Preparation of viromimetic rod-like nanoparticle vaccines (RLNVax) and study of their humoral immune activation efficacy.

Virus-like nanoparticle vaccines can efficiently activate the humoral immune response by cross-linking B cell receptors with their surface multivalent antigen arrays. This structurally dependent mechanism makes it crucial to regulate and optimize structural parameters to enhance the efficacy of nanoparticle vaccines. In this study, we prepared nanoparticle vaccines with different aspect ratios by chemically modifying antigen proteins onto the surfaces of poly(amino acid) nanoparticles of various shapes (spherical, ellipsoidal, and rod-like). This allowed us to investigate the impact of structural anisotropy on the humoral immune activation efficacy of nanoparticle vaccines. Furthermore, the end-group molecules of poly(amino acid) materials possess aggregation-induced emission (AIE) properties, which facilitate monitoring the dynamics of nano-assemblies within the body. Results showed that rod-like nanoparticle vaccines (RLNVax) with a higher aspect ratio (AR = 5) exhibited greater lymph node draining efficiency and could elicit more effective B cell activation compared to conventional isotropic spherical nanoparticle vaccines. In a murine subcutaneous immunization model using ovalbumin (OVA) as a model antigen, RLNVax elicited antigen-specific antibody titers that were about 64 times and 4.6 times higher than those induced by free antigen proteins and spherical nanoparticle vaccines, respectively. Additionally, when combined with an aluminum adjuvant, antibody titers elicited by RLNVax were further enhanced by 4-fold. These findings indicate that the anisotropic rod-like structure is advantageous for improving the humoral immune activation efficacy of nanoparticle vaccines, providing significant insights for the design and optimization of next-generation nanoparticle vaccines.

Pickering emulsion-guided monomeric delivery of monophosphoryl lipid A for enhanced vaccination.

Immunological adjuvants are vaccine components that enhance long-lasting adaptive immune responses to weakly immunogenic antigens. Monophosphoryl lipid A (MPLA) is a potent and safe vaccine adjuvant that initiates an early innate immune response by binding to the Toll-like receptor 4 (TLR4). Importantly, the binding and recognition process is highly dependent on the monomeric state of MPLA. However, current vaccine delivery systems often prioritize improving the loading efficiency of MPLA, while neglecting the need to maintain its monomeric form for optimal immune activation. Here, we introduce a Pickering emulsion-guided MPLA monomeric delivery system (PMMS), which embed MPLA into the oil-water interface to achieve the monomeric loading of MPLA. During interactions with antigen-presenting cells, PMMS functions as a chaperone for MPLA, facilitating efficient recognition by TLR4 regardless of the presence of lipopolysaccharide-binding proteins. At the injection site, PMMS efficiently elicited local immune responses, subsequently promoting the migration of antigen-internalized dendritic cells to the lymph nodes. Within the draining lymph nodes, PMMS enhanced antigen presentation and maturation of dendritic cells. In C57BL/6 mice models, PMMS vaccination provoked potent antigen-specific CD8+ T cell-based immune responses. Additionally, PMMS demonstrated strong anti-tumor effects against E.G7-OVA lymphoma. These data indicate that PMMS provides a straightforward and efficient strategy for delivering monomeric MPLA to achieve robust cellular immune responses and effective cancer immunotherapy.

Development of an engineered extracellular vesicles-based vaccine platform for combined delivery of mRNA and protein to induce functional immunity.

mRNA incorporated in lipid nanoparticles (LNPs) became a new class of vaccine modality for induction of immunity against COVID-19 and ushered in a new era in vaccine development. Here, we report a novel, easy-to-execute, and cost effective engineered extracellular vesicles (EVs)-based combined mRNA and protein vaccine platform (EVX-M+P vaccine) and explore its utility in proof-of-concept immunity studies in the settings of cancer and infectious disease. As a first example, we engineered EVs, natural nanoparticle carriers shed by all cells, to contain ovalbumin mRNA and protein (EVOvaM+P vaccine) to serve as cancer vaccine against ovalbumin-expressing melanoma tumors. EVOvaM+P administration to mice with established melanoma tumors resulted in tumor regression associated with effective humoral and adaptive immune responses. As a second example, we generated engineered EVs that contain Spike (S) mRNA and protein to serve as a combined mRNA and protein vaccine (EVSpikeM+P vaccine) against SARS-CoV-2 infection. EVSpikeM+P vaccine administration in mice and baboons elicited robust production of neutralizing IgG antibodies against RBD (receptor binding domain) of S protein and S protein specific T cell responses. Our proof-of-concept study describes a new platform with an ability for rapid development of combination mRNA and protein vaccines employing EVs for deployment against cancer and other diseases.

