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1.
Vaccines (Basel) ; 9(8)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34451973

RESUMO

Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of viral Spike S1 protein encoding mRNA incorporated in a lipid nanoparticle and stabilized by polyethylene glycol (PEG). The mRNA vaccines are novel in many respects, including cellular uptake and the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences, encoding the SARS-CoV-2 Spike protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to the induction of virus-neutralizing antibodies and cytotoxic T-lymphocytes, will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side effects. The mRNA-based vaccines may lead to an anaphylactic reaction. This reaction may be triggered by PEG. The intracellular routing of PEG and potential presentation in the context of CD1 will be discussed. Adenovirus vector-based vaccines have been associated with thrombocytopenic thrombosis events. The anti-platelet factor 4 antibodies found in these patients could be generated due to conformational changes of relevant epitopes presented to the immune system.

2.
BMC Infect Dis ; 21(1): 677, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34256735

RESUMO

BACKGROUND: SARS-CoV-2 has swept across the globe, causing millions of deaths worldwide. Though most survive, many experience symptoms of COVID-19 for months after acute infection. Successful prevention and treatment of acute COVID-19 infection and its associated sequelae is dependent on in-depth knowledge of viral pathology across the spectrum of patient phenotypes and physiologic responses. Longitudinal biobanking provides a valuable resource of clinically integrated, easily accessed, and quality-controlled samples for researchers to study differential multi-organ system responses to SARS-CoV-2 infection, post-acute sequelae of COVID-19 (PASC), and vaccination. METHODS: Adults with a history of a positive SARS-CoV-2 nasopharyngeal PCR are actively recruited from the community or hospital settings to enroll in the Northern Colorado SARS-CoV-2 Biorepository (NoCo-COBIO). Blood, saliva, stool, nasopharyngeal specimens, and extensive clinical and demographic data are collected at 4 time points over 6 months. Patients are assessed for PASC during longitudinal follow-up by physician led symptom questionnaires and physical exams. This clinical trial registration is NCT04603677 . RESULTS: We have enrolled and collected samples from 119 adults since July 2020, with 66% follow-up rate. Forty-nine percent of participants assessed with a symptom surveillance questionnaire (N = 37 of 75) had PASC at any time during follow-up (up to 8 months post infection). Ninety-three percent of hospitalized participants developed PASC, while 23% of those not requiring hospitalization developed PASC. At 90-174 days post SARS-CoV-2 diagnosis, 67% of all participants had persistent symptoms (N = 37 of 55), and 85% percent of participants who required hospitalization during initial infection (N = 20) still had symptoms. The most common symptoms reported after 15 days of infection were fatigue, loss of smell, loss of taste, exercise intolerance, and cognitive dysfunction. CONCLUSIONS: Patients who were hospitalized for COVID-19 were significantly more likely to have PASC than those not requiring hospitalization, however 23% of patients who were not hospitalized also developed PASC. This patient-matched, multi-matrix, longitudinal biorepository from COVID-19 survivors with and without PASC will allow for current and future research to better understand the pathophysiology of disease and to identify targeted interventions to reduce risk for PASC. Registered 27 October 2020 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT04603677 .


Assuntos
Bancos de Espécimes Biológicos , Teste para COVID-19/métodos , COVID-19/complicações , SARS-CoV-2/genética , Sobreviventes , Adulto , Idoso , COVID-19/sangue , COVID-19/epidemiologia , COVID-19/patologia , COVID-19/virologia , Colorado/epidemiologia , Progressão da Doença , Feminino , Seguimentos , Hospitalização , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Manejo de Espécimes , Adulto Jovem
3.
Vaccines (Basel) ; 9(4)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916180

RESUMO

The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopathological evaluation of tissues post-challenge. The results from this preliminary evaluation provide insight into the immunological responses occurring as a result of vaccination with the proposed vaccine candidate and the impact that adjuvant formulations, specifically developed to promote Th1 type immune responses, have on vaccine efficacy and protection against infection following challenge with live SARS-CoV-2. This data may have utility in the development of effective vaccine candidates broadly. Furthermore, the results of this preliminary evaluation suggest that preparation of a whole virion vaccine for COVID-19 using this specific photochemical method may have potential utility in the preparation of one such vaccine candidate.

