In vitro evaluation of CD40-targeting aptamer-based DNA vaccine adjuvants using HD11 cell culture

Introduction

Salmonella, a gram-negative, rod-shaped, facultatively anaerobic bacterium in the Enterobacteriaceae family, is a zoonotic pathogen responsible for human gastrointestinal disease. Contaminated poultry products, particularly meat and eggs, are the primary source of Salmonella infection in humans (1). Nontyphoidal S. enterica serovars, such as Typhimurium and Enteritidis, can infect multiple host species, complicating their transmission dynamics. Although these serotypes rarely cause noticeable symptoms in poultry, infected birds can silently carry and shed the bacteria, contaminating their meat and eggs. These asymptomatic carriers are particularly problematic because they are difficult to detect, yet they play a significant role in spreading the pathogen within flocks and into human food chains. The persistent intestinal colonization by S. enterica in poultry is a key factor driving its transmission (2). Vaccinating chickens, along with other control strategies, is a key approach to reducing Salmonella prevalence in poultry flocks, thereby lowering the risk of human infections. Despite extensive research, there is still a need for Salmonella vaccines that are both safer and more effective (2–6). Effective vaccines should control Salmonella colonization in the intestines, prevent organ invasion, and decrease fecal shedding, thereby limiting the spread of infection within flocks and contamination of their products and environment (2, 3).

One promising approach to enhance vaccine efficacy is to strategically engage and activate immune cells, particularly professional antigen-presenting cells (APCs), by directly targeting the antigen to CD40 on APCs (7–9). Initially identified for its crucial role in regulating humoral immune responses, CD40 is now widely recognized as a central regulator of both cellular and humoral immunity (10). It is a costimulatory, integral glycoprotein that is mostly on APC such as dendritic cells and macrophages, and expressed throughout all stages of B cell development and differentiation, playing a crucial role in immune function (11–14). CD40L, the primary ligand for CD40 (also known as CD154, gp39, T-BAM, or TRAP), is a type II transmembrane protein primarily expressed on activated CD4+ T cells and platelets, underscoring its role in immune regulation and intercellular communication. Beyond the immune system, CD40 is also expressed in endothelial cells, fibroblasts, hematopoietic progenitors, platelets, and basal epithelial cells, demonstrating the broad physiological impact of CD40 signaling (10, 15–17). Given its central role in immune activation, CD40 has emerged as an attractive target for vaccine development, as it facilitates rapid engagement and activation of APCs, leading to a more robust and sustained immune response (11, 18–20). Targeting CD40 in vaccine design holds promise for improving T cell and B cell responses, stimulating dendritic cells for enhanced antigen presentation, increasing the pro-inflammatory activity of macrophages, promoting B cell maturation, inducing isotype switching to more effective antibodies (IgG and IgA), and promoting cytokine release to amplify the immune response.

Previous studies using a monoclonal antibody (2C5) targeting chicken CD40 have demonstrated enhanced immune activation in chickens against various antigens (8, 14, 18, 20), but their high production costs and complexity limit their widespread application, particularly in veterinary and agricultural settings. On the other hand, DNA aptamers, with their high specificity, stability, and costeffective production, offer an attractive alternative for targeting CD40 in vaccine development. In addition, rolling Circle Amplification (RCA), an isothermal enzymatic process, has emerged as a powerful tool for producing long single-stranded DNA molecules with tandemly repeated sequences (21, 22). RCA has been effectively used to construct poly-aptamer-drug systems for targeted drug delivery and probe labeling, demonstrating its simplicity, versatility, efficiency, and applicability across various biomedical applications (23–26). Given its potential to form aptamer-based complexes with multiple target-binding units, RCA is a promising method for enhancing the efficacy of vaccine platforms.

Our previous study investigated a novel vaccine adjuvant strategy leveraging RCA products displaying anti-CD40 DNA aptamers. We conjugated the M2e epitope peptide from the influenza virus as a model hapten to the most promising Aptamer RCA (Aptamer RCA II), and evaluated the conjugate in a chicken challenge trial. The results showed that Aptamer RCA II significantly enhanced antiM2e IgG antibody production as compared to the M2e epitope alone (11). Building on these findings, our current study focused on developing a CD40-targeting vaccine complex in which anti-Salmonella DNA aptamers were employed to present formalin-killed Salmonella as an antigen. In this study, we performed an initial evaluation of two vaccine complex designs in the HD11 macrophage cell line to identify the final design suitable for evaluation in a chicken vaccination trial.

