Vaccines composed of whole cells or outer membrane envelope are available in some countries to prevent human leptospirosis, and clinical trials have been reported [20
]. In view of their disadvantages, especially their inability to elicit longer-term protection against different serovars of pathogenic leptospires, efforts have been focused on developing subunit vaccines[24
]. During recent years, Hap1[25
] (also known as LipL32[26
]), LipL41, OmpL1[27
] and Lig[28
] proteins have been identified as promising vaccine candidates for preclinical trials.
The availability of complete genome sequence information for many pathogens and the development of sophisticated computer programs have led to a new paradigm in vaccine development. Now it is possible to screen potential vaccine candidate genes in a reverse manner starting from the genome. This reverse vaccinology was first applied to MenB[30
] and is now applied routinely in vaccine development, as in the search for vaccines against S. pneumoniae, Streptococcus agalactiae, Staphylococcus aureus, Porphyromonas gingivalis, Chlamydia pneumoniae
and other microorganisms[10
]. Bioinformatics analysis is the first important strategy of reverse vaccinology. Gram-negative bacteria have five subcellular location sites: cytoplasm, inner membrane, outer membrane, periplasm and extracellular space. The surface-exposed proteins, i.e. those located in sites other than the cytoplasm, are the most suitable vaccine candidates because they are more susceptible to antibody recognition and can therefore elicit protective immune responses. Many sophisticated computer programs have been developed to predict the subcellular locations of putative proteins in the whole genome [31
]. Analyzing the gene transcription profile using DNA microarrays provides a second vaccine candidate selection strategy in reverse vaccinology. A gene having a fluorescent signal above the median value corresponds to an expression level higher than 5–10 mRNA copies per genome[34
]. Those highly expressed genes could be potential vaccine candidates[34
]. Finally, other approaches such as proteomic technology can be used to screen vaccine candidates. Using combined these strategies, genes encoding potential vaccine antigens can eventually be identified.
In our preliminary selection, all genes in L. interrogans
strain #56601 were searched using P-CLASSIFIER, a system for predicting the subcellular locations of proteins on the basis of amino acid subalphabets and a combination of multiple support vector machines[33
]. Moreover, four topologies were predicted by the corresponding programs. Proteins predicted to be surface-exposed and having any of these four topologies were screened as preliminary vaccine candidates. All proteins with more than four predicted transmembrane spanning regions were removed from the list of candidates, not only because they are likely to be completely embedded in the cell membrane and therefore inaccessible to antibodies, but also because they are difficult to express in E. coli
]. We retained the genes shared by the two sequenced serovars and subtracted genes that had human homologues. The reason we subtracted human homologues is they are likely to cause problems of autoimmunity[35
]. Finally, we narrowed the list of vaccine candidates to 616 genes in the genome of L. interrogans
In order to explore vaccine candidates that could generate cross-protection against the diverse serovars of leptospires, we applied CGH to identify genes that are conserved among the ten pathogenic strains involved in most infections[36
]. This approach allowed us to refine the vaccine candidate shortlist further by eliminating antigens that were not conserved among these serovars. The 565 vaccine candidates not only presumably surface-exposed but also conserved among the ten prevalent serovars in China were identified as the result of this approach.
Transcriptome analysis was performed using DNA microarrays of L. interrogans in order to assess the transcription levels of all genes in the genome. A graph of the signal obtained for each gene gave a diagonal distribution reflecting the expression level of that gene. After subtracting genes with transcriptional levels below the median, we were left with 226 genes as vaccine candidates.
Applying the theory of reverse vaccinology, 226 genes had been identified as potential vaccine candidates against L. interrogans
combined bioinformatics, CGH and transcriptional analysis. Among them, 60.6% (137/226) have COG annotations; thus, nearly 40% either have an unknown function or have no COG annotation. This group of gene products offers great promise as it comprises a pool of previously unexploited vaccine targets. To evaluate our results, we compared our candidates with those identified by others. Gamberini et al. (2005) found approximately 20% potential surface proteins using in silico
approach, and sixteen proteins were recognized by antibodies present in human sera[15
]. However, only three of them (LA0222, LA2637 and LA2741) appear in our final set. This is not unexpected, since 206 genes encoding hypothetical or unknown proteins were selected from approximately 20% of the genome for cloning and expression. Nally et al. (2005) characterized 32 proteins in outer membrane vesicles of L. interrogans
serovar Copenhageni by two-dimensional gel electrophoresis, including previously-described outer membrane proteins (OMPs); in addition, unknown, hypothetical and putative OMPs were also identified[17
]. Interestingly, only two proteins (LA0222 and LA2637) are represented among the sixteen proteins found by Gamberini and co-workers. There is an overlap of eight genes between our result and that of Nally et al. (2005) (LA0222, LA0505, LA0616, LA1495, LA2024, LA2295, LA2637 and LA3091). The reasons responsible for the discrepancies among the results may be due to differing methodologies. Genomics, transcriptional profiling and proteomics have emerged in the post genomic era with potential to speed up the vaccine discovery research process. It should be pointed out that those methods have their respective advantages and limitations, and can be complementally utilized in the development of the novel vaccines. Genomics involves the use of various softwares to predict sublocalization of proteins. However, some algorithms have limited accuracy. Although transcriptome analysis uses gene chip array to measure gene expression but suffers from the fact that mRNA levels may not reflect protein levels. Expression of a transcribed gene may be regulated at the level of translation. It is believed that the proteome maps of microorganisms are important to understand cellular status at the protein level, which cannot be deciphered from genome or transcriptome analysis[37
]. Proteomics of outer membrane can rapidly identify almost all proteins in outer membrane. However, some of the proteins identified in membrane preparations are in fact typical cytoplasmic proteins[10
]. Moreover, one of the major disadvantages of subproteomic studies by 2-D gel electrophoresis and mass spectrometry is the potential for contamination via leaky fractionation or lysis[39
]. Nally et al. (2005) also revealed that outer membrane vesicles contain small amounts of inner membrane or cytoplasmic proteins in their proteomic study[17
]. It is worth mentioning here that mainly surface-exposed proteins such as LipL32 (LA2637)[26
], LipL41 (LA0616)[27
], LipL45 (LA2295)[41
] and LipL21 (LA0011)[42
] have higher transcriptional levels in our results; this suggests that the genes with higher transcriptional levels identified in our current research may be preferable for development as vaccine candidates.
This is the first time that CGH and transcription analysis have been used to identify potential candidates for vaccines against L. interrogans
. Our present work corroborates previous studies, showing the advantages of reverse vaccinology[8
]. The next step following our present research is to verify whether the selected vaccine candidates are surface-exposed and to evaluate the protective activities of these proteins. Such studies will lead to the development of safe and effective new vaccines against leptospirosis in the future.