Enhancement of subunit vaccine delivery with zinc-carnosine coordination polymer through the addition of mannan.

Vaccines represent a pivotal health advancement for preventing infection. However, because carrier systems with repeated administration can invoke carrier-targeted immune responses that diminish subsequent immune responses (e.g., PEG antibodies), there is a continual need to develop novel vaccine platforms. Zinc carnosine microparticles (ZnCar MPs), which are composed of a one-dimensional coordination polymer formed between carnosine and the metal ion zinc, have exhibited efficacy in inducing an immune response against influenza. However, ZnCar MPs' limited suspendability hinders clinical application. In this study, we address this issue by mixing mannan, a polysaccharide derived from yeast, with ZnCar MPs. We show that the addition of mannan increases the suspendability of this promising vaccine formulation. Additionally, since mannan is an adjuvant, we illustrate that the addition of mannan increases the antibody response and T cell response when mixed with ZnCar MPs. Mice vaccinated with mannan + OVA/ZnCar MPs had elevated serum IgG and IgG1 levels in comparison to vaccination without mannan. Moreover, in the mannan + OVA/ZnCar MPs vaccinated group, mucosal washes demonstrated increased IgG, IgG1, and IgG2c titers, and antigen recall assays showed enhanced IFN-γ production in response to MHC-I and MHC-II immunodominant peptide restimulation, compared to the vaccination without mannan. These findings suggest that the use of mannan mixed with ZnCar MPs holds potential for subunit vaccination and its improved suspendability further promotes clinical translation.

Comparative study of acetalated-dextran microparticle fabrication methods for a clinically translatable subunit-based influenza vaccine.

The most common influenza vaccines are inactivated viruses produced in chicken eggs, which is a time-consuming production method with variable efficacy due to mismatches of the vaccine strains to the dominant circulating strains. Subunit-based vaccines provide faster production times in comparison to the traditional egg-produced vaccines but often require the use of an adjuvant to elicit a highly protective immune response. However, the current FDA approved adjuvant for influenza vaccines (MF59) elicits a primarily helper T-cell type 2 (Th2)-biased humoral immune response. Adjuvants that can stimulate a Th1 cellular response are correlated to have more robust protection against influenza. The cyclic dinucleotide cGAMP has been shown to provide a potent Th1 response but requires the use of a delivery vehicle to best initiate its signalling pathway in the cytosol. Herein, acetalated dextran (Ace-DEX) was used as the polymer to fabricate microparticles (MPs) via double-emulsion, electrospray, and spray drying methods to encapsulate cGAMP. This study compared each fabrication method's ability to encapsulate and retain the hydrophilic adjuvant cGAMP. We compared their therapeutic efficacy to Addavax, an MF59-like adjuvant, and cGAMP Ace-DEX MPs provided a stronger Th1 response in vaccinated BALB/c mice. Furthermore, we compared Ace-DEX MPs to spray dried MPs composed from a commonly used polymer for drug delivery, poly(lactic-co-glycolic acid) (PLGA). We observed that all Ace-DEX MPs elicited similar humoral and cellular responses to the PLGA MPs. Overall, the results shown here indicate Ace-DEX can perform similarly to PLGA as a polymer for drug delivery and that spray drying can provide an efficient way to produce MPs to encapsulate cGAMP and stimulate the immune system.

Zinc Carnosine Metal-Organic Coordination Polymer as a Potent Broadly Active Influenza Vaccine Platform with In Vitro Shelf-Stability.

Current seasonal influenza vaccines are limited in that they need to be reformulated every year in order to account for the constant mutation of the virus. Hemagglutinin (HA) immunogens have been developed using a computationally optimized broadly reactive antigen (COBRA) methodology, which are able to elicit an antibody response that neutralizes antigenically distinct influenza strains; however, subunit proteins are not immunogenic enough on their own to generate a substantial immune response. Due to this, different delivery strategies and adjuvants can be used to improve immunogenicity. Recently, we reported a new coordination polymer composed of the dipeptide carnosine and zinc (ZnCar) that is able to deliver protein antigens along with CpG to generate a potent immune response. In the present work, ZnCar was used to deliver the COBRA HA immunogen Y2 and the adjuvant CpG. We incorporated Y2 into ZnCar using two different methods to assess which would be the most immunogenic. Mice vaccinated with Y2 and CpG complexed with ZnCar showed an improved humoral and cellular response when compared to mice vaccinated with soluble Y2 and CpG. Further, we demonstrate in vitro that when Y2 and CpG are coordinated with ZnCar, they are protected from degradation at 40 °C for 3 months or 24 °C for 6 months. Overall, ZnCar shows promise as a delivery vehicle for subunit vaccines, given its superior immunogenicity and in vitro storage stability.