Materials and methods

Selection of Salmonella aptamers

For antigen presentation in this vaccine design, six candidate aptamers with a reported affinity for Salmonella were selected from the literature: 33 (27), S8-7 (28), C4 (29), St2p (30), St1 (31), and Sal26 (32). The sequences of these aptamers are shown in Table 1. Each aptamer was synthesized with a fluorescein amidite (FAM) fluorescence label at the 5′ end (Integrated DNA Technologies, IDT, Coralville, Iowa, USA). To quantitatively assess the affinity and binding specificity of the aptamers, 50 μL of each 100 μM FAM-labeled aptamer was mixed with 450 μL of overnight cultures (109 CFU/mL) of three Salmonella strains commonly associated with poultry: SE, Salmonella Typhimurium (ST), and Salmonella Kentucky (SK), resulting in a final aptamer concentration of 10 μM in each reaction. The Salmonella strains used were obtained from John Kirkpatrick Skeeles Poultry Health Laboratory (PHL) at the University of Arkansas System Division of Agriculture. The mixtures were incubated at room temperature for 1 h. with gentle shaking at 120 rpm using a Labnet Rocker 25. After 1 h of incubation, the bacterial culture was centrifuged (5,000 g) for 2 min, the supernatant was discarded, and the pellet was resuspended in sterile phosphate-buffered saline (PBS). The centrifugation and resuspension process was repeated 3 times to wash away any unbound aptamers thoroughly. Following the final wash, the aptamer-Salmonella complexes were resuspended in 1,000 μL of PBS. A 100 μL aliquot of each aptamer-Salmonella suspension was transferred to a black 96-well plate in triplicate, and fluorescence emission using an OD of 450/500 was measured in a dark room using a plate reader (Biotek synergy, Winooski, Vermont, USA). This assay was repeated 4 times, and the resulting data were analyzed to identify the top 3 aptamers with the highest binding affinity across all 3 serovars.

In vitro evaluation of CD40-targeting aptamer-based DNA vaccine adjuvants using HD11 cell culture - Image 1

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RCA template and primer design

For the selected Salmonella aptamers (33, S8-7, and C4), which exhibited the highest affinity across multiple Salmonella serovars (see Results and discussion section), the DNA sequences of the aptamers were converted to reverse-complement and linked with spacer sequences to create an oligonucleotide template for AntiSalmonella RCA. Additional sequences were incorporated at the 5′ and 3′ ends to serve as primer-binding regions for the circularization of the template and the RCA reaction. Both Aptamer RCA II and Anti-Salmonella RCA templates were synthesized with a phosphate group at the 5′ end to facilitate ligation, thereby producing circular templates for the RCA reaction. The DNA sequences of the RCA templates are shown in Table 1. For the primers for both RCA reactions, we designed primers with and without GC-rich sequences (50 nt) added to the 5′ end of each primer. This GC clamp was used to link the Aptamer RCA II and Anti-Salmonella RCA products through stable hybridization, as demonstrated in Figure 1B. All primers and templates, synthesized by Integrated DNA Technologies (IDT, Coralville, Iowa, USA), are detailed in Table 1.

Two spacer complement sequences were designed to develop two distinct vaccine constructs. The first construct used a linear spacer complement that matched the spacer regions of both Aptamer RCA II and Anti-Salmonella RCA, enabling efficient hybridization with the spacer regions of each RCA product (Figure 1B). In the second construct, three distinct spacer complements were created, each carrying one of the three selected Salmonella aptamers at the 5′ end. These modified spacers were combined in equal proportions and added to the Aptamer RCA II (Figure 1C). Unlike the first construct, the primer design for the second construct did not include a GC clamp, as the Anti-Salmonella RCA was directly integrated into the spacer complement. This approach was intended to facilitate more targeted interactions with Salmonella antigens, potentially enhancing the specificity, stability, and efficacy of the resulting vaccine complex.

