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1.
PLoS Pathog ; 16(9): e1008871, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936831

RESUMO

Deconvolution of syphilis pathogenesis and selection of candidate syphilis vaccinogens requires detailed knowledge of the molecular architecture of the Treponema pallidum outer membrane (OM). The T. pallidum OM contains a low density of integral OM proteins, while the spirochete's many lipoprotein immunogens are periplasmic. TP0751, a lipoprotein with a lipocalin fold, is reportedly a surface-exposed protease/adhesin and protective antigen. The rapid expansion of calycin/lipocalin structures in the RCSB PDB database prompted a comprehensive reassessment of TP0751. Small angle X-ray scattering analysis of full-length protein revealed a bipartite topology consisting of an N-terminal, intrinsically disordered region (IDR) and the previously characterized C-terminal lipocalin domain. A DALI server query using the lipocalin domain yielded 97 hits, 52 belonging to the calycin superfamily, including 15 bacterial lipocalins, but no Gram-negative surface proteins. Surprisingly, Tpp17 (TP0435) was identified as a structural ortholog of TP0751. In silico docking predicted that TP0751 can bind diverse ligands along the rim of its eight-stranded ß-barrel; high affinity binding of one predicted ligand, heme, to the lipocalin domain was demonstrated. qRT-PCR and immunoblotting revealed very low expression of TP0751 compared to other T. pallidum lipoproteins. Immunoblot analysis of immune rabbit serum failed to detect TP0751 antibodies, while only one of five patients with secondary syphilis mounted a discernible TP0751-specific antibody response. In opsonophagocytosis assays, neither TP0751 nor Tpp17 antibodies promoted uptake of T. pallidum by rabbit peritoneal macrophages. Rabbits immunized with intact, full-length TP0751 showed no protection against local or disseminated infection following intradermal challenge with T. pallidum. Our data argue that, like other lipoprotein lipocalins in dual-membrane bacteria, TP0751 is periplasmic and binds small molecules, and we propose that its IDR facilitates ligand binding by and offloading from the lipocalin domain. The inability of TP0751 to elicit opsonic or protective antibodies is consistent with a subsurface location.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Imunização , Lipoproteínas/imunologia , Sífilis/imunologia , Treponema pallidum/imunologia , Animais , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Humanos , Lipoproteínas/genética , Domínios Proteicos , Dobramento de Proteína , Coelhos , Sífilis/genética , Sífilis/patologia , Sífilis/prevenção & controle , Treponema pallidum/genética , Treponema pallidum/patogenicidade
2.
Rev Bras Parasitol Vet ; 29(3): e005820, 2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32756774

RESUMO

Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.


Assuntos
Proteínas de Bactérias , Doenças do Cão , Ehrlichia canis , Ehrlichiose/veterinária , Proteínas Recombinantes , Testes Sorológicos/veterinária , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Brasil , Linhagem Celular , Doenças do Cão/diagnóstico , Cães , Ehrlichia canis/genética , Ehrlichiose/diagnóstico , Escherichia coli/genética , Expressão Gênica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
3.
BMC Infect Dis ; 20(1): 459, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611401

