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.     

Induction of protective and specific antibodies against cocaine by intranasal immunisation using a glyceride adjuvant

The goal of this study was to investigate an intranasal cocaine vaccine containing the mucosal adjuvant macrogol-6-glycerol capylocaprate (RhinoVax). Cocaine-KLH conjugate was prepared and administered in two formulations. Ten mice were immunised intranasally using RhinoVax as adjuvant and ten subcutaneously using aluminium hydroxide as an adjuvant. A negative control group (n=10) received unconjugated KLH with RhinoVax intranasally. Specific cocaine antibodies in serum were measured following primary and booster immunisation. Relative antibody responses in serum indicated that the immunisation was successful. Animals were then challenged with cocaine either intranasally or intraperitoneally with subsequent measurement of drug distribution into the serum, brain and olfactory bulb. The cocaine-immunised groups revealed significantly lower cocaine levels in the brain compared to the negative control group. The inhibition of cocaine distribution to the brain in the intranasal immunised group was comparable to that of the subcutaneous immunised group. This was unexpected because the cocaine specific antibody levels in serum were fivefold lower in the intranasal immunised group. However, the presence of mucosal cocaine specific antibodies after intranasal immunisation could play an important role in hindering direct access of cocaine into the brain via the olfactory bulb.     

Immunogenicity of a West Nile Virus DIII-Cholera Toxin A2/B Chimera after Intranasal Delivery

West Nile virus (WNV) causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII) region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT) CTA₂/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA₂/B, DIII, DIII mixed with CTA₂/B, or CTA₂/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA₂/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA₂/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA₂/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA₂/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate.     

Safety of intranasally administered archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) vaccine in mice

The safety profile of a recently described novel archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system capable of eliciting robust antigen-specific mucosal and systemic immune responses was evaluated in female Balb/c mice (10/group) using ovalbumin (OVA) antigen. Mice were intranasally immunized (0, 7, and 21 days) with a vaccine comprising 1 microg OVA (0.05 mg/kg body weight) formulated in 0.04 mg total polar lipids extract (2.17 mg/kg body weight) of Methanobrevibacter smithii constituting the AMVAD system. Control groups were similarly immunized with 10-fold higher AMVAD vaccine dose (0.54 mg OVA and 21.7 mg lipid per kg), saline, 10 microg OVA in saline, or 0.04 or 0.4 mg lipid constituting empty AMVAD (no OVA) in saline, or were naive mice. Clinical signs, rectal temperature, and body weight were monitored once daily or as appropriate. Half the mice in each group were euthanized at 2 days after the first immunization. Blood was collected for clinical chemistry analyses. Major organs (heart, lungs, kidneys, liver, spleen, thymus, and brain) were examined macroscopically and histologically. The remaining mice were euthanized at 29 days and blood and organs collected for analyses as done at 2 days. Feces collected at 27 days, and sera, bile, and nasal lavage at 29 days, were assayed for antibody responses. Based on clinical symptoms, temperature, body weight changes, serum clinical chemistry, and tissue histopathology, there were no overt toxicities associated with OVA/AMVAD or empty AMVAD vaccines. There were no antibodies elicited against the lipids comprising the AMVAD system. These results demonstrate that at 10-fold excess dose of that required for vaccine efficacy, intranasally administered AMVAD vaccine appears to be relatively safe.     

A step-by-step approach to study the influence of N-acetylation on the adjuvanticity of N,N,N-trimethyl chitosan (TMC) in an intranasal nanoparticulate influenza virus vaccine

Recently we reported that reacetylation of N,N,N-trimethyl chitosan (TMC) reduced the adjuvant effect of TMC in mice after intranasal (i.n.) administration of whole inactivated influenza virus (WIV) vaccine. The aim of the present study was to elucidate the mechanism of this lack of adjuvanticity. Reacetylated TMC (TMC-RA, degree of acetylation 54%) was compared with TMC (degree of acetylation 17%) at six potentially critical steps in the induction of an immune response after i.n. administration in mice. TMC-RA was degraded in a nasal wash to a slightly larger extent than TMC. The local i.n. distribution and nasal clearance of WIV were similar for both TMC types. Fluorescently labeled WIV was taken up more efficiently by Calu-3 cells when formulated with TMC-RA compared to TMC and both TMCs significantly reduced transport of WIV over a Calu-3 monolayer. Murine bone-marrow derived dendritic cell activation was similar for plain WIV, and WIV formulated with TMC-RA or TMC. The inferior adjuvant effect in mice of TMC-RA over that of TMC might be caused by a slightly lower stability of TMC-RA-WIV in the nasal cavity, rather than by any of the other factors studied in this paper.     

