Ethics statement

The Purdue University Animal Care and Use Committee approved all experiments. All experiments performed on animals were in accordance with current guidelines and regulations. Six- to eight-week-old female C57BL/6 J (RRID: IMSR_JAX:000664) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). They were housed in ventilated cages with ad libitum access to food and water, with three to four animals per box. The room was kept at 20 ± 2 °C and 50 ± 15% relative humidity, with a light/dark cycle of 12 h. An acclimatization period of one week was given to the mice before the onset of the experiments. Mice received 20 μl of vaccine through intradermal injection into the pinna of the ear. The vaccines were administered again after 7 or 21 days. Inhalational isoflurane was used to induce anesthesia in mice. The Purdue University Animal Care and Use Committee approved the euthanasia of all animals using the carbon dioxide (CO2) inhalation method, followed by cervical dislocation. Blood and tissue samples were collected 10 days after the second immunization, unless otherwise specified.

Vaccine formulations

Nano-11 was prepared as previously described28. Briefly, in two sequential chemical processes, phytoglycogen (PG) nanoparticles from sweet corn encoding the sugary-1 mutant gene were conjugated with octenyl succinic anhydride and (3-chloro-2-hydroxypropyl)-trimethylammonium chloride to create PG-OS-CHPTAC (Nano-11). Endotoxin-free OVA was purchased from InvivoGen (San Diego, CA) and ADU-S100 from ChemiTek (MIW815; Indianapolis, IN). Each reagent was resuspended in a sterile solution consisting of 10 mm Tris-saline (pH 7.4). The adsorption efficiency of ADU-S100 to Nano-11 is over 80%, as determined by ultraperformance liquid chromatography/tandem mass spectrometry56. The intradermal vaccines had a final volume of 20 μl and were formulated by first combining 4 mg/ml Nano-11 with 250 μg/ml ADU-S100 to make the combination adjuvant (NanoST) for 1 h at room temperature, followed by the adsorption of 500 μg/ml OVA to Nano-11, ADU-S100, or NanoST for 1 h at room temperature. The final adjuvant and antigen doses per vaccine consisted of Nano-11 (80 μg), ADU-S100 (5 μg), OVA (10 μg), or B16 lysate (50 μg).

A B16 (RRID: CVCL_0158) whole lysate was prepared by freeze-thaw cell lysis. B16 cells were collected, washed five times with sterile phosphate-buffered saline (PBS) buffer, and assessed for their viability. Cell lysis was achieved by transferring 1 ml of cells (9 × 106 cells per ml) to a sterile cryogenic vial and subjecting it to eight freeze-thaw cycles between the gas phase of liquid nitrogen (4 min) and a 37 °C (5 min) water bath. The cells were centrifuged at 300 × g for 5 min, and the supernatant was filtered through a 40-micron cell strainer (Corning, Corning, NY). A micro-BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA) was utilized to determine the protein concentration of the lysate. Aliquots of the lysate were stored at −80 °C. The B16 lysate vaccine was formulated by mixing 2.5 mg/ml of B16 lysate to NanoST for 1 h at room temperature.

The physical characterization of the vaccine formulations was determined by utilizing a zetasizer (Nano ZS90, Malvern, UK) as previously described57. Briefly, the Nano-11 ± ADU-S100 ± OVA or B16 lysate vaccines were prepared as described above. A 0.1% and 0.01% of the final vaccine concentration in 10 mM Tris-saline (pH 7.4) were used to measure the particle size and zeta potential, respectively. Each test group consisted of three independent vaccine formulations. Three readings were taken for each of the vaccine formulations to determine the Z-average hydrodynamic diameter and zeta-potential measurements.

Cell lines

DC2.4 cells (RRID: CVCL_J409)58 were generously donated by Dr. Kenneth Rock (UMass Chan Medical School, MA). Dr. Marulasiddappa Suresh (University of Wisconsin-Madison, WI) kindly supplied the B3Z CD8+ T cell hybridoma (RRID: CVCL_6277)59,60. DC2.4 and B3Z cells were cultured in complete RPMI (RPMI 1640 supplemented with 2 mm L-glutamine, 55 μm 2-mercaptoethanol, 1× non-essential amino acids, 10 mm HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin) with 10% FBS. The B3Z cells were kept under selection by supplementing the culture media with 500 μg/ml geneticin (G418 sulfate, Thermo Fisher Scientific, MA). The OVA-expressing B16-OVA (RRID: CVCL_WM78; generously donated by Dr. Matthew Olson, Purdue University) and EG.7-OVA tumor cells (RRID: CVCL_3505) were maintained in a selection environment with 0.4 mg/ml of G418 in RPMI 1640 supplemented with 2 mm L-glutamine, 55 μm 2-mercaptoethanol, 10 mm HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin, 4.5 g/L glucose, 1 mm sodium pyruvate, and 10% FBS.

