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Immunopotentiating nano-chitosan as potent vaccine carter for efficacious prophylaxis of filarial antigens
International Journal of Biological Macromolecules 73 (2015) 131–137
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Immunopotentiating nano-chitosan as potent vaccine carter for efficacious prophylaxis of filarial antigens Balasubramaniyan Malathi a , Santhanam Mona b , Devasena Thiyagarajan a,∗ , Perumal Kaliraj b,∗ a b
Anna University, Guindy, Chennai 600025, Tamil Nadu, India Centre for Biotechnology, Anna University, Guindy, Chennai 600025, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 17 October 2014 Received in revised form 11 November 2014 Accepted 20 November 2014 Available online 26 November 2014 Keywords: Chitosan nanospheres Thioredoxin Abundant larval transcript-2 Lymphatic filariasis PBMC
a b s t r a c t Lymphatic filariasis (LF), a morbid vector-borne parasitic infection affects millions in tropical areas. Complete eradication can only be achieved by the development of a potent vaccine. Among the various filarial antigens that have been characterized, antigens Brugia malayi thioredoxin (TRX) and abundant larval transcript (ALT) have produced recognizable level of protection in Jirds, thereby evidenced to be good vaccine candidates. In this study an attempt was made to enhance their immunoprophylactic activity by encapsulating them in natural polysaccharide chitosan forming nanospheres (CN). High encapsulation efficiency for TRX (93%) in CN (TCN) and ALT-2 (90%) in CN (ACN) was achieved. Morphological studies confirmed the spherical and uniform distribution of nanospheres to be 220 nm. The electrostatic interaction between chitosan and the antigens were confirmed using differential scanning calorimetry and FT-IR. The study revealed the immunostimulatory property of chitosan providing enhanced level of proliferation for encapsulated antigens in peripheral blood mononuclear cells from endemic normal personals, at low concentration (TCN mean stimulation index (SI) = 4.23 ± 0.15 and ACN (SI) = 4.05 ± 0.33) compared to stimulation obtained by antigens alone. Hence, our study demonstrated that natural macromolecule derived CN can be used as efficacious immunostimulatory vaccine carter for LF thereby diminishing pathological sequel. © 2014 Elsevier B.V. All rights reserved.
1. Introduction A neglected tropical disease, human lymphatic filariasis (LF) is a debilitating parasitic infection standing second to malaria [1]. The primary etiology of this obnoxious disease is Wuchereia bancrofti and Brugia malayi, while a very small fraction of this infection is caused by Brugia timori. It mostly occurs in tropical and subtropical regions of world where mosquitoes thrive and act as a vector by transmitting these filarial worms [2]. It is an enervating parasitic infection that has affected around 160 million people in 83 countries with a potential risk of infecting billions of people [3]. There were several efforts to cope and cure LF related disabilities by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) that proved to be inadequate (WHO, 2013). The chief reason why control over LF has been eluding us is due to the complex, evolving
∗ Corresponding authors at: Anna University, Guindy, Chennai 600025, Tamil Nadu, India. Tel.: +91 044 22359151/ +91 44 22350772 E-mail addresses: [email protected], [email protected] (D. Thiyagarajan), [email protected] (P. Kaliraj). http://dx.doi.org/10.1016/j.ijbiomac.2014.11.014 0141-8130/© 2014 Elsevier B.V. All rights reserved.
and adapting lifestyle of parasites. In order to eradicate LF, the fortification against these infectious pathogens through vaccination would be a better alternate approach. Various filarial antigens like abundant larval trasnscript-2 (ALT-2), thioredoxin peroxidase (TPX), Venom allergen antigen homologue (VAH) and thioredoxin (TRX) have been identified that induces protective immunity against LF [4–7]. TRX is a protein, expressed throughout the life cycle of B. malayi and possesses antioxidant properties with conceivable part in the immune evasion mechanism [8]. TRX has shown 63% protection against murine model of filariasis with infective parasitic larvae L3 [9]. The highly stage specific ALT protein secretes abundantly in the L3 phase [10,11]. ALT plays a crucial role in modulating the host immune response and upholds infection through their immunomodulating action [4,7,12]. Due to these factors ALT is considered to be a potent vaccine candidate giving 75% protection among the normal populace [10,13]. When antigens are immunized directly, they are prone to rapid degradation which decreases its bioavailability, thereby leading to short lived and localized immune efficacy. This enigma can be overcome by providing nanocarriers to encapsulate these antigens
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which can act as adjuvants as well. These nanocarriers by creating a depot of antigens provide sustained release and thus infests protection in the in vivo environment [14,15]. Such nanocarriers can be synthesized using natural biomacromolecule chitosan, a linear incompletely N-deacetylated derivative of chitin which also has added advantages such as being nontoxic and biodegradable [16,17]. It aids in transmucosal absorption of peptides and proteins by loosening the tight junctions of the membranes [18,19]. Therefore, it reduces the rate of muco-ciliary clearance and aids in higher antigen availability [20–22]. Chitosan is also known to have elevated levels in the uptake of antigenpresenting cells (APC) [23]. Hence, there is an urgent need to enhance the bioavailability of the filarial antigens and to elevate the immune response that could be achieved by encapsulating them in chitosan nanospheres. Therefore, the aim of our study undertaken is to develop chitosan nanospheres (CN) encapsulating filarial immunoactive as a potent vaccine carrier.
2. Materials and methods 2.1. Production and purification of filarial antigen The Bm.TRX construct was over expressed as 20 kDa protein in E. coli (GJ1158) strain using NaCl induction and the recombinant protein was purified by ion-exchange chromatography using Q-sepharose (Amersham Pharmacia Biotech) under nondenaturing conditions [9]. Bm.ALT-2 protein was over expressed as 25 kDa protein in E. coli BL21 (DE3) strain using isopropyl-d-thiogalactopyranoside (IPTG) induction and purified by hydrophobic interaction chromatography using phenyl sepharose (GE Healthcare Bio-Sciences Ltd) [24]. The expressed proteins were confirmed with immunoblots using mouse anti-TRX sera and mouse anti-ALT2 sera.
2.2. Preparation of chitosan nanosphere Chitosan nanoparticles (CN) were prepared by ionotropic gelation method reported by Calvo et al. [25], with slight modification. The chitosan solution was prepared by dissolving high molecular weight chitosan in 2% aqueous acetic acid, while; the tripolyphosphate (TPP) solution of 1 mg/ml concentration was prepared using deionized water. The chitosan nanoparticles were formed when TPP solution was incorporated to chitosan solution under continuous stirring (chitosan:TPP ratio of 3:0.144 w/w) at room temperature. The nanoparticles were obtained by centrifuging at 8000 rpm for 30 min followed by washing twice with distilled water.