4.
Sci Rep ; 11(1): 9040, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907221

RESUMO

The nontuberculous mycobacteria (NTM) Mycobacterium avium is a clinically significant pathogen that can cause a wide range of maladies, including tuberculosis-like pulmonary disease. An immunocompromised host status, either genetically or acutely acquired, presents a large risk for progressive NTM infections. Due to this quietly emerging health threat, we evaluated the ability of a recombinant fusion protein ID91 combined with GLA-SE [glucopyranosyl lipid adjuvant, a toll like receptor 4 agonist formulated in an oil-in-water stable nano-emulsion] to confer protection in both C57BL/6 (wild type) and Beige (immunocompromised) mouse models. We optimized an aerosol challenge model using a clinical NTM isolate: M. avium 2-151 smt, observed bacterial growth kinetics, colony morphology, drug sensitivity and histopathology, characterized the influx of pulmonary immune cells, and confirmed the immunogenicity of ID91 in both mouse models. To determine prophylactic vaccine efficacy against this M. avium isolate, mice were immunized with either ID91 + GLA-SE or bacillus Calmette-Guérin (BCG). Immunocompromised Beige mice displayed a delayed influx of innate and adaptive immune cells resulting in a sustained and increased bacterial burden in the lungs and spleen compared to C57BL/6 mice. Importantly, both ID91 + GLA-SE and BCG vaccines significantly reduced pulmonary bacterial burden in both mouse strains. This work is a proof-of-concept study of subunit vaccine-induced protection against NTM.


Assuntos
Vacina BCG/administração & dosagem , Modelos Animais de Doenças , Hospedeiro Imunocomprometido/imunologia , Mycobacterium avium/patogenicidade , Tuberculose/prevenção & controle , Vacinas de Subunidades/administração & dosagem , Animais , Vacina BCG/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium avium/metabolismo , Tuberculose/imunologia , Tuberculose/microbiologia , Vacinação , Vacinas de Subunidades/imunologia
5.
Vox Sang ; 116(10): 1076-1083, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33835489

RESUMO

BACKGROUND AND OBJECTIVES: Convalescent plasma (CP) has been embraced as a safe therapeutic option for coronavirus disease 2019 (COVID-19), while other treatments are developed. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not transmissible by transfusion, but bloodborne pathogens remain a risk in regions with high endemic prevalence of disease. Pathogen reduction can mitigate this risk; thus, the objective of this study was to evaluate the effect of riboflavin and ultraviolet light (R + UV) pathogen reduction technology on the functional properties of COVID-19 CP (CCP). MATERIALS AND METHODS: COVID-19 convalescent plasma units (n = 6) from recovered COVID-19 research donors were treated with R + UV. Pre- and post-treatment samples were tested for coagulation factor and immunoglobulin retention. Antibody binding to spike protein receptor-binding domain (RBD), S1 and S2 epitopes of SARS-CoV-2 was assessed by ELISA. Neutralizing antibody (nAb) function was assessed by pseudovirus reporter viral particle neutralization (RVPN) assay and plaque reduction neutralization test (PRNT). RESULTS: Mean retention of coagulation factors was ≥70%, while retention of immunoglobulins was 100%. Starting nAb titres were low, but PRNT50 titres did not differ between pre- and post-treatment samples. No statistically significant differences were detected in levels of IgG (P ≥ 0·3665) and IgM (P ≥ 0·1208) antibodies to RBD, S1 and S2 proteins before and after treatment. CONCLUSION: R + UV PRT effects on coagulation factors were similar to previous reports, but no significant effects were observed on immunoglobulin concentration and antibody function. SARS-CoV-2 nAb function in CCP is conserved following R + UV PRT treatment.


