Methods for Analysis of Nanoparticle Immunosuppressive Properties.

Adverse drug effects on immune system function represent a significant concern in the pharmaceutical industry, because 10-20% of drug withdrawal from the market is attributed to immunotoxicity. Immunosuppression is one such adverse effect. The traditional immune function test used to estimate materials' immunosuppression is T cell dependent antibody response (TDAR). This method involves a 28-day in vivo study evaluating the animal's antibody titer to a known antigen (Keyhole Limpet Hemocyanin; KLH) with and without challenge. Due to the limited quantities of novel drug candidates, an in vitro method called human lymphocyte activation (HuLA) assay has been developed to substitute the traditional TDAR assay during early preclinical development. In this test, leukocytes isolated from healthy donors vaccinated with the current year's flu vaccine are incubated with Fluzone in the presence or absence of nanoparticles. The antigen-specific lymphocyte proliferation is then measured by ELISA analyzing incorporation of BrdU into DNA of the proliferating cells. Here we describe the experimental procedures for investigating immunosuppressive properties of nanoparticles by both TDAR and HuLA assays, discuss the in vitro-in vivo correlation of these methods, and show a case study using the iron oxide nanoparticle formulation, Feraheme.

Hybrid mRNA Nano Vaccine Potentiates Antigenic Peptide Presentation and Dendritic Cell Maturation for Effective Cancer Vaccine Therapy and Enhances Response to Immune Checkpoint Blockade.

Cancer vaccines combined with immune checkpoint blockades (ICB) represent great potential application, yet the insufficient tumor antigen presentation and immature dendritic cells hinder improved efficacy. Here, a hybrid nano vaccine composed by hyper branched poly(beta-amino ester), modified iron oxide nano adjuvant and messenger RNA (mRNA) encoded with model antigen ovalbumin (OVA) is presented. The nano vaccine outperforms three commercialized reagents loaded with the same mRNA, including Lipofectamine MessengerMax, jetPRIME, and in vivo-jetRNA in promoting dendritic cells' transfection, maturation, and peptide presentation. In an OVA-expressing murine model, intratumoral administration of the nano vaccine significantly induced macrophages and dendritic cells' presenting peptides and expressing co-stimulatory CD86. The nano vaccine also elicited strong antigen-specific splenocyte response and promoted CD8+ T cell infiltration. In combination with ICB, the nano vaccine aroused robust tumor suppression in murine models with large tumor burdens (initial volume >300 mm3 ). The hybrid mRNA vaccine represents a versatile and readily transformable platform and augments response to ICB.

Preparation and Evaluation of Polyacrylate Microgels and Their Adjuvant Activities Using Ovalbumin as a Model Antigen.

As vaccine adjuvants, polyacrylate materials can induce a specific immune response in the body and have been widely studied in recent years due to their advantages, such as their safety, effectiveness, and low required dosage. In this study, a series of polyacrylates with hydrophobic physical crosslinking and chemical crosslinking were prepared using precipitation polymerization, and their structures were characterized by nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectroscopy. The optimal reaction conditions were determined according to the effect of reaction time, azodiisobutyronitrile, Span 60, allyl pentaerythritol, and octadecyl methacrylate (OMA) contents on the viscosity of the polyacrylate microgel, combined with the effects of allyl pentaerythritol and OMA contents on the subcutaneous immune safety of the polyacrylate microgel in BALB/c mice. The polyacrylate microgels with different OMA contents showed good biological safety. In addition, in vivo immunity experiments were carried out in mice to analyze the adjuvant properties of ovalbumin as a model antigen. Based on the titer results of the IgG1 and IgG2a antibodies, with 1 wt % OMA content, the polyacrylate microgel vaccine could optimally induce the body to produce an immune response type dominated by Th2-type humoral immune response and supplemented by Th1-type cellular immune response.

Effect of Antigen Structure in Subunit Vaccine Nanoparticles on Humoral Immune Responses.

