RESUMO
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.
Assuntos
Leptospira , Leptospirose , Cricetinae , Humanos , Camundongos , Animais , Hidróxido de Alumínio , Reprodutibilidade dos Testes , Leptospirose/prevenção & controle , Vacinas Bacterianas , Antígenos de Bactérias/genética , Proteínas Recombinantes , Escherichia coli , Imunoglobulina G , EpitoposRESUMO
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.
Assuntos
Vacinas Bacterianas/administração & dosagem , Leptospirose/prevenção & controle , Animais , Vacinas Bacterianas/imunologia , Biologia Computacional , Humanos , Imunidade Inata , Leptospira/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.
Assuntos
Expressão Gênica , Vetores Genéticos , Mycobacterium bovis/genética , Regiões Promotoras Genéticas , Animais , Antígenos de Helmintos/biossíntese , Antígenos de Helmintos/genética , Fusão Gênica Artificial , Clonagem Molecular , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Mutagênese , Micobacteriófagos/genética , Mycobacterium smegmatis/genética , Plasmídeos , Reação em Cadeia da Polimerase , Schistosoma mansoni/genética , Análise de Sequência de DNARESUMO
The development of diagnostic tests which can readily differentiate between vaccinated and tuberculosis-infected individuals is crucial for the wider utilization of bacillus Calmette-Guérin (BCG) as vaccine in humans and animals. BCG_0092 is an antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both animals and humans infected with the tubercle bacilli. We carried out bioinformatics analyses of the BCG_0092 and designed a diagnostic test by using the predicted MHC class I epitopes. In addition, we performed a knockout of this gene by homologous recombination in the BCG vaccine strain to allow differentiation of vaccinated from infected individuals. For that, the flanking sequences of the target gene (BCG_0092)were cloned into a suicide vector. Spontaneous double crossovers, which result in wild type revertants or knockouts were selected using SacB. BCG_0092 is present only in members of the Mycobacterium tuberculosis complex. Eight predicted MHC class I epitopes with potential for immunological diagnosis were defined, allowing the design of a specific diagnostic test. The strategy used to delete the (BCG_0092) gene from BCG was successful. The knockout genotype was confirmed by PCR and by Southern blot. The mutant BCG strain has the potential of inducing protection against tuberculosis without interfering with the diagnostic test based on the use of selected epitopes from BCG_0092.
Assuntos
Adjuvantes Imunológicos , Epitopos de Linfócito T/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/diagnóstico , Tuberculose/imunologia , Vacina BCG/imunologia , Biologia Computacional , Epitopos de Linfócito T/análise , Técnicas de Inativação de Genes , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Hipersensibilidade Tardia/imunologia , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/genética , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/imunologiaRESUMO
The development of diagnostic tests which can readily differentiate between vaccinated and tuberculosis-infected individuals is crucial for the wider utilization of bacillus Calmette-Guérin (BCG) as vaccine in humans and animals. BCG_0092 is an antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both animals and humans infected with the tubercle bacilli. We carried out bioinformatics analyses of the BCG_0092 and designed a diagnostic test by using the predicted MHC class I epitopes. In addition, we performed a knockout of this gene by homologous recombination in the BCG vaccine strain to allow differentiation of vaccinated from infected individuals. For that, the flanking sequences of the target gene (BCG_0092)were cloned into a suicide vector. Spontaneous double crossovers, which result in wild type revertants or knockouts were selected using SacB. BCG_0092 is present only in members of the Mycobacterium tuberculosis complex. Eight predicted MHC class I epitopes with potential for immunological diagnosis were defined, allowing the design of a specific diagnostic test. The strategy used to delete the (BCG_0092) gene from BCG was successful. The knockout genotype was confirmed by PCR and by Southern blot. The mutant BCG strain has the potential of inducing protection against tuberculosis without interfering with the diagnostic test based on the use of selected epitopes from BCG_0092.
Assuntos
Humanos , Adjuvantes Imunológicos , Epitopos de Linfócito T/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/diagnóstico , Tuberculose/imunologia , Vacina BCG/imunologia , Biologia Computacional , Epitopos de Linfócito T/análise , Técnicas de Inativação de Genes , Antígenos de Histocompatibilidade Classe I/imunologia , Hipersensibilidade Tardia/imunologia , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controleRESUMO
The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Espectrometria de Massas , Mycobacterium avium/patogenicidade , Mycobacterium bovis/patogenicidade , Tuberculina/isolamento & purificação , Tuberculose Aviária/imunologia , Tuberculose Bovina/imunologia , Animais , Bovinos , Espectrometria de Massas/métodos , Mycobacterium avium/imunologia , Mycobacterium bovis/imunologia , Linfócitos T/imunologia , Tuberculose Aviária/patologia , Tuberculose Bovina/patologiaRESUMO
Mycobacterium bovis BCG has the potential to be an effective live vector for multivalent vaccines. However, most mycobacterial cloning vectors rely on antibiotic resistance genes as selectable markers, which would be undesirable in any practical vaccine. Here we report the use of auxotrophic complementation as a selectable marker that would be suitable for use in a recombinant vaccine. A BCG auxotrophic for the amino acid leucine was constructed by knocking out the leuD gene by unmarked homologous recombination. Expression of leuD on a plasmid not only allowed complementation, but also acted as a selectable marker. Removal of the kanamycin resistance gene, which remained necessary for plasmid manipulations in Escherichia coli, was accomplished by two different methods: restriction enzyme digestion followed by re-ligation before BCG transformation, or by Cre-loxP in vitro recombination mediated by the bacteriophage P1 Cre Recombinase. Stability of the plasmid was evaluated during in vitro and in vivo growth of the recombinant BCG in comparison to selection by antibiotic resistance. The new system was highly stable even during in vivo growth, as the selective pressure is maintained, whereas the conventional vector was unstable in the absence of selective pressure. This new system will now allow the construction of potential recombinante vaccine strains using stable multicopy plasmid vectors without the inclusion of antibiotic resistance markers.
Assuntos
Vacina BCG/genética , Marcadores Genéticos/genética , Vetores Genéticos/genética , Animais , Vacina BCG/imunologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Hidroliases/genética , Canamicina , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/genética , Mycobacterium smegmatis/genética , Plasmídeos/genética , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Humoral and cellular immune responses of mice inoculated with recombinant Mycobacterium bovis BCG expressing the MSP1a antigen of Anaplasma marginale were evaluated. The msp1a gene was amplified by PCR and cloned into the mycobacterial expression vectors pUS2000 and pMIP12. Immunization of isogenic BALB/c mice with the rBCG/pUS2000-msp1a construct induced significant seroconversion to MSP1a (p<0.001), which was 26 times above pre-immunization levels at day 63 post-initial immunization and which remained stable for the duration of the experiment (6 months). In contrast, rBCG/pMIP12-msp1a induced seroconversion at a level of 6 times above pre-immunization values, which peaked at day 63. Western blot analysis showed that sera derived from mice vaccinated with either rBCG construct recognized both native and recombinant forms of A. marginale MSP1a. In contrast to the humoral response data, immunization with rBCG/pMIP12-msp1a was found to induce a markedly stronger cellular response than that recorded for BCG/pUS2000-msp1a. These observations clearly demonstrated the immunogenicity of recombinant BCG expressing the MSP1a antigen and suggested that the immune responses were influenced by the level of antigen expression. The results of this research warrant studies of recombinant M. bovis BCG expressing MSP1a in cattle to test for protective antibody production for control of bovine anaplasmosis.
Assuntos
Vacina BCG/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Mycobacterium bovis/imunologia , Anaplasma marginale , Animais , Formação de Anticorpos/imunologia , Vacina BCG/genética , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Bovinos , Clonagem Molecular , Feminino , Imunidade Celular/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/genéticaRESUMO
The Sm14 antigen of Schistosoma mansoni was cloned and expressed in Mycobacterium bovis BCG as a fusion with the Mycobacterium fortuitum beta-lactamase protein under the control of its promoter, pBlaF*; the protein was localized in the bacterial cell wall. The rBCG-Sm14 strain was shown to be relatively stable in cultured murine and bovine monocytes in terms of infectivity, bacterial persistence, and plasmid stability. The immunization of mice with rBCG-Sm14 showed no induction of anti-Sm14 antibodies; however, splenocytes of immunized mice released increased levels of gamma interferon upon stimulation with recombinant Sm14 (rSm14), indicating an induction of a Th1-predominant cellular response against Sm14. Mice immunized with one or two doses of rBCG-Sm14 and challenged with live S. mansoni cercaria showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of rSm14 purified from Escherichia coli. The data presented here further enhance the status of Sm14 as a promising candidate antigen for the control of schistosomiasis and indicate that a one-dose regimen of rBCG-Sm14 could be considered a convenient means to overcome many of the practical problems associated with the successful implementation of a multiple-dose vaccine schedule in developing countries.