Spatiotemporal coordination of antigen presentation and co-stimulatory signal for enhanced anti-tumor vaccination.

Controlled-release systems enhance anti-tumor effects by leveraging local antigen persistence for antigen-presenting cells (APCs) recruitment and T cell engagement. However, constant antigen presentation alone tends to induce dysfunction in tumor-specific CD8+ T cells, neglecting the synergistic effects of co-stimulatory signal. To address this, we developed a soft particle-stabilized emulsion (SPE) to deliver lipopeptides with controlled release profiles by adjusting their hydrophobic chain lengths: C6-SPE (fast release), C10-SPE (medium release), and C16-SPE (slow release). Following administration, C6-SPE release antigen rapidly, inducing early antigen presentation, whereas C16-SPE's slow-release delays antigen presentation. Both scenarios missed the critical window for coordinating with the expression of CD86, leading to either T cell apoptosis or suboptimal activation. In contrast, C10-SPE achieved a spatiotemporally synergetic effect of the MHC-I-peptide complex and co-stimulatory signal (CD86), leading to effective dendritic cell (DC) activation, enhanced T cell activation, and tumor regression in EG7-OVA bearing mice. Additionally, co-delivery of cytosine-phosphate-guanine (CpG) with SPE provided a sustained expression of the CD86 window for DC activation, improving the immune response and producing robust anti-tumor effects with C6-SPE comparable to C10-SPE. These findings highlight that synchronizing the spatiotemporal dynamics of antigen presentation and APC activation may confer an optimal strategy for enhanced vaccinations.

Separable nanocomposite hydrogel microneedles for intradermal and sustained delivery of antigens to enhance adaptive immune responses.

Vaccines play a critical role in combating infectious diseases and cancers, yet improving their efficacy remains challenging. Here, we introduce a separable nanocomposite hydrogel microneedle (NHMN) patch designed for intradermal and sustained delivery of ovalbumin (OVA), a model antigen, to enhance adaptive immune responses. The NHMN patch consists of an array of OVA-loaded microneedles made from photo-cross-linked methacrylated hyaluronic acid and laponite (LAP), supported by a hyaluronic acid backing. The incorporation of LAP not only enhances the mechanical strength of the pure hydrogel microneedles but also significantly prolongs OVA release. Furthermore, in vitro cell experiments demonstrate that NHMNs effectively activate dendritic cells without compromising cell viability. Upon skin penetration, NHMNs detach from the backing as the hyaluronic acid rapidly dissolves upon contact with the skin interstitial fluid, thereby acting as antigen reservoirs to release antigens to abundant skin dendritic cells. NHMNs containing 0.5% w/v LAP achieved a 15-day OVA release in vivo. Immunization studies demonstrate that the intradermal and sustained release of OVA via NHMNs elicited stronger and longer-lasting adaptive immune responses compared to conventional bolus injection. Given its easy to use, painless and minimally invasive features, the NHMN patch shows promise in improving vaccination accessibility and efficacy against a range of diseases. STATEMENT OF SIGNIFICANCE: The study introduces a separable nanocomposite hydrogel microneedle (NHMN) patch. This patch consists of an array of ovalbumin (OVA, a model antigen)-loaded microneedles made from photo-cross-linked methacrylated hyaluronic acid and laponite, with a hyaluronic acid backing, designed for intradermal and sustained delivery of antigens. This patch addresses several key challenges in traditional vaccination methods, including poor antigen uptake and presentation, and rapid systematic clearance. The incorporation of laponite enhances mechanical strength of microneedles, promotes dendritic cell activation, and significantly slows down antigen release. NHMN-based vaccination elicits stronger and longer-lasting adaptive immune responses compared to conventional bolus injection. This NHMN patch holds great potential for improving the efficacy, accessibility, and patient comfort of vaccinations against a range of diseases.

