Bacillus Anthracis Protective Antigen Research Articles

Animal-to-Human Dose Translation of ANTHRASIL for Treatment of Inhalational Anthrax in Healthy Adults, Obese Adults, and Pediatric Subjects.

Anthrax Immune Globulin Intravenous (AIGIV [ANTHRASIL]), was developed for the treatment of toxemia associated with inhalational anthrax. It is a plasma product collected from individuals vaccinated with anthrax vaccine and contains antitoxin IgG antibodies against Bacillus anthracis protective antigen. A pharmacokinetic (PK) and exposure-response model was constructed to assess the PKs of AIGIV in anthrax-free and anthrax-exposed rabbits, non-human primates and anthrax-free humans, as well as the relationship between AIGIV exposure and survival from anthrax, based on available preclinical/clinical studies. The potential effect of anthrax on the PKs of AIGIV was evaluated and estimates of survival odds following administration of AIGIV protective doses with and without antibiotic co-treatment were established. As the developed PK model can simulate exposure of AIGIV in any species for any dosing scenario, the relationship between the predicted area under the concentration curve of AIGIV in humans and the probability of survival observed in preclinical studies was explored. Based on the simulation results, the intravenous administration of 420 U (units of potency as measured by validated Toxin Neutralization Assay) of AIGIV is expected to result in a > 80% probability of survival in more than 90% of the human population. Additional simulations suggest that exposure levels were similar in healthy and obese humans, and exposure in pediatrics is expected to be up to approximately seven-fold higher than in healthy adults, allowing for doses in pediatric populations that ranged from one to seven vials. Overall, the optimal human dose was justified based on the PK/pharmacodynamic (PD) properties of AIGIV in animals and model-based translation of PK/PD to predict human exposure and efficacy.

Recombinant full-length Bacillus Anthracis protective antigen and its 63 kDa form elicits protective response in formulation with addavax.

Bacillus anthracis is the causative agent for the lethal disease anthrax, primarily affecting animals and humans in close contact with an infected host. The pathogenicity of B. anthracis is attributed to the secreted exotoxins and their outer capsule. The host cell-binding exotoxin component "protective antigen" (PA) is reported to be a potent vaccine candidate. The aim of our study is to produce several PA constructs and analyze their vaccine potential. We have designed the various subunit, PA-based recombinant proteins, i.e., full-length Protective antigen (PA-FL), C-terminal 63 kDa fragment (PA63), Protective antigen domain 1-domain 4 chimeras (PA-D1-4) and protective antigen domain 4 (PA-D4) and analyzed their vaccine potential with different human-compatible adjuvants in the mouse model. We have optimized the process and successfully expressed our recombinant antigens as soluble proteins, except full-length PA. All the recombinant antigen formulations with three different adjuvants i.e., Addavax, Alhydrogel, and Montanide ISA 720, were immunized in different mouse groups. The vaccine efficacy of the formulations was analyzed by mouse serum antigen-specific antibody titer, toxin neutralization assay, and survival analysis of mouse groups challenged with a lethal dose of B. anthracis virulent spores. We have demonstrated that the PA-FL addavax and PA63 addavax formulations were most effective in protecting spore-challenged mice and serum from the mice immunized with PAFL addavax, PA-FL alhydrogel, PA63 addavax, and PA63 alhydrogel formulations were equivalently efficient in neutralizing the anthrax lethal toxin. The higher levels of serum Th1, Th2, and Th17 cytokines in PA-FL addavax immunized mice correspond to the enhanced protection provided by the formulation in challenged mice. We have demonstrated that the PA-FL addavax and PA63 addavax formulations exhibit equivalent efficiency as vaccine formulation both in a mouse model of anthrax and mammalian cell lines. However, PA63 is a smaller antigen than PA-FL and more importantly, PA63 is expressed as a soluble protein in E. coli, which imparts a translational advantage to PA63-based formulation. Thus, the outcome of our study has significant implications for the development of protective antigen-based vaccine formulations for human use against the lethal disease anthrax.

Impact of HLA Polymorphism on the Immune Response to Bacillus Anthracis Protective Antigen in Vaccination versus Natural Infection.

The causative agent of anthrax, Bacillus anthracis, evades the host immune response and establishes infection through the production of binary exotoxins composed of Protective Antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). The majority of vaccination strategies have focused upon the antibody response to the PA subunit. We have used a panel of humanised HLA class II transgenic mouse strains to define HLA-DR-restricted and HLA-DQ-restricted CD4+ T cell responses to the immunodominant epitopes of PA. This was correlated with the binding affinities of epitopes to HLA class II molecules, as well as the responses of two human cohorts: individuals vaccinated with the Anthrax Vaccine Precipitated (AVP) vaccine (which contains PA and trace amounts of LF), and patients recovering from cutaneous anthrax infections. The infected and vaccinated cohorts expressing different HLA types were found to make CD4+ T cell responses to multiple and diverse epitopes of PA. The effects of HLA polymorphism were explored using transgenic mouse lines, which demonstrated differential susceptibility, indicating that HLA-DR1 and HLA-DQ8 alleles conferred protective immunity relative to HLA-DR15, HLA-DR4 and HLA-DQ6. The HLA transgenics enabled a reductionist approach, allowing us to better define CD4+ T cell epitopes. Appreciating the effects of HLA polymorphism on the variability of responses to natural infection and vaccination is vital in planning protective strategies against anthrax.

Solution structures of Bacillus anthracis protective antigen proteins using small angle neutron scattering and protective antigen ion channel formation kinetics

We are studying the structures of bacterial toxins that form ion channels and enable macromolecule transport across membranes. For example, the crystal structure of the Staphylococcus aureus alpha-hemolysin channel in its functional state was confirmed using neutron reflectometry (NR) with the protein reconstituted in membranes tethered to a solid support. This method, which provides sub-nanometer structural information, could also test putative structures of the Bacillus anthracis Protective Antigen 63 (PA63) channel, locate where B.

Solution Structures of Bacillus anthracis Protective Antigen Proteins Using Small Angle Neutron Scattering and Protective Antigen 63 Ion Channel Formation Kinetics.

We are studying the structures of bacterial toxins that form ion channels and enable macromolecule transport across membranes. For example, the crystal structure of the Staphylococcus aureus α-hemolysin (α-HL) channel in its functional state was confirmed using neutron reflectometry (NR) with the protein reconstituted in membranes tethered to a solid support. This method, which provides sub-nanometer structural information, could also test putative structures of the Bacillus anthracis protective antigen 63 (PA63) channel, locate where B. anthracis lethal factor and edema factor toxins (LF and EF, respectively) bind to it, and determine how certain small molecules can inhibit the interaction of LF and EF with the channel. We report here the solution structures of channel-forming PA63 and its precursor PA83 (which does not form channels) obtained with small angle neutron scattering. At near neutral pH, PA83 is a monomer and PA63 a heptamer. The latter is compared to two cryo-electron microscopy structures. We also show that although the α-HL and PA63 channels have similar structural features, unlike α-HL, PA63 channel formation in lipid bilayer membranes ceases within minutes of protein addition, which currently precludes the use of NR for elucidating the interactions between PA63, LF, EF, and potential therapeutic agents.

Immunogenicity and Biodistribution of Anthrax DNA Vaccine Delivered by Intradermal Electroporation.

Anthrax is a lethal bacterial disease caused by gram-positive bacterium Bacillus anthracis and vaccination is a desirable method to prevent anthrax infections. In the present study, DNA vaccine encoding a protective antigen of Bacillus anthracis was prepared and we investigated the influence of DNA electrotransfer in the skin on the induced immune response and biodistribution. The tdTomato reporter gene for the whole animal in vivo imaging was used to assess gene transfer efficiency into the skin as a function of electrical parameters. Compared to that with 25 V, the transgene expression of red fluorescent protein increased significantly when a voltage of 90 V was used. Delivery of DNA vaccines expressing Bacillus anthracis protective antigen domain 4 (PAD4) with an applied voltage of 90 V induced robust PA-D4-specific antibody responses. In addition, the in vivo fate of anthrax DNA vaccine was studied after intradermal administration into the mouse. DNA plasmids remained at the skin injection site for an appropriate period of time after immunization. Intradermal administration of DNA vaccine resulted in detection in various organs (viz., lung, heart, kidney, spleen, brain, and liver), although the levels were significantly reduced. Our results offer important insights into how anthrax DNA vaccine delivery by intradermal electroporation affects the immune response and biodistribution of DNA vaccine. Therefore, it may provide valuable information for the development of effective DNA vaccines against anthrax infection.

