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1.
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
2.
Nat Commun ; 10(1): 2712, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221971

RESUMO

Clostridium difficile (C. difficile) incidence has tripled over the past 15 years and is attributed to the emergence of hypervirulent strains. While it is clear that C. difficile toxins cause damaging colonic inflammation, the immune mechanisms protecting from tissue damage require further investigation. Through a transcriptome analysis, we identify IL-33 as an immune target upregulated in response to hypervirulent C. difficile. We demonstrate that IL-33 prevents C. difficile-associated mortality and epithelial disruption independently of bacterial burden or toxin expression. IL-33 drives colonic group 2 innate lymphoid cell (ILC2) activation during infection and IL-33 activated ILC2s are sufficient to prevent disease. Furthermore, intestinal IL-33 expression is regulated by the microbiota as fecal microbiota transplantation (FMT) rescues antibiotic-associated depletion of IL-33. Lastly, dysregulated IL-33 signaling via the decoy receptor, sST2, predicts C. difficile-associated mortality in human patients. Thus, IL-33 signaling to ILC2s is an important mechanism of defense from C. difficile colitis.


Assuntos
Clostridium difficile/imunologia , Enterocolite Pseudomembranosa/imunologia , Imunidade Inata , Interleucina-33/metabolismo , Linfócitos/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antibacterianos/efeitos adversos , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Clostridium difficile/patogenicidade , Colo/citologia , Colo/imunologia , Colo/microbiologia , Colo/patologia , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/mortalidade , Enterocolite Pseudomembranosa/terapia , Transplante de Microbiota Fecal , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Perfilação da Expressão Gênica , Humanos , Interleucina-33/imunologia , Linfócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Virulência/imunologia , Adulto Jovem
3.
World J Microbiol Biotechnol ; 35(6): 94, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31187291

RESUMO

Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis (CF) patients. Therefore, it is necessary to develop appropriate strategies for preventing colonization by this bacterium and/or neutralizing virulence factors. In this study, we formulated the encapsulation of exotoxin A into PLGA nanoparticles. The biological activities of the nanovaccine candidate were also characterized. Based on the results, ETA-PLGA can act as a suitable immunogen to stimulate the humoral and cellular immune response. The antibodies raised against ETA-PLGA significantly decreased bacterial titer in the spleens of the immunized mice after challenge with PAO1 strain, compared to the control groups. The encapsulation of PLGA into ETA led to a significantly higher production of INF-γ, TNF-α, IL-4, and IL-17A cytokine responses compared to the ETA group. ETA-PLGA enhanced IgG responses in immunized mice compared to ETA antigen. We concluded that encapsulation of Pseudomonas aeruginosa ETA to PLGA nanoparticles can increase its functional activity by decreasing the bacterial dissemination.


Assuntos
ADP Ribose Transferases/imunologia , Toxinas Bacterianas/imunologia , Exotoxinas/imunologia , Imunização , Nanoconjugados , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/imunologia , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/patogenicidade , Vacinas Conjugadas , Fatores de Virulência/imunologia , ADP Ribose Transferases/uso terapêutico , Animais , Toxinas Bacterianas/uso terapêutico , Citocinas/metabolismo , Modelos Animais de Doenças , Exotoxinas/uso terapêutico , Feminino , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/uso terapêutico , Infecções por Pseudomonas/imunologia , Baço/imunologia , Baço/microbiologia , Fatores de Virulência/uso terapêutico
4.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096563

RESUMO

High immunogenicity and systemic toxicity are the main obstacles limiting the clinical use of the therapeutic agents based on Pseudomonas aeruginosa exotoxin A. In this work, we studied the immunogenicity, general toxicity and antitumor effect of the targeted toxin DARPin-LoPE composed of HER2-specific DARPin and a low immunogenic exotoxin A fragment lacking immunodominant human B lymphocyte epitopes. The targeted toxin has been shown to effectively inhibit the growth of HER2-positive human ovarian carcinoma xenografts, while exhibiting low non-specific toxicity and side effects, such as vascular leak syndrome and liver tissue degradation, as well as low immunogenicity, as was shown by specific antibody titer. This represents prospects for its use as an agent for targeted therapy of HER2-positive tumors.


