Preparation and in-vitro characterization of alginate microspheres incorporating leptospiral antigens as a delivery system and adjuvant

Document Type: Original Articles


1 Department of Microbiology, Faculty of Basic Sciences, Islamic Azad University, Karaj Branch, Karaj, Iran

2 Department of Microbiology, Razi Vaccine and Serum Research Institute, Karaj, Iran

3 Department of Venom and Human Sera, Razi Vaccine and Serum Research Institute, Karaj, Iran


Leptospirosis is one of the most prevalent zoonotic diseases worldwide. Currently, multivalent whole-cell leptospiral vaccines can induce protection against leptospirosis. Therefore, preparation and formulation of new generations of vaccines that can stimulate long-term immunity for leptospirosis control are essential. The aim of this study was to prepare and characterize alginate microspheres as an antigen delivery system for immunization against leptospirosis. We used five Leptospira interrogans serovars, namely, Icterohaemorrhagiae, Grippotyphosa, Serjo harjo, Pomona, and Canicola, for antigen preparation. Alginate microspheres containing leptospiral antigen (LA) were prepared by an emulsification method and evaluated with respect to morphology, size distribution, loading efficiency (LE), loading capacity (LC), and release profile. The effects of concentration of alginate and emulsifiers and stirring rate on characteristics of microspheres were investigated. The optimal condition parameters for the preparation of LA-loaded alginate microspheres were estimated. The optimum concentrations obtained for alginate and emulsifiers were 3.65% (w/v), Span 80 (0.24% w/v), and Tween 80 (3.85% w/v), respectively. Moreover, the appropriate homogenization rate was 500 rpm. Our results showed mean particle size of 200 μm, 97.41% LE, and 8% LC for the microspheres. Sufficient release profile was observed for in-vitro release test of LA from alginate microspheres over an extended period of time (216 hour). Therefore, alginate microspheres technologically seem to be a promising antigen delivery system for leptospiral vaccine.


Main Subjects

Article Title [French]

Préparation et caractérisation in-vitro des microsphères d’alginate chargées d’antigènes leptospiraux en tant que système de délivrance et adjuvant

Abstract [French]

La leptospirose est l’une des zoonoses les plus courantes à travers le monde. Actuellement, la protection contre la leptospirose peut être induite par un vaccin constitué de germes leptospiraux entiers. De ce fait, la préparation et la formulation d’une nouvelle génération de vaccins capable de favoriser une immunité à long terme sont essentielles pour le contrôle de cette maladie. L’objectif de cette étude était de préparer et caractériser un nouveau système de délivrance composé de microsphères d’alginate pour l’immunisation contre la leptospirose. Pour la préparation des antigènes, 5 sérotypes de Leptospira interrogans à savoir Icterohaemorrhagiae, Grippotyphosa, Serjo harjo, Pomona, et Canicola on été utilisés. Les microsphères d’alginate contenant les antigènes leptospiraux (AL) ont été préparées par émulsification et ensuite analysées en termes de morphologie, granulométrie, efficacité de chargement (EC), capacité de chargement (CC) et profil de libération. De plus, l’influence des concentrations en alginate et en émulsifiant a également été étudiée. Enfin, les conditions optimales de préparation pour les microsphères d’alginate chargées d’AL ont été déterminées. Selon nos résultats, les concentrations optimales de préparation étaient respectivement de 3,65% p/v (pourcentage de poids par volume) d’alginate dans l’émulsifiant, 0,24% v/v de Span 80 et 3,85% v/v de Tween 80. De plus, le taux d’homogénéisation approprié a été obtenu à 500 rpm. La taille moyenne des particules générées était de 200 nm avec un EC de 97,41% et un CC de 8%. Les testes de libération des AL à partir des microsphères d’alginate montrent un profil de libération étendu sur une période prolongée de 216 heures. Pa conséquent, sur le plan technique, les microsphères d’alginate semblent représenter un système de délivrance prometteur pour le vaccin leptospiral.

Keywords [French]

  • Leptospira spp
  • Antigène leptospiral
  • Alginate
  • Microsphères
  • Délivrance d’antigène
  • Immunisation
Adler, B., Moctezuma, A.D., 2009. Leptospira and leptospirosis. Veterinary Journal of Microbiology 4, 4382-4392.

Anal, A.K., Bhopatkar, D., Tokura, S., Tamura, H., Stevens, W.F., 2003. Chitosan-alginate multilayer beads for gastric passage and controlled intestinal release of protein. Drug Dev Ind Pharm 29, 713-724.

Andre-Fontaine, G., Branger, C., Gray, A.W., Klaasen, H.L., 2003. Comparison of the efficacy of three commercial bacterins in preventing canine leptospirosis. Vet Rec 153, 165-169.

Arenas-Gamboa, A.M., Ficht, T.A., Kahl-McDonagh, M.M., Rice-Ficht, A.C., 2008. Immunization with a single dose of a microencapsulated Brucella melitensis mutant enhances protection against wild-type challenge. Infect Immun 76, 2448-2455.

Behboudi, S., Morein, B., Rönnberg, B., 1995. Isolation and quantification of Quillaja saponaria Molina saponins and lipids in iscom-matrix and iscoms. Vaccine 13, 1690-1696.

Cho, N.-H., Seong, S.-Y., Chun, K.-H., Kim, Y.-H., Chan Kwon, I., Ahn, B.-Y., Jeong, S.Y., 1998. Novel mucosal immunization with polysaccharide–protein conjugates entrapped in alginate microspheres. Journal of Controlled Release 53, 215-224.