Assuntos
Anticorpos Neutralizantes , COVID-19 , Anticorpos Antivirais , COVID-19/terapia , Humanos , Imunização Passiva , Riboflavina , SARS-CoV-2 , Tecnologia , Raios Ultravioleta
6.
Vaccine ; 38(45): 7156-7165, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-32978002

RESUMO

Although vaccination with BCG prevents disseminated forms of childhood tuberculosis (TB), it does not protect against pulmonary infection or Mycobacterium tuberculosis (Mtb) transmission. In this study, we generated a complete deletion mutant of the Mtb Esx-5 type VII secretion system (Mtb Δesx-5). Mtb Δesx-5 was highly attenuated and safe in immunocompromised mice. When tested as a vaccine candidate to boost BCG-primed immunity, Mtb Δesx-5 improved protection against highly virulent Mtb strains in the murine and guinea pig models of TB. Enhanced protection provided by heterologous BCG-prime plus Mtb Δesx-5 boost regimen was associated with increased pulmonary influx of central memory T cells (TCM), follicular helper T cells (TFH) and activated monocytes. Conversely, lower numbers of T cells expressing exhaustion markers were observed in vaccinated animals. Our results suggest that boosting BCG-primed immunity with Mtb Δesx-5 is a potential approach to improve protective immunity against Mtb. Further insight into the mechanism of action of this novel prime-boost approach is warranted.


Assuntos
Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculose , Sistemas de Secreção Tipo VII , Animais , Antígenos de Bactérias , Vacina BCG , Cobaias , Imunização Secundária , Camundongos , Mycobacterium tuberculosis/genética , Tuberculose/prevenção & controle , Vacinação
7.
Pathogens ; 9(8)2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32781698

RESUMO

Nontuberculous mycobacteria (NTM) represent an increasingly prevalent etiology of soft tissue infections in animals and humans. NTM are widely distributed in the environment and while, for the most part, they behave as saprophytic organisms, in certain situations, they can be pathogenic, so much so that the incidence of NTM infections has surpassed that of Mycobacterium tuberculosis in developed countries. As a result, a growing body of the literature has focused attention on the critical role that drug susceptibility tests and infection models play in the design of appropriate therapeutic strategies against NTM diseases. This paper is an overview of the in vitro and in vivo models of NTM infection employed in the preclinical phase for early drug discovery and vaccine development. It summarizes alternative methods, not fully explored, for the characterization of anti-mycobacterial compounds.

8.
Sci Rep ; 10(1): 7651, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32377001

RESUMO

Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; however, conventional methods to analyze data are subjective and time-consuming. To address these issues, we have developed a novel flow cytometry analysis pipeline to identify a plethora of cell populations efficiently. Coupled with feature engineering and immunological context, researchers can immediately extrapolate novel discoveries through easy-to-understand plots. The R-based pipeline uses Fluorescence Minus One (FMO) controls or distinct population differences to develop thresholds for positive/negative marker expression. The continuous data is transformed into binary data, capturing a positive/negative biological dichotomy often of interest in characterizing cells. Next, a filtering step refines the data from all identified cell phenotypes to populations of interest. The data can be partitioned by immune lineages and statistically correlated to other experimental measurements. The pipeline's modularity allows customization of statistical testing, adoption of alternative initial gating steps, and incorporation of other datasets. Validation of this pipeline through manual gating of two datasets (murine splenocytes and human whole blood) confirmed its accuracy in identifying even rare subsets. Lastly, this pipeline can be applied in all disciplines utilizing flow cytometry regardless of cytometer or panel design. The code is available at https://github.com/aef1004/cyto-feature_engineering.


Assuntos
Citodiagnóstico/métodos , Suscetibilidade a Doenças/imunologia , Citometria de Fluxo , Animais , Biomarcadores , Células Sanguíneas/metabolismo , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Camundongos , Mycobacterium tuberculosis/imunologia , Fenótipo , Tuberculose/diagnóstico , Tuberculose/imunologia , Tuberculose/microbiologia
9.
Curr Protoc Cytom ; 93(1): e74, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32421215