Raxibacumab: a panacea for anthrax disease?

When anthrax exposure is suspected, recommended post-exposure prophylaxis is 60 days of antibiotics (ciprofloxacin or doxycycline) combined with anthrax vaccination. Anthrax Vaccine Adsorbed (AVA) is approved for human use in the USA, and Anthrax Vaccine Precipitated (AVP) is approved in the UK. The major antigen in these vaccines is the Bacillus anthracis protective antigen; this molecule is secreted by B anthracis and creates a pore in the host membrane that binds then separately translocates lethal factor and oedema factor into the cell.

DEVELOPMENT OF STABLE ANTHRAX ANTIGEN FOR PLANT VIRUS-BASED VACCINE

A stable genetically modified recombinant anthrax antigen containing 3rd and 4th Bacillus anthracis protective antigen domains has been obtained. Complexes of this antigen with structurally modified plant virus particles are a perspective basis for a new generation anthrax vaccine development.

<p>Crystalline and Amorphous Preparation of Aluminum Hydroxide Nanoparticles Enhances Protective Antigen Domain 4 Specific Immunogenicity and Provides Protection Against Anthrax</p>

IntroductionAluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis.Methods and ResultsHerein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150–200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2- and Th1-type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel.ConclusionThese results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response.

The biological role of the enigmatic C3larvinAB toxin of the honey bee pathogenic bacterium Paenibacillus larvae.

Paenibacillus larvae is the causative agent of the notifiable epizootic American foulbrood, a fatal bacterial disease of honey bee larvae. The species P. larvae has been classified into four differentially virulent and prevalent genotypes (ERIC I-IV), which also differ in their virulence factor equipment. Recently, a novel P. larvae toxin, the C3-like C3larvin, has been described. Genome analysis now revealed that the C3larvin gene is actually a part of a toxin locus encompassing two genes encoding a binary AB toxin with the A subunit being C3larvin (C3larvinA) and a putative B subunit (C3larvinB) encoded by the second gene. Sequence and structural analyses demonstrated that C3larvinB is a homologue of the Bacillus anthracis protective antigen (PA), the B subunit of anthrax toxin. The C3larvinAB toxin locus was interrupted by point mutations in all analysed P. larvae ERIC I and ERIC II strains. Only one P. larvae ERIC III/IV strain harboured an uninterrupted toxin locus comprising full-length genes for C3larvinA and B. Exposure bioassays did not substantiate a role as virulence factor for C3larvinAB in P. larvae ERIC I/II. However, the PA homologue C3larvinB had an influence on the virulence of the unique P. larvae strain expressing the functional C3larvinAB locus.

Bacillus anthracis Protective Antigen Shows High Specificity for a UV Induced Mouse Model of Cutaneous Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) accounts for the majority of non-melanoma skin cancer related deaths, particularly in immunosuppressed persons. Identification of biomarkers that could be used to identify or treat SCC would be of significant benefit. The anthrax toxin receptors, Tumor Endothelial Marker 8 (TEM8) and Capillary Morphogenesis Gene 2 (CMG2), are endothelial receptors involved in extracellular matrix homeostasis and angiogenesis that are selectively upregulated on numerous tumors. One method of targeting these receptors is Protective Antigen (PA), a protein produced by B. anthracis that mediates binding and translocation of anthrax toxins into cells. PA targeted toxins have been demonstrated to selectively inhibit tumor growth and angiogenesis, but tumor selectivity of PA is currently unknown. In this work fluorescently labeled PA was shown to maintain receptor dependent binding and internalization in vitro. Utilizing a human papillomavirus transgenic mouse model that develops cutaneous SCC in response to ultraviolet irradiation we identified tumor uptake of PA in vivo. The intravenously administered PA resulted in tumor specific localization, with exclusive tumor detection 24 h post injection. Ex vivo analysis identified significantly higher fluorescence in the tumor compared to adjacent healthy tissue and major clearance organs, demonstrating low non-specific uptake and rapid clearance. While both TEM8 and CMG2 were observed to be overexpressed in SCC tumor sections compared to control skin, the intravenously administered PA was primarily co-localized with TEM8. These results suggest that PA could be systemically administered for rapid identification of cutaneous SCC, with potential for further therapeutic development.