Dobakhti, F., Ajdary, S., Taghikhani, M., Rafiei, S., Bayati, K., Rafiee-Tehrani, M., 2006. Immune Response Following Oral Immunization with BCG Encapsulated in Alginate Microspheres. Iran J Immunol 3, 114-120.

Faisal, S.M., Yan, W., McDonough, S.P., Chang, C.F., Pan, M.J., Chang, Y.F., 2009a. Leptosome-entrapped leptospiral antigens conferred significant higher levels of protection than those entrapped with PC-liposomes in a hamster model. Vaccine 27, 6537-6545.

Faisal, S.M., Yan, W., McDonough, S.P., Chang, Y.F., 2009b. Leptospira immunoglobulin-like protein A variable region (LigAvar) incorporated in liposomes and PLGA microspheres produces a robust immune response correlating to protective immunity. Vaccine 27, 378-387.

Flick-Smith, H.C., Eyles, J.E., Hebdon, R., Waters, E.L., Beedham, R.J., Stagg, T.J., Miller, J., Alpar, H.O., Baillie, L.W., Williamson, E.D., 2002. Mucosal or parenteral administration of microsphere-associated Bacillus anthracis protective antigen protects against anthrax infection in mice. Infect Immun 70, 2022-2028.

Florindo, H.F., Pandit, S., Goncalves, L.M., Alpar, H.O., Almeida, A.J., 2008. Streptococcus equi antigens adsorbed onto surface modified poly-epsilon-caprolactone microspheres induce humoral and cellular specific immune responses. Vaccine 26, 4168-4177.

Gupta, R.K., 1998. Aluminum compounds as vaccine adjuvants. Advanced Drug Delivery Reviews 32, 155-172.

Jin, M., Zheng, Y., Hu, Q., 2009. Preparation and characterization of bovine serum albumin alginate/chitosan microspheres for oral administration. Pharmacological Sciences 4, 215-220.

Kim, B., Bowersock, T., Griebel, P., Kidane, A., Babiuk, L.A., Sanchez, M., Attah-Poku, S., Kaushik, R.S., Mutwiri, G.K., 2002. Mucosal immune responses following oral immunization with rotavirus antigens encapsulated in alginate microspheres. Journal of Controlled Release 85, 191-202.

Klimpel, G.R., Matthias, M.A., Vinetz, J.M., 2003. Leptospira interrogans Activation of Human Peripheral Blood Mononuclear Cells: Preferential Expansion of TCRγδ+ T Cells vs TCRαβ+ T Cells. The Journal of Immunology 171, 1447-1455.

Krasaekoopt, W., Bhandari, B., Deeth, H., 2004. The influence of coating materials on some properties of alginate beads and survivability of microencapsulated probiotic bacteria. International Dairy Journal 14, 737-743.

Lemoine, D., Wauters, F., Bouchend'homme, S., Préat, V., 1998. Preparation and characterization of alginate microspheres containing a model antigen. International Journal of Pharmaceutics 176, 9-19.

Rodrigues, A.P., Hirsch, D., Figueiredo, H.C.P., Logato, P.V.R., Moraes, Â.M., 2006. Production and characterisation of alginate microparticles incorporating Aeromonas hydrophila designed for fish oral vaccination. Process Biochemistry 41, 638-643.

Sangeetha, S., Nagasamy Venkatesh, D., Adhiyaman, R., Santhi, K., Suresh, B., 2007. Formulation of Sodium Alginate Nanospheres Containing Amphotericin B for the Treatment of Systemic Candidiasis. Tropical Journal of Pharmaceutical Research 6, 653-659.

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.

Stevenson, B., Choy, H.A., Pinne, M., Rotondi, M.L., Miller, M.C., Demoll, E., Kraiczy, P., Cooley, A.E., Creamer, T.P., Suchard, M.A., Brissette, C.A., Verma, A., Haake, D.A., 2007. Leptospira interrogans endostatin-like outer membrane proteins bind host fibronectin, laminin and regulators of complement. PLoS One 2, e1188.

Tafaghodi, M., Sajadi Tabasi, S.A., Jaafari, M.R., 2006. Formulation, characterization and release studies of alginate microspheres encapsulated with tetanus toxoid. J Biomater Sci Polym Ed 17, 909-924.

Tafaghodi, M., Sajadi Tabasi, S.A., Payan, M., 2007. Alginate Microsphere as a Delivery System and Adjuvant for Autoclaved Leishmania major and Quillaja Saponin: Preparation and Characterization. Iranian Journal of Pharmacological Sciences 3, 61-68.

Wan, L.S., Heng, P.W., Chan, L.W., 1992. Drug encapsulation in alginate microspheres by emulsification. J Microencapsul 9, 309-316.

Wan, L.S.C., Heng, P.W.S., Chan, L.W., 1994. Surfactant effects on alginate microspheres. International Journal of Pharmaceutics 103, 267-275.

Yan, W., Faisal, S.M., McDonough, S.P., Divers, T.J., Barr, S.C., Chang, C.F., Pan, M.J., Chang, Y.F., 2009. Immunogenicity and protective efficacy of recombinant Leptospira immunoglobulin-like protein B (rLigB) in a hamster challenge model. Microbes Infect 11, 230-237.

Yeh, M.-K., Liu, Y.-T., Chen, J.-L., Chiang, C.-H., 2002. Oral immunogenicity of the inactivated Vibrio cholerae whole-cell vaccine encapsulated in biodegradable microparticles. Journal of Controlled Release 82, 237-247.

Zheng, C.H., Gao, J.Q., Zhang, Y.P., Liang, W.Q., 2004. A protein delivery system: biodegradable alginate-chitosan-poly(lactic-co-glycolic acid) composite microspheres. Biochem Biophys Res Commun 323, 1321-1327.