Efficient Antigen Delivery Research Articles

Nanoparticles in Subunit Vaccines: Immunological Foundations, Categories, and Applications.

Subunit vaccines, significant in next-generation vaccine development, offer precise targeting of immune responses by focusing on specific antigens. However, this precision often comes at the cost of eliciting strong and durable immunity, posing a great challenge to vaccine design. To address this limitation, recent advancements in nanoparticles (NPs) are utilized to enhance antigen delivery efficiency and boost vaccine efficacy. This review examines how the physicochemical properties of NPs influence various stages of the immune response during vaccine delivery and analyzes how different NP types contribute to immune activation and enhance vaccine performance. It then explores the unique characteristics and immune activation mechanisms of these NPs, along with their recent advancements, and highlights their application in subunit vaccines targeting infectious diseases and cancer. Finally, it discusses the challenges in NP-based vaccine development and proposes future directions for innovation in this promising field.

Unlocking Intracellular Protein Delivery by Harnessing Polymersomes Synthesized at Microliter Volumes using Photo-PISA.

Efficient delivery of therapeutic proteins and vaccine antigens to intracellular targets is challenging due to generally poor cell membrane permeation and endolysosomal entrapment causing degradation. Herein, these challenges are addressed by developing an oxygen-tolerant photoinitiated polymerization-induced self-assembly (Photo-PISA) process, allowing for the microliter-scale (10µL) synthesis of protein-loaded polymersomes directly in 1536-well plates. High-resolution techniques capable of analysis at a single particle level are employed to analyze protein encapsulation and release mechanisms. Using confocal microscopy and super-resolution stochastic optical reconstruction microscopy (STORM) imaging, their ability to deliver proteins into the cytosol following endosomal escape is subsequently visualized. Lastly, the adaptability of these polymersomes is exploited to encapsulate and deliver a prototype vaccine antigen, demonstrating its ability to activate antigen-presenting cells and support antigen cross-presentation for applications in subunit vaccines and cancer immunotherapy. This combination of ultralow volume synthesis and efficient intracellular delivery holds significant promise for unlocking the high throughput screening of a broad range of otherwise cost-prohibitive or early-stage therapeutic protein and vaccine antigen candidates that can be difficult to obtain in large quantities. The versatility of this platform for rapid screening of intracellular protein delivery can result in significant advancements across the fields of nanomedicine and biomedical engineering.

Bacterial ghosts engineered with lipidated antigens as an adjuvant-free vaccine for Chlamydia abortus

Bacterial ghosts (BGs) provide novel vaccine delivery platforms because of their inherent adjuvant properties and efficient antigen delivery capabilities. However, effective engineering strategies are required to modify them for different antigens. In this study, the Escherichia coli (E. coli) ghost was modified by using a lpp’-ompA chimera, a widely used bacterial surface display vector, with a protective antigen macrophage infectivity potentiator (MIP) of Chlamydia abortus (C. abortus), and its protective effect was evaluated in a mouse model. The MIP fusion protein accumulated at 1.2% of the ghost total protein mass and a significant portion of the protein was modified into lipoproteins upon translocation to the BG surface. Lipidated MIP-modified recombinant E. coli ghosts (rECG-lpp’-MIP) effectively promoted antigen-presenting cells (APCs) uptake of antigens and stimulated APCs activation in vivo and in vitro. Immunization with rECG-lpp’-MIP and no adjuvant induced intense specific humoral responses as well as Th1-biased cellular immune responses, which significantly improved the efficiency of C. abortus infection clearance in mice and reduced pathological damage to the uterus. In summary, this study demonstrates that recombinant E. coli ghosts modified with lipidated antigens could help to develop an effective C. abortus vaccine and aid in the development of a universal adjuvant-free vaccine platform.

A Magnetically Driven Biodegradable Microsphere with Mass Production Capability for Subunit Vaccine Delivery and Enhanced Immunotherapy.