Biomimetic nanomedicine cocktail enables selective cell targeting to enhance ovarian Cancer chemo- and immunotherapy.

Ovarian cancer is one of the deadliest cancers, and combined chemo- and immunotherapies are potential strategies to combat it. However, the anti-cancer efficacy of the combined therapies may be limited by the non-selective co-delivery of chemotherapy and immunotherapy. Herein, a combined chemo- and immunotherapy is designed to selectively target ovarian tumor (ID8) cells and dendritic cells (DCs) using ID8 cell membrane (IM) and bacterial outer membrane vesicles (OMVs), respectively. Doxorubicin (DOX) and Ovalbumin (OVA) peptide (OVA257-264) are chosen as model chemotherapy and immunotherapy agents, respectively. A DNA nanocube capable of easily loading DOX or OVA257-264 is chosen as the carrier. Firstly, the DNA nanocube is used to load DOX or OVA257-264 to prepare cube-DOX or cube-OVA. This nanocube was then encapsulated with IM to form IM@Cube-DOX and with OMV to form OMV@Cube-OVA. IM@Cube-DOX can be selectively taken up by ID8 cells, leading to effective cell killing, while OMV@Cube-OVA targets and activates DC2.4 cells in vitro. Both IM@Cube-DOX and OMV@Cube-OVA show increased accumulation at ID8 tumors in C57BL/6 mice. Combined IM@Cube-DOX + OMV@Cube-OVA therapy demonstrates better anti-tumor efficacy than non-selective delivery methods such as OMV@(Cube-DOX + Cube-OVA) or IM@(Cube-DOX + Cube-OVA) in ID8-OVA tumor-bearing mice. In conclusion, this study demonstrates a biomimetic delivery strategy that enables selective drug delivery to tumor cells and DCs, thereby enhancing the anti-tumor efficacy of combined chemo- and immunotherapy through the selective delivery strategy.

Nitroxide radical conjugated ovalbumin theranostic nanosystem for enhanced dendritic cell-based immunotherapy and T1 magnetic resonance imaging.

Melanoma, known for its aggressive metastatic nature, presents a formidable challenge in cancer treatment, where conventional therapies often fall short. This study introduces a pioneering approach utilizing metal-free nanosystem as tumor vaccines, spotlighting their potential in revolutionizing melanoma treatment. This work employed organic nitroxides, specifically 4-carboxy-TEMPO, in combination with chitosan (CS), to create a novel nanocomposite material - the CS-TEMPO-OVA nanovaccines. This composition not only improves biocompatibility and extends blood circulation time of TEMPO but also marks a significant departure from traditional gadolinium-based contrast agents in MRI technology, addressing safety concerns. CS-TEMPO-OVA nanovaccines demonstrate excellent biocompatibility at both the cellular and organoid level. They effectively stimulate bone marrow-derived dendritic cells (BMDCs), which in turn promote the maturation and activation of T cells. This ultimately leads to a strong production of essential cytokines. These nanovaccines serve a dual purpose as both therapeutic and preventive. By inducing an immune response, activating cytotoxic T cells, and promoting macrophage M1 polarization, they effectively inhibit melanoma growth and enhance survival in mouse models. When combined with αPD-1, the CS-TEMPO-OVA nanovaccines significantly bolster the infiltration of cytotoxic T lymphocytes (CTLs) within tumors, sparking a powerful systemic antitumor response that effectively curbs tumor metastasis. The ability of these nanovaccines to control both primary (subcutaneous) and metastatic B16-OVA tumors highlights their remarkable efficacy. Furthermore, the CS-TEMPO-OVA nanovaccine can be administered in vivo via both intravenous and intramuscular routes, both of which effectively enhance the T1 contrast of magnetic resonance imaging in tumor tissue. This study offers invaluable insights into the integrated application of these nanovaccines in both clinical diagnostics and treatment, marking a significant stride in cancer research and patient care.

Identification of a novel DEC-205 binding peptide to develop dendritic cell-targeting nanovaccine for cancer immunotherapy.

