Evaluation of mucoadhesive carrier adjuvant: Toward an oral anthrax vaccine

Abstract

The aim of present study was to evaluate the potential of mucoadhesive alginate-coated chitosan microparticles (A-CHMp) for oral vaccine against anthrax. The zeta potential of A-CHMp was − 29.7 mV, and alginate coating could prevent the burst release of antigen in simulated gastric fluid. The results indicated that A-CHMp was mucoadhesive in nature and transported it to the peyer's patch upon oral delivery. The immunization studies indicated that A-CHMp resulted in the induction of potent systemic and mucosal immune responses, whereas alum-adjuvanted rPA could induce only systemic immune response. Thus, A-CHMp represents a promising acid carrier adjuvant for oral immunization against anthrax.

Keywords::

• alginate
• chitosan
• microparticles
• mucosal vaccination

Introduction

The causative agent of anthrax, B. anthracis, invades the host through mucosal (gastrointestinal, respiratory, and cutaneous routes) routes. The optimal immune response at both systemic and mucosal fronts is desirable to counteract the evasion of anthrax through cutaneous and mucosal (inhalational or gastrointestinal) sites. It has been reported that aluminum-based vaccine could offer significant protection against anthrax infection in systemic circulation but fails to induce specific mucosal immune response (Ivins et al. 1998, Fellows et al. 2001). However, current evidence clearly shows that only vaccines given by mucosal routes can effectively stimulate two distinct layers of protection consisting of both mucosal and systemic immunity (Mangal et al. 2011, Pawar et al. 2009).

The oral administration of the vaccine may constitute an important advance on the prophylaxis, specially, of the gastrointestinal anthrax. Oral route is most preferred for immunization as it offers the advantages of high compliance, minimal side effects, no fear of needle-borne infection, and induction of systemic as well as of mucosal immune response Despite the obvious need and apparent merits, the success in the field of oral vaccination is limited due to several constraints including, extremes of pH, enzymatic barrier and intestinal epithelium barrier which hamper the access of susceptible bio-macromolecule to gut immune inductive sites (Malik et al. 2010). Therefore, an ideally designed oral vaccine should overcome these limitations and induce both humoral and cellular counterparts of immunity along with the mucosal immune response against the specific antigen (Malik et al. 2010). We hypothesized that oral antigen delivery mediated through suitable carrier system may prove efficient in protecting the antigen in harsh gastric environment and inducing protective immunity in both systemic and mucosal compartments.

Polymeric particles are of special interest for the oral delivery of susceptible bioactive agents. They are reported to offer stability to the encapsulated content and also enhance the uptake of drug, protein, and peptide through gut epithelium (Mishra et al. 2010). It has been suggested that chitosan might be valuable for the delivery of drugs through gastrointestinal tract (Lopez et al. 2000, Mangal et al. 2011, Pawar et al. 2009, He et al. 1998, Shimoda et al. 2001), but its solubility in acidic environment may hamper its use particularly for oral vaccine delivery. The coating of chitosan particles with an acid-resistant polymer such as alginate may prove useful for delivering the antigens through oral route.

The primary aim of this study was to prepare and characterize rPA-associated A-CHMp. The study also includes understanding the impact of antigen dose on the magnitude of immune response following oral vaccination. The efficacy of the A-CHMp was tested against alum-adjuvanted vaccine and free rPA. The specific immunity in serum and secretions was quantified using ELISA. The protective efficacy of serum and secretory antibodies was evaluated using toxin neutralization assay.

Materials and methods

Materials

Chitosan was purchased from Fluka with the deacetylation value of 80% (according to supplier's specifications), and sodium alginate was procured from Sigma Chemical Co. (St. Louis, MO). Protein was estimated using BCA-protein estimation kit (Bangalore Genei Pvt Ltd., India). Recombinant protective antigen (rPA: Mol. Wt., 83 kDa) and lethal factor (LF) was kindly provided by Dr. Yogendra Singh (IGIB, New Delhi). Fluorescent isothiocynate–bovine serum albumin (FITC–BSA) and ELISA kits were procured from Sigma Chemicals Co. (St. Louis, MO, USA). All the others reagents and chemicals were of analytical grade and purchased from the local suppliers (HiMedia Laboratories Pvt. Ltd., Central Drug House, and Loba Chemie Pvt. Ltd.) unless otherwise mentioned.