Subunit vaccines have emerged as a promising strategy in immunotherapy for combating viral infections and cancer. Nevertheless, the clinical application of subunit vaccines is hindered by limitations in antigen delivery efficiency, characterized by rapid clearance and inadequate cellular uptake. Here, a novel subunit vaccine delivery system utilizing ovalbumin@magnetic nanoparticles (OVA@MNPs) encapsulated within biodegradable gelatin methacryloyl (GelMA) microspheres was proposed to enhance the efficacy of antigen delivery. OVA@MNPs-loaded GelMA microspheres, denoted as OMGMs, can be navigated through magnetic fields to deliver subunit vaccines into the lymphatic system efficiently. Moreover, the biodegradable OMGMs enabled the sustained release of subunit vaccines, concentrating OVA around lymph nodes and enhancing the efficacy of induced immune response. OMGMs were produced through a microfluidic droplet generation technique, enabling mass production. In murine models, OMGMs successfully accumulated antigens in lymph nodes abundant in antigen-presenting cells, leading to enhanced cellular and humoral immunity and pronounced antitumor effects with a single booster immunization. In conclusion, these findings highlight the promise of OMGMs as a practical subunit vaccination approach, thus addressing the limitations associated with antigen delivery efficiency and paving the way for advanced immunotherapeutic strategies.

Strategies for developing self-assembled nanoparticle vaccines against SARS-CoV-2 infection.

In the recent history of the SARS-CoV-2 outbreak, vaccines have been a crucial public health tool, playing a significant role in effectively preventing infections. However, improving the efficacy while minimizing side effects remains a major challenge. In recent years, there has been growing interest in nanoparticle-based delivery systems aimed at improving antigen delivery efficiency and immunogenicity. Among these, self-assembled nanoparticles with varying sizes, shapes, and surface properties have garnered considerable attention. This paper reviews the latest advancements in the design and development of SARS-CoV-2 vaccines utilizing self-assembled materials, highlighting their advantages in delivering viral immunogens. In addition, we briefly discuss strategies for designing a broad-spectrum universal vaccine, which provides insights and ideas for dealing with possible future infectious sarbecoviruses.

Endogenous capsid-forming protein ARC for self-assembling nanoparticle vaccines

The application of nanoscale scaffolds has become a promising strategy in vaccine design, with protein-based nanoparticles offering desirable avenues for the biocompatible and efficient delivery of antigens. Here, we presented a novel endogenous capsid-forming protein, activated-regulated cytoskeleton-associated protein (ARC), which could be engineered through the plug-and-play strategy (SpyCatcher3/SpyTag3) for multivalent display of antigens. Combined with the self-assembly capacity and flexible modularity of ARC, ARC-based vaccines elicited robust immune responses against Mpox or SARS-CoV-2, comparable to those induced by ferritin-based vaccines. Additionally, ARC-based nanoparticles functioned as immunostimulants, efficiently stimulating dendritic cells and facilitating germinal center responses. Even without adjuvants, ARC-based vaccines generated protective immune responses in a lethal challenge model. Hence, this study showed the feasibility of ARC as a novel protein-based nanocarrier for multivalent surface display of pathogenic antigens and demonstrated the potential of exploiting recombinant mammalian retrovirus-like protein as a delivery vehicle for bioactive molecules.Graphic

Stimulator of Interferon Genes-Activated Biomimetic Dendritic Cell Nanovaccine as a Chemotherapeutic Booster to Enhance Systemic Fibrosarcoma Treatment.

Fibrosarcoma, a malignant mesenchymal tumor, is characterized by aggressive invasiveness and a high recurrence rate, leading to poor prognosis. Anthracycline drugs, such as doxorubicin (DOX), represent the frontline chemotherapy for fibrosarcoma, but often exhibit suboptimal efficacy. Recently, exploiting the stimulator of interferon genes (STING)-mediated innate immunity has emerged as a hopeful strategy for cancer treatment. Integrating chemotherapy with immunomodulators in chemo-immunotherapy has shown potential for enhancing treatment outcomes. Herein, we introduce an advanced dendritic cell (DC) nanovaccine, cGAMP@PLGA@CRTM (GP@CRTM), combined with low-dose DOX to enhance fibrosarcoma chemo-immunotherapy. The nanovaccine consists of poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating the STING agonist 2,3-cGAMP (cGAMP@PLGA, GP) as its core, and a calreticulin (CRT) high-expressing fibrosarcoma cell membrane (CRTM) as the shell. Exposing CRT on the vaccine surface aids in recruiting DCs and stimulating uptake, facilitating efficient simultaneous delivery of STING agonists and tumor antigens to DCs. This dual delivery method effectively activates the STING pathway in DCs, triggering sustained immune stimulation. Simultaneously, low-dose DOX reduces chemotherapy-related side effects, directly kills a subset of tumor cells, and increases tumor immunogenicity, thus further amplifying immune therapeutic performance. Hence, these findings demonstrate the potential of DC nanovaccine GP@CRTM as a booster for chemotherapy. Synergistically combining low-dose DOX with the DC nanovaccine emerges as a powerful chemo-immunotherapy strategy, optimizing systemic fibrosarcoma therapy.

