The development of a vaccine would be essential for the control of schistosomiasis, which is recognized as the most important human helminth infection in terms of morbidity and mortality. A new approach of oral vaccination with DNA-chitosan nanoparticles appears interesting because of their great stability and the ease of target accessibility, besides chitosan immunostimulatory properties. Here we described that chitosan nanoparticles loaded with plasmid DNA encoding Rho1-GTPase protein of
Schistosomiasis is an important parasitic disease, caused by trematode worms of the genus Schistosoma, affecting more than 207 million people worldwide, with a further 700 million individuals living at risk of infection [
Currently, schistosomiasis control strategies are predominantly based on the treatment of infected individuals with safe and effective drugs [
Desirable characteristics of a schistosomiasis vaccine candidate include not only the capacity to reduce worm burden and fecundity but also the capacity to downregulate granulomatous responses to eggs that become trapped in the host liver and intestines and cause morbidity [
The identification of a specific antigen is a crucial task in the development of an effective vaccine. However, the antigens tested until now were found to induce insufficient levels of protection during the preclinical studies [
Despite current challenges to improve delivery and immunogenicity, DNA vaccination has several major advantages over traditional vaccines or over other types of investigational vaccine platforms [
To date, most gene delivery strategies have concentrated on the parenteral route of delivery and oral administration has been largely ignored. The main advantages presented by oral gene delivery are the ease of target accessibility and enhanced patient compliance owing to the noninvasive delivery method. For effective oral immunization, antigens and plasmids must be protected from the acidic and proteolytic environment of the gastrointestinal tract, efficiently taken up by cells of the gut associated lymphoid tissue (GALT), and an appropriate immune response must be induced. The interaction of plasmid DNA (pDNA) with a biodegradable cationic polymer to form nanoparticles offers a way to protect pDNA from degradation [
In this work, chitosan-based nanoparticles as adjuvant for mucosal vaccination were chosen. Chitosan (CH) has been considered an attractive gene carrier because it is nontoxic and biodegradable and has mucoadhesive properties. CH is able to form complexes easily with DNA [
Imidazole-modified CH was prepared as previously described [
A plasmid (6176 pb), NT-GFP fusion TOPO TA (Invitrogen), containing Rho1-GTPase (1–579) cDNA fragment under the cytomegalovirus (CMV) early promoter was constructed previously in our laboratory, following the fabricant’s recommendations. The cloning was verified by sequencing using GFP primers and confirmed the correct insertion of the Rho1-GTPase fragment in frame with GFP sequence.
The plasmid was amplified in DH5
Rho1-GTPase was expressed as His-fusion from pET-DEST42 (Invitrogen). The cloning was done using Gateway System (Invitrogen) and protein expression was carried out. The Rho1-GTPase was purified using the Ni-NTA His-Bind affinity chromatography. The His-Rho1-GTPase is hereafter referred to as SmRho.
Nanoparticles were prepared as previously described by mixing [
Complexes were prepared at various N/P molar ratios as previously described; 2.5
To characterize the size and zeta potential of the CH-DNA complexes as prepared, these were formed at different N/P molar ratios as described in Section
To characterize the size and zeta potential of the nanoparticles when in the gastrointestinal tract conditions, the complexes were suspended in solutions that simulate the gastric (SGF) and intestinal fluid (SIF). For this purpose, complexes were prepared as described previously at N/P molar ratios of 12 and 18. After stabilization, complexes were diluted to 1 mL using either SGF or SIF (described in USP XXIV) at 37°C. After 15 min in these solutions, nanoparticles were characterized as described previously.
Six-week-old female C57BL6 mice were purchased from the Federal University of Minas Gerais (UFMG) animal facility. All protocols involving animals were approved by the local Ethics Committee on Animal Care (CETEA-UFMG number 204/2009). Animals had free access to food and water, with 12 h light/dark cycle.
Five groups were submitted to different treatments, as follows: control group—phosphate buffer saline (PBS) ( suspension of CH nanoparticles ( solution of naked recombinant plasmid DNA (50 suspension of modified CH (CHimi)—plasmid DNA (25 suspension of modified CH (CHimi)—plasmid DNA (25
In order to evaluate the adjuvant effect of CH, chitosan nanoparticles without DNA were prepared (Group II). And with the aim to evaluate the improvement of gene delivery by CH modified (Chimi), Chimi-DNA nanoparticles were administered to groups IV and V while group III received DNA without CH. The best route of administration was also analyzed comparing groups IV and V.