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.     

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.     

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.     

TMC–MCC (N-trimethyl chitosan–mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines

In this study, for the first time, TMC/MCC complex nanoparticles as a delivery system and as an adjuvant were developed and evaluated to obtain systemic and mucosal immune responses against nasally administered tetanus toxoid (TT). Nanoparticles were developed by complexation between the oppositely charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic) and mono-N-carboxymethyl chitosan (MCC, polyampholytic) without using any crosslinker for mucosal vaccination. The cellular viability was found to be higher with TMC/MCC complex compared to that of MCC and TMC alone. Size, zeta potential and morphology of the nanoparticles were investigated as a function of preparation method. Nanoparticles with high loading efficacy (95%) and positively charged surface were obtained with an average particle size of 283 ± 2.5 nm. The structural integrity of the TT in the nanoparticles was confirmed by SDS–PAGE electrophoresis analysis. Cellular uptake studies indicated that FITC-BSA loaded nanoparticles were effectively taken up into the mouse Balb/c monocyte macrophages. Mice were nasally immunized with TT loaded TMC/MCC complex nanoparticles and compared to that of TMC and MCC nanoparticles. TMC/MCC complex nanoparticles were shown to induce both the mucosal and systemic immune response indicating that this newly developed system has potential for mucosal administration of vaccines.     

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.     

In vitro and in vivo study of N-trimethyl chitosan nanoparticles for oral protein delivery

In this study, the effects of alginate modification on absorption properties of FITC-BSA loaded TMC nanoparticles were investigated on an in vitro model of GI epithelium (Caco-2 cells). The feasibility of applying TMC nanoparticles loaded with a model vaccine urease in oral vaccination was also studied. Alginate modified TMC nanoparticles showed higher FITC-BSA permeate efficiency than non-modified TMC nanoparticles. However, alginate modification barely had any effect on TMC nanoparticles' property of decreasing TEER or enhancing drug paracellular transport. Mice s.c. immunized with urease loaded TMC nanoparticles showed highest systematic immune response (IgG levels) but the lowest mucosal response (secretory IgA levels). In the contrast, mice i.g. immunized with urease loaded TMC nanoparticles showed much higher antibody titers of both IgG and secretory IgA than those with urease solution or urease co-administrated with TMC solution. These results indicated that TMC nanoparticles are potential carriers for oral protein and vaccine delivery.     

In vivo induction of mucosal immune responses by intranasal administration of chitosan microspheres containing Bordetella bronchiseptica DNT.

In vitro immune-stimulating activities of Bordetella bronchiseptica dermonecrotoxin (BBD)-loaded in chitosan microspheres (CMs) were reported with a mouse alveolar macrophage cell line (RAW264.7). Based on the report, in vivo activity of immune-induction was investigated by intranasal administration of the BBD-loaded CMs into mice. BBD was loaded into the CMs prepared by an ionic gelation process with tripolyphosphate. Mice were immunized by direct administration of the BBD-loaded CMs into the nasal cavity. After immunization of the mice, BBD-specific immune responses (IgG and IgA titers) were measured in sera, nasal wash, and saliva by ELISA. BBD-specific IgA titers in the nasal cavity were time- and dose-dependently increased by the administration. Similar phenomena were observed in the analysis of systemic IgA and IgG in sera. However, the antibody in saliva was undetectable by ELISA. These results suggested that direct vaccination via the nasal cavity was effective for targeting nasal-associated lymphoid tissues, and that CMs were an efficient adjuvant in nasal mucosal immunity for atrophic rhinitis vaccine.     

