Failure of LIC13435 Protein of Leptospira interrogans Serovar Copenhageni to Confer Protection in Immunized Hamsters

Leptospirosis is a re-emergent zoonosis characterized by an acute febrile and systemic illness in humans caused by pathogenic spirochetes belonging to the genus Lepstospira. This disease has global distribution, and it is more frequent in tropical and subtropical areas. The complete genomic sequence of Leptospira species offered the possibility to identify potential vaccine candidates for leptospirosis, since environmental control measures are difficult to implement and there is not an ideal vaccine available for human use. Secreted and surface exposed molecules are potential targets for inducing protective immune response in the host. Although we selected six predicted sequences coding for putative outer membrane proteins with unknown function to be analyzed as vaccinal candidates against leptospirosis and for biological characterization, only the lic13435 gene was expressed and purified. The lic13435 gene is specific for pathogenic leptospires suggesting a possible virulence and/or pathogenicity associated function. The recombinant protein was purified and tested as vaccine candidate against leptospirosis. The immunization with the recombinant protein was able to produce a significant immune response in hamsters. Nevertheless, the animals were not protected against leptospirosis. Journal of Vaccines & Vaccination J o ur al of Va ccines & Vainati o n


Introduction
Leptospirosis, a widespread zoonosis caused by spirochetes belonging to the genus Leptospira, remains a major public and veterinary health problem in developing countries [1,2]. Mortality remains significant, related both to delays in diagnosis due to lack of infrastructure and adequate clinical diagnosis, and to other poorly understood reasons that may include inherent pathogenicity of some leptospiral strains or genetically determined host immunopathological responses [3]. Moreover, the overall disease burden is underestimated, since leptospirosis is a significant cause of undifferentiated fever frequently observed in other disease symptoms [4].
Since environmental control measurements are difficult to implement and there is not a universal available vaccine for human use, the complete genomic sequence of Leptospira species provides a window of opportunity to identify potential vaccine candidates for leptospirosis.
In the present study we have selected six genes (lic10359, lic11211, lic13435, lic10291, lic20087, lic10544) coding for predicted outermembrane proteins, which function still have not been determined. All genes were cloned but only recombinant LIC13435 protein (rLIC13435  ) could be expressed and characterized. Orthologous proteins can also be observed in sequenced genome of pathogenic Leptospira but not in the saprophytic L biflexa, suggesting that this protein may play an important role during the infection. The lic13435 gene was amplified from several pathogenic leptospiral strains and transcripts were also detected only in pathogenic retro-transcribed RNAs tested. The recombinant protein was expressed and purified in heterologous system. Peptide sequencing confirmed the correct purified protein and, the circular dichroism spectrum indicated that the protein was structured as predicted. Although the ELISA test indicates that the recombinant protein is very immunogenic and immunoblot detection indicates specificity of the antiserum generated, in an animal challenge model, it did not confer protection against leptospirosis.

In silico identification of surface proteins
Protein coding gene sequences were identified from the L interrogans sorovar Copenhageni genome by using GeneMark progam [18]. Six putative protein, presenting signal peptide according to Smart program [19], and/or predicted to be an outer membrane protein according to Psort program [20] were selected (LIC10359, LIC11211, LIC13435, LIC10291, LIC20087 and LIC10544). Blast analyses [21] were performed to identify orthologous genes in pathogenic Leptospira sequenced genomes.
Bacterianas, FMVZ, Universidade de São Paulo. They were cultured as described [13]. Escherichia coli DH5 α was used as the cloning host strain, E. coli (DE3) Star pLysS (Novagen, Gibbstown, NJ, United States) and E. coli BL21 SI [22] was tested for expression strains of the recombinant proteins. E. coli cells were grown in 2YT or 2YT ON (2YT whithout NaCl) medium supplemented with ampicillin and/or chloramphenicol.

DNA isolation for PCR analysis
Leptospira cultures were harvested by centrifugation at 5000g for 30 min and gently washed twice in sterile phosphate-buffered saline (PBS). Genomic DNA was isolated from the pellets with a guanidinedetergent lysing solution (DNAzol Reagent, Invitrogen, Carlsbad, CA, United States), according to the manufacturer's recommendation.

RNA extraction and reverse transcriptase-PCR (RT-PCR) analysis
The total RNA extraction was done by the acid guanidinium thiocyanate phenol-chloroform method using TRIzol Reagent (Invitrogen) according to the manufacturer's instruction. SuperScript™ III First-Strand Synthesis System for RT-PCR (Invitrogen) was used for cDNA synthesis. The amplification was performed with the same oligonucleotides as in cloning. The amplified products were loaded onto 1.0% agarose gels for electrophoresis and visualized by ethidium bromide staining. E. coli cells were grown in 2YT or 2YT ON (2YT whithout NaCl) medium supplemented with ampicillin and/or chloramphenicol.