Cancer vaccine is regarded as an effective immunotherapy approach mediated by dendritic cells (DCs) which are crucial for antigen presentation and the initiation of adaptive immune responses. However, lack of DC-targeting properties significantly hampers the efficacy of cancer vaccines. Here, by using the phage display technique, peptides targeting the endocytic receptor DEC-205 primarily found on cDC1s were initially screened. An optimized hydrolysis-resistant peptide, hr-8, was identified and conjugated to PLGA-loaded antigen (Ag) and CpG adjuvant nanoparticles, resulting in a DC-targeting nanovaccine. The nanovaccine hr-8-PLGA@Ag/CpG facilitates dendritic cell maturation and improves antigen cross-presentation. The nanovaccine can enhance the antitumor immune response mediated by CD8+ T cells by encapsulating the nanovaccine with either exogenous OVA protein antigen or endogenous gp100/E7 antigenic peptide. As a result, strong antitumor effects are observed in both anti-PD-1 responsive B16-OVA and anti-PD-1 non-responsive B16 and TC1 immunocompetent tumor models. In summary, this study presents the initial documentation of a nanovaccine that targets dendritic cells via the novel DEC-205 binding peptide. This approach offers a new method for developing cancer vaccines that can potentially improve the effectiveness of cancer immunotherapy.

Ionic Liquid-Based Immunization Patch for the Transdermal Delivery of Antigens.

Herein, we report a transdermal patch prepared using an ionic liquid-based solid in oil (IL-S/O) nanodispersion and a pressure-sensitive adhesive (PSA) to deliver the macromolecular antigenic protein, ovalbumin (OVA). The IL-S/O nanodispersion and a PSA were first mixed at an equal weight ratio, then coated onto a release liner, and covered with a support film. To evaluate the effect of the PSA, three types of PSAs, DURO-TAK 87-4098, DURO-TAK 87-4287, and DURO-TAK 87-235A, were used to obtain the corresponding IL-S/O patches SP-4098, SP-4287, and SP-235A, respectively. The prepared IL-S/O patches were characterized for surface morphology, viscoelasticity, and moisture content. In vitro skin penetration and in vivo immunization studies of the IL-S/O patches were performed using Yucatan micropig skin and the C57BL/6NJc1 mice model, respectively. The SP-4098 and SP-4287 delivered 5.49-fold and 5.47-fold higher amounts of drug compared with the aqueous formulation. Although both patches delivered a similar amount of drug, SP-4287 was not detached fully from the release liner after 30 days, indicating low stability. Mice immunized with the OVA-containing SP-4098 produced a 10-fold increase in anti-OVA IgG compared with those treated with an aqueous formulation. These findings suggested that the IL-S/O patch may be a good platform for the transdermal delivery of antigen molecules.

Surface-Engineered Polygonatum Sibiricum Polysaccharide CaCO3 Microparticles as Novel Vaccine Adjuvants to Enhance Immune Response.

Micro- and nanoparticles delivery systems have been widely studied as vaccine adjuvants to enhance immunogenicity and sustain long-term immune responses. Polygonatum sibiricum polysaccharide (PSP) has been widely studied as an immunoregulator in improving immune responses. In this study, we synthesized and characterized cationic modified calcium carbonate (CaCO3) microparticles loaded with PSP (PEI-PSP-CaCO3, CTAB-PSP-CaCO3), studied the immune responses elicited by PEI-PSP-CaCO3 and CTAB-PSP-CaCO3 carrying ovalbumin (OVA). Our results demonstrated that PEI-PSP-CaCO3 significantly enhanced the secretion of IgG and cytokines (IL-4, IL-6, IFN-γ, and TNF-α) in vaccinated mice. Additionally, PEI-PSP-CaCO3 induced the activation of dendritic cells (DCs), T cells, and germinal center (GC) B cells in draining lymph nodes (dLNs). It also enhanced lymphocyte proliferation, increased the ratio of CD4+/CD8+ T cells, and elevated the frequency of CD3+ CD69+ T cells in spleen lymphocytes. Therefore, PEI-PSP-CaCO3 microparticles induced a stronger cellular and humoral immune response and could be potentially useful as a vaccine delivery and adjuvant system.

Impact of antigen loading in tolerogenic nanoparticles to mitigate Th2-mediated allergic lung inflammation.