RESUMO

Flow cytometry allows the visualization of physical, functional, and/or biological properties of cells including antigens, cytokines, size, and complexity. With increasingly large flow cytometry panels able to analyze up to 50 parameters, there is a need to standardize flow cytometry protocols to achieve high-quality data that can be input into analysis algorithms. Without this clean data, algorithms may incorrectly categorize the cell populations present in the samples. In this protocol, we outline a comprehensive methodology to prepare samples for polychromatic flow cytometry. The use of multiple washing steps and rigorous controls creates high-quality data with good separation between cell populations. Experimental data acquired using this protocol can be analyzed via computational algorithms that perform end-to-end analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of single-cell suspension for flow cytometry Support Protocol 1: Lung preparation Support Protocol 2: Counting cells on a flow cytometer Basic Protocol 2: Surface and intracellular flow cytometry staining Support Protocol 3: Single-color bead controls.


Assuntos
Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Animais , Contagem de Células , Espaço Intracelular/metabolismo , Pulmão/citologia , Camundongos Endogâmicos C57BL , Análise de Célula Única , Baço/citologia , Linfócitos T/imunologia
10.
Vet Immunol Immunopathol ; 215: 109884, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31420066

RESUMO

Many vaccines against childhood diseases are administered early after birth, but vaccine development studies frequently test efficacy in adult rather than in neonatal animal models. In countries with endemic tuberculosis (TB), Bacillus Calmette-Guerin (BCG) is administered as part of the neonatal vaccine regimen because it prevents against the disseminated form of TB in children, although it has variable efficacy against pulmonary TB. Several promising new vaccines against TB are currently being tested in adult animal models. Here we evaluated neonatal piglets as an animal model to test vaccine efficacy. For this purpose, minipigs were vaccinated or not with BCG 48 h after birth and their immune response followed longitudinally until adolescence. We characterized the memory and activation phenotype of T cells, cytokine profile, and monocyte activation in response to BCG stimulation from 4 weeks of age into adolescence- age of 24 weeks. Immunological responses in vaccinated and non-vaccinated animals were further monitored upon infection with a low dose exposure to Mycobacterium tuberculosis strain HN878 via the aerosol route. Comparing the immunological response elicited by BCG vaccination in minipigs vs similar studies in infants, suggest that minipigs have the potential to serve as an effective neonatal animal model for vaccine development.


Assuntos
Vacina BCG/imunologia , Modelos Animais de Doenças , Mycobacterium tuberculosis/imunologia , Porco Miniatura/imunologia , Tuberculose Pulmonar/imunologia , Animais , Animais Recém-Nascidos , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Imunogenicidade da Vacina , Memória Imunológica , Imunofenotipagem , Estudos Longitudinais , Ativação Linfocitária , Masculino , Monócitos/imunologia , Suínos , Tuberculose/imunologia , Tuberculose Pulmonar/prevenção & controle
11.
Front Immunol ; 9: 461, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29568298

RESUMO

The purpose of vaccination against tuberculosis and other diseases is to establish a heightened state of acquired specific resistance in which the memory immune response is capable of mediating an accelerated and magnified expression of protection to the pathogen when this is encountered at a later time. In the earliest studies in mice infected with Mycobacterium tuberculosis, memory immunity and the cells that express this were definable both in terms of kinetics of emergence, and soon thereafter by the levels of expression of markers including CD44, CD62L, and the chemokine receptor CCR7, allowing the identification of effector memory and central memory T cell subsets. Despite these initial advances in knowledge, more recent information has not revealed more clarity, but instead, has created a morass of complications-complications that, if not resolved, could harm correct vaccine design. Here, we discuss two central issues. The first is that we have always assumed that memory is induced in the same way, and consists of the same T cells, regardless of whether that immunity is generated by BCG vaccination, or by exposure to M. tuberculosis followed by effective chemotherapy. This assumption is almost certainly incorrect. Second, a myriad of additional memory subsets have now been described, such as resident, stem cell-like, tissue specific, among others, but as yet we know nothing about the relative importance of each, or whether if a new vaccine needs to induce all of these, or just some, to be fully effective.


