RESUMO
The development of a vaccine for tuberculosis requires a combination of antigens and adjuvants capable of inducing appropriate and long-lasting T cell immunity. We evaluated Mtb72F formulated in AS02A in the cynomolgus monkey model. The vaccine was immunogenic and caused no adverse reactions. When monkeys were immunized with bacillus Calmette-Guérin (BCG) and then boosted with Mtb72F in AS02A, protection superior to that afforded by using BCG alone was achieved, as measured by clinical parameters, pathology, and survival. We observed long-term survival and evidence of reversal of disease progression in monkeys immunized with the prime-boost regimen. Antigen-specific responses from protected monkeys receiving BCG and Mtb72F/AS02A had a distinctive cytokine profile characterized by an increased ratio between 3 Th1 cytokines, IFN-gamma, TNF, and IL-2 and an innate cytokine, IL-6. To our knowledge, this is an initial report of a vaccine capable of inducing long-term protection against tuberculosis in a nonhuman primate model, as determined by protection against severe disease and death, and by other clinical and histopathological parameters.
Assuntos
Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Adjuvantes Imunológicos/química , Animais , Citocinas/metabolismo , Progressão da Doença , Haplorrinos , Sistema Imunitário , Interferon gama/metabolismo , Interleucina-6/metabolismo , Macaca fascicularis , Mycobacterium tuberculosis/metabolismo , Fatores de Tempo , Resultado do Tratamento , Tuberculose/microbiologia , Vacinas contra a Tuberculose/químicaRESUMO
Drug resistance surveillance identified six untreated leprosy patients in the Philippines with Mycobacterium leprae folP1 mutations which confer dapsone resistance. Five patients share a village of residence; four who carried the mutation, Thr53Val, were also linked by M. leprae variable-number tandem repeat (VNTR) strain types. In India, folP1 mutations were detected in two relapse patients with a history of dapsone treatment. Mutations were not found in the rifampin target gene rpoB. These findings indicate that dapsone resistance is being transmitted.
Assuntos
Dapsona/uso terapêutico , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Hanseníase/transmissão , Epidemiologia Molecular/métodos , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/patogenicidade , Proteínas de Bactérias/genética , Humanos , Índia , Hanseníase/genética , Mutação , Mycobacterium leprae/genética , Filipinas , Rifampina/uso terapêuticoRESUMO
Buruli ulcer, caused by Mycobacterium ulcerans, is a destructive infection that most commonly affects the skin. Animal models for Buruli ulcer include guinea pigs, rats, mice, and armadillos, but each is limited in replicating the spectrum of human disease. Here, a cynomolgus monkey was infected with two concentrations of M. ulcerans (1.0 and 2.2 x 10(8)) by intradermal inoculation, 3 months apart. All injection sites developed papules that progressed to ulcers with undermined borders within 2-4 weeks. The rate of progression and size of the ulcers were proportional to the numbers of organisms inoculated. Biopsies from ulcer edges showed ulceration, robust inflammatory cell infiltrates, granulomatous-like responses, mild edema, and extracellular acid-fast bacilli. The ulcers healed spontaneously between Weeks 8 and 12, with no signs of systemic infection. This report, the first to describe a non-human primate experimentally infected with M. ulcerans, suggests that cynomolgus monkeys are modestly susceptible and develop some of the clinical and histologic features of Buruli ulcer.
Assuntos
Macaca fascicularis/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium ulcerans , Dermatopatias Bacterianas/microbiologia , Dermatopatias Bacterianas/patologia , Animais , Injeções Intradérmicas , MasculinoRESUMO
Cynomolgus monkeys, as animal models of scrub typhus, are typically infected with Orientia tsutsugamushi by intradermal inoculation. However, the clinical and histological features at the O. tsutsugamushi inoculation sites, akin to "eschars" at chigger inoculation sites in humans, have not been fully characterized. We intradermally inoculated one medial thigh of six cynomolgus monkeys with semi-purified O. tsutsugamushi (Karp). Within 7 days, two animals developed scrub typhus-like eschars and four had dusky plaques, accompanied by inguinal lymphadenopathy. Biopsies of eschars and an enlarged regional lymph node resembled human disease and stained positively for O. tsutsugamushi by Giemsa, anti-Karp fluorescent antibody, or streptavidin alkaline phosphatase. O. tsutsugamushi-specific IgM and IgG antibody levels measured in both of two monkeys rose steadily after infection. This pilot study shows that cynomolgus intradermally inoculated with O. tsutsugamushi replicate the localized cutaneous pathogenesis of human scrub typhus infections, strengthening the value of this animal model.