RESUMO

BACKGROUND: Extra pulmonary manifestation of tuberculosis (TB) accounts for approximately one-half of TB cases in HIV-infected individuals with pleural TB as the second most common location. Even though mycobacteria are cleared, mycobacterial antigens may persist in infected tissues, causing sustained inflammation and chronicity of the disease. The aim of this study was to explore various mycobacterial antigens in pleural effusions, the impact of HIV infection and CD4+ T-cell depletion on the presence of antigens, and the diagnostic potential of antigens for improved and rapid diagnosis of pleural TB. METHODS: Pleural fluid specimens were collected from patients presenting with clinically suspected pleural TB, and processed routinely for culture, cytology, and adenosine deaminase activity analysis. HIV status and CD4+ T-cell counts were recorded. Pleural fluid mononuclear cells (PFMC) were isolated, and cell smears were stained with acid-fast staining and immunocytochemistry for various mycobacterial antigens. Real-time and nested-PCR were performed. Patients were categorized as pleural TB or non-TB cases using a composite reference standard. Performance of the mycobacterial antigens as diagnostic test was assessed. RESULTS: A total of 41 patients were enrolled, of which 32 were classified as pleural TB and 9 as non-TB. Thirteen patients had culture confirmed pleural TB, 26 (81%) were HIV-TB co-infected, and 64% had < 100 CD4+ T-cells/microL. Both secreted and cell-wall mycobacterial antigens were detected in PFMC. Lipoarabinomannan (LAM) was the most frequently detected antigen. There was no direct correlation between positive culture and antigens. Cases with low CD4+ T-cell counts had higher bacterial and antigen burden. By combining detection of secreted antigen or LAM, the sensitivity and specificity to diagnose pleural TB was 56 and 78%, respectively, as compared to 41 and 100% for culture, 53 and 89% for nested PCR, and 6 and 100% for real-time PCR. CONCLUSION: Mycobacterial antigens were detectable in PFMC from tuberculous pleural effusions, even in cases where viable mycobacteria or bacterial DNA were not always detected. Thus, a combination of secreted antigen and LAM detection by immunocytochemistry may be a complement to acid-fast staining and contribute to rapid and accurate diagnosis of pleural TB.


Assuntos
Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Linfócitos T CD4-Positivos/imunologia , Coinfecção/diagnóstico , Testes Diagnósticos de Rotina/métodos , Lipopolissacarídeos/genética , Lipopolissacarídeos/imunologia , Mycobacterium tuberculosis/imunologia , Derrame Pleural/microbiologia , Tuberculose Pleural/diagnóstico , Adulto , Idoso , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Contagem de Linfócito CD4 , Coinfecção/microbiologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Derrame Pleural/patologia , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Adulto Jovem
4.
BMC Infect Dis ; 20(1): 469, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32615981

RESUMO

BACKGROUND: Interferon-γ release assays (IGRA) with Resuscitation promoting factor (Rpf) proteins enhanced tuberculosis (TB) screening and diagnosis in adults but have not been evaluated in children. Children often develop paucibacillary TB and their immune response differs from that of adults, which together affect TB disease diagnostics and immunodiagnostics. We assessed the ability of Rpf to identify infection among household TB-exposed children in The Gambia and investigated their ability to discriminate Mycobacterium tuberculosis complex (MTBC) infection from active TB disease in children. METHODS: Detailed clinical investigations were done on 93 household TB-exposed Gambian children and a tuberculin skin test (TST) was administered to asymptomatic children. Venous blood was collected for overnight stimulation with ESAT-6/CFP-10-fusion protein (EC), purified protein derivative and RpfA, B, C, D and E. Interferon gamma (IFN-γ) production was measured by ELISA in supernatants and corrected for the background level. Infection status was defined by IGRA with EC and TB disease by mycobacterial confirmation and/or clinical diagnosis. We compared IFN-γ levels between infected and uninfected children and between infected and TB diseased children using a binomial logistic regression model while correcting for age and sex. A Receiver Operating Characteristics analysis was done to find the best cut-off for IFN-γ level and calculate sensitivity and specificity. RESULTS: Interferon gamma production was significantly higher in infected (IGRA+, n = 45) than in uninfected (IGRA-, n = 20) children after stimulation with RpfA, B, C, and D (P = 0.03; 0.007; 0.03 and 0.003, respectively). Using RpfB and D-specific IFN-γ cut-offs (33.9 pg/mL and 67.0 pg/mL), infection was classified with a sensitivity-specificity combination of 73-92% and 77-72% respectively, which was similar to and better than 65-75% for TST. Moreover, IFN-γ production was higher in infected than in TB diseased children (n = 28, 5 bacteriologically confirmed, 23 clinically diagnosed), following RpfB and D stimulation (P = 0.02 and 0.03, respectively). CONCLUSION: RpfB and RpfD show promising results for childhood MTBC infection screening, and both performed similar to and better than the TST in our study population. Additionally, both antigens appear to discriminate between infection and disease in children and thus warrant further investigation as screening and diagnostic antigens for childhood TB.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Citocinas/imunologia , Testes de Liberação de Interferon-gama/métodos , Tuberculose Latente/diagnóstico , Tuberculose Latente/epidemiologia , Programas de Rastreamento/métodos , Mycobacterium tuberculosis/imunologia , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Características da Família , Feminino , Gâmbia/epidemiologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Tuberculose Latente/microbiologia , Masculino , Sensibilidade e Especificidade , Teste Tuberculínico
5.
Nat Commun ; 11(1): 3363, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620750

