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International Journal of Pharmaceutics: X logoLink to International Journal of Pharmaceutics: X
. 2026 Oct 6;12:100679. doi: 10.1016/j.ijpx.2026.100679

A n-heneicosane solid emulsion as a single-dose subunit vaccine elicits broad and durable immune responses in an aged and a Pox virus model

Luis Ontiveros-Padilla a,1, Stephen A Ehrenzeller a,1, Connor T Murphy a, Denzel D Middleton a, Grace L Williamson a, Alexandra M Lopez a, Eric M Bachelder a, Kristy M Ainslie a,b,c,⁎
PMCID: PMC13675617  PMID: 42892869

Abstract

The development of single-dose vaccines capable of inducing durable humoral and cellular immune responses would eliminate the need for additional booster doses, which is particularly important for resource-limited settings or in a pandemic scenario. Here, we report the development of a sustained-release n-heneicosane solid emulsion designed to enhance subunit vaccine immunogenicity after a single administration. Fast- and slow-release emulsions were formulated by varying surfactant content and adding CpG to increase the adjuvant activity. The emulsions were characterized by release kinetics, cytotoxicity, and innate immune activation in dendritic cells. The slow-release formulation demonstrated prolonged antigen release and promoted pro-inflammatory cytokine production without inducing significant cytotoxicity. In vivo, a single subcutaneous immunization with antigen-loaded n-heneicosane emulsions elicited robust and long-lasting antigen-specific antibody responses and cellular immune responses in mice. A Sustained IgG, IgG1 and IgG2c responses was observed for 165 days after a single immunization, while an IFN-γ and IL-2 cellular response in splenocytes was observed after 180 days post-immunization. In an Orthopoxvirus subunit vaccine model, the emulsion platform induced high levels of IgG, IgG1 and IgG2a antibody responses at higher or comparable levels than a prime-boost immunization with the MF-59-like adjuvant Addavax. Finally, strong humoral and cellular responses were also generated following a single immunization in 18-month-old mice, highlighting the potential of this platform to overcome age-associated immune dysfunction. Overall, these findings demonstrate that n-heneicosane emulsions function as an effective single-dose vaccine delivery system capable of promoting durable adaptive immunity across multiple subunit vaccine models and aged groups.

Keywords: n-heneicosane solid emulsion, Single-dose subunit vaccine, Long-lasting antibody response, Memory T cell response, Orthopoxvirus, Aged mice

Graphical Abstract

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1. Introduction

Subunit vaccines offer significant safety and manufacturing advantages over live-attenuated or inactivated platforms; however, they inherently have low immunogenicity and require the use of potent adjuvants and repeated dosing to achieve protective immunity (Kozak and Hu, 2023). This has been made apparent by numerous studies that have demonstrated that the COVID-19 vaccines were significantly more effective when booster doses were administered (Adams et al., 2022; Lewis et al., 2023; Rahman et al., 2023). While prime–boost regimens are effective, they present major challenges for global immunization, including poor patient compliance, increasing costs and logistical barriers in resource-limited settings (Orenstein and Ahmed, 2017; Plotkin, 2020). These limitations become particularly serious in the context of emerging infectious diseases, such as those caused by Orthopoxviruses (e.g., smallpox, mpox), a viral family responsible for more than 400 million deaths over the course of the 20th century (Shchelkunova and Shchelkunov, 2022). They are further compounded when vaccinating elderly populations, where immunosenescence reduces both the magnitude and durability of vaccine-induced immune responses (Crooke et al., 2019). Consequently, there is a critical need for vaccine technologies capable of eliciting robust and long-lasting immunity with a single immunization.

A very important determinant in the rational development of vaccines, is the kinetics of antigen exposure, as studies have demonstrated that prolonged antigen release enhances germinal center reactions, promoting affinity maturation, durable antibody responses, and the generation of memory T cells. Sustained antigen presentation can, in part, recapitulate the immunological benefits of booster immunizations, providing a compelling strategy to enable single-dose vaccination (Cirelli and Crotty, 2017; Tam et al., 2016; Turner et al., 2020). To achieve this important feature, a range of biomaterial-based delivery systems (e.g., polymeric microparticles, lipid nanoparticles, and injectable hydrogels) have been developed to modulate antigen release profiles (Grego et al., 2021; Irvine et al., 2015; Roth et al., 2020). Moreover, controlled-release platforms and self-assembling depots have demonstrated improved immunogenicity in preclinical models by extending antigen exposure over days to weeks (Boopathy et al., 2019; McHugh et al., 2015; Roth et al., 2020).

