RESUMEN
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.
Asunto(s)
Leptospira , Leptospirosis , Cricetinae , Humanos , Ratones , Animales , Hidróxido de Aluminio , Reproducibilidad de los Resultados , Leptospirosis/prevención & control , Vacunas Bacterianas , Antígenos Bacterianos/genética , Proteínas Recombinantes , Escherichia coli , Inmunoglobulina G , EpítoposRESUMEN
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.
Asunto(s)
Leptospira interrogans , Leptospirosis , Animales , Antígenos Bacterianos , Vacuna BCG , Vacunas Bacterianas , Cricetinae , Epítopos , Leptospira interrogans/genética , Leptospirosis/prevención & controlRESUMEN
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.
Asunto(s)
Vacunas Bacterianas/administración & dosificación , Leptospirosis/prevención & control , Animales , Vacunas Bacterianas/inmunología , Biología Computacional , Humanos , Inmunidad Innata , Leptospira/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
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.
Asunto(s)
Análisis Costo-Beneficio/métodos , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/economía , Vacunas Meningococicas/uso terapéutico , Brasil , Humanos , Programas de Inmunización , Infecciones Meningocócicas/economíaRESUMEN
Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed (p < 0.05, fold change >1.25 or < -1.25) across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with trimethyllysine and acetyllysine occurs to a different degree in response to mammalian host signals encountered during persistent renal colonization. These results provide novel insights into differential protein and PTMs present in response to mammalian host signals which can be used to further define the unique equilibrium that exists between pathogenic leptospires and their reservoir host of infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Interacciones Huésped-Parásitos , Leptospira interrogans/metabolismo , Leptospira interrogans/patogenicidad , Leptospirosis/microbiología , Procesamiento Proteico-Postraduccional , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Reservorios de Enfermedades/microbiología , Femenino , Humanos , Leptospira interrogans/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Lisina/análogos & derivados , Proteoma , Ratas , Ratas Sprague-Dawley , Zoonosis/microbiologíaRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Leptospira/inmunología , Leptospirosis/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/administración & dosificación , Cricetinae , Modelos Animales de Enfermedad , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Análisis de Supervivencia , Resultado del Tratamiento , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunologíaRESUMEN
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.
Asunto(s)
Vacunas Bacterianas/inmunología , Leptospira/inmunología , Animales , Antígenos Bacterianos/inmunología , Humanos , Leptospira/genética , Leptospirosis/inmunología , Leptospirosis/microbiología , Vacunas Sintéticas/inmunologíaRESUMEN
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.
Asunto(s)
Expresión Génica , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5 , Rinotraqueítis Infecciosa Bovina , Pichia/metabolismo , Proteínas del Envoltorio Viral , Proteínas Virales , Animales , Bovinos , Herpesvirus Bovino 1/metabolismo , Vacunas contra Herpesvirus/farmacología , Rinotraqueítis Infecciosa Bovina/sangre , Rinotraqueítis Infecciosa Bovina/diagnóstico , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificación , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
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.
Asunto(s)
Animales Domésticos/microbiología , Animales Salvajes/microbiología , Reservorios de Enfermedades/veterinaria , Genoma Bacteriano , Leptospira interrogans/genética , Leptospirosis/veterinaria , Animales , Brasil , Bovinos/microbiología , Perros/microbiología , Cobayas/microbiología , Leptospira interrogans/clasificación , Leptospira interrogans/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Vacunas Bacterianas/inmunología , Leptospirosis/inmunología , Leptospirosis/prevención & control , Polisacáridos Bacterianos/inmunología , Vacunas de Subunidad/inmunología , Animales , Formación de Anticuerpos/efectos de los fármacos , Antígenos Bacterianos/inmunología , Células CHO , Muerte Celular/efectos de los fármacos , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Femenino , Humedad , Inmunidad Humoral/efectos de los fármacos , Inmunización , Leptospira interrogans/fisiología , Leptospirosis/microbiología , Mesocricetus , Nitrógeno/análisis , Polisacáridos Bacterianos/farmacología , Ácido Pirúvico/análisisRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Reactores Biológicos , Leptospira/química , Leptospirosis/prevención & control , Proteínas Recombinantes/inmunología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cricetinae , Femenino , Leptospira/genética , Leptospirosis/inmunología , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de SupervivenciaRESUMEN
Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.
Asunto(s)
Algoritmos , Bauhinia/química , Modelos Moleculares , Lectinas de Plantas/química , Semillas/química , Secuencia de Aminoácidos , Glicosilación , Datos de Secuencia Molecular , Lectinas de Plantas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteolisis , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
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.
