European Journal of Pharmacology 747 (2015) 174–180
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Biodegradable poly-l-lactide based microparticles as controlled release delivery system for filarial vaccine candidate antigens Gandhirajan Anugraha, Jayaprakasam Madhumathi, Parasurama Jawaharlal Jeya Prita, Perumal Kaliraj n Centre for Biotechnology, Anna University, Chennai 600025, India
art ic l e i nf o
a b s t r a c t
Article history: Received 3 October 2014 Received in revised form 2 December 2014 Accepted 5 December 2014 Available online 13 December 2014
Modern recombinant vaccines are less immunogenic than conventional vaccines which require adjuvants to enhance the effect of a vaccine. Alum is being used as a standard adjuvant for protein based vaccines to augment immune response in several diseases. However, the problem associated with alum is it requires multiple doses at specific time intervals to achieve the adequate level of immunity. Currently the adjuvanticity of Poly-l-lactide microparticles as single dose immunization was explored to overcome multiple immunization and reported to be effective for several diseases. In this regard we adsorbed filarial recombinant chimeric multivalent vaccine candidates such as TV and FEP on to PLA by double emulsion method and analyzed the characterization of PLA encapsulated microparticles and evaluated its immune responses in mice. The efficacy of single dose of PLA encapsulated proteins was investigated in comparison with single dose of alum or protein alone. In mice, single dose of PLA encapsulated antigens such as TV and FEP elicited significantly high antibody titer of 50,000 and 64,000 respectively than single dose of alum adsorbed TV/FEP (6000/9000) and single dose of protein TV/FEP (3000/4000) alone. Further PLA encapsulated antigens induced higher levels of cellular proliferation together with significant (Po 0.0001) levels of cytokine response [PLA-TV induced high levels of IL-4 (Th2) and IFN-γ (Th1) cytokines whereas PLA-FEP showed high levels of IL-5(Th2) and IFN-γ (Th1)] indicating a balanced response elicited by PLA antigens. Overall strong humoral and cellular responses were observed for PLA encapsulated antigens compared with single dose of alum adsorbed or protein alone. & 2014 Elsevier B.V. All rights reserved.
Keywords: Lymphatic filariasis Controlled release delivery system Poly-l-lactide Microsphere Adjuvants Immune response Chemical compounds studied in this article: Poly-l-lactide (PLA) Dichloromethane Polyvinyl alcohol (PVA) Acetonitrile
1. Introduction Lymphatic Filariasis, a mosquito borne parasitic infection caused by three closely related nematode worms, Wuchereria bancrofti, Brugia malayi and Brugia timori which damage the lymphatic system leading to painful swelling and disfigurement. Filariasis is the second largest cause of permanent and long-term disability in the world (Gyapong et al., 2005). It has a major social and economic impact in many tropical and subtropical countries. Current antifilarial drugs and exposure control measures have several limitations (Bockarie and Deb, 2010; Hoerauf et al., 2011; Taylor et al., 2010) and hence there is a need for additional strategy for elimination of lymphatic filariasis. In recent times, several recombinant vaccine candidate antigens have been reported to elicit promising results (Thirugnanam et al., 2007; Vanam et al., 2009a; Anand et al., 2008, 2011; Dakshinamoorthy et al., 2013)
n
Corresponding author. Tel.: þ 91 44 22350772; fax: þ 91 44 22352642. E-mail address: [email protected] (P. Kaliraj).
http://dx.doi.org/10.1016/j.ejphar.2014.12.004 0014-2999/& 2014 Elsevier B.V. All rights reserved.
which provided an excellent hope for developing vaccine against lymphatic filariasis. Although the recombinant protein vaccines elicited significant protection, they require adjuvant to induce optimum immune response. Currently, alum (U.S. FDA approved adjuvant) is widely used to increase immune response for protein based vaccines (Schmidt et al., 2007). However, multiple injections at specific time intervals are the general requirements to achieve the adequate level of immunity for alum adsorbed antigens, moreover it is a poor stimulator of cellular (Th1) immune responses, which are also important for protection (Schmidt et al., 2007; Petrovsky and Aguilar, 2004). In developing countries, limited access to medical care, lack of awareness and of floating population plays a crucial role in following the multiple immunization coverage to obtain desired response. Hence, an injectable formulation should be designed to ensure long-lasting immunity in a single dose vaccine which releases an antigen in a controlled manner as booster dose would be an improved strategy for immunization coverage. Currently, biodegradable microparticles based on poly-l-lactide (PLA) an FDA-approved polymer, is used for controlled-release
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delivery of protein-based vaccine antigens. The role of PLA as adjuvant has been reported by several investigators (Coombes et al., 1999; Gupta et al., 1998; Witschi and Doelker, 1998) and was reported to elicit strong and long-lasting immune response in single immunization. PLA is advantageous over other FDA approved polymer e.g. Poly(lactide-co-glycolide) (PLGA) because of its hydrophobicity and very low surface tension which are essential for efficient antigen adsorption (Absolom et al., 1987; Coombes et al., 1996) and its particulate form helps for more efficient antigen delivery to dendritic cells (Lavelle et al., 1999). These polymers undergo hydrolysis when injected into the body and forming biologically compatible and metabolizable moieties (lactic acid) due to their polyester nature. Finally they are removed from the body through the citric acid cycle. Recently, we have reported protective efficacy of filarial recombinant chimeric multivalent vaccine candidate namely TV (a 39 kDa fusion protein which comprising two filarial key vaccine candidate antigens such as Thioredoxin (TRX) and Venom Allergen Homologue (VAH)] (Anugraha et al., 2013) and another recombinant chimeric mutiepitope vaccine candidate such as Filarial epitope protein (FEP). FEP, a multi epitope protein (synthesized and expressed as 25 kDa protein) comprising immunodominant epitopes from multistage antigens such as Thioredoxin (TRX) (Madhumathi et al., 2010a), Transglutaminase (TGA) (Vanam et al., 2009b; Madhumathi et al., unpublished data), and Abundant Larval Transcript-2 (ALT-2) (Gregory et al., 1997; Madhumathi et al., 2010b, 2010c) and proved as potential vaccine candidate (Anugraha et al., unpublished data). In this present study we proposed to explore the use of PLA as a delivery system for filarial antigens and characterized the effective immune response for a single administration of PLA encapsulated antigens. To analyze this possibility adsorption of the recombinant Fusion protein (TV) and Filarial epitope protein (FEP) on to PLA and the immune responses of the same were studied in comparison with single dose of alum adjuvant or protein alone in mice model.
2. Materials and methods 2.1. Preparation and characterization of polymer particles The poly lactide (M.W. 103 kDa) (Sigma-Aldrich, Bangalore, India) microparticles were prepared using double emulsion (w/o/w) solvent evaporation method as described by Katare and group (Katare et al., 2005) with optimized modifications. Briefly, recombinant proteins (TV and FEP) were dissolved in 0.5 ml of an aqueous solution of 10% (w/v) polyvinyl alcohol (M.W. 30 to 70 kDa; 87–90% hydrolyzed). This primary aqueous phase was vigorously mixed through sonication, with an oily phase consisting of 250 mg of PLA dissolved in 5 ml of dichloromethane (50 mg/ml of dichloromethane). The resultant waterin-oil emulsion was then added, during vigorous agitation, to 75 ml of 5% (w/v) polyvinyl alcohol. The microparticles formed through overnight solvent evaporation in a magnetic stirrer were washed and harvested by centrifugation (40,695g, 20 min), and lyophilized to obtain the form of free-flowing powder. Size and surface morphology of the particles were analyzed using scanning electron microscope (Carl Zeiss, Germany). 2.2. Estimation of protein encapsulation efficiency in microparticles To estimate the protein content of particles, accurately weighed particles were dissolved in acetonitrile to solubilize the polymer (10 mg/ml). The suspension was centrifuged at 9037g for 10 min and the resultant pellet was dried at 37 1C. After the addition and incubation (1 h at 37 1C) of 100 μl PBS buffer to the pellete, the reaction mixture was centrifuged at 9037g for 10 min and the
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supernatant were collected. The procedure was repeated with 100 μl of 0.1 M NaOH. The protein content of the supernatant was estimated by BCA (Bicinchoninic acid) assay (Smith et al., 1985) which is a more sensitive assay for estimation. 10 μl of protein sample was added to 190 μl of BCA working reagent (Pierce, Rockford, USA) and incubated at 37 1C, in a dark room for 30 min and the absorbance was measured at 540–590 nm. The encapsulation efficiency was calculated as the percentage weight of antigen per unit weight of polymer (μg antigen/mg of PLA particles). 2.3. In vitro release assay from microparticles The release of proteins from microparticles was determined by incubation of accurately weighed particles (approx. 10 mg) in 100 ml PBS buffer at 37 1C in continuous shaking. The suspension was centrifuged and the supernatant was removed for analysis. At weekly intervals fresh PBS buffer (100 ml) was added to the pellet and resuspended by gentle vortexing and returned to the shaker. The process was repeated for up to 3 months. Released samples were estimated by BCA method as described earlier. 