Despite these advances, current slow-release vaccine systems face important limitations. Many platforms require complex fabrication processes, exhibit limited stability, or lack a precise tunability of release kinetics, compromising their potential as translational technologies. Furthermore, only a limited number of systems have successfully demonstrated the ability to replace conventional prime-boost regimens with a single administration while maintaining durable humoral and cellular immunity (Alter et al., 2021; Cleland, 1999; Graf et al., 2023). Therefore, there remains a need for simple, scalable, and tunable delivery systems that can reliably induce long-lived immune responses after a single dose.

In parallel, the selection of appropriate adjuvants is critical to shaping vaccine-induced immunity. Toll-like receptor (TLR) agonists are known for effectively activating the innate immune response and enhancing the immune response versus a co-immunized antigen (Duthie et al., 2011; Kawai et al., 2024), particularly, CpG oligodeoxynucleotides (CpG ODN), have emerged as powerful adjuvants capable of promoting Th1-biased immune responses through activation of TLR-9 signaling pathways (Bode et al., 2011; Yi et al., 1996; Zhao et al., 2013). CpG has been shown to enhance both antibody and cellular responses, including the induction of cytotoxic T lymphocytes and memory T cells, and has been incorporated into clinically approved vaccines (Brazolot Millan et al., 1998; Rothenfusser et al., 2004; Wang et al., 2024). Importantly, the efficacy of CpG is closely linked to its biodistribution and persistence, suggesting that integration with sustained-release delivery systems may further amplify its immunostimulatory effects (Kasturi et al., 2011; Palma and Cho, 2007). However, strategies that effectively combine controlled antigen release with CpG-mediated immune activation in a single-dose format remain poorly explored.

Lipid-based delivery systems represent an attractive class of materials for vaccine development due to their biocompatibility, scalability, and established clinical use. In particular, solid lipid scaffolds provide a unique opportunity to engineer temperature-dependent phase transitions and diffusion-controlled release, enabling prolonged antigen retention and presentation (Bolhassani, 2023; Javid-Naderi and Mousavi Shaegh, 2025). However, the application of solid lipid emulsions as sustained-release vaccine adjuvants has received limited attention.

Here, we developed a solid emulsion platform based on n-heneicosane, a long-chain alkane, designed to function as a tunable, slow-release adjuvant system for subunit vaccines. By modulating surfactant composition, we engineered formulations with distinct antigen release kinetics, enabling control over the duration of antigen exposure and immune stimulation. We hypothesized that this system could mimic the effects of prime–boost vaccination through a single administration by sustaining antigen availability and enhancing CpG-mediated innate immune activation.

2. Methods

2.1. Materials

Unless otherwise specified, all chemicals were purchased and used unmodified from Sigma Aldrich (St. Louis, MO) and all assays, biologics, and disposables were purchased from Thermo Fisher Scientific (Waltham, MA). To remove potential endotoxin, all glassware was soaked overnight in a 0.1 M sodium hydroxide solution and all metal instruments were kept in a 260 °C oven prior to use. Endotoxin content was determined to be less than 0.1 EU per dose by limulus amoebocyte lysate-based assay prior to in vivo studies.

2.2. Preparation and characterization of the n-heneicosane emulsion

n-Heneicosane (AmBeed; Buffalo Grove, IL) was melted at 50 °C and mixed with SPAN 80 (0.2% w/w–5% w/w relative to n-Heneicosane) via magnetic stirring at 500 rpm. The 0.2% w/w SPAN 80 and 5% w/w SPAN 80 formulations are hereafter referred to as “fast-release” and “slow-release”, respectively. Then, sterile water (20% w/w relative to n-Heneicosane) with or without ovalbumin (OVA) (Invivogen; San Diego, CA), B5R protein (BEI Resources; Manassas, VA), and/or CpG ODN 1826 (InvivoGen; San Diego, CA) was added dropwise to the oil-surfactant mixture and stirred at 50 °C, 500 rpm for 30 s. The resulting emulsion was vortexed vigorously for 10 s and returned to heat to prevent premature solidification. The crude emulsion was then extruded through a 0.2 μm filter using a Northern Lipids Inc. Lipex 10 mL extruder (Burnay, BC, Canada) and used immediately after for downstream studies.