Human THP-1 cells (TIB-202; American Type Culture Collection, Manassas, VA) were cultured in complete RPMI with 10% FBS. Cells were seeded at a density of 5 × 105 cells per well in a 96-well plate, stimulated with Nano-11 (80 μg/ml) and ADU-S100 (5 μg/ml) for 24 and 48 h at 37 °C with 5% CO2, and then processed for flow cytometry analysis. Similarly, the NF-ĸB-SEAP and IRF-Luc reporter THP1-Dual cells (RRID: CVCL_X599; InvivoGen, San Diego, CA) were grown at a density of 1 × 105 cells per well in a 96-well plate in complete RPMI supplemented with 10% FBS, 100 μg/ml zeocin, 100 μg/ml normocin, and 10 μg/ml blasticidin. THP1-Dual cells were subsequently stimulated with Nano-11 (80 μg/ml) and ADU-S100 (5 μg/ml) for 24 and 48 h at 37 °C with 5% CO2, and the supernatants were collected to quantify activation of the NF-ĸB and IRF pathways.

Antigen cross-presentation in vitro

The B3Z CD8+ T cell hybridoma cell line that expresses β-galactosidase under the control of the IL-2 promoter was utilized to detect antigen cross-presentation of the OVA 257–264 (SIINFEKL, InvivoGen, San Diego, CA) peptide59. Briefly, DC2.4 cells at 1 × 105 cells per well on 96-well round bottom plates were cultured in complete RPMI containing 10% FBS. The DC2.4 cells were stimulated with 10, 100, or 1000 μg/ml OVA adsorbed to 80 μg/ml Nano-11 or the combination adjuvant comprised of 80 μg/ml Nano-11 and 5 μg/ml ADU-S100 (NanoST) for 3 h at 37 °C with 5% CO2. Cells in media only or treated with 0.5 μg/ml OVA 257–264 (SIINFEKL) served as negative and positive controls, respectively. The cells were centrifuged at 300 × g for 5 min and then washed three times with 200 μl of sterile PBS/0.1% BSA. Following fixation with 200 μl of PBS/0.2% paraformaldehyde for 15 min at 4 °C, cells were washed twice with complete RPMI. Each well was seeded with 1 × 105 B3Z cells in complete RPMI containing 10% FBS for 16 h at 37 °C with 5% CO2. Following centrifugation at 300 × g for 5 min, cells were resuspended in 200 μl of lysis buffer containing 9 mm MgCl2, 0.125% NP40, and 0.15 mm chlorophenol red-β-D-galactopyranoside (CPRG, Sigma-Aldrich, St. Louis, MO). After 2, 4, 6, 8, 12, and 24 h, the conversion of CPRG by β -galactosidase was quantified by measuring the absorbance at 590 nm (OD 590) using a microplate reader (BioTek Instruments, Winooski, VT).

Antigen-specific cytotoxic T cell assay in vivo

Splenocytes were isolated from naïve C57BL/6 J mice and treated with red blood cell ACK lysis buffer to create a single-cell suspension. A total of 2 × 107 cells/ml were pulsed with 1 μm of SIINFEKL (pulsed) or left untreated (control) in HBSS and then incubated for 1 h at 37 °C with 5% CO2. The cells were centrifuged at 300 × g for 5 min, resuspended in complete RPMI 10% FBS, centrifuged once more, and resuspended in PBS/0.1% BSA. SIINFEKL-pulsed cells at a concentration of 5 × 106 cells/ml were stained with the fluorescent dye CFSE at 2.5 μm (CFSEhi), whereas the control cells were stained with 0.25 μm (CFSElow), immediately vortexed, and incubated for 10 min at 37 °C with 5% CO2. A 5-min centrifugation at 300 × g was followed by a resuspension in complete RPMI 10% FBS, additional centrifugation, and a final resuspension in PBS. Untreated cells and cells pulsed with SIINFEKL were combined at a 1:1 ratio to provide a final cell concentration of 2 × 108 cells/ml. One hundred μl (1 × 107 of each cell population) was injected into the tail vein of mice that had undergone two intradermal vaccinations with PBS, OVA, Nano-11 + OVA, or NanoST + OVA, 21 days apart. The vaccinated mice were euthanized 4 h later, and the spleen and draining lymph node (superficial parotid) were isolated and processed into single-cell suspensions. Target lysis of CFSE-labeled cells was quantified by flow cytometry. The total events corresponding to both fluorescent intensities (CFSEhi and CFSElow) were determined by flow cytometry. The percentage of lysis for each mouse was calculated as follows: % Lysis = 100-[(Total CFSEhi cells/ Total CFSElow cells) vaccinated mice × 100 × (Total CFSElow cells/ Total CFSEhi cells) PBS].

