TonB-dependent receptor epitopes expressed in M. bovis BCG induced significant protection in the hamster model of leptospirosis.

Leptospirosis is an emerging infectious disease caused by pathogenic Leptospira spp. A universal vaccine against leptospirosis is likely to require highly conserved epitopes from pathogenic leptospires that are exposed on the bacterial surface and that generate a protective and sterilizing immune response. Our group recently identified several genes predicted to encode TonB-dependent receptors (TBDR) in Leptospira interrogans using a reverse vaccinology approach. Three leptospiral TBDRs were previously described and partially characterized as ferric-citrate, hemin, and cobalamin transporters. In the current study, we designed a fusion protein composed of predicted surface-exposed epitopes from three conserved leptospiral TBDRs. Based on their three-dimensional structural models and the prediction of immunogenic regions, nine putative surface-exposed fragments were selected to compose a recombinant chimeric protein. A Mycobacterium bovis BCG strain expressing this chimeric antigen encoded in the pUP500/PpAN mycobacterial expression vector was used to immunize Syrian hamsters. All animals (20/20) vaccinated with recombinant BCG survived infection with an endpoint dose of L. interrogans (p < 0.001). No animal survived in the negative control group. Immunization with our recombinant BCG elicited a humoral immune response against leptospiral TBDRs, as demonstrated by ELISA and immunoblot. No leptospiral DNA was detected by lipL32 qPCR in the kidneys of vaccinated hamsters. Similarly, no growth was observed in macerated kidney cultures from the same animals, suggesting the induction of a sterilizing immune response. Design of new vaccine antigens based on the structure of outer membrane proteins is a promising approach to overcome the impact of leptospirosis by vaccination. KEY POINTS: • Predicted surface-exposed epitopes were identified in three leptospiral TBDRs. • An M. bovis BCG strain expressing a chimeric protein (rTBDRchi) was constructed. • Hamsters vaccinated with rBCG:TBDRchi were protected from lethal leptospirosis.

Secretory expression of bovine herpesvirus type 1/5 glycoprotein E in Pichia pastoris for the differential diagnosis of vaccinated or infected cattle.

Bovine herpesvirus (BoHV) glycoprotein E (gE) is a non-essential envelope glycoprotein and the deletion of gE has been used to develop BoHV-1 and BoHV-5 differential vaccine strains. The DIVA (Differentiation of Infected from Vaccinated Animals) strategy, using marker vaccines based on gE-negative BoHV strains, allows the identification of vaccinated or infected animals in immunoassays designed to detect anti-gE antibodies. In this study a codon optimized synthetic sequence of gE containing highly conserved regions from BoHV-1 and BoHV-5 was expressed in Pichia pastoris. Following expression, the recombinant gE (rgE) was secreted and purified from the culture medium. The rgE was identified by Western blotting (WB) using sera from cattle naturally infected with BoHV-1 and/or BoHV-5, or sera from bovines experimentally infected with wild-type BoHV-5. Sera collected from cattle vaccinated with a BoHV-5 gI/gE/US9¯ marker vaccine failed to recognise rgE. Expression of rgE, based on a sequence containing highly conserved regions from BoHV-1 and BoHV-5, in P. pastoris enabled the production of large quantities of rgE suitable for use in immunoassays for the differentiation vaccinated or infected cattle.

Reverse Vaccinology: An Approach for Identifying Leptospiral Vaccine Candidates.

Leptospirosis is a major public health problem with an incidence of over one million human cases each year. It is a globally distributed, zoonotic disease and is associated with significant economic losses in farm animals. Leptospirosis is caused by pathogenic Leptospira spp. that can infect a wide range of domestic and wild animals. Given the inability to control the cycle of transmission among animals and humans, there is an urgent demand for a new vaccine. Inactivated whole-cell vaccines (bacterins) are routinely used in livestock and domestic animals, however, protection is serovar-restricted and short-term only. To overcome these limitations, efforts have focused on the development of recombinant vaccines, with partial success. Reverse vaccinology (RV) has been successfully applied to many infectious diseases. A growing number of leptospiral genome sequences are now available in public databases, providing an opportunity to search for prospective vaccine antigens using RV. Several promising leptospiral antigens were identified using this approach, although only a few have been characterized and evaluated in animal models. In this review, we summarize the use of RV for leptospirosis and discuss the need for potential improvements for the successful development of a new vaccine towards reducing the burden of human and animal leptospirosis.