Subunit vaccines offer numerous attractive features, including good safety profiles and well-defined components with highly characterized properties because they do not contain whole pathogens. However, vaccine platforms based on one or few selected antigens are often poorly immunogenic. Several advances have been made in improving the effectiveness of subunit vaccines, including nanoparticle formulation and/or co-administration with adjuvants. Desolvation of antigens into nanoparticles is one approach that has been successful in eliciting protective immune responses. Despite this advance, damage to the antigen structure by desolvation can compromise the recognition of conformational antigens by B cells and the subsequent humoral response. Here, we used ovalbumin as a model antigen to demonstrate enhanced efficacy of subunit vaccines by preserving antigen structures in nanoparticles. An altered antigen structure due to desolvation was first validated by GROMACS and circular dichroism. Desolvant-free nanoparticles with a stable ovalbumin structure were successfully synthesized by directly cross-linking ovalbumin or using ammonium sulfate to form nanoclusters. Alternatively, desolvated OVA nanoparticles were coated with a layer of OVA after desolvation. Vaccination with salt-precipitated nanoparticles increased OVA-specific IgG titers 4.2- and 22-fold compared to the desolvated and coated nanoparticles, respectively. In addition, enhanced affinity maturation by both salt precipitated and coated nanoparticles was displayed in contrast to desolvated nanoparticles. These results demonstrate both that salt-precipitated antigen nanoparticles are a potential new vaccine platform with significantly improved humoral immunity and a functional value of preserving antigen structures in vaccine nanoparticle design.

Endogenous/Exogenous Nanovaccines Synergistically Enhance Dendritic Cell-Mediated Tumor Immunotherapy.

Traditional dendritic cell (DC)-mediated immunotherapy is usually suppressed by weak immunogenicity in tumors and generally leads to unsatisfactory outcomes. Synergistic exogenous/endogenous immunogenic activation can provide an alternative strategy for evoking a robust immune response by promoting DC activation. Herein, Ti3 C2 MXene-based nanoplatforms (termed MXP) are prepared with high-efficiency near-infrared photothermal conversion and immunocompetent loading capacity to form endogenous/exogenous nanovaccines. Specifically, the immunogenic cell death of tumor cells induced by the photothermal effects of the MXP can generate endogenous danger signals and antigens release to boost vaccination for DC maturation and antigen cross-presentation. In addition, MXP can deliver model antigen ovalbumin (OVA) and agonists (CpG-ODN) as an exogenous nanovaccine (MXP@OC), which further enhances DC activation. Importantly, the synergistic strategy of photothermal therapy and DC-mediated immunotherapy by MXP significantly eradicates tumors and enhances adaptive immunity. Hence, the present work provides a two-pronged strategy for improving immunogenicity and killing tumor cells to achieve a favorable outcome in tumor patients.

Tyrosinase-Related Protein2 Peptide with Replacement of N-Terminus Residue by Cysteine Binds to H-2Kb and Induces Antigen-Specific Cytotoxic T Lymphocytes after Conjugation with CpG-DNA.

Recent studies have shown the potent efficacy of peptide-based vaccines for cancer immunotherapy. Immunological performance is optimized through the co-delivery of adjuvant and antigenic peptide molecules to antigen-presenting cells simultaneously. In our previous study, we showed that a conjugate consisting of 40-mer CpG-DNA and an antigenic ovalbumin peptide through disulfide bonding could efficiently induce ovalbumin-specific cytotoxic T lymphocyte (CTL) responses in vivo. In this study, based on the conjugation design, we prepared a conjugate consisting of 30-mer CpG-DNA (CpG30) and a cancer antigenic peptide of Tyrosinase-related protein 2 (TRP2180-188) using a cysteine residue attached at the N-terminus of TRP2180-188. However, the immunization of mice with this conjugate did not induce efficient TRP2180-188-specific immune responses. It was thought that the resultant peptide (10-mer) cleaved from the conjugate might be too long to fit into the H-2Kb molecule because the optimal length for binding to it is 8-9 amino acids. We newly designed a conjugate consisting of CpG30 and the C-TRP2181-188 peptide (9-mer), in which the N-terminal serine residue of TRP2180-188 is replaced by a cysteine. By adjusting the peptide length, we succeeded in inducing strong TRP2180-188 peptide-specific CTL activity upon immunization with the CpG30-C-TRP2181-188 conjugate. Furthermore, various CpG30-C-TRP2181-188 conjugates having other CpG-DNA sequences or cysteine analogues also induced the same level of CTL activity. Therefore, CpG-C-peptide conjugates prepared by replacement of the amino acid residue at the N-terminus with a cysteine residue could be a new and effective platform for peptide vaccines for targeting specific antigens of cancers and infectious diseases.