Production of rolling circle amplification (RCA) products

To produce the RCA product, the RCA primer was annealed to the template by mixing 2.5 μL of 10 μM primer and template each with 45 μL of TE buffer (pH 8.0), resulting in a final concentration of 0.5 μM in a 50 μL reaction solution (we compared the two primer concentrations, 0.5 vs. 5 μM, for RCA products, which was then tested in HD11 assay. Since there was no significant difference, we used 0.5 μM consistently throughout the study. See Supplementary Table S1.) This mixture was heated to 95 °C for 10 min, then gradually cooled to 56 °C for 5 min to promote primer-template binding. Ligation was performed with Hi-T4 DNA ligase (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions, yielding a circular template. The RCA reaction was performed in a final volume of 60 μL containing the ligated template–primer complex, 2 μL of phi29 DNA polymerase (10 U/μL; New England Biolabs, Ipswich, MA, USA), 8 μL of 10× phi29 DNA polymerase buffer (New England Biolabs), 0.8 μL of 100 × bovine serum albumin (BSA; New England Biolabs), and 10 μL of 10 mM dNTPs (New England Biolabs). The mixture was incubated at 30 °C for 16 h., followed by heat inactivation at 65 °C for 10 min. phi29 DNA polymerase was used due to its high processivity and strand displacement activity, which are crucial for efficient RCA amplification (23, 33–35). RCA products were quantified using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Hybridization was performed by adding 5 μL of a 10 μM linear spacer complement to 50 μg of RCA product, followed by heating at 95 °C for 10 min and gradual cooling to facilitate stable complex formation. Verification of RCA amplification was performed using 1% agarose gel electrophoresis.

In vitro evaluation of CD40-targeting aptamer-based DNA vaccine adjuvants using HD11 cell culture - Image 3

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Assembly of the vaccine complex

The first vaccine complex was assembled by combining 50 μg of Aptamer RCA II with 50 μg of Anti-Salmonella RCA and 10 μL of the spacer complement. The mixture was heated at 95 °C for 10 min and gradually cooled to room temperature to promote hybridization of the complementary GC clamps, which had been incorporated during primer design to ensure proper construct assembly.

In the second vaccine design, three spacer complements containing each of the three Salmonella aptamers were mixed in equal proportions, and 5 μL of this combined spacer mixture was added to 50 μg of Aptamer RCA II. The reaction was similarly heat-treated at 95 °C for 10 min and gradually cooled to facilitate binding. The viscosity of the RCA products was reduced by heat treatment at 95 °C for 10 min after spacer addition, as shown in Figure 1A. In the final step, formalin-inactivated SE cells were attached to the Salmonella RCA component via aptamer binding to generate the complete vaccine complex. The resulting RCA vaccine complexes were designated RCA_v3 (GC clamp) and RCA_v5 (modified space complements with attached antiSalmonella aptamers), respectively, as illustrated in Figure 1.

Quantification of Salmonella binding to RCA vaccine construct

To quantify the number of SE cells that bind to anti-Salmonella RCA products in both vaccine constructs, we used DNase I digestion to release the captured target cells as previously described (36). An overnight culture of SE was prepared, washed three times, and resuspended in 1,000 μL of sterile PBS. Four experimental groups were prepared and labeled: RCA_v3 without DNase I, RCA_v3 with DNase I, RCA_v5 without DNase I, and RCA_v5 with DNase I. All RCA constructs were synthesized using primers modified with biotin at the 5′ end to enable binding to streptavidin-coated magnetic beads. Each tube containing the designated vaccine construct received 100 μL of the SE suspension and was incubated at room temperature for 1 h. with gentle shaking at 120 rpm using a Labnet Rocker 25. In parallel, a 10-fold dilution of the SE culture was plated to determine the starting bacterial concentration. After the initial incubation, 200 μL of preequilibrated streptavidin-coated magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) were added to each tube, followed by a 1 h incubation at room temperature with gentle shaking. Tubes were then washed five times with sterile PBS using a magnetic rack to remove unbound bacteria. DNase I treatment was performed by resuspending the washed pellet in 80 μL sterile water, 10 μL 10 × DNase I buffer, and 10 μL DNase I (New England Biolabs) to facilitate DNA digestion and release of bound Salmonella. Control tubes without DNase I received 90 μL of sterile water and 10 μL of 10 × buffer. All tubes were incubated at 37 °C for 25 min. Following incubation, the supernatant was removed using a magnetic rack, and 10-fold serial dilutions were prepared; 50 μL from each dilution was plated on LB agar. Plates were incubated at 37 °C for 24 h, and colony counts were used to estimate the number of bacteria bound to each RCA construct.