A therapeutic human antibody against the domain 4 of the Bacillus anthracis protective antigen shows protective efficacy in a mouse model

Since Bacillus anthracis is a high-risk pathogen and a potential tool for bioterrorism, numerous therapeutic methods including passive immunization have been actively developed. Using a human monoclonal antibody phage display library, we screened new therapeutic antibodies for anthrax infection against protective antigen (PA) of B. anthracis. Among 5 selected clones of antibodies based on enzyme-linked immunosorbent assay (ELISA) results, 7B1 showed neutralizing activity to anthrax lethal toxin (LT) by inhibiting binding of the domain 4 of PA (PD4) to its cellular receptors. Through light chain shuffling process, we improved the productivity of 7B1 up to 25 folds. The light chain shuffled 7B1 antibody showed protective activity against LT both in vitro and in vivo. Furthermore, the antibody also conferred protection of mice from 3 × LD50 challenges of fully virulent anthrax spores. Our result expands the possibility of developing a new therapeutic antibody for anthrax cure.

Lactoferrin adsorption onto silver nanoparticle interface: Implications of corona on protein conformation, nanoparticle cytotoxicity and the formulation adjuvanticity

Nanoparticle, in biological milieu, tends to bind a variety of biomolecules, especially protein, forming an interfacial corona that provides biological identity to nanoparticle. Understanding the protein corona is likely to provide insight in nanoparticle bio-distribution, nanoparticle-mediated cytotoxicity and potential nanoparticle-mediated applications. Thus, the bovine lactoferrin (BLf) corona onto silver nanoparticle (AgNP) interface is studied with reference to BLf conformational stability and the resulting complex’s cytotoxic and adjuvant properties. The adsorption of BLf onto AgNP interface is reflected by change in the particle-specific surface plasmon resonance (SPR). Additionally, the molecular dynamics (MD) simulation deciphered the binding energy and molecular recognition in BLf and AgNP complex. Protein adsorption onto the interface is majorly driven by van der Waals interactions and hydrogen bonds, without any significant effect on the protein conformation and stability. BLf retains bacteriostatic property in the conjugate, and the presence of BLf corona ameliorated the particle-mediated cytotoxicity, despite the enhanced cellular internalization. Additionally, BLf adsorbed AgNP formulation shows better adjuvanticity than AgNP only or BLf only formulation, as demonstrated using Bacillus anthracis protective antigen protein, in vivo.

Development and application of a Bacillus anthracis protective antigen domain-1 in-house ELISA for the detection of anti-protective antigen antibodies in cattle in Zambia.

In Zambia, anthrax outbreaks among cattle are reported on nearly an annual basis. Presently, there is a lack of serological assays and information to develop an anthrax management and control strategy. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) based on recombinant protective antigen domain 1 (rPA-D1) of Bacillus anthracis was developed and used to detect anti-PA antibodies in cattle in Zambia. An antigen coating of 10 ng/well and a serum dilution of 1:100 were determined to be the optimal rPA-D1 ELISA titration conditions. The intra- and inter-assay % coefficients of variation were less than 10% and 15%, respectively. The rPA-D1 ELISA could detect seroconversion in the cattle 1 month after anthrax vaccination. In a cross-sectional study conducted in the Western Province, Zambia, 187 serum samples from 8 herds of cattle were screened for anti-PA antibodies using the rPA-D1 ELISA. The seropositive rate of the serum samples was 8%, and the mean anti-PA antibody was 0.358 ELISA units. Additionally, we screened 131 cattle serum samples from Lusaka, which is a nonendemic area, and found no significant association between the antibody levels and sampling area (endemic versus nonendemic area). Conversely, significant differences were observed between the anti-PA antibody levels and herds, anti-PA antibody levels and vaccination status and anti-PA antibody levels and vaccination timing. Collectively, these findings suggest that the rPA-D1 ELISA is a useful tool for the detection of anti-PA antibodies in cattle in Zambia. The low proportion of seropositive sera indicates that there is inadequate cattle vaccination in the Western Province and, in addition to other epidemiological factors, this may precipitate the anthrax outbreak recurrence.