2.3. Loading efficiency of antigens in CN TRX loaded chitosan nanoparticles (TCN) and ALT-2 encapsulated chitosan nanoparticles (ACN) were formed by stirring the protein solution (1:3 w/w) along with chitosan solution for 30 min before incorporating TPP solution followed by centrifugation and washes. The entrapment efficiency was estimated indirectly by analysing the supernatant for antigen content and calculated using the following equation:
entrapment efficiency(%) = total antigen added − free antigen present in supernatant × 100 total antigen added
(1)
2.4. Evaluation of integrity of antigens after encapsulation The encapsulated antigens structural and functional integrities were analysed using immunoblot [26]. CN was dissolved in 20% (v/v) acetic acid and centrifuged at 8000 rpm for 30 min at 4 ◦ C to separate the antigens. The nature and size of thus obtained antigens were compared with pure antigens. 2.5. Characterization of chitosan nanoparticles 2.5.1. Size, zeta potential and morphology The particle size distribution was analysed using particle size analyser (PSA). The zeta potential of CN was analysed in demineralized water at neutral pH by Malvern 2000 zetasizer (Herrenberg, Germany). Surface morphology of the CN was examined using Scanning electron micrographs (SEM-Tescan Vega3). The TEM images were obtained using JEOL JEM-200CX at 60 kV. 2.5.2. Physicochemical characterization Differential scanning calorimetric analysis were carried out to characterize the thermal behaviour of CN and filarial antigen encapsulated CN. The DSC thermo-grams was recorded between 30 ◦ C and 400 ◦ C at a scan rate of 20 ◦ C/min using SII DSC 6220 EXSTAR apparatus. FT-IR analysis was carried out using Perkin-Elmer spectrum spectrometer for CN, TCN and ACN at scans/min ranging from 400 cm−1 to 4000 cm−1 . 2.6. In vitro release studies In order to evaluate the filarial antigen’s release, the encapsulated CN were suspended in phosphate buffer saline (PBS, pH 7.4) and then incubated at 37 ◦ C under mild agitation in different vials [27]. At predetermined time intervals, a vial was taken out and centrifuged at 8000 rpm for 30 min to separate the released antigens from encapsulated material. The released antigens were estimated using Bradford method. The time dependent release was determined using the following equation: % release =
concentration of antigen in the supernatant at time t total encapsulated antigen
× 100
(2)
2.7. Isolation of peripheral blood mononuclear cells (PBMCs) Human peripheral blood mononuclear cells (PBMCs) from healthy donors (Endemic normal) were isolated from the heparinized venous blood by gradient centrifugation over Lymphoprep (Nycomed Pharma AS, Oslo, Norway) [28]. The cells were washed, resuspended in RPMI 1640 media supplemented with 10% FBS and gentamicin (80 g/ml). Tryphan blue exclusion test for cell viability was also performed. 2.8. Cellular particle uptake in PBMCs PBMCs were seeded at a concentration of 0.5 × 106 cells in flat bottomed petri dish with sterile cover slips and incubated for 24 h at 37 ◦ C in presence of 5%CO2 . Fluorescein isothiocyanate (FITC) loaded CN at various time duration (5, 10 and 24 h)) were incubated with PBMC cells, then washed twice with PBS and fixed with 3.7% paraformaldehyde followed by wash and fixation with 4 ,6-diamidino-2-phenylindole (DAPI). The fixed cells were further imaged with ZEN CARL ZEISS confocal microscope.
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Fig. 1. Title: Validation of the structural and functional integrity of purified and encapsulated filarial recombinant protein. Legend: The structural integrity of the encapsulated antigens were compared with the naïve antigens through immunoblot. Molecular marker (lane1), naive rTRX (lane 2), encapsulated rTRX (lane 3), naïve ALT-2 (lane 4) and encapsulated ALT-2 (lane 5).
2.9. Immunostimulation effect on human PBMCs The PBMCs were seeded in 96-well tissue culture plates at a concentration of 0.2 × 106 cells/well in RPMI 1640. The cells were stimulated with TRX, ALT-2, TCN, ACN, CN at defined concentrations (0.1 to 25 g/ml) and media alone as unstimulated cells. ConA(1 g/ml) acted as positive control and CN served as control. The cultures were incubated for 72 h in humidified atmosphere at 37 ◦ C with 5% CO2 . After 72 h, the cell proliferation was measured by MTT assay (Cell Titer 96 R aqueous one solution cell proliferation assay, Promega, Madison, WI). Stimulation index (SI) was calculated using Eq. (3). Results were calculated by repeating the studies three times and expressed as mean SI ± SD. stimulation index (SI) =
absorbance of cells stimulated with antigen absorbance of unstimulated cells
(3)
2.10. Statistical analysis Statistical analyses of data were carried out using one way ANOVA with 95% confidence interval (P-value <0.05) followed by Tukey’s test for individual column comparison. In all circumstances, statistical analysis was conducted using GraphPad Prism software version5. 3. Results & discussion 3.1. Production and purification of filarial antigen The recombinant Bm.TRX without histidine tag expressed as 20 kDa protein was purified using ion-exchange chromatography. Bm.ALT-2 protein was expressed as 25 kDa protein and refined using HIC. The confirmation of the TRX and ALT-2 was done using immunoblot (Fig. 1) using mouse anti-TRX sera and mouse antiALT2 sera respectively. The molecular weights expressed are in correlation with the results of previous studies [9,24]. 3.2. Preparation of empty and antigen encapsulated chitosan nanospheres The chitosan nanoparticles were formed on addition of TPP solution at a very slow rate to the chitosan solution under continuous
stirring for 30 min. The formation of nano-particle was mainly due to electrostatic cross linking between the positively charged amino groups present in the polysaccharide chitosan and the negatively charged phosphate group present in the polyanionic TPP as shown in Fig. 2.1 [25]. The encapsulation efficiency of TRX was 93% and that of ALT-2 was 90%. High encapsulation has occurred due to the fact that the protein was stirred for 30 min allowing more exposure for electrostatic interaction before addition of TPP (Fig. 2.2). Also, molecular weight of protein plays an important role since molecular weight of protein and encapsulation efficiency are inversely proportional to each other. Chitosan of high molecular weight have long linear molecular chains which possess more expanse of functional group facilitating in the formation of complexes with acidic group of the proteins upon gelation with TPP groups [29]. 3.3. Evaluation of structural integrity of antigens after encapsulation The integrity of the encapsulated antigens was analysed using immunoblot along with purified antigens as shown in Fig. 1. The encapsulation did not alter the integrity or molecular weight of the antigens as it was similar to that of the pure antigens. The CN were prepared using ionotropic gelation, which is a mild technique not involving harsh organic solvents or mechanical shear. There is only reversible electrostatic interaction between the protein and chitosan therefore no solvent based harm or other undesirable side effects occur to the proteins [25]. 3.4. Characterization of chitosan nanoparticles The PSA (Fig. 3.1) and SEM (Fig. 3.2) depicts the uniform size distribution of CN having a mean diameter of 220 nm. The zeta potential which plays an important role in biological application of CN showed a positive value around 25 ± 16 mV. It increases the interaction of CN with negatively charged cell membranes, thereby increasing cellular uptake [30,31]. TEM image (Fig. 3.3) elucidates the spherical structure of the polymeric nanomaterial. The chitosan-antigen interaction was measured by observing the phase transition temperature of the carrier with and without the proteins. Fig. 4.1 shows the transitional temperature of empty chitosan carrier at 119 ◦ C. When TRX was loaded inside chitosan, a double endothermic peak at 114 ◦ C and 119 ◦ C
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Fig. 2. Title: Electrostatic interaction. Fig. 2 1-Legend: The picture shows the electrostatic interaction between positively charged amine group of chitosan and negatively charged TPP. Fig. 2 2-Legend: The picture shows the electrostatic interaction between the mine group of chitosan and oxygen atom of protein. There is also formation of electrostatic linkage between C O of protein and OH group of chitosan.