RESUMO

Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.


Assuntos
Regulação Bacteriana da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Doenças Negligenciadas/imunologia , Orientia tsutsugamushi/genética , Tifo por Ácaros/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Genoma Bacteriano , Células Endoteliais da Veia Umbilical Humana , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Sequências Repetitivas Dispersas/genética , Camundongos , Doenças Negligenciadas/microbiologia , Orientia tsutsugamushi/imunologia , Orientia tsutsugamushi/patogenicidade , Proteômica , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , RNA-Seq , Tifo por Ácaros/microbiologia , Transcrição Genética , Sequenciamento Completo do Exoma
6.
PLoS Pathog ; 16(7): e1008591, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32645118

RESUMO

Reactive arthritis, an autoimmune disorder, occurs following gastrointestinal infection with invasive enteric pathogens, such as Salmonella enterica. Curli, an extracellular, bacterial amyloid with cross beta-sheet structure can trigger inflammatory responses by stimulating pattern recognition receptors. Here we show that S. Typhimurium produces curli amyloids in the cecum and colon of mice after natural oral infection, in both acute and chronic infection models. Production of curli was associated with an increase in anti-dsDNA autoantibodies and joint inflammation in infected mice. The negative impacts on the host appeared to be dependent on invasive systemic exposure of curli to immune cells. We hypothesize that in vivo synthesis of curli contributes to known complications of enteric infections and suggest that cross-seeding interactions can occur between pathogen-produced amyloids and amyloidogenic proteins of the host.


Assuntos
Artrite Infecciosa/imunologia , Proteínas de Bactérias/imunologia , Febre Tifoide/imunologia , Animais , Anticorpos Antinucleares/imunologia , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Infecciosa/metabolismo , Proteínas de Bactérias/biossíntese , Intestino Grosso/imunologia , Intestino Grosso/microbiologia , Camundongos , Febre Tifoide/metabolismo
7.
Nat Commun ; 11(1): 3545, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669564

RESUMO

Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections.


Assuntos
Proteínas de Bactérias/imunologia , Proteção Cruzada/imunologia , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Aderência Bacteriana/imunologia , Linhagem Celular , Clonagem Molecular , Modelos Animais de Doenças , Feminino , Humanos , Imunogenicidade da Vacina , Camundongos , Testes de Neutralização , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Sorogrupo , Infecções Estreptocócicas/microbiologia , Vacinas Estreptocócicas/administração & dosagem , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia
8.
PLoS One ; 15(6): e0233695, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479551