Preparation of A-CHMp

The A-CHMp was prepared using the method previously described by Borges et al. with minor modifications (2005). Briefly, 0.25% w/v chitosan was dissolved in acetic acid solution (2% v/v). Then, 1.8 mL of sodium sulfate solution (10% w/v) was added with probe sonication to 100 mL of this solution. The resulting suspension was then centrifuged at 3500 rpm for 30 min to collect the Mps. These particles were washed with deionized water and then freeze-dried. For antigen loading, suspension of CHMp (0.4% w/v) was incubated with rPA (0.25% w/v) in phosphate buffer saline (PBS, pH 7.4) under mild agitation for 2 h. The mixture was then centrifuged at 1600 rpm for 10 min to remove the unassociated antigen. Resultant suspension of antigen-loaded Mps was mixed with sodium alginate (1% w/v) in equal volume and kept under magnetic stirring for 20 min to perform alginate coating. Particles were separated using centrifugation for 10 min at 2000 rpm. These particles were then suspended in 10 mL of 0.524 mM CaCl₂ in 50 mM HEPES buffer solution and agitated for 10 min to affect the cross-linking of alginate adsorbed on to the surface of CHMp.

Alum-adsorbed rPA was formulated following the procedure reported by Berthold et al. (2005) with slight modifications. Briefly, the rPA was added to the aluminum hydroxide adjuvant (Al(OH)₃), mixed using refrigerated shaker (MaxQ 4000) at 50 rpm (4°C) to affect adsorption, and then incubated overnight. After adsorption, the final volume was adjusted with a required volume of 0.8% NaCl, (pH, 6.5).

Characterization of Mps

Morphology, size, and zeta potential

The surface morphology of the particles was observed using scanning electron microscopy (SEM) (JEOL 6100, Japan). The freeze-dried powder of Mps was placed on the sample holders, sputter coated with gold. The particles were observed under scanning electron microscope. The zeta potential and particle size (in quintet) were evaluated using Zetasizer Nano ZS 90 (Malvern, UK).

FTIR spectroscopic analysis

The freeze-dried particles were analyzed using FTIR spectroscopy. The IR spectra of the samples were recorded using a Fourier-transformed infrared spectrophotometer instrument FT/IR Thermo Nicolet-380 (USA).

Differential scanning calorimetry (DSC)

DSC were recorded for further confirmation of the presence of alginate coating over CHMp. DSC spectra were recorded using a differential scanning calorimeter (DSC-2000, Dupont, USA). Particles were placed onto pans and heated from 25 to 350°C under a nitrogen influx of 20 cc/min. The temperature gradient at the rate of 10°C/min was maintained till it reached 350°C.

Loading efficiency and loading capacity

The CHMp and A-CHMp were evaluated for the loading efficiency and loading capacity. The aliquots of the particles suspension were collected, centrifuged at 14,000 rpm for 30 min, and the protein in the supernatant was determined using BCA-protein assay. Empty CHMp and A-CHMp were also treated under similar conditions and were used as negative control to normalize the background interference for the correction of the OD value and estimated using BCA protein assay.

SDS–PAGE analysis

The structural integrity of rPA loaded in A-CHMp was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Briefly, rPA-loaded A-CHMp was incubated overnight in PBS (pH 7.4) at 37°C under mild agitation (50 rpm). The particles were centrifuged at 4000 rpm for 30 min and released antigen was separated from supernatant and mixed with SDS loading buffer. Sample was heated at 100°C for 5 min and then allowed to cool down to an ambient temperature. SDS–PAGE was run after loading native rPA, rPA extracted from formulation, and marker in separate lane onto SDS electrophoresis assembly (Bio-Rad, USA) using 5% stacking gel and 10% separation gel, run at 60–110 V until the dye band reached the gel bottom. After migration, the gel was removed and stained with Coomassie blue to locate the respective position of proteins, which was then destained.