Draft genome of the Leptospira interrogans strains, Acegua, RCA, Prea, and Capivara, obtained from wildlife maintenance hosts and infected domestic animals.

In the present paper, we announce new draft genomes of four Leptospira interrogans strains named Acegua, RCA, Prea, and Capivara. These strains were isolated in the state of Rio Grande do Sul, Brazil, from cattle, dog, Brazilian guinea pig, and capybara, respectively.

Mannosylated LigANI produced in Pichia pastoris protects hamsters against leptospirosis.

The C-terminal region of the Leptospiral immunoglobulin-like A protein (LigA) contains six carboxy-terminal Ig-like repeat domains (LigANI). Subunit vaccine preparations based on recombinant LigANI produced in Escherichia coli, are promising vaccine candidates, albeit with variable efficacy. In the present study, LigANI was expressed in the methylotrophic yeast Pichia pastoris using a 12 L bioreactor to produce mannosylated LigANI (mLigANI) for use in a vaccine preparation against leptospirosis. Hamsters immunized with a mLigANI vaccine preparation produced a significant IgG antibody response (P < 0.001) and were protected (83.3 %; P < 0.001) against lethal challenge with 36× LD50 of a virulent strain of L. interrogans serovar Copenhageni. A vaccine preparation based on demannosylated mLigANI (nmLigANI) elicited an immune response in hamsters, but did not afford protection. The production of mLigANI in bioreactor by P. pastoris yielded ~50 mg L(-1) of recombinant protein. P. pastoris is a potential platform for the production of leptospiral antigens on an industrial scale. The results demonstrate that LigANI secreted by P. pastoris on mannosylated form (mLigANI) protect hamsters as subunit vaccine of L. interrogans lethal infection.

Characterization of a virulent Leptospira interrogans strain isolated from an abandoned swimming pool.

Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.

Challenges for the development of a universal vaccine against leptospirosis revealed by the evaluation of 22 vaccine candidates.

Leptospirosis is a neglected disease of man and animals that affects nearly half a million people annually and causes considerable economic losses. Current human vaccines are inactivated whole-cell preparations (bacterins) of Leptospira spp. that provide strong homologous protection yet fail to induce a cross-protective immune response. Yearly boosters are required, and serious side-effects are frequently reported so the vaccine is licensed for use in humans in only a handful of countries. Novel universal vaccines require identification of conserved surface-exposed epitopes of leptospiral antigens. Outer membrane ß-barrel proteins (ßb-OMPs) meet these requirements and have been successfully used as vaccines for other diseases. We report the evaluation of 22 constructs containing protein fragments from 33 leptospiral ßb-OMPs, previously identified by reverse and structural vaccinology and cell-surface immunoprecipitation. Three-dimensional structures for each leptospiral ßb-OMP were predicted by I-TASSER. The surface-exposed epitopes were predicted using NetMHCII 2.2 and BepiPred 2.0. Recombinant constructs containing regions from one or more ßb-OMPs were cloned and expressed in Escherichia coli. IMAC-purified recombinant proteins were adsorbed to an aluminium hydroxide adjuvant to produce the vaccine formulations. Hamsters (4-6 weeks old) were vaccinated with 2 doses containing 50 - 125 µg of recombinant protein, with a 14-day interval between doses. Immunoprotection was evaluated in the hamster model of leptospirosis against a homologous challenge (10 - 20× ED50) with L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. Of the vaccine formulations, 20/22 were immunogenic and induced significant humoral immune responses (IgG) prior to challenge. Four constructs induced significant protection (100%, P < 0.001) and sterilizing immunity in two independent experiments, however, this was not reproducible in subsequent evaluations (0 - 33.3% protection, P > 0.05). The lack of reproducibility seen in these challenge experiments and in other reports in the literature, together with the lack of immune correlates and commercially available reagents to characterize the immune response, suggest that the hamster may not be the ideal model for evaluation of leptospirosis vaccines and highlight the need for evaluation of alternative models, such as the mouse.

Attenuated nephritis in inducible nitric oxide synthase knockout C57BL/6 mice and pulmonary hemorrhage in CB17 SCID and recombination activating gene 1 knockout C57BL/6 mice infected with Leptospira interrogans.