Nanovaccines: Immunogenic tumor antigens, targeted delivery, and combination therapy to enhance cancer immunotherapy.

Nanovaccines have been designed to overcome the limitations associated with conventional vaccines. Effective delivery methods such as engineered carriers or smart nanoparticles (NPs) are critical requisites for inducing self-tolerance and optimizing vaccine immunogenicity with minimum side effects. NPs can be used as adjuvants, immunogens, or nanocarriers to develop nanovaccines for efficient antigen delivery. Multiloaded nanovaccines carrying multiple tumor antigens along with immunostimulants can effectively increase immunity against tumor cells. They can be biologically engineered to boost interactions with dendritic cells and to allow a gradual and constant antigen release. Modifying NPs surface properties, using high-density lipoprotein-mimicking nanodiscs, and developing nano-based artificial antigen-presenting cells such as dendritic cell-derived-exosomes are amongst the new developed technologies to enhance antigen-presentation and immune reactions against tumor cells. The present review provides an overview on the different perspectives, improvements, and barriers of successful clinical application of current cancer therapeutic and vaccination options. The immunomodulatory effects of different types of nanovaccines and the nanoparticles incorporated into their structure are described. The advantages of using nanovaccines to prevent and treat common illnesses such as AIDS, malaria, cancer and tuberculosis are discussed. Further, potential paths to develop optimal cancer vaccines are described. Given the immunosuppressive characteristics of both cancer cells and the tumor microenvironment, applying immunomodulators and immune checkpoint inhibitors in combination with other conventional anticancer therapies are necessary to boost the effectiveness of the immune response.

An Overview of Nanoparticle-Based Delivery Platforms for mRNA Vaccines for Treating Cancer.

With its unique properties and potential applications, nanoparticle-based delivery platforms for messenger RNA (mRNA) vaccines have gained significant attention in recent years. Nanoparticles have the advantages of enhancing immunogenicity, targeting delivery, and improving stability, providing a new solution for drug and vaccine delivery. In some clinical studies, a variety of nanoparticle delivery platforms have been gradually applied to a wide range of vaccine applications. Current research priorities are exploring various types of nanoparticles as vaccine delivery systems to enhance vaccine stability and immunogenicity. Lipid nanoparticles (LNPs) have shown promising potential in preclinical and clinical studies on the efficient delivery of antigens to immune cells. Moreover, lipid nanoparticles and other nanoparticles for nucleic acids, especially for mRNA delivery systems, have shown vast potential for vaccine development. In this review, we present various vaccine platforms with an emphasis on nanoparticles as mRNA vaccine delivery vehicles. We describe several novel nanoparticle delivery platforms for mRNA vaccines, such as lipid-, polymer-, and protein-based nanoparticles. In addition, we provide an overview of the anti-tumor immunity of nanovaccines against different tumors in cancer immunotherapy. Finally, we outline future perspectives and remaining challenges for this promising technology of nanoparticle-based delivery platforms for vaccines.