n-Heneicosane emulsions were imaged using scanning electron microscopy (SEM) (Hitachi S-4700 Cold Cathode Field Emission; Ibaraki, Japan; UNC CHANL). To prepare samples, a droplet of the emulsion was spread on an SEM sample mount affixed with adhesive carbon tape. The mount was then submerged in liquid nitrogen for 1 min and lyophilized for at least 48 h prior to imaging. Following preparation, emulsions were allowed to solidify at room temperature and were stored in sterile syringes until use.

2.3. OVA release studies

n- Heneicosane emulsions with OVA content were prepared in triplicate as described above and allowed to solidify at 25 °C following extrusion. The emulsions were then layered with 1 mL of PBS (pH 7.4) and incubated at 37 °C. Time points were collected at 0, 1, 6, 48, 168, 336, 504, 672, 840, 1008, and 1176 h. At each timepoint, 1 mL of PBS was removed, stored at 4 °C prior to downstream analysis, and replaced with 1 mL of fresh, pre-warmed PBS. Cumulative OVA release was measured via Bradford's Assay and percent release was calculated based on the known initial loading of the emulsion.

2.4. Cell culture and treatments

DC2.4 cells (ATCC; Manassas, VA) were cultured in RPMI 1640 media (Corning) supplemented with 10% v/v FBS and 1% v/v penicillin-streptomycin. All cells were maintained at 37 °C, 5% CO2, and 100% humidity for the duration of the experiments. 25,000 DC2.4 cells/well were plated in a flat bottom cell culture 96-well plate and incubated overnight. The emulsion treatments were then diluted in culture media before stimulating the cells for 48 h. After 48 h, viability was assessed via LDH release assay (Thermo). To assess the immunostimulatory properties of the emulsion, cells were treated with the specific concentration of adjuvant or emulsion and supernatants were collected after 48 h and used to determine cytokine secretion levels. The concentration of TNF-α and IL-6 was determined via ELISA kit (R & D Systems; Minneapolis, MN).

2.5. In vivo immunizations & sample collection

All animal procedures were reviewed and approved by the UNC IACUC and were performed in accordance with NIH guidelines for the care and use of laboratory animals. For the initial study using OVA as a model antigen, 6–8-week-old C57BL/6 mice (n = 5) were immunized subcutaneously with PBS, CpG + endotoxin-free ovalbumin (OVA) (Invivogen; San Diego, CA) (10 μg + 20 μg), fast-release emulsion + OVA (20 μg), slow-release emulsion + OVA (20 μg), slow-release emulsion + CpG + OVA (10 μg + 20 μg) or Addavax + OVA (1:1 v/v and 20 μg). Before immunization, the pre-filled syringe containing the solid emulsion was briefly warmed in a 56 °C heating block only until completely melted. The formulation was immediately removed from the heat source and administered after reaching an injectable temperature. Thus, animals were not injected with material at 56 °C and the mice were injected with 50 μL per flank (100 μL total). No evidence of local tissue damage or injection-site abnormalities was observed following administration. AddaVax formulations were prepared via 1:1 v/v mixing AddaVax (Invivogen) with OVA according to the manufacturer's protocol. Submandibular bleeds were subsequently collected biweekly until day 165. Mice were homologous boosted on day 165 to stimulate cellular responses. On day 180, mice were humanely euthanized to collect spleens which were subsequently processed into single cell suspensions as previously described (Batty et al., 2023).

For the subsequent immunization study, 6–8 week old BALB/c mice (n = 5) were immunized subcutaneously as described above with PBS, CpG + B5R (10 μg + 10 μg), slow-release emulsion + B5R (10 μg), slow-release emulsion + CpG + B5R (10 μg + 10 μg) or intramuscularly with Addavax + B5R (1:1 v/v and 10 μg) on a prime-boost schedule. Submandibular bleeds were subsequently collected biweekly until day 56.