RESUMEN
The seroprevalence of Toxocara canis antibodies in children aged from 1 to 12 yr old was evaluated in Pelotas City, Rio Grande do Sul, Brazil. Human toxocariasis or visceral larva migrans (VLM) was diagnosed with the use of an ELISA based on the T. canis excretory-secretory (TES) antigens; Western blotting was used to confirm the ELISA-positive results. From 427 samples, 50.6% were positive for the presence of anti-TES antibodies. A confirmatory test (Western blot) was carried out on a sample of the ELISA-positive sera (n = 70), and all were positive. The Western blots had specific banding pattern characteristics, where the 30-kDa fraction demonstrated the highest reactivity. This fraction could be important for the specific diagnosis of toxocariasis.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Toxocara canis/inmunología , Toxocariasis/epidemiología , Factores de Edad , Animales , Antígenos Helmínticos/inmunología , Western Blotting , Brasil/epidemiología , Niño , Preescolar , Intervalos de Confianza , Perros , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lactante , Masculino , Oportunidad Relativa , Estudios SeroepidemiológicosRESUMEN
The aim of this work was to evaluate the utilization of analysis of the distribution of relaxation time (DRT) using a dynamic light back-scattering technique as alternative method for the determination of the concentration regimes in aqueous solutions of biopolymers (xanthan, clairana and tara gums) by an analysis of the overlap (c*) and aggregation (c**) concentrations. The diffusion coefficients were obtained over a range of concentrations for each biopolymer using two methods. The first method analysed the behaviour of the diffusion coefficient as a function of the concentration of the gum solution. This method is based on the analysis of the diffusion coefficient versus the concentration curve. Using the slope of the curves, it was possible to determine the c* and c** for xanthan and tara gum. However, it was not possible to determine the concentration regimes for clairana using this method. The second method was based on an analysis of the DRTs, which showed different numbers of relaxation modes. It was observed that the concentrations at which the number of modes changed corresponded to the c* and c**. Thus, the DRT technique provided an alternative method for the determination of the critical concentrations of biopolymers.
Asunto(s)
Gomas de Plantas/química , Polisacáridos Bacterianos/química , Difusión , Luz , Concentración Osmolar , Dispersión de Radiación , SolucionesRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Leptospira interrogans serovar icterohaemorrhagiae/inmunología , Leptospirosis/inmunología , Factores de Transcripción/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/administración & dosificación , Cricetinae , Modelos Animales de Enfermedad , Inmunización Secundaria , Leptospirosis/prevención & control , Factores de Transcripción/administración & dosificación , Vacunación , Vacunas de ADN/inmunología , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
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.
Asunto(s)
Ratas , Secuencia de Bases , Genoma Bacteriano , Técnicas In Vitro , Mucosa Intestinal , Leptospira interrogans serovar icterohaemorrhagiae/genética , Leptospira interrogans serovar icterohaemorrhagiae/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Área Urbana , Enfermedad de Weil , Cricetinae , Técnicas Histológicas , Métodos , Piscinas , VirulenciaRESUMEN
AIMS: To investigate the effects and study the underlying cell death mechanisms of diaryl diselenides, including: diphenyl diselenide (C(6)H(5)Se)(2); 4-chlorodiphenyl diselenide (4-ClC(6)H(4)Se)(2); 3-(trifluoromethyl)-diphenyl diselenide (3-CF(3)C(6)H(4)Se)(2) and 4-methoxydiphenyl diselenide (4-MeOC(6)H(4)Se)(2), on the human colon adenocarcinoma cell line HT-29. MAIN METHODS: The viability of HT-29 cells after exposure to the diaryl diselenides and its substituted structures was based on the MTT assay. To verify if cell death was mediated throughout apoptosis mechanisms, flow cytometry and real-time PCR (qPCR) analyses were conducted. KEY FINDINGS: The MTT assay and flow cytometry analyses showed that (3-CF(3)C(6)H(4)Se)(2) and (4-MeOC(6)H(4)Se)(2) induced cytotoxicity through apoptosis mechanisms in HT-29 cells. qPCR revealed there was an up-regulation of pro-apoptotic (Bax, casapase-9, caspase-8, apoptosis-inducing factor (AIF) and Endonuclease G (EndoG)) and cell-cycle arrest genes (p53 and p21) and down-regulation of anti-apoptotic (Bcl-2 and survivin) and Myc genes. SIGNIFICANCE: These results demonstrate that (3-CF(3)C(6)H(4)Se)2 and (4-MeOC(6)H(4)Se)(2) have the potential to induce apoptosis in HT-29 cells through the activation of caspase-dependent and independent pathways and through cell-cycle arrest.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Compuestos de Selenio/farmacología , Adenocarcinoma/patología , Caspasas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/patología , Regulación hacia Abajo/efectos de los fármacos , Citometría de Flujo , Humanos , Reacción en Cadena de la Polimerasa , Compuestos de Selenio/química , Regulación hacia Arriba/efectos de los fármacosRESUMEN
A major limitation in the clinical management and experimental research of leptospirosis is the poor performance of the available methods for the direct detection of leptospires. In this study, we compared real-time PCR (qPCR), targeting the lipL32 gene, with the immunofluorescent imprint method (IM) for the detection and quantification of leptospires in kidney samples from the rat and hamster experimental models of leptospirosis. Using a virulent strain of Leptospira interrogans serovar Copenhageni, a chronic infection was established in the rat model, which were euthanized 28 days post-infection, while the hamster model simulated an acute infection and the hamsters were euthanized eight days after inoculation. Leptospires in the kidney samples were detected using culture isolation, qPCR and the IM, and quantified using qPCR and the IM. In both the acute and chronic infection models, the correlation between quantification by qPCR and the IM was found to be positive and statistically significant (P<0.05). Therefore, this study demonstrates that the IM is a viable alternative for not only the detection but also the quantification of leptospires, particularly when the use of qPCR is not feasible.
Asunto(s)
Riñón/metabolismo , Leptospira interrogans/metabolismo , Leptospirosis/microbiología , Microscopía Fluorescente/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Cricetinae , Femenino , Infecciones , Leptospirosis/patología , Mesocricetus , Ratas , Ratas Wistar , Análisis de Secuencia de ADNRESUMEN
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.