Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.917
Filtrar
1.
Nat Immunol ; 21(7): 746-755, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32514064

RESUMO

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.


Assuntos
Infecções Bacterianas/imunologia , Toxinas Bacterianas/imunologia , Hidroxicolesteróis/metabolismo , Interferons/isolamento & purificação , Fagócitos/imunologia , Estreptolisinas/imunologia , Animais , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Fagócitos/citologia , Fagócitos/metabolismo , Cultura Primária de Células , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Estreptolisinas/administração & dosagem , Estreptolisinas/metabolismo
2.
Vet Immunol Immunopathol ; 224: 110059, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32408182

RESUMO

There are currently no licensed vaccines against Clostridium perfringens which causes necrotic enteritis in poultry. Chitosan nanoparticles were formulated with native (CN) or toxoids (CT) of extracellular proteins (ECP) of C. perfringens, both surface-tagged with Salmonella flagellar proteins. In a pH stability assay, CN and CT nanoparticles released 6% and 0% of their protein at 8.0 pH. In a protein release assay, CN and CT nanoparticles released 16% and 10% of their protein respectively at 7.4 pH after 24 h. CN and CT nanoparticles incubated at 100 µg/mL PBS with Chicken RBCs released 1% and 0% hemoglobin respectively. Ninety broilers were randomly assigned to treatments; sham-vaccinated (Control), CN-vaccinated (CN), and CT-vaccinated (CT). Each bird was orally gavaged with 50 µg vaccine in 0.5 mL PBS or 0.5 mL PBS only on d 0, 3, 7 and 14 of age. At 21 d of age, the CN group had higher anti-ECP IgA than control (P < 0.05). At 21 d of age, the CN and CT group had higher anti-ECP IgA than control (P < 0.05). At 17 d of age, the CN group had higher anti-flagellar IgG than control (P < 0.05). At 10 d of age, the CN group had higher anti-flagellar IgA than control (P < 0.05). Splenic T cells from chickens in the CN and CT group ex-vivo stimulated with 0.05 mg/mL ECP, had higher proliferation control (P < 0.05, P < 0.01 respectively). Splenic T cells from chickens in the CN and CT groups ex-vivo stimulated with 0.1 mg/mL ECP had proliferation than control (P < 0.05). Pooled serum from 17 d of age CN and CT-vaccinated birds partially neutralized toxins in 50 µg of ECP (P < 0.05). Pooled serum from 28 d of age CN-vaccinated birds also partially neutralized toxins in 50 µg of ECP. The result from this study indicates the potential for chitosan loaded with Clostridium perfringens extracellular proteins to be applied to necrotic enteritis challenge studies.


Assuntos
Vacinas Bacterianas/imunologia , Quitosana/química , Infecções por Clostridium/veterinária , Enterocolite Necrosante/veterinária , Nanopartículas/química , Administração Oral , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Galinhas/imunologia , Galinhas/microbiologia , Infecções por Clostridium/imunologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens , Enterocolite Necrosante/imunologia , Enterocolite Necrosante/prevenção & controle , Flagelos/imunologia , Imunogenicidade da Vacina , Imunoglobulina A/análise , Imunoglobulina G/sangue , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/prevenção & controle , Salmonella , Vacinas Atenuadas/imunologia
3.
Mol Immunol ; 123: 88-96, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32447084

RESUMO

The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens.


Assuntos
Bacillus subtilis/metabolismo , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Vacinas Bacterianas/uso terapêutico , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/imunologia , Animais , Bacillus subtilis/genética , Toxinas Bacterianas/metabolismo , Vacinas Bacterianas/química , Vacinas Bacterianas/genética , Infecções por Clostridium/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Vacinação/métodos , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/uso terapêutico
4.
PLoS One ; 15(4): e0230782, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294093