Biomaterial-assisted local oxygenation safeguards the prostimulatory phenotype and functions of human dendritic cells in hypoxia.

Dendritic cells (DCs), professional antigen-presenting cells, function as sentinels of the immune system. DCs initiate and fine-tune adaptive immune responses by presenting antigenic peptides to B and T lymphocytes to mount an effective immune response against cancer and pathogens. However, hypoxia, a condition characterized by low oxygen (O2) tension in different tissues, significantly impacts DC functions, including antigen uptake, activation and maturation, migration, as well as T-cell priming and proliferation. In this study, we employed O2-releasing biomaterials (O2-cryogels) to study the effect of localized O2 supply on human DC phenotype and functions. Our results indicate that O2-cryogels effectively mitigate DC exposure to hypoxia under hypoxic conditions. Additionally, O2-cryogels counteract hypoxia-induced inhibition of antigen uptake and migratory activity in DCs through O2 release and hyaluronic acid (HA) mediated mechanisms. Furthermore, O2-cryogels preserve and restore DC maturation and co-stimulation markers, including HLA-DR, CD86, and CD40, along with the secretion of proinflammatory cytokines in hypoxic conditions. Finally, our findings demonstrate that the supplemental O2 released from the cryogels preserves DC-mediated T-cell priming, ultimately leading to the activation and proliferation of allogeneic CD3+ T cells. This work emphasizes the potential of local oxygenation as a powerful immunomodulatory agent to improve DC activation and functions in hypoxia, offering new approaches for cancer and infectious disease treatments.

Transplant Tolerance, Not Only Clonal Deletion.

The quest to understand how allogeneic transplanted tissue is not rejected and how tolerance is induced led to fundamental concepts in immunology. First, we review the research that led to the Clonal Deletion theory in the late 1950s that has since dominated the field of immunology and transplantation. At that time many basic mechanisms of immune response were unknown, including the role of lymphocytes and T cells in rejection. These original observations are reassessed by considering T regulatory cells that are produced by thymus of neonates to prevent autoimmunity. Second, we review "operational tolerance" induced in adult rodents and larger animals such as pigs. This can occur spontaneously especially with liver allografts, but also can develop after short courses of a variety of rejection inhibiting therapies. Over time these animals develop alloantigen specific tolerance to the graft but retain the capacity to reject third-party grafts. These animals have a "split tolerance" as peripheral lymphocytes from these animals respond to donor alloantigen in graft versus host assays and in mixed lymphocyte cultures, indicating there is no clonal deletion. Investigation of this phenomenon excludes many mechanisms, including anti-donor antibody blocking rejection as well as anti-idiotypic responses mediated by antibody or T cells. This split tolerance is transferred to a second immune-depleted host by T cells that retain the capacity to effect rejection of third-party grafts by the same host. Third, we review research on alloantigen specific inhibitory T cells that led to the first identification of the CD4+CD25+T regulatory cell. The key role of T cell derived cytokines, other than IL-2, in promoting survival and expansion of antigen specific T regulatory cells that mediate transplant tolerance is reviewed. The precise methods for inducing and diagnosing operational tolerance remain to be defined, but antigen specific T regulatory cells are key mediators.

Evaluation of the therapeutic efficacy of human bone marrow mesenchymal stem cells with COX-2 silence and TGF-ß3 overexpression in rabbits with antigen-induced arthritis.

