ABSTRACT
Leptospirosis, caused by bacteria of the genus Leptospira, is a direct zoonosis with wide geographical distribution. The implications in terms of public health and the economical losses caused by leptospirosis justify the use of a vaccine against Leptospira in human or animal populations at risk. In this study, we used the external membrane protein LipL32 as a model antigen, as it is highly immunogenic. The LipL32 coding sequence was cloned into several expression vectors: (i) pTarget, to create a DNA vaccine; (ii) pUS973, pUS974, and pUS977 for expression in BCG (rBCG); and (iii) pAE, to express the recombinant protein in Escherichia coli, for a subunit vaccine. Mice were immunized with the various constructs, and the immune response was evaluated. The highest humoral immune response was elicited by the subunit vaccine (rLipL32). However, with rBCG, the titer was still rising at the end of the experiment. The serum of vaccinated animals was able to recognize LipL32 on the membrane of the Leptospira, detected by indirect immunofluorescence. A monoclonal antibody anti-LipL32 was shown to inhibit the growth of Leptospira in vitro, indicating potential protection induced by the LipL32 antigen.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/immunology , Leptospira/immunology , Leptospirosis/prevention & control , Lipoproteins/immunology , Animals , Antibodies, Bacterial/immunology , Antibody Formation , Bacterial Outer Membrane Proteins/genetics , Bacterial Vaccines/genetics , Female , Lipoproteins/genetics , Mice , Mice, Inbred BALB C , Vaccines, DNA/genetics , Vaccines, DNA/immunology , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccines, Synthetic/immunologyABSTRACT
Foram obtidos dois hibridomas secretores de anticorpos monoclonais (MAbs) que reagem com uma lipoproteína (LipL32) da membrana externa de leptospiras patogênicas. Para a produção dos hibridomas, células do baço de camundongos BALB/c, imunizados com LipL32 recombinante (rLipL32), foram fusionadas com células SP2/O-Ag14, selecionadas em meio HAT e testadas em ELISA indireto. Um dos MAbs secretados pelos hibridomas é do isotipo IgG2b e o outro do isotipo IgM. A especificidade dos MAbs foi confirmada em ELISA indireto e immunoblotting usando rLipL32 purificada, Escherichia coli (E. coli) expressando LipL32 e sorovares patogênicos e saprófitas. Os dois MAbs reagiram com a maioria dos sorovares patogênicos e não reagiram com sorovares saprófitas. Os MAbs possuem potencial para uso em testes de diagnóstico de leptospirose.
ABSTRACT
Two hybridomas secreting monoclonal antibodies (MAbs) that react with a lipoprotein (LipL32) of the outer membrane of pathogenic Leptospira were obtained. For hybridoma production, spleen cells from BALB/c mice imunized with recombinant LipL32 (rLipL32) were fused to SP2/O-Ag14 cells, selected in HAT medium and screened in an indirect ELISA. One MAb produced was of the IgG2b isotype and the other was an IgM. MAbs specificity was confirmed by indirect ELISA and immunoblotting using purified rLipL32 and whole-cell antigen preparations from Escherichia coli (E. coli) expressing LipL32 and from pathogenic and non-pathogenic serovars. Both Mabs reacted with most of the pathogenic serovars tested and none reacted with non-pathogenic Leptospira. The MAbs described have potential for use in diagnostic tests for leptospirosis.
Foram obtidos dois hibridomas secretores de anticorpos monoclonais (MAbs) que reagem com uma lipoproteína (LipL32) da membrana externa de leptospiras patogênicas. Para a produção dos hibridomas, células do baço de camundongos BALB/c, imunizados com LipL32 recombinante (rLipL32), foram fusionadas com células SP2/O-Ag14, selecionadas em meio HAT e testadas em ELISA indireto. Um dos MAbs secretados pelos hibridomas é do isotipo IgG2b e o outro do isotipo IgM. A especificidade dos MAbs foi confirmada em ELISA indireto e immunoblotting usando rLipL32 purificada, Escherichia coli (E. coli) expressando LipL32 e sorovares patogênicos e saprófitas. Os dois MAbs reagiram com a maioria dos sorovares patogênicos e não reagiram com sorovares saprófitas. Os MAbs possuem potencial para uso em testes de diagnóstico de leptospirose.