Thermoresponsive nanosponges: Efficient antigen delivery carriers and adjuvants for in vivo vaccination

BackgroundVaccination is an effective disease prevention strategy involving immune cell stimulation and formation in response to foreign substances. However, the poor immunogenicity and deliverability of certain antigens necessitate novel methods to establish continuous immunity. Therefore, for efficient antigen delivering and simultaneously performing as a nanoadjuvant, herein, we prepared core (polylactic acid)-shell (poloxamer) thermoresponsive nanosponges (TNSs). MethodsBy simple nanoprecipitation, we prepared the thermoresponsive nanosponges (TNSs) with core-to-shell ratios of 1:2 and 1:20. Thereafter, antigens (ovalbumin, OVA) were loaded into TNSs by swelling behavior. ResultsThe TNSs were prepared with different sizes using differing core-to-shell ratios: TNS1:2 (160 nm) and TNS1:20 (67 nm). When loaded with the model antigen ovalbumin (OVA), TNS1:2 (289 nm) showed a seven-fold higher IL-2 secretion response cross-presentation ability than TNS1:20 (74 nm) and was used in subsequent experiments. TNS1:2 promoted dendritic cell maturation and exhibited high OVA encapsulation (optimal loading: 50 wt%), sufficient long-term stability, and controlled OVA release under physiological conditions. It was not toxic to dendritic cells even at high concentrations. Compared to free OVA, 50 wt% OVA loaded TNS1:2 exhibited better cellular uptake and a superior ability to elicit immune cell IL-2 secretion. The OVA antigen-specific activation of immune cells in mice was examined by monitoring serum IgG levels, cross-presentation to splenocyte CD8+ T-cells, and IFN-γ cytokine levels. ConclusionsThe results highlight TNSs as promising nanoadjuvants and carriers for antigen delivery in vaccine applications.

Comparative immunogenic and immunoprotective activities of PCV2d Cap and Rep antigens delivered by an efficient eukaryotic expression system engineered into a Salmonella vaccine vector

Porcine circovirus type 2 (PCV2) stands as a predominant etiological agent in porcine circovirus-associated diseases. To manage the spread of the disease, it is necessary to develop a next-generation vaccine expressing PCV2 antigens that target the prevailing genotype such as PCV2d. A bacterial-mediated vaccine delivery by live-attenuated Salmonella has attracted interest for its low-cost production and highly effective vaccine delivery. Thus, in this study, we utilized the advantages of the Salmonella-mediated vaccine delivery by cloning PCV2d cap and rep into a eukaryotic expression plasmid pJHL204 and electroporation into an engineered live-attenuated Salmonella Typhimurium JOL2500 (Δlon, ΔcpxR, ΔsifA, Δasd). The eukaryotic antigen expression by JOL2995 (p204:cap) and JOL2996 (p204:rep) was confirmed in vitro and in vivo which showed efficient antigen delivery. Furthermore, vaccination of mice model with the vaccine candidates elicited humoral and cell-mediated immune responses as depicted by high levels of PCV2-specific antibodies, CD4+ and CD8+ T cells, and neutralizing antibodies, especially by JOL2995 (p204:cap) which correlated with the significant decrease in the viral load in PCV2d-challenged mice. Interestingly, JOL2996 (p204:rep) may not have elicited high levels of neutralizing antibodies and protective efficacy, but it elicited considerably higher cell-mediated immune responses. This study demonstrated Salmonella-mediated vaccine delivery system coupled with the eukaryotic expression vector can efficiently deliver and express the target PCV2d antigens for strong induction of immune response and protective efficacy in mice model, further supporting the potential application of the Salmonella-mediated vaccine delivery system as an effective novel approach in vaccine strategies for PCV2d.

Metal-Free Atom Transfer Radical Polymerization to Prepare Recylable Micro-Adjuvants for Dendritic Cell Vaccine.

In the development of dendritic cell (DC) vaccines, the maturation of DCs is a critical stage. Adjuvants play a pivotal role in the maturation of DCs, with a major concern being to ensure both efficacy and safety. This study introduces an innovative approach that combines high efficacy with safety through the synthesis of micro-adjuvants grafted with copolymers of 2-(methacrylamido) glucopyranose (MAG) and methacryloxyethyl trimethyl ammonium chloride (DMC). The utilization of metal-free surface-initiated atom transfer radical polymerization enables the production of safe and recyclable adjuvants. These micrometer-sized adjuvants surpass the optimal size range for cellular endocytosis, enabling the retrieval and reuse of them during the ex vivo maturation process, mitigating potential toxicity concerns associated with the endocytosis of non-metabolized nanoparticles. Additionally, the adjuvants exhibit a "micro-ligand-mediated maturation enhancement" effect for DC maturation. This effect is influenced by the shape of the particle, as evidenced by the distinct promotion effects of rod-like and spherical micro-adjuvants with comparable sizes. Furthermore, the porous structure of the adjuvants enables them to function as cargo-carrying "micro-shuttles", releasing antigens upon binding to DCs to facilitate efficient antigen delivery.