In vitro release study

The release rate of antigen from CHMp and A-CHMp was determined in simulated intestinal fluid (SIF: pH, 6.8) and simulated gastric fluid (SGF: pH, 1.2) prepared according to USP25 NF20. The Mps were incubated with the release medium (SIF and SGF) and kept at 37°C in a shaker bath at 50 rpm. The samples were withdrawn at regular time intervals, and the amount of sample withdrawn was replaced with the similar volume of particles suspension kept under similar conditions to maintain the sink condition. The samples were centrifuged at 14,000 rpm for 30 min and the protein content was determined using BCA-protein assay. Simultaneously, control CHMp and A-CHMp suspensions were also subjected to the same conditions and kept as blank so as to remove background interference and estimated using the BCA-protein assay.

In vitro mucoadhesion measurements

A Franz diffusion cell (Permeager, USA) with a donor chamber was modified as described by Rossi et al. (1999) and Bonferoni et al. (1999). Briefly, in the donor chamber, a stream of buffer was maintained through two holes. The incoming buffer flux was regulated by means of an HPLC pump (model 300, Gynkotek, Munich, Germany). The outcoming buffer was collected in a beaker and continuously stirred. Rat jejunum tissue was placed between the donor and acceptor chambers of the cell laying on a filter paper disc imbibed in HBSS, in turn, placed on a Parafilm membrane (impermeable to fluids). The receptor chamber of the cell was filled with distilled water whose only function was to keep the jejunum tissue thermoregulated. The A-CHMp suspension (500 μl) was placed on the excised rat jejunum (area = 2 cm²) tissue, and physiologic solution (NaCl 0.9% w/v) at 37°C was fluxed at 0.7 mL/min over the formulation to mimic the washing action of intestine fluids. Five hundred-microliter samples of the fluid outcoming from the donor chamber were withdrawn at fixed times. The amount of FITC-BSA ‘‘washed away’’ was determined in a receptor beaker at defined times by means of a spectrofluorimetric method. The amount of FITC-BSA was not removed by the buffer stream adhered and interacted with the biological substrate providing an indirect measure of the mucoadhesion.

Peyer's patch uptake study

Fluorescence microscopy was performed to confirm the deposition of particulate carriers in peyer's patch. FITC-BSA was used as a fluorescent marker and was entrapped into the Mps. Mice were administered with 100 μl of 7.5% w/v sodium bicarbonate solution to neutralize the gastric acid. The fluorescent-loaded A-CHMp was then administered orally to BALB/c mice. After 2 h the mice was sacrificed, its intestine was removed, and peyer's patches were identified as gray nodule and isolated to determine the uptake of the carrier. The peyer's patch-dominated region of the intestine was microtomed, and the section(s) were observed under a fluorescent microscope (Nikon Eclips E-200, Japan). Mice receiving FITC-BSA in PBS (pH 7.4) was kept as control.

Immunization studies

Female BALB/c mice (CDRI, Lucknow) 8–10 weeks old, weighing 20–25 g, were used to assess the immunogenicity of developed formulations, as they are well established for the immunization studies. Mice were housed in group (n = 6) one week before the experiments for acclimatization, with free access to food and water. The Institutional Animals Ethical Committee of Dr. Hari Singh Gour University approved the protocols which were conducted following the guidelines of the Council for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. Animals were withdrawn of any food intake 2 h before immunization. Mice were pre-administered with 100 μl of 7.5% sodium bicarbonate solution prior to the administration of rPA-loaded A-CHMp in order to neutralize the gastric acid. Group I was kept control, and rPA-loaded A-CHMp (PBS: pH, 7.4) was administered orally in four different doses (equivalent to 10, 25, 40 and 55 μg of rPA) to four separate groups (II–V); group VI received 100 μg of soluble rPA (PBS: pH, 7.4) orally, while group VII was administered with 10 μg of alum-adsorbed rPA subcutaneously (s.c.). All the groups received a booster dose on day 21.

