Free Ovalbumin Research Articles

A novel fluoropolymer as a protein delivery vector with robust adjuvant effect for cancer immunotherapy.

The inefficient treatment using protein-based nanovaccines is largely attributed to their inadequate immunogenicity. Herein, we developed a novel fluoropolymer (PF) via ring-opening polymerization and constructed a fluoropolymer-based nanovaccine for tumor immunotherapy. Due to the existence of fluoroalkyl chains, PF not only played a crucial role in tumor antigen delivery but also exhibited a remarkable adjuvant effect in enhancing the immunogenicity of nanovaccines. The nanovaccines formed by mixing PF with a model antigen ovalbumin (OVA) enhanced the uptake of antigen proteins by dendritic cells (DCs) and promoted the maturation and antigen presentation of DCs. Compared with free OVA, PF/OVA showed better efficacy in both pre-cancer prevention and tumor treatment. Furthermore, the proportion of CD4+ T and CD8+ T cells was significantly increased in lymph nodes and tumors of mice immunized with PF/OVA. Additionally, there was a great enhancement in the levels of key anti-tumor cytokines (TNF-α and IFN-γ) in the serum of the PF/OVA immunized mice. Our research has shown that fluoropolymer PF applied as a protein vector and adjuvant has great potential for the development of nanovaccines with robust immunogenicity.

Dual-Responsive Glycopolymers for Intracellular Codelivery of Antigen and Lipophilic Adjuvants.

Traditional approaches to vaccines use whole organisms to trigger an immune response, but they do not typically generate robust cellular-mediated immunity and have various safety risks. Subunit vaccines composed of proteins and/or peptides represent an attractive and safe alternative to whole organism vaccines, but they are poorly immunogenic. Though there are biological reasons for the poor immunogenicity of proteins and peptides, one other key to their relative lack of immunogenicity could be attributed to the poor pharmacokinetic properties of exogenously delivered proteins and peptides. For instance, peptides often aggregate at the site of injection and are not stable in biological fluids, proteins and peptides are rapidly cleared from circulation, and both have poor cellular internalization and endosomal escape. Herein, we developed a delivery system to address the lack of protein immunogenicity by overcoming delivery barriers as well as codelivering immune-stimulating adjuvants. The glycopolymeric nanoparticles (glycoNPs) are composed of a dual-stimuli-responsive block glycopolymer, poly[2-(diisopropylamino)ethyl methacrylate]-b-poly[(pyridyl disulfide ethyl methacrylate)-co-(methacrylamidoglucopyranose)] (p[DPA-b-(PDSMA-co-MAG)]). This polymer facilitates protein conjugation and cytosolic release, the pH-responsive release of lipophilic adjuvants, and pH-dependent membrane disruption to ensure cytosolic delivery of antigens. We synthesized p[DPA-b-(PDSMA-co-MAG)] by reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by the formation and physicochemical characterization of glycoNPs using the p[DPA-b-(PDSMA-co-MAG)] building blocks. These glycoNPs conjugated the model antigen ovalbumin (OVA) and released OVA in response to elevated glutathione levels. Moreover, the glycoNPs displayed pH-dependent drug release of the model hydrophobic drug Nile Red while also exhibiting pH-responsive endosomolytic behavior as indicated by a red blood cell hemolysis assay. GlycoNPs coloaded with OVA and the toll-like receptor 7/8 (TLR-7/8) agonist Resiquimod (R848) activated DC 2.4 dendritic cells (DCs) significantly more than free OVA and R848 and led to robust antigen presentation of the OVA epitope SIINFEKL on major histocompatibility complex I (MHC-I). In sum, the dual-stimuli-responsive glycopolymer introduced here overcomes major protein and peptide delivery barriers and could vastly improve the immunogenicity of protein-based vaccines.

Reduction-Sensitive Protein Nanogels Enhance Uptake of Model and Tumor Lysate Antigens In Vitro by Mouse- and Human-Derived Dendritic Cells.