Allergic disease is a major global health concern that imposes significant life-altering and economic burdens on affected individuals. However, there is still no cure. Polymer-based nanoparticles (NP) have shown the potential to induce antigen (Ag)-specific immune tolerance in various Th1/17 and Th2-mediated immune disorders including autoimmunity and allergy. Common methods by which Ags are associated with NPs are through surface conjugation or encapsulation. However, these Ag delivery strategies can be associated with several caveats that dampen their effectiveness such as uncontrolled Ag loading, a high Ag burst release, and an increased immune recognition profile. We previously developed Ag-polymer conjugate NPs (acNPs) to overcome those noted limitations, while allowing for controlled delivery of precise quantities of Ag to innate immune cells for Ag-specific CD4 T cell modulation. Here, we utilized ovalbumin (OVA) protein-poly(lactic-co-glycolic acid) (PLGA) conjugate NPs (acNP-OVA) to elucidate the impact of Ag loading on the induction of Th2 tolerance using a prophylactic and therapeutic OVA/ALUM-induced mouse model of allergic lung inflammation (ALI) in comparison to Ag-encapsulated PLGA NPs (NP(Ag)). We demonstrate that acNP-OVA formulations reduced OVA-specific IgE and inhibited Th2 cytokine secretions in an Ag loading-dependent manner when administered prophylactically. Administration of acNP-OVA to pre-sensitized mice did not affect OVA-specific IgE and Th2 cytokines tended to be reduced, however, there was no clear Ag loading dependency. acNP-OVA with medium-to-low Ag loadings were well tolerated, while formulations with high Ag loadings, including NP(Ag) resulted in anaphylaxis. Overall, our results clarify the relationship between Ag loading and Ag-specific IgE and Th2 cytokine responses in a murine model of ALI, which provides insight useful for future design of tolerogenic NP-based immunotherapies.

Transdermal microarrayed electroporation for enhanced cancer immunotherapy based on DNA vaccination.

Despite the tremendous clinical potential of nucleic acid-based vaccines, their efficacy to induce therapeutic immune response has been limited by the lack of efficient local gene delivery techniques in the human body. In this study, we develop a hydrogel-based organic electronic device (µEPO) for both transdermal delivery of nucleic acids and in vivo microarrayed cell electroporation, which is specifically oriented toward one-step transfection of DNAs in subcutaneous antigen-presenting cells (APCs) for cancer immunotherapy. The µEPO device contains an array of microneedle-shaped electrodes with pre-encapsulated dry DNAs. Upon a pressurized contact with skin tissue, the electrodes are rehydrated, electrically triggered to release DNAs, and then electroporate nearby cells, which can achieve in vivo transfection of more than 50% of the cells in the epidermal and upper dermal layer. As a proof-of-concept, the µEPO technique is employed to facilitate transdermal delivery of neoantigen genes to activate antigen-specific immune response for enhanced cancer immunotherapy based on a DNA vaccination strategy. In an ovalbumin (OVA) cancer vaccine model, we show that high-efficiency transdermal transfection of APCs with OVA-DNAs induces robust cellular and humoral immune responses, including antigen presentation and generation of IFN-γ+ cytotoxic T lymphocytes with a more than 10-fold dose sparing over existing intramuscular injection (IM) approach, and effectively inhibits tumor growth in rodent animals.

An antibacterial, multifunctional nanogel for efficient treatment of neutrophilic asthma.