were observed. The double peak and shift of the peak to lower melting temperature can be attributed towards the strong interaction between polyelectrolytes of the encapsulated TRX and the chitosan amino groups [32,33]. While ALT-2 encapsulated chitosan has shift towards higher temperature giving endothermic peak at 130◦ . This shift can be attributed towards the ionic interaction between ALT-2 and chitosan. The isoelectric point (pI) of TRX and ALT-2 calculated using Schrödinger Software (2013 version) was found to be 5.276 and 4.06 respectively. The net charge of the amphoteric proteins depends on its pI and the pH of the buffer. At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge [34]. As both the antigens had pI below the pH (7.4) of the solutions during synthesis, they carried net negative charge. For all proteins; the farther the pH of its solution is from their pI the greater is their net charge [34]. ALT-2 has a lower pI than TRX, thereby causing it to have more negative charge. This leads to increased ionic activity between the negatively charged ALT2 antigens toward positively charged chitosan nanoparticles. This causes comparatively high stability of the chitosan-ALT complex to chitosan-TRX complex [32,33]. FT-IR spectra of CN, TCN and ACN are shown in Fig. 4.2. The spectra of CN contain broad peak at 3275 cm−1 representing an enhanced NH2 and OH band stretching, which are close to the values reported for chitosan nanospheres [35]. Previous
Fig. 3. Title: Characterization of synthesized chitosan nanospheres (CN). Legend:(1) Size evaluation of synthesized CN by PSA, displaying a mean diameter of 220 nm. (2) Confirmation of the size and uniform distribution by SEM. (3) Confirmation of spherical morphology by TEM.
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Fig. 5. Title: In vitro filarial antigen release (TRX and ALT-2) from encapsulated CN. Legend: Though there is sustained release of antigens in PBS pH 7.4 (mean ± SD, n = 3), the release of TRX was faster compared to the release of ALT-2.
molecular interactions between chitosan and the antigens. Knaul et al. [39] have stated that the interaction between the phosphoric and ammonium ion shows peaks around 1630 and 1534 cm−1 . We have also obtained similar peaks at 1637 and 1528 cm−1 , indicating the association of tripolyphosphoric groups with ammonium groups of chitosan thereby enhancing the inter and intra molecular interactions. The amide I and II peaks of the proteins alone forms at 1643 and 1547 cm−1 respectively [40]. The shift in our peak at 1645 and 1560 cm−1 for TCN and 1660 and 1553 cm−1 for ACN leads us to assume that the amide I and amide II groups of proteins forms linkage with phosphoric ion of TPP.
3.5. In vitro release studies The antigens were encapsulated in chitosan nanospheres and then utilized for release studies (Fig. 5) in PBS (pH 7.4). Though there is a burst release initially, a sustained release is observed in the later phase. TRX showed a rapid release upto 15% whereas, ALT showed less than 10% release in the same duration. The initial burst release of antigens is due to desorption of antigens loosely dispersed near the surface of the CN. Later there is gradual release of TRX (19%) and ALT (12%) antigens over 24 h. The slow gradual sustained release occurs primarily due to slow degradation of polymer and subsequent release of the protein. As the proteins have long molecular chain, it is difficult for them to diffuse through the chitosan surface pores in short duration of time [41]. Thus, it is evident that CN has high stability and gives sustained release.
3.6. Cellular particle uptake in PBMCs Fig. 4. Title: Physico-chemical characterization of empty and filarial antigen encapsulated CN. Legend: (1) DSC thermograms of CN (chitosan nanospheres), TCN (TRX encapsulated chitosan nanospheres) and ACN (ALT-2 encapsulated chitosan nanospheres). (2) FTIR analysis of CN (chitosan nanospheres), TCN (TRX encapsulated chitosan nanospheres) and ACN (ALT-2 encapsulated chitosan nanospheres).
reports reveal the possible formation of hydrogen bonding of NH2 group also [36,37]. Both chitosan and proteins contain C O and NH2 groups, therefore, there are possibilities for the formation of two types of hydrogen bonds. When antigens were encapsulated inside, the NH peak shifts to higher frequency, attributing to the formation of hydrogen bond between the amine hydrogen atoms of chitosan and oxygen atom of proteins and between C O of protein and OH group of chitosan. Molecular interaction between chitosan and the protein may also involve van der Waals interactions [38]. The peak at 2937 cm−1 in CN represents unreacted NH groups, which disappears in TCN and ACN indicating
The main requirement for stimulation of immune system is internalization of the antigen. Fig. 6 portrays the cellular uptake of CN by PBMCs. The confocal image analysis of PBMC along with CN enclosing FITC showed that the localization of the carrier is time reliant. Comparatively the internalization at 24 h of carrier was found to be higher in this study. As the carrier was transported into the cells, CN proves to be a promising delivery agent. The positive surface charge of chitosan along with smaller size enhances interaction with cellular membrane and its subsequent cellular uptake. Literature states the presence of electrostatic interaction between positively charged amine groups of chitosan and negatively charge cell membrane and hence modifying endocytosis process [30,31,42]. These interactions and the relatively smaller size of CN have led to higher potential of cellular conveyance. This time-dependent uptake shows the increased bioavailability of antigens when encapsulated in CN compared to that of naïve antigen alone.