Metal‐Free Atom Transfer Radical Polymerization to Prepare Recylable Micro‐Adjuvants for Dendritic Cell Vaccine

AbstractIn the development of dendritic cell (DC) vaccines, the maturation of DCs is a critical stage. Adjuvants play a pivotal role in the maturation of DCs, with a major concern being to ensure both efficacy and safety. This study introduces an innovative approach that combines high efficacy with safety through the synthesis of micro‐adjuvants grafted with copolymers of 2‐(methacrylamido) glucopyranose (MAG) and methacryloxyethyl trimethyl ammonium chloride (DMC). The utilization of metal‐free surface‐initiated atom transfer radical polymerization enables the production of safe and recyclable adjuvants. These micrometer‐sized adjuvants surpass the optimal size range for cellular endocytosis, enabling the retrieval and reuse of them during the ex vivo maturation process, mitigating potential toxicity concerns associated with the endocytosis of non‐metabolized nanoparticles. Additionally, the adjuvants exhibit a “micro‐ligand‐mediated maturation enhancement” effect for DC maturation. This effect is influenced by the shape of the particle, as evidenced by the distinct promotion effects of rod‐like and spherical micro‐adjuvants with comparable sizes. Furthermore, the porous structure of the adjuvants enables them to function as cargo‐carrying “micro‐shuttles”, releasing antigens upon binding to DCs to facilitate efficient antigen delivery.

Ultrasmall AuCe nanozyme adjuvant boosted ROS for three-pronged tumor immunotherapy

Catalytic tumor immunotherapy presents a new combination of nanozymes and immunotherapy agents, but the final therapeutic outcomes are usually diminished by insufficient nanozyme activity, immunosuppressive tumor microenvironment (TME), and poor antigen delivery efficiency. Herein, a bimetallic nanozyme-based tumor immunotherapy nanovaccine is first proposed to address these limitations by attenuating the immunosuppression of TME and effectively delivering tumor-specific antigens and catalytic tumor nanomedicine. The tumor-specific antigen OVA-templated bimetallic AuCe nanozyme (denoted as ACO) is a nanodot composed of Au cluster and CeOx. The high Ce3+/Ce4+ ratio and charge transfer from Au to Ce synergistically enhance the peroxidase-like activity of ACO. The boosted reactive oxygen species level inhibits the proliferation of B16-OVA cells and simultaneously promotes the maturation of dendritic cells. The functionalization of PD-L1 inhibitory D-peptide on ACO (named ACOD) increases tumor targeting specificity and prolongs tumor site retention, facilitating apoptosis of primary and distant tumor cells and conquering immune evasion. Moreover, ACOD catalytic nanovaccine induces long-term immune memory against tumor recurrence and lung metastasis. Therefore, the bimetallic catalytic nanovaccine is a promising nanoformulation to eradicate cancer and safeguard against tumor relapse.

MRNA Vaccine Nanoplatforms and Innate Immunity.

mRNA-based vaccine technology has been significantly developed and enhanced, particularly highlighted by the authorization of mRNA vaccines for addressing the COVID-19 pandemic. Various biomaterials are developed in nano-scales and applied as mRNA vaccine delivery platforms. However, how these mRNA nanoplatforms influence immune responses has not been thoroughly studied. Hence, we have reviewed the current understanding of various mRNA vaccine platforms. We discussed the possible pathways through which these platforms moderate the host's innate immunity and contribute to the development of adaptive immunity. We shed light on their development in reducing biotoxicity and enhancing antigen delivery efficiency. Beyond the built-in adjuvanticity of mRNA vaccines, we propose that supplementary adjuvants may be required to fine-tune and precisely control innate immunity and subsequent adaptive immune responses.

Self-adjuvant Astragalus polysaccharide-based nanovaccines for enhanced tumor immunotherapy: a novel delivery system candidate for tumor vaccines.

The study of tumor nanovaccines (NVs) has gained interest because they specifically recognize and eliminate tumor cells. However, the poor recognition and internalization by dendritic cells (DCs) and insufficient immunogenicity restricted the vaccine efficacy. Herein, we extracted two molecular-weight Astragalus polysaccharides (APS, 12.19 kD; APSHMw, 135.67 kD) from Radix Astragali and made them self-assemble with OVA257-264 directly forming OVA/APS integrated nanocomplexes through the microfluidic method. The nanocomplexes were wrapped with a sheddable calcium phosphate layer to improve stability. APS in the formed nanocomplexes served as drug carriers and immune adjuvants for potent tumor immunotherapy. The optimal APS-NVs were approximately 160 nm with uniform size distribution and could remain stable in physiological saline solution. The FITC-OVA in APS-NVs could be effectively taken up by DCs, and APS-NVs could stimulate the maturation of DCs, improving the antigen cross-presentation efficiency in vitro. The possible mechanism was that APS can induce DC activation via multiple receptors such as dectin-1 and Toll-like receptors 2 and 4. Enhanced accumulation of APS-NVs both in draining and distal lymph nodes were observed following s.c. injection. Smaller APS-NVs could easily access the lymph nodes. Furthermore, APS-NVs could markedly promote antigen delivery efficiency to DCs and activate cytotoxic T cells. In addition, APS-NVs achieve a better antitumor effect in established B16-OVA melanoma tumors compared with the OVA+Alum treatment group. The antitumor mechanism correlated with the increase in cytotoxic T cells in the tumor region. Subsequently, the poor tumor inhibitory effect of APS-NVs on the nude mouse model of melanoma also confirmed the participation of antitumor adaptive immune response induced by NVs. Therefore, this study developed a promising APS-based tumor NV that is an efficient tumor immunotherapy without systemic side effects.

Zinc-Organometallic Framework Vaccine Controlled-Release Zn2+ Regulates Tumor Extracellular Matrix Degradation Potentiate Efficacy of Immunotherapy.

Tumor extracellular matrix (ECM) not only forms a physical barrier for T cells infiltration, but also regulates multiple immunosuppressive pathways, which is an important reason for immunotherapy failure. The cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway plays a key role in activating CD8+ T cells, maintaining CD8+ T cells stemness and enhancing the antitumor effect. Herein, a zinc-organometallic framework vaccine (ZPM@OVA-CpG) prepared by self-assembly, which achieves site-directed release of Zn2+ in dendritic cell (DC) lysosomes and tumor microenvironment under acidic conditions, is reported. The vaccine actively targets DC, significantly enhances cGAS-STING signal, promotes DC maturation and antigen cross-presentation, and induces strong activation of CD8+ T cells. Meanwhile, the vaccine reaches the tumor site, releasing Zn2+ , significantly up-regulates the activity of matrix metalloproteinase-2, degrades various collagen components of tumor ECM, effectively alleviates immune suppression, and significantly enhances the tumor infiltration and killing of CD8+ T cells. ZPM@OVA-CpG vaccine not only solves the problem of low antigen delivery efficiency and weak CD8+ T cells activation ability, but also achieves the degradation of tumor ECM via the vaccine for the first time, providing a promising therapeutic platform for the development of efficient novel tumor vaccines.

Promoting immunity with novel targeting antigen delivery vehicle based on bispecific nanobody.