OBJECTIVE: Mesenchymal stem cells (MSCs), especially genetically modified MSCs, have become a promising therapeutic approach for the treatment of rheumatoid arthritis (RA) through modulating immune responses. However, most MSCs used in the treatment of RA are modified based on a single gene. In this study, we evaluated the therapeutic effects of human BMSCs (hBMSCs) with COX-2 silence and TGF-ß3 overexpression in the treatment of RA in a rabbit model. MATERIALS AND METHODS: hBMSCs were cotransfected with shCOX-2 and TGF-ß3 through lentiviral vector delivery. After SPIO-Molday ION Rhodamine-B™ (MIRB) labeling, lenti-shCOX2-TGF-ß3 hBMSCs, lenti-shCOX2 hBMSCs, lenti-TGF-ß3 hBMSCs, hBMSCs without genetic modification, or phosphate-buffered saline (PBS) were injected into the knee joint of rabbits with antigen-induced arthritis (AIA). The diameter of the knee joint and soft-tissue swelling score (STS) were recorded, and the levels of inflammatory mediators, including interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) were evaluated by ELISA. Clinical 3.0T MR imaging (MRI) was used to track the distribution and dynamic migration of hBMSCs in the joint. Histopathological and immunohistochemical assays were conducted to localize labeled hBMSCs and assess the alteration of synovial hyperplasia, inflammatory cell infiltration, and cartilage damage. RESULTS: COX-2 silencing and TGF-ß3 overexpression in hBMSCs were confirmed through real-time PCR and Western blot analyses. Reduced joint diameter, soft-tissue swelling (STS) score, and PGE2, IL-1ß, and TNF-α expression were detected 4 weeks after injection of MIRB-labeled lenti-shCOX2-TGF-ß3 hBMSCs into the joint in rabbits with AIA. Eight weeks after hBMSC injection, reduced inflammatory cell infiltration, improved hyperplasia of the synovial lining, recovered cartilage damage, and increased matrix staining were observed in joints injected with lenti-shCOX2-TGF-ß3 hBMSCs and lenti-shCOX2 hBMSCs. Slight synovial hyperplasia, no surface fibrillation, and strong positive expression of collagen II staining in chondrocytes and cartilage matrix were detected in the joints 12 weeks after injection of lenti-shCOX2-TGF-ß3 hBMSCs. In addition, hBMSCs were detected by MRI imaging throughout the process of hBMSC treatment. CONCLUSION: Intra-articular injection of hBMSCs with COX-2 silence and TGFß3 overexpression not only significantly inhibited joint inflammation and synovium hyperplasia, but also protected articular cartilage at the early stage. In addition, intra-articular injection of hBMSCs with COX-2 silence and TGFß3 overexpression promoted chondrocyte and matrix proliferation. This study provides an alternative therapeutic strategy for the treatment of RA using genetically modified hBMSCs.

Tolerance Induced by Antigen-Loaded PLG Nanoparticles Affects the Phenotype and Trafficking of Transgenic CD4+ and CD8+ T Cells.

We have shown that PLG nanoparticles loaded with peptide antigen can reduce disease in animal models of autoimmunity and in a phase 1/2a clinical trial in celiac patients. Clarifying the mechanisms by which antigen-loaded nanoparticles establish tolerance is key to further adapting them to clinical use. The mechanisms underlying tolerance induction include the expansion of antigen-specific CD4+ regulatory T cells and sequestration of autoreactive cells in the spleen. In this study, we employed nanoparticles loaded with two model peptides, GP33-41 (a CD8 T cell epitope derived from lymphocytic choriomeningitis virus) and OVA323-339 (a CD4 T cell epitope derived from ovalbumin), to modulate the CD8+ and CD4+ T cells from two transgenic mouse strains, P14 and DO11.10, respectively. Firstly, it was found that the injection of P14 mice with particles bearing the MHC I-restricted GP33-41 peptide resulted in the expansion of CD8+ T cells with a regulatory cell phenotype. This correlated with reduced CD4+ T cell viability in ex vivo co-cultures. Secondly, both nanoparticle types were able to sequester transgenic T cells in secondary lymphoid tissue. Flow cytometric analyses showed a reduction in the surface expression of chemokine receptors. Such an effect was more prominently observed in the CD4+ cells rather than the CD8+ cells.

Loading Effect of Chitosan Derivative Nanoparticles on Different Antigens and Their Immunomodulatory Activity on Dendritic Cells.

Drug carrier nanoparticles (NPs) were prepared by the polyelectrolyte method, with chitosan sulfate, with different substituents and quaternary ammonium chitosan, including C236-HACC NPs, C36-HACC NPs, and C6-HACC NPs. To evaluate whether the NPs are suitable for loading different antigens, we chose bovine serum albumin (BSA), ovalbumin (OVA), and myoglobin (Mb) as model antigens to investigate the encapsulation effect of the NPs. The characteristics (size, potential, and encapsulation efficiency) of the NPs were measured. Moreover, the NPs with higher encapsulation efficiency were selected for the immunological activity research. The results showed that chitosan derivative NPs with different substitution sites had different loading effects on the three antigens, and the encapsulation rate of BSA and OVA was significantly better than that of Mb. Moreover, the NPs encapsulated with different antigens have different immune stimulating abilities to DCS cells, the immune effect of OVA-coated NPs was significantly better than that of BSA-coated NPs and blank NPs, especially C236-HACC-OVA NPs. Furthermore, we found that C236-HACC-OVA NPs could increase the phosphorylation level of intracellular proteins to activate cell pathways. Therefore, C236-HACC NPs are more suitable for the loading of antigens similar to the OVA structure.

