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1.
J Immunol Methods ; 529: 113679, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38679364

RESUMO

The type II autoimmune subtype of Chronic Spontaneous Urticaria (CSU) is characterized by the presence of IgG autoantibodies targeting IgE or the IgE high-affinity receptor (FcεRI) on mast cells and basophils. In evaluation of CSU patients, indirect basophil activation testing (BAT), has been utilized, involving the mixing of patient serum with heterologous peripheral blood donors, followed by flow cytometric assessment of basophil markers. However, the reliability of the indirect BAT results hinges on the quality of the donor basophils utilized. In this study, we introduce an innovative approach where multiple potential basophil donors undergo rigorous BAT characterization alongside control samples. By selecting and pooling donors with optimal performance, we significantly enhance the inter-assay reproducibility of the indirect BAT test.


Assuntos
Basófilos , Urticária Crônica , Citometria de Fluxo , Humanos , Basófilos/imunologia , Urticária Crônica/imunologia , Urticária Crônica/diagnóstico , Urticária Crônica/sangue , Citometria de Fluxo/métodos , Reprodutibilidade dos Testes , Teste de Degranulação de Basófilos/métodos , Adulto , Feminino , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Masculino , Autoanticorpos/sangue , Autoanticorpos/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Pessoa de Meia-Idade , Receptores de IgE/imunologia , Doadores de Sangue
2.
Heliyon ; 9(2): e13103, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36712916

RESUMO

Despite a growing amount of data around the kinetics and durability of the antibody response induced by vaccination and previous infection, there is little understanding of whether or not a given quantitative level of antibodies correlates to protection against SARS-CoV-2 infection or reinfection. In this study, we examine SARS-CoV-2 anti-spike receptor binding domain (RBD) antibody titers and subsequent SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) tests in a large cohort of US-based patients. We analyzed antibody test results in a cohort of 22,204 individuals, 6.8% (n = 1,509) of whom eventually tested positive for SARS-CoV-2 RNA, suggesting infection or reinfection. Kaplan-Meier curves were plotted to understand the effect of various levels of anti-spike RBD antibody titers (classified into discrete ranges) on subsequent RT-PCR positivity rates. Statistical analyses included fitting a Cox proportional hazards model to estimate the age-, sex- and exposure-adjusted hazard ratios for S antibody titer, using zip-code positivity rates by week as a proxy for COVID-19 exposure. It was found that the best models of the temporally associated infection risk were those based on log antibody titer level (HR = 0.836 (p < 0.05)). When titers were binned, the hazard ratio associated with antibody titer >250 Binding Antibody Units (BAU) was 0.27 (p < 0.05, 95% CI [0.18, 0.41]), while the hazard ratio associated with previous infection was 0.20 (p < 0.05, 95% CI [0.10, 0.39]). Fisher exact odds ratio (OR) for Ab titers <250 BAU showed OR = 2.84 (p < 0.05; 95% CI: [2.30, 3.53]) for predicting the outcome of a subsequent PCR test. Antibody titer levels correlate with protection against subsequent SARS-CoV-2 infection or reinfection when examining a cohort of real-world patients who had the spike RBD antibody assay performed.

3.
Indian J Cancer ; 2021 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-35017366

RESUMO

BACKGROUND: The comparison of triclosan-coated sutures (TCS) was made with conventional nonantimicrobial-coated sutures (NCS) to reduce surgical site infection (SSI). This study demonstrates the efficacy and economic outcome of TCS versus NCS for SSIs in mastectomy in India. METHODS: In this retrospective analysis, 100 patients were included for both conditions-TCS and NCS-from a private and public hospital in India. A systematic literature search of available evidence for both SSI incidences and TCS efficacy data in India were gathered. We collected cost data from a private and public hospital, respectively, for mastectomy in India. The cost-effectiveness of TCS in comparison with the conventional NCS was calculated using a decision-tree deterministic model. We performed a one-way sensitivity analysis to compare TCS with NCS. RESULTS: Cost savings with the use of TCS increased with an increase in SSI incidence and an increase in efficacy for mastectomies in both public and private hospitals. We found a base cost saving of Indian rupees (INR) 27,299 at a private hospital and INR 2,958 at a public hospital for mastectomies. The incremental cost of TCS suture was 0.01% in a private hospital whereas 0.17% in a public hospital. CONCLUSION: The use of TCS resulted in reduced SSI incidence and cost savings for mastectomy in India.