For the aged mice study, 18-month-old C57/BL6J mice (n = 5) were obtained from the National Institute of Aging and were immunized subcutaneously as described above with PBS, CpG + OVA (10 μg + 25 μg), slow-release emulsion + OVA (25 μg), slow-release emulsion + CpG + OVA (10 μg + 25 μg) or Addavax + OVA (1:1 v/v and 25 μg). Submandibular bleeds were collected biweekly until day 84, when mice were homologous boosted to evaluate the cellular immune response. On day 96, spleens were harvested and processed into single cell suspensions as previously described.

2.6. Determination of antibody titers

OVA-specific and B5R-specific antibody titers in the collected sera were determined by a previously described indirect ELISA method (Hendy et al., 2024; Roque 3rd et al., 2024). Briefly, high-binding 384-well plates (Greiner Bio-One; Kremsmünster, Austria) were coated with OVA (10 μg/mL) or B5R (1 μg/mL) in PBS and incubated at 4 °C overnight. Plates were then blocked with 3% w/v instant nonfat dry milk in PBS (blocking buffer; Food Lion; Salisbury NC) for 1 h at room temperature (RT) and incubated with sera samples across a range of dilutions for 2 h at RT. Plates were subsequently incubated with IgG, IgG1, and IgG2c-specific HRP-conjugated detection antibodies (Southern Biotech; Birmingham AL) for 30 min at RT. 3,3,5,5-tetramethylbenzidine (TMB) was then added to initiate the detection reaction, and development was quenched with 2 N sulfuric acid. A minimum of three washes with 0.05% v/v Tween 20 in PBS (PBST) were performed between each step. Absorbances at 450 and 570 nm were read on a plate reader (SpectraMax M2 Microplate Reader; Molecular Devices; Sunnyvale, CA), with the 570 nm absorbance subtracted from the 450 nm absorbance to correct for background. Titer was determined using a plot of correct absorbance against serum dilution factor as previously reported (Eckshtain-Levi et al., 2022; Frey et al., 1998).

2.7. Antigen recall

Antigen recall was assessed by ELISPOT, as previously described (Batty et al., 2023; Eckshtain-Levi et al., 2022; Hendy et al., 2024; Roque 3rd et al., 2024). Splenocytes from immunized mice were plated at 1 × 106 cells per well in a multi-screen-IP 0.45 μm filter 96-well plate (Sigma Aldrich; St. Louis, MO) pre-coated with the desired capture antibody. Cells were then incubated with 10 μg/mL OVA protein, SIINFEKL or OVA323–339 peptides for 36 h, and ELISPOTs were developed according to the manufacturer's protocol (BD Biosciences; San Jose, CA). Spots were counted with the AID Classic ELISPOT Reader (AID Autoimmun Diagnostika GmbH; Straßberg, Germany).

2.8. Statistical analysis

Figures and statistical analyses were prepared with GraphPad Prism 10. For in vitro adjuvanticity experiments, a normal (Gaussian) distribution was assumed, and all the data were compared via ordinary one-way ANOVA with Tukey's multiple comparisons test For in vivo vaccination studies, the data was fit to a full model and were compared via ordinary two-way ANOVA with Tukey's multiple comparisons test.

3. Results and discussion

3.1. In vitro characterization of the n-heneicosane emulsion

We first evaluated the physicochemical and immunological properties of the n-heneicosane solid emulsions by modulating surfactant content to generate fast- and slow-release formulations. Scanning Electron Microscopy (SEM) revealed that the lyophilized n-heneicosane emulsions formed a porous structure with irregular surface topography (Fig. S1) Consistent with the design rationale, the slow-release emulsion exhibited a markedly prolonged release profile of ovalbumin (OVA), sustaining antigen release over several weeks, whereas the emulsion with lower content of surfactant (fast-release formulation) showed a rapid initial release with limited long-term retention (Fig. 1A). These findings demonstrate that compositional changes in the formulation enable precise control over antigen release kinetics, a critical parameter to modulate adaptive immune responses (Skakuj et al., 2021).

Fig. 1.