Humoral Response to the Acetalated Dextran M2e Vaccine is Enhanced by Antigen Surface Conjugation.

The influenza A virus causes substantial morbidity and mortality worldwide every year and poses a constant threat of an emergent pandemic. Seasonal influenza vaccination strategies fail to provide complete protection against infection due to antigenic drift and shift. A universal vaccine targeting a conserved influenza epitope could substantially improve current vaccination strategies. The ectodomain of the matrix 2 protein (M2e) of influenza is a highly conserved epitope between virus strains but is also poorly immunogenic. Administration of M2e and the immunostimulatory stimulator of interferon genes (STING) agonist 3'3'-cyclic guanosine-adenosine monophosphate (cGAMP) encapsulated in microparticles made of acetalated dextran (Ace-DEX) has previously been shown to be effective for increasing the immunogenicity of M2e, primarily through T-cell-mediated responses. Here, the immunogenicity of Ace-DEX MPs delivering M2e was further improved by conjugating the M2e peptide to the particle surface in an effort to affect B-cell responses more directly. Conjugated or encapsulated M2e co-administered with Ace-DEX MPs containing cGAMP were used to vaccinate mice, and it was shown that two or three vaccinations could fully protect against a lethal influenza challenge, while only the surface-conjugated antigen constructs could provide some protection against lethal challenge with only one vaccination. Additionally, the use of a reducible linker augmented the T-cell response to the antigen. These results show the utility of conjugating M2e to the surface of a particle carrier to increase its immunogenicity for use as the antigen in a universal influenza vaccine.

Development of a broadly active influenza intranasal vaccine adjuvanted with self-assembled particles composed of mastoparan-7 and CpG.

Currently licensed vaccine adjuvants offer limited mucosal immunity, which is needed to better combat respiratory infections such as influenza. Mast cells (MCs) are emerging as a target for a new class of mucosal vaccine adjuvants. Here, we developed and characterized a nanoparticulate adjuvant composed of an MC activator [mastoparan-7 (M7)] and a TLR ligand (CpG). This novel nanoparticle (NP) adjuvant was co-formulated with a computationally optimized broadly reactive antigen (COBRA) for hemagglutinin (HA), which is broadly reactive against influenza strains. M7 was combined at different ratios with CpG and tested for in vitro immune responses and cytotoxicity. We observed significantly higher cytokine production in dendritic cells and MCs with the lowest cytotoxicity at a charge-neutralizing ratio of nitrogen/phosphate = 1 for M7 and CpG. This combination formed spherical NPs approximately 200 nm in diameter with self-assembling capacity. Mice were vaccinated intranasally with COBRA HA and M7-CpG NPs in a prime-boost-boost schedule. Vaccinated mice had significantly higher antigen-specific antibody responses (IgG and IgA) in serum and mucosa compared with controls. Splenocytes from vaccinated mice had significantly increased cytokine production upon antigen recall and the presence of central and effector memory T cells in draining lymph nodes. Finally, co-immunization with NPs and COBRA HA induced influenza H3N2-specific HA inhibition antibody titers across multiple strains and partially protected mice from a challenge against an H3N2 virus. These results illustrate that the M7-CpG NP adjuvant combination can induce a protective immune response with a broadly reactive influenza antigen via mucosal vaccination.

Micro and nanotechnologies: The little formulations that could.

The first publication of micro- and nanotechnology in medicine was in 1798 with the use of the Cowpox virus by Edward Jenner as an attenuated vaccine against Smallpox. Since then, there has been an explosion of micro- and nanotechnologies for medical applications. The breadth of these micro- and nanotechnologies is discussed in this piece, presenting the date of their first report and their latest progression (e.g., clinical trials, FDA approval). This includes successes such as the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines from Pfizer, Moderna, and Janssen (Johnson & Johnson) as well as the most popular nanoparticle therapy, liposomal Doxil. However, the enormity of the success of these platforms has not been without challenges. For example, we discuss why the production of Doxil was halted for several years, and the bankruptcy of BIND therapeutics, which relied on a nanoparticle drug carrier. Overall, the field of micro- and nanotechnology has advanced beyond these challenges and continues advancing new and novel platforms that have transformed therapies, vaccines, and imaging. In this review, a wide range of biomedical micro- and nanotechnology is discussed to serve as a primer to the field and provide an accessible summary of clinically relevant micro- and nanotechnology platforms.