RESUMO

Johne's disease (JD) is an infectious wasting condition of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) in domestic livestock of every country that has been investigated. Controlling JD is problematic due to the lack of sensitive, specific, efficient, and cost-effective diagnostic tests. A major challenge in the development of diagnostics like ELISA is the selection of an ideal antigen/(s) that is pathogen-specific and allows sensitive recognition. Therefore, the purpose of this study was to identify and use Mce-truncated protein-based ELISA assay for the diagnosis of MAP infection with high sensitivity and specificity. In silico epitope prediction by epitope mapping throughout the whole length of MAP2191 protein revealed that C-terminal portion of this protein presented potential T- and B-cell epitopes. Therefore, a novel Mce-truncated protein encoded by the selected region of MAP2191 gene was expressed, purified with Ni-NTA gel matrix and confirmed by SDS PAGE and western blot. A profiling ELISA assay was developed to evaluate sera from MAP infected and non-infected ruminant species for antibodies against Mce-truncated protein to infer the immunogenicity of this protein in the host. Using this Mce protein-based ELISA, 251 goats, 53 sheep, 117 buffaloes, and 33 cattle serum samples were screened and 49.4, 51.0, 69.2, and 54.6% animals, respectively, were found positive. Comparing with i-ELISA, the new Mce-based ELISA kit showed a relatively higher specificity but suffered from slightly reduced sensitivity. Mce-based ELISA excluded apparently false positive results of i-ELISA. Mce protein was found to be antigenic and Mce-ELISA test could be employed as a diagnostic test for JD in domestic livestock in view of the a relatively higher specificity and accuracy. The antigenic potential of Mce antigen can also be exploited for the development of a new vaccine for the control of MAP infection.


Assuntos
Doenças dos Bovinos/sangue , Mycobacterium avium/imunologia , Paratuberculose/sangue , Testes Sorológicos/veterinária , Doenças dos Ovinos/sangue , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/microbiologia , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Cabras , Mycobacterium avium/patogenicidade , Paratuberculose/diagnóstico , Paratuberculose/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/microbiologia
9.
Mol Immunol ; 124: 70-82, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32540517

RESUMO

Several vaccine candidates have been introduced for immunization against Pseudomonas aeruginosa strains. Despite extensive efforts in recent decades, there is no accurate immunogenic candidate against this pathogen in the market yet. Due to the rapid increase in several drug-resistant strains, P. aeruginosa has caused various health concerns worldwide. It encodes many specific virulence features, which can be used as an appropriate vaccine candidate. The primary stage of the pathogenesis of P. aeruginosa is the expression of many dynamic adhesive molecules, such as type IV pili (T4P), which acts as a principal colonization factor. It has been confirmed that three different subtypes of T4P, including type IVa (T4aP), type IVb (T4bP) and tight adherence (Tad) pili are expressed by P. aeruginosa. The IVa fimbriae type is almost the main cause of challenges to design an effective pili based-immunotherapy method. Nevertheless, in terms of heterogeneity, variability and hidden conserved binding site of T4aP, this attitude has been remained controversial and there is no permitted human study based on IVa pilin commercially. The engineered synthetic peptide-based vaccines are highly talented to mimic the target. In this research, for the first time, some dominant immunogenic features of the Flp protein, such as both B- and T-cell-associated epitopes, presence of IgE-associated epitopes, solvent-accessible surface area were evaluated by analytical immunoinformatics methods. In addition, we designed the engineered Flp pilin as an effective immunogenic substance against several clinically important P. aeruginosa strains. Moreover, by practical active immunization approaches, the humoral and cellular immune response against the extremely conserved region of the engineered synthetic Flp (EFlp) formulated in Montanide ISA 266 compared to the control group. The results of active immunization against EFlp significantly signified that EFlp-Montanide ISA 266 (EFLP-M) strongly could induce both humoral and cellular immune responses. We concluded that Flp pilin has therapeutic potential against numerous clinically significant P. aeruginosa strains and can be served as a novel immunogen for further investigations for development of effective immunotherapy methods against P. aeruginosa as a dexterous pathogen.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Biologia Computacional , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pseudomonas/prevenção & controle , Vacinação , Vacinas Sintéticas/imunologia
10.
PLoS One ; 15(6): e0234043, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555593