Assuntos
Epitopos de Linfócito B/imunologia , Xenoenxertos , Imunotoxinas/imunologia , Imunotoxinas/farmacologia , Proteínas Musculares/imunologia , Proteínas Nucleares/imunologia , Neoplasias Ovarianas/tratamento farmacológico , Receptor ErbB-2/imunologia , ADP Ribose Transferases/imunologia , ADP Ribose Transferases/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/farmacologia , Biomarcadores Tumorais , Carcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Epitopos de Linfócito B/genética , Exotoxinas/imunologia , Exotoxinas/farmacologia , Feminino , Humanos , Concentração Inibidora 50 , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Proteínas Musculares/genética , Proteínas Nucleares/genética , Neoplasias Ovarianas/patologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Baço/patologia , Fatores de Virulência/imunologia , Fatores de Virulência/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31061144

RESUMO

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Toxinas Bacterianas/imunologia , Escherichia coli Enterotoxigênica/imunologia , Enterotoxinas/imunologia , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Hormônios Gastrointestinais/imunologia , Peptídeos Natriuréticos/imunologia , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Reações Cruzadas , Escherichia coli Enterotoxigênica/genética , Enterotoxinas/administração & dosagem , Enterotoxinas/química , Enterotoxinas/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Vacinas contra Escherichia coli/administração & dosagem , Vacinas contra Escherichia coli/genética , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Suínos
6.
Microb Pathog ; 131: 259-269, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002964

RESUMO

Staphylococcus aureus (S.aureus) is a Gram-positive bacterium that causes many infections and diseases. This pathogen can cause many types of infections such as impetigo, toxic shock syndrome toxin (TSST1), pneumonia, endocarditis, and autoimmune diseases like lupus erythematosus and can infect other healthy individuals. In the pathogenic process, colonization is a main risk factor for invasive diseases. Various factors including the cell wall-associated factors and receptors of the epithelial cells facilitate adhesion and colonization of this pathogen. S. aureus has many enzymes, toxins, and strategies to evade from the immune system either by an enzyme that lyses cellular component or by hiding from the immune system via surface antigens like protein A and second immunoglobulin-binding protein (Sbi). The strategies of this bacterium can be divided into five groups: A: Inhibit neutrophil recruitment B: Inhibit phagocytosis C: Inhibit killing by ROS, D: Neutrophil killing, and E: Resistance to antimicrobial peptide. On the other hand, innate immune system via neutrophils, the most important polymorphonuclear leukocytes, fights against bacterial cells by neutrophil extracellular trap (NET). In this review, we try to explain the role of each factor in immune evasion.


Assuntos
Evasão da Resposta Imune , Neutrófilos/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade , Antígenos de Bactérias/imunologia , Proteínas de Bactérias , Toxinas Bacterianas/imunologia , Enterotoxinas , Interações Hospedeiro-Patógeno/imunologia , Humanos , Evasão da Resposta Imune/imunologia , Imunidade Inata , Fagocitose , Proteína Estafilocócica A , Superantígenos
7.
mSphere ; 4(2)2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996108

RESUMO

Pseudomonas aeruginosa is a common Gram-negative opportunistic pathogen that is intrinsically resistant to a wide range of antibiotics. The development of a broadly protective vaccine against P. aeruginosa remains a major challenge. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express P. aeruginosa antigens. A fusion between the S. enterica type III secretion effector protein SseJ and the P. aeruginosa antigen PcrV expressed under the control of the sseA promoter was translocated by Salmonella into host cells in vitro and elicited the generation of specific antibodies in mice. Mice immunized with attenuated Salmonella expressing this fusion had reduced bacterial loads in the spleens and lungs and lower serum levels of proinflammatory cytokines than control mice after P. aeruginosa infection. Importantly, immunized mice also showed significantly enhanced survival in this model. These results suggest that type III secretion effectors of S. enterica are appropriate carriers in the design of a live vaccine to prevent infections caused by P. aeruginosa IMPORTANCE The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen that causes infections in cystic fibrosis and hospitalized patients. Therapeutic treatments are limited due to the emergence and spread of new antibiotic-resistant strains. In this context, the development of a vaccine is a priority. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express and deliver the Pseudomonas antigen PcrV. This vaccine induced the generation of specific antibodies in mice and protected them from lethal infections with P. aeruginosa This is an important step toward the development of an effective vaccine for the prevention of infections caused by P. aeruginosa in humans.