HD11 assay to quantify macrophage activation by the RCA vaccine complexes

The HD11 chicken macrophage cell line was obtained from Dr. Luc R. Berghman’s lab at Texas A&M University. The cells were cultured using a T-75 tissue culture flask (VWR, Radnor, PA, USA) in Dulbecco’s Modification of Eagle’s Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 8% fetal bovine serum (FBS, Thermo Fisher Scientific), 5% chicken serum (Thermo Fisher Scientific), 1% GlutaMAX (Thermo Fisher Scientific), and 1% penicillin–streptomycin (Thermo Fisher Scientific).

HD11 chicken macrophage cells (passage 7) were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. Cells were seeded at a density of 1 × 106 cells per well in a 24-well tissue culture plate and allowed to adhere for 8 h. After adherence, the culture medium was replaced with 1 mL of the appropriate treatment, diluted in growth medium. Negative controls included growth medium alone and pre-immune mouse serum (1:300; Jackson ImmunoResearch, West Grove, PA, USA), while lipopolysaccharide (LPS; 5 μg/mL, Millipore Sigma, St. Louis, MO, USA) served as a positive control for macrophage activation. Additional treatment groups included Aptamer RCA II, RCA_v3, SE alone, RCA_v3 vaccine complex, and RCA_v5 vaccine complex. Each treatment was performed in 4 independent biological replicates (4 separate independent experiments per treatment). For each biological replicate, nitric oxide levels were quantified using 2 technical replicates. The plates were incubated for 18 h, centrifuged at 500 × g for 10 min, and the supernatant was collected for nitric oxide (NO) analysis. Nitric oxide levels in the supernatant were determined using the Griess assay as described by Green et al. (37) and Crippen et al. (38). A sodium nitrite standard curve (0–80 μM) was prepared by serial dilution of a 2 mM stock solution in unsupplemented growth medium. Standards and supernatant samples (100 μL each) were added in triplicate to a 96-well flat-bottom plate, followed by 50 μL of Reagent 1 (1% sulfanilamide in 2.5% phosphoric acid). Plates were incubated for 10 min at room temperature in the dark, after which 50 μL of Reagent 2 (0.1% naphthylenediamine in 2.5% phosphoric acid) was added and incubated for another 10 min. Absorbance was measured at 540 nm using a microplate reader (Biotek Synergy, Winooski, VT, USA), and NO concentrations were calculated from the standard curve.

Statistical analysis

All data were analyzed using one-way ANOVA in JMP Pro 18. Means were further separated using Tukey–Kramer HSD, and significance was set at p ≤ 0.05.

Results and discussion

Rationale for the design of the RCA vaccine complexes

In our previous study, we employed the streptavidin-biotin interaction to conjugate Aptamer RCA II with the M2e peptide, aiming to enhance immune responses against the viral pathogen in chickens. Avidin is a tetrameric glycoprotein originally isolated from egg white, and it exhibits exceptionally high affinity and specificity for biotin. This property underlies the avidin-biotin system, which has been widely adopted in molecular biology and vaccine development for conjugating functional components (39–41).

Although effective for molecular assembly, the avidin-biotin system does not stimulate the immune system and has raised regulatory concerns. Notably, the U.S. Food and Drug Administration (FDA) has cautioned against its extensive use, as excess biotin can interfere with clinical immunoassays, leading to inaccurate diagnostic results (39).

To address these safety and regulatory concerns, we devised two alternative conjugation strategies for Salmonella vaccine complexes. For RCA_v3 (Figure 1B), two complementary high-GC regions were incorporated directly at the 5′ end of the RCA primer to produce two biotinylated RCA products, which can be joined by hybridization, forming a GC clamp (42–44). For RCA_v5 (Figure 1C), we engineered a complementary spacer sequence within the Salmonella Aptamer RCA, allowing direct hybridization with Aptamer RCA II via a spacer region. These modifications eliminate the streptavidin-biotin component, mitigating its potential adverse effects and simplifying largescale poultry vaccination production.