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Fig. 6. Title: Confocal image of PBMCs incubated with CN particles. Fig. 6 Legend: Time dependent uptake of FITC loaded CN particles were observed for 24 h incubation with human PBMCs. The uptake was in the order 24 h > 10 h > 5 h.
Fig. 7. Title: Lymphoproliferation of human PBMCs using various filarial antigens. Fig. 7 Legend: Proliferation of EN PBMCs stimulated with TRX, ALT-2, CN, TCN and ACN. Con A was used as positive control. The data is represented as mean stimulation index (mean S.I) of 4 endemic normal individual’s ± SD. The dotted line drawn parallel to the X-axis is the cut off value (mean ± SD of CN control). The asterisks on top of TCN shows the significantly high value of it compared to cells stimulated with CN and TRX.
SI = 4.23 ± 0.15). In other case the ACN (mean SI = 4.05 ± 0.33) is on a par with ALT (mean SI = 4.01 ± 0.24). The TRX in carrier has comparatively higher release rate compared to ALT. This leads to increased exposure of TRX to PBMCs compared to ALT and thereby attributing the higher SI of TCN compared to ACN. The slow degradation of chitosan leads to sustained release of antigens in the media, thereby decreasing the count of stimulating antigens in the media. Therefore, the study shows that low concentration of encapsulated antigens can produce SI almost equal to or more compared to that obtained with high concentration of plain antigens. This evidences the adjuvant property of chitosan as well as its efficient carrier nature [43–46]. Thus nano-chitosan encapsulation of antigens can make filarial vaccine programme cost effective as low concentration of antigens is required to produce recognizable level of stimulation. Aditya et al. [47] also have ascertained the role of chitosan nanospheres in stimulating lymphocyte proliferation. These studies along with our results show a good indication for the usage of CN as resilient vaccine carrier as it can attain good level of stimulation at low concentration of immunoactives.
3.7. Immunostimulation effect on PBMCs
4. Conclusion
To study the lymphoproliferation of EN personal’s towards filarial antigens, their PBMCs were stimulated in vitro with TRX, ALT-2, TCN, ACN and CN. The proliferative responses are shown in Fig. 7. The positive control ConA induced significant proliferation of PBMCs collected from various EN subjects. The control CN had very low stimulation stating that the chitosan has no or very less effect on the cells. The proliferative response was significantly high (P < 0.05) for all the antigens when compared with CN (mean SI = 0.97 ± 0.16). A significant difference (P = 0.0116) was observed between TRX (mean SI = 3.56 ± 0.24) and TCN (mean
In this study, natural polysaccharide derived chitosan nanospheres were successfully formed by ionotropic gelation method having uniform distribution and spherical contour. The physicochemical characterization studies proved the formation of nanospheres and integration of filarial antigens TRX and ALT in them. The in vitro studies showed sustained release nature of the antigens thereby proving the stable nature of the carrier. The elevated proliferation of PBMCs with low concentration of antigens encapsulated in chitosan compared to stimulation with antigens alone evidences the adjuvant property of chitosan. These
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results showed that filarial antigens loaded CN can act as an ideal immunostimulatory carrier for human lymphatic Filariasis. Acknowledgments The authors are thankful to the Anna Centenary Research Fellowship, Anna University (Grant number CR/ACRF/Jan.2012/50), Tamilnadu, India, for their financial assistance. We gratefully acknowledge Christiana Immanuel for her support and suggestion during the work. We would like to sincerely thank the Directors, Centre for Biotechnology (CBT) and Centre for Nanoscience and Technology (CNST), Anna University, Chennai for financial and infrastructural support. References [1] E.A. Ottesen., Trop. Med. Int. Health 5 (2000) 591–594. [2] T. Supali., H. Wibowo, P. Ruckert, K. Fischer, I.S. Ismid, Y. Purnomo Djuardi, P. Fischer, Am. J. Trop. Med. Hyg. 66 (5) (2002) 560–565. [3] M.J. Taylor., A. Hoerauf, M. Bockarie, Lancet 376 (2010) 1175–1185. [4] J.E. Allen, J. Daub, D. Guilliano, A. McDonnell, M. Lizotte-Waniewski, D.W. Taylor, M. Blaxter, Infect. Immun. 68 (2000) 5454–5458. [5] S.B. Anand, V. Murugan, P.R. Prabhu, V. Anandharaman, M.V. Reddy, P. Kaliraj, Acta Trop. 107 (2008) 106–112. [6] S.B. Anand., M. Gnanasekar, M. Thangadurai, P.R. Prabhu, P. Kaliraj, K. Ramaswamy, Parasitol. Res. 101 (2007) 981–988. [7] A. Hoerauf., J. Satoguina, M. Saeftel, S. Specht, Parasite Immunol. 27 (2005) 417–429. [8] K. Kunchithapautham., B. Padmavathi, R.B. Narayanan, P. Kaliraj, A.L. Scott, Infect. Immun. 71 (7) (2003) 4119–4126. [9] J. Madhumathi., R.P. Prince, G. Anugraha, P. Kiran, D.N. Rao, M.V.R. Reddy, P. Kaliraj, Vaccine 28 (2010) 5038–5048. [10] W.F. Gregory., A.K. Atmadja, J.E. Allen, R.M. Maizels, Infect. Immun. 68 (2000) 4174–4179. [11] K.H. Porthouse., S.R. Chirgwin, S.U. Coleman, H.W. Taylor, T.R. Klei, Infect. Immun. 74 (4) (2006) 2366–2372. [12] N. Gomez-Escobar., W.F. Gregory, C. Britton, L. Murray, C. Corton, N. Hall, J. Daub, M.L. Blaxter, R.M. Maizels, Mol. Biochem. Parasitol. 125 (2002) 59–71. [13] M. Gnanasekar., K.V. Rao, Y.X. He, P.K. Mishra, T.B. Nutman, P. Kaliraj, K. Ramaswamy, Infect. Immun. 72 (2004) 4707–4715. [14] D.T. O’Hagan., E. De Gregorio, Drug Discovery Today 14 (2009) 541–551. [15] J. Panyam., V. Labhasetwar, Adv. Drug Delivery Rev. 55 (2003) 329–347. [16] R.A.A. Muzzarelli, Natural Chelating Polymers; Alginic Acid, Chitin and Chitosan, Pergamon Press, New York, NY, 1973, pp. 83. [17] J.P. Zikakis (Ed.), Chitin, Chitosan and Related Enzymes, Academic Press, Orlando, FL, 1984, p. XVII.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Immunopotentiating nano-chitosan as potent vaccine carter for efficacious prophylaxis of filarial antigens Balasubramaniyan Malathi a , Santhanam Mona b , Devasena Thiyagarajan a,∗ , Perumal Kaliraj b,∗ a b
Anna University, Guindy, Chennai 600025, Tamil Nadu, India Centre for Biotechnology, Anna University, Guindy, Chennai 600025, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 17 October 2014 Received in revised form 11 November 2014 Accepted 20 November 2014 Available online 26 November 2014 Keywords: Chitosan nanospheres Thioredoxin Abundant larval transcript-2 Lymphatic filariasis PBMC
