Mucosal genetic immunization through microsphere–based oral carriers

Abstract

Polymeric carriers in the form of cellulose acetate phthalate (CAP) and alginate (ALG) microspheres were used for encapsulation of plasmid DNA for oral mucosal immunization. Access into the intestinal mucosa by pVAX1 eukaryotic expression plasmid vectors carrying gene-coding sequences, either for the cholera enterotoxin B subunit (ctxB) immunostimulatory antigen or the green fluorescent protein (GFP), delivered from both types of microsphere carriers were examined in orally immunized BALB/c mice. Demonstration of transgene protein expression and IgA antibody responses at local mucosal sites suggest immunological response to a potential oral DNA vaccine formulated within the microsphere carriers.

Introduction

The portals of entry for pathogens include the mucous membranes of the respiratory, gastrointestinal and urogenital tracts.^1,2 About 90% of human infections occur at mucosal surfaces. Thus, targeting the mucosal sites where pathogens invade remains an attractive strategy for immunization.³ Cholera is an enteric disease, caused by pathogenic events following ingestion of food or water which have been contaminated with the causative agent, Vibrio cholerae. The pVAX-ctxB DNA vaccine, as described in a previous report,⁴ consists of the cholera enterotoxin B subunit gene of the Vibrio cholerae bacterium cloned into a mammalian expression plasmid DNA vector (pVAX1), designed for DNA vaccine development. Since oral regimens for cholera vaccination remain to be the practical approach in mass immunization strategies, especially in cases of epidemic outbreaks or in the population where cholera is endemic, the need exists for studies on oral genetic immunization strategies. Some of the drawbacks of the current oral cholera vaccines, which consist of whole cell bacterium and recombinant CTB proteins, include the need for cold-chain transport, higher cost and need for booster immunizations due to decline in protective efficiency after some period. On the other hand, DNA vaccination delivered into mucosal sites may provide additional advantage of cheaper cost of production, transport at ambient temperatures and potential for immune modulation through plasmid vector design.

Microsphere-based oral carriers for encapsulation of DNA vaccines provide attractive means for mucosal immunization. Oral DNA vaccine delivery using alginate microspheres as carriers serves as an approach to induce local immune responses within the mucosal epithelium along the intestinal tract as first line of defense against the pathogen.^5,6 The concept is to deliver DNA vaccines through the oral route using carriers which protect the DNA vaccine itself from stomach acidity while releasing the vaccine within the intestinal environment. Oral delivery of microencapsulated DNA may be beneficial in terms of: availability of the encapsulated material, sustainability of the release, and the ability to control the absorption rate of the encapsulation material.⁷ Furthermore, the encapsulated material is protected from rapid degradation and its bioactivity may be prolonged by controlled release from the microspheres.⁸

The mucosal membranes serve as a first line of defense against invading pathogens where the cells at the inductive sites carry on the functions of antigen recognition and T or B cell activation, and extravasation and differentiation of immune cells at the effector sites lead to production and secretion of immunoglobulin IgA (sIgA) and/or activation of specific cellular mediated immune (CMI) responses.^5,6

Transport and uptake of substances may occur within the intestinal epithelium through mechanisms which may involve both the Peyer’s patches (PP) and other non-PP tissue, such as enterocytes.^9,10 Previous studies have illustrated the attachment, localization and uptake of micro- or nanoparticles composed of alginate/chitosan complexes^9,11 within the Peyer’s patches (PP) in rats. The role of specialized phagocytic cells called M cells or microfold cells which are present in the PP tissues include engulfment and presentation of the immunogens to the lymphatic system.¹² Moreover, transcytosis occurring among the enterocytes or the absorptive cells of the intestinal epithelium offer transport of substances across the epithelium.¹⁰ Pertaining to GFP expression within the intestinal epithelium in this study, further investigation to determine as to which specific cells express the foreign gene in response to delivery of DNA from within alginate microspheres is needed; however, speculation remains toward the concept of involvement of both tissues of the Peyer’s patches and, to some degree, the enterocytes. A number of related vaccination studies^13,14 further evaluated the delivery and cellular uptake of DNA molecules from within microspheres indicating antigen recognition and immune response activation through increasing levels of antibodies and cytokines in serum and the mucosal surfaces.