Sample collection

Blood samples were collected periodically from the retro-orbital plexus under mild ether anesthesia on days 0, 14, 28, and 42. Serum was separated and stored at − 40°C until tested using ELISA for antibody titer. The gastric, vaginal, and salivary secretions were collected on days 0, 21, and 42. The vaginal washes were obtained according to the method described by Debin et al. (2002). Briefly, the vaginal tract of non-anesthetized mice was flushed with 50 μl of PBS containing 1% (w/v) BSA using a Gilson pipette. These 50-μl aliquots were withdrawn and used for repeated vaginal flushing of nine times. Intestinal lavage was performed using the technique given by Elson et al. (1984). Briefly, four doses of lavage solution [NaCl (25 mM), Na₂SO₄ (20 mM), and polyethylene glycol of molecular weight, 3350 (48.5 mM)] were given orally at 15-min interval using a blunt-tipped needle. After 30 min of the last dose of lavage solution, the mice were injected intraperitoneally (i.p.) with 0.2 mL pilocarpine (10 mg/mL) to induce the gastric motility, causing discharge of intestinal contents. The intestinal discharges occurred for next 20 min were collected regularly and carefully. Salivary secretion was collected using the method developed in our laboratory with a minor modification (Jain et al. 2005). A volume of 0.2 mL of pilocarpine (10 mg/mL) was given intraperitoneally to induce salivation. After 20 min, the salivary samples were then collected from mice using capillary tube. All secretions were stored with 100 mM phenylmethyl sulfonyl fluoride (PMSF) at − 40°C until tested for secretory antibody (sIgA) levels using ELISA.

Measurement of anti-PA IgG and IgA antibodies

Specific anti-PA antibodies in serum/secretions were estimated using ELISA kit (Sigma, USA). Each well of 96-well flat-bottom immunoplate (Nunc-Immuno Plate^® Fb 96 Mexisorb, NUNC) was coated with 100 ng of rPA in 100 μl of carbonate buffer (pH, 9.6) after overnight incubation at 4°C. The plates were then washed thrice with PBS-Tween 20 (PBS-T). The free sites were blocked by incubating with 200 μl of 1% w/v solution of BSA in PBS-T (Blocking buffer) for 2 h at 37 ± 1°C. The Maxisorb plates were then washed 6 times with PBS-T. One hundred microliters of serially diluted mice sera/secretions samples (PBS: pH, 7.4; 0.05% Tween-20; and 1% BSA) was added to each well and incubated for 2 h at 37°C. The wells were then washed with washing buffer, and specific antibodies in the serum/secretion samples were determined by the addition of 100 μl of horse-radish peroxidase-conjugated goat anti-mouse IgG and IgA (diluted to 1:1000 in PBS–BSA), respectively. The wells were washed five times with washing buffer. Then, 100 μl of tetramethylbenzidine (TMB-H₂O₂) was added as a substrate to produce color. After 15–20 min of incubation, 50 μl of H₂SO₄ (1N) was added in each well to stop the reaction. Color produced within 15 min was measured using ELISA plate reader (Bio-Rad) at 450 nm. End-point titer was expressed as the log reciprocal of the last dilution, which gave an optical density (OD) at 450 nm above the optical density (OD) of negative controls. Similarly, antibody isotyping response (IgG1 and IgG2a) was determined by ELISA using sigma isotyping kit (type II) following specifications as per the manufacturer's recommendations.