RESUMO

Understanding immune responses to native antigens in response to natural infections can lead to improved approaches to vaccination. This study sought to characterize the humoral immune response to anthrax toxin components, capsule and spore antigens in individuals (n = 46) from the Kayseri and Malatya regions of Turkey who had recovered from mild or severe forms of cutaneous anthrax infection, compared to regional healthy controls (n = 20). IgG antibodies to each toxin component, the poly-γ-D-glutamic acid capsule, the Bacillus collagen-like protein of anthracis (BclA) spore antigen, and the spore carbohydrate anthrose, were detected in the cases, with anthrax toxin neutralization and responses to Protective Antigen (PA) and Lethal Factor (LF) being higher following severe forms of the disease. Significant correlative relationships among responses to PA, LF, Edema Factor (EF) and capsule were observed among the cases. Though some regional control sera exhibited binding to a subset of the tested antigens, these samples did not neutralize anthrax toxins and lacked correlative relationships among antigen binding specificities observed in the cases. Comparison of serum binding to overlapping decapeptides covering the entire length of PA, LF and EF proteins in 26 cases compared to 8 regional controls revealed that anthrax toxin-neutralizing antibody responses elicited following natural cutaneous anthrax infection are directed to conformational epitopes. These studies support the concept of vaccination approaches that preserve conformational epitopes.


Assuntos
Antraz/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Epitopos/imunologia , Dermatopatias Bacterianas/imunologia , Adulto , Vacinas contra Antraz/imunologia , Especificidade de Anticorpos/imunologia , Bacillus anthracis/imunologia , Feminino , Humanos , Imunidade Humoral/imunologia , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Testes de Neutralização/métodos , Turquia , Adulto Jovem
5.
J Appl Microbiol ; 129(2): 443-452, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32118336

RESUMO

AIM: Category A classified Bacillus anthracis is highly fatal pathogen that causes anthrax and creates challenges for global security and public health. In this study, development of a safe and ideal next-generation subunit anthrax vaccine has been evaluated in mouse model. METHOD AND RESULTS: Protective antigen (PA) and BA3338, a surface layer homology (SLH) domain possessing protein were cloned, expressed in heterologous system and purified by IMAC. Recombinant PA and BA3338 with alum were administered in mouse alone or in combination. The humoral and cell-mediated immune response was measured by ELISA and vaccinated animals were challenged with B. anthracis spores via intraperitoneal route. The circulating IgG antibody titre of anti-PA and anti-BA3338 was found significantly high in the first and second booster sera. A significant enhanced level of IL-4, IFN-γ and IL-12 was observed in antigens stimulated supernatant of splenocytes of PA + BA3338 vaccinated animals. A combination of PA and BA3338 provided 80% protection against 20 LD50 lethal dose of B. anthracis spores. CONCLUSION: Both antigens induced admirable humoral and cellular immune response as well as protective efficacy against B. anthracis spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has been evaluated for the first time using BA3338 as a vaccine candidate alone or in combination with well-known anthrax vaccine candidate PA. The findings of this study demonstrated that BA3338 could be a co-vaccine candidate for development of dual subunit vaccine against anthrax.


Assuntos
Vacinas contra Antraz/administração & dosagem , Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Glicoproteínas de Membrana/imunologia , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Antraz/imunologia , Vacinas contra Antraz/imunologia , Anticorpos Antibacterianos/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Imunização/métodos , Camundongos , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/imunologia
6.
Curr Pharm Biotechnol ; 21(10): 973-979, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101119

RESUMO

BACKGROUND: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. OBJECTIVE: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. METHODS: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. RESULTS: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. CONCLUSION: The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.


Assuntos
Toxinas Bacterianas/genética , Biolística/métodos , Centella/genética , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Plantas Geneticamente Modificadas/genética , Animais , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Centella/metabolismo , Enterotoxinas/química , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Temperatura Alta , Plantas Geneticamente Modificadas/metabolismo , Triterpenos
7.
Int J Nanomedicine ; 15: 239-252, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021177

RESUMO

Introduction: Aluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis. Methods and Results: Herein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150-200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2- and Th1-type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel. Conclusion: These results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response.