In this report, the use of microsphere-based oral carrier approaches for the delivery of reporter and immunostimulatory genes inserted within the pVAX1 vector were investigated in mice. Microspheres were produced from polymers, namely cellulose acetate phthalate (CAP) and alginate (ALG), as encapsulating materials for plasmid DNA. The feasibility of encapsulation of DNA molecules into the sodium alginate material, a natural polysaccharide with inherent characteristics of safety, biodegradability and non-toxicity have allowed for investigation of its use as a microparticle or nanoparticle carrier of DNA into the experimental animal.¹⁵ Furthermore, the ability of alginate particles to protect encapsulated material from acidic pH such as in the stomach and potential for mucoadhesiveness or controlled release of material, provide means for delivery into intestinal environment as means for mucosal immunization. CAP may be protective as pH-sensitive erodible polymeric coating of DNA vaccines due to its enteric characteristic.¹⁶ Insolubility of CAP microspheres at pH < 3 such as in gastric conditions protects its contents, while solubility at pH > 5 such as in intestinal conditions causes CAP to release its core materials in a sustained release pattern.⁸

Results

The physical properties and pDNA encapsulation profile of both cellulose acetate phthalate (CAP) and alginate (ALG) microspheres used in this study as oral carriers are summarized in Table 1. Most importantly, the observed pDNA release in medium with basic environment suggests the ability of both microspheres to remain intact upon oral delivery while protecting and preventing the release of pDNA through membrane shrinkage in acidic pH similar to the stomach acidity.¹⁷ Moreover, the expression of transgene protein through in vivo delivery of pDNA into intestinal tissues through the oral microsphere carriers are described in the succeeding experiments.

Table 1. Physical characteristics and pDNA encapsulation profiles of microsphere-based oral carriers: cellulose acetate phthalate (CAP) and alginate (ALG) microspheres.

	Cellulose acetate phthalate: CAP	Alginate: ALG
Nature of substance	Synthetic polymer	Polysaccharide polymer
Microsphere formation process	Solvent evaporation	Water-in-oil emulsification
General morphology	Spherical with smooth surface	Spherical with some teardrop shapes; smooth surface
Average diameter size (µm)	44.33 ± 30.22	46.88 ± 3.07
Encapsulation efficiency	1.67% to 21.30%	72.9 to 74.4%
pDNA release in acid/base	Majority of pDNA release in basic environment	Majority of pDNA release in basic environment
Advantages	Non-toxic, pH-sensitive erodible polymer	Non-toxic, pH-sensitive: swell at basic pH, biodegradable

Transgene protein expression in mice mediated through plasmid DNA delivery from oral microsphere carriers, CAP and alginate microspheres were observed among intestinal tissue cells at the optimal dose of 100 µg pVAX-ctxB or pVAX-GFP as shown in Figures 1 and 2.

Figure 1. Percentage (%) of green fluorescent protein (GFP) expressing cells in mice post-oral administration of various doses of pVAX-GFP in microspheres. Numerical figures indicated after CAP or ALG denote the loaded amount of pVAX-GFP (µg) within microsphere preparations.

Figure 2. (A) CTB-expressing cells among mice intestinal tissue samples in untreated, empty CAP microspheres and pVAX-ctxB/CAP mice groups. Immunofluorescent staining with FITC-labeled anti-β cholera toxin antibody for cells obtained from mice, 24 or 48 h post-oral administration with 100 μg of pVAX-ctxB/CAP. (B) Percentage of intestinal cells expressing ctxB in untreated, empty microspheres, and encapsulated pVAX-ctxB with the time points of intestinal cells collection at 24 or 48 h post-oral administration of individual mouse. Each symbol shows the value for one mouse. Bar represents the mean of each group.

In Figure 1, the in vivo biodistribution of pDNA among various tissues after oral delivery of pVAX-GFP–loaded CAP and ALG microspheres demonstrated reporter protein GFP expression in intestinal tissue samples and to a lesser degree, in stomach cell samples. Other tissues which have been evaluated for GFP expression include blood, liver, spleen, and esophagus; however, since persistently no expression were detected among these organs, the data are not shown in the figure except for the expression in spleen tissue samples to serve as representative of the negative baseline expression.

For experimental studies with ALG microspheres, the number of mice for each dose group = 6 (except for 150 µg-dose group which had only 3); total number of mice, n = 18. On the other hand, for studies with CAP microspheres, the number of animals in negative control group = 6; Number of animals for each dose group = 3; total number of mice, n = 12. All measurements were done in triplicates. Results represent mean ± standard error with the basal levels of expression from negative controls subtracted from expression levels observed in dosed groups.

Statistical analyses using ANOVA with post hoc Tukey HSD were performed to compare GFP expression between mice groups which have been orally dosed with pDNA dose of 50-, 100- and 150 µg pVAX-GFP encapsulated within either CAP or ALG microspheres and the negative control groups; as well as comparison of each dose group (i.e. 50 µg) against the various other dose (i.e. 100- and 150- µg) groups.

