1. IntroductionLeptospirosis is the most widespread zoonotic disease and is considered one of the neglected and emerging diseases in the world [1,2]. In humans and animals, the disease is caused by pathogenic bacteria of the genus Leptospira and affects livestock in all parts of the world. More than 200 serovars of these pathogenic spirochetes have been identified [1,3]. L. borgpetersenii serogroup Sejroe is an important serogroup causing infection in Thailand [4,5]. The distribution pattern of Leptospira serovars across regions in Thailand is similar in cattle but different in buffaloes. Since Leptospira serovar Tarassovi and serogroup Sejroe are predominant serogroups in cattle [4,6], researchers have focused on vaccine development for preventing leptospiral infections in tropical environments such as South-East Asia and Latin American countries [7,8]. It is important to emphasize that strains from the Sejroe serogroup are host-adapted to bovine, leading to a chronic and silent disease affecting the reproductive tract of cows, recognized as Bovine Genital Leptospirosis [9]. In addition, serogroup/serovars prevalence in MAT-positive in cattle, buffaloes, and pigs in Thailand were Sejroe, Ranarum, Mini, Pomona, and Bataviae in cattle; Mini, Sejroe, Bratislava, Pomona, and Ranarum in buffaloes; Ranarum, Pomona, Bratislava, and Bataviae in pigs [10]; and serogroups Icterohaemorrhagiae, Canicola, Pomona, Bataviae and Sejroe in dogs [10,11]. The serogroup Pomona serovar Pomona had been reported to cause acute and severe leptospirosis in cattle by incidental infection [12,13] even though protective immunity against the acute disease caused by L. interrogans serovar Pomona was observed, stimulated by LRR proteins cloned from L. borgpetersenii serogroup Sejroe, the most prevalent leptospiral serovar in bovine livestock in Thailand and South-East Asia.Currently, leptospiral vaccine development is pursued by two major strategies: (1) whole-cell bacteria and (2) recombinant protein [7,14,15]. Commercial whole-cell leptospiral vaccines, which are widely used for veterinary purposes, induce antibodies against the leptospiral lipopolysaccharide (LPS). The humoral and cellular immunity stimulated by these bacterins is limited to serovars and/or serogroups included in the vaccine formulation [15,16,17]. The commercial leptospiral whole-cell-based vaccines contain four, six, or eight serovars for canine, swine, and bovine vaccines, respectively [12,18,19]. However, these multivalent whole-cell leptospiral vaccines usually do not achieve sufficient coverage against the spectrum of serovars important for animal health [15]. Although several research groups recently presented a novel approach for the preparation of whole-cell leptospiral vaccines with cross-protective immunity, the disadvantages of the bacterin vaccine are still a concern [20,21]. Adverse effects of the whole-cell vaccines are contaminated medium components resulting in serious side effects, the requirement for ongoing surveillance to identify new bacterin serovars for the preparation of multivalent whole-cell vaccines, and the maintenance of virulent strains in bacterin formulations [14,15,22].Given the limitations of whole-cell vaccines currently in use, the identification of suitable protein candidates has emerged as a major task for vaccine development [15]. Several recombinant protein vaccines for leptospirosis, including LipL32, LipL41, LigA, LigB, FcpA, and Ompl1 proteins, have been studied and found a wide range of immune responses [15,23,24,25,26,27,28,29,30,31,32,33]. However, the application of these results is complicated by numerous modifications to the recombinant proteins. The greatest challenge for vaccine development against leptospirosis is the prediction of antigens that provide sterilizing immunity with long-lasting responses [15]. Recent advances in reverse vaccinology (RV) could represent a promising approach to identifying leptospiral vaccine candidates and for the urgently required development of improved recombinant leptospiral vaccines [14]. Proteins containing leucine-rich repeats (LRRs) have been predicted and reported to function in bacterial host-pathogen interactions, membrane anchoring, invasion, and stimulation of host defense mechanisms (Kobe and Kajava, 2001; McDonald et al., 2003; Seepersaud et al., 2005; Ye et al., 2009). It is interesting to concentrate on the pathogenic Leptospira LRR proteins as immunogens. Pathogenic Leptospira LRRs are can compete with the functions of the host to adhere and invade host cells such as LRR20 [34,35]. The role of rLRR20 in leptospirosis revealed that rLRR20 was observed to colocalize with E-cadherin on the cell surface and activate the downstream transcription factor, beta-catenin, which subsequently promoted the expression of MMP7, a kidney injury biomarker [34,35]. Recently, some bioinformatics studies revealed that the pathogenic Leptospira strains possess more leucine-rich repeat (LRR) genes than the