RESUMO

Syphilis serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance and discordant results between tests make clinical decisions difficult. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. Our aim was to assess the varied from performance of T. pallidum-recombinant proteins TmpA, TpN17 and TpN47 for syphilis serodiagnosis. The proteins were evaluated using sera of 338 T. pallidum-negative, 173 T. pallidum-positive individuals and 209 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. In the liquid microarray analyses, the ROC curve varied from 99.0% for TmpA and TpN17 to 100% for TpN47. The sensitivity score yielded values of up to 90% for TpN17, 100% for TpN47 and 80.0% for TmpA. The lowest and highest specificity values were presented by TpN47 (91.9%) and TmpA antigens (100%), respectively. TpN47 showed the highest accuracy score (95.5%) among all the recombinant proteins assayed. For the ELISA, the ROC curve was 97.2%, 91.8% and 81.6% for TpN17, TmpA and TpN47, respectively. TpN17 and TmpA yielded a sensitivity of 69.9%, while TpN47 obtained a value of 53.8%. Specificity was almost 100% for all three proteins. No cross-reaction was observed for TpN17 in the serum samples from non-bacterial infections. Regarding leptospirosis-positive samples, cross-reactivity score was varied from 8.6 to 34.6%. This is most probably due to conservation of the epitopes in these proteins across bacteria. The use of recombinant proteins in immunoassays for syphilis diagnosis was showed provide greater reliability to results of the treponemal assays. Despite the low sensitivity, the proteins showed high diagnostic capacity due to the AUC values found. However, an improvement in sensitivity could be achieved when antigenic mixtures are evaluated.


Assuntos
Proteínas de Bactérias/imunologia , Proteínas Recombinantes/imunologia , Testes Sorológicos , Sífilis/diagnóstico , Treponema pallidum/imunologia , Reações Cruzadas , Sífilis/imunologia
11.
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
12.
PLoS One ; 15(6): e0235139, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574205

RESUMO

Viral infections complicated by a bacterial infection are typically referred to as coinfections or superinfections. Streptococcus pyogenes, the group A streptococcus (GAS), is not the most common bacteria associated with influenza A virus (IAV) superinfections but did cause significant mortality during the 2009 influenza pandemic even though all isolates are susceptible to penicillin. One approach to improve the outcome of these infections is to use passive immunization targeting GAS. To test this idea, we assessed the efficacy of passive immunotherapy using antisera against either the streptococcal M protein or streptolysin O (SLO) in a murine model of IAV-GAS superinfection. Prophylactic treatment of mice with antiserum to either SLO or the M protein decreased morbidity compared to mice treated with non-immune sera; however, neither significantly decreased mortality. Therapeutic use of antisera to SLO decreased morbidity compared to mice treated with non-immune sera but neither antisera significantly reduced mortality. Overall, the results suggest that further development of antibodies targeting the M protein or SLO may be a useful adjunct in the treatment of invasive GAS diseases, including IAV-GAS superinfections, which may be particularly important during influenza pandemics.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Imunoterapia/métodos , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Estreptolisinas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Coinfecção/microbiologia , Coinfecção/terapia , Coinfecção/virologia , Feminino , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Soros Imunes/imunologia , Soros Imunes/farmacologia , Vírus da Influenza A/fisiologia , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/terapia , Infecções por Orthomyxoviridae/virologia , Coelhos , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/terapia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/fisiologia , Estreptolisinas/antagonistas & inibidores , Estreptolisinas/metabolismo , Superinfecção/microbiologia , Superinfecção/terapia , Superinfecção/virologia
13.
Int J Nanomedicine ; 15: 3877-3886, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32581535

RESUMO

Introduction: Vaccine formulation with appropriate adjuvants is an attractive approach to develop protective immunity against pathogens. Calcium phosphate nanoparticles (CaPNs) are considered as ideal adjuvants and delivery systems because of their great potential for enhancing immune responses. In the current study, we have designed nanoparticle-based vaccine candidates to induce immune responses and protection against B. melitensis and B. abortus. Materials and Methods: For this purpose, we used three Brucella antigens (FliC, 7α-HSDH, BhuA) and two multi-epitopes (poly B and poly T) absorbed by CaPNs. The efficacy of each formulation was evaluated by measuring humoral, cellular and protective responses in immunized mice. Results: The CaPNs showed an average size of about 90 nm with spherical shape and smooth surface. The CaPNs-adsorbed proteins displayed significant increase in cellular and humoral immune responses compared to the control groups. In addition, our results showed increased ratio of specific IgG2a (associated with Th1) to specific IgG1 (associated with Th2). Also, immunized mice with different vaccine candidate formulations were protected against B. melitensis 16M and B. abortus 544, and showed same levels of protection as commercial vaccines (B. melitensis Rev.1 and B. abortus RB51) except for BhuA-CaPNs. Discussion: Our data support the hypothesis that these antigens absorbed with CaPNs could be effective vaccine candidates against B. melitensis and B. abortus.