Assuntos
Macaca fascicularis/microbiologia , Doenças dos Macacos/patologia , Orientia tsutsugamushi/fisiologia , Tifo por Ácaros/patologia , Pele/patologia , Animais , Anticorpos Antibacterianos/sangue , Modelos Animais de Doenças , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Injeções Intradérmicas , Linfonodos/microbiologia , Doenças Linfáticas/etiologia , Doenças Linfáticas/microbiologia , Doenças Linfáticas/patologia , Masculino , Projetos Piloto , Tifo por Ácaros/complicações , Tifo por Ácaros/imunologiaRESUMO
The search for new and improved tuberculosis (TB) vaccines has focused on IFN-γ both for selecting antigens and for evaluating vaccine delivery strategies. The essential role of IFN-γ in endogenous host protection is well established, but it is still uncertain whether this also holds true for vaccine protection. Here we evaluate the H56 fusion protein vaccine as a BCG booster in a non-human primate (NHP) model of TB that closely recapitulates human TB pathogenesis. To date, only a handful of novel adjuvants have been tested in the NHP model of TB, and therefore we administered H56 in 3 novel cationic liposome adjuvants of increasing immunogenicity (CAF01, CAF04, CAF05) and compared them to H56 in the IC31® adjuvant previously reported to promote protection in this model. The individual clinical parameters monitored during infection (weight, ESR, X-ray) all correlated with survival, and boosting BCG with H56 in all adjuvants resulted in better survival rates compared to BCG alone. The adjuvants promoted IFN-γ-responses of increasing intensity as measured by ELISPOT in the peripheral blood, but the level of vaccine-specific IFN-γ production did not correlate with or predict disease outcome. This study's main outcome underscores the importance of the choice of adjuvant for TB subunit vaccines, and secondly it highlights the need for better correlates of protection in preclinical models of TB.
Assuntos
Adjuvantes Imunológicos/farmacologia , Vacina BCG/imunologia , Imunização Secundária/métodos , Macaca fascicularis , Proteínas Recombinantes de Fusão/imunologia , Tuberculose/prevenção & controle , Animais , Modelos Animais de Doenças , Interferon gama/metabolismo , Análise de Sobrevida , Tuberculose/metabolismoRESUMO
In a randomized, double-blind, double-dummy controlled study, 22 men with erythema nodosum leprosum (ENL) received six capsules containing either 100 mg (group A, n = 12) or 300 mg (group B, n = 10) of thalidomide daily for one week. A six-week, four capsules per day taper followed, in which group A received 50 mg/day of thalidomide in weeks 2 and 3, then dummy capsules in weeks 4 through 7, while group B had gradual decrements every two weeks. Both regimens caused comparable improvement in 19 patients at day 7 (group A [12 of 12] versus group B [7 of 10]; P = 0.08), but slower tapering in group B showed less re-emergence of ENL through week 7 (P = 0.02, versus group A). Most patients developed new lesions soon after stopping treatment. Slower tapering from a higher initial thalidomide dose may improve clinical ENL responses, but high recurrence rates after discontinuation indicates further assessment is needed to identify better tapering regimens.
Assuntos
Eritema Nodoso/tratamento farmacológico , Hansenostáticos/administração & dosagem , Hanseníase Virchowiana/tratamento farmacológico , Talidomida/administração & dosagem , Adolescente , Adulto , Relação Dose-Resposta a Droga , Método Duplo-Cego , Humanos , Interleucina-6/sangue , Hansenostáticos/efeitos adversos , Linfócitos , Masculino , Pessoa de Meia-Idade , Neopterina/urina , Talidomida/efeitos adversos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Monthly doses of rifampin, ofloxacin, and minocycline (ROM) are expected to be effective treatment for multi-bacillary leprosy. Patients with MB leprosy received ROM (n = 10) or World Health Organization multi-drug therapy (MDT) (n = 11). Treatment with ROM was given as 24 consecutive monthly observed doses of rifampin (600 mg), ofloxacin (400 mg), and minocycline (100 mg). Treatment with MDT was given as 24 consecutive monthly observed doses of rifampin (600 mg) and clofazimine (300 mg), and unobserved daily dapsone (100 mg) and clofazimine (50 mg). Twenty patients completed the 24-month regimens with > 99% compliance. Treatments with ROM and MDT were safe, tolerable, and caused similar improvements in lesions, bacterial indices, and histology. All MDT recipients developed clofazimine-induced pigmentation. Six ROM and nine MDT recipients assessed at five or more years after completion of treatment had no evidence of relapse. Twenty-four months of treatment with ROM is a safe, well-tolerated, and convenient regimen that may provide an alternate therapy to MDT for MB leprosy. Larger trials with sufficient follow-up would better define the role of ROM.
