DNA Vaccine Carrier Research Articles

Enhancing Mucosal Immune Response of Newcastle Disease Virus DNA Vaccine Using N-2-Hydroxypropyl Trimethylammonium Chloride Chitosan and N,O-Carboxymethyl Chitosan Nanoparticles as Delivery Carrier.

Because mucosal sites are the entry ports of pathogens, immunization via mucosal routes can extremely enhance the immunity. To elevate the potential of N-2-hydroxypropyl trimethylammonium chloride chitosan (N-2-HACC) and N,O-carboxymethyl chitosan (CMC) nanoparticles as a mucosal immune delivery carrier for DNA vaccines, we prepared the NDV F gene plasmid DNA with C3d6 molecular adjuvant (pVAX I-F(o)-C3d6) encapsulated in the N-2-HACC-CMC nanoparticles (N-2-HACC-CMC/pFDNA-C3d6 NPs). The N-2-HACC-CMC/pFDNA-C3d6 NPs had regular spherical morphology and low toxicity with a mean diameter of 309.7 ± 6.52 nm, zeta potential of 49.9 ± 4.93 mV, encapsulation efficiency of 92.27 ± 1.48%, and loading capacity of 50.75 ± 1.35%. The N-2-HACC-CMC had high stability and safety. The pVAX I-F(o)-C3d6 could be sustainably released from the N-2-HACC-CMC/pFDNA-C3d6 NPs after an initial burst release. Immunization intranasally of chickens with N-2-HACC-CMC/pFDNA-C3d6 NPs not only produced higher anti-NDV IgG and sIgA antibody than chickens in other groups did, but also significantly stimulated lymphocyte proliferation and triggered higher the IL-2, IL-4, and IFN-γ levels. These findings indicated that the N-2-HACC-CMC could be used as an efficient delivery carrier for the mucosal immunity of Newcastle disease virus DNA vaccine. The work laid a basis for the quaternized chitosan nanoparticles as efficient mucosal immunity delivery carrier for DNA vaccines and had immense application promise and potential for vaccines and drugs.

Single-walled carbon nanotubes as delivery vehicles enhance the immunoprotective effect of a DNA vaccine against spring viremia of carp virus in common carp

Spring viremia of carp virus (SVCV) is highly contagious and pathogenic to cyprinid fish, causing enormous economic losses in aquaculture. Efficient and economic prophylactic measure against is the most pressing desired for the common carp farming industry. In this research, single-walled carbon nanotubes (SWCNTs) as a candidate DNA vaccine carrier was administrated via bath (1, 5, 10, 20, 40 mg L−1) or injection (1, 4, 8, 12, 20 μg) in common carp juvenile, and the different immune treatments to induce immunoprotective effect was analyzed. The results showed that higher levels of transcription and expression of G gene could be detected in muscle, spleen and kidney tissues via bath administration or intramuscular injection in SWCNTs-pEGFP-G treatment groups compared with naked pEGFP-G treatment groups. Meanwhile, complement activity, superoxide dismutase activity, alkaline phosphatase activity, immune-related genes (especially the TNF-α) and antibody levels were significantly enhanced in fish immunized with DNA vaccine combined with SWCNTs. The relative percentage survival were significantly enhanced in fish bathed with SWCNTs-pEGFP-G vaccine and the relative percentage survival reached to 57.5% in SWCNTs-pEGFP-G group than that of naked pEGFP-G (40.0%) at the highest vaccine dose (40 mg L−1) after 22 days of post infection, and fish in bath immunization group at a concentration of 40 mg L−1 could reach the similar relative percentage survival in injection group at a dose of 12 μg. This study suggest that ammonium-functionalized SWCNTs is the promising carrier for DNA vaccine and might be used to vaccinate large-scale juvenile fish by bath administration approach in aquaculture.

Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus

BackgroundCationic derivatives of polyprenols (trimethylpolyprenylammonium iodides – PTAI) with variable chain length between 6 and 15 isoprene units prepared from naturally occurring poly-cis-prenols were tested as DNA vaccine carriers in chickens and mice. This study aimed to investigate if PTAI could be used as an efficient carrier of a DNA vaccine.MethodsSeveral vaccine mixtures were prepared by combining different proportions of the vaccine plasmid (carrying cDNA encoding a vaccine antigen, hemagglutinin from H5N1 influenza virus) and various compositions of PTAI. The vaccines were delivered by intramuscular injection to either chickens or mice. The presence of specific antibodies in sera collected from the immunized animals was analyzed by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test.ResultsThe mixtures of PTAI with helper lipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), DC-cholesterol [{3ß-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) induced strong humoral response to the antigen encoded by the DNA vaccine plasmid.ConclusionThe animal immunization results confirmed that PTAI compositions, especially mixtures of PTAI with DOPE and DC-cholesterol, do work as effective carriers of DNA vaccines, comparable to the commercially available lipid transfection reagent.