Assuntos
Antígenos de Bactérias/química , Vacina contra Brucelose/química , Vacina contra Brucelose/imunologia , Nanopartículas/química , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Proteínas de Bactérias/imunologia , Brucella abortus/imunologia , Brucella melitensis/imunologia , Brucelose/imunologia , Brucelose/prevenção & controle , Fosfatos de Cálcio/química , Sistemas de Liberação de Medicamentos , Feminino , Imunidade Humoral , Imunização , Imunoglobulina G/imunologia , Proteínas de Membrana Transportadoras/imunologia , Camundongos Endogâmicos BALB C
14.
PLoS Negl Trop Dis ; 14(5): e0008326, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463817

RESUMO

Salmonella and Shigella species are food- and water-borne pathogens that are responsible for enteric infections in both humans and animals and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics require the development of broadly protective therapies. Those bacteria utilize a Type III Secretion System (T3SS), necessary for their pathogenicity. The structural proteins composing the T3SS are common to all virulent Salmonella and Shigella spp., particularly the needle-tip proteins SipD (Salmonella) and IpaD (Shigella). We investigated the immunogenicity and protective efficacy of SipD and IpaD administered by intranasal and intragastric routes, in a mouse model of Salmonella enterica serotype Typhimurium (S. Typhimurium) intestinal challenge. Robust IgG (in all immunization routes) and IgA (in intranasal and oral immunization routes) antibody responses were induced against both proteins. Mice immunized with SipD or IpaD were protected against lethal intestinal challenge with S. Typhimurium or Shigella flexneri (100 Lethal Dose 50%). We have shown that SipD and IpaD are able to induce a cross-protection in a murine model of infection by Salmonella and Shigella. We provide the first demonstration that Salmonella and Shigella T3SS SipD and IpaD are promising antigens for the development of a cross-protective Salmonella-Shigella vaccine. These results open the way to the development of cross-protective therapeutic molecules.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Proteção Cruzada , Disenteria Bacilar/prevenção & controle , Proteínas de Membrana/imunologia , Infecções por Salmonella/prevenção & controle , Vacinas contra Salmonella/imunologia , Vacinas contra Shigella/imunologia , Administração Intranasal , Administração Oral , Animais , Anticorpos Antibacterianos/análise , Modelos Animais de Doenças , Feminino , Imunoglobulina A/análise , Imunoglobulina G/análise , Camundongos Endogâmicos BALB C , Vacinas contra Salmonella/administração & dosagem , Salmonella typhimurium/imunologia , Vacinas contra Shigella/administração & dosagem , Shigella flexneri/imunologia , Análise de Sobrevida
15.
Bioinformatics ; 36(13): 4065-4069, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32374823