Assuntos
Receptores de Hialuronatos/imunologia , Memória Imunológica , Selectina L/imunologia , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Receptores CCR7/imunologia , Linfócitos T/imunologia , Tuberculose , Animais , Humanos , Linfócitos T/patologia , Tuberculose/imunologia , Tuberculose/patologia , Tuberculose/prevenção & controle
12.
Tuberculosis (Edinb) ; 109: 123-133, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29559116

RESUMO

Transmission of Mycobacterium tuberculosis bacilli from one individual to another is the basis of the disease process. While considerable emphasis has been placed on the role of host mechanisms of resistance in establishing or preventing new infection, far less has been expended on understanding possible factors operative at the bacterial level. In this study we established a panel of clinical isolates of M. tuberculosis strains obtained from the Western Cape region of South Africa, each of which had been carefully tracked in terms of their degree of transmission in the community. Each of the panel were used to infect guinea pigs with 15-20 bacilli by aerosol exposure and the course of the infection then determined. Strains with different degrees of transmission could not be distinguished in terms of their capacity to grow in the main target organs of infected animals. However, rather surprisingly, while strains with no evidence of transmission [NOT] in general caused moderate to severe lung damage, this parameter in animals infected with highly transmitted [HT] strains was mostly mild. In terms of TH1 immunity these signals were strongest in these latter animals, as was IL-17 gene expression, whereas minimal signals for regulatory molecules including IL-10 and FoxP3 were seen across the entire panel. In terms of T cell numbers, responses of both CD4 and CD8 were both far faster and far higher in animals infected with the HT strains. At the gene expression level we observed a major three-fold difference [both up and down] between NOT and HT strains, but in terms of proteins of key interest only a few [including PD-L1 and HIF-3] showed major differences between the two groups. Overall, it was apparent that NOT strains were far more inflammatory that HT strains, and appeared to trigger a much larger number of genes, possibly explaining the observed damage to the lungs and progressive pathology. In contrast, the HT strains, while equally virulent, were more immunogenic and developed much stronger T cell responses, while keeping lung damage to a minimum. Hence, in terms of trying to explain the capacity of these strains to cause transmission, these results are clearly paradoxical.


Assuntos
Pulmão/microbiologia , Mycobacterium tuberculosis/patogenicidade , Linfócitos T/microbiologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/transmissão , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Cobaias , Interações Hospedeiro-Patógeno , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/isolamento & purificação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/microbiologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/metabolismo , Virulência
13.
Clin Vaccine Immunol ; 24(12)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29046306

RESUMO

As yet, very few vaccine candidates with activity in animals against Mycobacterium tuberculosis infection have been tested as therapeutic postexposure vaccines. We recently described two pools of mycobacterial proteins with this activity, and here we describe further studies in which four of these proteins (Rv1738, Rv2032, Rv3130, and Rv3841) were generated as a fusion polypeptide and then delivered in a novel yeast-based platform (Tarmogen) which itself has immunostimulatory properties, including activation of Toll-like receptors. This platform can deliver antigens into both the class I and class II antigen presentation pathways and stimulate strong Th1 and Th17 responses. In mice this fusion vaccine, designated GI-19007, was immunogenic and elicited strong gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses; despite this, they displayed minimal prophylactic activity in mice that were subsequently infected with a virulent clinical strain. In contrast, in a therapeutic model in the guinea pig, GI-19007 significantly reduced the lung bacterial load and reduced lung pathology, particularly in terms of secondary lesion development, while significantly improving survival in one-third of these animals. In further studies in which guinea pigs were vaccinated with BCG before challenge, therapeutic vaccination with GI-19007 initially improved survival versus that of animals given BCG alone, although this protective effect was gradually lost at around 400 days after challenge. Given its apparent ability to substantially limit bacterial dissemination within and from the lungs, GI-19007 potentially can be used to limit lung damage as well as facilitating chemotherapeutic regimens in infected individuals.


Assuntos
Técnicas de Transferência de Genes , Mycobacterium tuberculosis/imunologia , Saccharomyces cerevisiae/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Feminino , Cobaias , Interferon gama/metabolismo , Interleucina-17/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/genética , Profilaxia Pós-Exposição/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sobrevida , Resultado do Tratamento , Tuberculose/imunologia , Tuberculose/patologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/genética , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
14.
Tuberculosis (Edinb) ; 106: 91-98, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28802411

RESUMO

In endemic countries more than 20% of tuberculosis (TB) cases are in infants and children. Current animal models study TB during adulthood but animal models for infant TB are scarce. Here we propose that minipigs can be used as an animal model to study adult, adolescent and infant TB including natural transmission. In these studies, two-month old minipigs (representing infant age in humans) and six-month old minipigs (representing adolescence in humans) were infected via the aerosol route with hyper-virulent clinical strain W-Beijing Mycobacterium tuberculosis (Mtb) HN878 and were monitored for 11 or 36 weeks post-challenge, respectively. In the same studies, infected and unchallenged animals were housed together. Viable bacteria were recovered from pulmonary and thoracic lymph nodes from both -infected and their initially unchallenged natural contacts. Bacillary load, gross lesions and histopathology revealed similarities to the spectrum of disease observed in human TB. The study did not reach terminal end point, thus it was not possible to annotate definitive clinical symptoms of active TB. The results demonstrated that minipigs are experimental hosts of Mtb HN878, and the pathology developed in their lungs resembles pathological findings described in human TB. Importantly, within communities of Mtb infected minipigs natural transmission occurs.


Assuntos
Mycobacterium tuberculosis/patogenicidade , Tuberculose dos Linfonodos/microbiologia , Tuberculose dos Linfonodos/transmissão , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/transmissão , Aerossóis , Fatores Etários , Animais , Carga Bacteriana , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Exposição por Inalação , Pulmão/microbiologia , Pulmão/patologia , Linfonodos/microbiologia , Linfonodos/patologia , Suínos , Porco Miniatura , Fatores de Tempo , Tuberculose dos Linfonodos/patologia , Tuberculose Pulmonar/patologia
15.
Microbiol Spectr ; 4(6)2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-28087940

RESUMO

Immunological memory is a central feature of the adaptive immune system and a prerequisite for generating effective vaccines. Understanding long-term memory responses to Mycobacterium tuberculosis will thus provide us with valuable insights that can guide us in the search for a novel vaccine against tuberculosis (TB). For many years, triggering CD4 T cells and, in particular, those secreting interferon-γ has been the goal of most TB vaccine research, and numerous data from animals and humans support the key role of this subset in protective immunity. More recently, we have learned that the memory response required for effective control of M. tuberculosis is much more complex, probably involving several phenotypically different CD4 T cell subsets as well as other cell types that are yet to be defined. Herein, we describe recent insights into memory immunity to TB in the context of both animal models and the human infection. With the increasing amount of data generated from clinical testing of novel TB vaccines, we also summarize recent knowledge of vaccine-induced memory immunity.


Assuntos
Imunidade Adaptativa , Memória Imunológica , Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Modelos Animais de Doenças , Humanos , Vacinas contra a Tuberculose/imunologia
16.
J Leukoc Biol ; 99(4): 605-17, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26590147

RESUMO

Increasing information has shown that many newly emerging strains of Mycobacterium tuberculosis, including the highly prevalent and troublesome Beijing family of strains, can potently induce the emergence of Foxp3(+)CD4 Tregs Although the significance of this is still not fully understood, we have previously provided evidence that the emergence of this population can significantly ablate the protective effect of BCG vaccination, causing progressive fatal disease in the mouse model. However, whether the purpose of this response is to control inflammation or to directly dampen the acquired immune response is still unclear. In the present study, we have shown, using both cell depletion and adoptive transfer strategies, that Tregs can have either properties. Cell depletion resulted in a rapid, but transient, decrease in the lung bacterial load, suggesting release or temporary re-expansion of effector immunity. Transfer of Tregs into Rag2(-/-)or marked congenic mice worsened the disease course and depressed cellular influx of effector T cells into the lungs. Tregs from infected donors seemed to preferentially depress the inflammatory response and granulocytic influx. In contrast, those from BCG-vaccinated and then challenged donors seemed more focused on depression of acquired immunity. These qualitative differences might be related to increasing knowledge reflecting the plasticity of the Treg response.