a b s t r a c t Lymphatic filariasis (LF), a morbid vector-borne parasitic infection affects millions in tropical areas. Complete eradication can only be achieved by the development of a potent vaccine. Among the various filarial antigens that have been characterized, antigens Brugia malayi thioredoxin (TRX) and abundant larval transcript (ALT) have produced recognizable level of protection in Jirds, thereby evidenced to be good vaccine candidates. In this study an attempt was made to enhance their immunoprophylactic activity by encapsulating them in natural polysaccharide chitosan forming nanospheres (CN). High encapsulation efficiency for TRX (93%) in CN (TCN) and ALT-2 (90%) in CN (ACN) was achieved. Morphological studies confirmed the spherical and uniform distribution of nanospheres to be 220 nm. The electrostatic interaction between chitosan and the antigens were confirmed using differential scanning calorimetry and FT-IR. The study revealed the immunostimulatory property of chitosan providing enhanced level of proliferation for encapsulated antigens in peripheral blood mononuclear cells from endemic normal personals, at low concentration (TCN mean stimulation index (SI) = 4.23 ± 0.15 and ACN (SI) = 4.05 ± 0.33) compared to stimulation obtained by antigens alone. Hence, our study demonstrated that natural macromolecule derived CN can be used as efficacious immunostimulatory vaccine carter for LF thereby diminishing pathological sequel. © 2014 Elsevier B.V. All rights reserved.
1. Introduction A neglected tropical disease, human lymphatic filariasis (LF) is a debilitating parasitic infection standing second to malaria [1]. The primary etiology of this obnoxious disease is Wuchereia bancrofti and Brugia malayi, while a very small fraction of this infection is caused by Brugia timori. It mostly occurs in tropical and subtropical regions of world where mosquitoes thrive and act as a vector by transmitting these filarial worms [2]. It is an enervating parasitic infection that has affected around 160 million people in 83 countries with a potential risk of infecting billions of people [3]. There were several efforts to cope and cure LF related disabilities by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) that proved to be inadequate (WHO, 2013). The chief reason why control over LF has been eluding us is due to the complex, evolving
∗ Corresponding authors at: Anna University, Guindy, Chennai 600025, Tamil Nadu, India. Tel.: +91 044 22359151/ +91 44 22350772 E-mail addresses: [email protected], [email protected] (D. Thiyagarajan), [email protected] (P. Kaliraj). http://dx.doi.org/10.1016/j.ijbiomac.2014.11.014 0141-8130/© 2014 Elsevier B.V. All rights reserved.
and adapting lifestyle of parasites. In order to eradicate LF, the fortification against these infectious pathogens through vaccination would be a better alternate approach. Various filarial antigens like abundant larval trasnscript-2 (ALT-2), thioredoxin peroxidase (TPX), Venom allergen antigen homologue (VAH) and thioredoxin (TRX) have been identified that induces protective immunity against LF [4–7]. TRX is a protein, expressed throughout the life cycle of B. malayi and possesses antioxidant properties with conceivable part in the immune evasion mechanism [8]. TRX has shown 63% protection against murine model of filariasis with infective parasitic larvae L3 [9]. The highly stage specific ALT protein secretes abundantly in the L3 phase [10,11]. ALT plays a crucial role in modulating the host immune response and upholds infection through their immunomodulating action [4,7,12]. Due to these factors ALT is considered to be a potent vaccine candidate giving 75% protection among the normal populace [10,13]. When antigens are immunized directly, they are prone to rapid degradation which decreases its bioavailability, thereby leading to short lived and localized immune efficacy. This enigma can be overcome by providing nanocarriers to encapsulate these antigens
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which can act as adjuvants as well. These nanocarriers by creating a depot of antigens provide sustained release and thus infests protection in the in vivo environment [14,15]. Such nanocarriers can be synthesized using natural biomacromolecule chitosan, a linear incompletely N-deacetylated derivative of chitin which also has added advantages such as being nontoxic and biodegradable [16,17]. It aids in transmucosal absorption of peptides and proteins by loosening the tight junctions of the membranes [18,19]. Therefore, it reduces the rate of muco-ciliary clearance and aids in higher antigen availability [20–22]. Chitosan is also known to have elevated levels in the uptake of antigenpresenting cells (APC) [23]. Hence, there is an urgent need to enhance the bioavailability of the filarial antigens and to elevate the immune response that could be achieved by encapsulating them in chitosan nanospheres. Therefore, the aim of our study undertaken is to develop chitosan nanospheres (CN) encapsulating filarial immunoactive as a potent vaccine carrier.
2. Materials and methods 2.1. Production and purification of filarial antigen The Bm.TRX construct was over expressed as 20 kDa protein in E. coli (GJ1158) strain using NaCl induction and the recombinant protein was purified by ion-exchange chromatography using Q-sepharose (Amersham Pharmacia Biotech) under nondenaturing conditions [9]. Bm.ALT-2 protein was over expressed as 25 kDa protein in E. coli BL21 (DE3) strain using isopropyl-d-thiogalactopyranoside (IPTG) induction and purified by hydrophobic interaction chromatography using phenyl sepharose (GE Healthcare Bio-Sciences Ltd) [24]. The expressed proteins were confirmed with immunoblots using mouse anti-TRX sera and mouse anti-ALT2 sera.
2.2. Preparation of chitosan nanosphere Chitosan nanoparticles (CN) were prepared by ionotropic gelation method reported by Calvo et al. [25], with slight modification. The chitosan solution was prepared by dissolving high molecular weight chitosan in 2% aqueous acetic acid, while; the tripolyphosphate (TPP) solution of 1 mg/ml concentration was prepared using deionized water. The chitosan nanoparticles were formed when TPP solution was incorporated to chitosan solution under continuous stirring (chitosan:TPP ratio of 3:0.144 w/w) at room temperature. The nanoparticles were obtained by centrifuging at 8000 rpm for 30 min followed by washing twice with distilled water.