A novel form of immunotherapy using antigen peptides conjugated on PD-L1 antibody.

Immune checkpoint inhibitors (ICIs), including programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 have shown promising cancer clinical outcomes. However, IC therapy has low patient response rates (10%-15%). Thus, ICIs and sufficient antigen combinations into the tumor microenvironment (TME) is important to produce strong tumor-specific adaptive immune responses. Mice were treated with cisplatin, and human cancer cells were exposed to inflammatory cytokines, to confirm increased PD-L1 and major histocompatibility complex (MHC) I expression by tumor cells or dendritic cells. TC-1, CT26, B16-F1, or B16-F10 tumor cells, and bone marrow-derived dendritic cells, were treated with interferon (IFN)-ß, IFN-γ, or tumor necrosis factor-α to identify the molecular mechanisms underlying tumor PD-L1 and MHC I upregulation, and to examine MHC I, CD40, CD80, CD86, or PD-L1 levels, respectively. For synergistic combination therapy, αPD-L1 monoclonal antibody (mAb) covalently linked to the long E7 peptide was generated. Chemotherapy shifted the TME to express high PD-L1 and MHC I, resulting in targeted ICI cargo delivery and enhanced generation and activation of tumor antigen-specific T cells. Synergistic effects of vaccination and IC blockade in the TME were demonstrated using an anti-PD-L1 mAb covalently conjugated to the E7 long peptide.

Transcriptome Signature of Vγ9Vδ2 T Cells Treated With Phosphoantigens and Notch Inhibitor Reveals Interplay Between TCR and Notch Signaling Pathways.

Gamma delta (γδ) T cells, especially the Vγ9Vδ2 subtype, have been implicated in cancer therapy and thus have earned the spotlight in the past decade. Although one of the most important properties of γδ T cells is their activation by phosphoantigens, which are intermediates of the Mevalonate and Rohmer pathway of isoprenoid biosynthesis, such as IPP and HDMAPP, respectively, the global effects of such treatments on Vγ9Vδ2 T cells remain elusive. Here, we used the high-throughput transcriptomics approach to elucidate the transcriptional changes in human Vγ9Vδ2 T cells upon HDMAPP, IPP, and anti-CD3 treatments in combination with interleukin 2 (IL2) cytokine stimulation. These activation treatments exhibited a dramatic surge in transcription with distinctly enriched pathways. We further assessed the transcriptional dynamics upon inhibition of Notch signaling coupled with activation treatments. We observed that the metabolic processes are most affected upon Notch inhibition via GSI-X. The key effector genes involved in gamma-delta cytotoxic function were downregulated upon Notch blockade even in combination with activation treatment, suggesting a transcriptional crosstalk between T-cell receptor (TCR) signaling and Notch signaling in Vγ9Vδ2 T cells. Collectively, we demonstrate the effect of the activation of TCR signaling by phosphoantigens or anti-CD3 on the transcriptional status of Vγ9Vδ2 T cells along with IL2 stimulation. We further show that the blockade of Notch signaling antagonistically affects this activation.

Dendritic cells support a proliferative antigen-specific T-cell response in the presence of poly(lactic-co-glycolic acid).

Biomaterials are known to modulate immune cell functions, which subsequently determine the host inflammatory and immune responses. Poly(lactic-co-glycolic acid) or PLGA, a biodegradable and biocompatible biomaterial, induces a pro-inflammatory, mature phenotype in antigen presentation cells, namely dendritic cells (DCs) in vitro. In vivo, PLGA can boost the humoral immune response to a co-delivered model antigen, a phenomenon known as the PLGA-adjuvant effect. This study elucidates the link between PLGA's effect on the DC phenotype in vitro and its adjuvant effect in vivo using the CD11c-DTR mouse model. These mice undergo conditional ablation of DCs upon treatment with diphtheria toxin. To measure immune activation, the mice were first given ovalbumin (OVA)-reactive T cells from OT-II/OT-I mice. Later, the same mice received subcutaneous OVA-loaded PLGA scaffold implants. In response to the scaffold implants, OVA-reactive OT-II CD4+ T cells showed decreased proliferation in the absence of CD11c+ DCs, indicating an attenuation of the PLGA-adjuvant effect. Furthermore, PLGA may also influence the antigen cross-presentation function of DCs, as evident with the lowered OVA-reactive OT-I CD8+ T-cell response. Understanding the immunomodulatory ability of biomaterials in the context of DCs will aid in designing improved DC-based immunotherapies against infectious diseases and cancer.