saprophytic strains [36,37]. Moreover, Nitipan et al. reported the presence of seven pairs of the conserved LRR genes in the serovar Hardjo-bovis strain JB197 by the analysis of the L. borgpetersenii genome [37]. Identifying immunogenic epitopes in LRR proteins is interesting.The immunogenic epitopes were identified by in silico studies from two leptospiral LRR proteins, the rKU_Sej_LRR_2012M and rKU_Sej_LRR_2271 proteins, from L. borgpetersenii serogroup Sejroe genome [37,38,39]. In addition, the two identified LRR proteins have cross-reactive immunity with rabbit hyperimmune sera against Leptospira serovars Icterohaemorrhagiae, Javanica, serogroup Sejroe, and serovars Bratislava, Icterohaemorrhagiae, and serogroup Sejroe, respectively [38,40].Further evaluation of leptospiral LRR proteins, the rKU_Sej_LRR_2012M and the rKU_Sej_LRR_2271 proteins from L. borgpetersenii serogroup Sejroe, was performed as a first step to the vaccine development in cattle. The rKU_Sej_LRR_2012M and rKU_Sej_LRR_2271 proteins were chosen because they were cloned from L. borgpetersenii serogroup Sejroe, which is the most prevalent leptospiral serogroup in bovine livestock in Thailand and South-East Asia. It is important to emphasize that strains from the Sejroe serogroup are host-adapted to bovine, leading to a chronic and silent disease affecting the reproductive tract of cows, recognized as Bovine Genital Leptospirosis (BGL) [9]. Even though a cross-protective immunity against the acute disease caused by L. interrogans serovar Pomona was observed, stimulated by LRR proteins cloned from L. borgpetersenii serogroup Sejroe, the most prevalent leptospiral serovar in bovine livestock in Thailand and South-East Asia. This study investigates the LRR proteins as potent vaccine candidates for protecting cattle leptospirosis by evaluating cross-protective immunity in the hamster model challenged with L. interrogans serovar Pomona. 4. DiscussionThe greatest challenge for the development of a vaccine against leptospirosis is to identify antigens that provide long-lasting, cross-protective, and sterilizing immunity. To develop a fully protective, sterilizing immune response to a leptospiral vaccine, mixed strong humoral and cell-mediated immune responses are obligatory [14,15,16,20,29,30,45,48,49,50]. Since Leptospira is an extracellular bacterium, the predominant immunological effector response is humoral immunity, whereby IgG antibodies would inactivate the Leptospira because of complement-mediated lysis and/or via opsonization for phagocytosis. Although Leptospira strains from serogroup Sejroe are host-adapted to bovine, leading to a chronic and silent disease affecting the reproductive tract of cows, recognized as Bovine Genital Leptospirosis (BGL) [9], the incidence of BGL in Thailand is underestimated and no precise data have been reported in Thailand [51]. On the other hand, Pomona is incidental in ruminants and associated with an acute disease, whereas the serogroup Pomona serovar Pomona was frequently reported seropositive in cattle, buffaloes, pigs, and dogs in Thailand [10,11]. In addition, the serovar Pomona had been reported to cause acute and severe leptospirosis in cattle by incidental infection [12,13], especially in multi-host ecologically systems as in Thai rural agriculture areas [41]. Therefore, the hamster model for acute leptospirosis against heterologous L. interrogans serovar Pomona was set to evaluate vaccine candidate proficiencies of two LRR proteins cloned from L. borgpetersenii serogroup Sejroe.Proteins containing leucine-rich repeats (LRRs) have been predicted and reported to function in bacterial host-pathogen interactions, membrane anchoring, and invasions, such as proteins Internalin A, B and J, YopM, and LRR20 [34,52,53,54,55,56,57,58,59]. LRR proteins containing the LPXTG motif, a cell wall anchoring motif [LPXTG cell wall anchor domain (IPR019931), https://www.ebi.ac.uk/interpro/entry/InterPro/IPR019931/ (accessed on 1 Febuary 2019)], have been reported as virulence factor such as internalin J (InlJ) [55]. Leucine-rich repeats of bacterial surface proteins also serve as common pattern recognition motifs of host cell receptors, as reported in human scavenger receptor gp340 by LrrG and E-cadherin by rLRR20 [34,60]. Both rKU_Sej_LRR_2012M and rhKU_Sej_LRR_2271 proteins contain the LPXAG motif, and the two leptospiral LRR proteins characterized in this study had been reported to exhibit rapid induction of specific humoral immune responses in immunized rabbits as well as in hamsters of this report [8,37,38,39,40]. Although this report did not investigate the virulence of rKU_Sej_LRR_2012M and rhKU_Sej_LRR_2271 proteins in pathogenic Leptospira infection, it has been reported that the LRR domain-containing protein family is vital for the virulence of pathogenic Leptospira species [34,35]. Therefore, two leptospiral LRR proteins investigated in this report are of interest as candidates for the development of a leptospirosis vaccine.