As the most potent professional antigen presenting cells, dendritic cells (DCs) have been targeted in strategies to enhance vaccination efficacy. To date, targeted delivery has been mainly used for cancer therapy, with few studies focusing on vaccine antigens for animal epidemic diseases. In this study, we selected a series of mouse DC-specific nanobodies from a non-immunized camel. The four candidate nanobodies identified (Nb4, Nb13, Nb17, and Nb25), which showed efficient endocytosis of bone marrow-derived DCs, were evaluated as potential vaccine antigen targeted delivery vehicles. First, green fluorescent protein (GFP) was selected and four corresponding DCNb-GFP fusions were constructed for verification. Nb17-GFP was effective at promoting antibody production, inducing a cellular immune response, and increasing the IL-4 level. Second, foot-and-mouth disease virus (FMDV) and a FMDV-specific nanobody (Nb205) were selected and four bispecific nanobody DCNb-Nb205 fusions were generated to investigate the feasibility of a novel targeting antigen delivery vehicle. The resulting bispecific nanobody, Nb17-Nb205, could not only deliver FMDV particles instead of antigenic peptide, but also induced the production of specific antibodies, a cellular immune response, and IFN-γ and IL-4 levels upon immunization with a single subcutaneous injection. In conclusion, our results demonstrate the potential of bispecific nanobody as a novel and efficient DC-specific antigen delivery vehicle. This highlights the potential to expand targeted delivery to the field of animal epidemic diseases and provides a reference for the general application of nanotechnology in viral diseases.

Engineered Curli Nanofilaments as a Self-Adjuvanted Antigen Delivery Platform.

Proteinaceous nanoparticles constitute efficient antigen delivery systems in vaccine formulations due to their size and repetitive nature that mimic most invading pathogens and promote immune activation. Nonetheless, the coadministration of an adjuvant with subunit nanovaccines is usually required to induce a robust, long-lasting, and protective immune response. Herein, the protein Curli-specific gene A (CsgA), which is known to self-assemble into nanofilaments contributing to bacterial biofilm, is exploited to engineer an intrinsically immunostimulatory antigen delivery platform. Three repeats of the M2e antigenic sequence from the influenza A virus matrix 2 protein are merged to the N-terminal domain of engineered CsgA proteins. These chimeric 3M2e-CsgA spontaneously self-assemble into antigen-displaying cross-β-sheet nanofilaments that activate the heterodimeric toll-like receptors 2 and 1. The resulting nanofilaments are avidly internalized by antigen-presenting cells and stimulate the maturation of dendritic cells. Without the need of any additional adjuvants, both assemblies show robust humoral and cellular immune responses, which translate into complete protection against a lethal experimental infection with the H1N1 influenza virus. Notably, these CsgA-based nanovaccines induce neither overt systemic inflammation, nor reactogenicity, upon mice inoculation. These results highlight the potential of engineered CsgA nanostructures as self-adjuvanted, safe, and versatile antigen delivery systems to fight infectious diseases.

Asymmetric Assembly and Self-Adjuvanted Antigen Delivery Platform for Improved Antigen Uptake and Antitumor Effect

The development of effective tumor vaccines is an important direction in the field of cancer prevention/immunotherapy. Efficient antigen delivery is essential for inducing effective antitumor responses for tumor vaccines. Lumazine synthase (BLS) from Brucella spp. is a decameric protein with delivery and adjuvant properties, but its application in tumor vaccines is limited. Here, we developed an antigen delivery platform by combining a BLS asymmetric assembly and the Plug-and-Display system of SpyCatcher/SpyTag. An asymmetric assembly system consisting of BLSke and BLSdr was developed to equally assemble two molecules. Then, the MHC-I-restricted ovalbumin peptide (OVA(257-264) SIINFEKL) was conjugated with BLSke, and a cell-penetrating peptide (CPP) KALA was conjugated with BLSdr using the SpyCatcher/SpyTag system. KALA modification enhanced internalization of OVA peptides by DCs as well as promoted the maturation of DCs and the cross-presentation of SIINFEKL. Moreover, the immunotherapy of a KALA-modified vaccine suppressed tumor growth and enhanced CD8+ T cell responses in E.G7-OVA tumor-bearing mice. In the prophylactic model, KALA-modified vaccination showed the most significant protective effect and significantly prolonged the survival period of tumor challenged mice. In conclusion, the asymmetric assembly platform equally assembles two proteins or peptides, avoiding their spatial or functional interference. This asymmetric assembly and Plug-and-Display technology provide a universal platform for rapid development of personalized tumor vaccines.