Assuntos
Adjuvantes Imunológicos/farmacologia , Hidróxido de Alumínio/química , Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Nanopartículas Metálicas/química , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacocinética , Hidróxido de Alumínio/imunologia , Hidróxido de Alumínio/farmacologia , Animais , Antraz/imunologia , Vacinas contra Antraz/química , Vacinas contra Antraz/imunologia , Vacinas contra Antraz/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Difusão Dinâmica da Luz , Feminino , Humanos , Camundongos , Células RAW 264.7 , Espectroscopia de Infravermelho com Transformada de Fourier , Células Th1/imunologia
8.
mSphere ; 5(1)2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941807

RESUMO

Protective antigen (PA) is a component of anthrax toxin that can elicit toxin-neutralizing antibody responses. PA is also the major antigen in the current vaccine to prevent anthrax, but stability problems with recombinant proteins have complicated the development of new vaccines containing recombinant PA. The relationship between antigen physical stability and immunogenicity is poorly understood, but there are theoretical reasons to think that this parameter can affect immune responses. We investigated the immunogenicity of anthrax PA, in the presence and absence of the soluble von Willebrand factor A domain of the human form of receptor capillary morphogenesis protein 2 (sCMG2), to elicit antibodies to PA in BALB/c mice. Prior studies showed that sCMG2 stabilizes the 83-kDa PA structure to pH, chemical denaturants, temperature, and proteolysis and slows the hydrogen-deuterium exchange rate of histidine residues far from the binding interface. In contrast to a vaccine containing PA without adjuvant, we found that mice immunized with PA in stable complex with sCMG2 showed markedly reduced antibody responses to PA, including toxin-neutralizing antibodies and antibodies to domain 4, which correlated with fewer toxin-neutralizing antibodies. In contrast, mice immunized with PA in concert with a nonbinding mutant of sCMG2 (D50A) showed anti-PA antibody responses similar to those observed with PA alone. Our results suggest that addition of sCMG2 to a PA vaccine formulation is likely to result in a significantly diminished immune response, but we discuss the multitude of factors that could contribute to reduced immunogenicity.IMPORTANCE The anthrax toxin PA is the major immunogen in the current anthrax vaccine (anthrax vaccine adsorbed). Improving the anthrax vaccine for avoidance of a cold chain necessitates improvements in the thermodynamic stability of PA. We address how stabilizing PA using sCMG2 affects PA immunogenicity in BALB/c mice. Although the stability of PA is increased by binding to sCMG2, PA immunogenicity is decreased. This study emphasizes that, while binding of a ligand retains or improves conformational stability without affecting the native sequence, epitope recognition or processing may be affected, abrogating an effective immune response.


Assuntos
Vacinas contra Antraz/imunologia , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Imunogenicidade da Vacina , Receptores de Peptídeos/imunologia , Fator de von Willebrand/metabolismo , Animais , Antraz/imunologia , Antraz/prevenção & controle , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Fator de von Willebrand/imunologia
9.
J Bacteriol ; 202(7)2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-31932311

RESUMO

Type II toxin-antitoxin (TA) systems are small genetic elements composed of a toxic protein and its cognate antitoxin protein, the latter counteracting the toxicity of the former. While TA systems were initially discovered on plasmids, functioning as addiction modules through a phenomenon called postsegregational killing, they were later shown to be massively present in bacterial chromosomes, often in association with mobile genetic elements. Extensive research has been conducted in recent decades to better understand the physiological roles of these chromosomally encoded modules and to characterize the conditions leading to their activation. The diversity of their proposed roles, ranging from genomic stabilization and abortive phage infection to stress modulation and antibiotic persistence, in conjunction with the poor understanding of TA system regulation, resulted in the generation of simplistic models, often refuted by contradictory results. This review provides an epistemological and critical retrospective on TA modules and highlights fundamental questions concerning their roles and regulations that still remain unanswered.


Assuntos
Antitoxinas/genética , Antitoxinas/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Evolução Biológica , Sistemas Toxina-Antitoxina , Estudos de Associação Genética , Genoma Bacteriano , Fenótipo , Sistemas Toxina-Antitoxina/genética , Sistemas Toxina-Antitoxina/imunologia
10.
Plant Mol Biol ; 102(1-2): 159-169, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31820286