Assuntos
Imunidade Celular/imunologia , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Linfócitos T Reguladores/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinação , Animais , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/patologia , Tuberculose/genética
17.
PLoS One ; 10(9): e0136500, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26368806

RESUMO

To date, most new vaccines against Mycobacterium tuberculosis, including new recombinant versions of the current BCG vaccine, have usually been screened against the laboratory strains H37Rv or Erdman. In this study we took advantage of our recent work in characterizing an increasingly large panel of newly emerging clinical isolates [from the United States or from the Western Cape region of South Africa], to determine to what extent vaccines would protect against these [mostly high virulence] strains. We show here that both BCG Pasteur and recombinant BCG Aeras-422 [used here as a good example of the new generation BCG vaccines] protected well in both mouse and guinea pig low dose aerosol infection models against the majority of clinical isolates tested. However, Aeras-422 was not effective in a long term survival assay compared to BCG Pasteur. Protection was very strongly expressed against all of the Western Cape strains tested, reinforcing our viewpoint that any attempt at boosting BCG would be very difficult to achieve statistically. This observation is discussed in the context of the growing argument made by others that the failure of a recent vaccine trial disqualifies the further use of animal models to predict vaccine efficacy. This viewpoint is in our opinion completely erroneous, and that it is the fitness of prevalent strains in the trial site area that is the centrally important factor, an issue that is not being addressed by the field.


Assuntos
Vacina BCG/imunologia , Mycobacterium tuberculosis/imunologia , Potência de Vacina , Animais , Feminino , Cobaias , Camundongos , Camundongos Endogâmicos C57BL
18.
Antimicrob Agents Chemother ; 59(11): 6904-12, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26303795

RESUMO

Over the last 10 years, Mycobacterium abscessus group strains have emerged as important human pathogens, which are associated with significantly higher fatality rates than any other rapidly growing mycobacteria. These opportunistic pathogens are widespread in the environment and can cause a wide range of clinical diseases, including skin, soft tissue, central nervous system, and disseminated infections; by far, the most difficult to treat is the pulmonary form. Infections with M. abscessus are often multidrug-resistant (MDR) and require prolonged treatment with various regimens and, many times, result in high mortality despite maximal therapy. We report here the evaluation of diverse mouse infection models for their ability to produce a progressive high level of infection with M. abscessus. The nude (nu/nu), SCID (severe combined immunodeficiency), gamma interferon knockout (GKO), and granulocyte-macrophage colony-stimulating factor (GMCSF) knockout mice fulfilled the criteria for an optimal model for compound screening. Thus, we set out to assess the antimycobacterial activity of clarithromycin, clofazimine, bedaquiline, and clofazimine-bedaquiline combinations against M. abscessus-infected GKO and SCID murine infection models. Treatment of GKO and SCID mice with a combination of clofazimine and bedaquiline was the most effective in decreasing the M. abscessus organ burden.


Assuntos
Antibacterianos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Mycobacterium/efeitos dos fármacos , Animais , Claritromicina/farmacologia , Clofazimina/farmacologia , Diarilquinolinas/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Camundongos , Camundongos Knockout , Camundongos SCID , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium
19.
BMC Microbiol ; 15: 39, 2015 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-25887904