4.
Immunology ; 152(1): 150-162, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28502122

RESUMO

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4+ and CD8+ T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG-C, a liposome-based formulation containing the M. tuberculosis antigen ESAT-6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T-cell response. Humanized mice provide a crucial pre-clinical platform for evaluating human T-cell immune responses in vaccine development against M. tuberculosis.


Assuntos
Vacina BCG/administração & dosagem , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Oligodesoxirribonucleotídeos/administração & dosagem , Tuberculose Pulmonar/prevenção & controle , Animais , Vacina BCG/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/microbiologia , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Cobaias , Humanos , Antígenos Comuns de Leucócito/sangue , Antígenos Comuns de Leucócito/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Mycobacterium tuberculosis/metabolismo , Oligodesoxirribonucleotídeos/imunologia , Fenótipo , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Vacinação
5.
Vaccine ; 34(25): 2798-805, 2016 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-27131285

RESUMO

The use of novel vaccine delivery systems allows for the manipulation of the adaptive immune systems through the use of molecular adjuvants that target specific innate pathways. Such strategies have been used extensively for vaccines against cancer and multiple pathogens such as Mycobacterium tuberculosis. In the current study we used heat killed non-pathogenic recombinant Saccharomyces cerevisiae expressing M. tuberculosis antigen Rv1886c (fbpB, mpt59, Ag85B) as a delivery system in conjunction with its ability to stimulate innate immunity to determine its ability to induce immunity. We established that the recombinant yeast induced activated antigen specific T cells are capable of reducing the mycobacterial burden. Inoculation of the recombinant yeast after vaccination with BCG resulted in a systemic alteration of the phenotype of the immune response although this was not reflected in an increase in the reduction of the mycobacterial burden. Taken together the data suggest that heat killed yeast can induce multiple cytokines required for induction of protective immunity and can function as a vehicle for delivery of M. tuberculosis antigens in a vaccine formulation. In addition, while it can enhance the effector memory response induced by BCG, it had little effect on central memory responses.


Assuntos
Aciltransferases/imunologia , Adjuvantes Imunológicos/administração & dosagem , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Saccharomyces cerevisiae/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Citocinas/imunologia , Feminino , Temperatura Alta , Imunização Secundária , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia
6.
Immunology ; 142(1): 111-23, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24350616

RESUMO

In order to ensure an ample supply of quality candidate tuberculosis (TB) subunit vaccines for clinical trials, it is imperative to develop new immunostimulatory adjuvants. High Mobility Box Group 1 (HMGB1), a member of the alarmin group of immunostimulatory proteins, is released by antigen-presenting cells under various conditions and has been shown to induce T helper type 1 cytokines. We report that HMGB1 is effective as an adjuvant to enhance the protective efficacy and cellular immune response of TB subunit vaccines and that it is not dependent on the interaction between HMGB1 and receptor for advanced glycation end products, a major receptor for HMGB1. In the mouse model of TB, HMGB1 protein, when formulated with dioctadecylammonium bromide and 6000 MW early secretory antigenic target (ESAT-6), was protective as a subunit vaccine but did not protect as molecular adjuvant in an ESAT-6-based DNA formulation. We then evaluated the immunoprophylactic and protective potential of a fusion protein of HMGB1 and ESAT-6. The HMGB1-ESAT-6 fusion protein induced strong antigen-specific T helper type 1 cytokines at 30 days post-immunization. The fusion protein vaccine enhanced activated and effector memory CD4 and CD8 T-cell responses in the lungs and spleens of mice at 80 days post vaccination. Vaccination with the HMGB1-ESAT-6 fusion protein also resulted in elevated numbers of poly-functional CD4 T cells co-expressing interleukin-2, interferon-γ and tumour necrosis factor-α. The potent cell-mediated immune response generated by the fusion protein correlated with protection against subsequent challenge with Mycobacterium tuberculosis in the mouse TB model.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Proteína HMGB1/farmacologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/prevenção & controle , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos de Bactérias/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Proteína HMGB1/administração & dosagem , Proteína HMGB1/imunologia , Imunidade Celular/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/microbiologia , Fatores de Tempo , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Vacinas de Subunidades Antigênicas/imunologia
7.
Immunol Cell Biol ; 90(10): 945-54, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22801575