The different formulations were administered orally with a gavage-feeding needle (groups I–IV) and intramuscularly with an injection in the tibialis anterior muscle (group V). The primary immunization was followed by two immunizations with an interval of two weeks. Seven days after the last immunization mice were challenged and then after 50 days mice were sacrificed.
Blood samples were taken through tail veins from four mice of each experimental group at two-week intervals, and the sera were prepared by centrifugation and stored at −20°C until further analysis. Serum was collected from immunized and control mice to measure kinetics of specific antibodies.
A measurement of specific anti-SmRho antibodies was performed using an indirect enzyme-linked immunoabsorbent assay (ELISA). Maxisorp 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with 10
Seven days after the last immunization, mice were challenged through percutaneous exposure of abdominal skin to water containing 25 cercariae (LE strain) for one hour. 50 days after challenge, mice were sacrificed and adult worms were perfused from their portal veins [
Antigenic preparations were obtained from schistosome eggs (SEA) and adult worms (SWAP), prepared as soluble supernatant fluids from buffered saline homogenates of the respective life-cycle stages [
Cytokine experiments were performed using splenocyte cultures from individual mice of control and experimental groups. Splenocytes were isolated from macerated spleens of individual mice at day 50 post infection. Cells were washed twice with sterile PBS and were plated at a concentration of 1 × 106 cells per well in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS, 100 U/mL of penicillin G sodium, 100
Liver fragments from mice (3 mice per group) of control and experimental groups immunized and infected were collected 50 days post infection in order to evaluate the effect of immunization on granuloma formation. Liver fragments were fixed in 10% paraformaldehyde. Fragments processed for paraffin embedding and histopathological sections were cut using a microtome at 5
Statistical analysis was performed using the ANOVA test and the GraphPad Prism 5 software package. The Bonferroni test was used to compare subgroups with the level of significance set at
To determine the minimum amount of CHimi polymer required to complex plasmid DNA fully, varying amounts of polymer were mixed with DNA solutions with a fixed amount of plasmid DNA, and the resulting complexes were evaluated for their electrophoretic mobility. The modified polymers halt DNA mobility at the same N/P molar ratio as for nonmodified CH, which was found to be 2 (Figure
Electrophoretic retention of DNA by CH (a) and CHimi (b). Unless otherwise mentioned, lanes assignments correspond to N/P molar ratios tested and are as follows: Lane M: gene ruler 1 kb DNA ladder; lane DNA: plasmid DNA solution; lane 0.5–24: N/P molar ratios.
The size of CH-DNA nanoparticles was determined by light scattering. Particles with N/P molar ratios raging from 1 to 24 were evaluated and results presented on Figure
Mean particle size (a) and zeta potential (b) of CH-based complexes as a function of N/P molar ratio (measurements performed at 25°C, pH 5.5; average ± SD,
Nanoparticles were also characterized in terms of zeta potential (Figure
In order to evaluate the nanoparticles prepared as a candidate to oral delivery system for DNA vaccines, the stability of CH-DNA complexes in media that simulates the stomach and intestinal environment was tested. For this propose size and zeta potential of the particles suspended in SGF pH = 1.2 or SIF pH = 6.8, both at 37°C, were measured using nanoparticles with N/P molar ratios of 12 and 18. The results illustrated in Figure
Zeta potential (a) and mean size (b) of CH-based complexes prepared in N/P molar ratios (R) 12 and 18 at different media. The measures were realized at 25°C in acetate buffer pH 5.5 and at a 37°C in SGF and SIF.
Further
Serum anti-rSmRho-specific IgG levels of mice immunized orally with DNA (a) or CHimi-DNA (b) and intramusculary with CHimi-DNA nanoparticles (c). Sera of immunized mice were collected at days 15 (one week after the first immunization), 30, 45, 60, 75, and 95 and assayed by ELISA. The results are presented as the mean absorbance measured at 450 nm for each group. Statistically significant differences of vaccinated mice compared with the control group, PBS, in each time evaluated, are indicated by (*) for
To investigate the protective activity induced by vaccination with CH and CHimi-DNA nanoparticles in murine model of
Protective effect and liver granuloma size induced by C57BL/6 mice vaccination with CH-DNA nanoparticles and challenged with 25
Groups | Total worms mean ± SD | Percentage reduction of worm burden | Hepatic granuloma area ( | Percentage reduction of granuloma area |
---|---|---|---|---|
Control | 19.0 ± 2.4 | — | 109743 ± 47427 | — |
CH | 10.1 ± 0.81* | 47* | 83269 ± 50226 | 24,1 |
DNA | 13.8 ± 4.5 | 27 | 82352 ± 46222* | 24,9* |
CHimi-DNA | 16.5 ± 3.6 | 23 | 69085 ± 40416* | 37,0* |
Chimi-DNA (i.m.) | 17.5 ± 4.1 | 8 | 108222 ± 50046 | 1,39 |
*Statistically significant compared with the control group (
To evaluate the effect of the proposed vaccine on reducing granuloma reactions, histological analysis was performed by digital morphometry. Seven days after the third immunization, mice were challenged with 25 cercariae. After 50 days of challenge infection, mice were sacrificed and liver samples were taken for histological analysis. Hematoxylin and eosin stained liver sections were then used to measure the size of individual granulomas. CHimi-DNA nanoparticles vaccination by oral route reduced liver granuloma area by 37,0% (Table
Cytokine experiments were performed using splenocyte cultures from individual mice immunized with CHimi-DNA nanoparticles. Production (IFN-
Cytokine profiles of mice immunized with CH-DNA nanoparticles. Splenocytes isolated from mice immunized with Chimi-DNA nanoparticles (a), CHimi-DNA (i.m.) (b), DNA (c), or CH (d) were assayed as IL-10 (A), IL-4 (B) and INF-
Schistosomiasis is a chronic debilitating parasitic disease that represents a major health problem in endemic areas, such as various parts of South America, Africa, and Southeast Asia [
In the present study, we found that it is possible to induce some arms of a protective immunity against experimental schistosomiasis in mice using CH and CHimi-DNA nanoparticles administered by oral route. This vaccination strategy offers many technical advantages, including a simple production process and the possibility of administration without the need of the use of needles, which facilitates administration mainly in development areas [
Electrophoretic retention of DNA was assessed for complexes prepared with increasing amounts of polymer. The modified polymer halts DNA mobility at the same N/P molar ratio (primary amines to plasmid DNA phosphate groups ratio) as nonmodified CH, which was found to be 2. Upon proving the ability of the modified polymers to complex DNA, it was important to evaluate their size and charge. For N/P molar ratios >3, no differences were observed in the behavior of CHimi-DNA or CH-DNA nanoparticles in terms of mean particle size and zeta potential. Large particles and polydisperse populations were observed only at lower N/P molar ratios. Other authors have also reported increased size at molar ratio 1 (the condition at which the zeta potential of the complexes is close to neutrality), which could be explained by the instability of the colloidal system. The dimension range referred to in the literature for CH-based complexes with DNA is quite wide and found to be strongly dependent on preparation conditions [
To evaluate the behavior of these nanoparticles in media at different pHs and at physiological temperature, DNA-CH complexes were prepared at the N/P molar ratio of 12 and 18 and submitted to SGF
After characterization, the immunization with nanoparticles was realized to investigate the effect of CH-DNA nanoparticles by oral delivery on immune response in mice and their potential to treat the immunopathological responses or prevent infection.
Our results demonstrated that the antipathological response induced against infection with cercariae is probably not related to the antibodies against rSmRho, because the assessment to the antibodies production by immunized mice showed low levels of rSmRho specific antibodies, although these levels had been significative at determined times during the period evaluated. On the other hand, the groups that showed the higher reduction in granuloma areas, CHimi-DNA (37%) and DNA (24.9%), were groups in which the formulation administered carried the plasmid DNA that codifies the SmRho protein. According to this, studies realized by other authors had suggested the involvement of this protein in process of maturation and egg deposition by
The modulation of immunopathological responses of
with rSmRho and also with SEA-stimulation. IL-10 plays a key regulatory role in facilitating the shift from a Th1 to Th2 response and preventing the development of severe pathology due to excessive Th1 and/or Th2 responses [
To determine whether CHimi-DNA nanoparticles conferred protection against
The lower rate of protection obtained by CHimi-DNA group can be explained by the number of CH nanoparticles in the formulation to be much less than the CH immunized group and also because the modified CH has a structure not so similar with the polysaccharides present in schistosoma tegument.
Thus, we showed that the candidate of vaccine based on CHimi-DNA nanoparticles is able to modulate the granuloma area, which represents the major pathological response in schistosomiasis, and therefore, it can be useful in preventing or reducing such injuries of the disease, mainly in underdeveloped countries. Furthermore, the results obtained from these works, although being preliminaries data, suggest an important role of CH in inducing protection against infection of
The authors declare that they have no conflict of interests.
This paper was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG/Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Pró-reitoria de Pesquisa da Universidade Federal de Minas Gerais. Work in the laboratory of Ana Paula Pêgo was supported by FEDER funds through the