Fig. 1

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In vitro characterization of the n-Heneicosane emulsion. (A) Release profile of OVA in the fast and slow-release emulsions at pH 7.4. DC2.4 cells were stimulated for 48 h with fast and slow-release emulsions, with or without CpG (2.5 μg/mL) in them; (B) LDH release in supernatants was measured, as well as (C) TNF-α and (D) IL-6 concentrations via ELISA. Data is presented as mean ± SD (n = 3). Statistical significance is presented as ns = not significant and **p < 0.01, for an ordinary one-way ANOVA with Turkey's multiple comparisons test.

Both formulations exhibited minimal cytotoxicity in DC2.4 cells, as indicated by low LDH release after stimulation (Fig. 1B), supporting the biocompatibility of the system. In parallel, the emulsions induced measurable pro-inflammatory cytokine production, including TNF-α and IL-6 (Fig. 1C–D), indicative of innate immune activation. It is important to note that the addition of CpG enhanced cytokine secretion, consistent with its established role as a TLR-9 agonist (Bauer et al., 2001). These results suggest that the n-heneicosane emulsion can function not only as a delivery system but also as an immunostimulatory scaffold, both are desirable features in the rational vaccine design.

The n-heneicosane emulsion provides a simpler and more tunable platform, which compared to widely studied polymeric systems (such as PLGA microparticles), often exhibit burst release and heterogeneous degradation kinetics (Makadia and Siegel, 2011; Xu et al., 2017). In addition, recent studies have demonstrated that co-localization of antigen and adjuvant enhances the activation of the immune system (Shen et al., 2026), further supporting the design of this system.

3.2. A single immunization of n-heneicosane emulsion induces long-lasting antibody and cellular responses

We next assessed whether the observed sustained antigen release could translate into an enhanced in vivo immunogenicity with a single immunization. Mice were immunized with OVA +/− CpG-loaded formulations, and the antibody and cellular immune responses were monitored longitudinally for more than 5 months. A single immunization with both, fast or slow-release emulsions + CpG elicited robust and long-lasting OVA-specific IgG and responses that persisted for over 160 days, however, the slow-release + CpG formulation induced higher and more durable IgG titers (Fig. 2A). Immunization with soluble OVA and CpG elicited comparably weaker total IgG titers, which waned by 92 days post-immunization. Analysis of IgG subclasses revealed presence of anti-OVA IgG1 titers (Fig. S2) but also a long-lasting IgG2c response induced by both fast and slow-release + CpG emulsions, with higher titers in the mice immunized with the slow-release + CpG emulsion (Fig. 2B). This antibody profile is indicative of a strong Th1-biased immune response consistent with CpG-mediated signaling (Coffman et al., 2010).

Fig. 2.

Fig. 2

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OVA-specific serum antibody titers and cellular responses following a single immunization with n-Heneicosane emulsions. C57BL/6 mice (n = 5) were immunized subcutaneously with PBS, CpG + OVA, fast-release emulsion + OVA, fast-release emulsion + CpG + OVA, slow-release emulsion + OVA, and slow-release emulsion + CpG + OVA.Sera were collected at the indicated timepoints and assessed for OVA-specific (A) total IgG and (B) IgG2c. On day 165, mice were homologous boosted and spleens were collected at day 180. Splenocytes were stimulated with either OVA protein alone, SIINFEKL peptide, or OVA 323–339 peptide and the production of (C) IL-2 and (D) IFN-γ was measured via enzyme-linked immunosorbent spot (ELISpot). Data is presented as median ± SEM. Statistical significance is presented as *p < 0.05, and ****p < 0.0001 for an ordinary two-way ANOVA with Turkey's multiple comparisons test.