There was no significant (p > 0.05) amount of GFP expression detected in the spleen samples. The trend of higher levels of expression in intestines compared with that in stomach was observed for 100 μg pVAX-GFP post-24 h of oral administration for both ALG and CAP encapsulation. There was no statistical difference between the levels of expression (p > 0.05); however, in ALG, there was a trend of increasingly higher GFP expression in 100 μg pVAX-GFP compared with 50 μg pVAX-GFP followed by a decrease in the expression for 150 μg pVAX-GFP encapsulated in ALG. The similarity of the expression trends between ALG and CAP was the sustained expression that increased as the dose increased.

Figure 2 shows results of in vivo delivery studies of encapsulated pVAX-ctxB DNA vaccine at 100 µg dose in CAP microspheres. Concurrent findings suggest an optimal dose of 100 µg pDNA for both adequate protein expression among transfected cells in vivo and for induction of measurable induction of immunoglobulin responses at local mucosal sites. As reported in other studies, administration of 100 μg pDNA dose in mice showed adequate gene expression for reporter gene expression studies^18,19 and induction of immune response.²⁰ Cox et al.²¹ reported that 50 μg and 100 μg pDNA doses showed detectable neutralizing antibody response in mice, whereas in a rotavirus DNA vaccine study, mice orally administered with 50 μg of pDNA induced potent immune response.²²

Flow cytometric analyses showing CTB expressing cells from intestinal tissue samples were conducted with the following number of mice: untreated (n = 3), empty microspheres (n = 4), 100 μg 24h (n = 5) and 100 μg 48 h (n = 6). Total n = 18. All measurements were done in triplicates. Results represent mean ± standard error with the basal levels of expression from negative controls subtracted from expression levels observed in dosed groups.

In this report, the mice intestinal cells expressing CTB protein was detected by the anti-β cholera toxin-FITC antibody. The percentage of CTB expression was determined from the fluorescence level of FITC-positive cells.

CTB expression was demonstrated after 24 h among 1.3% of intestinal cells in orally immunized mice group detected through immunofluorescent techniques. CTB-expressing cells detected by FITC-labeled antibody were detected among mice intestinal tissue samples as previous results from DNA delivery from within ALG or CAP microspheres which indicate higher transgene expression in the region in contrast to all other organs.

Statistical analyses using ANOVA with post hoc Tukey HSD were performed to compare CTB expression in vivo within intestinal tissue samples among mice from the different dosed time-point as compared with negative control and mice dosed with empty microspheres. Although the results observed were not statistically significant, in vivo CTB expression at the intestinal regions showed a trend of higher expression in CAP-encapsulated pVAX-ctxB groups as compared with the negative control and mock treatment groups (Fig. 2).

Intestinal fluid IgA measurements were performed on the samples obtained upon animal sacrifice or at the endpoint of the study period. However, the measured IgA levels from dosed or treated mice showed no increase compared with the IgA levels from negative control or untreated mice samples (data not shown). Additional samples for the other time-points were not collected in this study. ELISA measurements of stool secretory IgA (sIgA) levels were performed at day 7, 14, and day 21 among mice dosed with en(pVAX–GFP) in ALG microspheres (Table 2). At day 7, sIgA levels in stool among dosed mice were about 5-fold higher than controls which indicates the peak time-point for IgA antibody level rises observed with delivery of en(pVAX–GFP) in ALG microspheres. At days 14 and 21, dosed mice have shown 1.4-fold to 1.9-fold higher sIgA levels as compared with controls. Stool IgA levels in mice delivered with en(pVAX-GFP) in CAP microspheres were measured in samples collected at week 2, 4, and 6 post-oral administration (Table 2). Compared with the negative control group, the CAP-encapsulated pDNA group showed similar IgA level at week 2 with 0.01-fold difference, and showed a 1.27-fold increase at week 4, while at week 6 there was only 1.17-fold increase. This indicates that its peak time-point was at week 4.

Table 2. Fold differences of titers for faecal secretory IgA (sIgA) levels among dosed mice groups against controls.

Fold differencesa of fecal sIgA levels
pVAX-ctxB/CAP	pVAX-ctxB/ALG
Week 2: 0.01	Week 1: 6.3
Week 4: 1.27	Week 2: 1.6
Week 6: 1.17	Week 3: 1.9

^a Mean titers of sIgA levels among vaccinated mice compared with mean titers of the negative control group

Discussion

Among mice orally administered with plasmid pVAX-GFP in CAP or ALG microsphere carriers, biodistribution studies showed a trend of higher percentages of GFP-expressing cells in the intestines followed by stomach as compared with expression in the liver, spleen and blood. These findings illustrate successful delivery of DNA material within the intestinal mucosa and as a potential approach for genetic mucosal immunization through the oral routes.

Results of immunization studies suggest a trend in the humoral IgA response in mice groups administered with CAP- and ALG-encapsulated pVAX-ctxB. Interestingly, the levels of IgA concentration in all mice groups in CAP-encapsulated pVAX-ctxB studies peaked at week 4 post-oral administration. The mice group orally immunized with CAP-encapsulated pVAX-ctxB was found to show an overall trend of producing the highest concentrations of IgA compared with control groups. Although the levels are low, this still indicates the potential of the CAP-encapsulated pVAX-ctxB in eliciting immune response against the cholera toxin. A study on rotavirus DNA vaccine only started to show significant faecal IgA response at week 6 after oral administration.²² Even so, there was no significant difference between the groups at sampling times of weeks 2, 4, and 6. This may be due to the environment in which the samples were processed for ELISA assays at each particular week. On the other hand, studies on oral immunization with ALG-encapsulated pVAX-ctxB in mice showed peak increase in sIgA levels at week 1 while some decline from week 2 and onwards. Although variability in the IgA titers were observed from one time-point to another, more specifically in week 2, the sIgA levels among vaccinated animals show relatively higher titers as compared with the negative control. The variability for each time point may reflect the limitation in standardized sample processing of the stools prior to performing the ELISA procedure which can be augmented through additional replicates on either the same or other animal group. In contrast, for serum IgA levels, no notable differences or fold-increases were observed when comparing the vaccinated animals and the negative control (data not shown). The antibody responses against the vaccine, therefore, can be observed at local sites such as the mucosal surfaces of the intestines which were measured in stool samples and intraluminal fluid, rather than serum samples.

Moreover, the assessments of secretory IgA or sIgA, from either fecal samples or intestinal lavage fluids, have been employed in various studies.^23,24 Findings by Tokuhara et al.²⁵ suggest that protection against CT-induced diarrheal disease is correlated with specific secretory or sIgA immunoglobulins against CTB and not the serum counterparts, thus highlighting the advantages of mucosal immunization for cholera and other enteric diseases.

Cellulose acetate phthalate and alginate, categorized as microsphere-based oral carriers, provide an alternative approach for delivery of antigens into intestinal cells as means for mucosal genetic immunization to stimulate local sIgA production at mucosal sites and for gene delivery among cells of the mucosal system networks. Further research needs to be undertaken to improve both the immunogenicity of vaccine construct, the efficiency of encapsulating microspheres, as well as more standardized assessment protocols for immune induction of both antibody and cellular responses during vaccination studies against cholera or other disease.

Methods

Cellulose acetate phthalate (CAP) (Fluka) microspheres were produced through solvent evaporation method. On the other hand, alginate microspheres were produced through water-in-oil emulsification method from sodium alginate (FMC BioPolymer). Characterization of the physical characteristics and the capacity for plasmid DNA encapsulation for both microsphere carriers are shown in Table 1.^18,26 The sequences and eukaryotic expression of plasmid vectors, namely the pVAX-ctxB and pVAX-GFP, have also been described in previous studies.^4,18

Female BALB/c mice (Institute for Medical Research) of 6–8 weeks old were dosed by performing oral gavage to administer microsphere carriers encapsulating pVAX-ctxB or pVAX-GFP as described previously.^18,26 All animal experiments were performed in compliance with the Universiti Putra Malaysia Guidelines for Animal Experimentation.

Tissue biodistribution of both microsphere carriers were assessed in mice through intracellular reporter protein expression among various organs collected upon necropsy at 24 h post-oral administration of CAP and ALG microspheres carrying the plasmid DNA, pVAX–GFP. Doses of 50, 100, and 150 μg pVAX–GFP were examined among mice groups as proof-of-concept for pDNA delivery through microsphere carriers. GFP-positive cells were identified using flow cytometric analysis of cell suspensions from each organ obtained from mice orally dosed with pVAX-GFP encapsulated within CAP or ALG microspheres.