Toxin neutralization assay (TNA)

The lethal toxin (Letx) neutralization ability of various sera and secretion samples was measured by determining the capacity of anti-PA antibody to prevent cytotoxicity of Letx to J774A.1 cells (Singh et al. 1989). A volume of 0.2 mL of cell suspension (6 × 10⁵−8 × 10⁵ cells/mL) was plated onto 96-well cell culture plates. Samples were serially diluted with PBS (pH 7.4) containing 0.05% Tween-20 and 1% BSA. The protective antigen (rPA) and lethal factor (LF) were added to the antiserum dilutions at final concentrations of 5 and 2 μg/mL, respectively. After incubation, 10 μl of antiserum–toxin mixture was added to J774A.1 cell suspension. The plates were incubated for 5 h at 37°C under 5% CO₂. MTT assay was conducted in order to monitor the cell viability by taking the absorbance at 540 nm (Mosmann 1983). The end point was defined as the highest antibody sample dilution that exhibits OD above that of the control. Neutralizing-antibody titer was expressed as the reciprocal end-point dilution.

Statistical analysis

The results were expressed as mean ± standard deviation. Student's t-test was carried out for statistical analysis of data obtained, and the statistical significance was designated as p < 0.05. Multiple comparisons were made using one-way analysis of variance (ANOVA) followed by post hoc analysis by applying Tukey–Karmer post-test.

Results

Size, morphology, and zeta potential analysis

The SEM photomicrograph revealed that both uncoated and coated CHMp had smooth surface and almost spherical shape (Figures 1A and 1B). The particle size and zeta potential of CHMp were found to be 667 ± 119 nm and 34.22 ± 3.98 mV, respectively (Table I). The coating CHMp with alginate resulted in the complete inversion of zeta potential of the CHMp (−29.7 ± 5.5 mV). This gives a qualitative indication of coating of a positively charged CHMp with a negatively charged alginate. The antigen was loaded onto preformed CHMp, and high percent antigen loading was recorded (Table I). This adsorption of antigen was mainly caused by the ionic interaction between amine group of positively charged chitosan and carboxyl group of negatively charged antigen substrate. The results also indicated that the alginate coating over antigen-bearing CHMp did not affect the antigen loading to a large extent (Table I).

Figure 1. SEM photomicrograph. (A) CHMps, (B) A-CHMp.

Table I. Table showing particle size, zeta potential, % loading efficiency and % loading capacity.

System	Zeta potential (mV)	Particle size (nm)	P.D.I*	% LE	% LC
Chitosan particles	34.22 ± 3.98	667 ± 119	0.241 ± 0.092	–	–
Antigen-adsorbed particles	28.82 ± 5.27	691 ± 125	0.272 ± 0.072	79.44 ± 7.11	36.78 ± 7.34
Alginate-coated chitosan particles	‐29.7 ± 5.5	714 ± 167	0.293 ± 0.132	70.23 ± 4.13	31.61 ± 2.11

Results are expressed as mean ± SD (n = 5).

P.D.I*: polydispersity index; % LE: % loading efficiency; % LC: % loading capacity.

FTIR analysis

The FTIR spectrum of native chitosan showed peaks near 1650 cm^{− 1} and 1430 cm^{− 1} indicating the presence of C = O stretching and N-H stretching of amide, respectively. The CHMp also demonstrated an absorption peak near 1650 cm^{− 1}, whereas the peak due to N-H stretching was almost disappeared (Figure 2A), which may be attributed to the interaction of counter ion with the primary amino groups of the chitosan (Xu and Du 2003). The native sodium alginate demonstrated a strong peak near 1640 cm^{− 1} and 1450 cm^{− 1} which correspond to the carboxylate salt present in the gluronic and mannuronic acid residue and carboxylate group, respectively. The similar peaks were observed in the case of A-CHMp, which indicates the association of alginate with CHMp.

Figure 2. FT-IR spectra. (A) Figure Ccomparing FTIR spectra of chitosan and CHMps; (B) Figure Ccomparing FTIR spectra of CHMps, sodium alginate, and A-CHMps.