Assuntos
Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Salmonella/administração & dosagem , Sistemas de Secreção Tipo III/genética , Animais , Anticorpos Antibacterianos/sangue , Carga Bacteriana , Citocinas/sangue , Feminino , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa , Baço/microbiologia , Sistemas de Secreção Tipo III/imunologia , Vacinas Atenuadas/administração & dosagem
8.
Chemosphere ; 226: 439-446, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30951938

RESUMO

Cylindrospermopsin (CYN), a cyanobacterial toxin, is an important water pollutant with broad biological activity. It has been known mainly from tropical areas, but the area of occurrence of its producers is spreading to temperate climates. It can be found in high concentrations in the environment as well as in purified drinking waters. The aim of the study is to bring a basic information on the ability of CYN to interfere with mammalian innate immunity cells and thus increase the understanding of the immunomodulatory potency of CYN. This study investigated whether immune cells can be a target of CYN either alone or in combination with a model immunomodulatory agent, lipopolysaccharide (LPS). We examined the effects on cellular viability and inflammation signaling of CYN on murine macrophage-like RAW 264.7 cells. Macrophages were treated either with pure toxin (1 µM) or together with a known stimulator of immunologically active cells, bacterial or cyanobacterial LPS. CYN has had a significant effect on production on pro-inflammatory mediator tumor necrosis factor α (TNF-α) which correlates with its effect on reactive oxygen species (ROS) production. We found that CYN potentiated the effect of bacterial and cyanobacterial LPS that was documented by activation of inflammatory signaling pathways including mitogen-activated protein kinase p38 as well as consequent expression of inducible nitric oxide synthase (iNOS) and increased production of pro-inflammatory mediators such as nitric oxide (NO), TNF-α, interleukin-6 (IL-6). Our study brings one of the first information that contributes to the elucidation of immunomodulatory role of CYN in macrophages under normal and pro-inflammatory conditions.


Assuntos
Toxinas Bacterianas/imunologia , Imunidade Inata/imunologia , Imunomodulação/genética , Macrófagos/efeitos dos fármacos , Toxinas Marinhas/imunologia , Microcistinas/imunologia , Uracila/análogos & derivados , Animais , Camundongos , Transdução de Sinais , Uracila/imunologia
9.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30873936

RESUMO

Staphylococcus aureus is a formidable pathogen capable of causing infections in different sites of the body in a variety of vertebrate animals, including humans and livestock. A major contribution to the success of S. aureus as a pathogen is the plethora of virulence factors that manipulate the host's innate and adaptive immune responses. Many of these immune modulating virulence factors are secreted toxins, cofactors for activating host zymogens, and exoenzymes. Secreted toxins such as pore-forming toxins and superantigens are highly inflammatory and can cause leukocyte cell death by cytolysis and clonal deletion, respectively. Coagulases and staphylokinases are cofactors that hijack the host's coagulation system. Exoenzymes, including nucleases and proteases, cleave and inactivate various immune defense and surveillance molecules, such as complement factors, antimicrobial peptides, and surface receptors that are important for leukocyte chemotaxis. Additionally, some of these secreted toxins and exoenzymes can cause disruption of endothelial and epithelial barriers through cell lysis and cleavage of junction proteins. A unique feature when examining the repertoire of S. aureus secreted virulence factors is the apparent functional redundancy exhibited by the majority of the toxins and exoenzymes. However, closer examination of each virulence factor revealed that each has unique properties that have important functional consequences. This chapter provides a brief overview of our current understanding of the major secreted virulence factors critical for S. aureus pathogenesis.