Gene transfection efficiency into dendritic cells is influenced by the size of cationic liposomes/DNA complexes

Cationic liposomes have attracted recent attention as DNA vaccine carriers that can target dendritic cells (DCs). In general, cationic liposome/DNA complexes (lipoplexes) are taken up by various cells via clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, or phagocytosis, with the mode of endocytosis determining further intracellular trafficking pathways. Moreover, the physicochemical properties of cationic lipoplexes, including lipid composition, shape, size, and charge, influence transfection efficiency, affecting uptake and subsequent intracellular pathways. To develop cationic liposomes as potential DNA vaccine carriers, the objective of this study was to study the effect of lipoplex size on DNA transfection efficiency in DCs. We explored the size-dependent endocytosis pathway and the intracellular trafficking of cationic lipoplexes using bone marrow derived dendritic cells (BMDCs). Our results indicated that small-sized lipoplexes (approximately 270nm diameter) were taken up by BMDCs via caveolae-mediated endocytosis, which led to a non-degradative pathway, whereas larger-sized lipoplexes (approximately 500nm diameter) were taken up by BMDCs via clathrin-mediated endocytosis and micropinocytosis, which led to a lysosomal degradation pathway. These findings suggest that, by regulating the size of lipoplexes, it may be possible to develop cationic liposomes as DNA vaccine therapies for targeting DCs.

Enhanced Spacer Length between Mannose Mimicking Shikimoyl and Quinoyl Headgroups and Hydrophobic Region of Cationic Amphiphile Increases Efficiency of Dendritic Cell Based DNA Vaccination: A Structure-Activity Investigation.

In the field of dendritic cell based genetic immunization, previously we showed that liposomes of cationic amphiphiles containing mannose-mimicking shikimoyl headgroup are promising DNA vaccine carriers for dendritic cell (DC) transfection. The present structure-activity study reports on the influence of spacer length (between mannose-mimicking headgroups and quaternary nitrogen centers) in modulating the DC-transfection efficiencies. Further, we report on the anti-melanoma immune response inducing properties of the promising cationic amphiphiles in syngeneic C57BL/6J mice under prophylactic settings.

Incorporation of a Nuclear Localization Signal in pH Responsive LAH4-L1 Peptide Enhances Transfection and Nuclear Uptake of Plasmid DNA.

The major intracellular barriers associated with DNA delivery using nonviral vectors are inefficient endosomal/lysosomal escape and poor nuclear uptake. LAH4-L1, a pH responsive cationic amphipathic peptide, is an efficient DNA delivery vector that promotes the release of nucleic acid into cytoplasm through endosomal escape. Here we further enhance the DNA transfection efficiency of LAH4-L1 by incorporating nuclear localizing signal (NLS) to promote nuclear importation. Four NLSs were investigated: Simian virus 40 (SV40) large T-antigen derived NLS, nucleoplasmin targeting signal, M9 sequence, and the reverse SV40 derived NLS. All peptides tested were able to form positively charged nanosized complexes with DNA. Significant improvement in DNA transfection was observed in slow-dividing epithelial cancer cells (Calu-3), macrophages (RAW264.7), dendritic cells (JAWSII), and thymidine-induced growth-arrested cells, but not in rapidly dividing cells (A549). Among the four NLS-modified peptides, PK1 (modified with SV40 derived NLS) and PK2 (modified with reverse SV40 derived NLS) were the most consistent in improving DNA transfection; up to a 10-fold increase in gene expression was observed for PK1 and PK2 over the unmodified LAH4-L1. Additionally PK1 and PK2 were shown to enhance cellular uptake as well as nuclear entry of DNA. Overall, we show that the incorporation of SV40 derived NLS, in particular, to LAH4-L1 is a promising strategy to improve DNA delivery efficiency in slow-dividing cells and dendritic cells, with development potential for in vivo applications and as a DNA vaccine carrier.