Assuntos
Hansenostáticos/administração & dosagem , Hanseníase/tratamento farmacológico , Minociclina/administração & dosagem , Ofloxacino/administração & dosagem , Rifampina/administração & dosagem , Adolescente , Adulto , Clofazimina/administração & dosagem , Dapsona/administração & dosagem , Quimioterapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
RESULTS: HSP65 + IL-12 DNA vaccine showed higher protective efficacy compared with BCG in both mouse and monkey models of TB. It induced the TB-specific CTL in the mouse model of TB, while little level of activity was observed after the injection of BCG. It also showed strong therapeutic efficacy against MDR-TB. In the monkey model, the vaccine augmented the production of IFN-γ and IL-2 from PBL and the therapeutic effect was correlated with the level of IL-2. We next evaluated the potential of DNA vaccine encoding a granulysin, which is an important defensive molecule expressed by human T cells. We found that granulysin-encoding vaccine induced the differentiation of the CTL in vitro and in vivo. It also showed therapeutic efficacy against TB in the monkey as well as the mouse model. The DNA vaccine encoding a Ksp37 also induced the TB-specific CTL in vitro and in vivo in the mouse model. It augmented the production of IL-2, IFN-γ and IL-6 from T cells and spleen cells. A synergistic effect on the activation of the TB-specific CTL was observed by the combination of Ksp37 DNA vaccine with granulysin DNA vaccine. PURPOSE AND METHODS: Emergence of the multi-drug resistant (MDR) Mycobacterium tuberculosis (TB) is a big problem in the world. We have developed novel TB vaccines [DNA vaccines encoding HSP65 + IL-12, granulysin or killer-specific secretory protein of 37kDa (Ksp37)] using Hemagglutinating virus of Japan -envelope (HVJ-E). It is suggested that the activity of the TB-specific CTL is one of the most important factor for the resistance to TB and immunity for TB in chronic human TB disease. Therefore, we examined the level of activation of the TB-specific CTL after the administration of these vaccines. CONCLUSION: These data indicate that our novel vaccines (HSP65 + IL-12 DNA, granulysin and Ksp37) have a capability to activate the TB-specific CTL and will be very strong protective and therapeutic vaccines against TB.
Assuntos
Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Tuberculose/terapia , Vacinas de DNA/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Proteínas de Bactérias/genética , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/imunologia , Chaperonina 60/genética , Modelos Animais de Doenças , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-2/metabolismo , Japão , Macaca fascicularis , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas de DNA/administração & dosagemRESUMO
PURPOSE: Multi-drug resistant tuberculosis (MDR-TB) and extremely drug resistant (XDR) TB are big problems in the world. We have developed novel TB therapeutic vaccines, HVJ-Envelope/HSP65 + IL-12 DNA vaccine (HSP65-vaccine), granulysin vaccine and killer specific secretory protein of 37kDa (Ksp37) vaccine. METHODS AND RESULTS: HSP65 vaccine showed strong therapeutic effect against both MDR-TB and XDR-TB in mice. Intradermal immunization of HSP65-vaccine showed stronger therapeutic effect against TB than intramuscular or subcutaneous immunization. Furthermore, the synergistic therapeutic effect was observed when the vaccine was administrated in combination with Isoniazid (INH), which is a first line drug for chemotherapy. The combination of types of vaccines (HSP65- and granulysin- vaccines) also showed synergistic therapeutic effect. In the monkey model, granulysin-vaccine prolonged the survival period after the infection of TB and long-term survival was observed in vaccine-treated group. We examined the potential of two kinds of novel DNA vaccines (Ksp37-vaccine and granulysin-vaccine). Both vaccines augmented in vivo differentiation of CTL against TB. We measured the amount of Ksp37 protein in human serum and revealed that the level of Ksp37 protein of patients with tuberculosis was lower than that of healthy volunteers. Therefore, we established Ksp37 transgenic mice as well as granulysin transgenic mice to elucidate the function of those proteins. Both transgenic mice were resistant to TB infection. CONCLUSION: These data indicate the potential of combinational therapy; the combination of two DNA vaccines or combination of DNA vaccine with antibiotic drug. Thus, it will provide a novel strategy for the treatment of MDR-TB.
Assuntos
Antituberculosos/uso terapêutico , Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/terapia , Vacinas de DNA/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Proteínas de Bactérias/genética , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/imunologia , Chaperonina 60/genética , Terapia Combinada/métodos , Modelos Animais de Doenças , Interleucina-12/genética , Interleucina-12/imunologia , Macaca fascicularis , Camundongos Transgênicos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Resultado do Tratamento , Vacinas contra a Tuberculose/administração & dosagem , Vacinas de DNA/administração & dosagemRESUMO
Cynomolgus monkeys are a useful model for human tuberculosis, but susceptibility to M. leprae is unknown. A cynomolgus model of leprosy could increase understanding of pathogenesis-importantly, neuritis and nerve-damaging reactions. We administered viable Mycobacterium leprae to 24 cynomolgus monkeys by three routes, with a median follow-up period of 6 years (range = 1-19 years) involving biopsies, nasal smears, antiphenolic glycolipid-1 (PGL-1) antibody serology, and lepromin skin testing. Most developed evanescent papules at intradermal M. leprae inoculation sites that, on biopsy, showed a robust cellular immune response akin to a lepromin skin test reaction; many produced PGL-1 antibodies. At necropsy, four monkeys, without cutaneous or gross neurological signs of leprosy but with elevated PGL-1 antibodies, including three with nasal smears (+) for acid fast bacilli (AFB), showed histological features, including AFB, suggestive of leprosy at several sites. Overall, however, cynomolgus monkeys seem minimally susceptible to leprosy after experimental M. leprae administration.
Assuntos
Hanseníase/imunologia , Macaca fascicularis , Doenças dos Macacos/microbiologia , Mycobacterium leprae/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Biópsia/veterinária , Modelos Animais de Doenças , Feminino , Glicolipídeos/imunologia , Histocitoquímica/veterinária , Antígeno de Mitsuda , Hanseníase/microbiologia , Masculino , Doenças dos Macacos/imunologiaRESUMO
A simple serodiagnostic test based on the Mycobacterium leprae-specific phenolic glycolipid I(PGL-I), for individuals with leprosy is nearly universally positive in leprosy patients with high bacillary loads but cannot be used as a stand-alone diagnostic test for the entire spectrum of the disease process. For patients with early infection with no detectable acid-fast bacilli in lesions or with low or no antibody titer to PGL-I, as in those at the tuberculoid end of the disease spectrum, this diagnostic approach has limited usefulness. To identify additional M. leprae antigens that might enhance the serological detection of these individuals, we have examined the reactivity patterns of patient sera to PGL-I, lipoarabinomannan (LAM), and six recombinant M. leprae proteins (ML1877, ML0841, ML2028, ML2038, ML0380, and ML0050) by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Overall, the responses to ML2028 (Ag85B) and ML2038 (bacterioferritin) were consistently high in both multibacillary and paucibacillary groups and weak or absent in endemic controls, while responses to other antigens showed considerable variability, from strongly positive to completely negative. This analysis has given a clearer understanding of some of the differences in the antibody responses between individuals at opposite ends of the disease spectrum, as well as illustrating the heterogeneity of antibody responses toward protein, carbohydrate, and glycolipid antigens within a clinical group. Correlating these response patterns with a particular disease state could allow for a more critical assessment of the form of disease within the leprosy spectrum and could lead to better patient management.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Glicolipídeos/imunologia , Hanseníase/diagnóstico , Hanseníase/imunologia , Lipopolissacarídeos/imunologia , Mycobacterium leprae/imunologia , Adolescente , Adulto , Idoso , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologia , Fatores de Tempo , Adulto JovemRESUMO
Despite the reduction in the number of leprosy cases registered worldwide as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains stable in many countries. This indicates that Mycobacterium leprae, the causative agent of leprosy, is still being transmitted and that, without an earlier diagnosis, transmission will continue and infection will remain a health problem. The current means of diagnosis of leprosy is based on the appearance of clinical symptoms, which in many cases occur after significant and irreversible nerve damage has occurred. Our recent work identified several recombinant antigens that are specifically recognized by leprosy patients. The goal of the present study was to produce and validate the reactivity of a chimeric fusion protein that possesses the antibody binding properties of several of these proteins. The availability of such a chimeric fusion protein will simplify future test development and reduce production costs. We first identified the antibody binding regions within our top five antigen candidates by performing enzyme-linked immunosorbent assays with overlapping peptides representing the amino acid sequences of each protein. Having identified these regions, we generated a fusion construct of these components (protein advances diagnostic of leprosy [PADL]) and demonstrated that the PADL protein retains the antibody reactivity of the component antigens. PADL was able to complement a protein that we previously produced (the leprosy IDRI [Infectious Disease Research Institute] diagnostic 1 [LID-1] protein) to permit the improved diagnosis of multibacillary leprosy and that had a good ability to discriminate patients with multibacillary leprosy from control individuals. A serological diagnostic test consisting of these antigens could be applied within leprosy control programs to reduce transmission and to limit the appearance of leprosy-associated disabilities and stigmatizing deformities by directing treatment.