The aims of this study were to investigate the frequency of pulmonary hemorrhage (PH) in mice unable to produce functional B and T lymphocytes and to explore the effect of an inducible nitric oxide synthase gene (Inos) knockout (KO) on the frequency/severity of interstitial nephritis in vivo. We studied the outcome of infection by the virulent Leptospira interrogans serovar Copenhageni strain Cop. The animals used were Inos KO mice, recombination activating gene 1 (Rag1) KO mice, CB17 severe combined immunodeficiency (SCID) mice, and the respective wild-type (WT) C57BL/6 and BALB/c controls. The Inos KO and WT mice survived with no clinical symptoms of leptospirosis. The frequency and severity of nephritis was significantly lower in the Inos KO mice. All of the Rag1 KO and SCID animals died of acute leptospirosis, whereas all of the WT mice survived. PH was observed in 57 and 94% of Rag1 KO mice and in 83 and 100% of SCID mice, using inoculum doses of 10(7) and 10(6) leptospires, respectively. There was no evidence of PH in the WT controls. In conclusion, the loss of the Inos gene had a negligible effect on the outcome of leptospiral infection, although we observed a reduced susceptibility for interstitial nephritis in this group. Of note, the absence of functional B- and T-cell lymphocytes did not preclude the occurrence of PH. These data provide evidence that PH in leptospirosis may not be related only to autoimmune mechanisms.

Characterization of the immunogenic and antigenic potential of putative lipoproteins from Leptospira interrogans.

The search for a vaccine capable of conferring heterologous protection, through the identification of conserved and cross-protective antigens, remains an ongoing priority in leptospirosis research. In the present study, an in silico analysis was used to identify potentially protective lipoproteins from Leptospira interrogans serovar Copenhageni. Eight putative lipoproteins were selected (LIC10009, LIC10054, LIC10091, LIC11058, LIC11567, LIC13059, LIC13305, and LIC20172), cloned and expressed in Escherichia coli and purified by affinity chromatography. The recombinant proteins were used to inoculate mice and the subsequent humoral immune response was evaluated by ELISA. Seven of the potential lipoproteins induced a significant IgG response. Furthermore, all of the recombinant proteins were recognized by antibodies present in the sera of severe leptospirosis patients. These putative lipoproteins exhibited potential for further evaluation as prospective vaccine candidates.

Recombinant vaccines against leptospirosis.

Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine.

High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast Pichia pastoris.

BACKGROUND: Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins. RESULTS: The vaccine candidates LigANI and LipL32 were cloned and expressed in P. pastoris as secreted proteins. Large-scale expression resulted in a yield of 276 mg/L for LigANI and 285 mg/L for LipL32. The recombinant proteins were glycosylated and were recognized by antibodies present in the sera of patients with severe leptospirosis. CONCLUSIONS: The expression of LigANI and LipL32 in P. pastoris resulted in a significant increase in yield compared to expression in E. coli. In addition, the proteins were secreted, allowing for easy purification, and retained the antigenic characteristics of the native proteins, demonstrating their potential application as subunit vaccine candidates.

An overview of human leptospirosis vaccine design and future perspectives.

Introduction: It's been 20 years since the first report of a recombinant vaccine that protected against leptospirosis. Since then, numerous recombinant vaccines have been evaluated; however, no recombinant vaccine candidate has advanced to clinical trials. With the ever-increasing burden of leptospirosis, there is an urgent need for a universal vaccine against leptospirosis.Areas covered: This review covers the most promising vaccine candidates that induced significant, reproducible, protection and how advances in the field of bioinformatics has led to the discovery of hundreds of novel protein targets. The authors also discuss the most recent findings regarding the innate immune response and host-pathogen interactions and their impact on the discovery of novel vaccine candidates. In addition, the authors have identified what they believe are the most challenging problems for the discovery and development of a universal vaccine and their potential solutions.Expert opinion: A universal vaccine for leptospirosis will likely only be achieved using a recombinant vaccine as the bacterins are of limited use due to the lack of a cross-protective immune response. Although there are hundreds of novel targets, due to the lack of immune correlates and the need for more research into the basic microbiology of Leptospira spp., a universal vaccine is 10-15 years away.

Distribution of the leptospiral immunoglobulin-like (lig) genes in pathogenic Leptospira species and application of ligB to typing leptospiral isolates.