2.3. Loading efficiency of antigens in CN TRX loaded chitosan nanoparticles (TCN) and ALT-2 encapsulated chitosan nanoparticles (ACN) were formed by stirring the protein solution (1:3 w/w) along with chitosan solution for 30 min before incorporating TPP solution followed by centrifugation and washes. The entrapment efficiency was estimated indirectly by analysing the supernatant for antigen content and calculated using the following equation:
entrapment efficiency(%) = total antigen added − free antigen present in supernatant × 100 total antigen added
(1)
2.4. Evaluation of integrity of antigens after encapsulation The encapsulated antigens structural and functional integrities were analysed using immunoblot [26]. CN was dissolved in 20% (v/v) acetic acid and centrifuged at 8000 rpm for 30 min at 4 ◦ C to separate the antigens. The nature and size of thus obtained antigens were compared with pure antigens. 2.5. Characterization of chitosan nanoparticles 2.5.1. Size, zeta potential and morphology The particle size distribution was analysed using particle size analyser (PSA). The zeta potential of CN was analysed in demineralized water at neutral pH by Malvern 2000 zetasizer (Herrenberg, Germany). Surface morphology of the CN was examined using Scanning electron micrographs (SEM-Tescan Vega3). The TEM images were obtained using JEOL JEM-200CX at 60 kV. 2.5.2. Physicochemical characterization Differential scanning calorimetric analysis were carried out to characterize the thermal behaviour of CN and filarial antigen encapsulated CN. The DSC thermo-grams was recorded between 30 ◦ C and 400 ◦ C at a scan rate of 20 ◦ C/min using SII DSC 6220 EXSTAR apparatus. FT-IR analysis was carried out using Perkin-Elmer spectrum spectrometer for CN, TCN and ACN at scans/min ranging from 400 cm−1 to 4000 cm−1 . 2.6. In vitro release studies In order to evaluate the filarial antigen’s release, the encapsulated CN were suspended in phosphate buffer saline (PBS, pH 7.4) and then incubated at 37 ◦ C under mild agitation in different vials [27]. At predetermined time intervals, a vial was taken out and centrifuged at 8000 rpm for 30 min to separate the released antigens from encapsulated material. The released antigens were estimated using Bradford method. The time dependent release was determined using the following equation: % release =
concentration of antigen in the supernatant at time t total encapsulated antigen
× 100
(2)
2.7. Isolation of peripheral blood mononuclear cells (PBMCs) Human peripheral blood mononuclear cells (PBMCs) from healthy donors (Endemic normal) were isolated from the heparinized venous blood by gradient centrifugation over Lymphoprep (Nycomed Pharma AS, Oslo, Norway) [28]. The cells were washed, resuspended in RPMI 1640 media supplemented with 10% FBS and gentamicin (80 g/ml). Tryphan blue exclusion test for cell viability was also performed. 2.8. Cellular particle uptake in PBMCs PBMCs were seeded at a concentration of 0.5 × 106 cells in flat bottomed petri dish with sterile cover slips and incubated for 24 h at 37 ◦ C in presence of 5%CO2 . Fluorescein isothiocyanate (FITC) loaded CN at various time duration (5, 10 and 24 h)) were incubated with PBMC cells, then washed twice with PBS and fixed with 3.7% paraformaldehyde followed by wash and fixation with 4 ,6-diamidino-2-phenylindole (DAPI). The fixed cells were further imaged with ZEN CARL ZEISS confocal microscope.
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Fig. 1. Title: Validation of the structural and functional integrity of purified and encapsulated filarial recombinant protein. Legend: The structural integrity of the encapsulated antigens were compared with the naïve antigens through immunoblot. Molecular marker (lane1), naive rTRX (lane 2), encapsulated rTRX (lane 3), naïve ALT-2 (lane 4) and encapsulated ALT-2 (lane 5).
2.9. Immunostimulation effect on human PBMCs The PBMCs were seeded in 96-well tissue culture plates at a concentration of 0.2 × 106 cells/well in RPMI 1640. The cells were stimulated with TRX, ALT-2, TCN, ACN, CN at defined concentrations (0.1 to 25 g/ml) and media alone as unstimulated cells. ConA(1 g/ml) acted as positive control and CN served as control. The cultures were incubated for 72 h in humidified atmosphere at 37 ◦ C with 5% CO2 . After 72 h, the cell proliferation was measured by MTT assay (Cell Titer 96 R aqueous one solution cell proliferation assay, Promega, Madison, WI). Stimulation index (SI) was calculated using Eq. (3). Results were calculated by repeating the studies three times and expressed as mean SI ± SD. stimulation index (SI) =
absorbance of cells stimulated with antigen absorbance of unstimulated cells
(3)
2.10. Statistical analysis Statistical analyses of data were carried out using one way ANOVA with 95% confidence interval (P-value <0.05) followed by Tukey’s test for individual column comparison. In all circumstances, statistical analysis was conducted using GraphPad Prism software version5. 3. Results & discussion 3.1. Production and purification of filarial antigen The recombinant Bm.TRX without histidine tag expressed as 20 kDa protein was purified using ion-exchange chromatography. Bm.ALT-2 protein was expressed as 25 kDa protein and refined using HIC. The confirmation of the TRX and ALT-2 was done using immunoblot (Fig. 1) using mouse anti-TRX sera and mouse antiALT2 sera respectively. The molecular weights expressed are in correlation with the results of previous studies [9,24]. 3.2. Preparation of empty and antigen encapsulated chitosan nanospheres The chitosan nanoparticles were formed on addition of TPP solution at a very slow rate to the chitosan solution under continuous
stirring for 30 min. The formation of nano-particle was mainly due to electrostatic cross linking between the positively charged amino groups present in the polysaccharide chitosan and the negatively charged phosphate group present in the polyanionic TPP as shown in Fig. 2.1 [25]. The encapsulation efficiency of TRX was 93% and that of ALT-2 was 90%. High encapsulation has occurred due to the fact that the protein was stirred for 30 min allowing more exposure for electrostatic interaction before addition of TPP (Fig. 2.2). Also, molecular weight of protein plays an important role since molecular weight of protein and encapsulation efficiency are inversely proportional to each other. Chitosan of high molecular weight have long linear molecular chains which possess more expanse of functional group facilitating in the formation of complexes with acidic group of the proteins upon gelation with TPP groups [29]. 3.3. Evaluation of structural integrity of antigens after encapsulation The integrity of the encapsulated antigens was analysed using immunoblot along with purified antigens as shown in Fig. 1. The encapsulation did not alter the integrity or molecular weight of the antigens as it was similar to that of the pure antigens. The CN were prepared using ionotropic gelation, which is a mild technique not involving harsh organic solvents or mechanical shear. There is only reversible electrostatic interaction between the protein and chitosan therefore no solvent based harm or other undesirable side effects occur to the proteins [25]. 3.4. Characterization of chitosan nanoparticles The PSA (Fig. 3.1) and SEM (Fig. 3.2) depicts the uniform size distribution of CN having a mean diameter of 220 nm. The zeta potential which plays an important role in biological application of CN showed a positive value around 25 ± 16 mV. It increases the interaction of CN with negatively charged cell membranes, thereby increasing cellular uptake [30,31]. TEM image (Fig. 3.3) elucidates the spherical structure of the polymeric nanomaterial. The chitosan-antigen interaction was measured by observing the phase transition temperature of the carrier with and without the proteins. Fig. 4.1 shows the transitional temperature of empty chitosan carrier at 119 ◦ C. When TRX was loaded inside chitosan, a double endothermic peak at 114 ◦ C and 119 ◦ C
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Fig. 2. Title: Electrostatic interaction. Fig. 2 1-Legend: The picture shows the electrostatic interaction between positively charged amine group of chitosan and negatively charged TPP. Fig. 2 2-Legend: The picture shows the electrostatic interaction between the mine group of chitosan and oxygen atom of protein. There is also formation of electrostatic linkage between C O of protein and OH group of chitosan.