Delivery of small molecule mast cell activators for West Nile Virus vaccination using acetalated dextran microparticles.

Recently, there has been increasing interest in the activation of mast cells to promote vaccine efficacy. Several mast cell activating (MCA) compounds have been reported such as M7 and Compound 48/80 (C48/80). While these MCAs have been proven to be efficacious vaccine adjuvants, their translatability is limited by batch-to-batch variability, challenging large-scale manufacturing, and poor in vivo stability for the M7 peptide. Due to this, high throughput screening was performed to identify small molecule MCAs. Several potent MCAs were identified via this screening, but the in vivo translatability of the compounds was limited due to their poor aqueous solubility. To enhance the delivery of these MCAs we encapsulated them in acetalated dextran (Ace-DEX) microparticles (MPs). We have previously utilized Ace-DEX MPs for vaccine delivery due to their passive targeting to phagocytic cells, acid sensitivity, and tunable degradation. Four different MCA loaded MPs were combined with West Nile Virus Envelope III protein (EDIII) and their vaccine adjuvant activities were compared in vivo. MPs containing the small molecule MCA ST101036 produced the highest anti-EDIII IgG titers of all the MCAs tested. Further, ST101036 MPs produced higher titers than ST101036 formulated with PEG as a cosolvent which highlights the benefit of Ace-DEX MPs over a conventional formulation technique. Finally, in a mouse model of West Nile Virus infection ST101036 MPs produced similar survival to soluble M7 (80-90%). Overall, these data show that ST101036 MPs produce a robust antibody response against EDIII and survival emphasizing the benefits of using Ace-DEX as a delivery platform for the poorly soluble ST101036.

Vinyl Sulfone-functionalized Acetalated Dextran Microparticles as a Subunit Broadly Acting Influenza Vaccine.

Influenza is a global health concern with millions of infections occurring yearly. Seasonal flu vaccines are one way to combat this virus; however, they are poorly protective against influenza as the virus is constantly mutating, particularly at the immunodominant hemagglutinin (HA) head group. A more broadly acting approach involves Computationally Optimized Broadly Reactive Antigen (COBRA). COBRA HA generates a broad immune response that is capable of protecting against mutating strains. Unfortunately, protein-based vaccines are often weekly immunogenic, so to help boost the immune response, we employed the use of acetalated dextran (Ace-DEX) microparticles (MPs) two ways: one to conjugate COBRA HA to the surface and a second to encapsulate cGAMP. To conjugate the COBRA HA to the surface of the Ace-DEX MPs, a poly(L-lactide)-polyethylene glycol co-polymer with a vinyl sulfone terminal group (PLLA-PEG-VS) was used. MPs encapsulating the STING agonist cGAMP were co-delivered with the antigen to form a broadly active influenza vaccine. This vaccine approach was evaluated in vivo with a prime-boost-boost vaccination schedule and illustrated generation of a humoral and cellular response that could protect against a lethal challenge of A/California/07/2009 in BALB/c mice.

Sustained delivery of CpG oligodeoxynucleotide by acetalated dextran microparticles augments effector response to Computationally Optimized Broadly Reactive Antigen (COBRA) influenza hemagglutinin.

A subunit or protein-based influenza vaccine can be a safer alternative to live attenuated vaccine (Flumist) and require fewer boosts than an inactivated vaccine (e.g. Fluzone). However, to form an effective subunit vaccine, an adjuvant is often needed. In this work we used electrospray to encapsulate the hydrophilic adjuvant CpG into microparticles made from the hydrophobic biodegradable polymer acetalated dextran. To understand the rate of particle degradation on CpG release, polymer that was slow (21 h at phagosomal pH 5) and fast (0.25 h at pH 5) degrading was used to encapsulate the adjuvant. The slow-degrading particles exhibited the greatest degree of innate immune stimulation of antigen-presenting cells in vitro. In mice, the broadly acting Computationally Optimized Broadly Reactive Antigen (COBRA) Y2 influenza hemagglutinin (HA) antigen was used with CpG particles, soluble CpG, or MF-59 like adjuvant Addavax. Particles and soluble CpG elicited similar induction of anti-HA antibodies and protection against lethal influenza challenge, but the sustained release particles elicited the highest levels antibody effector functions. These results demonstrate a suitable method for encapsulation of CpG oligonucleotide in a hydrophobic particle matrix, and suggest that sustained release of CpG from Ace-DEX microparticles could potentially be used to induce potent antibody effector functions.