Characterization of the binding efficiency of selected Salmonella-specific aptamers

To incorporate whole killed Salmonella cells into the vaccine complex, it was essential to capture and anchor them to the CD40-targeting component. In this study, we evaluated the potential of anti-Salmonella DNA aptamers for this purpose. Based on a literature review, six previously reported DNA aptamers that bind various Salmonella serotypes were selected (Table 1). These aptamers were tested for binding affinity against three relevant serovars—SE, ST, and SK—to identify those with the strongest binding capacity against these 3 serovars.

Various methods have been employed to assess aptamer-cell binding interactions, including flow cytometry (45–47), SELEX-based assay (28, 48, 49), and confocal imaging (46). Additionally, Joshi et al. (27) employed Electrophoretic Mobility Shift Analysis (EMSA), mass spectrometry, and DNase footprinting to study aptamer binding to ST outer membrane protein (OMP). In our study, we employed a fluorescence spectrophotometry-based assay, chosen for its ease of use, rapid turnaround, high sensitivity and selectivity, and cost-effectiveness (50, 51).

The results, presented in Supplementary Figure S1, show that aptamers St2p, Sal26, and St1 demonstrated minimal binding affinity across all three Salmonella serovars (≤2.750), often at very low levels for ST. In contrast, aptamers S8-7, 33, and C4 exhibited stronger binding affinity across all serovars, with aptamer C4 displaying the highest binding affinity, particularly to ST (19.000), consistent with the findings by Moon et al. (29). Aptamers 33 and S8-7 demonstrated moderate binding compared to C4, with binding values ranging from 6.000 to 9.750, in agreement with the previous result (28, 72).

These findings informed the selection of aptamers S8-7, 33, and C4 for further application in vaccine design. Their ability to bind multiple Salmonella serovars suggests potential for broad-spectrum vaccine applications, addressing a critical need for comprehensive Salmonella control in the poultry industry.

Verification of the optimized high-yield RCA products

Agarose gel electrophoresis is one of the most common methods for validating and visualizing RCA product formation (23, 52–56). Other visualization techniques include fluorescence-based methods such as microscopy, flow cytometry, and fluorescence spectroscopy, using dye-labeled probes (24, 53, 57–60), and nanoparticle-based platforms, including magnetic beads and quantum dots (61, 62).

In this study, RCA products were assessed on a 1.0% TAE agarose gel (Figure 2). Lane A contains a 25-kb DNA ladder as a molecular weight reference. Lanes B and E, corresponding to 10x diluted anti-Salmonella RCA and Aptamer RCA II, respectively, display high-molecularweight bands (> 25 kb), confirming successful amplification (52, 53, 55). Control lanes included RCA reactions performed without primers (lanes C and F) and without templates (lanes D and G) for both RCA constructs. These lanes showed only low-molecular-weight bands, indicating the absence of RCA amplification. Lane H, containing a 50 bp ladder, provided size approximation for smaller bands. The presence of distinct high-molecular-weight bands in the experimental lanes and their absence in the controls confirms the successful generation of high-yield RCA products.

In vitro evaluation of CD40-targeting aptamer-based DNA vaccine adjuvants using HD11 cell culture - Image 5

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Quantification of Salmonella binding to RCA constructs for vaccine dose selection

To optimize the vaccine complex for SE binding, we quantified the colony-forming units (CFU) of SE bound to each vaccine construct (RCA_v3 and RCA_v5, with or without DNase I treatment). It was accomplished using streptavidin magnetic beads to capture biotinylated RCA products containing bound SE. The strong, specific interaction between biotin and streptavidin made streptavidin-coated magnetic beads an effective tool for isolating biotin-labeled DNA– probe hybrids (63). The initial SE dose used for binding was 4.6 × 108 CFU. For RCA_v3, DNase I treatment markedly increased bacterial binding from 9.6 ± 0.5 × 105 CFU in the untreated sample to 3.1 ± 0.2 × 106 CFU, representing an approximate increase of 2.1 × 106 CFU. Similarly, RCA_v5 exhibited enhanced binding following DNase I treatment, increasing from 4.3 ± 0.4 × 105 CFU in the untreated group to 3.2 ± 0.3 × 106 CFU, a difference of about 2.8 × 106 CFU (Supplementary Figure S2). These results represent the mean ± standard deviation of 4 independent experiments conducted on separate days using independently prepared samples, without additional technical replication (p < 0.05). Based on these binding results, and consistent with previous studies reporting that Salmonella doses within the range of 105 –108 CFU induce robust immune responses in poultry, a dose of 1 × 107 CFU was selected for vaccination (64–66). However, future studies may incorporate fluorescent labeling and flow cytometry–based approaches to further characterize RCA–bacteria binding dynamics in addition to CFU-based viability quantification.