Immunofluorescence detection of CTB protein in vivo was performed in BALB/c mice which were orally administered with preparations of CAP microspheres loaded with 100 μg pVAX-ctxB for each animal through oral gavage. The mice in the control groups were not dosed and in another group, mice were orally administered with CAP microspheres which were not loaded with plasmid DNA. The mice were fasted for 12 h with free access to water before the terminal procedure. Mice were fasted 12 h post delivery of microspheres to clear the GI organs from digested food particles as preparation for facilitated processing of epithelial cells at the luminal lining of the gastrointestinal tract and to keep presence of food debris to the minimum in the processed samples. The mice were then euthanized using carbon dioxide in a chamber and were ultimately sacrificed by cervical dislocation at 24 or 48 h post-oral administration. Intestines were collected from each animal and isolated cells were disaggregated by adapting a method described by Perret et al.²⁷ Intracellular antibody staining was performed at 5 μg/ml FITC-labeled anti-β cholera toxin antibody (Abcam Inc.) according to the manufacturer’s protocol. The cells were analyzed using a flow cytometer (BD FACSCalibur™, Becton Dickinson) for the percentage of FITC-expressing cells to determine the percentage of cells expressing the CTB protein.

Oral immunization studies with pVAX-ctxB DNA vaccine delivered through both microsphere carriers were performed. IgA ELISA measurements were performed on faecal samples obtained from mice groups at 1 or 2 weeks intervals to assess mucosal IgA production. The mice were grouped according to the treatment as follows: negative control group (n = 12), and another group designated as pVAX-ctxB/CAP group for mice orally dosed with 100 μg pVAX-ctxB encapsulated in CAP microcapsules dispersed in nuclease-free water (n = 8); total number of mice = 20. Similarly, oral administration of 50 and 100 μg pVAX-ctxB encapsulated in ALG microspheres, designated as pVAX-ctxB/ALG group, was performed in mice, including appropriate control groups. The number of mice for each group = 3; total number of mice = 9. Mice were isolated in individualized compartments in cages for a brief period whereby around 4 to 5 fresh fecal pellets were collected from individual mice. Fecal pellet samples were collected and weighed, then further processed according to manufacturer’s recommendation for stool preparation for secretory IgA detection using the IBL Secretory IgA (sIgA) ELISA (Saliva, Stool) Enzyme Immunoassay kit (IBL International GMBH). The stool supernatant was resuspended in wash buffer to a dilution of 1:250. Additionally, intestinal fluid samples were also collected upon animal sacrifice. The level of antibody produced post-oral administration of pVAX-ctxB was determined through IgA Mouse ELISA Kit (E-90A; Immunology Consultants Laboratory [ICL], Inc.).

Standards were prepared according to manufacturer’s protocol and in duplicates for each concentration. A hundred microlitres of appropriately diluted sample was pipetted into each well and each sample was performed in duplicates. The micro titer plate was incubated at room temperature for 60 min while being covered with Parafilm (BrandGmbH, KG, Wertheim). After the samples were removed, the wells were washed by filling each of them with 200 μl of appropriately diluted wash solution. The plate was then inverted to pour out the solution and then sharply striked on absorbent paper to remove residual solution. The wells were washed repeatedly for another three times. Each well was added with 100 μl of appropriately diluted HRP-antimouse IgA conjugate (ICL) and the plate was incubated at room temperature for 30 min. The plate was covered with Parafilm (Brand GmbH) and dark paper to keep the reaction in dark. The wells were washed and blotted with absorbent paper again for four times. A hundred microlitres of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (ICL) was added into each well. The plate was incubated at room temperature for 10 min and covered with Parafilm (Brand GmbH) and a dark paper. After that, 100 μl of stop solution (0.3 M sulfuric acid; ICL) was pipetted into each well.

IgA levels were quantified based on the absorbance readings measured using a microplate reader (Dynex MRX II, Dynex Technologies, Dynex Technologies, Inc.) and plotted on a four-parameter logistics standard curve, which served as reference for calculated values of IgA levels in µg/ml.

Results are presented as mean ± standard deviation and representative of three independent experiments. Statistical comparisons were made using one-way ANOVA with post hoc Tukey HSD analysis at 95% confidence level using SPSS 20.0 software package (SPSS, Inc.). Statistical comparisons were made using ANOVA with post hoc Tukey HSD with reference to mean IgA level responses of each group of negative controls which include untreated mice group, mice which received water only or empty/unloaded CAP only; as compared with treated group with pVAX-ctxB encapsulated in CAP (pVAX-ctxB/CAP) or unencapsulated pVAX-ctxB group. Furthermore, comparisons were also made within the group and among the different groups for the IgA responses observed for each group on week 2, 4 and 6 of the study period for CAP encapsulated pDNA treatment and week 1, 2, and 3 for ALG encapsulated pDNA treatment.