Microparticle Delivery of a STING Agonist Enables Indirect Activation of NK Cells by Antigen-Presenting Cells.

Natural killer (NK) cells are an important member of the innate immune system and can participate in direct tumor cell killing in response to immunotherapies. One class of immunotherapy is stimulator of interferon gene (STING) agonists, which result in a robust type I interferon (IFN-I) response. Most mechanistic studies involving STING have focused on macrophages and T cells. Nevertheless, NK cells are also activated by IFN-I, but the effect of STING activation on NK cells remains to be adequately investigated. We show that both direct treatment with soluble STING agonist cyclic di-guanosine monophosphate-adenosine monophosphate (cGAMP) and indirect treatment with cGAMP encapsulated in microparticles (MPs) result in NK cell activation in vitro, although the former requires 100× more cGAMP than the latter. Additionally, direct activation with cGAMP leads to NK cell death. Indirect activation with cGAMP MPs does not result in NK cell death but rather cell activation and cell killing in vitro. In vivo, treatment with soluble cGAMP and cGAMP MPs both cause short-term activation, whereas only cGAMP MP treatment produces long-term changes in NK cell activation markers. Thus, this work indicates that treatment with an encapsulated STING agonist activates NK cells more efficiently than that with soluble cGAMP. In both the in vitro and in vivo systems, the MP delivery system results in more robust effects at a greatly reduced dosage. These results have potential applications in aiding the improvement of cancer immunotherapies.

Metal-Organic Coordination Polymer for Delivery of a Subunit Broadly Acting Influenza Vaccine.

A zinc-carnosine (ZnCar) metal-organic coordination polymer was fabricated in biologically relevant N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) buffer for use as a vaccine platform. In vitro, ZnCar exhibited significantly less cytotoxicity than a well-established zeolitic imidazolate framework (ZIF-8). Adsorption of CpG on the ZnCar surface resulted in enhanced innate immune activation compared to soluble CpG. The model antigen ovalbumin (OVA) was encapsulated in ZnCar and exhibited acid-sensitive release in vitro. When injected intramuscularly on days 0 and 21 in C57BL/6 mice, OVA-specific serum total IgG and IgG1 were significantly greater in all groups with ZnCar and antigen compared to soluble controls. Th1-skewed IgG2c antibodies were significantly greater in OVA and CpG groups delivered with ZnCar for all time points, regardless of whether the antigen and adjuvant were co-formulated in one material or co-delivered in separate materials. When broadly acting Computationally Optimized Broadly Reactive Antigen (COBRA) P1 influenza hemagglutinin (HA) was ligated to ZnCar via its His-tag, significantly greater antibody levels were observed at all time points compared to soluble antigen and CpG. ZnCar-formulated antigen elicited increased peptide presentation to B3Z T cells in vitro and production of IL-2 after ex vivo antigen recall of splenocytes isolated from vaccinated mice. Overall, this work displays the formation of a zinc-carnosine metal-organic coordination polymer that can be applied as a platform for recombinant protein-based vaccines.

Multiplexed electrospray enables high throughput production of cGAMP microparticles to serve as an adjuvant for a broadly acting influenza vaccine.

Subunit vaccines employing designer antigens such as Computationally Optimized Broadly Reactive Antigen (COBRA) hemagglutinin (HA) hold the potential to direct the immune response toward more effective and broadly-neutralizing targets on the Influenza virus. However, subunit vaccines generally require coadministration with an adjuvant to elicit a robust immune response. One such adjuvant is the stimulator of interferon genes (STING) agonist cyclic dinucleotide 3'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). We have shown that encapsulation of cGAMP in acetalated dextran (Ace-DEX) microparticles through electrospray results in significantly greater biological activity. Electrospray is a continuous manufacturing process which achieves excellent encapsulation efficiency. However, the throughput of electrospray with a single spray head is limited. Here we report the development of a multiplexed electrospray apparatus with an order of magnitude greater throughput than a single-head apparatus. Physicochemical characterization and evaluation of adjuvant activity in vitro and in vivo indicated that microparticles produced with the higher throughput process are equally suited for use as a potent vaccine adjuvant to induce a balanced immune response to COBRA HA antigens.