Macrophage activation by RCA-based vaccine complexes

The production of nitric oxide by chicken macrophages plays a key role in the innate immune response as a defense mechanism against bacterial invasion (38). The HD11 macrophage cell line is a well-established model for assessing macrophage activation via nitric oxide production (11, 67, 68). In this study, HD11 cells were treated with vaccine formulations to evaluate their immunostimulatory potential. Medium alone and mouse IgG served as negative controls and induced minimal nitric oxide production (Figure 3). In contrast, lipopolysaccharide (LPS), used as a positive control, elicited the highest level of nitric oxide production, consistent with its known role as a potent macrophage activator (69, 70).

The RCA complex without inactivated SE (Aptamer RCA II and RCA_v3) and inactivated SE alone showed moderate levels of macrophage activation, with no significant difference between them.

However, both RCA_v3 and RCA_v5 vaccine complexes demonstrated significantly higher macrophage activation compared to the RCA complex without inactivated SE (RCA only) (p < 0.05). Although the difference between RCA_v3 and RCA_v5 was not statistically significant, RCA_v3 induced a numerically greater response (Figure 3). This in vitro activation of macrophages, evidenced by increased nitric oxide production, highlights the potential of the RCA-based vaccine complexes (RCA_v3 and RCA_v5) to engage CD40 receptors on antigen-presenting cells. The enhanced NO production following stimulation with RCA constructs incorporating inactivated SE highlights the importance of combining antigen presentation with CD40 targeting to boost immune activation, an effect that may be critical for initiating protective adaptive immunity in vivo.

In vitro evaluation of CD40-targeting aptamer-based DNA vaccine adjuvants using HD11 cell culture - Image 7

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It is important to note that in our previous study (11), we included NC RCA product, which is the same type of RCA product but using two random aptamer sequences (which were not enriched during SELEX cycles) in the HD11 assay as a negative control. Indeed, the result showed that NC RCA failed to activate HD11 cells, suggesting that the activation by Aptamer RCA II, RCA_v3, and RCA_v5 shown in this study (Figure 3) is due to the specific aptamers (SEQ3 and SEQ4) present in these RCA products. Although we cannot exclude the possibility of HD11 activation through other pathways or receptors (without further experimental data), the specificity of SEQ3 and SEQ4 for chCD40 and their requirement for activating HD11 strongly suggest that CD40 receptors are the major pathway of HD11 activation in the current study.

Conclusion

In this study, DNA-based Aptamer RCA vaccine complexes targeting CD40 were developed and evaluated for their potential to control Salmonella colonization in chickens. The results demonstrated their ability to activate macrophage cells in vitro (Figure 3). The results highlight the vaccine’s potential as an effective strategy for controlling Salmonella colonization in broiler chickens, which is a critical issue in poultry production and food safety. By combining the specificity and stability of DNA aptamers with the scalability of the high-yield RCA process, this novel vaccine design may provide a promising solution to control Salmonella in the poultry industry.

While both RCA_v3 and RCA_v5 vaccine complex designs showed promising results in vitro, a future study evaluating vaccine efficacy in a chicken vaccination study with Salmonella challenge is necessary to demonstrate their efficacy in chickens. Future research could also include measuring immune responses, such as IgM, IgY, and IgA responses, and cell uptake analyses (including macrophage and dendritic cell binding/uptake assays). Overall, this study highlights a novel, easy-to-make vaccination strategy for controlling Salmonella in poultry, with potential applications in veterinary and agricultural settings.

This article was originally published in Frontiers in Veterinary Science. 13:1735450. doi: 10.3389/fvets.2026.1735450. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY).