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.     

Nanoparticulate Systems for Nasal Delivery of Drugs: A Real Improvement over Simple Systems?

This review discusses the possible benefits of using nanoparticles for nasal delivery of drugs and vaccines. It considers the various factors affecting particle transport across the nasal tissue. The evidence for the improved transport of drugs, such as peptides and proteins, across the nasal epithelium when formulated in a nanoparticulate system, as compared to an optimal solution formulation, is not convincing. For instance it has been shown that a chitosan solution and especially a chitosan powder formulation was superior in enhancing the nasal absorption of insulin as compared to chitosan nanoparticles. On the other hand, the use of nanoparticles for vaccine delivery seems beneficial in that good immune responses are achieved. This could be due to the fact that small particles can be transported preferentially by the lymphoid tissue of the nasal cavity (NALT). However, apparently no studies have been published comparing directly other adjuvant nasal systems with nanoparticulate systems.     

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.     

Mucoadhesive glycol chitosan nanoparticles for intranasal delivery of hepatitis B vaccine: enhancement of mucosal and systemic immune response

In this study, for the first time, glycol chitosan (GC) nanoparticles (NPs) were prepared and evaluated to obtain systemic and mucosal immune responses against nasally administered hepatitis B surface antigen (HBsAg). Size, zeta potential and morphology of the NPs were investigated as a function of preparation method. NPs with high loading efficacy (?>?95%) and positively charged surface were obtained with an average particle size of approximately 200?nm. The structural integrity of HBsAg in NPs was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and further confirmed by measuring the in vitro antigenicity using an enzyme immunoassay. During in vivo studies, GC NPs showed the lowest nasal clearance rate and better mucosal uptake when compared with chitosan (CS) NPs. The immunogenicity of NPs-based delivery system(s) was assessed by measuring anti-HBsAg antibody titer in mice serum and secretions after intranasal administration. The alum-based HBsAg vaccine injected subcutaneously was used as positive control. Results indicated that alum-based HBsAg induced strong humoral but negligible mucosal immunity. However, GC NPs induced stronger immune response at both of the fronts as compared to generated by CS NPs. This study demonstrates that this newly developed system has potential for mucosal administration of vaccines.     

Dried influenza vaccines: over the counter vaccines

Since last year influenza pandemic has struck again after 40 years, this is the right moment to discuss the different available formulation options for influenza vaccine. Looking back to the last 4 decades, most vaccines are still formulated as liquid solution. These vaccines have shown a poor stability profile and are limited in their potential route of administration. An ideal solution to these problems can be to convert vaccines into a dry and stable powder formulation. Recently, drying and delivery technologies have shown promising results regarding the vaccine stability. Therefore, in future we can envisage a stable dry influenza vaccine. Also with regard to the route of administration developments are accelerating and the potential of non-invasive administration has been demonstrated.     

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.     

Results obtained with the NIVGRIP inactivated influenza vaccine applicable by nasal and/or oral route

The NIVGRIP inactivated influenza vaccine prepared in the "Stefan S. Nicolau" Institute of Virology has proved its efficacy in the specific prophylaxis of influenza. The vaccine can be administered by nasal and/or oral route and has a very low reactogenicity. Application of the NIVGRIP vaccine results in seroconversion rates of 50-70% and in rises in the levels of local neutralizing antibodies in 75-80% of the vaccines. Numerous epidemiological surveys have ascertained that the protection rate obtained by nasal administration of the NIVGRIP vaccine ranges from 50 to 90%, being comparable with that of other commercially available vaccines. The advantages of the use of an inactivated, whole virus vaccine applicable by nonparenteral routes are discussed.     

Immunité et vaccinations antivirales : exemple de la muqueuse respiratoire

ObjectiveAs the mucosal surfaces of the respiratory tract represent a major portal of entry for most human viruses and many bacteria, they seem to be a critical component of the mammalian immunologic repertoire. Thus, vaccines stimulating this local immunity could represent an interesting approach to prevent these infections. After detailing the different mechanisms implied in this mucosal immunity, the aim of this study is to analyze the basis of such a vaccination and the different vaccines available to mucosal respiratory tract use.Mucosal immunityThe major antibody isotype in external secretions is secretory immunoglobin A (S-IgA); the role of IgM (S-IgM) and IgG (S-IgG) are actually questionned. It is, however, interesting that the major effector cells in the mucosal surfaces are not IgA B cells, but T lymphocytes that may represent up to 80% of the entire mucosal lymphoid cell population.Immunoprophylaxis by the mucosal routePassive antibodies were shown to protect against mucosal viral infections, such as those caused by RSV, but very high quantities of passive antibodies are needed to restrict virus replication on mucosal surface.In general, factors which favor development of mucosal antibody and cell mediated immune responses include the oral or respiratory immunization and the replicating nature of the vaccine agents. However, to date only a few vaccines have become available to mucosal respiratory tract use, and cold-adapted influenza virus vaccines is the only one available using nasal route. Other parenteral licensed vaccines have not been recommended for mucosal administration. Some of them have been experimentally used with nasal administration of replicating agents (varicella and measles vaccines) or non replicating agents (influenza inactivated vaccine), but have been found to induce a very low mucosal response.ConclusionBased on the experience with existing vaccines, the development of mucosal immunity or administration of vaccines via the mucosal route is clearly not a prerequisite today for control or prevention of most viral infectious respiratory diseases or diseases with respiratory tract as a route of contamination. But the example of live attenuated intranasal influenza vaccine inducing both systemic and local immune response without immunopathology, is promising for the future of the mucosal immunization against respiratory viral infections.     

Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein

This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.     

Nasal administration of carbamazepine using chitosan microspheres: in vitro/in vivo studies

The nasal route is used both for local therapies and, more recently, for the systemic administration of drugs, as well as for the delivery of peptides and vaccines. In this study the nasal administration of Carbamazepine (CBZ) has been studied using microspheres constituted by chitosan hydrochloride (CH) or chitosan glutamate (CG). Blank microspheres were also prepared as a comparison. The microspheres were produced using a spray-drying technique and characterized in terms of morphology (scanning electron microscopy, SEM), drug content, particle size (laser diffraction method) and thermal behaviour (differential scanning calorimetry, DSC). In vitro drug release studies were performed in phosphate buffer (pH 7.0). In vivo tests were carried out in sheep using the microparticles containing chitosan glutamate, chosen on the basis of the results of in vitro studies. The results were compared to those obtained after the nasal administration of CBZ (raw material) alone. For the evaluation of in vivo data statistical analysis was carried out using the unpaired t-test. Spray-drying was a good technique of preparation of CBZ-loaded microspheres. The loading of the drug into the polymeric network always led to an increase in the dissolution rate compared to CBZ raw material. The microspheres obtained using chitosan glutamate had the best behaviour both in vitro and in vivo. They increased the drug concentration in the serum when compared to the nasal administration of the pure drug (Cmax 800 and 25 ng/ml for microspheres and pure drug, respectively). The results obtained indicate that the loading of CBZ in chitosan glutamate microspheres increases the amount of the drug absorbed through the nose.     

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.     

Generation of Toxoplasma gondii GRA1 protein and DNA vaccine loaded chitosan particles: preparation, characterization, and preliminary in vivo studies

Chitosan microparticles as carriers for GRA-1 protein vaccine were prepared and characterized with respect to loading efficiency and GRA-1 stability after short-term storage. Chitosan nanoparticles as carriers for GRA-1 pDNA vaccine were prepared and characterized with respect to size, zeta potential, and protection of the pDNA vaccine against degradation by DNase I. Both protein and pDNA vaccine preparations were tested with regard to their potential to elicit GRA-1-specific immune response after intragastric administration using different prime/boost regimen. The immune response was measured by determination of IgG2a and IgG1 antibody titers. It was shown that priming with GRA1 protein vaccine loaded chitosan particles and boosting with GRA1 pDNA vaccine resulted in high anti-GRA1 antibodies, characterized by a mixed IgG2a/IgG1 ratio. These results showed that oral delivery of vaccines using chitosan as a carrier material appears to be beneficial for inducing an immune response against Toxoplasma gondii. The type of immune response, however, will largely depend on the prime/boost regimen and the type of vaccine used.     

Brain uptake of an anti-ischemic agent by nasal administration of microparticles

N(6)-cyclopentyladenosine (CPA) has neuronal anti-ischemic properties, but it is not absorbed into the brain from the bloodstream, where it shows poor stability and induces side effects. Microparticulate drug delivery systems designed for CPA nasal administration and constituted by mannitol or chitosan, were prepared by spray-drying and characterized. Mannitol-lecithin microparticles showed high CPA dissolution rate, whereas chitosan microparticles controlled its release rate. In vitro mucoadhesion studies indicated that CPA-loaded chitosan microparticles had higher mucoadhesive properties compared to mannitol particles. Ex vivo studies on sheep nasal mucosa showed that mannitol microparticles promoted CPA permeation across the mucosa, whereas chitosan microparticles controlled CPA permeation rate in comparison with CPA raw material. In vivo studies were carried out on rats. No CPA was detected in rat cerebrospinal fluid (CSF) and brain sections after intravenous administration. In contrast, after nasal administration of loaded microparticles CPA was found in the CSF at concentrations ranging from high nM to microM values and up to two order of magnitude higher than those obtained at systemic level. CPA was also found in the olfactory bulb at concentrations around 0.1 ng/mg of tissue. These results can open new perspectives for the employment of CPA against brain damages following stroke.     

Alternative routes of influenza vaccine delivery

The global emergence of virulent avian influenza and the concomitant raised threat of an influenza pandemic has increased interest in the development of improved influenza vaccines. Whereas conventional influenza vaccines are delivered by parenteral injection, an intranasal influenza vaccine has been marketed since 2003. Many other technologies are in development for intranasal, oral, epidermal and topical influenza vaccines. This editorial summarises the advances in clinical development of technologies for needle-free influenza vaccine delivery.     

Alternative routes of influenza vaccine delivery

The global emergence of virulent avian influenza and the concomitant raised threat of an influenza pandemic has increased interest in the development of improved influenza vaccines. Whereas conventional influenza vaccines are delivered by parenteral injection, an intranasal influenza vaccine has been marketed since 2003. Many other technologies are in development for intranasal, oral, epidermal and topical influenza vaccines. This editorial summarises the advances in clinical development of technologies for needle-free influenza vaccine delivery.     

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.     

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.     

Development of a novel adjuvanted nasal vaccine: C48/80 associated with chitosan nanoparticles as a path to enhance mucosal immunity

In a time in which mucosal vaccines development has been delayed by the lack of safe and effective mucosal adjuvants, the combination of adjuvants has started to be explored as a strategy to obtain potent vaccine formulations. This study describes a novel adjuvant combination as an effective approach for a nasal vaccine – the association of the mast cell activator compound 48/80 with chitosan based nanoparticles. It was hypothesized that mucoadhesive nanoparticles would promote the cellular uptake and prolong the antigen residence time on nasal cavity. Simultaneously, mast cell activation would promote a local microenvironment favorable to the development of an immune response. To test this hypothesis, two different C48/80 loaded nanoparticles (NPs) were prepared: Chitosan-C48/80 NP (Chi-C48/80 NP) and Chitosan/Alginate-C48/80 NP (Chi/Alg-C48/80 NP). The potential as a vaccine adjuvant of the two delivery systems was evaluated and directly compared. Both formulations had a mean size near 500 nm and a positive charge; however, Chi-C48/80 NP was a more effective adjuvant delivery system when compared with Chi/Alg-C48/80 NP or C48/80 alone. Chi-C48/80 NP activated mast cells at a greater extent, were better internalized by antigen presenting cells than Chi/Alg-C48/80 NP and successfully enhanced the nasal residence time of a model antigen. Superiority of Chi-C48/80 NP as adjuvant was also observed in vivo. Therefore, nasal immunization of mice with Bacillus anthracis protective antigen (PA) adsorbed on Chi-C48/80 NP elicited high levels of serum anti-PA neutralizing antibodies and a more balanced Th1/Th2 profile than C48/80 in solution or Chi/Alg-C48/80 NP. The incorporation of C48/80 within Chi NP also promoted a mucosal immunity greater than all the other adjuvanted groups tested, showing that the combination of a mast cell activator and chitosan NP could be a promising strategy for nasal immunization.     

Pharmaceutical aspects of intranasal delivery of vaccines using particulate systems

The nasal route offers a promising opportunity for the delivery of vaccines. This review analyses the opportunities and novel delivery strategies based on particulate systems for the nasal delivery of vaccines, including liposomes, proteosomes, virosomes, nano- and microparticulate systems, with and without adjuvants. The influence of pharmaceutical aspects of the particulate formulations on nasal delivery is analysed. Recently developed delivery devices for nasal vaccination are also described. Potential barriers to clinical and commercial success of some novel intranasal vaccines are critically evaluated. Although particulate systems may offer potential in the nasal delivery of vaccines by enhancing uptake by antigen-presenting cells, the real success in enhancement of vaccine delivery can only be achieved by careful design and manipulation of physicochemical properties of particulate vaccine delivery systems.     

Intranasal vaccination against plague, tetanus and diphtheria

Plague is an extremely virulent and potentially lethal infection caused by the bacterium Y. pestis. The current vaccine used to immunise against plague often fails to engender solid (100%) protection against inhalational infection with Y. pestis. Similarly, logistical factors favour the development of non-parenteral immunisation protocols to counter plague. Recently an improved parenteral vaccination strategy for plague, based on the recombinant subunit approach, has entered clinical trails. The Yersinia pestis subunit antigens (F1 and V) have been successfully incorporated into novel vaccine delivery systems such as biodegradable microspheres composed of poly-L-(lactide) (PLLA). Intranasal and intratracheal administration of PLLA microencapsulated F1 and V serves to protect experimental animals from inhalational and subcutaneous challenge with virulent Y. pestis bacilli. Liposomes have also been used to improve the immunogenicity of intranasally administered Y. pestis antigens, and the effectiveness of this approach to plague immunisation has been evaluated. Tetanus and diphtheria still cause many deaths worldwide. The maintenance of protective immunity to diphtheria and tetanus requires booster injections of the currently licensed toxoid vaccines. Consequently, many people remain unprotected. Improved coverage may well result from the development of effective non-invasive vaccines that could be readily distributed and potentially self-administered. To this end, the intranasal and inhalational routes of administration have been extensively investigated. Tetanus and diphtheria toxoids have been delivered intranasally to experimental animals using a wide variety of adjuvants (enterotoxin derivatives), penetration enhancers (cyclodextrins, bile salts, surfactants, cationic polymers) and delivery systems (microspheres and liposomes). As compared with parenteral vaccination, nasal immunisation has been shown favourably effective in small animal models, and a limited number of early phase clinical trails. As a caveat to this, adjuvantisation of toxoid/subunit molecules appears to be a requisite for elicitation of appreciable immunological responses, following nasal administration of acellular immunogens. Testing in larger animal models and humans is needed to ascertain if the promising results obtained in rodents can be reciprocated without compromising safety.