Asthma is a chronic and heterogeneous disease affecting the lungs and respiratory tract. In particular, the neutrophil subtype of asthma was described as persistent, more severe, and corticosteroid-resistant. Growing evidence suggested that nontypeable Haemophilus influenzae (NTHi) infection contributes to the development of neutrophilic asthma, exacerbating clinical symptoms and increasing the associated medical burden. In this work, arginine-grafted chitosan (CS-Arg) was ionically cross-linked with tris(2-carboxyethyl) phosphine (TCEP), and a highly-efficient antimicrobial agent, poly-ε-L-Lysine (ε-PLL), was incorporated to prepare ε-PLL/CS-Arg/TCEP (ECAT) composite nanogels. The results showed that ECAT nanogels exhibited highly effective inhibition against the proliferation of NTHi, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). In addition, ECAT nanogels could effectively inhibit the formation of mucins aggregates in vitro, suggesting that the nanogel might have the potential to destroy mucin in respiratory disease. Furthermore, in the ovalbumin (OVA)/NTHi-induced Balb/c mice model of neutrophilic asthma, the number of neutrophils in the alveolar lavage fluid and the percentage of inflammatory cells in the blood were effectively reduced by exposure to tower nebulized administration of ECAT nanogels, and reversing airway hyperresponsiveness (AHR) and reducing inflammation in neutrophilic asthma mice. In conclusion, the construction of ECAT nanogels was a feasible anti-infective and anti-inflammatory therapeutic strategy, which demonstrated strong potential in the clinical treatment of neutrophilic asthma.

Minimally invasive nasal infusion (MINI) approach for CNS delivery of protein therapeutics: A case study with ovalbumin.

One of the primary obstacles in treating central nervous system (CNS) disorders lies in the limited ability of disease-modifying drugs to cross the blood-brain barrier (BBB). Our previously described Minimally Invasive Nasal Depot (MIND) technique has proven successful in delivering various drugs to the brain in rat models via a trans-olfactory mucosal approach. In this study, we introduce a novel Minimally Invasive Nasal Infusion (MINI) delivery approach for administering ovalbumin, a model protein, utilizing a programmable infusion pump (iPRECIO SMP-310R) in a mouse model. This research highlights the significant role of olfactory mucosa in nose-to-brain delivery, with an efficacy of nearly 45% compared to intracerebroventricular (ICV) administration. This demonstrates its potential as an alternative procedure for treating CNS diseases, offering a greater safety profile relative to the highly invasive clinical routes traditionally adopted for CNS drug delivery.

Boosting antigen-specific T cell activation with lipid-stabilized protein nanoaggregates.

Vaccines based on protein antigens have numerous advantages over inactivated pathogens, including easier manufacturing and improved safety. However, purified antigens are weakly immunogenic, as they lack the spatial organization and the associated 'danger signals' of the pathogen. Formulating vaccines as nanoparticles enhances the recognition by antigen presenting cells, boosting the cell-mediated immune response. This study describes a nano-precipitation method to obtain stable protein nanoaggregates with uniform size distribution without using covalent cross-linkers. Nanoaggregates were formed via microfluidic mixing of ovalbumin (OVA) and lipids in the presence of high methanol concentrations. A purification protocol was set up to separate the nanoaggregates from OVA and liposomes, obtained as byproducts of the mixing. The nanoaggregates were characterized in terms of morphology, ζ-potential and protein content, and their interaction with immune cells was assessed in vitro. Antigen-specific T cell activation was over 6-fold higher for nanoaggregates compared to OVA, due in part to the enhanced uptake by immune cells. Lastly, a two-dose immunization with nanoaggregates in mice induced a significant increase in OVA-specific CD8+ T splenocytes compared to soluble OVA. Overall, this work presents for the first time the microfluidic production of lipid-stabilized protein nanoaggregates and provides a proof-of-concept of their potential for vaccination.

Oral biomimetic virus vaccine hydrogel for robust abscopal antitumour efficacy.

Remarkable progress has been made in tumour immunotherapy in recent decades. However, the clinical outcomes of therapeutic interventions remain unpredictable, largely because of inefficient immune responses. To address this challenge and optimise immune stimulation, we present a novel administration route for enhancing the bioavailability of immunotherapeutic drugs. Our approach involves the development of an oral tumour vaccine utilising virus-like particles derived from the Hepatitis B virus core (HBc) antigen. The external surfaces of these particles are engineered to display the model tumour antigen OVA, whereas the interiors are loaded with cytosine phosphoguanosine oligodeoxynucleotide (CpG ODN), resulting in a construct called CpG@OVAHBc with enhanced antigenicity and immune response. For oral delivery, CpG@OVAHBc is encapsulated in a crosslinked dextran hydrogel called CpG@OVAHBc@Dex. The external hydrogel shield safeguards the biomimetic virus particles from degradation by gastric acid and proteases. Upon exposure to intestinal flora, the hydrogel disintegrates, releasing CpG@OVAHBc at the intestinal mucosal site. Owing to its virus-like structure, CpG@OVAHBc exhibits enhanced adhesion to the mucosal surface, facilitating uptake by microfold cells (M cells) and subsequent transmission to antigen-presenting cells. The enzyme-triggered release of this oral hydrogel ensures the integrity of the tumour vaccine within the digestive tract, allowing targeted release and significantly improving bioavailability. Beyond its efficacy, this oral hydrogel vaccine streamlines drug administration, alleviates patient discomfort, and enhances treatment compliance without the need for specialised injection methods. Consequently, our approach expands the horizons of vaccine development in the field of oral drug administration.