RESUMO

BACKGROUND: Macrophages are the primary effector cells responsible for killing Mycobacterium tuberculosis (MTB) through various mechanisms, including apoptosis. However, MTB can evade host immunity to create a favorable environment for intracellular replication. MTB-infected human macrophages produce interleukin-32 (IL-32). IL-32 is a pro-inflammatory cytokine and has several isoforms. We previously found that IL-32γ reduced the burden of MTB in human macrophages, in part, through the induction of caspase-3-dependent apoptosis. However, based on our previous studies, we hypothesized that caspase-3-independent death pathways may also mediate IL-32 control of MTB infection. Herein, we assessed the potential roles of cathepsin-mediated apoptosis, caspase-1-mediated pyroptosis, and apoptosis-inducing factor (AIF) in mediating IL-32γ control of MTB infection in THP-1 cells. RESULTS: Differentiated human THP-1 macrophages were infected with MTB H37Rv alone or in the presence of specific inhibitors to caspase-1, cathepsin B/D, or cathepsin L for up to four days, after which TUNEL-positive cells were quantified; in addition, MTB was quantified by culture as well as by the percentage of THP-1 cells that were infected with green fluorescent protein (GFP)-labeled MTB as determined by microscopy. AIF expression was inhibited using siRNA technology. Inhibition of cathepsin B/D, cathepsin L, or caspase-1 activity significantly abrogated the IL-32γ-mediated reduction in the number of intracellular MTB and of the percentage of GFP-MTB-infected macrophages. Furthermore, inhibition of caspase-1, cathepsin B/D, or cathepsin L in the absence of exogenous IL-32γ resulted in a trend toward an increased proportion of MTB-infected THP-1 cells. Inhibition of AIF activity in the absence of exogenous IL-32γ also increased intracellular burden of MTB. However, since IL-32γ did not induce AIF and because the relative increases in MTB with inhibition of AIF were similar in the presence or absence of IL-32γ, our results indicate that AIF does not mediate the host-protective effect of IL-32γ against MTB. CONCLUSIONS: The anti-MTB effects of IL-32γ are mediated through classical caspase-3-dependent apoptosis as well as caspase-3-independent apoptosis.


Assuntos
Apoptose , Interleucinas/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/imunologia , Carga Bacteriana , Linhagem Celular , Citoplasma/microbiologia , Humanos
20.
Proc Natl Acad Sci U S A ; 112(16): 5111-6, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25820174

RESUMO

Silencing of interleukin-32 (IL-32) in a differentiated human promonocytic cell line impairs killing of Mycobacterium tuberculosis (MTB) but the role of IL-32 in vivo against MTB remains unknown. To study the effects of IL-32 in vivo, a transgenic mouse was generated in which the human IL-32γ gene is expressed using the surfactant protein C promoter (SPC-IL-32γTg). Wild-type and SPC-IL-32γTg mice were infected with a low-dose aerosol of a hypervirulent strain of MTB (W-Beijing HN878). At 30 and 60 d after infection, the transgenic mice had 66% and 85% fewer MTB in the lungs and 49% and 68% fewer MTB in the spleens, respectively; the transgenic mice also exhibited greater survival. Increased numbers of host-protective innate and adaptive immune cells were present in SPC-IL-32γTg mice, including tumor necrosis factor-alpha (TNFα) positive lung macrophages and dendritic cells, and IFN-gamma (IFNγ) and TNFα positive CD4(+) and CD8(+) T cells in the lungs and mediastinal lymph nodes. Alveolar macrophages from transgenic mice infected with MTB ex vivo had reduced bacterial burden and increased colocalization of green fluorescent protein-labeled MTB with lysosomes. Furthermore, mouse macrophages made to express IL-32γ but not the splice variant IL-32ß were better able to limit MTB growth than macrophages capable of producing both. The lungs of patients with tuberculosis showed increased IL-32 expression, particularly in macrophages of granulomas and airway epithelial cells but also B cells and T cells. We conclude that IL-32γ enhances host immunity to MTB.


Assuntos
Interleucinas/metabolismo , Mycobacterium tuberculosis/patogenicidade , Tuberculose/imunologia , Tuberculose/prevenção & controle , Imunidade Adaptativa/imunologia , Animais , Antígenos Ly/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Humanos , Imunidade Inata/imunologia , Interferon gama , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Linfonodos/imunologia , Linfonodos/patologia , Macrófagos Alveolares/imunologia , Camundongos Transgênicos , Mutação/genética , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteína C Associada a Surfactante Pulmonar/metabolismo , Sítios de Splice de RNA/genética , Linfócitos T Reguladores/imunologia , Transfecção , Transgenes , Tuberculose/microbiologia , Fator de Necrose Tumoral alfa/metabolismo , Virulência/imunologia
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