In silico and microarray-based genomic approaches to identifying potential vaccine candidates against Leptospira interrogans

Abstract

BackgroundCurrently available vaccines against leptospirosis are of low efficacy, have an unacceptable side-effect profile, do not induce long-term protection, and provide no cross-protection against the different serovars of pathogenic leptospira. The current major focus in leptospirosis research is to discover conserved protective antigens that may elicit longer-term protection against a broad range of Leptospira. There is a need to screen vaccine candidate genes in the genome of Leptospira interrogans.ResultsBioinformatics, comparative genomic hybridization (CGH) analysis and transcriptional analysis were used to identify vaccine candidates in the genome of L. interrogans serovar Lai strain #56601. Of a total of 4727 open reading frames (ORFs), 616 genes were predicted to encode surface-exposed proteins by P-CLASSIFIER combined with signal peptide prediction, α-helix transmembrane topology prediction, integral β-barrel outer membrane protein and lipoprotein prediction, as well as by retaining the genes shared by the two sequenced L. interrogans genomes and by subtracting genes with human homologues. A DNA microarray of L. interrogans strain #56601 was constructed for CGH analysis and transcriptome analysis in vitro. Three hundred and seven differential genes were identified in ten pathogenic serovars by CGH; 1427 genes had high transcriptional levels (Cy3 signal ≥ 342 and Cy5 signal ≥ 363.5, respectively). There were 565 genes in the intersection between the set encoding surface-exposed proteins and the set of 307 differential genes. The number of genes in the intersection between this set of 565 and the set of 1427 highly transcriptionally active genes was 226. These 226 genes were thus identified as putative vaccine candidates. The proteins encoded by these genes are not only potentially surface-exposed in the bacterium, but also conserved in two sequenced L. interrogans. Moreover, these genes are conserved among ten epidemic serovars in China and have high transcriptional levels in vitro.ConclusionOf the 4727 ORFs in the genome of L. interrogans, 226 genes were identified as vaccine candidates by bioinformatics, CGH and transcriptional analysis on the basis of the theory of reverse vaccinology. The proteins encoded by these genes might be useful as vaccine candidates as well as for diagnosis of leptospirosis.

The OmpA-Like Protein Loa22 Is Essential for Leptospiral Virulence

“…One such determinant, loa22, is up-regulated during host infection [17] and encodes a lipoprotein with an OmpA domain [14] that is strongly recognized by sera from human leptospirosis patients [15]. Furthermore, Loa22 is conserved among pathogenic Leptospira [14–16], suggesting that it may play a specific role in disease pathogenesis. However, its role has not been elucidated until now, because targeted mutagenesis was not previously feasible in pathogenic Leptospira .…”

Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen

“…possess a much higher number of lipoprotein genes than that of other bacterial genomes. Analysis of the genome sequences of L. interrogans led to the detection of approximately 145 putative lipoproteins 98 and several putative extracellular and outer membrane proteins 99, 100 .…”

“…The L. interrogans and borgpetersenii genomes share 2708 ORFs, of which 656 are not present in the L. biflexa genome 16, 17 (Box 2). Strategies to refine the number of target candidates include sequencing of a wider representation of pathogenic Leptospira genomes and bioinformatic analysis and selection of ORFs which are highly conserved among these genomes and encode outer membrane proteins 100 . Yet the major barrier in pursuing this strategy is the lack of in vitro correlates for immunity against leptospirosis.…”

Recombinant vaccines against Leptospirosis

“…Research has therefore concentrated on identifying virulence determinants such as the surface-exposed antigens recognized by sera collected from leptospirosis patients, [66][67][68] and bioinformatics analysis of the Leptospira genome sequences has identified homologs to vaccine candidates in other pathogens, Figure 1. 69,70 Many leptospiral proteins have since been tested as potential vaccine candidates and the most promising antigens used in vaccine preparations (subunit, DNA, Adenovirus and BCG vaccine constructs), to date are described, see Table 1.…”