RESUMO

New approaches consisting of 'multistage' vaccines against (TB) are emerging that combine early antigenic proteins with latency-associated antigens. In this study, HspX was tested for its potential to elicit both short- and long-term protective immune responses. HspX is a logical component in vaccine strategies targeting protective immune responses against primary infection, as well as against reactivation of latent infection, because as previously shown, it is produced during latency, and as our studies show, it elicits protection within 30 days of infection. Recent studies have shown that the current TB vaccine, bacilli Calmette-Guerin (BCG), does not induce strong interferon-γ T-cell responses to latency-associated antigens like HspX, which may be in part why BCG fails to protect against reactivation disease. We therefore tested HspX protein alone as a prophylactic vaccine and as a boost to BCG vaccination, and found that HspX purified from M. tuberculosis cell lysates protected mice against aerosol challenge and improved the protective efficacy of BCG when used as a booster vaccine. Native HspX was highly immunogenic and protective, in a dose-dependent manner, in both short- and long-term infection models. Based on these promising findings, HspX was produced as a recombinant protein in E. coli, as this would enable facile purification; however, recombinant HspX (rHspX) alone consistently failed to protect against aerosol challenge. Incubation of rHspX with mycobacterial cell lysate and re-purification following incubation restored the capacity of the protein to confer protection. These data suggest the possibility that the native form may chaperone an immunogenic and protective antigen that is mycobacteria-specific.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Chaperonas Moleculares/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Escherichia coli/genética , Feminino , Humanos , Imunidade Ativa/genética , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Transgenes/genética
8.
PLoS One ; 7(7): e40836, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22808273

RESUMO

HLA-B-associated transcript 3 (BAT3), also known as Scythe or BAG6, is a nuclear protein implicated in the control of apoptosis and natural killer (NK) cell-dendritic cell (DC) interaction. We demonstrate that BAT3 modulates the immune response by regulating the function of macrophages. BAT3 is released by macrophages in vitro and it down-regulates nitric oxide and proinflammatory cytokines release in IFN-γ and LPS stimulated macrophages. Furthermore, Mycobacterium tuberculosis-derived protein ESAT-6 (Rv3875) induced transient increase in the expression and release of BAT3 in macrophages. We show that induction of apoptosis by ESAT-6 is dependent on the cleavage of BAT3 by caspase-3 and proteasomal degradation. Our results also indicate that BAT3 regulates ESAT-6-induced apoptosis by interacting with anti-apoptotic protein BCL-2. Taken together, the data suggest that BAT3 plays a role in the early immune response to M. tuberculosis infection and may be a key protein associated with the fate of antigen presenting cells during infection.