RESUMO

MOTIVATION: The outbreak of COVID-2019 initiated at Wuhan, China has become a global threat by rapid transmission and severe fatalities. Recent studies have uncovered whole genome sequence of SARS-CoV-2 (causing COVID-2019). In addition, lung metagenomic studies on infected patients revealed overrepresented Prevotella spp. producing certain proteins in abundance. We performed host-pathogen protein-protein interaction analysis between SARS-CoV-2 and overrepresented Prevotella proteins with human proteome. We also performed functional overrepresentation analysis of interacting proteins to understand their role in COVID-2019 severity. RESULTS: It was found that overexpressed Prevotella proteins can promote viral infection. As per the results, Prevotella proteins, but not viral proteins, are involved in multiple interactions with NF-kB, which is involved in increasing clinical severity of COVID-2019. Prevotella may have role in COVID-2019 outbreak and should be given importance for understanding disease mechanisms and improving treatment outcomes. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Proteínas de Bactérias/imunologia , Infecções por Coronavirus/complicações , Interações Hospedeiro-Patógeno , Pneumonia Viral/complicações , Prevotella , Mapeamento de Interação de Proteínas , Infecções por Bacteroidaceae/complicações , Betacoronavirus , China , Humanos , NF-kappa B , Pandemias
16.
Zhonghua Yu Fang Yi Xue Za Zhi ; 54(5): 539-545, 2020 May 06.
Artigo em Chinês | MEDLINE | ID: mdl-32388956

RESUMO

Objective: The cellular immunity of 5 Mycobacterium tuberculosis recombinant proteins and their compositions was evaluated. Method: A total of 88 fresh venous blood from peripheral heparin anticoagulant population, 42 of which were from tuberculosis patients treated by The Tuberculosis Prevention and Treatment Center of Changping District, Beijing, and 46 of healthy volunteers were provided by the Infection Diseases of Chinese Center for Disease Control and Prevention. Healthy volunteers without a history of tuberculosis exposure and any clinical signs and symptoms. Using the Mycobacterium tuberculosis standard strain H37Rv DNA as a template, complete genes of the selected 5 recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c by PCR amplified; 5 recombinant proteins were cloned, expressed and purified as stimulants by genetic recombination and protein purification techniques, and the effector T cell enzyme-linked immunospot assay (ELISPOT) was used to detect cellular immunity in the population. Results: The recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c were successfully cloned, expressed and purified; And the sensitivities were 50.00%, 71.43%, 69.04%, 73.81% and 76.19%, and the specificities were 86.96%, 76.09%, 71.74%, 39.13% and 36.96%. In addition, the positive predictive value, negative predictive value, area under the curve and Youden index were 52.46% to 77.78%, 62.96% to 74.47%, 0.511 to 0.754 and 0.129 to 0.475, respectively. Except for Rv1411c and Rv3418c, the number of spot-forming cell (SFC) detected by Rv3874, Rv3875 and Rv2031c in tuberculosis patients was higher than healthy volunteers, and the differences were statistically significant (P<0.001). Among the 26 compositions composed of 5 recombinant proteins, the sensitivity was 80.95% to 95.24%, and the specificity was 68.89% to 24.44%. As the number of recombinant proteins in the composition increases, the sensitivity gradually increased, but the specificity decreased. Conclusion: The recombinant proteins of Mycobacterium tuberculosis Rv3874, Rv3875 and Rv2031c have strong ability to stimulate T cells to produce immune response, and have certain antigenicity. The efficacy of Rv1411c and Rv3418c alone as diagnostic antigens is not ideal, and the composition composed of multi-component antigens has certain application value. This article provides experimental evidence for the immune diagnosis of tuberculosis and the preparation of new anti-tuberculosis vaccines.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Imunidade Celular , Proteínas Recombinantes/imunologia , Tuberculose/imunologia , Pequim , Humanos , Mycobacterium tuberculosis
17.
Int J Infect Dis ; 96: 240-243, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32339714