Cloning, expression and purification of recombinant proteins
The nucleotide sequence encoding the six selected genes was amplified by PCR using genomic DNA purified from L. interrogans serovar Copenhageni strain Fiocruz L1-130 isolated from a patient in Salvador, Brazil, as template [23]. The primer sequences with the respective restriction sites are shown in the Table 1. The coding DNA fragments exclude the sequence predicted to be the signal peptide. The amplification product was cloned in Plasmid pGEM-T Easy (Promega, Madison, WI, United States) and then subcloned in pAE expression plasmid, a vector with a promoter derived from T7 phage that adds an N-terminal hexa-histidine tag to the recombinant protein [24].
Correct insertions were confirmed by sequencing and the construct was transformed into E. coli BL21 SI or E. coli (DE3) Star pLysS. Bacteria were cultured in 1 liter of 2YT or 2YT ON medium containing the respective antibiotic, until the optical density reached 0.6 at 600 nm. The culture was then induced for 3 h at 30°C with the addition of 0.3 mM of NaCl in case of E. coli BL21 SI, or 10mM IPTG (isopropylbeta-D-thiogalactopyranoside) in the case of E. coli (DE3) Star pLysS. The cells were collected by centrifugation and resuspended in 100 ml of PBS, pH 7.4, and lysed in a French Press Disrupter (Thermo Spectronic, Waltham, MA, United States). The soluble and insoluble fractions were isolated by centrifugation at 10,000 ×g, at 4°C for 20 min. The supernatant was loaded onto a 5ml of Ni 2+ charged chelating Sepharose (GE Healthcare, Buckinghamshire, United Kingdon) column (1 cm diameter) equilibrated with PBS and the purification was performed according to described protocols [14]. After purification, the proteins were extensively dialyzed against PBS for imidazol removal before protein assays.

Protein sequencing
The N-terminal portion of the purified protein was sequenced by Edman degradation [25] using a Shimadzu PPSQ-21 (Kyoto, Japan) automated protein sequencer, following the manufacturer's standard instructions. The sequencing was performed by Laboratório de Bioquímica e Biofisica, Instituto Butantan, Brazil.

Circular dichroism spectroscopic studies
Secondary structure of the purified protein was assessed by circular dichroism (CD) spectroscopy as described [26]. Briefly, CD measurements were performed using a Jasco J-810 Spectropolarimeter at 20°C (Japan Spectroscopic, Tokyo, Japan) equipped with a Peltier unit for temperature control. Purified recombinant protein was diluted at a concentration of 20 mM and dialyzed into 10 mM sodium phosphate buffer before analysis. Far-UV CD spectra were measured using a 1 mm path length cell at 0.5 nm intervals over the wavelength range from 190 to 260 nm. Five scans were averaged for each sample from which an averaged blank spectrum was subtracted.

Immunoblot analysis
Samples were fractionated on a 15% SDS-PAGE and electroblotted onto a nitrocellulose membrane. The membrane was incubated with 10% non-fat dried milk in PBS-T overnight at 4°C. After the incubation, the membrane was washed three times for 10 min with PBS-T, and then incubated with the serum of interest in 10% nonfat dried milk-PBS-T for 2 h. Following a repeat of the PBS-T wash as described above, the membrane was incubated with an appropriate antibody-peroxidase conjugate in 5% non-fat dried milk-PBS-T for 1h. After this, the membrane was washed again with PBS-T, and detected with ECL reagent (GE Healthcare). The recombinant proteins LipL32 [13,27] and TlyC [28], a leptospiral extra-cellular matrix (ECM) binding proteins, were used as control.

Hamster challenge assays
Groups of 10 hamsters, 4-6 weeks old male Golden Syrian, were intradermally immunized with 50 µg of purified recombinant protein, in addition to 500 µg of aluminum ion (added in the form of Al(OH) 3 ), twice at 2 weeks intervals. Two weeks after the second immunization, the hamsters were challenged with approximately 1 LD 50 (6x10 3 ) of L. interrogans serovar Copenhageni str. Fiocruz L1-130 (ATCC BAA-1198) injected intraperitoneally. The animals were monitored daily for 21 days after challenge, the surviving hamsters were sacrificed and their kidneys were removed and tested for the presence of Leptospira by culture in EMJH medium. The positive control group was immunized with the commercial vaccine (FarrowSure B, Pfizer, New York, United States) and the negative control group was immunized with saline (PBS). The experiment was three times reproduced.
All animal experiments were approved by Ethics Committee of Universidade de São Paulo and of Instituto Butantan, São Paulo, Brazil.

LIC13435 is present in pathogenic Leptospira genome
Though we selected six predicted sequences coding for putative outer membrane proteins with unknown function, from Leptospira interrogans serovar Copenhageni genome, only the gene of LIC13435 could be expressed and purified. Orthologous genes of Leptospira interrogans serovar Copenhageni lic13435 sequence (GenBank Accession AE016823.1) can be observed in sequenced genomes of pathogenic L. interrogans serovar Lai 56601 (GenBank Accession AE010300.2), L. borgpetersenii serovar hardjo-bovis JB197 (GenBank Accession CP000350.1) and L. borgpetersenii serovar hardjo-bovis L550 (GenBank Accession CP000348.1), but not in saprofitic L. biflexa serovar Patoc. Therefore, this protein seems to be specific of pathogenic Leptospira species. The amino acid sequence alignment (Figure 1) shows that the four putative Leptospira proteins share 71% -100% of amino acid identity. It was clear using bioinformatics tools, the presence of a Expression, purification and secondary structure of rLIC13435  The lic13435 gene was amplified and the 513bp DNA insert cloned and expressed in E. coli BL21 (SI) in its soluble fraction. The purification was performed by metal chelating chromatography and the final yield was approximately 15 -20 mg of purified protein per liter of induced culture (Figure 3a). Protein N-terminal characterization by Edman sequencing confirmed the correct purified protein. The circular dichroism spectra indicated a predominant α-helix secondary structure, consistent with the computational analysis prediction (NetSurf, http:// www.cbs.dtu.dk/services/NetSurfP/) [29] (Figure 3b).

rLIC13435 25-190 generates high titers and specific antisera
The anti-rLIC13435  serum was produced by intraperitoneal    with approximately 20.2 KDa. b Circular Dichroism spectrum of rLIC13435 25-190. Far UV spectra indicates that rLIC13435  has regular secondary sctructure, composed mainly of a-helix.     immunized hamsters after challenge with virulent leptospires. Ten hamsters were immunized with rLIC13435  , saline (PBS), or commercial vaccine and then challenged with L. interrogans serovar Copenhageni str. Fiocruz L1-130. None of the hamsters immunized with rLIC13435  or saline survived while commercial vaccine had effective protection.
immunization of hamster. ELISA test indicates that the recombinant protein is immunogenic. The titer after second immunization was approximately 64.000 (Figure 4). The anti-rLIC13435  serum seems to be specific, it does not recognize recombinant LipL32 and TlyC, two well characterized leptospiral surface proteins but reacts strongly with rLIC13435   (Figure 5). rLIC13435 is not protective against leptospirosis Taking advantage of the titers presented by immunized hamsters (see above), they were challenged intraperitoneally with 6x10 3 virulent L. interrogans serovar Copenhageni. Although the serum titre after second immunization was approximately 64.000 ( Figure 5), none of the hamsters immunized with rLIC13435  or with saline survived, while a commercial veterinary vaccine had effective protection as expected ( Figure 6).

Discussion
Identification of genes that are regulated during interaction with host tissues and the mechanisms that regulate these infectionassociated genes represent an important step toward the understanding of leptospiral pathogenesis. Efforts have been focused on discovering cross-species-conserved or cross-serovar-conserved protective antigens that may elicit longer-term protection against a broad range of Leptospira. Currently avaiable leptospiral vaccines have low efficacy, are serovar specific, and generally produce only short-term immunity in domestic livestock [17]. Surface associated molecules are potential targets for inducing protective immune responses in the host. Reverse vaccinomology approach was first described for N. meningitides [30]. This pioneering work was designed for high throughput cloning and expression of putative cell exposed antigens selelcted from in silico analysis. This kind of project needs a whole human and physical support not found in most of the laboratories aiming to use similar approaches. Small scale assaying of in silico selected antigens is a feasible alternative to some groups, like ours. Therefore, we have identified six potential targets by in silico analysis of the genome sequence of L. interrogans serovar Copenhageni. However, we were only successful to produce the recombinant protein of LIC13435 (rLIC13435  ). Sequence analysis of the deduced protein revealed the presence of a typical signal peptide with a hydrophobic core ( Figure 1). Orthologous of this gene are present in sequenced pathogenic genomes of Leptospira, which prompted us to examine the gene/protein conservation among other important Leptospira strains. Genomic and retro-transcribed PCR indicate that this gene is conserved and transcribed among pathogenic Leptospira tested. The recombinant protein was expressed and purified from soluble fraction by Ni 2+ affinity chromatography. Peptide sequencing confirmed the correct purified protein. Circular dichroism characterized the secondary structure as being mainly composed of α-helix.
The immunization of hamsters generated specific antisera ( Figure  4 and Figure 5) against rLIC13435  . Instead of using this serum to characterize the antigen localization, we took advantage of the presence of immunized animals to directly challenge them with pathogenic Leptospira in a more straightforward approach. Unfortunately the recombinant protein was not protective in the challenge assays. In conclusion, our findings suggest that, although the presence of the lic13435 gene and its transcripts in pathogenic Leptospira, the immunogenicity of the recombinant protein, LIC13435 does not present potential as vaccine antigen against leptospirosis.