Although both LRR proteins characterized in this report exhibited rapid induction of specific humoral immune responses in immunized hamsters, only the rhKU_Sej_LRR_2271 protein induced antibody production 3 weeks after the first and the second immunization in hamsters. The high levels of IgG production against the rhKU_Sej_LRR_2271 protein from 2271 immunized hamsters also prominently promoted the complement-mediated killing of Leptospires per bactericidal assays. The strength of bactericidal activity exhibited by sera from 2012-vaccinated animals was less intense than the action exposed by sera from the 2271 immunized hamsters. In addition, the 2012 vaccines demonstrated only 50% protective efficacy against challenging virulent L. interrogans serovar Pomona in hamsters. The rKU_Sej_LRR_2012M protein showed poor proficiency as a leptospiral vaccine candidate under the challenging condition with virulent L. interrogans serovar Pomona. However, this report has not been performed and challenged with different pathogenic serovars and serogroups and awaits further studies.

The rKU_Sej_LRR_2012M (2012) protein was produced from two overlapping LRR genes of L. borgpetersenii serogroup Sejroe, the KU_Sej_R21N_2012 (NCBI accession: JN627491.1) and KU_Sej_R21C_2012 (NCBI accession: JN627492.1) genes to produce the KU_Sej_R21_2012M gene with a deletion at A346 of the gene “KU_Sej_R21_2012 (NCBI accession: JN627495)” [39]. The gene “KU_Sej_R21_2012” from L. borgpetersenii serogroup Sejroe genome is an orthologous gene of the LBJ_2012 gene of L. borgpetersenii serovar Hardjo-bovis str. JB197 [40]. Since the BLAST results showed no significant similarity found for the alignment between the “KU_Sej_R21_2012 (NCBI accession: JN627495)” gene and L. interrogans serovar Pomona (taxid:44276), and only two genomes from L. interrogans serovar Bataviae strain 1489 and serovar Canicola strain 782 showed 74.53% identity with the KU_Sej_R21_2012 gene (Supplementary Data S1); therefore, the 2012 protein provided poor cross-protective efficacy against challenging with virulent L. interrogans serovar Pomona in this report. In addition, Sripattanakul et al. reported recently that the rKU_Sej_LRR_2012M (2012) protein could be detected by rabbit hyperimmune sera against L. borgpetersenii serovar Canicola, Mini, and Tarassovi in both line-blot and ELISA, but the 2012 LRR protein could not be detected by rabbit hyperimmune sera against L. interrogans serovar Pomona by both techniques [11]. It implied a poor cross-immunity between serogroup Sejroe and serovar Pomona induced by the 2012 protein. Therefore, further investigation on the 2012 potential as a vaccine candidate against L. borgpetersenii, L. mayottensis, L. weilii, L. santarosai, and L. interrogans serovar Bataviae and serovar Canicola could present a commendable task.Although the rhKU_Sej_LRR_2271 protein promoted intense humoral immune response and sera bactericidal action from 2271 immunized hamsters, only 75% protective efficacy in immunized hamsters against challenging with heterogeneous virulent strain L. interrogans serovar Pomona was attained. In addition, sterilizing immunity was not achieved. This could be explained by the result of a BLAST search of the KU_R21_2271 gene (NCBI accession: JX522460), which yielded no significant similarity for the alignment with the genome of L. interrogans serovar Pomona (taxid:44276), whereas the KU_R21_2271 sequence similarities were 75.58% to 78.19% identity with 75 genes from other L. interrogans strains (Supplementary Data S2), and 98.04% to 99.84% identity to 36 sequences from L. borgpetersenii genomes (Supplementary Data S3). The BLAST data imply better prospects for the 2271 protein as a vaccine candidate against either L. borgpetersenii or L. interrogans serovars other than serovar Pomona, as the results of not fully cross-protection was achieved by the induction of the 2271 protein. The fully 100% cross-protective against the heterogeneous strain of serovar Pomona was not attended from the 2271 antigen; nevertheless, the sera from 2271 immunized hamsters provided great bactericidal action.The intense humoral immune response and bactericidal ability of the 2271 antigen are in concordance with previous results from Tansiri et al., having demonstrated that the 2271 protein contains promiscuous T-cell epitopes, which were in silico computationally proposed to have potential binding both MHC class I and II alleles and successfully forming the pMHC/TCR complex [8]. The rhKU_Sej_LRR_2271 protein promiscuous T-cell