RESUMO

OBJECTIVES: Between-person variability in T-cell-specific interferon-gamma release assay (IGRA) responses and discordance between IGRA test formats are poorly understood. METHODS: We evaluated the IFN-γ responses (QuantiFERON-TB Gold-In-Tube [QFT-GIT] and TSPOT-TB) stratified according to the Mycobacterium tuberculosis spoligotype of the culture isolate obtained from the same patients with confirmed active tuberculosis (n = 91). We further analysed differences within the RD-1-encoding ESX-1 region between the different strain types using whole genome sequencing. RESULTS: In HIV-uninfected patients, TSPOT.TB and QFT-GIT IFN-γ responses were 5-fold (p < 0.01) and 2-fold higher (p < 0.05) for those infected with family 33 compared to the LAM strain (additionally, TSPOT.TB responses were 5.6-fold [p < 0.05] and 2.6-fold higher [p < 0.05] for the patients infected with the family 33 versus the X strain and Beijing versus the LAM strain, respectively). Multivariate analysis revealed that strain type (determined by spoligotyping) was independently associated with the magnitude of the IGRA response (varied by IGRA test type) and this is likely explained by variability in the ESX-1 region of Mycobacteriumtuberculosis (determined by next-generation sequencing). CONCLUSIONS: These data have implications for the understanding of between-person heterogeneity in IGRA responses, Mycobateriumtuberculosis-specific host immunity, and the discordance between different IGRA test formats.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Interferon gama/metabolismo , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/imunologia , Adulto , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Pequim , Feminino , Genótipo , Humanos , Testes de Liberação de Interferon-gama , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Linfócitos T/imunologia
18.
Am J Trop Med Hyg ; 103(1): 260-265, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32314688

RESUMO

Infection by Helicobacter pylori is a major risk factor for gastric cancer (GC), the second leading cause of cancer-related death worldwide. Although biomarkers such as pepsinogens (PGs) and soluble urokinase plasminogen activator receptor (suPAR) may have diagnostic and/or prognostic value in patients with GC, their levels may be affected by H. pylori infection. The aim of this study was to investigate the association of the presence of antibodies to H. pylori and cytotoxin-associated gene A (CagA) with plasma levels of PGs and suPAR in a cohort of Guatemalan GC patients and controls. To this end, levels of suPAR, Pepsinogens I and II (PGI and PGII), and antibodies to H. pylori and CagA toxin were determined by ELISA in plasma samples from 67 GC patients and 136 matched healthy controls. Seropositivity for CagA was significantly higher in patients with GC than in controls. Pepsinogens II and suPAR levels were higher and PGI/PGII ratios were lower in GC patients than in controls. There was a significant association of H. pylori seropositivity status with increased levels of PGII and lower PGI/PGII ratios, particularly in the control (non-GC) population. The levels of suPAR were not significantly affected by H. pylori or CagA seropositivity status. These results suggest that the seropositivity status for H. pylori and CagA need to be taken into account during the GC diagnostic process.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Infecções por Helicobacter/epidemiologia , Helicobacter pylori/imunologia , Pepsinogênio A/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Neoplasias Gástricas/microbiologia , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Guatemala/epidemiologia , Infecções por Helicobacter/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Soroepidemiológicos , Neoplasias Gástricas/sangue
19.
Mem Inst Oswaldo Cruz ; 115: e190396, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32321154

RESUMO

BACKGROUND: Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis. OBJECTIVES: We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection. METHODS: An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge. FINDINGS: rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05). MAIN CONCLUSIONS: NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.


Assuntos
Antígenos de Bactérias/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Vacinas Bacterianas/administração & dosagem , Leptospirose/prevenção & controle , Fatores de Transcrição/administração & dosagem , Vacinas de DNA/administração & dosagem , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Cricetinae , Modelos Animais de Doenças , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Leptospira interrogans/imunologia , Leptospirose/imunologia , Nanopartículas , Fatores de Transcrição/imunologia , Vacinas de DNA/imunologia
20.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32152195

RESUMO

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 × 105 CFU (80,000 50% lethal dose [LD50]) and intranasal challenge with 5 × 103 CFU (50 LD50) of virulent Y. pestis This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Nanopartículas/administração & dosagem , Vacina contra a Peste/imunologia , Peste/imunologia , Yersinia pestis/imunologia , Imunidade Adaptativa/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Feminino , Imunidade Inata/imunologia , Imunização/métodos , Imunoglobulina G/imunologia , Masculino , Camundongos , Ativadores de Plasminogênio/imunologia , Células Th1/imunologia , Células Th2/imunologia , Vacinação/métodos
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