Assuntos
Antígenos de Bactérias/metabolismo , Apoptose , Proteínas de Bactérias/metabolismo , Macrófagos/citologia , Macrófagos/microbiologia , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/fisiologia , Proteínas Nucleares/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Inibidores de Caspase/farmacologia , Linhagem Celular , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Mycobacterium tuberculosis/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Solubilidade
9.
Tuberculosis (Edinb) ; 92(1): 105-11, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21962569

RESUMO

The guinea pig model of tuberculosis is used extensively in different locations to assess the efficacy of novel tuberculosis vaccines during pre-clinical development. Two key assays are used to measure protection against virulent challenge: a 30 day post-infection assessment of mycobacterial burden and long-term post-infection survival and pathology analysis. To determine the consistency and robustness of the guinea pig model for testing vaccines, a comparative assessment between three sites that are currently involved in testing tuberculosis vaccines from external providers was performed. Each site was asked to test two "subunit" type vaccines in their routine animal model as if testing vaccines from a provider. All sites performed a 30 day study, and one site also performed a long-term survival/pathology study. Despite some differences in experimental approach between the sites, such as the origin of the Mycobacterium tuberculosis strain and the type of aerosol exposure device used to infect the animals and the source of the guinea pigs, the data obtained between sites were consistent in regard to the ability of each "vaccine" tested to reduce the mycobacterial burden. The observations also showed that there was good concurrence between the results of short-term and long-term studies. This validation exercise means that efficacy data can be compared between sites.


Assuntos
Mycobacterium tuberculosis/efeitos dos fármacos , Vacinas contra a Tuberculose/farmacologia , Tuberculose/prevenção & controle , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Cobaias , Camundongos , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Vacinas contra a Tuberculose/imunologia
11.
Infect Immun ; 79(2): 716-23, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21134965

RESUMO

Purified protein derivative (PPD) is a widely used reagent for the diagnosis of Mycobacterium tuberculosis infection. Recently, the molecular composition of PPD was defined, with hundreds of mycobacterial protein representatives making up PPD. Which, if any, of these specific products drive the potency of PPD remains in question. In this study, two proteins (DnaK and GroEL2) previously identified as dominant proteins in PPD were tested for the capacity to induce delayed-type hypersensitivity (DTH) responses in H37Rv-infected or BCG-vaccinated guinea pigs. These two proteins were used in pull-down assays to identify interacting PPD products. Six proteins were identified as interacting partners with DnaK and GroEL2, i.e., Rv0009, Rv0475, Rv0569, Rv0685, Rv2626c, and Rv2632c. These six proteins were tested alone and in combination with DnaK and GroEL2 for the capacity to induce a DTH response in the guinea pig model. From these studies, two cocktails, DnaK/GroEL2/Rv0009 and DnaK/GroEL2/Rv0685, were found to induce DTH responses in H37Rv-infected or BCG-vaccinated guinea pigs that were indistinguishable from DTH responses driven by a PPD injection. The mechanism by which DTH responses were induced was elucidated by histologic examination, analysis of activated CD4(+)/CD8(+) T cells, and cytokine mRNA expression at the site of the DTH response. PPD and the protein cocktails tested induced strong DTH responses in H37Rv-infected guinea pigs. Ex vivo phenotyping of T cells at the DTH site indicated that this response is mediated by activated CD4(+) and CD8(+) T cells, with increases in gamma interferon and tumor necrosis factor alpha, but not interleukin-10, at the site of the DTH response. Our results demonstrate for the first time that the PPD response can be mimicked at the molecular level with defined protein cocktails. The use of this defined product will allow a more thorough understanding of the DTH response and may provide a platform for more rapid and sensitive second-generation skin test reagents for the diagnosis of M. tuberculosis infection.