Assuntos
Toxinas Bacterianas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Animais , Toxinas Bacterianas/classificação , Toxinas Bacterianas/imunologia , Humanos , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/imunologia
10.
World J Gastroenterol ; 25(9): 1050-1066, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30862994

RESUMO

BACKGROUND: The bacteria Campylobacter jejuni (C. jejuni) is commonly associated with Guillane-Barré syndrome (GBS) and irritable bowel syndrome (IBS), but studies have also linked it with Miller Fisher syndrome, reactive arthritis and other disorders, some of which are autoimmune. It is possible that C. jejuni and its toxins may be cross-reactive with some human tissues and food antigens, potentially leading to autoimmune responses. AIM: To measure the immune reactivity of C. jejuni and C. jejuni cytolethal distending toxin (Cdt) antibodies with tissue and food antigens to examine their role in autoimmunities. METHODS: Using enzyme-linked immunosorbent assay (ELISA) methodology, specific antibodies made against C. jejuni and C. jejuni Cdt were applied to a variety of microwell plates coated with 45 tissues and 180 food antigens. The resulting immunoreactivities were compared to reactions with control wells coated with human serum albumin (HSA) which were used as negative controls and with wells coated with C. jejuni lysate or C. jejuni Cdt which served as positive controls. RESULTS: At 3 SD above the mean of control wells coated with HSA or 0.41 OD, the mouse monoclonal antibody made against C. jejuni showed moderate to high reactions with zonulin, somatotropin, acetylcholine receptor, ß-amyloid and presenilin. This immune reaction was low with an additional 25 tissue antigens including asialoganglioside, and the same antibody did not react at all with another 15 tissue antigens. Examining the reaction between C. jejuni antibody and 180 food antigens, we found insignificant reactions with 163 foods but low to high immune reactions with 17 food antigens. Similarly, we examined the reaction of C. jejuni Cdt with the same tissues and food antigens. The strongest reactions were observed with zonulin, intrinsic factor and somatotropin. The reaction was moderate with 9 different tissue antigens including thyroid peroxidase, and reaction was low with another 10 different antigens, including neuronal antigens. The reaction of C. jejuni Cdt antibody with an additional 23 tissue antigens was insignificant. Regarding the reaction of C. jejuni Cdt antibody with different food antigens, 160 out of 180 foods showed insignificant reactions, while 20 foods showed reactions ranging from low to high. CONCLUSION: Our findings indicate that C. jejuni and its Cdt may play a role in inflammation and autoimmunities beyond the gut.


Assuntos
Anticorpos Antibacterianos/imunologia , Doenças Autoimunes/imunologia , Infecções por Campylobacter/imunologia , Campylobacter jejuni/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Doenças Autoimunes/microbiologia , Autoimunidade , Toxinas Bacterianas/imunologia , Toxina da Cólera/imunologia , Reações Cruzadas/imunologia , Proteínas na Dieta/imunologia , Hormônio do Crescimento Humano/imunologia , Humanos , Fator Intrínseco/imunologia
11.
Cell Physiol Biochem ; 52(2): 280-301, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30816675

RESUMO

Acid sphingomyelinase hydrolyzes sphingomyelin to ceramide and phosphorylcholine. Ceramide molecules spontaneously interact with each other and generate ceramide-enriched membrane domains. These ceramide-enriched domains further fuse, forming large ceramideenriched platforms that participate in the organization of receptors and in the amplification of signaling molecules. Recent studies have suggested several bacteria and bacterial toxins that stimulate the activation and the translocation of acid sphingomyelinase, which leads to the release of ceramide. The acid sphingomyelinase/ceramide system also regulates the internalization of bacteria into the host cell, the subsequent cytokine release, inflammatory response, and initiation of host cell apoptosis. In addition, ceramide has been implicated in the fusion of phagosomes and lysosomes upon bacterial infection. Thus, this system modulates the reorganization of cell membrane receptors and intracellular signaling molecules during bacteria-host interactions. The acid sphingomyelinase and ceramide system may thus serve as a novel therapeutic target for treating infections.


Assuntos
Infecções Bacterianas/imunologia , Toxinas Bacterianas/imunologia , Ceramidas/imunologia , Transdução de Sinais/imunologia , Esfingomielina Fosfodiesterase/imunologia , Animais , Infecções Bacterianas/patologia , Ativação Enzimática/imunologia , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Lisossomos/imunologia , Lisossomos/microbiologia , Fagossomos/imunologia , Fagossomos/microbiologia
12.
Anaerobe ; 56: 78-87, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30771460

RESUMO

Clostridium chauvoei is the etiologic agent of blackleg in cattle, inducing fever, severe myonecrosis, oedemic lesions and ultimately death of infected animals. The pathogen often results in such rapid death that antibiotic therapy is futile and thus vaccination is the only efficient strategy in order to control the disease. The ß-barrel pore forming leucocidin Clostridium chauvoei toxin A (CctA) is one of the best characterised toxins of C. chauvoei and has been shown to be an important virulence factor. It has been reported to induce protective immunity and is conserved across C. chauvoei strains collected from diverse geographical locations for more than 50 years. The aim of this study was to identify the location of the CctA toxin during liquid culture fermentation and to use CctA to develop an in vitro assay to replace the current guinea pig challenge assay for vaccine potency in standard batch release procedures. We report that CctA is fully secreted in C. chauvoei culture and show that it is found abundantly in the supernatant of liquid cultures. Sera from cattle vaccinated with a commercial blackleg vaccine revealed strong haemolysin-neutralizing activity against recombinant CctA which reached titres of 1000 times 28 days post-vaccination. Similarly, guinea pig sera from an official potency control test reached titres of 600 times 14 days post-vaccination. In contrast, ELISA was not able to specifically measure anti-CctA antibodies in cattle serum due to strong cross-reactions with antibodies against other proteins present pre-vaccination. We conclude that haemolysin-neutralizing antibodies are a valuable measurement for protective immunity against blackleg and have the potential to be a suitable replacement of the guinea pig challenge potency test, which would forego the unnecessary challenge of laboratory animals.


Assuntos
Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Doenças dos Bovinos/prevenção & controle , Infecções por Clostridium/veterinária , Clostridium chauvoei/imunologia , Animais , Toxinas Bacterianas/metabolismo , Vacinas Bacterianas/administração & dosagem , Bovinos , Infecções por Clostridium/prevenção & controle , Clostridium chauvoei/metabolismo , Meios de Cultura/química , Ensaio de Imunoadsorção Enzimática , Cobaias , Leucocidinas/imunologia , Leucocidinas/metabolismo , Testes de Neutralização , Fatores de Virulência/imunologia
13.
Virulence ; 10(1): 166-179, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30806148

RESUMO

Clostridium perfringens α-toxin is one of the major virulence factors during C. perfringens infection, causing hemolysis of erythrocytes in various species. Here, genetically engineered Lactobacillus casei (pPG-α/L. casei 393) constitutively expressing the toxoid of C. perfringens α-toxin was generated and its immunogenicity in mice for induction of protective immunity against the α-toxin was evaluated via oral immunization. The α-toxoid was constitutively expressed by pPG-α/L. casei 393 without a specific inducer, as confirmed by western blotting, laser confocal microscopy, and flow cytometry. In an experiment on BALB/c mice to evaluate the oral immunogenicity of pPG-α/L. casei 393, significant levels of a specific secretory IgA (sIgA) antibody in the intestinal mucus and feces and an IgG antibody in the serum of the probiotic vaccine group were detected after booster immunization (p < 0.05) as compared with the pPG/L. casei 393 and PBS control groups. These antibodies effectively neutralized C. perfringens natural α-toxin. Moreover, significantly higher levels of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and interferon (IFN) γ in the serum and increased proliferation of spleen lymphocytes obtained from mice orally immunized with pPG-α/L. casei 393 were detected. With a commercial C. perfringens type A inactivated vaccine as a control, immune protection provided by the probiotic vaccine against C. perfringens α-toxin was evaluated, and 90% and 80% protection rates were observed, respectively. Therefore, strain pPG-α/L. casei 393 effectively elicited mucosal, humoral, and cellular immunity, suggesting that pPG-α/L. casei 393 is a promising candidate for development of a vaccine against C. perfringens α-toxin.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Infecções por Clostridium/prevenção & controle , Lactobacillus casei/genética , Fosfolipases Tipo C/imunologia , Administração Oral , Animais , Anticorpos Antibacterianos/sangue , Toxinas Bacterianas/genética , Proteínas de Ligação ao Cálcio/genética , Infecções por Clostridium/imunologia , Clostridium perfringens , Citocinas/sangue , Feminino , Imunidade Celular , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados/imunologia , Probióticos , Fosfolipases Tipo C/genética
14.
Analyst ; 144(7): 2264-2274, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30810119