Peptides and proteins represent an emerging class of powerful therapeutics. Peptide and protein nanogels are attractive carriers for the transport and delivery of biologically active peptides and proteins because they allow essentially quantitative encapsulation of these biologics. One interesting field of use of peptide and protein nanogels is the transport of antigens and adjuvants in cancer immunotherapy. This study demonstrates the use of reduction-sensitive protein nanogels for the delivery of ovalbumin and oxidized tumor lysate-based antigens to mouse and human-donor-derived dendritic cells. Challenging mouse-derived and human dendritic cells with reduction-sensitive ovalbumin nanogels was found to significantly enhance antigen uptake as compared to the use of the corresponding free protein antigen. The experiments with mouse-derived dendritic cells further showed that the administration of ovalbumin in the form of reduction-sensitive nanogels enhanced dendritic cell maturation as well as the presentation of the SIINFEKL epitope as compared to experiments that use free ovalbumin. In addition to ovalbumin as a model antigen, the feasibility of reduction-sensitive nanogels was also demonstrated for the delivery of oxidized, whole tumor lysate-based cancer antigens. In experiments with dendritic cells harvested from human donors, dendritic cell uptake of the oxidized tumor lysate antigen was significantly enhanced in experiments that used oxidized tumor lysate nanogels as compared to the free antigen.

Association of a vaccine adjuvant with endogenous HDL increases lymph uptake and dendritic cell activation

Vaccines are a powerful health intervention but there is still an unmet need for effective preventative and therapeutic vaccines for many diseases such as cancer and infections. Interstitial (e.g. subcutaneous (SC)) injection in nano-sized carriers such as high density lipoproteins (HDLs) can improve the access of vaccine subunit antigens or adjuvants to target immune cells in the lymphatics and potentiate vaccination responses such as cytotoxic T lymphocyte (CTL) responses (Kuai et al., 2016, 2018; Qian et al., 2016). Here we examined how cholesterol conjugation to the vaccine adjuvant CpG, and incorporation into HDL, changes lymphatic absorption and association with, and processing by, dendritic cells (DCs), ultimately influencing adjuvant efficacy. We investigated the lymphatic disposition of cholesterol conjugated CpG incorporated into HDL (HDL(Chol-CpG-Cy5)) relative to free cholesterol conjugated CpG (Chol-CpG-Cy5) and unconjugated CpG (free CpG-Cy5) after SC administration in rats and mice. HDL (Chol-CpG-Cy5) and Chol-CpG-Cy5 differentially altered CpG absorption into lymph vs. blood, but surprisingly resulted in similarly higher LN accumulation relative to free CpG. The mechanism of access of Chol-CpG-Cy5 into lymph might be partly due to association with endogenous HDL at the injection site followed by transport into lymph in association with the HDL. To measure CpG association with and processing by DCs and the strength of the immune response, mice were vaccinated with free ovalbumin (OVA) co-administered with the different CpG constructs. There were significant changes in DC activation that were reflective of the trend in LN accumulation at 24 h post-vaccination. However, T cell responses at 24 h and 7 days post-vaccination were not significantly different across the CpG groups although the response was less variable for Chol-CpG-Cy5 compared to free CpG Cy5 and also HDL(Chol-CpG-Cy5) – despite similar LN accumulation with the latter. Overall, our data indicate that cholesterol conjugation and incorporation into HDL increases adjuvant lymph disposition and DC activation.

Poly(ethylene glycol)-Mediated Assembly of Vaccine Particles to Improve Stability and Immunogenicity.

We report the one-step assembly of vaccine particles by encapsulating ovalbumin (OVA) and cytosine-phosphate-guanine oligodeoxynucleotides (CpG) into poly(ethylene glycol) (PEG)-mediated zeolitic imidazolate framework-8 nanoparticles (OVA-CpG@ZIF-8 NPs), where PEG improves the stability and dispersity of ZIF-8 NPs and the NPs protect the encapsulated OVA and CpG to circumvent the cold chain issue. Compared with free OVA and OVA-encapsulated ZIF-8 (OVA@ZIF-8) NPs, OVA-CpG@ZIF-8 NPs can enhance antigen uptake, cross-presentation, dendritic cell (DC) maturation, production of specific antibody and cytokines, and CD4+ T and CD8+ T cell activation. More importantly, the vaccine particles retain their bioactivity against enzymatic degradation, elevated temperatures, and long-term storage at ambient temperature. The study highlights the importance of PEG-mediated ZIF-8 NPs as a vaccine delivery system for the promising application of effective and cold chain-independent vaccination against diseases.

Improving emulsion stability based on ovalbumin-carboxymethyl cellulose complexes with thermal treatment near ovalbumin isoelectric point

Ovalbumin (OVA) is an important protein emulsifier. However, it is unstable near the isoelectric point pH, which limits its applications in the food industry. Polysaccharides may be explored to tackle this challenge by improving its pH-dependent instability. In this work, carboxymethyl cellulose (CMC) was used as a model polysaccharide to mix with OVA near its isoelectric point (pH 4.7) with subsequent mild heating at 60 °C for 30 min. The molecular interactions between OVA and CMC were comprehensively studied via a series of characterizations, including turbidity, zeta potential, intrinsic fluorescence, surface hydrophobicity, circular dichroism (CD) spectra and Fourier transform infrared spectroscopy (FTIR). The droplet sizes of the emulsions prepared by OVA-CMC were measured to analyze emulsifying property and stability. The results indicated that free OVA was easily aggregated due to loss of surface charges, while complexing with CMC significantly inhibited OVA aggregation before and after heating owing to the strong electrostatic repulsion. In addition, OVA exposed more hydrophobic clusters after heating, which resulted in the growth of surface hydrophobicity. Altogether, the heated OVA-CMC complexes presented the best emulsifying property and stability. Our study demonstrated that complexing OVA with CMC not only greatly improved its physicochemical properties but also significantly enhanced its functionality as a food-grade emulsifying agent, expanding its applications in the food industry, as development of emulsion-based acidic food products.

Nano-vesicles based on phospholipid-like amphiphilic polyphosphazenes to orally deliver ovalbumin antigen for evoking anti-tumor immune response

Aimed at evoking an adequate anti-tumor immune response via oral administration route, this study constructed functionally and structurally mimicking-bacteria-membrane (MBM) nano-vesicle (RGD-PEOP) to orally deliver ovalbumin (OVA) antigen. In terms of simulating bacterial membrane structure, we creatively designed this nano-vesicle to have phospholipid-like octadecylphosphoethanolamine groups in vesicle membrane to improve OVA loading by means of specific interactions including salt bridge and hydrogen bond interaction. For simulating bacterial membrane function, the RGD peptide was modified onto the nano-vesicle surface, and the resulting vector displayed a good transport ability with a 3.4-fold higher than free OVA. In vitro and in vivo assay showed that the expression of co-stimulatory molecules and MHC class II complexes was significantly enhanced by MBM nano-vesicle. IFN-γ and IL-4 levels also increased several folds in the MBM nano-vesicle group. Consequently, MBM nano-vesicle achieved the highest in vivo inhibition rate of 69% against E.G7-OVA tumors among all the oral groups. These results suggest that this MBM nano-vesicle may be a promising vector to orally deliver OVA antigen for cancer immunotherapy. STATEMENT OF SIGNIFICANCE: Developing an effective non-bacterial carrier for oral cancer immunotherapy remains challenging. This work constructed a mimicking-bacteria-membrane nano-vesicle based on phospholipid-like amphiphilic polyphosphazenes for oral delivery of ovalbumin antigen. With the considerable capability to load ovalbumin antigen and target M cells, the nano-vesicle produced remarkable tumor suppression in vivo by evoking anti-tumor immune response.