Self-assembled protein vesicles as vaccine delivery platform to enhance antigen-specific immune responses.

Self-assembling protein nanoparticles are beneficial platforms for enhancing the often weak and short-lived immune responses elicited by subunit vaccines. Their benefits include multivalency, similar sizes as pathogens and control of antigen orientation. Previously, the design, preparation, and characterization of self-assembling protein vesicles presenting fluorescent proteins and enzymes on the outer vesicle surface have been reported. Here, a full-size model antigen protein, ovalbumin (OVA), was genetically fused to the recombinant vesicle building blocks and incorporated into protein vesicles via self-assembly. Characterization of OVA protein vesicles showed room temperature stability and tunable size. Immunization of mice with OVA protein vesicles induced strong antigen-specific humoral and cellular immune responses. This work demonstrates the potential of protein vesicles as a modular platform for delivering full-size antigen proteins that can be extended to pathogen antigens to induce antigen specific immune responses.

Induction of antigen-specific immunity by mesoporous silica nanoparticles incorporating antigen peptides.

Mesoporous silica nanoparticles (MSNs) are physically and chemically stable inorganic nanomaterials that have been attracting much attention as carriers for drug delivery systems in the field of nanomedicine. In the present study, we investigated the potential of MSN vaccines that incorporate antigen peptides for use in cancer immunotherapy. In vitro experiments demonstrated that fluorescently labeled MSNs accumulated in a line of mouse dendritic cells (DC2.4 cells), where the particles localized to the cytosol. These observations could suggest that MSNs have potential for use in delivering the loaded molecules into antigen-presenting cells, thereby stimulating the host acquired immune system. In vivo experiments demonstrated prolonged survival in mice implanted with ovalbumin (OVA)-expressing lymphoma cells (E.G7-OVA cells) following subcutaneous inoculation with MSNs incorporating OVA antigen peptides. Furthermore, OVA-specific immunoglobulin G antibodies and cytotoxic T lymphocytes were detected in the serum and the spleen cells, respectively, of mice inoculated with an MSN-OVA vaccine, indicating the induction of antigen-specific responses in both the humoral and cellular immune systems. These results suggested that the MSN therapies incorporating antigen peptides may serve as novel vaccines for cancer immunotherapy.

Bilaterally Aligned Electroosmotic Flow Generated by Porous Microneedle Device for Dual-Mode Delivery.

Here, a novel porous microneedle (PMN) device with bilaterally aligned electroosmotic flow (EOF) enabling controllable dual-mode delivery of molecules is developed. The PMNs placed at anode and cathode compartments are modified with anionic poly-2-acrylamido-2-methyl-1-propanesulfonic acid and cationic poly-(3-acrylamidopropyl) trimethylammonium, respectively. The direction of EOF generated by PMN at the cathode compartment is, therefore, reversed from cathode to anode, countering the unwanted cathodal suctioning of interstitial fluid caused by reverse iontophoresis. With the bilateral alignment of EOF, the versatility of the proposed device is evaluated by delivering molecules with different charges and sizes using Franz cell. In addition, a 3D printed probe device is developed to ease practical handling and minimize electrical stimulation by integrating two PMNs in closed proximity. Finally, the performance of the integrated probe device is demonstrated by dual delivery of a variety of molecules (methylene blue, rhodamine B, and fluorescein isothiocyanate-dextran) using pig skin and vaccination using mice with delivered ovalbumin.

Alpha-galactosylceramide improves the potency of mRNA LNP vaccines against cancer and intracellular bacteria.

Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8+ T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8+ T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections.