Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B

This work investigates the preparation and in vivo efficacy of plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. Chitosan pDNA nanoparticles were prepared using a complex coacervation process. Prepared nanoparticles were characterized for size, shape, surface charge, plasmid loading and ability of nanoparticles to protect DNA against nuclease digestion and for their transfection efficacy. Nasal administration of nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level. However, intramuscular administration of naked DNA and alum adsorbed HBsAg did not elicit sIgA titre in mucosal secretions that was induced by nasal immunization with chitosan nanoparticles. Similarly, cellular responses (cytokine levels) were poor in case of alum adsorbed HBsAg. Chitosan nanoparticles thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. The study signifies the potential of chitosan nanoparticles as DNA vaccine carrier and adjuvant for effective immunization through non-invasive nasal route.     

Stable dry powder formulation for nasal delivery of anthrax vaccine

There is a current biodefense interest in protection against anthrax. Here, we developed a new generation of stable and effective anthrax vaccine. We studied the immune response elicited by recombinant protective antigen (rPA) delivered intranasally with a novel mucosal adjuvant, a mast cell activator compound 48/80 (C48/80). The vaccine formulation was prepared in a powder form by spray-freeze-drying (SFD) under optimized conditions to produce particles with a target size of D(50) = 25 μm, suitable for delivery to the rabbit nasal cavity. Physicochemical properties of the powder vaccines were characterized to assess their delivery and storage potential. Structural stability of rPA was confirmed by circular dichroism and attenuated total reflectance-Fourier transform infrared spectroscopy, whereas functional stability of rPA and C48/80 was monitored by cell-based assays. Animal study was performed using a unit-dose powder device for direct nasal application. Results showed that C48/80 provided effective mucosal adjuvant activity in rabbits. Freshly prepared SFD powder vaccine formulations or powders stored for over 2 years at room temperature elicited significantly elevated serum PA-specific and lethal toxin neutralization antibody titers that were comparable to that induced by intramuscular immunization with rPA. Nasal delivery of this vaccine formulation may be a viable alternative to the currently licensed vaccine or an attractive vaccine platform for other mucosally transmitted diseases.     

Antigen-adjuvant nanoconjugates for nasal vaccination: an improvement over the use of nanoparticles?

Entrapment of antigens in mucoadhesive nanoparticles prepared from N-trimethyl chitosan (TMC) has been shown to increase their immunogenicity. However, because of their large size compared to soluble antigens, particles poorly diffuse through the nasal epithelium. The aim of this work was to study whether nasal vaccination with a much smaller TMC-antigen nanoconjugate would result in higher antibody responses as compared to TMC nanoparticles. TMC was covalently linked to a model antigen, ovalbumin (OVA), using thiol chemistry. For comparison, TMC/OVA nanoparticles and solutions of OVA and a physical mixture of TMC and OVA were made. As shown previously for TMC/OVA nanoparticles, TMC-OVA conjugate prolonged the nasal residence time of the antigen. TMC-OVA conjugate diffused significantly better through a monolayer of lung carcinoma (Calu-3) cells than TMC/OVA nanoparticles did. Moreover, nasal immunization of mice with the conjugate resulted in significantly more OVA positive DCs in the cervical lymph nodes as compared to TMC/OVA nanoparticles. Mice nasally immunized with TMC-OVA conjugate produced high levels of secretory IgA in nasal washes and higher titers of OVA-specific IgG than mice immunized with TMC/OVA nanoparticles after a priming dose. Moreover, as compared to TMC/OVA nanoparticles, TMC-OVA conjugate induced a more balanced IgG1/IgG2a response. In conclusion, the TMC-antigen nanoconjugate improves nasal delivery and immunogenicity of the antigen. This suggests that efficient codelivery of antigen and adjuvant to DCs, rather than a particulate form of the antigen/adjuvant combination, is decisive for the immunogenicity of the antigen.     

Protective immune responses to meningococcal C conjugate vaccine after intranasal immunization of mice with the LTK63 mutant plus chitosan or trimethyl chitosan chloride as novel delivery platform

Chitosan and its derivative N-trimethyl chitosan chloride (TMC), given as microparticles or powder suspensions, and the non-toxic mucosal adjuvant LTK63, were evaluated for intranasal immunization with the group C meningococcal conjugated vaccine (CRM-MenC). Mice immunized intranasally with CRM-MenC formulated with chitosan or TMC and the LTK63 mutant, showed high titers of serum and mucosal antibodies specific for the MenC polysaccharide. Neither significant differences were observed between microparticle formulations and powder suspensions nor when LTK63 was pre-associated to the delivery system or not. The bactericidal activity measured in serum of mice immunized intranasally with the conjugated vaccine formulated with the delivery systems and the LT mutant was superior to the activity in serum of mice immunized sub-cutaneously. Importantly, intranasal but not parenteral immunization, induced bactericidal antibodies at the nasal level, when formulated with both delivery system and adjuvant.     

Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles

Alginate coated chitosan nanoparticles were previously developed with the aim of protecting the antigen, adsorbed on the surface of those chitosan nanoparticles, from enzymatic degradation at mucosal surfaces. In this work, this new delivery system was loaded with the recombinant hepatitis B surface antigen (HBsAg) and applied to mice by the intranasal route. Adjuvant effect of the delivery system was studied by measuring anti-HBsAg IgG in serum, anti-HBsAg sIgA in faeces extracts or nasal and vaginal secretions and interferon-gamma production in supernatants of the spleen cells. The mice were primed with 10 microg of the vaccine associated or not with nanoparticles and associated or not with 10 microg CpG oligodeoxynucleotide (ODN) followed by two sequential boosts at three week intervals. The association of HBsAg with the alginate coated chitosan nanoparticles, administered intranasally to the mice, gave rise to the humoral mucosal immune response. Humoral systemic immune response was not induced by the HBsAg loaded nanoparticles alone. The generation of Th1-biased antigen-specific systemic antibodies, however, was observed when HBsAg loaded nanoparticles were applied together with a second adjuvant, the immunopotentiator, CpG ODN. Moreover, all intranasally vaccinated groups showed higher interferon-gamma production when compared to na?ve mice.     

Chitosan for mucosal vaccination.

The striking advantage of mucosal vaccination is the production of local antibodies at the sites where pathogens enter the body. Because vaccines alone are not sufficiently taken up after mucosal administration, they need to be co-administered with penetration enhancers, adjuvants or encapsulated in particles. Chitosan easily forms microparticles and nanoparticles which encapsulate large amounts of antigens such as ovalbumin, diphtheria toxoid or tetanus toxoid. It has been shown that ovalbumin loaded chitosan microparticles are taken up by the Peyer's patches of the gut associated lymphoid tissue (GALT). This unique uptake demonstrates that chitosan particulate drug carrier systems are promising candidates for oral vaccination. Additionally, after co-administering chitosan with antigens in nasal vaccination studies, a strong enhancement of both mucosal and systemic immune responses is observed. This makes chitosan very suitable for nasal vaccine delivery. In conclusion, chitosan particles, powders and solutions are promising candidates for mucosal vaccine delivery. Mucosal vaccination not only reduces costs and increases patient compliance, but also complicates the invasion of pathogens through mucosal sites.     

壳聚糖衍生物在口鼻黏膜疫苗中的应用

壳聚糖是一种有效的黏膜疫苗佐剂和递送载体,但因其水溶性差,应用受到一定限制.通过对壳聚糖进行不同的化学修饰可得到各类壳聚糖衍生物,这些衍生物不仅溶解性较好,而且保持了壳聚糖良好的生物相容性、生物降解性、免疫刺激活性等优势,为黏膜疫苗,尤其是经口、鼻途径递送的疫苗提供了新型候选佐剂和递送载体.此文对修饰壳聚糖的主要方法以及其衍生物在口鼻黏膜疫苗中的应用做一综述.     

Chitosan-based delivery systems for mucosal vaccines

Introduction: Mucosal vaccine development faces several challenges and opportunities. Critical issues for effective mucosal vaccination include the antigen-retention period that enables interaction with the lymphatic system, choice of adjuvant that is nontoxic and induces the required immune response and possibly an ability to mimic mucosal pathogens. Chitosan-based delivery systems are reviewed here as they address these issues and hence represent the most promising candidates for the delivery of mucosal vaccines. Areas covered: A comprehensive literature search was conducted, to locate relevant studies published within the last 5 years. Mucosal delivery via nasal and oral routes is evaluated with respect to chitosan type, dosage forms, co-adjuvanting with novel adjuvants and modulation of the immune system. Expert opinion: It is concluded that chitosan derivatives offer advantageous opportunities such as nanoparticle and surface charge manipulation that facilitate vaccine targeting. Nevertheless, these technologies represent a longer-term goal. By contrast, chitosan (unmodified form) with or without a co-adjuvant has significant toxicology and human data to support safe mucosal administration, and thus has the potential for earlier product introduction into the market.     

Tetanus Toxoid Loaded Nanoparticles from Sulfobutylated Poly(Vinyl Alcohol)-Graft-Poly(Lactide-co-Glycolide): Evaluation of Antibody Response After Oral and Nasal Application in Mice

Purpose. Aim of the study was the evaluation of the potential of novel tetanus toxoid (TT) loaded nanoparticles (NP) for electing an immune response in mice against TT. Methods. Six week-old female Balb/c mice were immunized by oral (p.o.), nasal (i.n.) and intraperitoneal (i.p.) application of TT NP loaded by adsorption. As polymer a novel polyester, sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide), SB(43)-PVAL-g-PLGA was used. Blood samples were collected 4 and 6 weeks after immunization and assayed for serum IgG- as well as IgA antibody titers by ELISA. NP formulations varying in size and loading were compared to alum adsorbates as well as to TT solutions. Results. Both, p.o. and i.n. administration of TT associated NP increased serum titers up to 3 × 10³ (IgG) and 2 × 10³ (IgA). While small NP induced significantly higher titers then larger ones after oral administration, intermediate NP induced antibodies after nasal application. Of the mucosal routes investigated, i.n. seems to be more promising compared to p.o. immunization. Conclusions. Antigen loaded NP prepared from surface modified polyesters combined with CT show considerable potential as a vaccine delivery system for mucosal immunization. The results warrant further experiments to explore in more detail the potential use of NP as mucosal vaccine delivery system.     

Bioadhesive delivery systems for mucosal vaccine delivery

Mucosal vaccine delivery potentially induces mucosal as well as systemic immune responses and may have advantages particularly for optimal protection against pathogens that infect the host through mucosal surfaces. However, the delivery of antigens through mucosal membranes remains a major challenge due to unfavorable physiological conditions (pH and enzymes) and significant biological barriers, which restrict the uptake of antigens. To improve mucosal vaccine delivery, the use of bioadhesive delivery systems offers numerous advantages, including protection from degradation, increasing concentration of antigen in the vicinity of mucosal tissue for better absorption, extending their residence time, and/or targeting them to sites of antigen uptake. Although some bioadhesives have direct immune stimulating properties, it appears most likely that successful mucosal vaccination will require the addition of vaccine adjuvants for optimal immune responses, particularly if they are to be used in an unprimed population. Thus, complex vaccine formulations and delivery strategies have to be carefully designed to appropriately stimulate immune response for the target pathogen. In addition, careful consideration is needed to define the “best” route for mucosal immunization for each individual pathogen.     

Intranasal administration of CpG oligonucleotides induces mucosal and systemic Type 1 immune responses and adjuvant activity to porcine reproductive and respiratory syndrome killed virus vaccine in piglets in vivo

Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvants for immune responses, particularly in mice. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in piglets are scarce. We have previously demonstrated that CpG ODN is a potent adjuvant to pseudorabies attenuated virus (PRV) vaccine when administered subcutaneously (SC) in newborn piglets. Herein, we evaluated intranasal (IN) delivery of CpG ODN with porcine reproductive and respiratory syndrome (PRRS) killed virus vaccine (PRRSV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, Peripheral blood mononuclear cells (PBMC) proliferation) and mucosal (IgA in feces, nasal and oral secretions) immune responses against antigen. CpG ODN stimulated both T-helper type1 (Type 1) (IgG2) and Type 2 (IgA) responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN may be effective as a mucosal adjuvant with commercial vaccine in husbandry animals.     

Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice.

High molecular weight (Mw) chitosan (CS) solutions have already been proposed as vehicles for nasal immunization. The aim of the present work was to investigate the potential utility of low Mw CS in the form of nanoparticles as new long-term nasal vaccine delivery vehicles. For this purpose, CS of low Mws (23 and 38 kDa) was obtained previously by a depolymerization process of the commercially available CS (70 kDa). Tetanus toxoid (TT), used as a model antigen, was entrapped within CS nanoparticles by an ionic cross-linking technique. TT-loaded nanoparticles were first characterized for their size, electrical charge, loading efficiency and in vitro release of antigenically active toxoid. The nanoparticles were then administered intranasally to conscious mice in order to study their feasibility as vaccine carriers. CS nanoparticles were also labeled with FITC-BSA and their interaction with the rat nasal mucosa examined by confocal laser scanning microcopy (CLSM). Irrespective of the CS Mw, the nanoparticles were in the 350 nm size range, and exhibited a positive electrical charge (+40 mV) and associated TT quite efficiently (loading efficiency: 50-60%). In vitro release studies showed an initial burst followed by an extended release of antigenically active toxoid. Following intranasal administration, TT-loaded nanoparticles elicited an increasing and long-lasting humoral immune response (IgG concentrations) as compared to the fluid vaccine. Similarly, the mucosal response (IgA levels) at 6 months post-administration of TT-loaded CS nanoparticles was significantly higher than that obtained for the fluid vaccine. The CLSM images indicated that CS nanoparticles can cross the nasal epithelia and, hence, transport the associated antigen. Interestingly, the ability of these nanoparticles to provide improved access to the associated antigen to the immune system was not significantly affected by the CS Mw. Indeed, high and long-lasting responses could be obtained using low Mw CS molecules. Furthermore, the response was not influenced by the CS dose (70-200 microg), achieving a significant response for a very low CS dose. In conclusion, nanoparticles made of low Mw CS are promising carriers for nasal vaccine delivery.     

Microfold-cell targeted surface engineered polymeric nanoparticles for oral immunization

Present work was envisaged to develop novel M-cell targeted polymeric particles that are capable of protecting the antigen from harsh gastric conditions. Ulex europaeus agglutinin (UEA-1) lectin was anchored for selective delivery of antigen to gut-associated lymphoid tissue (GALT). In the present investigation, chitosan nanoparticles were prepared by ionic gelation followed by antigen (bovine serum albumin, BSA) adsorption. Developed nanoparticles were further coated by UEA-1 lectin conjugated alginate and characterized for size, shape, zeta-potential, entrapment efficiency, and in vitro release. The immunological response of the developed system were performed in Balb/c mice and compared with aluminium hydroxide gel-based conventional vaccine. Results indicated that immunization with UEA-1 lectin conjugated alginate-coated particles induces efficient systemic as well as mucosal immune responses against BSA compared to other formulations. Aluminium-based vaccine dominated throughout the study, while failed in case of mucosal antibody. Additionally, IgG1 and IgG2a isotypes were determined to confirm the TH1/TH2 mixed immune response. The developed formulation exhibited superior systemic response along with dominating mucosal immunity. These data demonstrate the potential of UEA-alginate-coated nanoparticles as effective delivery system via oral route.     

Chitosan-based delivery systems for mucosal vaccines

Introduction: Mucosal vaccine development faces several challenges and opportunities. Critical issues for effective mucosal vaccination include the antigen-retention period that enables interaction with the lymphatic system, choice of adjuvant that is nontoxic and induces the required immune response and possibly an ability to mimic mucosal pathogens. Chitosan-based delivery systems are reviewed here as they address these issues and hence represent the most promising candidates for the delivery of mucosal vaccines.

Areas covered: A comprehensive literature search was conducted, to locate relevant studies published within the last 5 years. Mucosal delivery via nasal and oral routes is evaluated with respect to chitosan type, dosage forms, co-adjuvanting with novel adjuvants and modulation of the immune system.

Expert opinion: It is concluded that chitosan derivatives offer advantageous opportunities such as nanoparticle and surface charge manipulation that facilitate vaccine targeting. Nevertheless, these technologies represent a longer-term goal. By contrast, chitosan (unmodified form) with or without a co-adjuvant has significant toxicology and human data to support safe mucosal administration, and thus has the potential for earlier product introduction into the market.     

以壳聚糖为佐剂的幽门螺杆菌疫苗免疫小鼠的TH免疫应答

目的探讨壳聚糖在幽门螺杆菌疫苗中的免疫佐剂效应。方法将20只Balb/c小鼠分为4组,分别以磷酸盐缓冲液、单纯幽门螺杆菌(Hp)抗原、Hp抗原+壳聚糖酸溶液、Hp抗原+壳聚糖颗粒进行口服免疫,用ELISA法检测胃黏膜中白细胞介素(IL)-2I、L-4,IL-10。结果胃黏膜内IL-10和IL-4的含量在以壳聚糖为佐剂组(255.25,237.05 pg/mg和14.70,14.48 pg/mg)显著高于无佐剂组(104.33和6.49 pg/mg)和对照组(67.13,4.19 pg/mg)(P<0.05~0.01)。结论以壳聚糖为佐剂的Hp疫苗可明显促进TH2细胞因子IL-4I、L-10的分泌,这可能在其免疫防御中起作用。     

Chitosan and its derivatives in mucosal drug and vaccine delivery.

Numerous studies have demonstrated that chitosan and their derivatives (N-trimethyl chitosan, mono-N-carboxymethyl chitosan) are effective and safe absorption enhancers to improve mucosal (nasal, peroral) delivery of hydrophylic macromolecules such as peptide and protein drugs and heparins. This absorption enhancing effect of chitosans is caused by opening of the intercellular tight junctions, thereby favouring the paracellular transport of macromolecular drugs. Chitosan nano- and microparticles are also suitable for controlled drug release. Association of vaccines to some of these particulate systems has shown to enhance the antigen uptake by mucosal lymphoid tissues, thereby inducing strong systemtic and mucosal immune responses against the antigens. The aspecific adjuvant activity of chitosans seems to be dependent on the degree of deacetylation and the type of formulation. From the studies reviewed it is concluded that chitosan and chitosan derivatives are promising polymeric excipients for mucosal drug and vaccine delivery.     

Microparticles for intranasal immunization

Of the several routes available for mucosal immunization, the nasal route is particularly attractive because of ease of administration and the induction of potent immune responses, particularly in the respiratory and genitourinary tracts. However, adjuvants and delivery systems are required to enhance immune responses following nasal immunization. This review focuses on the use of microparticles as adjuvants and delivery systems for protein and DNA vaccines for nasal immunization. In particular we discuss our own work on poly(lactide co-glycolide) (PLG) microparticles with entrapped protein or adsorbed DNA as a vaccine delivery system. The possible mechanisms involved in the enhancement of immune responses through the use of DNA adsorbed onto PLG microparticles are also discussed.     

Protective immune responses to meningococcal C conjugate vaccine after intranasal immunization of mice with the LTK63 mutant plus chitosan or trimethyl chitosan chloride as novel delivery platform

Chitosan and its derivative N-trimethyl chitosan chloride (TMC), given as microparticles or powder suspensions, and the non-toxic mucosal adjuvant LTK63, were evaluated for intranasal immunization with the group C meningococcal conjugated vaccine (CRM-MenC). Mice immunized intranasally with CRM-MenC formulated with chitosan or TMC and the LTK63 mutant, showed high titers of serum and mucosal antibodies specific for the MenC polysaccharide. Neither significant differences were observed between microparticle formulations and powder suspensions nor when LTK63 was pre-associated to the delivery system or not. The bactericidal activity measured in serum of mice immunized intranasally with the conjugated vaccine formulated with the delivery systems and the LT mutant was superior to the activity in serum of mice immunized sub-cutaneously. Importantly, intranasal but not parenteral immunization, induced bactericidal antibodies at the nasal level, when formulated with both delivery system and adjuvant.     

Quaternized chitosan nanoparticles loaded with the combined attenuated live vaccine against Newcastle disease and infectious bronchitis elicit immune response in chicken after intranasal administration

Newcastle disease (ND) and infectious bronchitis (IB) are important diseases, which cause respiratory diseases in chickens, resulting in severely economic losses in the poultry industry. In this study, N-2-hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC) and N,O-carboxymethyl chitosan (CMC) were synthesized as adjuvant and delivery carrier for vaccine antigens. N-2-HACC-CMC/NDV/IBV nanoparticles (NPs) (NDV/La Sota and IBV/H120 encapsulated in N-2-HACC-CMC NPs) and N-2-HACC-CMC/NDV-IBV NPs (the mixing of N-2-HACC-CMC/NDV NPs and N-2-HACC-CMC/IBV NPs in a ratio of 1:1) were prepared by the polyelectrolyte composite method, respectively. Both nanoparticles exhibited lower cytotoxicity and higher stability. Their bioactivities were maintained when they were stored at 37?°C for three weeks. Release assay in vitro showed that both NDV and IBV could be sustainably released from the nanoparticles after an initial burst release. In vivo immunization of chickens showed that N-2-HACC-CMC/NDV/IBV NPs or N-2-HACC-CMC/NDV-IBV NPs intranasally induced higher titers of IgG and IgA antibodies, significantly promoted proliferation of lymphocytes and induced higher levels of interleukine-2 (IL-2), IL-4 and interferon-γ (IFN-γ) than the commercially combined attenuated live vaccine did. This is the first study in the field of animal vaccines demonstrating that intranasal administration of chickens with antigens (NDV and IBV) encapsulated with chitosan derivative could induce humoral, cellular, and mucosal immune responses, which protected chickens from the infection of highly virulent NDV and IBV. This study indicated that N-2-HACC-CMC could be used as an efficient adjuvant and delivery carrier for further development of mucosal vaccines and drugs and could have an immense application potential in medicine.     

Evaluation of mucoadhesive nanoparticle based nasal vaccine

The main objective of this study was to prepare Hepatitis B surface antigen (HBsAg) loaded poly(lactic-co-glycolic acid) (PLGA), Trimethyl chitosan (TMC) as well as TMC-coated PLGA nanoparticles and compare their efficacy as nasal vaccine. The developed formulations were characterized for size, zeta potential, entrapment efficiency, mucin adsorption ability, Dentritic cells interaction, in vitro and in vivo studies. PLGA nanoparticles demonstrated negative zeta potential whereas TMC and PLGA?CTMC nanoparticles showed higher positive zeta potential. Results indicated that TMC and PLGA?CTMC nanoparticles demonstrated substantially higher mucin adsorption when compared to PLGA nanoparticles. The nanoparticles were nontoxic to isolated nasal epithelium. Immunogenicity increased as a function of particle size upon nasal administration. HBsAg encapsulated in PLGA?CTMC particles elicited a significantly higher secretory (IgA) immune response compared to that encapsulated in PLGA and TMC particles. Similar to in vivo immune response data, fluorescent-labelled nanoparticles of smaller size are taken up more efficiently by rat alveolar macrophages compared to those of larger size. Results indicated that alum based HBsAg induced strong humoral but less mucosal immunity. However, PLGA?CTMC nanoparticles induced stronger immune response at both of the fronts as compared to generated by PLGA or TMC nanoparticles. Present study demonstrates that PLGA?CTMC nanoparticles with specific size range can be a better carrier adjuvant for nasal subunit vaccines.