Differential scanning calorimetry

The DSC spectrum of chitosan (Figure 3) revealed an endothermic peak near 100°C and an exothermic peak near 250°C, which may be due to the evaporation of water and the onset of the chitosan degradation, respectively. The CHMp demonstrated two endothermic peaks at 235°C and 275°C, which may be related to the breakdown of weak unspecific interaction and the cleavage of electrostatic bond between sulfate ion and polymer, respectively. The native sodium alginate demonstrated an exothermic peak near 250°C which may be attributed to the breakdown of alginate. On the other hand, A-CHMp revealed no peak in this region; instead, a slow exothermic raise was manifested near 200°C, which may be attributed to the contribution of two phenomena: the exothermic behavior of alginate and the endothermic behavior of the CHMp. The results obtained were in line with the report previously published (Borges et al. 2005, Gonzalez-Rodriguez et al. 2002).

Figure 3. DSC spectra. Figure comparing DSC spectra of chitosan, CHMps, sodium alginate, and A-CHMps.

SDS–PAGE analysis

Previous studies have reported the use of biodegradable particles of PLGA or PLA as a vaccine carrier. Encapsulation of antigen in these carriers requires the use of organic solvent and high shearing conditions that may require the use of additional care to protect antigen (Jaganathan et al. 2005). However, in this study, the antigen was loaded onto chitosan particles by simple incubation of antigen with preformed particles to avoid encounter of antigen with such harsh conditions. SDS–PAGE (Figure 4) of the rPA isolated from the Mps demonstrated a band at the position (83 kDa) identical to that of native antigen. This confirmed that the preparation conditions did not cause any irreversible aggregation or cleavage of the protein.

Figure 4. SDS–PAGE Analysis. SDS–PAGE showing stability of antigen isolated from particulate formulation. Lane 1: Marker proteins (205-kDa myosin, rabbit muscle; 97-kDa phosphorylase B; 67-kDa BSA; 43-kDa ovalbumin; and 29-kDa carbonic anhydrase); Lane 2: native rPA (83 kDa); and Lane 3: rPA isolated from A-CHMps (83 kDa).

In vitro release studies

The in vitro release study indicated that CHMp exhibited burst release in both SGF and SIF (Figure 5). It was observed that approximately 80% and around 60% of the antigen was released within first 30 min in both SGF and SIF, respectively. This may be attributed to the rapid desorption of antigen into the release medium. However, A-CHMp could prevent the burst release of antigen in both SIF and SGF (Figure 5).

Figure 5. In vitro release. Graph showing percentage of antigen release with respect to time in SGF (pH, 1.2) and SIF (pH, 6.8).

In vitro mucoadhesion measurements

Figure 6 shows the amount of FITC-BSA washed away vs time of FITC-BSA and FITC-BSA associated with A-CHMp using the rat jejunum tissue as a biological substrate. The results showed that FITC–BSA washed away rapidly indicating no mucoadhesion. On the other hand, the washing rate of FITC–BSA associated with A-CHMp was significantly low as compared to that of FITC–BSA. This study indicated that the A-CHMp was mucoadhesive in nature and could prolong the residence time of antigen in gastrointestinal tract.

Figure 6. In vitro Mucoadhesion Measurement. Graph showing FITC–BSA amount washed away vs time.

Peyer's patch uptake study

It has been reported that particles smaller than 10 μm are selectively taken up by M cells (Eldridge et al. 1990, Smith et al. 1995). These M cells transport the antigen-loaded carrier to the underlying gut-associated lymphoid tissue (GALT), that is, peyer's patch. The results indicated that orally administered soluble FITC–BSA (PBS, pH 7.4) could not produce any fluorescence in the peyer's patch (Figure 7A). However, orally administered A-CHMp-associated FITC-BSA could demonstrate fluorescence in peyer's patch indicating the uptake/deposition of dye-loaded A-CHNp into the peyer's patch (Figure 7B).

Figure 7. Fluorescence photograph of mice peyer's patch. (A). Mice treated with FITC-BSA solution; (B). Mice treated with A-CHMps loaded with FITC–BSA. Hot-Spot indicated by arrows showing the uptake of A-CHMps by peyer's patch of mice.

Anti-PA antibody titer

The anti-PA IgG titer of animals immunized with different formulations is shown in Figure 8A.