RESUMO

KEY MESSAGE: A plant-based multiepitopic protein (LTBentero) containing epitopes from ETEC, S. typhimurium, and V. parahaemolyticus was produced in plants cells and triggered systemic and intestinal humoral responses in immunized mice. Around 200 million people suffer gastroenteritis daily and more than 2 million people die annually in developing countries due to such pathologies. Vaccination is an alternative to control this global health issue, however new low-cost vaccines are needed to ensure proper vaccine coverage. In this context, plants are attractive hosts for the synthesis and delivery of subunit vaccines. Therefore, in this study a plant-made multiepitopic protein named LTBentero containing epitopes from antigens of enterotoxigenic E. coli, S. typhimurium, and V. parahaemolyticus was produced and found immunogenic in mice. The LTBentero protein was expressed in tobacco plants at up to 5.29 µg g-1 fresh leaf tissue and was deemed immunogenic when administered to BALB/c mice either orally or subcutaneously. The plant-made LTBentero antigen induced specific IgG (systemic) and IgA (mucosal) responses against LTB, ST, and LptD epitopes. In conclusion, multiepitopic LTBentero was functionally produced in plant cells, being capable to trigger systemic and intestinal humoral responses and thus it constitutes a promising oral immunogen candidate in the fight against enteric diseases.


Assuntos
Toxinas Bacterianas/imunologia , Epitopos/imunologia , Imunização , Proteínas de Plantas/imunologia , Proteínas Recombinantes/imunologia , Vacinas de Plantas Comestíveis/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Toxinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/imunologia , Epitopos/genética , Feminino , Regulação da Expressão Gênica de Plantas , Imunoglobulina A , Imunoglobulina G , Camundongos , Camundongos Endogâmicos BALB C , Membrana Mucosa/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/metabolismo , Tabaco/genética , Vacinação , Vacinas de Plantas Comestíveis/genética
11.
Infect Immun ; 88(3)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31871095

RESUMO

The intracellularly active bacterial toxin TcdB is a major Clostridioides difficile virulence factor that contributes to inflammation and tissue damage during disease. Immunization with an inactive TcdB fragment prevents C. difficile infection (CDI)-associated pathology. The protective immune response against inactive TcdB involves development of antigen-specific memory B cells and long-lived plasma cells that encode TcdB-neutralizing antibodies. Unlike the response to inactive TcdB, very little is known about the host humoral immune response to C. difficile and TcdB during primary and recurrent infection. Here, we used a murine model of C. difficile disease recurrence to demonstrate that an initial infection induced a serum IgM and mucosal IgA response against the toxin, but a low serum IgG response, which is associated with a lack of protection against disease during reinfection. Infection induced a partial expansion of the T follicular helper cell compartment, essential for B cell memory responses, and, consistent with that, failed to significantly expand the memory B cell compartment. Further, infection failed to stimulate the memory B cell compartment in preimmunized mice, although they were protected against associated disease. These results delineate the key humoral immune events that follow primary and recurrent C. difficile infection and provide a compelling inverse correlation between B cell memory and disease recurrence.


Assuntos
Linfócitos B/imunologia , Infecções por Clostridium/imunologia , Clostridium difficile/imunologia , Imunização , Imunoglobulina G/sangue , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Toxinas Bacterianas/imunologia , Infecções por Clostridium/microbiologia , Imunoglobulina A/metabolismo , Camundongos , Membrana Mucosa/metabolismo
12.
Cell Host Microbe ; 26(6): 795-809.e5, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31784259

RESUMO

The host must develop tolerance to commensal microbes and protective responses to infectious pathogens, yet the mechanisms enabling a privileged relationship with commensals remain largely unknown. Skin colonization by commensal Staphylococcus epidermidis facilitates immune tolerance preferentially in neonates via induction of antigen-specific regulatory T cells (Tregs). Here, we demonstrate that this tolerance is not indiscriminately extended to all bacteria encountered in this early window. Rather, neonatal colonization by Staphylococcus aureus minimally enriches for antigen-specific Tregs and does not prevent skin inflammation upon later-life exposure. S. aureus α-toxin contributes to this response by stimulating myeloid cell production of IL-1ß, which limits S. aureus-specific Tregs. Loss of α-toxin or the IL-1 receptor increases Treg enrichment, whereas topical application of IL-1ß or α-toxin diminishes tolerogenic responses to S. epidermidis. Thus, the preferential activation of a key alarmin pathway facilitates early discrimination of microbial "foe" from "friend," thereby preventing tolerance to a common skin pathogen.