The family of leptospiral immunoglobulin-like (lig) genes comprises ligA, ligB and ligC. This study used PCR to demonstrate the presence of lig genes among serovars from a collection of leptospiral strains and clinical isolates. Whilst ligA and ligC appeared to be present in a limited number of pathogenic serovars, the ligB gene was distributed ubiquitously among all pathogenic strains. None of the lig genes were detected among intermediate or saprophytic Leptospira species. It was also shown that, similar to the previously characterized secY gene, a short specific PCR fragment of ligB could be used to correctly identify pathogenic Leptospira species. These findings demonstrate that ligB is widely present among pathogenic strains and may be useful for their reliable identification and classification.

Health impact and cost-effectiveness of introducing the vaccine (Bexsero) against MenB disease into the Brazilian immunization programme.

Invasive meningococcal disease (IMD) is associated with a high mortality and severe sequelae. The aim of the present study was to evaluate the potential cost-effectiveness of the Bexsero vaccine in Brazil. We used a cohort model to compare routine vaccination against MenB disease with no vaccination. Epidemiological and cost estimates were obtained from the Brazilian Health Information System. The cost per disability-adjusted life year (DALY) averted and incremental cost-effectiveness ratio (ICER) was estimated assuming a 3-dose vaccination schedule, at R$90 (£ 20.50) per vaccine dose, 82.0% vaccine efficacy against MenB disease and a vaccine uptake of 90.0%. We estimated that 1,527 MenB cases would be prevented and 78 deaths averted. This strategy would cost R$ 762,381, 000 (£ 174,059,503) with a R$ 4,364,280 (£ 996,410) reduction in disease treatment costs. However, at an ICER of 372,256 (£ 84,990) per DALY averted, a vaccination programme is unlikely to be cost-effective.

Rattus norvegicus as a model for persistent renal colonization by pathogenic Leptospira interrogans.

Leptospirosis continues to be a disease with a poorly understood pathogenesis. The experimental rat model is amenable for the investigation of leptospiral dissemination, tropism, persistence of renal colonization and factors related to disease resistance. In this study, Wistar rats were infected intraperitoneally with virulent Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. The detection of leptospires in tissue samples was based on culture, silver staining and immunofluorescence techniques. An inoculum of 10,000 leptospires induced colonization in 50% of rats and colonization persisted for the 4-month period of the study. Dissemination kinetics revealed that renal colonization took place 7-9 days after infection, with no underlying histopathology. The peak leptospiral load occurred on day 5 post-infection, followed by rapid clearance in all tissues except the kidneys, where dense leptospiral aggregates persisted in the renal tubules. We conclude that the experimental rat model is suitable for studies contributing towards the understanding of the mechanisms of colonization and resistance to severe disease in leptospirosis.

Evaluation of the EIE-IgM-Leptospirose assay for the serodiagnosis of leptospirosis.

Access to low-cost, effective diagnosis for leptospirosis is urgently needed in developing countries. The EIE-IgM-Leptospirose, a kit produced for public health laboratories in Brazil, was shown to have a sensitivity of 76% (77 of 102 patients) and 100% (102 of 102 patients) during acute and convalescent-phase leptospirosis, respectively, and a specificity of 93-100% (total healthy and patient control subjects evaluated, 486). These findings indicate that the assay will be useful for diagnosis of this emerging infectious disease in Brazil and other developing countries.

LigB subunit vaccine confers sterile immunity against challenge in the hamster model of leptospirosis.

Neglected tropical diseases, including zoonoses such as leptospirosis, have a major impact on rural and poor urban communities, particularly in developing countries. This has led to major investment in antipoverty vaccines that focus on diseases that influence public health and thereby productivity. While the true, global, impact of leptospirosis is unknown due to the lack of adequate laboratory diagnosis, the WHO estimates that incidence has doubled over the last 15 years to over 1 million cases that require hospitalization every year. Leptospirosis is caused by pathogenic Leptospira spp. and is spread through direct contact with infected animals, their urine or contaminated water and soil. Inactivated leptospirosis vaccines, or bacterins, are approved in only a handful of countries due to the lack of heterologous protection (there are > 250 pathogenic Leptospira serovars) and the serious side-effects associated with vaccination. Currently, research has focused on recombinant vaccines, a possible solution to these problems. However, due to a lack of standardised animal models, rigorous statistical analysis and poor reproducibility, this approach has met with limited success. We evaluated a subunit vaccine preparation, based on a conserved region of the leptospiral immunoglobulin-like B protein (LigB(131-645)) and aluminium hydroxide (AH), in the hamster model of leptospirosis. The vaccine conferred significant protection (80.0-100%, P < 0.05) against mortality in vaccinated animals in seven independent experiments. The efficacy of the LigB(131-645)/AH vaccine ranged from 87.5-100% and we observed sterile immunity (87.5-100%) among the vaccinated survivors. Significant levels of IgM and IgG were induced among vaccinated animals, although they did not correlate with immunity. A mixed IgG1/IgG2 subclass profile was associated with the subunit vaccine, compared to the predominant IgG2 profile seen in bacterin vaccinated hamsters. These findings suggest that LigB(131-645) is a vaccine candidate against leptospirosis with potential ramifications to public and veterinary health.

High serum nitric oxide levels in patients with severe leptospirosis.

Leptospirosis is a globally distributed zoonosis of major public health importance and is associated with severe disease manifestations such as acute renal failure and pulmonary haemorrhage syndrome. However, the extent to which the pathogenesis of leptospirosis mimics sepsis caused by Gram-negative bacteria remains unknown. The aim of this study was to evaluate serum levels of nitric oxide (NO) in patients diagnosed with severe leptospirosis. Sera from 35 confirmed cases of severe leptospirosis and 13 healthy subjects were analysed. Patients with severe leptospirosis had significantly higher NO levels compared to healthy individuals (30.82+/-10.90 microM versus 3.86+/-1.34 microM, P < 0.001), indicating that this immune mediator plays a role in the underlying systemic inflammatory response.

Xanthan gum as an adjuvant in a subunit vaccine preparation against leptospirosis.

Leptospiral immunoglobulin-like (Lig) proteins are of great interest due to their ability to act as mediators of pathogenesis, serodiagnostic antigens, and immunogens. Purified recombinant LigA protein is the most promising subunit vaccine candidate against leptospirosis reported to date, however, as purified proteins are weak immunogens the use of a potent adjuvant is essential for the success of LigA as a subunit vaccine. In the present study, we compared xanthan pv. pruni (strain 106), aluminium hydroxide (alhydrogel), and CpG ODN as adjuvants in a LigA subunit vaccine preparation. Xanthan gum is a high molecular weight extracellular polysaccharide produced by fermentation of Xanthomonas spp., a plant-pathogenic bacterium genus. Preparations containing xanthan induced a strong antibody response comparable to that observed when alhydrogel was used. Upon challenge with a virulent strain of L. interrogans serovar Copenhageni, significant protection (Fisher test, P < 0.05) was observed in 100%, 100%, and 67% of hamsters immunized with rLigANI-xanthan, LigA-CpG-xanthan, and rLigANI-alhydrogel, respectively. Furthermore, xanthan did not cause cytotoxicity in Chinese hamster ovary (CHO) cells in vitro. The use of xanthan as an adjuvant is a novel alternative for enhancing the immunogenicity of vaccines against leptospirosis and possibly against other pathogens.

A prime-boost strategy using the novel vaccine candidate, LemA, protects hamsters against leptospirosis.

Toward developing an effective vaccine capable of conferring heterologous protection, the putative lipoprotein LemA, which presents an M3 epitope similar to that of Listeria, was evaluated as a vaccine candidate in the hamster model of leptospirosis. LemA is conserved (>70% pairwise identity) among the pathogenic Leptospira spp., indicating its potential in stimulating a cross-protective immune response. Using different vaccination strategies, including prime-boost, DNA vaccine, and a subunit preparation, recombinant LemA conferred different levels of protection in hamsters. Significant protection against mortality was observed for the prime-boost and the DNA vaccine strategies, which showed 87.5% (P < 0.01) and 62.5% (P < 0.05) efficacy, respectively. Although the subunit vaccine preparation protected 50.0% of immunized hamsters, the level of protection was not significant. None of the hamsters in the control groups survived challenge with a virulent strain of Leptospira interrogans serogroup Icterohaemorrhagiae. Characterization of the immune response found that the strongest antibody response was stimulated by the subunit vaccine preparation, followed by the prime-boost strategy. The DNA vaccine failed to elicit an antibody response in immunized hamsters.