Enhanced antibody response to ovalbumin coupled to poly-dispersed acid functionalized single walled carbon nanotubes

Activated mouse B cells as compared to the resting B cells are known to internalize substantially more acid-functionalized single walled carbon nanotubes (AF-SWCNTs). It was hypothesized that the antigens coupled to AF-SWCNTs would also be taken up more efficiently by B cells. Further the enhanced uptake of the antigen by B cells may facilitate antigen presentation by B cells resulting in a better antibody response. Aim of this study was to test this hypothesis. Ovalbumin was chemically coupled to AF-SWCNTs that yielded a coupled product that had 0.08% of all carbon atoms in AF-SWCNTs occupied by ovalbumin. Coupling of ovalbumin to AF-SWCNTs was confirmed by staining the product with anti-ovalbumin antibodies. B cells incubated with ovalbumin-AF-SWCNT internalized more ovalbumin than the B cells incubated with free ovalbumin. Groups of mice were immunized subcutaneously with (a) free ovalbumin, (b) free ovalbumin and AF-SWCNTs at two different subcutaneous sites respectively on mice, and (c) ovalbumin-AF-SWCNT coupled product. In each case a primary immunization was followed by three weekly booster doses. It was found that the anti-ovalbumin antibody response assessed by ELISA, was highest in the group where ovalbumin coupled to AF-SWCNTs was used for immunization (p < 0.001). Antibody response in ovalbumin-AF-SWCNT group was comparable to the group where ovalbumin was used for immunization using complete and incomplete Freund’s adjuvant (primary and secondary immunizations respectively). We propose that AF-SWCNTs could be explored as an adjuvant to improve the antibody response especially in vaccine development.

Co-delivery of CpG and antigen using hyaluronic acid bioconjugates-decorated nanoparticles to promote maturation and activation of dendritic cells

Objective To study the maturation and activation effects of hyaluronic acid (HA) modified polymer nanoparticles co-delivering adjuvants and antigens on mouse bone marrow dendritic cells (BMDCs). Methods HA-modified polylactic acid-glycolic acid copolymer (PLGA) and cationic lipid DOTAP were used as nanocarriers (DOTAP-PLGA) to co-deliver adjuvant CpG with model antigen ovalbumin (OVA). In the drug-loaded nanocarriers, CpG was covalently bound to the surface of HA, and OVA was physically blended into DOTAP-PLGA nanocarriers. The nanoparticles were characterized by transmission electron microscopy and dynamic light scattering. The in vitro release of CpG and OVA in the nanoparticles was investigated. The uptake and distribution of nanoparticles in mouse BMDCs were studied by flow cytometry and laser scanning confocal microscopy. The maturation and cytokine expression of mouse BMDCs were evaluated by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results The CpG-HA-OVA-PLGA nanoparticles loading CpG and OVA were prepared. The average particle size was (305.1±2.2) nm and the polydispersity index was 0.203. A core-shell structure of the nanoparticles modified by HA was clearly observed by transmission electron microscopy. Cellular experiment results showed that CpG-HA-OVA-PLGA nanoparticles could be efficiently uptaken by mouse BMDCs, and promote lysosomal release of CpG and cytoplasmic delivery of antigen OVA. Compared with free OVA group and free OVA+CpG group, the CpG-HA-OVA-PLGA nanoparticles significantly up-regulated the expression of co-stimulatory molecules CD86 and CD40 (all P<0.01), major histocompatibility complex I (MHC-I) (P<0.01), and cytokine tumor necrosis factor-α (TNF-α) (P<0.01). Conclusions HA-modified CpG and OVA nanoparticle co-delivery vectors can effectively promote the maturation and activation of dendritic cells, which provides a basis for the development of novel vaccine vectors for the co-delivery of antigens and adjuvants. Key words: Nanoparticles; Co-delivery; CpG; Antigen; Dendritic cells

Controlled release of mTOR inhibitors to enhance CD8+ T cell memory

Abstract Recent studies demonstrate, they frequent, low-dose administration of rapamycin (Rapa) during vaccination can help maintain the plasticity of antigen-specific CD8+ T cells, polarizing the phenotypes of these cells toward central memory T cells (TCM). However, these effects required multiple, systemic doses of Rapa. Here we hypothesized that local, intra-LN controlled release of Rapa from these depots during vaccination would polarize T cells toward highly proliferative TCM during T cell expansion. to enhance T cell immunity. To test this idea, vaccine were formulated by mixing free ovalbumin (OVA) and PLGA microparticles loaded with a TLR9 agonist (CpG). Mice were first immunized with the vaccine, and then received daily i.p injection of Rapa at low doses (75 μg/kg) for 28 days. This regimen confirmed i.p injection of Rapa increased the percentage of OVA-specific CD8+ T cells in PBMCs by 2.4 times on day 14 and specific TCM levels by 4.3 times on day 49 compared to vaccine without Rapa. We next assess the effects of controlled release by using the vaccine above but treating mice with a single dose of controlled release depots loaded with defined doses of Rapa. In mice, this approach showed functional as similar as daily i.p injection of Rapa. While high dose of Rapa (20.9 μg to 36.4 μg per dose) suppressed the immunity, Rapa at a low dose (0.07 μg to 4.17 μg per dose) in MP significantly promote the CD8+ T cell immunity over 7 weeks, where specific CD8+ T cells was promoted by 2.9 times on day 14 and specific TCM on day 49 by 2.4 times of vaccine without Rapa. These result suggested that sustained release of immunomodulator in biomaterials depots directly in the LN microenvironment can significantly enhance the antigen specific CD8+ T cells immunity.

Self-assembled PEG-b-PDPA-b-PGEM copolymer nanoparticles as protein antigen delivery vehicles to dendritic cells: preparation, characterization and cellular uptake.

Antigen uptake by dendritic cells (DCs) is a key step for initiating antigen-specific T cell immunity. In the present study, novel synthetic polymeric nanoparticles were prepared as antigen delivery vehicles to improve the antigen uptake by DCs. Well-defined cationic and acid-responsive copolymers, monomethoxy poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate)-block-poly(2-(guanidyl) ethyl methacrylate) (mPEG-b-PDPA-b-PGEM, PEDG) were synthesized by reversible addition-fragmentation chain transfer polymerization of 2-(diisopropylamino)ethyl methacrylate) and N-(tert-butoxycarbonyl) amino ethyl methacrylate monomers, followed by deprotection of tert-butyl protective groups and guanidinylation of obtained primary amines. 1H NMR, 13C NMR and GPC results indicated the successful synthesis of well-defined PEDG copolymers. PEDG copolymers could self-assemble into nanoparticles in aqueous solution, which were of cationic surface charges and showed acid-triggered disassembly contributed by PGEM and PDPA moieties, respectively. Significantly, PEDG nanoparticles could effectively condense with negatively charged model antigen ovalbumin (OVA) to form OVA/PEDG nanoparticle formulations with no influence on its secondary and tertiary structures demonstrating by far-UV circular dichroism and UV–vis spectra. In vitro antigen cellular uptake by bone marrow DCs (BMDCs) indicated using PEDG nanoparticles as antigen delivery vehicles could significantly improve the antigen uptake efficiency of OVA compared with free OVA or the commercialized Alum adjuvant. Moreover, as the surface cationic charges of OVA/PEDG nanoparticle formulations reduced, the uptake efficiency decreased correspondingly. Collectively, our work suggests that guanidinylated, cationic and acid-responsive PEDG nanoparticles represent a new kind of promising antigen delivery vehicle to DCs and hold great potential to serve as immunoadjuvants in the development of vaccines.

Hyaluronic Acid-Modified Cationic Lipid-PLGA Hybrid Nanoparticles as a Nanovaccine Induce Robust Humoral and Cellular Immune Responses.

Here, we investigated the use of hyaluronic acid (HA)-decorated cationic lipid-poly(lactide-co-glycolide) acid (PLGA) hybrid nanoparticles (HA-DOTAP-PLGA NPs) as vaccine delivery vehicles, which were originally developed for the cytosolic delivery of genes. Our results demonstrated that after the NPs uptake by dendritic cells (DCs), some of the antigens that were encapsulated in HA-DOTAP-PLGA NPs escaped to the cytosolic compartment, and whereas some of the antigens remained in the endosomal/lysosomal compartment, where both MHC-I and MHC-II antigen presentation occurred. Moreover, HA-DOTAP-PLGA NPs led to the up-regulation of MHC, costimulatory molecules, and cytokines. In vivo experiments further revealed that more powerful immune responses were induced from mice immunized with HA-DOTAP-PLGA NPs when compared with cationic lipid-PLGA nanoparticles and free ovalbumin (OVA); the responses included antigen-specific CD4(+) and CD8(+) T-cell responses, the production of antigen-specific IgG antibodies and the generation of memory CD4(+) and CD8(+) T cells. Overall, these data demonstrate the high potential of HA-DOTAP-PLGA NPs for use as vaccine delivery vehicles to elevate cellular and humoral immune responses.

Supramolecular Glyco-nanoparticles Toward Immunological Applications.

Glyco-mimicking nanoparticles (glyco-NPs) with Förster resonance energy transfer (FRET) donor and acceptor groups formed via dynamic covalent bond of benzoboroxole and sugar from two complementary polymers are prepared. The glyco-NPs are proved to be quite stable under physiological conditions but sensitive to pH. So the glyco-NPs can be internalized by dendritic cells with integrity and nontoxicity and then dissociate within the acidic organelles. This particle dissociation is directly observed and visualized in vitro, for the first time via the FRET measurements and fluorescent microscopy. This feature makes controlled release of drug or protein by glyco-NPs possible, i.e., when model antigen Ovalbumin is loaded in the glyco-NPs, the released Ovalbumin in dendritic cells stimulates T cells more efficiently than the free Ovalbumin itself as a result of the enhanced antigen processing and presentation. Thus, the results enlighten a bright future of the glyco-NPs in immunotherapy.

Neutral polymer micelle carriers with pH-responsive, endosome-releasing activity modulate antigen trafficking to enhance CD8+ T cell responses

Synthetic subunit vaccines need to induce CD8+ cytotoxic T cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8+ cytotoxic T cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8+ T cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendent pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25–30nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non-pH-responsive micelle–ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC 2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4h post uptake. Subcutaneous immunization of mice with ovalbumin–polymer conjugates significantly enhanced antigen-specific CD8+ T cell responses (0.4% IFN-γ+ of CD8+) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells in the draining lymph nodes. As early as 90min post injection, ova–micelle conjugates were associated with 28% and 55% of dendritic cells and macrophages, respectively. After 24h, conjugates preferentially associated with dendritic cells, affording 30-, 3-, and 3-fold enhancements in uptake relative to free protein, physical mixture, and the non-pH-responsive conjugate controls, respectively. These results demonstrate the potential of pH-responsive polymeric micelles for use in vaccine applications that rely on CD8+ T cell activation.

Tunable T cell immunity towards a protein antigen using polymersomes vs. solid-core nanoparticles

Using poly(propylene sulfide) (PPS) and poly(ethylene glycol) (PEG) as components of a nanocarrier platform, we sought to compare immune responses induced by PPS-bl-PEG polymersomes (PSs; watery-core structures, with antigen incorporated within the PSs) and PEG-stabilized PPS nanoparticles (NPs; solid-core structures, with antigen conjugated upon the NP surface). We have previously shown strong CD8+ T cell responses to antigen conjugated to NPs via a disulfide link, and here we investigated the extent to which antigen incorporated within oxidatively-sensitive PSs could induce CD4+ or CD8+ T cell responses. C57BL/6 mice were subcutaneously immunized with free ovalbumin (OVA) as a model antigen, or equivalent doses of OVA-loaded into PSs, conjugated onto NPs, or given as a mixture of the two. Free CpG was used as an adjuvant. Antigen-loaded PSs induced enhanced frequencies of antigen-specific CD4+ T cells in the spleen, lymph nodes and lungs as compared to the NP formulation, whereas antigen-conjugated NPs induced stronger CD8+ T cell responses. Co-administration of both PSs and NPs elicited T cell immunity characteristic of the two nanocarriers at the same time, i.e. both strong CD4+ and CD8+ T cell responses. These results have important implications for particulate-based vaccine design and highlight the potential of using different antigen-delivery systems for the induction of both T helper and cytotoxic T lymphocyte immune responses.

The pH-Sensitive Fusogenic 3-Methyl-Glutarylated Hyperbranched Poly(Glycidol)-Conjugated Liposome Induces Antigen-Specific Cellular and Humoral Immunity

We examined the ability of a novel liposome, surface modified by 3-methyl-glutarylated hyperbranched poly(glycidol) (MGlu-HPG), to enhance antigen-specific immunity in vitro and in vivo and to function as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) encapsulated in MGlu-HPG-modified liposomes more effectively than free OVA or OVA encapsulated in unmodified liposomes. Immunization of mice with OVA-containing MGlu-HPG-modified liposomes induced antigen-specific splenocyte proliferation and production of gamma interferon (IFN-γ) more strongly than did immunization with free OVA or OVA encapsulated in unmodified liposomes. The immune responses induced by OVA encapsulated in MGlu-HPG-modified liposomes were significantly suppressed by addition of anti-major histocompatibility complex (MHC) class I and class II monoclonal antibodies, indicating the involvement of antigen presentation via MHC class I and II. Furthermore, delayed-type hypersensitivity responses and OVA-specific antibodies were induced more effectively in mice immunized with OVA encapsulated by MGlu-HPG-modified liposomes than with unencapsulated OVA or OVA encapsulated in unmodified liposomes. These results suggested that MGlu-HPG-modified liposomes effectively induced both cell-mediated and humoral immune responses. Collectively, this study is the first to demonstrate the induction of both cell-mediated and humoral immune responses in vivo by MGlu-HPG-modified liposomes.

Induction of antigen-specific immunity by pH-sensitive carbonate apatite as a potent vaccine carrier

The ability of carbonate apatite (CO 3Ap) to enhance antigen-specific immunity was examined in vitro and in vivo to investigate its utility as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) containing CO 3Ap more effectively than free OVA. Interestingly, mice immunized with OVA-containing CO 3Ap produced OVA-specific antibodies more effectively than mice immunized with free OVA. Furthermore, immunization of C57BL/6 mice with OVA-containing CO 3Ap induced the proliferation and antigen-specific production of IFN-γ by splenocytes more strongly than immunization with free OVA. Moreover, no significant differences were detected in the induction of delayed-type hypersensitivity responses, an immune reaction involving an antigen-specific, cell-mediated immune response between OVA-containing CO 3Ap and OVA-containing alumina salt (Alum), suggesting that CO 3Ap induced cell-mediated immune response to the same degree as Alum, which is commonly used for clinical applications. This study is the first to demonstrate the induction of antigen-specific immune responses in vivo by CO 3Ap.

PH-responsive polymer–antigen vaccine bioconjugates

Protein-based vaccines play an important role in controlling infectious disease but their full clinical impact has been limited by their inability to generate a coordinated cellular CD4+ and CD8+ immune response. Vaccines that better deliver antigens to the cytosolic MHC1 display system could in principle provide a better coordinated response. Here, controlled radical polymerization was employed to prepare a diblock copolymer containing an endosomal releasing segment based on poly(propylacrylic acid) (poly(PAA)) and a hydrophilic segment containing thiol-reactive disulfide moieties for antigen conjugation. Propylacrylic acid (PAA) was polymerized in the presence of a trithiocarbonate based RAFT chain transfer agent (CTA). The resultant poly(PAA) was then employed as a macroCTA in the copolymerization of pyridyl disulfide methacrylamide (PDSMA) (thiol-reactive monomer) and N,N-dimethylacrylamide (DMA) as a hydrophilic comonomer. Copolymer compositions and molecular weights were determined via1H NMR spectroscopy and size exclusion chromatography. Native polyacrylamide gel electrophoresis (PAGE) showed a complete disappearance of the bands corresponding to free thiolated ovalbumin after conjugation to the polymer at pyridyl disulfide to thiol ratios as low as 2.5. The ability of the poly[(PAA)-b-(DMA)co(PDSMA)]–ovalbumin conjugates to activate CTLs was evaluated in vivo, tumor protection using the EG7 tumor protection model. Tumors were visible in the PBS and free ovalbumin immunized mice by day 7 but were not visible in the polyPAA–ovalbumin conjugate immunized mice until day 18. The mice immunized with the polyPAA–ovalbumin conjugate had a survival rate at day 21 of 100% versus 20% for PBS and 40% for ovalbumin immunized mice.

Robust Stimulation of Humoral and Cellular Immune Responses following Vaccination with Antigen-Loaded β-Glucan Particles

β-Glucan particles (GPs) are purified Saccharomyces cerevisiae cell walls treated so that they are primarily β1,3-d-glucans and free of mannans and proteins. GPs are phagocytosed by dendritic cells (DCs) via the Dectin-1 receptor, and this interaction stimulates proinflammatory cytokine secretion by DCs. As the hollow, porous GP structure allows for high antigen loading, we hypothesized that antigen-loaded GPs could be exploited as a receptor-targeted vaccine delivery system. Ovalbumin (OVA) was electrostatically complexed inside the hollow GP shells (GP-OVA). Incubation of C57BL/6J mouse bone marrow-derived DCs with GP-OVA resulted in phagocytosis, upregulation of maturation markers, and rapid proteolysis of OVA. Compared with free OVA, GP-OVA was >100-fold more potent at stimulating the proliferation of OVA-reactive transgenic CD8+ OT-I and CD4+ OT-II T cells, as measured by in vitro [3H]thymidine incorporation using DCs as antigen-presenting cells. Next, immune responses in C57BL/6J mice following subcutaneous immunizations with GP-OVA were compared with those in C57BL/6J mice following subcutaneous immunizations with OVA absorbed onto the adjuvant alum (Alum/OVA). Vaccination with GP-OVA stimulated substantially higher antigen-specific CD4+ T-cell lymphoproliferative and enzyme-linked immunospot (ELISPOT) responses than that with Alum/OVA. Moreover, the T-cell responses induced by GP-OVA were Th1 biased (determined by gamma interferon [IFN-γ] ELISPOT assay) and Th17 biased (determined by interleukin-17a [IL-17a] ELISPOT assay). Finally, both the GP-OVA and Alum/OVA formulations induced strong secretions of IgG1 subclass anti-OVA antibodies, although only GP-OVA induced secretion of Th1-associated IgG2c antibodies. Thus, the GP-based vaccine platform combines adjuvanticity and antigen delivery to induce strong humoral and Th1- and Th17-biased CD4+ T-cell responses.

Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination

The presence of RGD on nanoparticles allows the targeting of β1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of PCL–PEG and incorporated in PLGA-based nanoparticles. RGD and RGDp significantly increased the transport of nanoparticles across an in vitro model of human M cells as compared to enterocytes. RGD, LDVp, LDVd and mannose enhanced nanoparticle uptake by macrophages in vitro. The intraduodenal immunization with RGDp-, LDVd- or mannose-labeled nanoparticles elicited a higher production of IgG antibodies than the intramuscular injection of free ovalbumin or intraduodenal administration of either non-targeted or RGD-nanoparticles. Targeted formulations were also able to induce a cellular immune response. In conclusion, the in vitro transport of nanoparticles, uptake by macrophages and the immune response were positively influenced by the presence of ligands at the surface of nanoparticles. These targeted-nanoparticles could thus represent a promising delivery system for oral immunization.