity (40). Similarly, our study shows that SNPs alone were not able to stimulate immune cells. Mn gluconate salt formulated in a microemulsion was previously recognized as immune adjuvant and was deemed pharmaceutically acceptable (WO1996032964A1). However, metals (such as aluminum) delivered by nanoparticles have shown to significantly enhance the adjuvant effect on humoral immune responses compared with metal salts (alum) in vivo (41) because of the specific nanomaterial’s shape and crystalline properties. To enhance adjuvancy, we therefore incorporated Mn4+ into SNPs. The Mn4+-SNP formulation used in this study was rationally designed with the aim to bring together multiple features, resulting in a highly effective adjuvant. The specific nanoparticle properties of a hollow structure (18), a small size of <100 nm (42), and a rough topology (19) have previously been shown to improve Ag delivery to immune cells, adjuvant effect, and consequent immune responses in vivo. We showed that Mn4+-SNPs significantly promoted ROS generation and effectively induced innate immune cell recruitment, DC maturation, and inflammasome activation, suggesting a potent adjuvant function of Mn4+-SNPs. We also demonstrate that Mn4+-SNPs possessed pH-regulating capability, thereby achieving cytosolic MHC I targeting of Ags for enhanced CD8+ T cell immune responses. The experimental therapeutic Mn4+-SNP-based HPV16 E7 vaccine led to eradication of established HPV16 E7-expressing solid TC-1 tumors. To the best of our knowledge, this therapeutic vaccine candidate for the first time demonstrates a significant shrinkage of well-healed E7-expressing transgenic skin grafts, providing supportive preclinical evidence for further clinical studies to treat advanced and recurrent HPV-associated disease in humans.

Prophylactic HPV vaccines (Cervarix and Gardasil) are based on virus-like particles of the major capsid protein L1. These vaccines can prevent infection with high-risk HPV genotypes and therewith associated malignancies but are not able to eliminate established HPV-induced disease because HPV-transformed cells lack the expression of viral capsid genes (43). To target established HPV-associated disease, candidate therapeutic HPV vaccines mostly include epitopes of the viral oncoproteins E6 and E7, which are constitutively expressed by HPV-transformed hyperproliferative cells. The immunogenicity of such well-defined tumor Ags typically is modest, and common adjuvants have failed to increase immunogenicity efficiently (44). To fill unmet clinical needs, the development of novel adjuvants or Ag delivery systems to elicit sufficient and desired immune responses is critical (45). Encouragingly, collaborative studies from engineers and biologists demonstrated that nanotechnology can enhance the efficacy of cancer vaccines by regulating their biodistribution and localization (11). The Mooney group used mesoporous silica microrod modified with polyethyleneimine to deliver CpG and GM-CSF, together with E7 peptide, leading to enhanced Ag-specific CD8+ T cells against established large TC-1 tumors (14). However, additional immune-stimulatory adjuvants were essential for the strong immunogenicity of the engineered silica material–based vaccine.

Our study identified that Mn4+-SNPs had an increased ability to activate innate immune cells compared with undoped SNPs. The doped oxidative Mn4+ enhanced ROS production, leading to inflammation, activation, and elevated proinflammatory cytokine secretion. Inflammation mediated the recruitment of innate immune cells, particularly neutrophils, which play a central role in the inflammatory process (29). ROS-associated inflammation activated DCs in vivo. Other transition metals, such as zinc (46), copper (47), magnesium (48), and iron (49), were also able to induce ROS and immune cell activation, but Mn communicated with innate immune cells differently compared with these metal ions. The chemistry of metal ions determines their unique affinities to biological proteins and consequent cell activation capability. For example, Mn2+ demonstrated a unique capability in stimulating the receptor function of integrin CD11b by increasing the affinity of this receptor to its ligands, comparing with Ca2+ or Mg2+ ions (50), thereby enhancing APC functionality.

Thirty years ago, Smialowicz et al. (51) found that mice treated with MnCl2 showed augmented NK cell activity mediated by enhanced production of IFN-α/β, but the detailed mechanism was not resolved. Very recently, Jiang’s research team reported that cytosolic Mn2+ (supplied as MnCl2) activated the cGAS–STING pathway potently, and induced type I IFN production and antiviral innate immunity (52). Mn2+ ions were released from organelles into the cytosol upon DNA virus infection and contributed to the recognition of dsDNA. Mn deficiency in mice impaired the host defense against DNA viruses. Interestingly, Mn2+ rather than other metal cations (Mg2+, Ca2+, Zn2+, Cu2+, and Fe2+) selectively produced type I IFNs in innate immune cells, supposedly by unique binding affinity of cGAS with Mn2+-ATP. In our study, the doped Mn4+-SNPs with a small diameter of ∼70 nm entered immune cells via the endocytosis pathway and were trapped in the acidic environment. Upon reaction with protons, soluble Mn2+ was released (Fig. 3A) and escaped to the cytosol. The cytosolic Mn2+ derived from Mn4+-SNPs likely facilitates STING activation as observed by Jiang’s laboratory (52), thereby contributing to its self-adjuvant potency.