RESUMO

Anthrax protective antigen (83 kDa, PA83) is an essential component of two major binary toxins produced by Bacillus anthracis, lethal toxin (LTx) and edema toxin (ETx). During infection, LTx and ETx contribute to immune collapse, endothelial dysfunction, hemorrhage and high mortality. Following protease cleavage on cell receptors or in circulation, the 20 kDa (PA20) N-terminus is released, activating the 63 kDa (PA63) form which binds lethal factor (LF) and edema factor (EF), facilitating their entry into their cellular targets. Several ELISA-based PA methods previously developed are primarily qualitative or semi-quantitative. Here, we combined protein immunocapture, tryptic digestion and isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS), to develop a highly selective and sensitive method for detection and accurate quantification of total-PA (PA83 + PA63) and PA83. Two tryptic peptides in the 63 kDa region measure total-PA and three in the 20 kDa region measure PA83 alone. Detection limits range from 1.3-2.9 ng mL-1 PA in 100 µL of plasma. Spiked recovery experiments with combinations of PA83, PA63, LF and EF in plasma showed that PA63 and PA83 were quantified accurately against the PA83 standard and that LF and EF did not interfere with accuracy. Applied to a study of inhalation anthrax in rhesus macaques, total-PA suggested triphasic kinetics, similar to that previously observed for LF and EF. This study is the first to report circulating PA83 in inhalation anthrax, typically at less than 4% of the levels of PA63, providing the first evidence that activated PA63 is the primary form of PA throughout infection.


Assuntos
Antígenos de Bactérias/sangue , Bacillus anthracis/imunologia , Toxinas Bacterianas/sangue , Imunoensaio/métodos , Limite de Detecção , Espectrometria de Massas , Animais , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Macaca mulatta
15.
Biochem Biophys Res Commun ; 509(2): 611-616, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30606479

RESUMO

Since Bacillus anthracis is a high-risk pathogen and a potential tool for bioterrorism, numerous therapeutic methods including passive immunization have been actively developed. Using a human monoclonal antibody phage display library, we screened new therapeutic antibodies for anthrax infection against protective antigen (PA) of B. anthracis. Among 5 selected clones of antibodies based on enzyme-linked immunosorbent assay (ELISA) results, 7B1 showed neutralizing activity to anthrax lethal toxin (LT) by inhibiting binding of the domain 4 of PA (PD4) to its cellular receptors. Through light chain shuffling process, we improved the productivity of 7B1 up to 25 folds. The light chain shuffled 7B1 antibody showed protective activity against LT both in vitro and in vivo. Furthermore, the antibody also conferred protection of mice from 3 × LD50 challenges of fully virulent anthrax spores. Our result expands the possibility of developing a new therapeutic antibody for anthrax cure.


Assuntos
Antraz/prevenção & controle , Anticorpos/uso terapêutico , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Sequência de Aminoácidos , Animais , Antraz/imunologia , Anticorpos/química , Anticorpos/imunologia , Antígenos de Bactérias/química , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/química , Linhagem Celular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Biblioteca de Peptídeos
16.
BMC Vet Res ; 15(1): 6, 2019 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-30606265

RESUMO

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection. RESULTS: In this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer. CONCLUSIONS: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression.