epitopes, LL17:171-LLFLPLIKILYVDRNKL-187 and SL19:209SLNSGIKALPFNYEKLVNL-227, which can bind to over three of MHC alleles, significantly increased interferon-gamma (IFNγ)-producing specific T-cell responses in the rhKU_Sej_LRR_2271 immunized rabbits compared to nonimmunized rabbits. The LL17 peptide can induce interferon-gamma-producing specific CD4+ T-cell responses in immunized rabbits [8]. Tansiri et al. showed that the 2271 protein induced both humoral and cell-mediated immune responses in vaccinated rabbits. However, the mechanisms by which 2271 confers protective immunity against leptospires in both humoral and cell-mediated immune responses in acute leptospirosis have to be further investigated.It is interesting to note that the rhKU_Sej_LRR_2271 protein can activate both Th1 and Th2 immune responses as a single protein. It has been previously reported that cell-mediated immunity is required for protection against bovine leptospirosis [16,48,50] and in a hamster model of acute leptospirosis [20,29,30,45,49,61]. The results in the present study are in agreement with the data reported by Tansiri et al. The 2271 antigen, including LL17 and SL19 peptides, stimulated splenocyte proliferative responses on cultured splenocytes isolated from the 2271 vaccinated hamster spleens. It was suggested that vaccines’ induction of lymphoproliferative responses indicates protective immunity through CMI responses [29,30,45]. The 2271 and its derived peptides in vitro stimulating Th1 cells were observed by IFNγ and IL-12 cytokine gene expressions in the whole spleen and the splenocytes of vaccinated hamsters. Although the IgG isotype levels against each antigen were not analyzed in this report, the Th1 immune response, which is thought to be responsible for protection against leptospirosis, has been observed in related studies [45,49,50,62,63]. These results strongly suggest that the 2271 vaccine elicited protective immunity through CMI actions.Since the 2271-immunized hamsters demonstrated elevated levels of specific IgG against the rhKU_Sej_LRR_2271 protein, it is conceivable that the solid humoral immune responses by the vaccine result in increased mRNA profiles of IL-4 and IL-10. This would clearly suggest that the 2271 vaccine was capable of protecting hamsters against experimental leptospiral infection by activating strong humoral immunity and significant accomplishments of CMI. However, the 2271 antigen could not confer 100% protection from lethality as small numbers of leptospires were detected by qRT-PCR in tissues from some vaccinated hamsters. Nevertheless, the 2271 vaccine could reduce severe inflammatory lesions in immunized hamsters, although no statistically significant differences were observed at a p-value leptospires in the liver, kidney, and urinary bladder when compared to unvaccinated hamsters. The lower number of leptospires in organs could be related to less severe tissue inflammation in immunized animals. The reduced number of leptospires in organs points to a possible control of the proliferation of leptospires after infection. Although the 2271 protein is supposed to function as bacterial host-pathogen interactions, membrane anchoring, and invasion, such as proteins Internalin A, B and J, YopM, and LRR20 [34,52,53,54,55,56,57,58,59], the 2271 vaccine could not provide a sterilizing immunity against challenging with virulent L. interrogans serovar Pomona in immunized hamsters. However, the protein alleviated severe inflammatory lesions in vital tissues; therefore, it is interesting to investigate further the 2271 vaccine activity against homologous Leptospira such as L. borgpetersenii and additional cross-immunity against heterologous strains from L. interrogans as previously studied [20,21,64].Although the immunized hamsters had reduced leptospiral colonization in the liver, kidney, and urinary bladder, as seen in bacterial culture and qPCR leptospiral quantification results, renal colonization was observed in the 2271 immunized hamsters that survived on 21 days post-challenge. Therefore, the purified recombinant KU_Sej_LRR_2271 protein alone is insufficient as a subunit vaccine against L. interrogans serovar Pomona. Further studies to combine with other antigens such as LigA, LigB, LipL32, and/or with leptosome–entrapped, PC-liposome entrapped antigens, PLGA microsphere, DNA, chimeric BCG vaccine delivery system, [23,26,27,28,29,30,45,49,50,65]. As previously reported, a similar technology may be applicable to construct a 2271- mutant similar to the fcpA- mutant [20,21].