Mimetic membrane system to carry multiple antigenic proteins from Leishmania amazonensis.
Liposomes have long been used as models for lipid membranes and for the reconstitution of a single or multiple proteins. Also, liposomes have adjuvant activity in vaccines against several protozoan or bacterial organisms. Thus, the main objective of the present study was to obtain a crude extract of detergent-solubilized proteins of Leishmania amazonensis amastigotes and reconstitute them into liposomes. Neutral and zwiterionic detergents were less efficient than an ionic detergent. In order to obtain efficient solubilization using only sodium dodecyl sulfate (SDS), the effects of detergent and protein concentration and incubation time were studied. The maximum of solubilized proteins was obtained instantaneously using a ratio of 0.5 mg/ml of protein to 0.1% (w/v) detergent at 4 degrees C. Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS) and cholesterol in a weight ratio of 5:1:4 were used for protein reconstitution into liposomes using the cosolubilization method, yielding 60% of incorporation. The incorporation of multiple parasite proteins results in a vesicular diameter of proteoliposomes of about 140 nm, presenting a final lipid weight ratio for DPPC, DPPS and cholesterol of 1:1:5, with high stability. The detergent-solubilized proteins of L. amazonensis amastigotes present in the proteoliposome, when analyzed by SDS-polyacrylamide gel electrophoresis, include a wide range of parasite-incorporated proteins. BALB/c mice inoculated with these proteoliposomes were able to produce antibodies against the proteins reconstituted in DPPC:DPPS:cholesterol liposomes and were partially resistant to infection with L. amazonensis promastigotes. These results indicate that this system can be used as a possible vaccine against L. amazonensis.
Santos
Fabiana R
FR
Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14049-900 Ribeirão Preto, São Paulo, Brasil.
Ferraz
Denise B
DB
Daghastanli
Katia R P
KR
Ramalho-Pinto
F Juarez
FJ
Ciancaglini
Pietro
P
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000276:immunology* / Q000302:isolation & purification; D040761:Biomimetic Materials / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry; D004337:Drug Carriers; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007891:Leishmania / Q000276:immunology*; D007896:Leishmaniasis / Q000276:immunology*; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D021962:Membrane Microdomains; D051379:Mice; D008807:Mice, Inbred BALB C; D010718:Phosphatidylserines / Q000737:chemistry; D011510:Proteolipids / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D004337:Drug Carriers; D008081:Liposomes; D010718:Phosphatidylserines; D011510:Proteolipids; C020485:proteoliposomes; D015060:1,2-Dipalmitoylphosphatidylcholine; C038694:dipalmitoylphosphatidylserine; D012967:Sodium Dodecyl Sulfate; D002784:Cholesterol