Abstract 2360: GP350 containing exosomes as DNA vaccine carriers

Abstract Background: Plasmid DNA vaccination is a safe and economical vaccine modality that has been demonstrated to elicit cellular and humoral immunity in preclinical models and human clinical trials. We have previously shown that enriched mature naive B cells, and not myeloid/DC lineages, are capable of expanding Ag specific CD8 T cells in vivo in mice, and in vitro with human cells. In this study, we sought a means to directly deliver plasmid DNA to B cells as a means to specifically activate this antigen-presenting cell type. Methods: Exosomes were isolated from EBV+ lymphoblastic cell lines (LCL) and from HEK293T cells that had been transfected to express the EBV protein GP350. Plasmid DNA was labeled with a fluorescent peptide nucleic acid (PNA) probe for detection by imaging and conventional cytometry. Exosomes were transfected with fluorescently labeled plasmid DNA and incubated with either human CD21 transgenic mouse splenocytes or human PBMC and assayed for uptake and activation by FACS. Plasmid DNA transfected exosomes were coincubated with human PBMC and evaluated for their ability to expand antigen-specific CD8+ T cells by different methods. Results: EBV LCL and HEK293T derived exosomes were successfully transfected with plasmid DNA as verified by image assisted flow cytometry. Transient transfection of HEK293T cells with plasmids encoding a transgene led to release of exosomes containing the transgene product. EBV LCL and HEK 293T-GP350 derived exosomes increased delivery of plasmid DNA to both human B lymphocytes and transgenic hCD21 mouse B lymphocytes, when compared to naked DNA alone, in an hCD21 dependent fashion. At 24h, plasmid DNA containing B lymphocytes exhibited increased activation and upregulation of CD80/86 costimulatory molecules. Further, human B lymphocytes treated with exosome-plasmid DNA complexes led to antigen mRNA production and expansion of Ag specific CD8 T cells without the need for APC subset enrichment. Studies comparing the immunogenicity of GP350-exosome mediated delivery versus naked DNA delivery are underway in different mouse models. Conclusion: GP350 containing exosomes can successfully increase delivery of plasmid DNA to B lymphocytes even in the presence of other phagocytic cells like macrophages or dendritic cells. This led to detectable antigen production and expansion of cognate Ag specific CD8 T cell expansion without the need for B cell enrichment. Studies examining in vivo immunogenicity of exosomal plasmid DNA delivery are underway, but results to date suggest this approach may be a simple method to boost vaccine efficacy through increased delivery of DNA directly to relevant antigen-presenting B cells. Citation Format: Viswa Teja Colluru, Douglas G. McNeel. GP350 containing exosomes as DNA vaccine carriers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2360.

Protective immunity of grass carp immunized with DNA vaccine against Aeromonas hydrophila by using carbon nanotubes as a carrier molecule.

To reduce the economic losses caused by diseases in aquaculture industry, more efficient and economic prophylactic measures should be urgently investigated. In this research, the effects of a novel functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for DNA vaccine administration in juvenile grass carp against Aeromonas hydrophila were studied. Our results showed that SWCNTs loaded with DNA vaccine induced a better protection to juvenile grass carp against A.hydrophila. Moreover, SWCNTs conjugated with DNA vaccine provided significantly protective immunity compared with free DNA vaccine. Thereby, SWCNTs may be considered as a potential efficient DNA vaccine carrier to enhance the immunological activity.

IgA response and protection following nasal vaccination of chickens with Newcastle disease virus DNA vaccine nanoencapsulated with Ag@SiO2 hollow nanoparticles.

Newcastle disease caused by ND virus (NDV) is a highly contagious disease of birds. Vaccine for effective protection of poultry animals from NDV infection is urgently needed. Mucosal immunity plays a very important role in the antiviral immune response. In this study, a NDV F gene-containing DNA vaccine encapsulated in Ag@SiO2 hollow nanoparticles (pFDNA-Ag@SiO2-NPs) with an average diameter of 500 nm were prepared to assess the mucosal immune response. These nanoparticles exhibited low cytotoxicity and did not destroy the bioactivity of plasmid DNA, which could be expressed in vitro. The plasmid DNA was sustainably released after an initial burst release. In vivo immunization showed that the intranasal immunization of chickens with pFDNA-Ag@SiO2-NPs induced high titers of serum antibody, significantly promoted lymphocyte proliferation and induced higher expression levels of IL-2 and IFN-γ in a dose-dependent manner. These results indicated that the Ag@SiO2 hollow nanoparticles could serve as an efficient and safe delivery carrier for NDV DNA vaccine to induce mucosal immunity. This study has provided promising results for the further development of mucosal vaccines encapsulated in inorganic nanoparticles.