Assuntos
Infecções por Actinobacillus/veterinária , Actinobacillus pleuropneumoniae/imunologia , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Infecções por Actinobacillus/imunologia , Infecções por Actinobacillus/prevenção & controle , Animais , Escherichia coli/genética , Engenharia Genética/métodos , Engenharia Genética/veterinária , Glicoconjugados/genética , Glicoconjugados/imunologia , Microrganismos Geneticamente Modificados/genética , Pleuropneumonia/imunologia , Pleuropneumonia/prevenção & controle , Pleuropneumonia/veterinária , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Doenças dos Suínos/prevenção & controle , Vacinas Conjugadas/imunologia
17.
Med Microbiol Immunol ; 208(2): 185-195, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30671633

RESUMO

Bacillus anthracis (BA), the etiological agent of anthrax, secretes protective antigen (PA), lethal factor (LF), and edema factor (EF) as major virulence mediators. Amongst these, PA-based vaccines are most effective for providing immunity against BA, but their low shelf life limits their usage. Previous studies showed that B-cell epitopes, ID II and ID III present in PA domain IV possess higher toxin neutralization activity and elicit higher antibody titer than ID I. Moreover, N-terminal region of both LF and EF harbors PA-binding sites which share 100% identity with each other. Here, in this study, we have developed an epitope-based chimeric vaccine (ID-LFn) comprising ID II-ID III region of PA and N-terminal region of LF. We have also evaluated its protective efficacy as well as stability and found it to be more stable than PA-based vaccine. Binding reactivities of ID-LFn with anti-PA/LF/EF antibodies were determined by ELISA. The stability of chimeric vaccine was assessed using circular dichroism spectroscopy. ID-LFn response was characterized by toxin neutralization, lymphocyte proliferation isotyping and cytokine profiling. The protective efficacy was analyzed by challenging ID-LFn-immunized mice with B. anthracis (pXO1+ and pXO2+). ID-LFn was found to be significantly stable as compared to PA. Anti-ID-LFn antibodies recognized PA, LF as well as EF. The T-cell response and the protective efficacy of ID-LFn were found to be almost similar to PA. ID-LFn exhibits equal protective efficacy in mice and possesses more stability as compared to PA along with the capability of recognizing PA, LF and EF at the same time. Thus, it can be considered as an improved vaccine against anthrax with better shelf life. ID-LFn, a novel multiepitope chimeric anthrax vaccine: ID-LFn comprises of immunodominant epitopes of domain 4 of PA and N-terminal homologous stretch of LF and EF. The administration of this protein as a vaccine provides protection against anthrax.


Assuntos
Vacinas contra Antraz/imunologia , Vacinas contra Antraz/isolamento & purificação , Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Epitopos/imunologia , Animais , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/química , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Dicroísmo Circular , Modelos Animais de Doenças , Estabilidade de Medicamentos , Epitopos/genética , Feminino , Camundongos Endogâmicos BALB C , Análise de Sobrevida , Linfócitos T/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação
18.
Biologicals ; 57: 55-60, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30635155

RESUMO

In this study, an ELISA was developed for simultaneous detection of antibodies against both the important toxins of B. anthracis i.e. protective antigen (PA) and lethal factor (LF). A chimera of PA and LF was made by fusion and cloned and expressed in E. coli. The purified recombinant protein was used in plate ELISA for serodiagnosis of anthrax. The chimera could detect antibodies against both the toxins of Bacillus anthracis. The human serum samples (n = 98) collected from anthrax endemic and non-endemic areas were tested employing ELISA. The ELISA gave sensitivity of 100% (95% Confidence Interval [CI], 92.13 to 100) and specificity of 97.78% (95% Confidence Interval [CI], 88.23 to 99.94) with a J index of 0.97. The efficiency of ELISA was found to be 98.9% with the positive predictive value (PPV) and negative predictive value (NPV) of 97.8% and 100%, respectively. The chimera of PA and LF could be a better diagnostic antigen for serodiagnosis as the assay detects antibodies against both the toxins in early as well delayed infection cases of anthrax. Therefore, it can be a very useful tool for the surveillance as well as for confirmation of cutaneous anthrax cases.