were observed. The double peak and shift of the peak to lower melting temperature can be attributed towards the strong interaction between polyelectrolytes of the encapsulated TRX and the chitosan amino groups [32,33]. While ALT-2 encapsulated chitosan has shift towards higher temperature giving endothermic peak at 130◦ . This shift can be attributed towards the ionic interaction between ALT-2 and chitosan. The isoelectric point (pI) of TRX and ALT-2 calculated using Schrödinger Software (2013 version) was found to be 5.276 and 4.06 respectively. The net charge of the amphoteric proteins depends on its pI and the pH of the buffer. At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge [34]. As both the antigens had pI below the pH (7.4) of the solutions during synthesis, they carried net negative charge. For all proteins; the farther the pH of its solution is from their pI the greater is their net charge [34]. ALT-2 has a lower pI than TRX, thereby causing it to have more negative charge. This leads to increased ionic activity between the negatively charged ALT2 antigens toward positively charged chitosan nanoparticles. This causes comparatively high stability of the chitosan-ALT complex to chitosan-TRX complex [32,33]. FT-IR spectra of CN, TCN and ACN are shown in Fig. 4.2. The spectra of CN contain broad peak at 3275 cm−1 representing an enhanced NH2 and OH band stretching, which are close to the values reported for chitosan nanospheres [35]. Previous
Fig. 3. Title: Characterization of synthesized chitosan nanospheres (CN). Legend:(1) Size evaluation of synthesized CN by PSA, displaying a mean diameter of 220 nm. (2) Confirmation of the size and uniform distribution by SEM. (3) Confirmation of spherical morphology by TEM.
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Fig. 5. Title: In vitro filarial antigen release (TRX and ALT-2) from encapsulated CN. Legend: Though there is sustained release of antigens in PBS pH 7.4 (mean ± SD, n = 3), the release of TRX was faster compared to the release of ALT-2.
molecular interactions between chitosan and the antigens. Knaul et al. [39] have stated that the interaction between the phosphoric and ammonium ion shows peaks around 1630 and 1534 cm−1 . We have also obtained similar peaks at 1637 and 1528 cm−1 , indicating the association of tripolyphosphoric groups with ammonium groups of chitosan thereby enhancing the inter and intra molecular interactions. The amide I and II peaks of the proteins alone forms at 1643 and 1547 cm−1 respectively [40]. The shift in our peak at 1645 and 1560 cm−1 for TCN and 1660 and 1553 cm−1 for ACN leads us to assume that the amide I and amide II groups of proteins forms linkage with phosphoric ion of TPP.
3.5. In vitro release studies The antigens were encapsulated in chitosan nanospheres and then utilized for release studies (Fig. 5) in PBS (pH 7.4). Though there is a burst release initially, a sustained release is observed in the later phase. TRX showed a rapid release upto 15% whereas, ALT showed less than 10% release in the same duration. The initial burst release of antigens is due to desorption of antigens loosely dispersed near the surface of the CN. Later there is gradual release of TRX (19%) and ALT (12%) antigens over 24 h. The slow gradual sustained release occurs primarily due to slow degradation of polymer and subsequent release of the protein. As the proteins have long molecular chain, it is difficult for them to diffuse through the chitosan surface pores in short duration of time [41]. Thus, it is evident that CN has high stability and gives sustained release.
3.6. Cellular particle uptake in PBMCs Fig. 4. Title: Physico-chemical characterization of empty and filarial antigen encapsulated CN. Legend: (1) DSC thermograms of CN (chitosan nanospheres), TCN (TRX encapsulated chitosan nanospheres) and ACN (ALT-2 encapsulated chitosan nanospheres). (2) FTIR analysis of CN (chitosan nanospheres), TCN (TRX encapsulated chitosan nanospheres) and ACN (ALT-2 encapsulated chitosan nanospheres).
reports reveal the possible formation of hydrogen bonding of NH2 group also [36,37]. Both chitosan and proteins contain C O and NH2 groups, therefore, there are possibilities for the formation of two types of hydrogen bonds. When antigens were encapsulated inside, the NH peak shifts to higher frequency, attributing to the formation of hydrogen bond between the amine hydrogen atoms of chitosan and oxygen atom of proteins and between C O of protein and OH group of chitosan. Molecular interaction between chitosan and the protein may also involve van der Waals interactions [38]. The peak at 2937 cm−1 in CN represents unreacted NH groups, which disappears in TCN and ACN indicating
The main requirement for stimulation of immune system is internalization of the antigen. Fig. 6 portrays the cellular uptake of CN by PBMCs. The confocal image analysis of PBMC along with CN enclosing FITC showed that the localization of the carrier is time reliant. Comparatively the internalization at 24 h of carrier was found to be higher in this study. As the carrier was transported into the cells, CN proves to be a promising delivery agent. The positive surface charge of chitosan along with smaller size enhances interaction with cellular membrane and its subsequent cellular uptake. Literature states the presence of electrostatic interaction between positively charged amine groups of chitosan and negatively charge cell membrane and hence modifying endocytosis process [30,31,42]. These interactions and the relatively smaller size of CN have led to higher potential of cellular conveyance. This time-dependent uptake shows the increased bioavailability of antigens when encapsulated in CN compared to that of naïve antigen alone.
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Fig. 6. Title: Confocal image of PBMCs incubated with CN particles. Fig. 6 Legend: Time dependent uptake of FITC loaded CN particles were observed for 24 h incubation with human PBMCs. The uptake was in the order 24 h > 10 h > 5 h.
Fig. 7. Title: Lymphoproliferation of human PBMCs using various filarial antigens. Fig. 7 Legend: Proliferation of EN PBMCs stimulated with TRX, ALT-2, CN, TCN and ACN. Con A was used as positive control. The data is represented as mean stimulation index (mean S.I) of 4 endemic normal individual’s ± SD. The dotted line drawn parallel to the X-axis is the cut off value (mean ± SD of CN control). The asterisks on top of TCN shows the significantly high value of it compared to cells stimulated with CN and TRX.
SI = 4.23 ± 0.15). In other case the ACN (mean SI = 4.05 ± 0.33) is on a par with ALT (mean SI = 4.01 ± 0.24). The TRX in carrier has comparatively higher release rate compared to ALT. This leads to increased exposure of TRX to PBMCs compared to ALT and thereby attributing the higher SI of TCN compared to ACN. The slow degradation of chitosan leads to sustained release of antigens in the media, thereby decreasing the count of stimulating antigens in the media. Therefore, the study shows that low concentration of encapsulated antigens can produce SI almost equal to or more compared to that obtained with high concentration of plain antigens. This evidences the adjuvant property of chitosan as well as its efficient carrier nature [43–46]. Thus nano-chitosan encapsulation of antigens can make filarial vaccine programme cost effective as low concentration of antigens is required to produce recognizable level of stimulation. Aditya et al. [47] also have ascertained the role of chitosan nanospheres in stimulating lymphocyte proliferation. These studies along with our results show a good indication for the usage of CN as resilient vaccine carrier as it can attain good level of stimulation at low concentration of immunoactives.
3.7. Immunostimulation effect on PBMCs
4. Conclusion
To study the lymphoproliferation of EN personal’s towards filarial antigens, their PBMCs were stimulated in vitro with TRX, ALT-2, TCN, ACN and CN. The proliferative responses are shown in Fig. 7. The positive control ConA induced significant proliferation of PBMCs collected from various EN subjects. The control CN had very low stimulation stating that the chitosan has no or very less effect on the cells. The proliferative response was significantly high (P < 0.05) for all the antigens when compared with CN (mean SI = 0.97 ± 0.16). A significant difference (P = 0.0116) was observed between TRX (mean SI = 3.56 ± 0.24) and TCN (mean
In this study, natural polysaccharide derived chitosan nanospheres were successfully formed by ionotropic gelation method having uniform distribution and spherical contour. The physicochemical characterization studies proved the formation of nanospheres and integration of filarial antigens TRX and ALT in them. The in vitro studies showed sustained release nature of the antigens thereby proving the stable nature of the carrier. The elevated proliferation of PBMCs with low concentration of antigens encapsulated in chitosan compared to stimulation with antigens alone evidences the adjuvant property of chitosan. These
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results showed that filarial antigens loaded CN can act as an ideal immunostimulatory carrier for human lymphatic Filariasis. Acknowledgments The authors are thankful to the Anna Centenary Research Fellowship, Anna University (Grant number CR/ACRF/Jan.2012/50), Tamilnadu, India, for their financial assistance. We gratefully acknowledge Christiana Immanuel for her support and suggestion during the work. We would like to sincerely thank the Directors, Centre for Biotechnology (CBT) and Centre for Nanoscience and Technology (CNST), Anna University, Chennai for financial and infrastructural support. References [1] E.A. Ottesen., Trop. Med. Int. Health 5 (2000) 591–594. [2] T. Supali., H. Wibowo, P. Ruckert, K. Fischer, I.S. Ismid, Y. Purnomo Djuardi, P. Fischer, Am. J. Trop. Med. Hyg. 66 (5) (2002) 560–565. [3] M.J. Taylor., A. Hoerauf, M. Bockarie, Lancet 376 (2010) 1175–1185. [4] J.E. Allen, J. Daub, D. Guilliano, A. McDonnell, M. Lizotte-Waniewski, D.W. Taylor, M. Blaxter, Infect. Immun. 68 (2000) 5454–5458. [5] S.B. Anand, V. Murugan, P.R. Prabhu, V. Anandharaman, M.V. Reddy, P. Kaliraj, Acta Trop. 107 (2008) 106–112. [6] S.B. Anand., M. Gnanasekar, M. Thangadurai, P.R. Prabhu, P. Kaliraj, K. Ramaswamy, Parasitol. Res. 101 (2007) 981–988. [7] A. Hoerauf., J. Satoguina, M. Saeftel, S. Specht, Parasite Immunol. 27 (2005) 417–429. [8] K. Kunchithapautham., B. Padmavathi, R.B. Narayanan, P. Kaliraj, A.L. Scott, Infect. Immun. 71 (7) (2003) 4119–4126. [9] J. Madhumathi., R.P. Prince, G. Anugraha, P. Kiran, D.N. Rao, M.V.R. Reddy, P. Kaliraj, Vaccine 28 (2010) 5038–5048. [10] W.F. Gregory., A.K. Atmadja, J.E. Allen, R.M. Maizels, Infect. Immun. 68 (2000) 4174–4179. [11] K.H. Porthouse., S.R. Chirgwin, S.U. Coleman, H.W. Taylor, T.R. Klei, Infect. Immun. 74 (4) (2006) 2366–2372. [12] N. Gomez-Escobar., W.F. Gregory, C. Britton, L. Murray, C. Corton, N. Hall, J. Daub, M.L. Blaxter, R.M. Maizels, Mol. Biochem. Parasitol. 125 (2002) 59–71. [13] M. Gnanasekar., K.V. Rao, Y.X. He, P.K. Mishra, T.B. Nutman, P. Kaliraj, K. Ramaswamy, Infect. Immun. 72 (2004) 4707–4715. [14] D.T. O’Hagan., E. De Gregorio, Drug Discovery Today 14 (2009) 541–551. [15] J. Panyam., V. Labhasetwar, Adv. Drug Delivery Rev. 55 (2003) 329–347. [16] R.A.A. Muzzarelli, Natural Chelating Polymers; Alginic Acid, Chitin and Chitosan, Pergamon Press, New York, NY, 1973, pp. 83. [17] J.P. Zikakis (Ed.), Chitin, Chitosan and Related Enzymes, Academic Press, Orlando, FL, 1984, p. XVII.
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