After a homologous boosting at day 165, splenocytes from mice immunized with the slow-release emulsion exhibited significantly elevated IL-2 and IFN-γ production upon antigen recall stimulation with either complete OVA protein, the OVA peptide, SIINFEKL, that binds specifically to MHCI, or OVA323–339, that binds specifically to MHCII (Fig. 2C–D), demonstrating robust CD8+ and CD4+ memory T cell responses, respectively. These findings indicate that sustained antigen exposure from a single dose is sufficient to prime long-lived adaptive immunity. This data has similarities with studies showing that extending antigen availability enhances germinal center persistence and promotes the development of high-affinity antibodies and memory T cells (Mesin et al., 2016; Victora and Nussenzweig, 2012). While delivery systems and implantable devices have been developed to mimic booster immunizations, these approaches often require complex fabrication and may face translational barriers. In contrast, the n-heneicosane emulsion achieves comparable durability of immune responses using a simple injectable formulation. Similar single-dose strategies based on self-assembling depots or injectable hydrogels have demonstrated promising results (Shi et al., 2024; Wang et al., 2025), but remain limited in their ability to precisely tune release kinetics and co-deliver immunostimulatory adjuvants.

3.3. A single immunization of n-heneicosane emulsion induces high antibody responses in a Pox virus and an aged mice model

As we previously identified the slow-release emulsion as the best formulation to achieve strong and long-lasting humoral and cellular responses, we decided to evaluate the translational relevance of this platform, assessing its performance in a Pox virus immunization model, using the vaccinia virus (VACV) surface antigen B5R. With just a single immunization, mice receiving the slow-release emulsion exhibited robust B5R-specific IgG antibody responses after 56 days post-injection, including both IgG1 and IgG2c subclasses (Fig. 3A–C), indicating a balanced immune profile (Th1/Th2). The magnitude of the IgG and IgG1 response induced by the n-heneicosane emulsion was comparable to or even greater than the response elicited by AddaVax and soluble CpG + B5R controls. This demonstrates that our single dose formulation can elicit an immune response that is comparable to current multi-dose vaccination regiments. It is important to note that, although antigen (B5R or OVA) and CpG stability following brief warming was not directly evaluated in the present study, both components remained biologically active as evidenced by the strong antigen-specific humoral and cellular responses observed after immunization.

Fig. 3.

Fig. 3

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B5R-specific antibody titers following a single immunization with n-Heneicosane + B5R emulsions. BALB/c mice (n = 5) were immunized subcutaneously with PBS, CpG + B5R, slow-release emulsion + B5R, slow-release emulsion + CpG + B5R, or Addavax + B5R intramuscularly on prime-boost schedule. Sera were collected at the indicated timepoints and assessed for B5R-specific (A) total IgG, (B) IgG1 and (C) IgG2a titers via ELISA. Data is presented as median ± SEM. Statistical significance is presented as **p < 0.01 and ***p < 0.001 for an ordinary two-way ANOVA with Turkey's multiple comparisons test.

These findings are particularly significant given the importance of neutralizing antibodies in protection against Orthopoxviruses (Edghill-Smith et al., 2005; Moss, 2011). These results are consistent with prior reports demonstrating that sustained antigen delivery enhances protective immunity against viral infections (Tam et al., 2016). However, most existing systems rely on polymeric scaffolds or nanoparticle-based carriers, which may introduce challenges related to scalability and stability.

Given the reduced vaccine responsiveness associated with aging, we next evaluated the performance of the n-heneicosane platform in an aged mice model. To determine whether this platform could overcome age-associated immune dysfunction, we evaluated its performance in 18-month-old mice. Aged mice receiving a single immunization with the slow-release emulsion exhibited sustained OVA-specific IgG, IgG1 and IgG2c antibody responses over time (Fig. 4A–C). Although antibody titers were modestly reduced compared to young mice (Fig. 2), they remained significantly higher than those induced by the soluble CpG + OVA control group. Importantly, antigen-specific cellular responses were preserved in aged animals, as evidenced by increased IL-2 and IFN-γ production upon antigen recall (Fig. 4D–E). These findings suggest that the n-heneicosane emulsion can partially overcome the age-related immunosenescence in vaccinations, a major limitation of current vaccine platforms (Goyani et al., 2024; Nikolich-Zugich, 2018). Previous studies have demonstrated that both sustained antigen exposure and TLR agonists can enhance immune responses in aged populations (Jones et al., 2010; Lefebvre and Haynes, 2013). These studies will facilitate translation of this platform toward clinical applications. However, few systems have successfully combined these strategies into a single-dose platform capable of inducing durable immunity. The ability of this system to maintain both humoral and cellular responses in aged mice highlights its potential for improving vaccine efficacy in high-risk populations.