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.     

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.     

A comparison of anionic nanoparticles and microparticles as vaccine delivery systems

The objective of this work was to conduct an in vivo comparison of nanoparticles and microparticles as vaccine delivery systems. Poly (lactide-co-glycolide) (PLG) polymers were used to create nanoparticles size 110 nm and microparticles of size 800-900 nm. Protein antigens were then adsorbed to these particles. The efficacy of these delivery systems was tested with two protein antigens. A recombinant antigen from Neisseria meningitides type B (MenB) was administered intramuscularly (i.m.) or intraperitonealy (i.p.). An antigen from HIV-1, env glycoprotein gp140 was administered intranasally (i.n.) followed by an i.m. boost. From three studies, there were no differences between the nanoparticles and micro-particles formulations. Both particles led to comparable immune responses in mice. The immune responses for MenB (serum bactericidal activity and antibody titers) were equivalent to the control of aluminum hydroxide. For the gp140, the LTK63 was necessary for high titers. Both nanoparticles and microparticles are promising delivery systems.     

Intranasal administration of dry powder anthrax vaccine provides protection against lethal aerosol spore challenge

The use of an aerosolizable form of anthrax as a biological weapon is considered to be among the most serious bioterror threats. Intranasal (IN) delivery of a dry powder anthrax vaccine could provide an effective and non-invasive administration alternative to traditional intramuscular (IM) or subcutaneous (SC) injection. We evaluated a dry powder vaccine based on the recombinant Protective Antigen (rPA) of Bacillus anthracis for vaccination against anthrax via IN immunization in a rabbit model. rPA powders were formulated and administered IN using a prototype powder delivery device. We compared serum IgG and toxin neutralizing antibody (TNA) titers of rabbits immunized IN with 10 microg rPA of a powder formulation with those immunized with the same dose of liquid rPA vaccine, delivered either IN or by IM injection. In addition, each group was tested for survival after aerosol spore challenge. Our results showed that IN vaccination with rPA powders elicited serum PA-specific IgG and TNA titers that were equivalent to those raised by liquid rPA administered IN. Serum PA-specific IgG and TNA titers after IN delivery were lower than for IM injection, however, after aerosol spore challenge, rabbits immunized IN with powders displayed 100% protection versus 63% for the group immunized IN with the liquid vaccine and 86% for the group immunized by IM injection. The results suggest that an IN powder vaccine based on rPA is at least as protective as a liquid delivered by IM injection.     

In vivo toxicity and immunogenicity of wheat germ agglutinin conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles for intranasal delivery to the brain

Biodegradable polymer-based nanoparticles have been widely studied to deliver therapeutic agents to the brain after intranasal administration. However, knowledge as to the side effects of nanoparticle delivery system to the brain is limited. The aim of this study was to investigate the in vivo toxicity and immunogenicity of wheat germ agglutinin (WGA) conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles (WGA-NP) after intranasal instillation. Sprague-Dawley rats were intranasally given WGA-NP for 7 continuous days. Amino acid neurotransmitters, lactate dehydrogenase (LDH) activity, reduced glutathione (GSH), acetylcholine, acetylcholinesterase activity, tumor necrosis factor α (TNF-α) and interleukin-8 (IL-8) in rat olfactory bulb (OB) and brain were measured to estimate the in vivo toxicity of WGA-NP. Balb/C mice were intranasally immunized by WGA-NP and then WGA-specific antibodies in serum and nasal wash were detected by indirect ELISA. WGA-NP showed slight toxicity to brain tissue, as evidenced by increased glutamate level in rat brain and enhanced LDH activity in rat OB. No significant changes in acetylcholine level, acetylcholinesterase activity, GSH level, TNF-α level and IL-8 level were observed in rat OB and brain for the WGA-NP group. WGA-specific antibodies in mice serum and nasal wash were not increased after two intranasal immunizations of WGA-NP. These results demonstrate that WGA-NP is a safe carrier system for intranasal delivery of therapeutic agents to the brain.     

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.