Figure 8. Anti-PA Antibody Titer. Graph showing anti-PA antibody titer in serum [A] and secretion [B]. Anti-PA antibody titer is expressed as the reciprocal dilution titers ± SE (n = 6), which gave an optical density (OD) above negative control. Abbreviation used in graph indicated the various groups used for the study and are as follows: rPA – Group fed with soluble rPA (100 μg), CN10 – Group fed with A-CHMps containing 10 μg rPA, CN25 – Group fed with A-CHMps containing 25 μg rPA; CN40 – Group fed with A-CHMps containing 40 μg rPA and CN55 – Group fed with A-CHMps containing 55 μg rPA, Alum-adsorbed rPA – Group injected s.c. with 10 μg of alum-adjuvanted rPA. Asterisk over bars indicated degree of significance. Where, [* = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant].

We first determined the optimal dose of rPA for oral immunization of mice. It was observed that oral delivery of free rPA-induced low plasma anti-PA antibody responses indicating that rPA itself was poorly immunogenic when administered orally. However, rPA in A-CHMp demonstrated substantially higher antibody titer in serum when compared to free rPA at all tested dose. It was also observed that the antibody response with A-CHMp was typically dose dependent and higher rPA dose resulted in the higher antibody titer. The ELISA results indicated that the serum antibody titer increased significantly when the dose of A-CHMp-associated rPA was increased from 10 to 25 μg. On the other hand, 25, 40, and 55 μg of A-CHMp-associated rPA demonstrated comparable serum IgG titer. The alum-adjuvanted rPA resulted in the induction of strongest antibody titer in serum when compared to all other formulations.

Specific mucosal immunity structured as sIgA can prevent the attachment of infectious pathogen to gastrointestinal (GI) mucosa (Jain et al. 2006, Medina and Guzmán 2000). Both nasal and oral immunizations are well established to be the most reliable strategy for inducing mucosal immunity for optimal protection of mucosal surfaces. The results indicated that alum-adsorbed rPA and free rPA could induce minimal antibody titer in all examined (local and distal) secretions. The A-CHMp-associated rPA resulted in the induction of substantially higher secretory antibody titer when compared to free rPA and alum-adsorbed rPA. The secretory antibody titer was also found to be dose dependent. It was observed that the secretory antibody titer was substantially higher in the case of CN25 when compared to CN10. However, CN25, CN40, and CN55 demonstrated comparable secretory antibody titer in local and distal secretions.

Neutralizing antibody titer

The functional significance of both plasma and secretory anti-PA antibody was analyzed using the in vitro toxin neutralization assay (Figures 9A and 9B) (Reuveny et al. 2001, Williamson et al. 1999). It was observed that A-CHMp demonstrated strong neutralizing antibody titer in serum as compared soluble rPA (Figure 9A). The neutralizing antibody titer increased amongst the group fed with various dosage of Mps-associated rPA following the order: soluble rPA< CN10 < CN25 < CN40 = CN55. Results indicated that the alum-adjuvanted rPA could induce substantially high neutralizing antibody titer as compared to A-CHMp at equivalent dose. The neutralizing ability of anti-PA sIgA of various secretions was also determined (Figure 9B). It was observed that A-CHMp could induce significantly high neutralizing antibody titer in various local and distal mucosal secretions when compared to alum-adjuvanted rPA and soluble rPA.

Figure 9. Neutralizing Antibody Titer. Graph showing neutralizing antibodies titer in serum [A] and secretions [B]. End-point titer is expressed as the reciprocal dilution titers ± SE (n = 6), which gave an optical density (OD) above negative control. Note: Free rPA and alum-adsorbed rPA could not generate detectable neutralizing antibody titer in examined secretions.

Antibody subtyping

To determine the type of immune response being stimulated, the levels of two subtypes of antibodies, that is, IgG1 and IgG2a, were assessed in mice sera on day 42 (Figure 10). IgG2a is indicative of Th1-type (cellular) response, whereas IgG1 is indicative of the presence of Th2 (humoral) response. The results indicated that IgG1 subtype constitute the major fraction and was significantly higher as compared to IgG2a subtype (p < 0.05). This indicated that the immune response was Th2 based (humoral) regardless of the formulation composition and the route of immunization in this study.