Mannosylated protamine as a novel DNA vaccine carrier for effective induction of anti-tumor immune responses

Gene immunotherapy has been developed as a promising strategy for inhibition of tumor growth. In the study, mannosylated protamine sulphate (MPS) was used as a novel DNA vaccine carrier to enhance transfection efficiency and anti-tumor immune responses. Anti-GRP DNA vaccine (pGRP) was selected as a model gene and condensed by MPS to form MPS/pGRP nanoparticles. The cellular uptake and transfection efficiency of MPS/pGRP nanoparticles in macrophages were evaluated. The effect of the nanoparticles in enhancing GRP-specific humoral immune response was then evaluated by nasal vaccination of nanoparticles in mice. The results demonstrated that both the cellular uptake and transfection efficiency of MPS nanoparticles in macrophages were higher than those of protamine nanoparticles. MPS/pGRP nanoparticles stimulated the production of higher titers (3.9×103) of specific antibodies against GRP than those of protamine/pGRP nanoparticles (6.4×102, p<0.01) and intramuscular injection pGRP solution (2.5×103, p<0.05). Furthermore, the inhibitory rate in MPS/pGRP nanoparticles group (65.80%) was significantly higher than that in protamine/pGRP nanoparticles group (35.13%) and pGRP solution group (43.39%). Hence, it is evident that MPS is an efficient targeting gene delivery carrier which could improve in vitro transfection efficiency as well as anti-tumor immunotherapy in mice.

Genetic Immunization With In Vivo Dendritic Cell-targeting Liposomal DNA Vaccine Carrier Induces Long-lasting Antitumor Immune Response

A major limiting factor retarding the clinical success of dendritic cell (DC)-based genetic immunizations (DNA vaccination) is the scarcity of biologically safe and effective carrier systems for targeting the antigen-encoded DNA vaccines to DCs under in vivo settings. Herein, we report on a potent, mannose receptor selective in vivo DC-targeting liposomes of a novel cationic amphiphile with mannose-mimicking shikimoyl head-group. Flow cytometric experiments with cells isolated from draining lymph nodes of mice s.c. immunized with lipoplexes of pGFP plasmid (model DNA vaccine) using anti-CD11c antibody-labeled magnetic beads revealed in vivo DC-targeting properties of the presently described liposomal DNA vaccine carrier. Importantly, s.c. immunizations of mice with electrostatic complex of the in vivo DC-targeting liposome and melanoma antigen-encoded DNA vaccine (p-CMV-MART1) induced long-lasting antimelanoma immune response (100 days post melanoma tumor challenge) with remarkable memory response (more than 6 months after the second tumor challenge). The presently described direct in vivo DC-targeting liposomal DNA vaccine carrier is expected to find future exploitations toward designing effective vaccines for various infectious diseases and cancers.

Recombinant Saccharomyces cerevisiae serves as novel carrier for oral DNA vaccines in Carassius auratus.

Oral delivery of DNA vaccines represents a promising vaccinating method for fish. Recombinant yeast has been proved to be a safe carrier for delivering antigen proteins and DNAs to some species in vivo. However, whether recombinant yeast can be used to deliver functional DNAs for vaccination to fish is still unknown. In this study, red crucian carp (Carassius auratus) was orally administrated with recombinant Saccharomyces cerevisiae harboring CMV-EGFP expression cassette. On day 5 post the first vaccination, EGFP expression in the hindgut was detected under fluorescence microscope. To further study whether the delivered gene could induce specific immune responses, the model antigen ovalbumin (OVA) was used as immunogen, and oral administrations were conducted with recombinant S. cerevisiae harboring pCMV-OVA mammalian gene expression cassette as gene delivery or pADH1-OVA yeast gene expression cassette as protein delivery. Each administration was performed with three different doses, and the OVA-specific serum antibody was detected in all the experimental groups by western blotting and enzyme-linked immunosorbent assay (ELISA). ELISA assay also revealed that pCMV-OVA group with lower dose (pCMV-OVA-L) and pADH1-OVA group with moderate dose (pADH1-OVA-M) triggered relatively stronger antibody response than the other two doses. Moreover, the antibody level induced by pCMV-OVA-L group was significantly higher than pADH1-OVA-M group at the same serum dilutions. All the results suggested that recombinant yeast can be used as a potential carrier for oral DNA vaccines and would help to develop more practical strategies to control infectious diseases in aquaculture.

Synthesis, characterization, and immune efficacy of layered double hydroxide@SiO2 nanoparticles with shell-core structure as a delivery carrier for Newcastle disease virus DNA vaccine.

Layered double hydroxide (LDH)@SiO2 nanoparticles were developed as a delivery carrier for the plasmid DNA expressing the Newcastle disease virus F gene. The LDH was hydrotalcite-like materials. The plasmid DNA encapsulated in the LDH@SiO2 nanoparticles (pFDNA-LDH@SiO2-NPs) was prepared by the coprecipitation method, and the properties of pFDNA-LDH@SiO2-NPs were characterized by transmission electron microscopy, zeta potential analyzer, Fourier transform infrared spectroscopy, and X-ray diffraction analysis. The results demonstrated that the pFDNA-LDH@SiO2-NPs had a regular morphology and high stability with a mean diameter of 371.93 nm, loading capacity of 39.66%±0.45%, and a zeta potential of +31.63 mV. A release assay in vitro showed that up to 91.36% of the total plasmid DNA could be sustainably released from the pFDNA-LDH@SiO2-NPs within 288 hours. The LDH@SiO2 nanoparticles had very low toxicity. Additionally, their high transfection efficiency in vitro was detected by fluorescent microscopy. Intranasal immunization of specific pathogen-free chickens with pFDNA-LDH@SiO2-NPs induced stronger cellular, humoral, and mucosal immune responses and achieved a greater sustained release effect than intramuscular naked plasmid DNA, and the protective efficacy after challenge with the strain F48E9 with highly virulent (mean death time of chicken embryos ≤60 hours, intracerebral pathogenicity index in 1 -day-old chickens >1.6) was 100%. Based on the results, LDH@SiO2 nanoparticles can be used as a delivery carrier for mucosal immunity of Newcastle disease DNA vaccine, and have great application potential in the future.

A KALA-modified lipid nanoparticle containing CpG-free plasmid DNA as a potential DNA vaccine carrier for antigen presentation and as an immune-stimulative adjuvant.

Technologies that delivery antigen-encoded plasmid DNA (pDNA) to antigen presenting cell and their immune-activation are required for the success of DNA vaccines. Here we report on an artificial nanoparticle that can achieve these; a multifunctional envelope-type nanodevice modified with KALA, a peptide that forms α-helical structure at physiological pH (KALA-MEND). KALA modification and the removal of the CpG-motifs from the pDNA synergistically boosted transfection efficacy. In parallel, transfection with the KALA-MEND enhances the production of multiple cytokines and chemokines and co-stimulatory molecules via the Toll-like receptor 9-independent manner. Endosome-fusogenic lipid envelops and a long length of pDNA are essential for this immune stimulation. Furthermore, cytoplasmic dsDNA sensors that are related to the STING/TBK1 pathway and inflammasome are involved in IFN-β and IL-1β production, respectively. Consequently, the robust induction of antigen-specific cytotoxic T-lymphoma activity and the resulting prophylactic and therapeutic anti-tumor effect was observed in mice that had been immunized with bone marrow-derived dendritic cells ex vivo transfected with antigen-encoding pDNA. Collectively, the KALA-MEND possesses dual functions; gene transfection system and immune-stimulative adjuvant, those are both necessary for the successful DNA vaccine.

PH-sensitive carbonate apatite nanoparticles as DNA vaccine carriers enhance humoral and cellular immunity

To demonstrate the potential of pH-sensitive carbonate apatite (CO3Ap) nanoparticles as DNA vaccine carriers to enhance vaccination efficacy, we examined the humoral and cellular immune responses of C57BL/6 mice immunized with the plasmid expression vector pCI-neo encoding the full-length soluble ovalbumin (OVA) (pCI-neo-sOVA), pCI-neo-sOVA/CO3Ap complexes, or pCI-neo/CO3Ap complexes as a control. Mice immunized with a low dose of pCI-neo-sOVA-loaded CO3Ap (10μg) produced ex vivo splenocyte proliferation after stimulation with CD8 T-cell but not CD4 T-cell epitopes and a delayed-type-hypersensitivity reaction more efficiently than mice in the other groups. Furthermore, mice receiving this immunization generated the same levels of OVA-specific antibodies and interferon (IFN)-γ secretion after CD8 T-cell and CD4 T-cell epitope challenges as those in mice treated with 100μg of free pCI-neo-sOVA, whereas mice injected with a high dose of pCI-neo-sOVA-loaded CO3Ap (100μg) or with control plasmids produced negligible levels of OVA-specific antibodies or IFN-γ. Therefore, our results showed that 10μg of pCI-neo-sOVA delivered by CO3Ap strongly elicited humoral and cellular immune responses. This study is the first to demonstrate the promising potential of CO3Ap nanoparticles for DNA vaccine delivery.

Protective efficacy of PLGA microspheres loaded with divalent DNA vaccine encoding the ompA gene of Aeromonas veronii and the hly gene of Aeromonas hydrophila in mice

In the present study, poly (lactic-co-glycolic) acid (PLGA) was used as a carrier for a divalent fusion DNA vaccine encoding the Aeromonas veronii outer membrane protein A (ompA) and Aeromonas hydrophila hemolysins (hly) protein. The recombinant pET-28a-ompA-hly was constructed by inserting the ompA gene and hly gene into a pET-28a expression vector. Loading of ompA-hly antigen module on PLGA microspheres were accomplished by water-in-oil-in-water (W/O/W) encapsulation. The molecular weight and specificity of pET-28a-ompA-hly were detected by dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The microspheres showed an average particle size of 100–150μm and a loading efficiency (LE) of 68.8%. Mice received ompA-hly antigen-loaded PLGA microspheres by intraperitoneal or intragastric administration mounted strong and sustained IgG response, which was significantly higher (p<0.05) than those achieved by pET-28a-ompA-hly antigen alone. OmpA-hly antigen-loaded PLGA microsphere vaccine uniquely conferred a long lasting (30 days) sterile immunity against challenge infection. Results indicated that ompA-hly antigen-loaded PLGA microsphere vaccine is a qualified candidate vector system for sterile protective immunity against A. hydrophila and A. veronii infections.

Dendritic cell targeted liposomes–protamine–DNA complexes mediated by synthetic mannosylated cholestrol as a potential carrier for DNA vaccine

To construct mannosylated liposomes/protamine/DNA (LPD) carriers for DNA vaccine targeting to dendritic cells (DCs), a mannosylated cholesterol derivative (Man-C6-Chol) was synthesized via simple ester linkage and amide bonds. Then, the Man-C6-Chol was applied to LPD formulation as a synthetic ligand. The physicochemical properties of mannosylated LPD (Man-LPD) were first evaluated, including the size and zeta potential, morphology and the ability to protect DNA against DNase I degradation. Man-LPD showed a small size with a stable viral-like structure. In comparison to non-mannose liposomes/LPD (Man-free liposomes/LPD), mannosylated liposomes/LPD (Man-liposomes/Man-LPD) exhibited higher efficiency in both intracellular uptake (2.3-fold) and transfection (4.5-fold) in vitro. Subsequent MTT assays indicated that the LPD carriers had low toxicity on the tested cells. Afterwards, the investigation into the maturation activation on primary bone marrow-derived DCs (BMDCs) showed that both Man-LPD and Man-free LPD induced remarkable up-regulation of CD80, CD86 and CD40 on BMDCs. Inspired by these studies, we can conclude that the synthetic mannosylated LPD targeting to DCs was a potential carrier for DNA vaccine.

Alginic Acid-Coated Chitosan Nanoparticles Loaded with Legumain DNA Vaccine: Effect against Breast Cancer in Mice

Legumain-based DNA vaccines have potential to protect against breast cancer. However, the lack of a safe and efficient oral delivery system restricts its clinical application. Here, we constructed alginic acid-coated chitosan nanoparticles (A.C.NPs) as an oral delivery carrier for a legumain DNA vaccine. First, we tested its characteristic in acidic environments in vitro. DNA agarose electrophoresis data show that A.C.NPs protected DNA better from degradation in acidic solution (pH 1.5) than did chitosan nanoparticles (C.NPs). Furthermore, size distribution analysis showed that A.C.NPs tended to aggregate and form micrometer scale complexes in pH<2.7, while dispersing into nanoparticles with an increase in pH. Mice were intragastrically administrated A.C.NPs carrying EGFP plasmids and EGFP expression was detected in the intestinal Peyer’s patches. Full-length legumain plasmids were loaded into different delivery carriers, including C.NPs, attenuated Salmonella typhimurium and A.C.NPs. A.C.NPs loaded with empty plasmids served as a control. Oral vaccination was performed in the murine orthotopic 4T1 breast cancer model. Our data indicate that tumor volume was significantly smaller in groups using A.C.NPs or attenuated Salmonella typhimurium as carriers. Furthermore, splenocytes co-cultured them with 4T1 cells pre-stimulated with CoCl2, which influenced the translocation of legumain from cytoplasm to plasma membrane, showed a 4.7 and 2.3 folds increase in active cytotoxic T lymphocytes (CD3+/CD8+/CD25+) when treated with A.C.NPs carriers compared with PBS C.NPs. Our study suggests that C.NPs coated with alginic acid may be a safe and efficient tool for oral delivery of a DNA vaccine. Moreover, a legumain DNA vaccine delivered orally with A.C.NPs can effectively improve autoimmune response and protect against breast cancer in mice.

AttenuatedSalmonella typhimuriumSV4089 as a Potential Carrier of Oral DNA Vaccine in Chickens

Attenuated Salmonella has been used as a carrier for DNA vaccine. However, in vitro and in vivo studies on the bacteria following transfection of plasmid DNA were poorly studied. In this paper, eukaryotic expression plasmids encoding avian influenza virus (AIV) subtype H5N1 genes, pcDNA3.1/HA, NA, and NP, were transfected into an attenuated Salmonella enteric typhimurium SV4089. In vitro stability of the transfected plasmids into Salmonella were over 90% after 100 generations. The attenuated Salmonella were able to invade MCF-7 (1.2%) and MCF-10A (0.5%) human breast cancer cells. Newly hatched specific-pathogen-free (SPF) chicks were inoculated once by oral gavage with 109 colony-forming unit (CFU) of the attenuated Salmonella. No abnormal clinical signs or deaths were recorded after inoculation. Viable bacteria were detected 3 days after inoculation by plating from spleen, liver, and cecum. Fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) were carried out for confirmation. Salmonella was not detected in blood cultures although serum antibody immune responses to Salmonella O antiserum group D1 factor 1, 9, and 12 antigens were observed in all the inoculated chickens after 7 days up to 35 days. Our results showed that live attenuated S. typhimurium SV4089 harboring pcDNA3.1/HA, NA, and NP may provide a unique alternative as a carrier for DNA oral vaccine in chickens.

Poly(2-aminoethyl methacrylate) with Well-Defined Chain Length for DNA Vaccine Delivery to Dendritic Cells

Poly(2-aminoethyl methacrylate) (PAEM) homopolymers with defined chain length and narrow molecular weight distribution were synthesized using atom transfer radical polymerization (ATRP), and a comprehensive study was conducted to evaluate the colloidal properties of PAEM/plasmid DNA polyplexes, the uptake and subcellular trafficking of polyplexes in antigen-presenting dendritic cells (DCs), and the biological performance of PAEM as a potential DNA vaccine carrier. PAEM of different chain length (45, 75, and 150 repeating units) showed varying strength in condensing plasmid DNA into narrowly dispersed nanoparticles with very low cytotoxicity. Longer polymer chain length resulted in higher levels of overall cellular uptake and nuclear uptake of plasmid DNA, but shorter polymer chains favored intracellular and intranuclear release of free plasmid from the polyplexes. Despite its simple chemical structure, PAEM transfected DCs very efficiently in vitro in media with or without serum and led to phenotypic maturation of DCs. When a model antigen-encoding ovalbumin plasmid was used, transfected DCs stimulated the activation of naïve CD8(+) T cells to produce high levels of interferon-γ. The efficiency of transfection, DC maturation, and CD8(+) T cell activation showed varying degrees of polymer chain-length dependence. These structurally defined cationic polymers may have much potential as efficient DNA vaccine carriers and immunostimulatory adjuvants. They may also serve as a model material system for elucidating structural and intracellular mechanisms of polymer-mediated DNA vaccine delivery.