Assuntos
Toxinas Bacterianas/imunologia , Receptores de Interleucina-1/metabolismo , Pele/microbiologia , Infecções Estafilocócicas/imunologia , Linfócitos T Reguladores/imunologia , Animais , Animais Recém-Nascidos , Toxinas Bacterianas/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Tolerância Imunológica , Camundongos , Receptores de Interleucina-1/imunologia , Transdução de Sinais/imunologia , Staphylococcus aureus/imunologia , Staphylococcus epidermidis/imunologia , Simbiose/imunologia , Virulência/imunologia
13.
Proc Natl Acad Sci U S A ; 116(49): 24808-24818, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31744876

RESUMO

Myxobacteria are an example of how single-cell individuals can transition into multicellular life by an aggregation strategy. For these and all organisms that consist of social groups of cells, discrimination against, and exclusion of, nonself is critical. In myxobacteria, TraA is a polymorphic cell surface receptor that identifies kin by homotypic binding, and in so doing exchanges outer membrane (OM) proteins and lipids between cells with compatible receptors. However, TraA variability alone is not sufficient to discriminate against all cells, as traA allele diversity is not necessarily high among local strains. To increase discrimination ability, myxobacteria include polymorphic OM lipoprotein toxins called SitA in their delivered cargo, which poison recipient cells that lack the cognate, allele-specific SitI immunity protein. We previously characterized 3 SitAI toxin/immunity pairs that belong to 2 families. Here, we discover 4 additional SitA families. Each family is unique in sequence, but share the characteristic features of SitA: OM-associated toxins delivered by TraA. We demonstrate that, within a SitA family, C-terminal nuclease domains are polymorphic and often modular. Remarkably, sitA loci are strikingly numerous and diverse, with most genomes possessing >30 and up to 83 distinct sitAI loci. Interestingly, all SitA protein families are serially transferred between cells, allowing a SitA inhibitor cell to poison multiple targets, including cells that never made direct contact. The expansive suites of sitAI loci thus serve as identify barcodes to exquisitely discriminate against nonself to ensure populations are genetically homogenous to conduct cooperative behaviors.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Myxococcales/genética , Myxococcales/metabolismo , Receptores de Superfície Celular/metabolismo , Alelos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Toxinas Bacterianas/classificação , Toxinas Bacterianas/imunologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Lipoproteínas , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Filogenia , Análise de Sequência
14.
Int Immunopharmacol ; 77: 105917, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31675617

RESUMO

Burkholderia lethal factor 1 (BLF1), a glutamine deamidase, is a key virulence factor that plays significant role in B. pseudomallei pathogenesis. To elucidate the BLF1 immunological responses, two truncated BLF1 structural units, BLF1-C (90-211 amino acids) with structural similarity to T. maritima Chemoreceptor glutamine deamidase (CheD) protein, and BLF1-N (1-89 amino acids) disparate to CheD were identified from the 23 kDa BLF1 protein. Both the components were devoid of toxicity in mice and elicited an antibody titer of 1:16,000 that reacted with the respective truncated proteins and BLF1. A549 cell lines supplemented with anti BLF1-N and BLF1-C antibodies exhibited 73.47% and 83.24% survival when treated with BLF1 toxin. Passive i.p. transfer with antibodies elicited by BLF1-C that contained LSGC active site resulted in 80% protection while anti BLF1-N (devoid of LSGC) antibodies provided 51.4% protection, establishing the role of BLF1-N terminal also in deamidase action. The truncated proteins also elicited cell mediated immune responses through proliferation of CD4+ T cells, IFN-γ and IL-4 cytokines but with bias towards Th2 subsets. BLF1-C and BLF1-N immunization resulted in 80% and 60% active protection when challenged with BLF1 toxin while the sham immunized mice exhibited severe histopathological changes like necrosis in liver, lung, spleen and kidney similar to that observed in melioidosis and were killed within 7 days post challenge. The higher level of active and passive protection by BLF1-C protein could be attributed to the comparatively higher level of immune responses and inclusion of LSGC residues.