2.4. Protein stability study The integrity of the antigens (TV and FEP) was analyzed in SDSPAGE after encapsulation in comparison with purified proteins (TV and FEP). The known amount of antigen encapsulated with PLA microparticles were incubated (separately) with 0.1% (w/v) SDSphosphate buffered saline (PBS; 0.01 M; pH 7.4) at 37 1C for 3 h with gentle shaking and then centrifuged at 18,074g for 25 min at 4–8 1C (Saini et al., 2011). The supernatants were checked in SDSPAGE gel. 2.5. Immunological studies in mice 2.5.1. Immunization protocol Six to eight weeks old BALB/c (H-2d) mice were purchased from King Institute, Chennai, Tamilnadu, India. All the experiments were followed as per ‘Indian Animal Ethics Committee’ regulations. For PLA encapsulated antigen (mentioned as PLA-TV and PLA-FEP) groups (5 mice/group), were immunized with 50 μg (an optimum concentration for microsphere encapsulated proteins (Madhumathi et al., 2010a) of protein equivalent in microspheres suspended in 100 μl of PBS buffer. Other groups were immunized with 50 μg of TV and FEP protein mixed with an equal volume of alum (Sigma-Aldrich, Bangalore, India) (mentioned as AlumþTV and AlumþFEP) and as well as antigens without alum [TV and FEP in Phosphate buffered saline (PBS) alone]. Three control groups were kept for this study. One group received alum alone, another received PBS alone and the third group with PLA microparticles alone. All groups were injected with single dose of respective antigens via subcutaneous route. A small amount of blood (approximately 50 μl) was taken from tail vein of immunized animals at weekly intervals up to three months since microsphere encapsulation provides long-lasting immunity due to sustained release of antigen and serum were separated to analyse the antibody levels by ELISA. 2.5.2. Analysis of total IgG and Isotype antibody levels Protein specific IgG levels in the sera were observed by ELISA. The 96-well microtiter plates (Nunc, Maxisorp, Nalge Nunc International, Denmark) were used and each wells were coated with 100 ng of TV and FEP proteins prepared in 100 μl of coating buffer separately (Prince et al., 2013). The plates were incubated over night at 4 1C or for 4 h at 37 1C. After washing and blocking with 5% skim milk powder, a serial dilution of antisera from all groups was carried out. The color was developed using p-nitrophenyl
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phosphate substrate (1 mg/ml) in substrate buffer (100 mM Tris–Cl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) and absorbance (OD) was read at 405 nm. For determining the antibody titer, the mean ODþ 3S.D. (Standard deviation) of the pre-immune serum was taken as the cut off value. The highest dilution of the antiserum that showed an OD value above the cut off value was taken as the antibody titer. For analysis of antibody isotypes, mice sera (1:100 dilution) from different immunization groups were incubated for 1 h at 37 1C, with respective proteins coated on ELISA plates. The IgG isotype binding was detected using secondary rabbit anti-mouse IgG specific for each subclass (Sigma-Aldrich, Bangalore, India) as per the manufacturer instructions (Madhumathi et al., 2010a). 2.5.3. T cell proliferation assay Single cell suspension of spleen cells were prepared from both immunized and control mice on day 80 post immunization (Anand et al., 2011). Approximately 2 106 cells/ml suspended in RPMI1640 supplemented with 10% heat inactivated FBS, gentamycin (80 μg/ml) (Ranbaxy Laboratories, India), 25 mM HEPES (USB, Amersham Pharmacia, UK) and 2 mM glutamine (USB, Amersham Pharmacia, UK) was cultured in triplicate in 96-well plates. Cells from Alum, PBS, TV, Alum þTV, PLA and PLA-TV immunized animals were cultured in triplicate wells were stimulated with TV, TRX and VAH antigens. Similarly, cells from Alum, PBS, FEP, Alumþ FEP, PLA and PLA-FEP immunized animals were stimulated with FEP antigen at varying concentrations (0.1, 1, 5 and 10 μg/ well) and with positive control Con A (Concanavalin A) (1 μg/well). Wells with medium alone were used as unstimulated controls. Plates were then incubated at 37 1C in a CO2 incubator. After 72 h, 3H-Thymidine (0.5 μCi per well, Amersham Biosciences) was added to each well and further incubated. Cells were harvested 18 h later and 3H-Thymidine uptake was measured in a liquid scintillation counter and expressed as stimulation index (SI) ¼ (counts per min. of stimulated cultures/counts per min. of unstimulated cultures)7 S.D. 2.5.4. Cytokine assay The culture supernatants from antigen-stimulated spleen cells were collected after 72 h (Sharmila et al., 2011) and tested for cytokines levels using sandwich ELISA for mouse IL-2, IL-4, IL-5, IL-10 and IFN-γ (E BioSciences, USA) as described by the manufacturer. Assay was performed in triplicates and the cytokine concentrations were calculated from standard curves and data expressed in pg/ml after subtracting the values of unstimulated culture.
2.6. Statistical analysis All statistical analysis was done with Graphpad prism software version 5. For multiple comparisons, Two-way ANOVA with Bonferroni post-test was performed. A probability (P) value o ¼0.05 was considered statistically significant.
3. Results 3.1. Characterization and encapsulation efficiency of PLA microparticles The scanning electron microscopic image of the PLA encapsulated filarial antigens showed spherical shape and with smooth surfaces (Fig. 1). The size of the antigen entrapped PLA particles were ranging from 1.677 0.06 to 7.78 70.13 mm as shown in Figure. The percentage encapsulation efficiency of TV and FEP antigens were 81.36% and 78%, respectively.
3.2. In vitro release assay In vitro release studies showed (Fig. 2) that there was a slow release of TV and FEP proteins from the encapsulated microparticles at the initial days upon contact with aqueous solutions such as PBS. Between 30% and 60% of encapsulated antigen was found to be released in the 56th day followed by no detectable release in vitro for the following 30 days. This suggested that there were unencapsulated proteins associated with the surface of the particles that were quickly released upon contact with the PBS but the microparticles themselves were stable in PBS and retained the entrapped antigen for at least a month.
3.3. Stability of the antigen The purified proteins such as TV (39 kDa), FEP (25 kDa) and in vitro release of respective proteins from PLA microparticles were run in SDS-PAGE. The SDS-PAGE resolved bands of in vitro released proteins (TV and FEP) from PLA particles revealed that microparticles preparation conditions did not cause any degradation of the antigen (Fig. 3) and proteins were found to be stable even after the encapsulation on to the PLA particles.
Fig. 1. Scanning electron microscopic images of protein entrapped by PLA microparticles. Size and surface morphology of the particles were analyzed using scanning electron microscope.
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significantly (Po 0.001) elevated levels of IgG2a and IgG1 than alum adsorbed FEP and FEP along with PBS (Fig. 5B). Overall response showed that PLA encapsulated filarial antigens elicited a balanced Th1(IgG2a, IgG2b)/Th2 (IgG1) response.
Fig. 2. In vitro release of proteins from PLA encapsulated microspheres. The in vitro release of TV and FEP proteins from PLA-TV and PLA-FEP microparticles at weekly intervals were determined.
Fig. 3. SDS-PAGE profile of PLA encapsulated filarial antigens. (A) Lane 1: molecular weight markers; Lane 2: Purified recombinant TV Protein (an arrow indicating 39 kDa protein); Lane 3: rTV antigen released in vitro from PLA encapsulated microparticle. (B) Lane 1: molecular weight markers; Lane 2: Purified recombinant FEP protein (an arrow indicating 25 kDa protein), Lane 3: rFEP antigen released in vitro from PLA encapsulated microparticle.
3.4. Immunological characterization of PLA encapsulated protein microparticles 3.4.1. Antibody titer in mice The total IgG antibody raised against PLA encapsulated proteins such as TV and FEP were measured post immunization at weekly intervals by ELISA and the results were represented as antibody titer (Fig. 4). The peak titer against PLA-TV and PLA-FEP were observed between 56th and 63th day after first immunization. But in groups immunized with a single dose of TV and FEP in PBS or single dose of alum adsorbed TV and FEP induced peak titers between 14th and 21th day itself after immunization. The peak titer against single dose of PLA encapsulated TV and FEP groups were 50,000 and 64,000, respectively. The peak titer against single dose of alum adsorbed TV and FEP were 6000 and 9000, respectively while that of TV and FEP in PBS were 3000 and 4000, respectively. Over all, PLA encapsulated proteins showed significantly (P o0.0001) high antibody titer than single dose of TV or FEP in PBS and single dose of alum adsorbed TV or FEP.
3.4.2. Analysis of isotype antibody The IgG subclass pattern of TV antigen immunized with different formulation was almost similar. But the IgG1 and IgG2b levels were found to be increased significantly (P o0.001) in PLATV immunized animals as compared with alum adsorbed TV group and with TV alone in PBS (Fig. 5A). Likewise, the isotype pattern of FEP was similar when adsorbed with PLA or alum and besides PLA encapsulated FEP showed
3.4.3. Splenocyte proliferation assay Splenocyte proliferation assay of mice immunized with single dose of TV/FEP, alum adsorbed TV/FEP or PLA encapsulated TV/FEP were stimulated with respective antigens (Fig. 6). The PLA encapsulated TV/FEP proteins showed significantly (P o0.001) high proliferative response than all groups for their corresponding antigens (Fig. 6A and B). The maximum proliferation was obtained in 1 μg/ml concentration in all antigens. However, there was no significant difference in the proliferative response of TV/FEP immunized cells and alum adsorbed TV/FEP immunized cells with their corresponding controls when stimulated with respective antigens. The proliferative responses of alum adsorbed TV/FEP proteins were comparatively lower than PLA encapsulated proteins. The results showed that cellular proliferative responses induced by PLA encapsulated proteins were much higher than that of alum adsorbed TV/FEP and single dose of TV/FEP probably due to long lasting immunity that triggers memory T cell formation. 3.4.4. Cytokine analysis The cytokine levels of TV/FEP proteins immunized with different formulation were measured by cytokine ELISA from culture supernatants of mice spleen cells stimulated with respective antigens (Table 1). The concentration of cytokines represented in the table was derived from standard curves and data expressed in pg/ml after deducting the values of unstimulated cultures. The levels of IL-4 (307.471.7) and IFN-γ (286.770.6) cytokines were significantly (Po0.0001) high in cells of PLA-TV antigen immunized animals when stimulated with respective antigen and PLA-FEP group induced significantly (Po0.0001) high levels of IL-5 (326.870.9) and IFN-γ (274.371.1) cytokines. There was no remarkable difference in the cytokine levels of animals immunized with TV/FEP with PBS and alum adsorbed TV/FEP antigens compared with controls.