In addition to activation of APCs, cross-presentation of vaccine-delivered HPV tumor Ags on MHC I of APCs is essential for priming of CD8+ cytotoxic T cells, which are key mediators for anti-tumor immunity (33). Thus, targeting the DC subset conventional type 1 DC (cDC1), which is highly specialized in cross-presentation, is a desired approach to enhance anti-tumor immunity. For example, Zeng et al. conjugated Clec9A mAbs to a nanoemulsion system for targeted delivery of HPV16 E6/E7 Ags to cross-presenting cDC1. This nanovaccine favored induction of Ag-specific CD8+ T cells to effectively target early stage TC-1 tumors (16). cDC1 have a unique ability to maintain endosomes and phagosomes in an alkaline rather than acidic pH, leading to limited Ag degradation in endocytic compartments and subsequent Ag escape to the cytosolic presentation pathway (53). For this reason, we developed a system in which we doped SNPs with Mn4+, which leads to the depletion of protons in the acidic endocytic compartments of APCs, resulting in endosome escape and cytosolic delivery of Ags to enhance cross-presentation in APCs. Additionally, type I IFNs directly stimulate DCs, promoting cross-priming of CD8+ T cells (54).

We also observed Mn4+-SNPs interacting with other innate immune cells, particularly with neutrophils and macrophages, which were recruited to the immunization sites. Similar observations are found at the injection sites with other adjuvants (55). However, the role of neutrophils and macrophages on CD8+ T cell priming is controversial and heavily depends on the biological setting. In a bacterial infection model, specific neutrophil depletion in vivo resulted in decreased CD8+ T cell responses to bacterial Ags (56). In contrast, in vaccine immunization experiments, the CD8+ T cell response was not affected by neutrophil depletion, in which various protein Ags were tested in three commercially available adjuvants (26), which is in line with the observations in our immunization system. CD169+ macrophages were found sufficient to prime CD8+ T cells after depletion of DCs when mice were immunized with adenoviral vectors, which selectively infect CD169+ macrophages (57). In a vaccination system, depletion of macrophages prior to immunization with a minimal peptide and polysaccharide poly-N-acetyl glucosamine adjuvant abolished tumor-specific CTL responses (58), although macrophages were found not essential for CD8+ T cell priming in our immunization system.

Systemic tumor-specific CD8+ T cells were identified as key players in the regression of TC-1 tumors (33). However, despite infiltration of E7-specific effector CD8+ T cells into established TC-1 tumors (59), the tumors still failed to reject. Therefore, the degree of full rejection of established TC-1 tumors not solely correlates with effector CD8+ T cells but also depends on the local immune-suppressive environment, for example downregulation of neoantigen and MHC I expression in the TC-1 tumor. Similarly, a full rejection of K14E7 skin graft requires more than promotion of vaccine-induced E7-specific effector CD8+ T cells. Depletion of mast cells (60) and NKT cells (61) that contribute to a local immune suppression enables full rejection of K14E7 skin grafts. In clinical trials testing therapeutic HPV immunization in patients with HPV+ cervical cancer, patients mounted anti-tumor immunity following immunization and concomitant depletion of myeloid suppressor cells (62). Therefore, a combination therapy of HPV vaccination and therapeutics targeting tumor immunosuppression is desirable for better treatment of patients (3).

Mn is an essential trace element in mammal tissues and regulates enzyme activities of Mn-dependent pyruvate carboxylase, superoxide dismutase, and arginase (63). Excessive Mn exposure was found harmful to the CNS with symptoms resembling Parkinson (64). Jiang’s laboratory reported that intranasal or i.v. administration of MnCl2 (0.44–0.87 Mn μg/g body weight) leads to potent stimulation of innate immunity against viral infection (52). In our study, the effective Mn dose (0.43–1.07 Mn μg/g) was comparable. These doses are in the physiological trace amount range of Mn in mammal tissues (0.3–2.9 μg/g) (63), suggesting that systemic toxicity is not expected to arise after immunization. In fact, no signs of adverse events at the local injection site or systemically were observed after administration of Mn4+-SNPs. No evidence of acute hepatic toxic injury was observed 1 wk after the second immunization (corresponding to 4 wk after the first dose). Additionally, we found that Mn4+-SNPs were degraded within 3 wk post–s.c. immunization; thus, it is unlikely that the nanoadjuvant deposits in the body. To aid clinical translation, the potential benefits of Mn4+-SNPs as therapeutic cancer vaccine acting as highly immunogenic stimulator and vaccine delivery platform will be evaluated systematically in future toxicity studies.

We thank the Translational Research Institute Flow Cytometry Core, Translational Research Institute Biological Research Facility, and Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis at The University of Queensland for service support.

This work was supported by the National Health and Medical Research Council Early Career Fellowship (APP1124265), a University of Queensland Early Career Research Grant and Foundation Research Excellence Award to M.Y., and the Queensland Biomedical Assistance Fund (2017003021, from Queensland Government/Admedus Vaccines [now Jinggang Medicine]) to I.H.F. and M.Y.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BMDC

bone marrow–derived DC

BMM

bone marrow macrophage

cDC1

conventional type 1 DC

Ct

cycle threshold

DC

dendritic cell

DCF-DA

dichlorofluorescin diacetate

dLN

draining LN

FITC-OVA

FITC-conjugated OVA conjugate

GSH

glutathione

HPV

human papillomavirus

ICP-OES

inductively coupled plasma optical emission spectrometry

K14E7

HPV16 E7 oncoprotein driven by the keratin 14 promoter

LN

lymph node

MHC I

MHC class I

MHC II

MHC class II

Mn

manganese

Mn4+-SNP

Mn4+-doped SNP

ROS

reactive oxygen species

SNP

silica nanoparticle

STEM

scanning TEM

TC-1

tissue culture-1

TEM

transmission electron microscopy

XPS

x-ray photoelectron spectroscopy.

1
Walboomers
,
J. M. M.
,
M. V.
Jacobs
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M. M.
Manos
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F. X.
Bosch
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J. A.
Kummer
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K. V.
Shah
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P. J. F.
Snijders
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The authors have no financial conflicts of interest.

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