Assuntos
Proteínas de Bactérias/imunologia , Hipersensibilidade Tardia/imunologia , Mycobacterium tuberculosis , Tuberculina/imunologia , Tuberculose/imunologia , Animais , Vacina BCG/imunologia , Regulação da Expressão Gênica/imunologia , Cobaias , Tuberculose/prevenção & controle
12.
Diagn Microbiol Infect Dis ; 66(2): 153-61, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19833469

RESUMO

We evaluated the diagnostic potential of a cocktail of 4 antigens encoded by regions of difference (RD) 1 and 2 of Mycobacterium tuberculosis, that is, early secretory antigenic target-6, culture filtrate protein-10 (CFP-10), CFP-21, and mycobacterial protein from species tuberculosis-64 (MPT-64) on the basis of antigen and antibody detection by enzyme-linked immunosorbent assay. Parallel detection of antigens and antibodies in the serum samples of pulmonary tuberculosis (PTB) patients resulted in higher sensitivity as compared to either of the single tests in both smear-positive (90%) and smear-negative (60%) PTB patients. In addition, combined detection of antigens and antibodies in the fluids of extrapulmonary tuberculosis (EPTB) patients could detect >90% of the patients with high specificity. These results demonstrate the ability of the combination of antigen and antibody detection assays based on the cocktail of RD antigens to diagnose a substantial number of PTB and EPTB cases with high specificity.


Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/diagnóstico , Humanos , Sensibilidade e Especificidade
13.
Expert Rev Vaccines ; 8(11): 1547-53, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19863247

RESUMO

Several new antituberculous vaccine candidates that are effective against primary infection in preclinical animal models have now entered the early phases of clinical trials. Many of these clinical trials involve subunit vaccines, recombinant bacillus Calmette-Guérin (BCG), or improvement of BCG immunity by boosting with subunit vaccines or recombinant viral vectors expressing immunodominant TB antigens. The burning question at this stage is: will the current vaccines be effective in the endemic world where the diverse and complex challenges of TB exist? These challenges include protection of those individuals who are already vaccinated with BCG, those already exposed to environmental mycobacteria and those infected with latent TB or HIV. This review focuses on the available BCG vaccine, new TB vaccines in the pipeline and what type of vaccines are actually needed in high-burden endemic countries.


Assuntos
Doenças Endêmicas , Vacinas contra a Tuberculose/imunologia , Tuberculose/epidemiologia , Tuberculose/prevenção & controle , Animais , Pesquisa Biomédica/tendências , Ensaios Clínicos como Assunto , Humanos , Vacinação/métodos , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologia
14.
Infect Immun ; 77(11): 4837-46, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19737892

RESUMO

The guinea pig model of tuberculosis is used extensively in assessing novel vaccines, since Mycobacterium bovis BCG vaccination effectively prolongs survival after low-dose aerosol infection with virulent M. tuberculosis. To better understand how BCG extends time to death after pulmonary infection with M. tuberculosis, we examined cytokine responses postvaccination and recruitment of activated T cells and cytokine response postinfection. At 10 weeks postvaccination, splenic gamma interferon (IFN-gamma) mRNA was significantly elevated compared to the levels at 5 weeks in ex vivo stimulation assays. At 15, 40, 60, and 120 days postinfection, T-cell activation (CD4+ CD62Llow and CD8+ CD62Llow) and mRNA expression of IFN-gamma, tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-10, IL-12, and eomesodermin were assessed. Our data show that at day 40, BCG-vaccinated guinea pigs had significantly increased levels of IFN-gamma mRNA expression but decreased TNF-alpha mRNA expression in their lungs compared to the levels in nonvaccinated animals. At day 120, a time when nonvaccinated guinea pigs succumbed to infection, low levels of IFN-gamma mRNA were observed even though there were increasing levels of IL-1, IL-12, and IL-10, and the numbers of activated T cells did not differ from those in BCG-vaccinated animals. BCG vaccination conferred the advantage of recruiting greater numbers of CD4+ CD62Llow T cells at day 40, although the numbers of CD8+ CD62Llow T cells were not elevated compared to the numbers in nonvaccinated animals. Our data suggest that day 40 postinfection may be a pivotal time point in determining vaccine efficacy and prolonged survival and that BCG promotes the capacity of T cells in the lungs to respond to infection.