4. Discussion Vaccination has been showing successful results for controlling or even complete eradication of many infectious diseases. A common problem in current vaccination coverage is the need for multiple administrations of antigens to achieve long-lasting immunity. Poly-l-lactide (PLA) polymer based particles have been investigated as an alternative vaccine delivery system (Boehm et al., 2002) which offer many advantages such as single point immunization for multidose vaccines, protection of antigen in vivo, flexibility of rate and profile of antigen release and coencapsulation of multiple antigens (Boehm et al., 2002; Shi et al., 2002; Jones, 2003; Peyre et al., 2004). Limitation in adjuvanticity of alum which requires multidose schedule to generate immune response against many recombinant protein based antigens has further necessitated research in the field of microencapsulated antigen. In this present study, we examined the role of two such PLA microencapsulated antigens TV (Anugraha et al., 2013) and FEP (Anugraha, unpublished data), which showed promising results in earlier studies, as a single dose vaccine. PLA encapsulation was carried out by double emulsion (w/o/w) solvent evaporation method. The conditions applied for microparticles preparation were followed as per protocol described by Katare et al. (2005) where they have obtained particle size ranging between 1 and 8 μm for tetanus antigens and reported to induce a long lasting immunity against tetanus. The size of the particle is
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Fig. 4. Antibody titer for TV and FEP in mice. (A) The antibody titer induced by mice immunized with one dose of TV or Alum þ TV or PLA-TV was measured by ELISA at different day intervals after immunization. (B) The antibody titer induced by mice immunized with one dose of FEP or Alumþ FEP or PLA-FEP was measured by ELISA at different day intervals after immunization. These results represent as mean 7S.D. antibody titer of ten mice from two experiments with five animals per group in each experiment.
Fig. 5. Isotype antibody levels of filarial proteins in different formulation. (A) The Isotype antibody levels induced against one dose of TV or Alum þ TV or PLA-TV (B) The antibody levels induced against one dose of FEP or Alum þFEP or PLA-FEP. The results represent as mean absorbance 7S.D. of ten mice from two experiments with five animals per group in each experiment. The asterisk (*) on top of the bars indicate a significantly (P o0.001) high absorbance value compared with other groups.
Fig. 6. Splenocyte proliferation assay in mice. T cell proliferation response of PLA encapsulated antigens. (A) PLA-TV protein immunized mice stimulated with respective antigens or ConA in comparison with alum control mice. (B) PLA-FEP protein immunized mice stimulated with the respective antigen or ConA in comparison with alum control mice. The data is represented as mean stimulation index (SI) 7 S.D. of ten mice from two experiments with five animals per group in each experiment. The asterisk (*) on top of the bars indicate a significantly (P o0.001) high absorbance values compared with control.
mainly influenced by amount of polymer, nature of solvent, the rate of stirring and the method used for preparation. Since, the particle size is an important factor for an efficient delivery of PLA encapsulated antigens to dendritic cells (DCs), which are major cells involved in antigen processing and presentation to MHC molecules. It has been reported that larger size particles (50– 150 mm) will not be taken up by antigen presenting cells or macrophages whereas the particles in the range of 10–70 mm will
have very little chance to be taken up by cells (Howie et al., 1993; Horisawa et al., 2002). The immune responses of single dose of PLA encapsulated antigens (TV and FEP) were studied in mice model in comparison with single dose of alum adsorbed antigens (Alumþ TV/Alum þFEP) or antigens (TV/FEP) alone. Analysis of humoral response showed that the antibody titer induced by PLA encapsulated antigens such as PLA-TV and PLAFEP were 50,000 and 64,000 respectively which was observed
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Table 1 Analysis of cytokine levels (pg/ml) in mice. Animal groups
Unstimulated T cells TV Alum þ TV PLA-TV FEP Alum þ FEP PLA-FEP
Cytokines (pg/ml) IL-2
IL-4
IL-5
IL-10
IFN-γ
2.17 0.5 10.577 0.4 13.6 7 0.3 15.9 7 0.8 10.4 7 0.9 12.54 7 0.3 11.6 7 0.7
1.6 7 0.1 80.9 7 0.7 97.3 7 1.0 307.4a 7 1.7 46.89 7 0.4 53.277 0.7 98.6 7 1.3
2.3 7 0.2 40.7 7 1.1 47.17 0.2 59.6 7 1.1 54.5 7 0.3 69.3 7 0.3 326.8a 7 0.9
1.2 7 0.3 22.5 7 0.4 20.4 7 1.6 30.56 7 1.2 10.6 7 0.5 13.8 7 0.3 28.9 7 0.6
2.7 7 0.3 48.47 0.3 62.7 7 0.4 286.7a 7 0.6 75.4 7 0.2 79.7 7 0.5 274.3a 7 1.1
The cytokine levels were measured in culture supernatants of spleen cells from mice immunized and stimulated in vitro with the corresponding antigen. Experiments were done thrice and the data is represented as mean concentration 7 S.D. a
P o0.0001 compared with other formulation.
between 56th and 63th day after immunization. In the case of alum adsorbed TV/FEP and TV/FEP antigen alone showed maximum antibody titer induction between 14th and 21st day after immunization (6000/9000 and 3000/4000 respectively), followed by very negligible induction was observed in the following days. These results showed that PLA encapsulated antigens were released slowly from the site of injection that leads to sustained communication with antigen presenting cells (Coombes et al., 1999) which induced a significantly (P o0.001) higher antibody titer than other groups indicating the enhanced adjuvanticity of PLA. Similar results were observed in in vitro protein release assay. Overall isotype antibody profile shows that PLA encapsulated filarial antigens (TV/FEP) elicited a high levels of balanced Th1 (IgG2a, IgG2b)/Th2 (IgG1) antibody response. In vivo studies from vast literature reports suggest that all mammalian stages of filarial parasites can be killed by ADCC where large amounts of antibody of different isotypes were involved (Lawrence, 2001). The murine IgG2a and IgG2b isotypes have the ability to fix complement and bind to protein antigens, whereas murine IgG1 is shown to be important because of their property of binding to mast cells and all these subtypes have been reported to participate in ADCC (Akiyama et al., 1984) which correlates with our results. T cells have also been shown to be important for host protection in murine infections with B. malayi and B. pahangi, However their precise role in host protection has not been clearly established (Babu et al., 1999; Vincent et al., 1980). In T cell proliferation assay, the groups immunized with PLA encapsulated TV/FEP proteins showed significantly (Po0.001) high proliferative response than all groups when stimulated with corresponding antigens. The difference in proliferative response is probably due to slow and continuous release of antigens from PLA, which generate persistent immune response since it triggers memory T cells while stimulating with antigen indicating strong recall response produced by the antigen. Other formulations are poorly immunogenic and require several booster doses to induce strong immune response. Previous studies using B. malayi adult worm extract (BmA) and its SDS-PAGE resolved 54–68 kDa fraction F6, also have showed similar proliferative response in mice model when encapsulated with PLA (Saini et al., 2011). The cytokine analysis showed that PLA-TV antigen induced significantly (P o0.0001) high levels of IL-4 and IFN-γ cytokines whereas PLA-FEP group showed significantly (P o0.0001) high levels of IL-5 and IFN-γ cytokines. There was no remarkable difference in the cytokine levels of animals immunized with TV/ FEP with PBS and alum adsorbed TV/FEP antigens compared with controls. The role of Th2 cell cytokine such as IL-4 and IL-5 in elimination of larval parasite has been demonstrated in Onchocerca volvulus infection in BALB/cBYJ mice (Lange et al., 1994). Similarly several recent studies in different animal models of filarial diseases have shown that Th1-type responses are also
involved in the host protection against filarial infection and IFN-γ has been shown to be required for the clearance of B. malayi in mice (Babu et al., 2000). The results indicate that both TV and FEP antigens elicited a balanced Th1/Th2 immune response when the proteins are encapsulated with PLA than other formulation. It is envisioned that the balanced Th1 and Th2 cell responses are sufficient to kill the invading helminths (Maizels and Yazdanbakhsh, 2003) and thus any vaccine candidate which is proved to be promising should be capable of eliciting both antibody and T cell responses in humans, especially in filariasis, since there is a trademark T cell suppression immediately after infection (Lawrence, 2001). In this study we observed that single immunization of PLA encapsulated antigens (both TV and FEP) induced a strong humoral as well as cellular response compared to single dose of other formulations. However, similar responses were achieved for TV (Anugraha et al., 2013) and FEP (Anugraha et al., unpublished data) proteins when the antigens are adsorbed in alum and given as multiple doses. These results show that PLA formulations are found to be more immunogenic and suitable for filarial antigens to generate long lasting immune response from single point immunization as it mounted strong and protective humoral as well as cellular response.