Fig. 4.

Fig. 4

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Antibody and cellular response of a single immunization with n-Heneicosane emulsion in aged mice. 18-month-old C57BL/6 mice (n = 5) were immunized subcutaneously with PBS, CpG + OVA and slow-release emulsion + CpG + OVA. Sera were collected at the indicated timepoints and assessed for OVA-specific (A) total IgG and (B) IgG1 and (C) IgG2c. On day 84 mice were homologous boosted, and spleens were collected at day 96. Splenocytes were stimulated with either OVA protein alone, SIINFEKL peptide, or OVA 323–339 peptide and the production of (D) IL-2 and (E) IFN-γ production in splenocytes from immunized mice via ELISPOT. Data is presented as median ± SEM. Statistical significance is presented as ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 for an ordinary two-way ANOVA with Turkey's multiple comparisons test.

4. Conclusion

We have developed n-heneicosane solid emulsions that can be easily tuned to possess both fast and slow release kinetics of antigen that exhibits minimal cytotoxicity and the production of inflammatory cytokines when combined with the TLR9 agonist, CpG, in vitro. When administered via a singular subcutaneous dose in mice, both formulations induce significant and long-lasting antigen-specific antibody titers, while the slow-release formulation produces a significant cellular response. Similarly, when combined with the vaccinia virus protein, B5R, the slow-release formulation induces an IgG and IgG subtype response that is comparable to or better than AddaVax, a multi-dose, MF59-like adjuvant, highlighting the effectiveness of our single dose subunit vaccine. Additionally, the slow-release formulation combined with CpG produced both significant humoral and cellular responses in an aged mouse model, demonstrating the formulations ability to invoke potent immune responses in immunosenescent populations.

By integrating sustained antigen delivery with CpG-mediated immune activation, this system addresses key limitations of current subunit vaccines and offers a simplified approach to achieving durable immune responses.

CRediT authorship contribution statement

Luis Ontiveros-Padilla: Investigation, Formal analysis, Data curation, Conceptualization, Writing – original draft. Stephen A. Ehrenzeller: Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Writing – original draft. Connor T. Murphy: Investigation, Writing – review & editing. Denzel D. Middleton: Supervision, Investigation, Formal analysis, Writing – review & editing. Grace L. Williamson: Investigation, Writing – review & editing. Alexandra M. Lopez: Investigation, Writing – review & editing. Eric M. Bachelder: Supervision, Conceptualization, Writing – review & editing. Kristy M. Ainslie: Supervision, Resources, Conceptualization, Writing – review & editing, Writing – original draft.

Ethics declaration

Animal subject

This study was conducted in accordance with the following guidelines for animal welfare and/or reporting: All animal procedures were reviewed and approved by the UNC IACUC and were performed in accordance with NIH guidelines for the care and use of laboratory animals.

This study was approved by the UNC IACUC.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ainslie, Bachelder, Ontiveros-Padilla, and Ehrenzeller are named inventors on a patent application covering the technology described in this manuscript. This patent has been filed but has not been licensed to any commercial entity.

The authors declare no other competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported, in part, by National Institutes of Health (NIH) NIAID CIVICs (site PI: Ainslie), R01AI167099 (PI: Ainslie). This work was performed in part at the Chapel Hill Analytical and Nanofabrication Laboratory, CHANL, a member of the North Carolina Research Triangle Nanotechnology Network, RTNN, which is supported by the National Science Foundation, Grant ECCS-2025064, as part of the National Nanotechnology Coordinated Infrastructure, NNCI. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpx.2026.100679.

Appendix A. Supplementary data

Supplementary material contains SEM images of the fast and slow-release n-Heneicosane emulsions, and a graph showing the production of OVA-specific IgG1 antibody between the fast and slow-release emulsions with or without CpG.
mmc1.docx (314.5KB, docx)

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material contains SEM images of the fast and slow-release n-Heneicosane emulsions, and a graph showing the production of OVA-specific IgG1 antibody between the fast and slow-release emulsions with or without CpG.
mmc1.docx (314.5KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from International Journal of Pharmaceutics: X are provided here courtesy of Elsevier

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