Assuntos
Toxinas Bacterianas , Células A549 , Animais , Anticorpos Antibacterianos/farmacologia , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/toxicidade , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/imunologia , Citocinas/sangue , Citocinas/imunologia , Feminino , Humanos , Camundongos Endogâmicos BALB C , Domínios Proteicos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
15.
PLoS One ; 14(11): e0224073, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31682624

RESUMO

The development of an effective subunit vaccine is frequently complicated by the difficulty of eliciting protective immune responses, often requiring the co-administration of an adjuvant. Heat-labile toxin (LT), an enterotoxin expressed by enterotoxigenic E. coli (ETEC) with an AB5 structure similar to cholera toxin, is a strong adjuvant. While the mucosa represents the natural route of exposure to LT and related toxins, the clinical utility of LT and similar adjuvants given by mucosal routes has been limited by toxicity, as well as the association between intranasal delivery of LT and Bell's palsy. Single and double amino acid mutants of LT, LT(R192G)/mLT and LT(R192G/L211A)/dmLT respectively, have been proposed as alternatives to reduce the toxicity associated with the holotoxin. In the present study, we compared mLT and dmLT given via a non-mucosal route (i.e. intradermally) to investigate their adjuvanticity when co-administrated with an enterotoxigenic E. coli vaccine candidate, CfaEB. Antigenicity (i.e. ability to elicit response against LT) and reactogenicity at the injection site were also evaluated. BALB/c mice were immunized by the intradermal route with CfaEB plus increasing doses of either mLT or dmLT (0.01 to 2.5 µg). Both adjuvants induced dose-dependent skin reactogenicity, with dmLT being less reactogenic than mLT. Both adjuvants significantly boosted the anti-CfaE IgG and functional hemagglutination inhibiting (HAI) antibody responses, compared to the antigen alone. In addition to inducing anti-LT responses, even at the lowest dose tested (0.01 µg), the adjuvants also prompted in vitro cytokine responses (IFN-γ, IL-4, IL-5, IL-10 and IL-17) that followed different patterns, depending on the protein used for stimulation (CfaE or LTB) and/or the dose used for immunization. The two LT mutants evaluated here, mLT and dmLT, are potent adjuvants for intradermal immunization and should be further investigated for the intradermal delivery of subunit ETEC vaccines.


Assuntos
Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Escherichia coli Enterotoxigênica/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração através da Mucosa , Animais , Anticorpos Antibacterianos/imunologia , Toxina da Cólera/imunologia , Feminino , Imunização/métodos , Injeções Intradérmicas/métodos , Camundongos , Camundongos Endogâmicos BALB C , Vacinação/métodos , Vacinas de Subunidades/imunologia
16.
Mol Immunol ; 116: 98-105, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31634816

RESUMO

Pseudomonas aeruginosa is a common nosocomial pathogen in burn patients, and rapidly acquires antibiotic resistance; thus, developing an effective therapeutic approach is the most promising strategy for combating infection. Type III secretion system (T3SS) translocates bacterial toxins into the cytosol of the targeted eukaryotic cells, which plays important roles in the virulence of P. aeruginosa infections in both acute pneumonia and burn wound models. The PcrV protein, a T3SS translocating protein, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targeting P. aeruginosa. In the present study, we evaluated the protective efficacy of chicken egg yolk antibodies (IgY) raised against recombinant PcrV (r-PcrV) in both acute pneumonia and burn wound models. R-PcrV protein was generated by expressing the pcrV gene (cloned in pET-28a vector) in E. coli BL-21. Anti-PcrV IgY was obtained by immunization of hen. Anti-PcrV IgY induced greater protection in P. aeruginosamurine acute pneumonia and burn wound models than control IgY (C-IgY) and PBS groups. Anti-PcrV IgY improved opsonophagocytic killing and inhibition of bacterial invasion of host cells. Taken together, our data provide evidence that anti-PcrV IgY can be a promising therapeutic candidate for combating P. aeruginosa infections.


Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Queimaduras/imunologia , Imunoglobulinas/imunologia , Pneumonia/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Queimaduras/microbiologia , Galinhas/imunologia , Galinhas/microbiologia , Modelos Animais de Doenças , Feminino , Imunização/métodos , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia/microbiologia , Vacinação/métodos , Virulência/imunologia
17.
Lett Appl Microbiol ; 69(5): 385-390, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31529707