Assuntos
Antraz/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes de Fusão/imunologia , Testes Sorológicos/métodos , Dermatopatias Bacterianas/diagnóstico , Animais , Antraz/imunologia , Antraz/microbiologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Bacillus anthracis/fisiologia , Toxinas Bacterianas/imunologia , Humanos , Índia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Dermatopatias Bacterianas/imunologia , Dermatopatias Bacterianas/microbiologia
19.
Microb Pathog ; 127: 225-232, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30528250

RESUMO

Along with robust immunogenicity, an ideal vaccine candidate should be able to produce a long lasting protection. In this regard, the frequency of memory B-cells is possibly an important factor in memory B-cell persistency and duration of immunological memory. On this basis, binding domains of tetanus toxin (HcT), botulinum type A1 toxin (HcA), and heat-labile toxin (LTB) were selected as antigen models that induced long-term, midterm and short-term immune memory, respectively. In the present study, the frequency of total memory B-cells after immunization with HcT, HcA and LTB antigens after 90 and 180 days, and also after one booster, in 190 days, was evaluated. The results showed a significant correlation between frequency of total memory B-cells and duration of humoral immunity. Compared to other antigens, the HcT antibody titers and HcT total memory B-cell populations were greater and persistent even after 6 months. At 6 months after the final immunization, all HcT- and HcA-immunized mice survived against tetanus and botulinum toxins, and also LT toxin binding to GM1 ganglioside was blocked in LTB-immunized mice. We conclude the frequency of memory B-cells and their duration are likely a key factor for vaccine memory duration.


Assuntos
Antígenos de Bactérias/imunologia , Subpopulações de Linfócitos B/imunologia , Toxinas Bacterianas/imunologia , Toxinas Botulínicas/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Memória Imunológica , Toxina Tetânica/imunologia , Animais , Antígenos de Bactérias/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Toxinas Botulínicas/administração & dosagem , Enterotoxinas/administração & dosagem , Proteínas de Escherichia coli/administração & dosagem , Camundongos , Toxina Tetânica/administração & dosagem , Fatores de Tempo
20.
Front Immunol ; 9: 2603, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30555457

RESUMO

Staphylococcus aureus (Sa), as one of the major human pathogens, has very effective strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant Sa (CA-MRSA) depends on the secretion of phenol-soluble modulin (PSM) peptide toxins e.g., by binding to and modulation of innate immune cells. Previously, by using mouse bone marrow-derived dendritic cells we demonstrated that PSMs in combination with various Toll-like receptor (TLR) ligands induce a tolerogenic DC phenotype (tDC) characterized by the production of IL-10 and impaired secretion of pro-inflammatory cytokines. Consequently, PSM-induced tDCs favored priming of CD4+CD25+FoxP3+ Tregs with suppressor function while impairing the Th1 response. However, the relevance of these findings for the human system remained elusive. Here, we analyzed the impact of PSMα3 on the maturation, cytokine production, antigen uptake, and T cell stimulatory capacity of human monocyte-derived DCs (moDCs) treated simultaneously with either LPS (TLR4 ligand) or Sa cell lysate (TLR2 ligand). Herein, we demonstrate that PSMs indeed modulate human moDCs upon treatment with TLR2/4 ligands via multiple mechanisms, such as transient pore formation, impaired DC maturation, inhibited pro- and anti-inflammatory cytokine secretion, as well as reduced antigen uptake. As a result, the adaptive immune response was altered shown by an increased differentiation of naïve and even CD4+ T cells from patients with Th1/Th17-induced diseases (spondyloarthritis and rheumatoid arthritis) into CD4+CD127-CD25hiCD45RA-FoxP3hi regulatory T cells (Tregs) with suppressor function. This Treg induction was mediated most predominantly by direct DC-T-cell interaction. Thus, PSMs from highly virulent Sa strains affect DC functions not only in the mouse, but also in the human system, thereby modulating the adaptive immune response and probably increasing the tolerance toward the bacteria. Moreover, PSMα3 might be a novel peptide for tolerogenic DC induction that may be used for DC vaccination strategies.


Assuntos
Células Dendríticas/imunologia , Monócitos/imunologia , Peptídeos/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Linfócitos T Reguladores/imunologia , Toxinas Bacterianas/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Citocinas/imunologia , Humanos , Tolerância Imunológica/imunologia , Ativação Linfocitária/imunologia , Receptores Toll-Like/imunologia
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