Acknowledgements The authors are thankful to University Grants Commission (UGC) and Council of Scientific and Industrial Research (CSIR) for their financial support. Dr. Rajavel Malligarjunan’s critical comments and positive feedbacks during the preparation of manuscript are gratefully acknowledged. References Absolom, D.R., Zingg, W., Neumann, A.W., 1987. Protein adsorption to polymer particles: role of surface properties. J. Biomed. Mater. Res. 21, 161–171. Akiyama, Y., Lubeck, M.D., Steplewski, Z., Koprowski, H., 1984. Induction of mouse IgG2a- and IgG3-dependent cellular cytotoxicity in human monocytic cells (U937) by immune interferon. Cancer Res. 44, 5127–5131. Anand, S.B., Kodumudi, K.N., Reddy, M.V., Kaliraj, P., 2011. A combination of two Brugia malayi filarial vaccine candidate antigens (BmALT-2 and BmVAH) enhances immune responses and protection in jirds. J. Helminthol. 85, 442–452. Anand, S.B., Murugan, V., Prabhu, P.R., Anandharaman, V., Reddy, M.V., Kaliraj, P., 2008. Comparison of immunogenicity, protective efficacy of single and cocktail DNA vaccine of Brugia malayi abundant larval transcript (ALT-2) and thioredoxin peroxidase (TPX) in mice. Acta Trop. 107, 106–112. Anugraha, G., Jeyaprita, P.J., Madhumathi, J., Sheeba, T., Kaliraj, P., 2013. Immune responses of B. malayi thioredoxin (TRX) and venom allergen homologue (VAH) chimeric multiple antigen for lymphatic filariasis. Acta Parasitol. 58, 468–477. Babu, S., Ganley, L.M., Klei, T.R., Shultz, L.D., Rajan, T.V., 2000. Role of gamma interferon and interleukin-4 in host defense against the human filarial parasite Brugia malayi. Infect. Immun. 68, 3034–3035.
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Babu, S., Shultz, L.D., Klei, T.R., Rajan, T.V., 1999. Immunity in experimental murine filariasis: roles of T and B cells revisited. Infect. Immun. 67, 3166–3167. Bockarie, M.J., Deb, R.M., 2010. Elimination of lymphatic filariasis: do we have the drugs to complete the job? Curr. Opin. Infect. Dis 23, 617–620. Boehm, G., Peyre, M., Sesardic, D., Huskisson, R.J., Mawas, F., Douglas, A., Xing, D., Merkle, H.P., Gander, B., Johansen, P., 2002. On technological and immunological benefits of multivalent single-injection microsphere vaccines. Pharm. Res. 19, 1330–1336. Coombes, A.G., Lavelle, E.C., Davis, S.S., 1999. Biodegradable lamellar particles of poly(lactide) induce sustained immune responses to a single dose of adsorbed protein. Vaccine 17, 2410–2422. Coombes, A.G., Lavelle, E.C., Jenkins, P.G., Davis, S.S., 1996. Single dose, polymeric, microparticle-based vaccines: the influence of formulation conditions on the magnitude and duration of the immune response to a protein antigen. Vaccine 14, 1429–1438. Dakshinamoorthy, G., Samykutty, A.K., Munirathinam, G., Reddy, M.V., Kalyanasundaram, R., 2013. Multivalent fusion protein vaccine for lymphatic filariasis. Vaccine 31, 1616–1622. Gregory, W.F., Blaxter, M.L., Maizels, R.M., 1997. Differentially expressed, abundant trans-spliced cDNAs from larval Brugia malayi. Mol. Biochem. Parasitol. 87, 85–95. Gupta, R.K., Singh, M., O’Hagan, D.T., 1998. Poly(lactide-co-glycolide) microparticles for the development of single-dose controlled-release vaccines. Adv. Drug Deliv. Rev. 32, 225–246. Gyapong, J.O., Kumaraswami, V., Biswas, G., Ottesen, E.A., 2005. Treatment strategies underpinning the global programme to eliminate lymphatic filariasis. Expert Opin. Pharmacother. 6, 179–200. Hoerauf, A., Pfarr, K., Mand, S., Debrah, A.Y., Specht, S., 2011. Filariasis in Africa— treatment challenges and prospects. Clin. Microbiol. Infect. 17, 977–985. Horisawa, E., Kubota, K., Tuboi, I., Sato, K., Yamamoto, H., Takeuchi, H., Kawashima, Y., 2002. Size-dependency of DL-lactide/glycolide copolymer particulates for intra-articular delivery system on phagocytosis in rat synovium. Pharm. Res. 19, 132–139. Howie, D.W., Manthey, B., Hay, S., Vernon-Roberts, B., 1993. The synovial response to intraarticular injection in rats of polyethylene wear particles. Clin. Orthop. Relat. Res, 352–357. Jones, D.H., 2003. Microencapsulation of vaccine antigens. Methods Mol. Med. 87, 211–222. Katare, Y.K., Muthukumaran, T., Panda, A.K., 2005. Influence of particle size, antigen load, dose and additional adjuvant on the immune response from antigen loaded PLA microparticles. Int. J. Pharm. 301, 149–160. Lange, A.M., Yutanawiboonchai, W., Scott, P., Abraham, D., 1994. IL-4- and IL-5dependent protective immunity to Onchocerca volvulus infective larvae in BALB/ cBYJ mice. J. Immunol. 153, 205–211. Lavelle, E.C., Yeh, M.K., Coombes, A.G., Davis, S.S., 1999. The stability and immunogenicity of a protein antigen encapsulated in biodegradable microparticles based on blends of lactide polymers and polyethylene glycol. Vaccine 17, 512–529. Lawrence, R.A., 2001. Immunity to filarial nematodes. Vet. Parasitol. 100, 33–44. Madhumathi, J., Pradiba, D., Prince, P.R., Jeyaprita, P.J., Rao, D.N., Kaliraj, P., 2010b. Crucial epitopes of Wuchereria bancrofti abundant larval transcript recognized in natural infection. Eur. J. Clin. Microbiol. Infect. Dis. 29, 1481–1486.
Madhumathi, J., Prince, P.R., Anugraha, G., Kiran, P., Rao, D.N., Reddy, M.V., Kaliraj, P., 2010a. Identification and characterization of nematode specific protective epitopes of Brugia malayi TRX towards development of synthetic vaccine construct for lymphatic filariasis. Vaccine 28, 5038–5048. Madhumathi, J., Prince, P.R., Rao, D.N., Kaliraj, P., 2010c. Dominant T-cell epitopes of filarial BmALT-2 and their cytokine profile in BALB/c mice. Parasite Immunol. 32, 760–763. Maizels, R.M., Yazdanbakhsh, M., 2003. Immune regulation by helminth parasites: cellular and molecular mechanisms. Nat. Rev. Immunol. 3, 733–744. Petrovsky, N., Aguilar, J.C., 2004. Vaccine adjuvants: current state and future trends. Immunol. Cell Biol. 82, 488–496. Peyre, M., Audran, R., Estevez, F., Corradin, G., Gander, B., Sesardic, D., Johansen, P., 2004. Childhood and malaria vaccines combined in biodegradable microspheres produce immunity with synergistic interactions. J. Controlled Release 99, 345–355. Prince, P.R., Madhumathi, J., Anugraha, G., Jeyaprita, P.J., Reddy, M.V., Kaliraj, P., 2013. Tandem antioxidant enzymes confer synergistic protective responses in experimental filariasis. J. Helminthol, 1–9. Saini, V., Verma, S.K., Sahoo, M.K., Kohli, D.V., Murthy, P.K., 2011. Sufficiency of a single administration of filarial antigens adsorbed on polymeric lamellar substrate particles of poly (L-lactide) for immunization. Int. J. Pharm. 420, 101–110. Schmidt, C.S., Morrow, W.J., Sheikh, N.A., 2007. Smart adjuvants. Expert Rev. Vaccines 6, 391–400. Sharmila, S., Christiana, I., Kiran, P., Reddy, M.V., Kaliraj, P., 2011. The adjuvant-free immunoprotection of recombinant filarial protein Abundant Larval Transcript-2 (ALT-2) in Mastomys coucha and the immunoprophylactic importance of its putative signal sequence. Exp. Parasitol. 129, 247–253. Shi, L., Caulfield, M.J., Chern, R.T., Wilson, R.A., Sanyal, G., Volkin, D.B., 2002. Pharmaceutical and immunological evaluation of a single-shot hepatitis B vaccine formulated with PLGA microspheres. J. Pharm. Sci. 91, 1019–1035. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M. D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. Taylor, M.J., Hoerauf, A., Bockarie, M., 2010. Lymphatic filariasis and onchocerciasis. Lancet 376, 1175–1185. Thirugnanam, S., Pandiaraja, P., Ramaswamy, K., Murugan, V., Gnanasekar, M., Nandakumar, K., Reddy, M.V., Kaliraj, P., 2007. Brugia malayi: comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT2 protein and prime-boost vaccine regimens in a jird model. Exp. Parasitol. 116, 483–491. Vanam, U., Pandey, V., Prabhu, P.R., Dakshinamurthy, G., Reddy, M.V., Kaliraj, P., 2009a. Evaluation of immunoprophylactic efficacy of Brugia malayi transglutaminase (BmTGA) in single and multiple antigen vaccination with BmALT-2 and BmTPX for human lymphatic filariasis. Am. J. Trop. Med. Hyg. 80, 319–324. Vanam, U., Prabhu, P.R., Pandey, V., Dakshinamurthy, G., Reddy, M.V., Perumal, K., 2009b. Immune responses generated by intramuscular DNA immunization of Brugia malayi transglutaminase (BmTGA) in mice. Parasitology 136, 887–894. Vincent, A.L., Sodeman, W.A., Winters, A., 1980. Development of Brugia pahangi in normal and nude mice. J. Parasitol. 66, 448. Witschi, C., Doelker, E., 1998. Influence of the microencapsulation method and peptide loading on poly(lactic acid) and poly(lactic-co-glycolic acid) degradation during in vitro testing. J. Controlled Release 51, 327–341.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Biodegradable poly-l-lactide based microparticles as controlled release delivery system for filarial vaccine candidate antigens Gandhirajan Anugraha, Jayaprakasam Madhumathi, Parasurama Jawaharlal Jeya Prita, Perumal Kaliraj n Centre for Biotechnology, Anna University, Chennai 600025, India
art ic l e i nf o
a b s t r a c t
Article history: Received 3 October 2014 Received in revised form 2 December 2014 Accepted 5 December 2014 Available online 13 December 2014
Modern recombinant vaccines are less immunogenic than conventional vaccines which require adjuvants to enhance the effect of a vaccine. Alum is being used as a standard adjuvant for protein based vaccines to augment immune response in several diseases. However, the problem associated with alum is it requires multiple doses at specific time intervals to achieve the adequate level of immunity. Currently the adjuvanticity of Poly-l-lactide microparticles as single dose immunization was explored to overcome multiple immunization and reported to be effective for several diseases. In this regard we adsorbed filarial recombinant chimeric multivalent vaccine candidates such as TV and FEP on to PLA by double emulsion method and analyzed the characterization of PLA encapsulated microparticles and evaluated its immune responses in mice. The efficacy of single dose of PLA encapsulated proteins was investigated in comparison with single dose of alum or protein alone. In mice, single dose of PLA encapsulated antigens such as TV and FEP elicited significantly high antibody titer of 50,000 and 64,000 respectively than single dose of alum adsorbed TV/FEP (6000/9000) and single dose of protein TV/FEP (3000/4000) alone. Further PLA encapsulated antigens induced higher levels of cellular proliferation together with significant (Po 0.0001) levels of cytokine response [PLA-TV induced high levels of IL-4 (Th2) and IFN-γ (Th1) cytokines whereas PLA-FEP showed high levels of IL-5(Th2) and IFN-γ (Th1)] indicating a balanced response elicited by PLA antigens. Overall strong humoral and cellular responses were observed for PLA encapsulated antigens compared with single dose of alum adsorbed or protein alone. & 2014 Elsevier B.V. All rights reserved.