Preventive cancer vaccination

The efficacy of preventive cancer vaccines was investigated by two intradermal injections with OVA, Nano-11 + OVA, NanoST + OVA, or PBS with a 21-day interval. Eleven days following the second injection, mice were inoculated subcutaneously in the flank with 1.0 × 106 B16-OVA, B16, or 2.5 × 106 E.G7-OVA cells suspended in 100 μl of PBS. The tumor size was measured every other day, and the body weight was assessed to determine the body condition score using Ullman-Cullere’s scoring method61. Using a caliper, the length (maximum longitudinal dimension) and width (maximum transverse dimension) of the subcutaneous tumor were measured in order to determine the approximate volume. Tumor volume was calculated using the ellipsoidal formula: V = ½ × (Length × Width2)57. Each tumor was measured by three observers to account for interobserver variability. The tumor endpoint measurements were taken when the tumors reached a volume of 2,000 mm3, when the tumors became ulcerated, when the body condition scores dropped below 2, or when the mice became moribund. Mice without palpable tumors at 32 days after tumor inoculation were considered tumor-free and euthanized.

Therapeutic cancer vaccination

To mimic therapeutic immunotherapy, mice were injected subcutaneously with 1.75 × 105 B16-OVA or 2.5 × 106 E.G7-OVA cells on day 0 to establish the tumor model. The B16-OVA-bearing mice were immunized with PBS, OVA, ADU-S100 + OVA, Nano-11 + OVA, or NanoST + OVA 5 days after tumor inoculation, followed by a second injection on day 12. A similar vaccination regimen was administered to E.G7-OVA-bearing mice at 1 and 21 days after tumor inoculation. The tumor endpoint measurements were recorded as described under the prophylactic cancer vaccine strategy. Mice without visible tumors were deemed tumor-free and euthanized 32 or 33 days post-tumor inoculation.

Immune checkpoint blockade with therapeutic cancer vaccination

Mice were injected subcutaneously with 3.5 × 105 B16-OVA or B16 cells on day 0 to establish the tumor model. The B16-OVA-bearing mice were intradermally immunized with NanoST ± OVA or B16 cell lysate on day 5, followed by a second vaccination on day 12. A second group of NanoST + B16 cell lysate or NanoST + OVA immunized mice were treated with anti-PD-1 (clone RMP1-14, Leinco) checkpoint inhibitors. Mice were treated with 200 μg of anti-PD-1 mAbs intraperitoneally 1, 3, and 6 days after the first vaccination. The tumor endpoint measurements were recorded as described under the prophylactic cancer vaccine strategy. Mice without visible tumors were deemed tumor-free and euthanized 32 days post-tumor inoculation.

Depletion of CD4+ and CD8+ T cells

To evaluate the contribution of CD4+ and CD8+ T cell responses to prophylactic-mediated vaccine protection against B16-OVA and E.G7-OVA tumor formation, C57BL/6 J mice were vaccinated with Nano-11 + OVA or NanoST + OVA, and 21 days later the vaccination was repeated. Mice received 300 μg of anti-mouse CD4-depleting mAbs (clone GK1.5, Leinco, St. Louis, MO) or anti-mouse CD8α-depleting mAbs (clone 2.43, Leinco) 9 days after the second immunization through intraperitoneal injection, and continued to receive 100 μg of αCD4 and αCD8α depletion treatment for 3 weeks on every fourth day. Two days following the initial depletion treatment, the flanks of mice were injected subcutaneously with 1.0 × 106 B16-OVA or 2.5 × 106 E.G7-OVA cells. The tumor growth and survival of mice were monitored for 32 days post tumor inoculation.

ELISA

OVA-specific IgG, IgG1, and IgG2c titers were measured by enzyme-linked immunosorbent assays (ELISA) on serum samples taken 10 days following the second vaccination, as previously reported26. In short, 96-well plates were coated with 1 μg/ml OVA overnight at 4 °C. After three washes with phosphate-buffered saline containing 0.05% TWEEN (PBST; Sigma-Aldrich), the plates were blocked at room temperature (RT) for 2 h with 200 μl PBST comprised of 1% BSA, followed by adding 100 μl of serially diluted serum samples to the wells in duplicate for 1 h at 37 °C. Wells were washed and incubated with 100 μl of peroxidase-conjugated goat anti-mouse IgG (1030-05), IgG1 (1073-05), or IgG2c (1079-05; SouthernBiotech, Birmingham, AL) for 1 h at 37 °C. Following a final wash, 100 μl of 3,3’,5,5’ tetramethylbenzidine substrate solution (Neogen, Lansing, MI) was added to all the wells and allowed to react in the dark for 10 min at RT. The reaction was stopped with the addition of 50 μl 2 m sulfuric acid, and absorbance at 450 nm (OD 450) was determined using the Synergy HT microplate reader (BioTek, Winooski, VT). End-point titers for OVA-specific antibodies were determined when the dilution at which OD 450 nm approached 0.2. The final OD values were obtained by subtracting the average value of three blank samples from the OD of the experimental test samples.