Development of an Intranasal Gel for the Delivery of a Broadly Acting Subunit Influenza Vaccine.

Influenza virus is a major cause of death on a global scale. Seasonal vaccines have been developed to combat influenza; however, they are not always highly effective. One strategy to develop a more broadly active influenza vaccine is the use of multiple rounds of layered consensus buildings to generate recombinant antigens, termed computationally optimized broadly reactive antigen (COBRA). Immunization with the COBRA hemagglutinin (HA) can elicit broad protection against multiple strains of a single influenza subtype (e.g., H1N1). We formulated a COBRA H1 HA with a stimulator of interferon genes agonist cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) into a nasal gel for vaccination against influenza. The gel formulation was designed to increase mucoadhesion and nasal retention of the antigen and adjuvant to promote a strong mucosal response. It consisted of a Schiff base-crosslinked hydrogel between branched polyethyleneimine and oxidized dextran. Following a prime-boost-boost schedule, an intranasal gel containing cGAMP and model antigen ovalbumin (OVA) led to the faster generation of serum IgG, IgG1, and IgG2c and significantly greater serum IgG1 levels on day 42 compared to soluble controls. Additionally, OVA-specific IgA was detected in nasal, vaginal, and fecal samples for all groups, except the vehicle control. When the COBRA HA was given intranasally in a prime-boost schedule, the mice receiving the gel containing the COBRA and cGAMP had significantly higher serum IgG and IgG2c at day 41 compared to all groups, and only this group had IgA levels above the background in vaginal, nasal, and fecal samples. Overall, this study indicates the utility of an intranasal gel for the delivery of COBRAs for the generation of serum and mucosal humoral responses.

STING Agonist Mitigates Experimental Autoimmune Encephalomyelitis by Stimulating Type I IFN-Dependent and -Independent Immune-Regulatory Pathways.

The cGAS-cyclic GMP-AMP (cGAMP)-stimulator of IFN genes (STING) pathway induces a powerful type I IFN (IFN-I) response and is a prime candidate for augmenting immunity in cancer immunotherapy and vaccines. IFN-I also has immune-regulatory functions manifested in several autoimmune diseases and is a first-line therapy for relapsing-remitting multiple sclerosis. However, it is only moderately effective and can induce adverse effects and neutralizing Abs in recipients. Targeting cGAMP in autoimmunity is unexplored and represents a challenge because of the intracellular location of its receptor, STING. We used microparticle (MP)-encapsulated cGAMP to increase cellular delivery, achieve dose sparing, and reduce potential toxicity. In the C57BL/6 experimental allergic encephalomyelitis (EAE) model, cGAMP encapsulated in MPs (cGAMP MPs) administered therapeutically protected mice from EAE in a STING-dependent fashion, whereas soluble cGAMP was ineffective. Protection was also observed in a relapsing-remitting model. Importantly, cGAMP MPs protected against EAE at the peak of disease and were more effective than rIFN-ß. Mechanistically, cGAMP MPs showed both IFN-I-dependent and -independent immunosuppressive effects. Furthermore, it induced the immunosuppressive cytokine IL-27 without requiring IFN-I. This augmented IL-10 expression through activated ERK and CREB. IL-27 and subsequent IL-10 were the most important cytokines to mitigate autoreactivity. Critically, cGAMP MPs promoted IFN-I as well as the immunoregulatory cytokines IL-27 and IL-10 in PBMCs from relapsing-remitting multiple sclerosis patients. Collectively, this study reveals a previously unappreciated immune-regulatory effect of cGAMP that can be harnessed to restrain T cell autoreactivity.

Historical Perspective of Clinical Nano and Microparticle Formulations for Delivery of Therapeutics.

Nano and micro-technologies are used for therapeutic delivery of biologics and small molecules in formulations ranging in size from one nanometer to 100 microns or more. Here we review the unique physiochemical properties of these technologies and how they lead to more beneficial drug pharmacokinetics and toxicity over conventional formulations.

Considerations for Size, Surface Charge, Polymer Degradation, Co-Delivery, and Manufacturability in the Development of Polymeric Particle Vaccines for Infectious Diseases.