Keywords: Lymphatic filariasis Controlled release delivery system Poly-l-lactide Microsphere Adjuvants Immune response Chemical compounds studied in this article: Poly-l-lactide (PLA) Dichloromethane Polyvinyl alcohol (PVA) Acetonitrile
1. Introduction Lymphatic Filariasis, a mosquito borne parasitic infection caused by three closely related nematode worms, Wuchereria bancrofti, Brugia malayi and Brugia timori which damage the lymphatic system leading to painful swelling and disfigurement. Filariasis is the second largest cause of permanent and long-term disability in the world (Gyapong et al., 2005). It has a major social and economic impact in many tropical and subtropical countries. Current antifilarial drugs and exposure control measures have several limitations (Bockarie and Deb, 2010; Hoerauf et al., 2011; Taylor et al., 2010) and hence there is a need for additional strategy for elimination of lymphatic filariasis. In recent times, several recombinant vaccine candidate antigens have been reported to elicit promising results (Thirugnanam et al., 2007; Vanam et al., 2009a; Anand et al., 2008, 2011; Dakshinamoorthy et al., 2013)
n
Corresponding author. Tel.: þ 91 44 22350772; fax: þ 91 44 22352642. E-mail address: [email protected] (P. Kaliraj).
http://dx.doi.org/10.1016/j.ejphar.2014.12.004 0014-2999/& 2014 Elsevier B.V. All rights reserved.
which provided an excellent hope for developing vaccine against lymphatic filariasis. Although the recombinant protein vaccines elicited significant protection, they require adjuvant to induce optimum immune response. Currently, alum (U.S. FDA approved adjuvant) is widely used to increase immune response for protein based vaccines (Schmidt et al., 2007). However, multiple injections at specific time intervals are the general requirements to achieve the adequate level of immunity for alum adsorbed antigens, moreover it is a poor stimulator of cellular (Th1) immune responses, which are also important for protection (Schmidt et al., 2007; Petrovsky and Aguilar, 2004). In developing countries, limited access to medical care, lack of awareness and of floating population plays a crucial role in following the multiple immunization coverage to obtain desired response. Hence, an injectable formulation should be designed to ensure long-lasting immunity in a single dose vaccine which releases an antigen in a controlled manner as booster dose would be an improved strategy for immunization coverage. Currently, biodegradable microparticles based on poly-l-lactide (PLA) an FDA-approved polymer, is used for controlled-release
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delivery of protein-based vaccine antigens. The role of PLA as adjuvant has been reported by several investigators (Coombes et al., 1999; Gupta et al., 1998; Witschi and Doelker, 1998) and was reported to elicit strong and long-lasting immune response in single immunization. PLA is advantageous over other FDA approved polymer e.g. Poly(lactide-co-glycolide) (PLGA) because of its hydrophobicity and very low surface tension which are essential for efficient antigen adsorption (Absolom et al., 1987; Coombes et al., 1996) and its particulate form helps for more efficient antigen delivery to dendritic cells (Lavelle et al., 1999). These polymers undergo hydrolysis when injected into the body and forming biologically compatible and metabolizable moieties (lactic acid) due to their polyester nature. Finally they are removed from the body through the citric acid cycle. Recently, we have reported protective efficacy of filarial recombinant chimeric multivalent vaccine candidate namely TV (a 39 kDa fusion protein which comprising two filarial key vaccine candidate antigens such as Thioredoxin (TRX) and Venom Allergen Homologue (VAH)] (Anugraha et al., 2013) and another recombinant chimeric mutiepitope vaccine candidate such as Filarial epitope protein (FEP). FEP, a multi epitope protein (synthesized and expressed as 25 kDa protein) comprising immunodominant epitopes from multistage antigens such as Thioredoxin (TRX) (Madhumathi et al., 2010a), Transglutaminase (TGA) (Vanam et al., 2009b; Madhumathi et al., unpublished data), and Abundant Larval Transcript-2 (ALT-2) (Gregory et al., 1997; Madhumathi et al., 2010b, 2010c) and proved as potential vaccine candidate (Anugraha et al., unpublished data). In this present study we proposed to explore the use of PLA as a delivery system for filarial antigens and characterized the effective immune response for a single administration of PLA encapsulated antigens. To analyze this possibility adsorption of the recombinant Fusion protein (TV) and Filarial epitope protein (FEP) on to PLA and the immune responses of the same were studied in comparison with single dose of alum adjuvant or protein alone in mice model.
2. Materials and methods 2.1. Preparation and characterization of polymer particles The poly lactide (M.W. 103 kDa) (Sigma-Aldrich, Bangalore, India) microparticles were prepared using double emulsion (w/o/w) solvent evaporation method as described by Katare and group (Katare et al., 2005) with optimized modifications. Briefly, recombinant proteins (TV and FEP) were dissolved in 0.5 ml of an aqueous solution of 10% (w/v) polyvinyl alcohol (M.W. 30 to 70 kDa; 87–90% hydrolyzed). This primary aqueous phase was vigorously mixed through sonication, with an oily phase consisting of 250 mg of PLA dissolved in 5 ml of dichloromethane (50 mg/ml of dichloromethane). The resultant waterin-oil emulsion was then added, during vigorous agitation, to 75 ml of 5% (w/v) polyvinyl alcohol. The microparticles formed through overnight solvent evaporation in a magnetic stirrer were washed and harvested by centrifugation (40,695g, 20 min), and lyophilized to obtain the form of free-flowing powder. Size and surface morphology of the particles were analyzed using scanning electron microscope (Carl Zeiss, Germany). 2.2. Estimation of protein encapsulation efficiency in microparticles To estimate the protein content of particles, accurately weighed particles were dissolved in acetonitrile to solubilize the polymer (10 mg/ml). The suspension was centrifuged at 9037g for 10 min and the resultant pellet was dried at 37 1C. After the addition and incubation (1 h at 37 1C) of 100 μl PBS buffer to the pellete, the reaction mixture was centrifuged at 9037g for 10 min and the
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supernatant were collected. The procedure was repeated with 100 μl of 0.1 M NaOH. The protein content of the supernatant was estimated by BCA (Bicinchoninic acid) assay (Smith et al., 1985) which is a more sensitive assay for estimation. 10 μl of protein sample was added to 190 μl of BCA working reagent (Pierce, Rockford, USA) and incubated at 37 1C, in a dark room for 30 min and the absorbance was measured at 540–590 nm. The encapsulation efficiency was calculated as the percentage weight of antigen per unit weight of polymer (μg antigen/mg of PLA particles). 2.3. In vitro release assay from microparticles The release of proteins from microparticles was determined by incubation of accurately weighed particles (approx. 10 mg) in 100 ml PBS buffer at 37 1C in continuous shaking. The suspension was centrifuged and the supernatant was removed for analysis. At weekly intervals fresh PBS buffer (100 ml) was added to the pellet and resuspended by gentle vortexing and returned to the shaker. The process was repeated for up to 3 months. Released samples were estimated by BCA method as described earlier. 2.4. Protein stability study The integrity of the antigens (TV and FEP) was analyzed in SDSPAGE after encapsulation in comparison with purified proteins (TV and FEP). The known amount of antigen encapsulated with PLA microparticles were incubated (separately) with 0.1% (w/v) SDSphosphate buffered saline (PBS; 0.01 M; pH 7.4) at 37 1C for 3 h with gentle shaking and then centrifuged at 18,074g for 25 min at 4–8 1C (Saini et al., 2011). The supernatants were checked in SDSPAGE gel. 2.5. Immunological studies in mice 2.5.1. Immunization protocol Six to eight weeks old BALB/c (H-2d) mice were purchased from King Institute, Chennai, Tamilnadu, India. All the experiments were followed as per ‘Indian Animal Ethics Committee’ regulations. For PLA encapsulated antigen (mentioned as PLA-TV and PLA-FEP) groups (5 mice/group), were immunized with 50 μg (an optimum concentration for microsphere encapsulated proteins (Madhumathi et al., 2010a) of protein equivalent in microspheres suspended in 100 μl of PBS buffer. Other groups were immunized with 50 μg of TV and FEP protein mixed with an equal volume of alum (Sigma-Aldrich, Bangalore, India) (mentioned as AlumþTV and AlumþFEP) and as well as antigens without alum [TV and FEP in Phosphate buffered saline (PBS) alone]. Three control groups were kept for this study. One group received alum alone, another received PBS alone and the third group with PLA microparticles alone. All groups were injected with single dose of respective antigens via subcutaneous route. A small amount of blood (approximately 50 μl) was taken from tail vein of immunized animals at weekly intervals up to three months since microsphere encapsulation provides long-lasting immunity due to sustained release of antigen and serum were separated to analyse the antibody levels by ELISA. 2.5.2. Analysis of total IgG and Isotype antibody levels Protein specific IgG levels in the sera were observed by ELISA. The 96-well microtiter plates (Nunc, Maxisorp, Nalge Nunc International, Denmark) were used and each wells were coated with 100 ng of TV and FEP proteins prepared in 100 μl of coating buffer separately (Prince et al., 2013). The plates were incubated over night at 4 1C or for 4 h at 37 1C. After washing and blocking with 5% skim milk powder, a serial dilution of antisera from all groups was carried out. The color was developed using p-nitrophenyl
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phosphate substrate (1 mg/ml) in substrate buffer (100 mM Tris–Cl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) and absorbance (OD) was read at 405 nm. For determining the antibody titer, the mean ODþ 3S.D. (Standard deviation) of the pre-immune serum was taken as the cut off value. The highest dilution of the antiserum that showed an OD value above the cut off value was taken as the antibody titer. For analysis of antibody isotypes, mice sera (1:100 dilution) from different immunization groups were incubated for 1 h at 37 1C, with respective proteins coated on ELISA plates. The IgG isotype binding was detected using secondary rabbit anti-mouse IgG specific for each subclass (Sigma-Aldrich, Bangalore, India) as per the manufacturer instructions (Madhumathi et al., 2010a). 2.5.3. T cell proliferation assay Single cell suspension of spleen cells were prepared from both immunized and control mice on day 80 post immunization (Anand et al., 2011). Approximately 2 106 cells/ml suspended in RPMI1640 supplemented with 10% heat inactivated FBS, gentamycin (80 μg/ml) (Ranbaxy Laboratories, India), 25 mM HEPES (USB, Amersham Pharmacia, UK) and 2 mM glutamine (USB, Amersham Pharmacia, UK) was cultured in triplicate in 96-well plates. Cells from Alum, PBS, TV, Alum þTV, PLA and PLA-TV immunized animals were cultured in triplicate wells were stimulated with TV, TRX and VAH antigens. Similarly, cells from Alum, PBS, FEP, Alumþ FEP, PLA and PLA-FEP immunized animals were stimulated with FEP antigen at varying concentrations (0.1, 1, 5 and 10 μg/ well) and with positive control Con A (Concanavalin A) (1 μg/well). Wells with medium alone were used as unstimulated controls. Plates were then incubated at 37 1C in a CO2 incubator. After 72 h, 3H-Thymidine (0.5 μCi per well, Amersham Biosciences) was added to each well and further incubated. Cells were harvested 18 h later and 3H-Thymidine uptake was measured in a liquid scintillation counter and expressed as stimulation index (SI) ¼ (counts per min. of stimulated cultures/counts per min. of unstimulated cultures)7 S.D. 2.5.4. Cytokine assay The culture supernatants from antigen-stimulated spleen cells were collected after 72 h (Sharmila et al., 2011) and tested for cytokines levels using sandwich ELISA for mouse IL-2, IL-4, IL-5, IL-10 and IFN-γ (E BioSciences, USA) as described by the manufacturer. Assay was performed in triplicates and the cytokine concentrations were calculated from standard curves and data expressed in pg/ml after subtracting the values of unstimulated culture.
2.6. Statistical analysis All statistical analysis was done with Graphpad prism software version 5. For multiple comparisons, Two-way ANOVA with Bonferroni post-test was performed. A probability (P) value o ¼0.05 was considered statistically significant.
3. Results 3.1. Characterization and encapsulation efficiency of PLA microparticles The scanning electron microscopic image of the PLA encapsulated filarial antigens showed spherical shape and with smooth surfaces (Fig. 1). The size of the antigen entrapped PLA particles were ranging from 1.677 0.06 to 7.78 70.13 mm as shown in Figure. The percentage encapsulation efficiency of TV and FEP antigens were 81.36% and 78%, respectively.
3.2. In vitro release assay In vitro release studies showed (Fig. 2) that there was a slow release of TV and FEP proteins from the encapsulated microparticles at the initial days upon contact with aqueous solutions such as PBS. Between 30% and 60% of encapsulated antigen was found to be released in the 56th day followed by no detectable release in vitro for the following 30 days. This suggested that there were unencapsulated proteins associated with the surface of the particles that were quickly released upon contact with the PBS but the microparticles themselves were stable in PBS and retained the entrapped antigen for at least a month.
3.3. Stability of the antigen The purified proteins such as TV (39 kDa), FEP (25 kDa) and in vitro release of respective proteins from PLA microparticles were run in SDS-PAGE. The SDS-PAGE resolved bands of in vitro released proteins (TV and FEP) from PLA particles revealed that microparticles preparation conditions did not cause any degradation of the antigen (Fig. 3) and proteins were found to be stable even after the encapsulation on to the PLA particles.
Fig. 1. Scanning electron microscopic images of protein entrapped by PLA microparticles. Size and surface morphology of the particles were analyzed using scanning electron microscope.
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significantly (Po 0.001) elevated levels of IgG2a and IgG1 than alum adsorbed FEP and FEP along with PBS (Fig. 5B). Overall response showed that PLA encapsulated filarial antigens elicited a balanced Th1(IgG2a, IgG2b)/Th2 (IgG1) response.
Fig. 2. In vitro release of proteins from PLA encapsulated microspheres. The in vitro release of TV and FEP proteins from PLA-TV and PLA-FEP microparticles at weekly intervals were determined.
Fig. 3. SDS-PAGE profile of PLA encapsulated filarial antigens. (A) Lane 1: molecular weight markers; Lane 2: Purified recombinant TV Protein (an arrow indicating 39 kDa protein); Lane 3: rTV antigen released in vitro from PLA encapsulated microparticle. (B) Lane 1: molecular weight markers; Lane 2: Purified recombinant FEP protein (an arrow indicating 25 kDa protein), Lane 3: rFEP antigen released in vitro from PLA encapsulated microparticle.
3.4. Immunological characterization of PLA encapsulated protein microparticles 3.4.1. Antibody titer in mice The total IgG antibody raised against PLA encapsulated proteins such as TV and FEP were measured post immunization at weekly intervals by ELISA and the results were represented as antibody titer (Fig. 4). The peak titer against PLA-TV and PLA-FEP were observed between 56th and 63th day after first immunization. But in groups immunized with a single dose of TV and FEP in PBS or single dose of alum adsorbed TV and FEP induced peak titers between 14th and 21th day itself after immunization. The peak titer against single dose of PLA encapsulated TV and FEP groups were 50,000 and 64,000, respectively. The peak titer against single dose of alum adsorbed TV and FEP were 6000 and 9000, respectively while that of TV and FEP in PBS were 3000 and 4000, respectively. Over all, PLA encapsulated proteins showed significantly (P o0.0001) high antibody titer than single dose of TV or FEP in PBS and single dose of alum adsorbed TV or FEP.
3.4.2. Analysis of isotype antibody The IgG subclass pattern of TV antigen immunized with different formulation was almost similar. But the IgG1 and IgG2b levels were found to be increased significantly (P o0.001) in PLATV immunized animals as compared with alum adsorbed TV group and with TV alone in PBS (Fig. 5A). Likewise, the isotype pattern of FEP was similar when adsorbed with PLA or alum and besides PLA encapsulated FEP showed
3.4.3. Splenocyte proliferation assay Splenocyte proliferation assay of mice immunized with single dose of TV/FEP, alum adsorbed TV/FEP or PLA encapsulated TV/FEP were stimulated with respective antigens (Fig. 6). The PLA encapsulated TV/FEP proteins showed significantly (P o0.001) high proliferative response than all groups for their corresponding antigens (Fig. 6A and B). The maximum proliferation was obtained in 1 μg/ml concentration in all antigens. However, there was no significant difference in the proliferative response of TV/FEP immunized cells and alum adsorbed TV/FEP immunized cells with their corresponding controls when stimulated with respective antigens. The proliferative responses of alum adsorbed TV/FEP proteins were comparatively lower than PLA encapsulated proteins. The results showed that cellular proliferative responses induced by PLA encapsulated proteins were much higher than that of alum adsorbed TV/FEP and single dose of TV/FEP probably due to long lasting immunity that triggers memory T cell formation. 3.4.4. Cytokine analysis The cytokine levels of TV/FEP proteins immunized with different formulation were measured by cytokine ELISA from culture supernatants of mice spleen cells stimulated with respective antigens (Table 1). The concentration of cytokines represented in the table was derived from standard curves and data expressed in pg/ml after deducting the values of unstimulated cultures. The levels of IL-4 (307.471.7) and IFN-γ (286.770.6) cytokines were significantly (Po0.0001) high in cells of PLA-TV antigen immunized animals when stimulated with respective antigen and PLA-FEP group induced significantly (Po0.0001) high levels of IL-5 (326.870.9) and IFN-γ (274.371.1) cytokines. There was no remarkable difference in the cytokine levels of animals immunized with TV/FEP with PBS and alum adsorbed TV/FEP antigens compared with controls.
4. Discussion Vaccination has been showing successful results for controlling or even complete eradication of many infectious diseases. A common problem in current vaccination coverage is the need for multiple administrations of antigens to achieve long-lasting immunity. Poly-l-lactide (PLA) polymer based particles have been investigated as an alternative vaccine delivery system (Boehm et al., 2002) which offer many advantages such as single point immunization for multidose vaccines, protection of antigen in vivo, flexibility of rate and profile of antigen release and coencapsulation of multiple antigens (Boehm et al., 2002; Shi et al., 2002; Jones, 2003; Peyre et al., 2004). Limitation in adjuvanticity of alum which requires multidose schedule to generate immune response against many recombinant protein based antigens has further necessitated research in the field of microencapsulated antigen. In this present study, we examined the role of two such PLA microencapsulated antigens TV (Anugraha et al., 2013) and FEP (Anugraha, unpublished data), which showed promising results in earlier studies, as a single dose vaccine. PLA encapsulation was carried out by double emulsion (w/o/w) solvent evaporation method. The conditions applied for microparticles preparation were followed as per protocol described by Katare et al. (2005) where they have obtained particle size ranging between 1 and 8 μm for tetanus antigens and reported to induce a long lasting immunity against tetanus. The size of the particle is
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Fig. 4. Antibody titer for TV and FEP in mice. (A) The antibody titer induced by mice immunized with one dose of TV or Alum þ TV or PLA-TV was measured by ELISA at different day intervals after immunization. (B) The antibody titer induced by mice immunized with one dose of FEP or Alumþ FEP or PLA-FEP was measured by ELISA at different day intervals after immunization. These results represent as mean 7S.D. antibody titer of ten mice from two experiments with five animals per group in each experiment.
Fig. 5. Isotype antibody levels of filarial proteins in different formulation. (A) The Isotype antibody levels induced against one dose of TV or Alum þ TV or PLA-TV (B) The antibody levels induced against one dose of FEP or Alum þFEP or PLA-FEP. The results represent as mean absorbance 7S.D. of ten mice from two experiments with five animals per group in each experiment. The asterisk (*) on top of the bars indicate a significantly (P o0.001) high absorbance value compared with other groups.
Fig. 6. Splenocyte proliferation assay in mice. T cell proliferation response of PLA encapsulated antigens. (A) PLA-TV protein immunized mice stimulated with respective antigens or ConA in comparison with alum control mice. (B) PLA-FEP protein immunized mice stimulated with the respective antigen or ConA in comparison with alum control mice. The data is represented as mean stimulation index (SI) 7 S.D. of ten mice from two experiments with five animals per group in each experiment. The asterisk (*) on top of the bars indicate a significantly (P o0.001) high absorbance values compared with control.
mainly influenced by amount of polymer, nature of solvent, the rate of stirring and the method used for preparation. Since, the particle size is an important factor for an efficient delivery of PLA encapsulated antigens to dendritic cells (DCs), which are major cells involved in antigen processing and presentation to MHC molecules. It has been reported that larger size particles (50– 150 mm) will not be taken up by antigen presenting cells or macrophages whereas the particles in the range of 10–70 mm will
have very little chance to be taken up by cells (Howie et al., 1993; Horisawa et al., 2002). The immune responses of single dose of PLA encapsulated antigens (TV and FEP) were studied in mice model in comparison with single dose of alum adsorbed antigens (Alumþ TV/Alum þFEP) or antigens (TV/FEP) alone. Analysis of humoral response showed that the antibody titer induced by PLA encapsulated antigens such as PLA-TV and PLAFEP were 50,000 and 64,000 respectively which was observed
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Table 1 Analysis of cytokine levels (pg/ml) in mice. Animal groups
Unstimulated T cells TV Alum þ TV PLA-TV FEP Alum þ FEP PLA-FEP
Cytokines (pg/ml) IL-2
IL-4
IL-5
IL-10
IFN-γ
2.17 0.5 10.577 0.4 13.6 7 0.3 15.9 7 0.8 10.4 7 0.9 12.54 7 0.3 11.6 7 0.7
1.6 7 0.1 80.9 7 0.7 97.3 7 1.0 307.4a 7 1.7 46.89 7 0.4 53.277 0.7 98.6 7 1.3
2.3 7 0.2 40.7 7 1.1 47.17 0.2 59.6 7 1.1 54.5 7 0.3 69.3 7 0.3 326.8a 7 0.9
1.2 7 0.3 22.5 7 0.4 20.4 7 1.6 30.56 7 1.2 10.6 7 0.5 13.8 7 0.3 28.9 7 0.6
2.7 7 0.3 48.47 0.3 62.7 7 0.4 286.7a 7 0.6 75.4 7 0.2 79.7 7 0.5 274.3a 7 1.1
The cytokine levels were measured in culture supernatants of spleen cells from mice immunized and stimulated in vitro with the corresponding antigen. Experiments were done thrice and the data is represented as mean concentration 7 S.D. a
P o0.0001 compared with other formulation.
between 56th and 63th day after immunization. In the case of alum adsorbed TV/FEP and TV/FEP antigen alone showed maximum antibody titer induction between 14th and 21st day after immunization (6000/9000 and 3000/4000 respectively), followed by very negligible induction was observed in the following days. These results showed that PLA encapsulated antigens were released slowly from the site of injection that leads to sustained communication with antigen presenting cells (Coombes et al., 1999) which induced a significantly (P o0.001) higher antibody titer than other groups indicating the enhanced adjuvanticity of PLA. Similar results were observed in in vitro protein release assay. Overall isotype antibody profile shows that PLA encapsulated filarial antigens (TV/FEP) elicited a high levels of balanced Th1 (IgG2a, IgG2b)/Th2 (IgG1) antibody response. In vivo studies from vast literature reports suggest that all mammalian stages of filarial parasites can be killed by ADCC where large amounts of antibody of different isotypes were involved (Lawrence, 2001). The murine IgG2a and IgG2b isotypes have the ability to fix complement and bind to protein antigens, whereas murine IgG1 is shown to be important because of their property of binding to mast cells and all these subtypes have been reported to participate in ADCC (Akiyama et al., 1984) which correlates with our results. T cells have also been shown to be important for host protection in murine infections with B. malayi and B. pahangi, However their precise role in host protection has not been clearly established (Babu et al., 1999; Vincent et al., 1980). In T cell proliferation assay, the groups immunized with PLA encapsulated TV/FEP proteins showed significantly (Po0.001) high proliferative response than all groups when stimulated with corresponding antigens. The difference in proliferative response is probably due to slow and continuous release of antigens from PLA, which generate persistent immune response since it triggers memory T cells while stimulating with antigen indicating strong recall response produced by the antigen. Other formulations are poorly immunogenic and require several booster doses to induce strong immune response. Previous studies using B. malayi adult worm extract (BmA) and its SDS-PAGE resolved 54–68 kDa fraction F6, also have showed similar proliferative response in mice model when encapsulated with PLA (Saini et al., 2011). The cytokine analysis showed that PLA-TV antigen induced significantly (P o0.0001) high levels of IL-4 and IFN-γ cytokines whereas PLA-FEP group showed significantly (P o0.0001) high levels of IL-5 and IFN-γ cytokines. There was no remarkable difference in the cytokine levels of animals immunized with TV/ FEP with PBS and alum adsorbed TV/FEP antigens compared with controls. The role of Th2 cell cytokine such as IL-4 and IL-5 in elimination of larval parasite has been demonstrated in Onchocerca volvulus infection in BALB/cBYJ mice (Lange et al., 1994). Similarly several recent studies in different animal models of filarial diseases have shown that Th1-type responses are also
involved in the host protection against filarial infection and IFN-γ has been shown to be required for the clearance of B. malayi in mice (Babu et al., 2000). The results indicate that both TV and FEP antigens elicited a balanced Th1/Th2 immune response when the proteins are encapsulated with PLA than other formulation. It is envisioned that the balanced Th1 and Th2 cell responses are sufficient to kill the invading helminths (Maizels and Yazdanbakhsh, 2003) and thus any vaccine candidate which is proved to be promising should be capable of eliciting both antibody and T cell responses in humans, especially in filariasis, since there is a trademark T cell suppression immediately after infection (Lawrence, 2001). In this study we observed that single immunization of PLA encapsulated antigens (both TV and FEP) induced a strong humoral as well as cellular response compared to single dose of other formulations. However, similar responses were achieved for TV (Anugraha et al., 2013) and FEP (Anugraha et al., unpublished data) proteins when the antigens are adsorbed in alum and given as multiple doses. These results show that PLA formulations are found to be more immunogenic and suitable for filarial antigens to generate long lasting immune response from single point immunization as it mounted strong and protective humoral as well as cellular response.
Acknowledgements The authors are thankful to University Grants Commission (UGC) and Council of Scientific and Industrial Research (CSIR) for their financial support. Dr. Rajavel Malligarjunan’s critical comments and positive feedbacks during the preparation of manuscript are gratefully acknowledged. References Absolom, D.R., Zingg, W., Neumann, A.W., 1987. Protein adsorption to polymer particles: role of surface properties. J. Biomed. Mater. Res. 21, 161–171. Akiyama, Y., Lubeck, M.D., Steplewski, Z., Koprowski, H., 1984. Induction of mouse IgG2a- and IgG3-dependent cellular cytotoxicity in human monocytic cells (U937) by immune interferon. Cancer Res. 44, 5127–5131. Anand, S.B., Kodumudi, K.N., Reddy, M.V., Kaliraj, P., 2011. A combination of two Brugia malayi filarial vaccine candidate antigens (BmALT-2 and BmVAH) enhances immune responses and protection in jirds. J. Helminthol. 85, 442–452. Anand, S.B., Murugan, V., Prabhu, P.R., Anandharaman, V., Reddy, M.V., Kaliraj, P., 2008. Comparison of immunogenicity, protective efficacy of single and cocktail DNA vaccine of Brugia malayi abundant larval transcript (ALT-2) and thioredoxin peroxidase (TPX) in mice. Acta Trop. 107, 106–112. Anugraha, G., Jeyaprita, P.J., Madhumathi, J., Sheeba, T., Kaliraj, P., 2013. Immune responses of B. malayi thioredoxin (TRX) and venom allergen homologue (VAH) chimeric multiple antigen for lymphatic filariasis. Acta Parasitol. 58, 468–477. Babu, S., Ganley, L.M., Klei, T.R., Shultz, L.D., Rajan, T.V., 2000. Role of gamma interferon and interleukin-4 in host defense against the human filarial parasite Brugia malayi. Infect. Immun. 68, 3034–3035.
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G. Anugraha et al. / European Journal of Pharmacology 747 (2015) 174–180
Babu, S., Shultz, L.D., Klei, T.R., Rajan, T.V., 1999. Immunity in experimental murine filariasis: roles of T and B cells revisited. Infect. Immun. 67, 3166–3167. Bockarie, M.J., Deb, R.M., 2010. Elimination of lymphatic filariasis: do we have the drugs to complete the job? Curr. Opin. Infect. Dis 23, 617–620. Boehm, G., Peyre, M., Sesardic, D., Huskisson, R.J., Mawas, F., Douglas, A., Xing, D., Merkle, H.P., Gander, B., Johansen, P., 2002. On technological and immunological benefits of multivalent single-injection microsphere vaccines. Pharm. Res. 19, 1330–1336. Coombes, A.G., Lavelle, E.C., Davis, S.S., 1999. Biodegradable lamellar particles of poly(lactide) induce sustained immune responses to a single dose of adsorbed protein. Vaccine 17, 2410–2422. Coombes, A.G., Lavelle, E.C., Jenkins, P.G., Davis, S.S., 1996. Single dose, polymeric, microparticle-based vaccines: the influence of formulation conditions on the magnitude and duration of the immune response to a protein antigen. Vaccine 14, 1429–1438. Dakshinamoorthy, G., Samykutty, A.K., Munirathinam, G., Reddy, M.V., Kalyanasundaram, R., 2013. Multivalent fusion protein vaccine for lymphatic filariasis. Vaccine 31, 1616–1622. Gregory, W.F., Blaxter, M.L., Maizels, R.M., 1997. Differentially expressed, abundant trans-spliced cDNAs from larval Brugia malayi. Mol. Biochem. Parasitol. 87, 85–95. Gupta, R.K., Singh, M., O’Hagan, D.T., 1998. Poly(lactide-co-glycolide) microparticles for the development of single-dose controlled-release vaccines. Adv. Drug Deliv. Rev. 32, 225–246. Gyapong, J.O., Kumaraswami, V., Biswas, G., Ottesen, E.A., 2005. Treatment strategies underpinning the global programme to eliminate lymphatic filariasis. Expert Opin. Pharmacother. 6, 179–200. Hoerauf, A., Pfarr, K., Mand, S., Debrah, A.Y., Specht, S., 2011. Filariasis in Africa— treatment challenges and prospects. Clin. Microbiol. Infect. 17, 977–985. Horisawa, E., Kubota, K., Tuboi, I., Sato, K., Yamamoto, H., Takeuchi, H., Kawashima, Y., 2002. Size-dependency of DL-lactide/glycolide copolymer particulates for intra-articular delivery system on phagocytosis in rat synovium. Pharm. Res. 19, 132–139. Howie, D.W., Manthey, B., Hay, S., Vernon-Roberts, B., 1993. The synovial response to intraarticular injection in rats of polyethylene wear particles. Clin. Orthop. Relat. Res, 352–357. Jones, D.H., 2003. Microencapsulation of vaccine antigens. Methods Mol. Med. 87, 211–222. Katare, Y.K., Muthukumaran, T., Panda, A.K., 2005. Influence of particle size, antigen load, dose and additional adjuvant on the immune response from antigen loaded PLA microparticles. Int. J. Pharm. 301, 149–160. Lange, A.M., Yutanawiboonchai, W., Scott, P., Abraham, D., 1994. IL-4- and IL-5dependent protective immunity to Onchocerca volvulus infective larvae in BALB/ cBYJ mice. J. Immunol. 153, 205–211. Lavelle, E.C., Yeh, M.K., Coombes, A.G., Davis, S.S., 1999. The stability and immunogenicity of a protein antigen encapsulated in biodegradable microparticles based on blends of lactide polymers and polyethylene glycol. Vaccine 17, 512–529. Lawrence, R.A., 2001. Immunity to filarial nematodes. Vet. Parasitol. 100, 33–44. Madhumathi, J., Pradiba, D., Prince, P.R., Jeyaprita, P.J., Rao, D.N., Kaliraj, P., 2010b. Crucial epitopes of Wuchereria bancrofti abundant larval transcript recognized in natural infection. Eur. J. Clin. Microbiol. Infect. Dis. 29, 1481–1486.
Madhumathi, J., Prince, P.R., Anugraha, G., Kiran, P., Rao, D.N., Reddy, M.V., Kaliraj, P., 2010a. Identification and characterization of nematode specific protective epitopes of Brugia malayi TRX towards development of synthetic vaccine construct for lymphatic filariasis. Vaccine 28, 5038–5048. Madhumathi, J., Prince, P.R., Rao, D.N., Kaliraj, P., 2010c. Dominant T-cell epitopes of filarial BmALT-2 and their cytokine profile in BALB/c mice. Parasite Immunol. 32, 760–763. Maizels, R.M., Yazdanbakhsh, M., 2003. Immune regulation by helminth parasites: cellular and molecular mechanisms. Nat. Rev. Immunol. 3, 733–744. Petrovsky, N., Aguilar, J.C., 2004. Vaccine adjuvants: current state and future trends. Immunol. Cell Biol. 82, 488–496. Peyre, M., Audran, R., Estevez, F., Corradin, G., Gander, B., Sesardic, D., Johansen, P., 2004. Childhood and malaria vaccines combined in biodegradable microspheres produce immunity with synergistic interactions. J. Controlled Release 99, 345–355. Prince, P.R., Madhumathi, J., Anugraha, G., Jeyaprita, P.J., Reddy, M.V., Kaliraj, P., 2013. Tandem antioxidant enzymes confer synergistic protective responses in experimental filariasis. J. Helminthol, 1–9. Saini, V., Verma, S.K., Sahoo, M.K., Kohli, D.V., Murthy, P.K., 2011. Sufficiency of a single administration of filarial antigens adsorbed on polymeric lamellar substrate particles of poly (L-lactide) for immunization. Int. J. Pharm. 420, 101–110. Schmidt, C.S., Morrow, W.J., Sheikh, N.A., 2007. Smart adjuvants. Expert Rev. Vaccines 6, 391–400. Sharmila, S., Christiana, I., Kiran, P., Reddy, M.V., Kaliraj, P., 2011. The adjuvant-free immunoprotection of recombinant filarial protein Abundant Larval Transcript-2 (ALT-2) in Mastomys coucha and the immunoprophylactic importance of its putative signal sequence. Exp. Parasitol. 129, 247–253. Shi, L., Caulfield, M.J., Chern, R.T., Wilson, R.A., Sanyal, G., Volkin, D.B., 2002. Pharmaceutical and immunological evaluation of a single-shot hepatitis B vaccine formulated with PLGA microspheres. J. Pharm. Sci. 91, 1019–1035. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M. D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. Taylor, M.J., Hoerauf, A., Bockarie, M., 2010. Lymphatic filariasis and onchocerciasis. Lancet 376, 1175–1185. Thirugnanam, S., Pandiaraja, P., Ramaswamy, K., Murugan, V., Gnanasekar, M., Nandakumar, K., Reddy, M.V., Kaliraj, P., 2007. Brugia malayi: comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT2 protein and prime-boost vaccine regimens in a jird model. Exp. Parasitol. 116, 483–491. Vanam, U., Pandey, V., Prabhu, P.R., Dakshinamurthy, G., Reddy, M.V., Kaliraj, P., 2009a. Evaluation of immunoprophylactic efficacy of Brugia malayi transglutaminase (BmTGA) in single and multiple antigen vaccination with BmALT-2 and BmTPX for human lymphatic filariasis. Am. J. Trop. Med. Hyg. 80, 319–324. Vanam, U., Prabhu, P.R., Pandey, V., Dakshinamurthy, G., Reddy, M.V., Perumal, K., 2009b. Immune responses generated by intramuscular DNA immunization of Brugia malayi transglutaminase (BmTGA) in mice. Parasitology 136, 887–894. Vincent, A.L., Sodeman, W.A., Winters, A., 1980. Development of Brugia pahangi in normal and nude mice. J. Parasitol. 66, 448. Witschi, C., Doelker, E., 1998. Influence of the microencapsulation method and peptide loading on poly(lactic acid) and poly(lactic-co-glycolic acid) degradation during in vitro testing. J. Controlled Release 51, 327–341.
