RESUMO
Leptospirosis is a bacterial disease caused by bacteria of the genus Leptospira affecting humans and animals. Untreated leptospirosis may result in severe kidney damage, meningitis, liver failure, respiratory distress, and even death. Virulent leptospirosis can rapidly enter kidney fibroblasts and induce a programmed cell death. Thus, it is a challenge for immunologists to develop an effective and safe leptospirosis vaccine.Here, we compared the commercial canine leptospira vaccine and recombinant proteins (OmpL1 and LipL41) with and without adjuvant in terms of immune response and challenge studies in hamsters and immune response studies alone in experimental dogs. The outer membrane proteins viz., lipL41 and OmpL1 of leptospira interrogans serovars icterohaemorrhagiae were amplified. The primers were designed in such a way that amplified products of OmpL1 and lipL41 were ligated and cloned simultaneously into a single vector. The cloned products were expressed in E.coli BL21 cells. The immunoprotection studies were conducted for both recombinant proteins and commercial vaccine. The challenge experiment studies revealed that combination of both rLip41 and rOmpL1 and commercial vaccine gave 83% and 87% protection, respectively. Histopathological investigation revealed mild sub lethal changes were noticed in liver and kidney in commercially vaccinated group alone. The immune responses against recombinant leptospiral proteins were also demonstrated in dogs.
RESUMO
El objetivo de este estudio fue determinar la diversidad molecular de las proteínas OmpL1, LipL32, LipL41, LigA y LigB y de los genes que las codifican mediante análisis bioinformáticos en diferentes cepas patógenas de Leptospira spp., a partir de la información disponible en las bases de datos. Se utilizaron las secuencias de aminoácidos de las proteínas OmpL1, LipL32, LipL41, LigA y LigB, así como las de los genes que las codifican en las cepas de Leptospira spp. registradas en The National Center for Biotechnology Information (NCBI). Los análisis de las proteínas y los genes se realizaron mediante los recursos Protein, Nucleotide y Gene del NCBI. La alineación de las secuencias consenso se realizó con las herramientas PSI-BLAST y BLASTn. El porcentaje de cobertura de las secuencias seleccionadas de los genes ompL1, lipL32, lipL41, ligA y ligB en cepas patógenas de Leptospira spp. es de 100% para ompL1, lipL32 y lipL41, 75% para ligA y 99% para ligB con porcentajes de identidad de 85, 98, 88, 90 y 80% respectivamente; el porcentaje de cobertura de las secuencias seleccionadas de las proteínas es de 100, 77, 99, 100 y 100% con porcentajes de identidad de 90, 99, 92, 63 y 60% respectivamente, lo cual indica que los genes y las proteínas, excepto las proteínas LigA y LigB, son bastante conservadas en los diferentes serovares patógenos de Leptospira spp. Según dichos resultados, se recomienda realizar análisis complementarios de estas proteínas con el fin de determinar si es viable su uso como candidatos vacunales.
The aim of this study was to determine the molecular diversity of OmpL1, LipL32, LipL41, LigA and LigB proteins and that of the genes that encode them using bioinformatic analysis in different pathogenic strains of Leptospira spp. based on the information available in databases. The amino acid sequences of OmpL1, LipL32, LipL41, LigA and LigB proteins were used, as well as the genes encoding them in strains of Leptospira spp. reported at The National Center for Biotechnology Information (NCBI). The analysis of proteins and genes were performed using the Protein, Nucleotide and Gene resources from the NCBI. The alignment of the consensus sequences was performed using the PSI-BLAST and BLASTn tools. The coverage percentage of the selected sequences ofthe ompL1, lipL32, lipL41, ligA and ligB genes in pathogenic strains of Leptospira spp. is 100% for ompL1, lipL32 and lipL41, 75% for ligA and 99% for ligB with identity percentages of 85, 98, 88, 90 and 80% respectively; the coverage percentage of the selected protein sequences is 100, 77, 99, 100 and 100% with identity percentages of 90, 99, 92, 63 and 60% respectively, indicating that genes and proteins, except LigA and LigB proteins, are highly conserved in various pathogenic serovars of Leptospira spp. According to these results, it is recommended that further analysis of these proteins be made in order to determine the feasibility of its use as vaccine candidates.
El objetivo de este estudo foi determinar a diversidade molecular das proteínas OmpL1, LipL32, LipL41, LigA e LigB e dos genes que as codificam mediante análises bioinformáticas em diferentes cepas patógenas de Leptospira spp. A partir da informação disponível nas bases de dados. Utilizaram-se as sequências de aminoácidos das proteínas OmpL1, LipL32, LipL41, LigA e LigB, assim como as dos genes que as codificam nas cepas de Leptospira spp. Reportadas em The National Center for Biotechnology Information (NCBI). As análises das proteínas e dos genes se realizaram mediante os recursos Protein, Nucleotide e Gene do NCBI. O alinhamento das sequências consenso se realizou com as ferramentas PSI-BLAST e BLASTn. A porcentagem de cobertura das sequências selecionadas dos genes ompL1, lipL32, lipL41, ligA e ligB em cepas patógenas de Leptospira spp. É de 100% para ompL1, lipL32 e lipL41, 75% para ligA e 99% para ligB com porcentagens de identidade de 85, 98, 88, 90 e 80% respectivamente; A porcentagem de cobertura das sequências selecionadas das proteínas é de 100, 77, 99, 100 e 100% com porcentagens de identidade de 90, 99, 92, 63 e 60% respectivamente, o que indica que os genes e as proteínas, exceto as proteínas LigA e LigB, são altamente conservadas nos diferentes serovares patogênicos de Leptospira spp. Segundo esses resultados, se recomenda realizar análises complementares destas proteínas com finalidade de determinar se é viável o seu uso como candidatos para vacina.
RESUMO
Objective To screen the efficient antigenic epitopes in genus-specific envelope proteins OmpL1 and LipL21 of Leptospira interrogans for further development of multiple antigenic peptide (MAP)vaccine.Methods Based on bioinformatic technique,the combined epitopes of T and B lymphcytes in OmpL1 and LipL21 molecules were screened.Nucleotide fragments of each epitopes were amplified by PCR and then constructed their phage display systems.Using antisera against rOmpL1,rLipL21,L.interrogans serogroup Icterohaemorrhagiae strain Lai and leptospirosis patients' sera as the first antibodies.respectively,Western blot assays were performed to determine the immunoreaetivity and reactive ability of the epitopes with different antisera.Resuits Four combined epitopes of OmpL1 and two combined epitopes of LipL21 were selected out by the predicting procedure.All the amplified epitope fragments were accurately inserted into the region at N end of phage PⅢ protein and successfully expressed.All of the antisera could recognize each of the epitopes.Based on the results of Western blot,the two LipL21 epitopes at 97-112 and 176-184 showed similar strong hybridization signals with any of the antisera,and the hybridization signals of four OmpL1 epitopes with the three antisera were 173-191,87-98,297-320 and 59-78,from strong to weak.Conclusion The six combined epitopes in this study are efficiently antigenic.And the epitopes at positions 97-112 and 176-184 in LipL21 as well as the epitopes at position 87-98 and 173-191 in OmpL1 have a potential for developing leptospiral MAP vaccine.