Assuntos
Vacina BCG/imunologia , Mycobacterium bovis/imunologia , Tuberculose/imunologia , Tuberculose/terapia , Animais , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Citometria de Fluxo , Cobaias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Tuberculose/veterinária
15.
Vaccine ; 27(33): 4412-23, 2009 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-19500523

RESUMO

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected approximately two billion individuals worldwide with approximately 9.2 million new cases and 1.6 million deaths annually. Current efforts are focused on making better BCG priming vaccines designed to induce a comprehensive and balanced immunity followed by booster(s) targeting a specific set of relevant antigens in common with the BCG prime. We describe the generation and immunological characterization of recombinant BCG strains with properties associated with lysis of the endosome compartment and over-expression of key Mtb antigens. The endosome lysis strain, a derivative of BCG SSI-1331 (BCG(1331)) expresses a mutant form of perfringolysin O (PfoA(G137Q)), a cytolysin normally secreted by Clostridium perfringens. Integration of the PfoA(G137Q) gene into the BCG genome was accomplished using an allelic exchange plasmid to replace ureC with pfoA(G137Q) under the control of the Ag85B promoter. The resultant BCG construct, designated AERAS-401 (BCG(1331) DeltaureC::OmegapfoA(G137Q)) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice. A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401. Compared to the parental BCG strain, vaccination of mice and guinea pigs with AFRO-1 resulted in enhanced immune responses. Mice vaccinated with AFRO-1 and challenged with the hypervirulent Mtb strain HN878 also survived longer than mice vaccinated with the parental BCG. Thus, we have generated improved rBCG vaccine candidates that address many of the shortcomings of the currently licensed BCG vaccine strains.


Assuntos
Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Toxinas Bacterianas/imunologia , Proteínas Hemolisinas/imunologia , Epitopos Imunodominantes/imunologia , Tuberculose/prevenção & controle , Animais , Vacina BCG/administração & dosagem , Vacina BCG/genética , Toxinas Bacterianas/genética , Linhagem Celular , Eritrócitos/microbiologia , Feminino , Genes Bacterianos , Cobaias , Proteínas Hemolisinas/genética , Hemólise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Plasmídeos , Ovinos , Tuberculose/imunologia
16.
Cell Microbiol ; 10(6): 1390-404, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18331466

RESUMO

High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein that acts as a pro-inflammatory cytokine and is released by monocytes and macrophages. Necrotic cells also release HMGB1 at the site of tissue damage which induces a variety of cellular responses, including the expression of pro-inflammatory mediators. This study investigated the secretion of HMGB1 in mycobacterial infection by macrophages in vitro and in the lungs of infected guinea pigs. We observed that infection by mycobacterium effectively induced HMGB1 release in both macrophage and monocytic cell cultures. Culture filtrate proteins from Mycobacterium tuberculosis induced maximum release of HMGB1 compared with different subcellular fractions of mycobacterium. We demonstrated that HMGB1 is released in lungs during infection of M. tuberculosis in guinea pigs and increased HMGB1 secretion in lungs of guinea pigs was delayed by prior vaccination with Mycobacterium bovis BCG. The secretion of cytokines like tumour necrosis factor alpha (TNF-alpha) and Interleukin-1beta was significantly increased when M. bovis BCG-infected cultures of J774A.1 cells were incubated with HMGB1. Among different mycobacterial toll-like receptor ligands, heat-shock protein 65 (HSP65) was found to be more potent in inducing HMGB1 secretion in RAW 264.7 cells. Pharmacological suppression of p38 or extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases with specific inhibitors failed to inhibit HSP65-induced HMGB1 release, but inhibition of c-Jun NH(2)-terminal kinase activation attenuated HMGB1 release. Inhibition of the inducible NO synthase and neutralizing antibodies against TNF-alpha also reduced HMGB1 release stimulated by HSP65. We conclude that HMGB1 is secreted by macrophages during tuberculosis and it may act as a signal of tissue or cellular injury and enhances immune response.