Carboxymethyl-ß-glucan/chitosan nanoparticles: new thermostable and efficient carriers for antigen delivery.

In the last few decades, nanotechnology has emerged as an important tool aimed at enhancing the immune response against modern antigens. Nanocarriers designed specifically for this purpose have been shown to provide protection, stability, and controlled release properties to proteins, peptides, and polynucleotide-based antigens. Polysaccharides are particularly interesting biomaterials for the construction of these nanocarriers given their wide distribution among pathogens, which facilitates their recognition by antigen-presenting cells (APCs). In this work, we focused on an immunostimulant beta-glucan derivative, carboxymethyl-ß-glucan, to prepare a novel nanocarrier in combination with chitosan. The resulting carboxymethyl-ß-glucan/chitosan nanoparticles exhibited adequate physicochemical properties and an important protein association efficiency, with ovalbumin as a model compound. Moreover, thermostability was achieved through the optimization of a lyophilized form of the antigen-loaded nanoparticles, which remained stable for up to 1 month at 40 ºC. Biodistribution studies in mice showed an efficient drainage of the nanoparticles to the nearest lymph node following subcutaneous injection, and a significant co-localization with dendritic cells. Additionally, subcutaneous immunization of mice with a single dose of the ovalbumin-loaded nanoparticles led to induced T cell proliferation and antibody responses, comparable to those achieved with alum-adsorbed ovalbumin. These results illustrate the potential of these novel nanocarriers in vaccination.

The Immunoenhancement Effects of Polyethylenimine-Modified Chinese Yam Polysaccharide-Encapsulated PLGA Nanoparticles as an Adjuvant.

BACKGROUND: Poly(lactic-co-glycolic acid) (PLGA) has been extensively applied for sustained drug delivery and vaccine delivery system. However, vaccines delivered by PLGA nanoparticles alone could not effectively activate antigen-presenting cells (APCs) to induce strong immune responses. PURPOSE: The aim of the present study was to design polyethylenimine (PEI)-modified Chinese yam polysaccharide (CYP)-encapsulated PLGA nanoparticles (CYPP-PEI) as a vaccine delivery system and evaluate the adjuvant activities in vitro and in vivo. MATERIALS AND METHODS: Cationic-modified nanoparticles exhibited high antigen absorption and could be efficiently taken by APCs to enhance the immune responses. Therefore, PEI-modified CYP-encapsulated PLGA nanoparticles (CYPP-PEI) were prepared. The storage stability and effective adsorption capacity for porcine circovirus-2 (PCV-2) antigen of these antigen-absorbed nanoparticles were measured for one month. Furthermore, the adjuvant activity of CYPP-PEI nanoparticles was evaluated on macrophages in vitro and through immune responses triggered by PCV-2 antigen in vivo. RESULTS: The PCV-2 absorbed CYPP-PEI nanoparticles showed excellent storage stability and high absorption efficiency of PCV-2 antigen. In vitro, CYPP-PEI nanoparticles promoted antigen uptake, enhanced surface molecular expressions of CD80 and CD86, and improved cytokine secretion of TNF-α, IFN-γ, and IL-12p70 in macrophages. After immunization with CYPP-PEI/PCV-2 formulation in mice, the expressions of surface activation markers on dendritic cells which located in draining lymph nodes were increased, such as MHCI, MHCII, and CD80. In addition, CYPP-PEI nanoparticles induced dramatically high PCV-2-specific IgG levels which could last for a long time and stimulated the secretion of subtype antibodies and cytokines. The results showed that CYPP-PEI could induce Th1/Th2 mixed but Th1-biased type immune responses. CONCLUSION: Polyethylenimine-modified Chinese yam polysaccharide-encapsulated PLGA nanoparticle was a potential vaccine delivery system to trigger strong and persistent immune responses.

Sustained-immunostimulatory nanocellulose scaffold to enhance vaccine efficacy.