Vaccines have advanced human health for centuries. To improve upon the efficacy of subunit vaccines they have been formulated into nano/microparticles for infectious diseases. Much progress in the field of polymeric particles for vaccine formulation has been made since the push for a tetanus vaccine in the 1990s. Modulation of particle properties such as size, surface charge, degradation rate, and the co-delivery of antigen and adjuvant has been used. This review focuses on advances in the understanding of how these properties influence immune responses to injectable polymeric particle vaccines. Consideration is also given to how endotoxin, route of administration, and other factors influence conclusions that can be made. Current manufacturing techniques involved in preserving vaccine efficacy and scale-up are discussed, as well as those for progressing polymeric particle vaccines toward commercialization. Consideration of all these factors should aid the continued development of efficacious and marketable polymeric particle vaccines.

Vaccine formulations in clinical development for the prevention of severe acute respiratory syndrome coronavirus 2 infection.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an unprecedented effort toward the development of an effective and safe vaccine. Aided by extensive research efforts into characterizing and developing countermeasures towards prior coronavirus epidemics, as well as recent developments of diverse vaccine platform technologies, hundreds of vaccine candidates using dozens of delivery vehicles and routes have been proposed and evaluated preclinically. A high demand coupled with massive effort from researchers has led to the advancement of at least 31 candidate vaccines in clinical trials, many using platforms that have never before been approved for use in humans. This review will address the approach and requirements for a successful vaccine against SARS-CoV-2, the background of the myriad of vaccine platforms currently in clinical trials for COVID-19 prevention, and a summary of the present results of those trials. It concludes with a perspective on formulation problems which remain to be addressed in COVID-19 vaccine development and antigens or adjuvants which may be worth further investigation.

Merozoite surface protein 2 adsorbed onto acetalated dextran microparticles for malaria vaccination.

Malaria remains a global health threat, with significant morbidity and mortality worldwide despite current interventions. The human disease is caused by five different parasitic species, with Plasmodium falciparum being the deadliest. As a result, vaccine research against P. falciparum is a global priority. Merozoite surface protein 2 (MSP2) is a promising vaccine antigen as MSP2-specific antibodies have been shown previously to be protective against malaria infection. In this study, the formulation of an MSP2 vaccine was explored to enhance antigen uptake and achieve both an antibody and Th1 immune response by adsorbing MSP2 antigen onto a biomaterial carrier system. Specifically, MSP2 antigen was adsorbed onto acetalated dextran (Ace-DEX) microparticles (MPs). IgG and IgG2a titers elicited by the Ace-DEX MP platform were compared to titer levels elicited by MSP2 adsorbed to an FDA-approved alum adjuvant, MSP2 alone, and PBS alone. Both adsorption of MSP2 to Ace-DEX MPs and to alum elicited antibody responses in vivo, but only the formulation containing Ace-DEX MPs was able to elicit a significant Th1-biased response needed to combat the intracellular pathogen. As such, MSP2 adsorbed to Ace-DEX MPs demonstrates promise as a malaria vaccine.

Injectable, Ribbon-Like Microconfetti Biopolymer Platform for Vaccine Applications.

Previously, high-aspect- ratio ribbon-like microconfetti (MC) composed of acetalated dextran (Ace-DEX) have been shown to form a subcutaneous depot for sustained drug release. In this study, MC were explored as an injectable vaccine platform. Production of MC by electrospinning followed by high-shear homogenization allowed for precise control over MC fabrication. Three distinct sizes of MC, small (0.67 × 10.2 µm2), medium (1.28 × 20.7 µm2), and large (5.67 × 90.2 µm2), were fabricated and loaded with the adjuvant, resiquimod. Steady release rates of resiquimod were observed from MC, indicating their ability to create an immunostimulatory depot in vivo. Resiquimod-loaded MC stimulated inflammatory cytokine production in bone marrow-derived dendritic cells without incurring additional cytotoxicity in vitro. Interestingly, even medium and large MC were able to be internalized by antigen-presenting cells and facilitate antigen presentation when ovalbumin was adsorbed onto their surface. After subcutaneous injection in vivo with adsorbed ovalbumin, blank MC of all sizes were found to stimulate a humoral response. Adjuvant activity of resiquimod was enhanced by loading it into MC and small- and medium-sized MC effectively induced a Th1-skewed immune response. Antigen co-delivered with adjuvant-loaded MC of various sizes illustrates a new potential vaccine platform.