Assuntos
Proteína HMGB1/metabolismo , Infecções por Mycobacterium/metabolismo , Mycobacterium tuberculosis , Animais , Vacina BCG/administração & dosagem , Proteínas de Bactérias/fisiologia , Linhagem Celular , Chaperonina 60 , Chaperoninas/fisiologia , Citocinas/biossíntese , Cobaias , Injeções Subcutâneas , Pulmão/metabolismo , Macrófagos/metabolismo , Camundongos , Infecções por Mycobacterium/imunologia , Mycobacterium tuberculosis/química , Vacinação
17.
Clin Immunol ; 125(2): 173-83, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17766185

RESUMO

Different combinations of ESAT-6, CFP-10, CFP-21, MPT-64, encoded by RD1 and RD2 of Mycobacterium tuberculosis were evaluated on the basis of antigenicity in PPD positive TB contacts and immunogenicity in C57BL/6J mice immunized with the combination of all four RD antigens. The peripheral blood mononuclear cells of TB contacts showed maximum recognition in response to the combination of ESAT-6+MPT-64 in terms of predominant lymphoproliferation, IFN-gamma levels and the number of responders. On the contrary, the combination of ESAT-6+CFP-21+MPT-64 was found to be most immunogenic based on both T-cell and antibody responses in immunized mice. Prophylactic potential of the selected combinations was assessed as supplementation vaccines to BCG against intravenous challenge with M. tuberculosis in mice. BCG supplementation with the selected combinations resulted in significantly greater protection as compared to BCG alone against experimental tuberculosis and thus appears to be a promising approach to enhance the protective efficacy of the existing vaccine.


Assuntos
Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Imunização/métodos , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Adulto , Animais , Antígenos de Bactérias/farmacologia , Vacina BCG/farmacologia , Proteínas de Bactérias/imunologia , Contagem de Colônia Microbiana , Feminino , Humanos , Imunização/normas , Interferon gama/metabolismo , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Tuberculose/microbiologia
18.
Microbes Infect ; 8(9-10): 2390-9, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16962360

RESUMO

Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.


Assuntos
Antígenos de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinas de DNA/imunologia , Animais , Formação de Anticorpos/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Feminino , Imunofenotipagem/métodos , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/genética , Linfócitos T Citotóxicos/imunologia , Tuberculose/microbiologia , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/uso terapêutico , Vacinas de DNA/genética , Vacinas de DNA/uso terapêutico
19.
Protein Expr Purif ; 48(2): 274-80, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16716602

RESUMO

Secreted proteins encoded by different regions of difference (RDs) from the genome of Mycobacterium tuberculosis have been considered as attractive candidates for vaccination against tuberculosis owing to their absence in most BCG strains. In this study, the structural gene for the RD2 locus encoding protein CFP21 was PCR amplified and expressed as a fusion protein with hexahistidine residues in Escherichia coli. Expression of CFP21 in E. coli under transcriptional regulation of the T7 promoter yielded a protein located within inclusion bodies. The inclusion bodies were solubilized in the presence of 8M urea and the protein was purified to homogeneity under denaturing conditions at low pH using nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradient dialysis against a decreasing concentration of urea. The purified protein was shown to have esterase activity. CFP21 protein was evaluated for immunogenicity in C57BL/6J mice. We observed an elevated T cell proliferative response and production of IFN-gamma and IL-12 (p40). CFP21 also induced an optimum level of cytotoxic T cell activity and induced a strong humoral response as indicated by higher levels of specific IgG1 and IgG2a antibody isotypes. In addition, a moderate level of protection was observed against experimental tuberculosis. This is the first report describing esterase activity of the M. tuberculosis complex-restricted protein CFP21 and its protective potential against experimental tuberculosis.


Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Expressão Gênica , Modelos Animais , Mycobacterium tuberculosis/imunologia , Animais , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Modelos Animais de Doenças , Escherichia coli/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Imunológicos , Mycobacterium tuberculosis/genética , Filogenia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Tuberculose/imunologia , Tuberculose/prevenção & controle
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