Figure 10. Antibody Subtyping. Graph showing anti-PA IgG1 and IgG2a titers. End-point titer is expressed as the reciprocal dilution titers ± SE (n = 6), which gave an optical density (OD) above negative control.

Discussion

It has been found that the upper respiratory tract and other mucosal tissues are affected after anthrax spore exposure through mucosal sites (Abramova et al. 1993, Grinberg et al. 2001). Therefore, optimal immune protection against anthrax (inhalational and gastrointestinal) requires two layers of defense in the form of mucosal and systemic immune response to check the infection in early stages (Welkos et al. 2001, 2002) and the cutaneous anthrax. In this study, we tested the efficacy of a carrier-based subunit vaccine for oral administration with the intent to induce mucosal and systemic immunity against anthrax.

It has been reported that recombinant antigens are less immunogenic in nature, and hence, they need adjuvant to enhance their immunogenicity (Boyaka et al. 2003). Polymeric particulate carriers are considered to be of special interest for this purpose as these carriers are reported to enhance the uptake of loaded antigen by antigen- presenting cells (APCs) and consequently the presentation other cells of immune system for the elicitation of strong immune response. The results in this study indicated that soluble rPA was poorly immunogenic when administered orally even at higher dose (100 μg). Our results showed that alum-adjuvanted rPA could induce substantially higher antibody titer in serum but it fails to induce specific secretory immune response (sIgA) at the mucosal level. However, A-CHMp could induce strong immunological response in both systemic and mucosal compartments. The results indicated that A-CHMp demonstrated mucoadhesive ability and may prolong the residence time of the antigen in GIT and subsequently promote the uptake of A-CHMp into GALT. Our study also confirmed the deposition of the selected carrier in peyer's patch. Therefore, It was concluded that the A-CHMp may enhance the uptake, presentation, and processing of antigen in the gut immune inductive sites for efficient immune induction. It is worth noting that strong neutralizing antibody titer was determined in serum and secretions of mice fed with A-CHMp-associated rPA. However, neither alum-adjuvanted rPA nor soluble rPA could induce neutralizing antibody titer in mucosal secretions. Thus, the potential benefit of the developed vaccine is the induction of specific s-IgA in various secretions that may also interfere with the germination of B. anthracis spores and/or favor spore uptake by phagocytic cells (Boyaka et al. 2003). It was also observed that the magnitude of immunological response in mice immunized with A-CHMp was typically antigen dose dependent and higher dose of rPA resulted into the potentiation of the anti-PA antibody titer in serum and secretions. To further characterize the nature of immune response, we analyzed the antibody sub-typing pattern. It was observed that IgG1 was the predominant subtype obtained in the serum of all animal groups immunized with rPA. This indicates that the developed vaccine resulted in the induction of humoral-based immune response. It was concluded that the immune mechanisms stimulated remain identical in mice immunized with different rPA-based formulations.

Conclusion

In summary, A-CHMp is an efficient mucoadhesive carrier adjuvant, which could prolong the residence time of loaded antigen in the GIT and subsequently resulted in better uptake by M cells. This consequently results in the prolonged activation of professional APC in GALT and much efficient immune induction. Our results clearly indicate that A-CHMp represents a promising mucoadhesive carrier adjuvant for effective anthrax vaccine that provides strong immune response in systemic and mucosal compartments.

Acknowledgement

We are thankful to Dr. Yogendra Singh (IGIB, New Delhi) for providing recombinant Protective Antigen and Lethal Factor and Raj Kurupati (IGIB, New Delhi) for his cooperation, support, and help. We are thankful to Indian Institute of Technology (Bombay), All India Institute of Medical Sciences (AIIMS) and Punjab University (PU) for providing SAIF facility. The authors would also like to acknowledge All India Council of Technical Education (AICTE) for providing Junior Research Fellowship (JRF) to carry out the research work.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.