Targeting Bacillus anthracis toxicity with a genetically selected inhibitor of the PA/CMG2 protein-protein interaction

The protein-protein interaction between the human CMG2 receptor and the Bacillus anthracis protective antigen (PA) is essential for the transport of anthrax lethal and edema toxins into human cells. We used a genetically encoded high throughput screening platform to screen a SICLOPPS library of 3.2 million cyclic hexapeptides for inhibitors of this protein-protein interaction. Unusually, the top 3 hits all contained stop codons in the randomized region of the library, resulting in linear rather than cyclic peptides. These peptides disrupted the targeted interaction in vitro; two act by binding to CMG2 while one binds PA. The efficacy of the most potent CMG2-binding inhibitor was improved through the incorporation of non-natural phenylalanine analogues. Cell based assays demonstrated that the optimized inhibitor protects macrophages from the toxicity of lethal factor.

Sublingual targeting of STING with 3′3′-cGAMP promotes systemic and mucosal immunity against anthrax toxins

Anthrax is caused by Bacillus anthracis, a zoonotic bacterial pathogen affecting humans and livestock worldwide. The current human anthrax vaccine, anthrax vaccine adsorbed (AVA), is an injected vaccine with a cumbersome administration schedule and fails to promote mucosal immunity. Bacterial enterotoxins, which stimulate production of the cyclic nucleotide cAMP are effective experimental mucosal vaccine adjuvants, but their inherent toxicity has precluded their use in humans. We investigated whether cyclic dinucleotides that target Stimulator of Interferon Gamma Genes (STING) in mammalian cells could represent an alternative to bacterial enterotoxins as adjuvant for sublingual immunization and promotion of mucosal immunity and secretory IgA responses in addition to systemic immunity. We found that sublingual immunization of mice with Bacillus anthracis protective antigen (PA) and the STING ligand 3′3′-cGAMP promotes PA-specific serum IgG Ab responses of the same magnitude as those induced after immunization with PA and the experimental adjuvant cholera toxin (CT). Interestingly, this STING ligand also promoted serum anti-PA IgA and IgA-producing cells in the bone marrow. Furthermore, the saliva of mice immunized with the STING ligand exhibited similar levels of PA-specific IgA Abs as groups immunized with CT as adjuvant. The adjuvant activity of 3′3′-cGAMP was associated with mixed Th1, Th2, and Th17 responses. This STING ligand also induced rapid IFN-β and IL-10 responses in sublingual tissues and cervical lymph nodes, and TGF-β responses in the cervical lymph nodes, which could contribute to promoting IgA responses after sublingual immunization.

Secrening Producer of Truncated Bacillus anthracis Protective Antigen in Bacillus subtilis

Secrening Producer of Truncated Bacillus anthracis Protective Antigen in Bacillus subtilis

Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model

The Sterne 34F2 live spore vaccine (SLSV) developed in 1937 is the most widely used veterinary vaccine against anthrax. However, literature on the immunogenicity of this vaccine in a target ruminant host is scarce. In this study, we evaluated the humoral response to the Bacillus anthracis protective antigen (rPA), a recombinant bacillus collagen-like protein of anthracis (rBclA), formaldehyde inactivated spores (FIS) prepared from strain 34F2 and a vegetative antigen formulation prepared from a capsule and toxin deficient strain (CDC 1014) in Boer goats. The toxin neutralizing ability of induced antibodies was evaluated using an in vitro toxin neutralization assay. The protection afforded by the vaccine was also assessed in vaccinates. Anti-rPA, anti-FIS and lethal toxin neutralizing titres were superior after booster vaccinations, compared to single vaccinations. Qualitative analysis of humoral responses to rPA, rBclA and FIS antigens revealed a preponderance of anti-FIS IgG titres following either single or double vaccinations with the SLSV. Antibodies against FIS and rPA both increased by 350 and 300-fold following revaccinations respectively. There was no response to rBclA following vaccinations with the SLSV. Toxin neutralizing titres increased by 80-fold after single vaccination and 700-fold following a double vaccination. Lethal challenge studies in naïve goats indicated a minimum infective dose of 36 B. anthracis spores. Single and double vaccination with the SLSV protected 4/5 and 3/3 of goats challenged with>800 spores respectively. An early booster vaccination following the first immunization is suggested in order to achieve a robust immunity. Results from this study indicate that this crucial second vaccination can be administered as early as 3 months after the initial vaccination.

Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding

Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins.

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 500nm 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.