An implantable scaffold-based vaccination system is a promising platform to generate robust immune responses by modulating the immune system. However, establishment of an effective vaccine using a biodegradable, cell-infiltrative scaffold remain challenging. Here we demonstrate a biodegradable, nanocellulose-based immune scaffold capable of sustainably activating immune cells to elicit cellular immunity. Cell-infiltrative nanocellulose hydrogels were used as a delivery carrier and cellular scaffold microenvironment. Nanofibrous hydrogels allowed for high cell infiltration and delivery of antigen-loaded nanocellulose while cells degraded the hydrogel matrix. Importantly, antigen-loaded nanocellulose hydrogels exhibited sustained activation of macrophages in vitro compared to free antigen and collagen scaffold. Histological observation revealed infiltration of macrophages and dendritic cells into the nanocellulose scaffold subcutaneously implanted in mice. In vivo fluorescence imaging indicated that the implanted scaffold released antigens at a zero-order release profile without burst diffusion. Antigen-loaded nanocellulose hydrogels increased interferon-γ-producing cells compared to free antigen injection, suggesting the enhancement of cellular immunity. Thus, nanocellulose immune scaffold may serve as a sustained-immunostimulatory vaccine platform by providing favorable microenvironments for immune cells thus enhancing vaccine efficacy.

A fast-dissolving microneedle array loaded with chitosan nanoparticles to evoke systemic immune responses in mice.

Microneedle (MN) arrays offer an alternative approach to hypodermic injection via syringe needles. In this work, polyvinylpyrrolidone (PVP)-based fast dissolving MN arrays were developed in which the needle tips were loaded with chitosan nanoparticles (NPs) for coencapsulation of a model antigen, ovalbumin (OVA), and an adjuvant, CpG oligodeoxynucleotides (CpG). After insertion into the skin, these MN arrays fully dissolved within 3 min to release antigen and adjuvant co-loaded NPs rapidly in the epidermal layer. Positively charged chitosan was proven to be an excellent carrier for negatively charged OVA and CpG, which formed nanocomplexes via simple electrostatic interactions and greatly enhanced the uptake efficiency of OVA in DC2.4 dendritic cells. Vaccination studies in mice further demonstrated that chitosan NPs effectively accumulated in peripheral lymph nodes, thus inducing greatly enhanced immune responses compared to those of free OVA. The antibody dose-response curve further demonstrated that MN immunization achieved comparable levels of immune responses as compared to conventional subcutaneous injections in a more convenient and less invasive way. Overall, a PVP-based fast dissolving MN array with chitosan NPs represents a promising and robust platform system for efficient transcutaneous vaccine delivery.

Modular Polymer Antigens To Optimize Immunity.

Subunit vaccines can have excellent safety profiles, but their ability to give rise to robust immune responses is often compromised. For glycan-based vaccines, insufficient understanding of B and T cell epitope combinations that yield optimal immune activation hinders optimization. To determine which antigen features promote desired IgG responses, we synthesized epitope-functionalized polymers using ring-opening metathesis polymerization (ROMP) and assessed the effect of B and T cell epitope loading. The most robust responses were induced by polymers with a high valency of B and T cell epitopes. Additionally, IgG responses were greater for polymers with T cell epitopes that are readily liberated upon endosomal processing. Combining these criteria, we used ROMP to generate a nontoxic, polymeric antigen that elicited stronger antibody responses than a comparable protein conjugate. These findings highlight principles for designing synthetic antigens that elicit strong IgG responses against inherently weak immune targets such as glycans.

Simultaneous in situ visualization and quantitation of dual antigens adsorbed on adjuvants using high content analysis.

Aim: Traditional antigenicity assay requires antigen recovery from the particulate adjuvants prior to analysis. An in situ method was developed for interrogating vaccine antigens with monoclonal antibodies while being adsorbed on adjuvants. Materials & methods: The fluorescence imaging-based high content analysis was used to visualize the antigen distribution on adjuvant agglomerates and to analyze the antigenicity for adsorbed antigens. Results: Simultaneous visualization and quantitation were achieved for dual antigens in a bivalent human papillomavirus vaccine with uniquely labeled antibodies. Good agreement was observed between the in situ multiplexed assays with well-established sandwich enzyme-linked immunosorbent assays. Conclusion: The streamlined procedures and the amenability for multiplexing make the in situ antigenicity analysis a favorable choice for in vitro functional assessment of bionanoparticles as vaccine antigens.