ELISpot

The ELISpot assay was performed to quantify the number of OVA-specific antibody-secreting cells (ASCs) in bone marrow cells extracted from the tibias and femurs of mice 10 days after the second vaccination, as previously described26. Briefly, MultiScreen IP filter plates (MAIPS4510; Sigma-Aldrich) were prepared by activating the filter membranes with 20 μl/well of 35% ethanol for 30 sec and then washing the plates with 300 μl/well of sterile PBS three times. The wells were coated overnight at 4 ˚C with 10 μg/ml of OVA (100 μl/well) in sterile PBS. Wells were washed with sterile PBS and then blocked with complete RPMI (RPMI 1640 supplemented with 2 mm L-glutamine, 55 μm beta-mercaptoethanol, 1 × non-essential amino acids, 10 mm HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin) with 10% FBS for 2 h at 37 °C, followed by incubating the serially diluted single-cell suspensions of bone marrow cells in duplicate at 37 °C with 5% CO2 for 24 h. The plates were then washed five times with PBST and incubated at RT for 2 h with biotin-labeled anti-mouse IgG (SouthernBiotech) in 1% FBS-containing PBST. This was followed by five washes, and then the plates were incubated with avidin-HRP conjugate (Thermo Fisher Scientific) in 1% FBS-containing PBST for 1 h at 37 °C. To initiate enzymatic activity, wells were exposed to 3-amino-9-ethylcarbazole (Sigma-Aldrich) treatment and protected from light for 10 min, followed by 15 washes with deionized water. The membrane tray was protected from light and dried at RT before the red-colored spots were quantified utilizing an ELISPOT reader (AID Diagnostika, Strassberg, Germany).

Flow cytometry

Single-cell suspensions were prepared from isolated splenocytes or lymph nodes and washed with Cell Staining Buffer (CSB; BioLegend, San Diego, CA). The staining protocols were conducted in accordance with the manufacturer’s instructions, and unless otherwise specified, all mAbs were obtained from BioLegend. Live, viable cells were treated for 30 min at 4 °C with an anti-mouse CD16/32 (clone 93) antibody. A total of 2 × 106 splenocytes were labeled with anti-mouse mAbs against CD3ε (clone 145-2C11), CD4 (clone GK1.5), CD8α (clone 53-6.7), CD185/CXCR5 (clone L138D7), CD279/PD-1 (clone 29 F.1A12), GL-7 (clone GL7), CD45R/B220 (clone RA3-6B2), and CD95/FAS (clone SA367H8) in CSB for 45 min at 4 °C. Identification of SIINFEKL-specific CD8+ T cells was achieved by utilizing a H-2K(b) chicken OVA 257–264 SIINFEKL tetramer (RRID: AB_3068342) donated from the NIH Tetramer Core Facility at Emory University (Atlanta, GA). A total of 1 × 106 splenocytes were utilized for tetramer staining. In order to perform intracellular cytokine staining (ICS), splenocytes were first cultured in complete RPMI supplemented with 10% FBS and 25 μg/ml OVA for 24 h at 37 °C with 5% CO2. Thereafter, the cells were stimulated at 37 °C with 5% CO2 for 6 h with PMA, ionomycin, and monensin in complete RPMI supplemented with 10% FBS. Subsequently, the splenocytes were labeled with mAbs following the same method as described above and then permeabilized using Perm Wash Buffer (BioLegend) in preparation for ICS with IFN-γ (clone XMG1.2) and IL-17A (clone TC11-18H10.1). The cells were washed with CSB prior to getting fixed using a 4% paraformaldehyde fixation buffer (BioLegend). Human THP-1 cells were labeled with anti-human CD11b (clone ICRF44), anti-human CD80 (clone 2D10), and anti-human CD86 (clone BU63) mAbs after 30 min of treatment with human TruStain FcX (BioLegend) at 4 °C. Flow cytometry was conducted using an Attune NxT flow cytometer (Invitrogen, Waltham, MA), and the data was analyzed using FlowJo software (FlowJo, Ashland, OR).

Statistical analysis

To identify statistically significant differences between experimental groups, a one- or two-way analysis of variance (ANOVA) test was conducted, followed by Tukey’s or Sidak’s multiple comparison test. The percentage of tumor-free mice and survival rate were determined using the log-rank (Mantel-Cox) curve comparison test. All statistics were performed using GraphPad Prism (version 9.2, San Diego, CA). The results are presented as mean ± SEM. A value of p < 0.05 indicated statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).