RESUMO

Clostridium perfringens is the main cause of sudden death in dogs and currently there is no vaccine to prevent it. In this study, a canine C. perfringens type A strain was used to prepare a vaccine. C. perfringens was inactivated by formaldehyde and adjuvants were added. The safety and immunological characteristics of the inactivated C. perfringens vaccine were evaluated in mice and dogs. The results showed that the C. perfringens vaccine was safe and had immunoprotective activity. The serum antibody titre of immunized mice reached up to 6·25 × 104 . Both single immunization of 4 ml and dual immunizations of 2 ml each provided good immune protection, with five of five immunized dogs surviving. This study also studied a detoxified crude α-toxin extract vaccine. The results showed that a single immunization with 0·5 ml of the detoxified crude α-toxin extract vaccine provided immune protection, with five of five immunized dogs surviving. The inactivated C. perfringens type A vaccine can be used to prevent canine C. perfringens infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium perfringens is the main cause of sudden death in dogs and currently there is no vaccine to prevent it. In this study, an inactivated canine C. perfringens vaccine and a detoxified crude α-toxin vaccine were prepared. The safety and protective effects of these vaccines were evaluated using mouse and dog models. The vaccines were shown to be safe and to provide immune protection effects that can be used to prevent canine C. perfringens infection.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/imunologia , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Infecções por Clostridium/microbiologia , Clostridium perfringens/genética , Cães , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunização , Camundongos , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/genética , Vacinas de Produtos Inativados/imunologia
18.
J Vet Med Sci ; 81(10): 1475-1484, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31527353

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is one of the major swine diseases responsible for a significant challenge in the global swine industry. The current PRRS inactivated vaccine only confers limited protection against PRRSV. Thus, using an appropriate adjuvant via a suitable administration route may help improve vaccine efficacy. In this study, the recombinant B subunit of the Escherichia coli heat-labile enterotoxin rLTB, was highly expressed in Pichia pastoris, through high-density fermentation. rLTB intranasal adjuvant properties were evaluated on an inactivated PRRS antigen in mice. Compared to the group immunized with solely PRRS antigen, a dose of 50 µg rLTB remarkably raised antigen-specific IgA antibodies at mucosal sites, and increased serum IgG antibodies, preferentially the IgG2a and IgG2b subclasses. Further, rLTB induced increases in Th1- (IFN-γ and IL-12) and Th17 (IL-6) cytokine profiles, but had little effect on Th2 cytokine profiles (IL-4 and IL-10). Moreover, there were no overt toxicities associated with intranasal rLTB administration. Our data provide evidence that the rLTB produced by P. pastoris fermentation portrays low toxicity, and its intranasal adjuvant effect involves immune system modulation to a Th1 profile.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Vacinas Virais/administração & dosagem , Administração Intranasal , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/imunologia , Enterotoxinas/administração & dosagem , Enterotoxinas/imunologia , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/imunologia , Camundongos , Síndrome Respiratória e Reprodutiva Suína/virologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Suínos
19.
Ann Agric Environ Med ; 26(3): 392-395, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559791

RESUMO

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/microbiologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Animais , Antraz/imunologia , Antraz/prevenção & controle , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Vacinas contra Antraz/isolamento & purificação , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/química , Bacillus anthracis/genética , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios Proteicos
20.
Biologicals ; 61: 38-43, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31416791

RESUMO

Tremendous efforts are being made to develop an anthrax vaccine with long term protection. The main component of traditional anthrax vaccine is protective antigen (PA) with the trace amount of other proteins and bacterial components. In this study, we developed a recombinant PA-LF chimera antigen of Bacillus anthracis by fusing the PA domain 2-4 with lethal factor (LF) domain 1 and evaluated its protective potential against B. anthracis in mouse model. The anti-PA-LF chimera serum reacted with both PA and LF antigen, individually. The chimera elicited a strong antibody titer in mice with predominance of IgG1 isotype followed by IgG2b, IgG2a and IgG3. Cytokines were assessed in splenocytes of immunized mice and a significant up-regulation in the expression of IL-4, IL-10, IFN-γ and TNF-α was observed. The PA-LF chimera immunized mice exhibited 80% survival after challenge with virulent spores of B. anthracis. Pathological studies showed normal architecture in vital organs (spleen, lung, liver and kidney) of recovered immunized mice on 20 DPI after spore challenge. These findings suggested that PA-LF chimera of B. anthracis elicited good humoral as well as cell mediated immune response in mice, and thus, can be a potent vaccine candidate against anthrax.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Antraz/imunologia , Antraz/patologia , Vacinas contra Antraz/genética , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Gerenciamento Clínico , Avaliação de Medicamentos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA