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1.
Nat Commun ; 11(1): 4994, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020485

RESUMO

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed.


Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Infecções Meningocócicas/imunologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Criança , Pré-Escolar , Mapeamento de Epitopos , Humanos , Lactente , Infecções Meningocócicas/prevenção & controle , Biblioteca de Peptídeos , Análise Serial de Proteínas , Ensaios de Anticorpos Bactericidas Séricos , Adulto Jovem
2.
Anticancer Res ; 40(11): 6387-6398, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109577

RESUMO

BACKGROUND/AIM: Helicobacter pylori (Hp) infection affects a substantial proportion of the world population and is a major risk factor of gastric cancer (GC). The caveats of common Hp-tests can be evaded by a serological biomarker test (GastroPanel®, Biohit Oyj, Helsinki), the most comprehensive Hp-test on the market. The clinical validation of Helicobacter pylori IgG ELISA of the new-generation GastroPanel® test is reported. The aim of the study is to validate the clinical performance of the Helicobacter pylori IgG ELISA test in diagnosis of biopsy-confirmed Hp-infection in gastroscopy referral patients. PATIENTS AND METHODS: A cohort of 101 patients (mean age=50.1 years) referred for gastroscopy at the outpatient Department of Gastroenterology (SM Clinic, St. Petersburg) were examined by two test versions to validate the new-generation GastroPanel®. All patients were examined by gastroscopy and biopsies, which were stained with Giemsa for specific identification of Hp in the antrum (A) and corpus (C). RESULTS: Biopsy-confirmed Hp-infection was found in 64% of patients, most often confined to antrum. The overall agreement between Hp IgG ELISA and gastric biopsies in Hp-detection was 91% (95%CI=84.1-95.8%). Hp IgG ELISA diagnosed biopsy-confirmed Hp (A&C) with sensitivity (SE) of 92.3%, specificity (SP) of 88.6%, positive predictive value (PPV) of 93.8% and negative predictive value (NPV) of 86.1%, with AUC=0.904 (95%CI=0.842-0.967). In ROC analysis for Hp detection (A&C), Hp IgG ELISA shows AUC=0.978 (95%CI=0.956-1.000). CONCLUSION: The Hp IgG ELISA test successfully concludes the clinical validation process of the new-generation GastroPanel® test, which retains the unrivalled diagnostic performance of all its four biomarkers, extensively documented for the first-generation test in different clinical settings.


Assuntos
Anticorpos Antibacterianos/isolamento & purificação , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Imunoglobulina G/isolamento & purificação , Adolescente , Adulto , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Biópsia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Gastrinas/genética , Gastrinas/isolamento & purificação , Gastrite Atrófica/diagnóstico , Gastrite Atrófica/genética , Gastrite Atrófica/microbiologia , Gastrite Atrófica/patologia , Gastroscopia/métodos , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Pepsinogênio A/genética , Pepsinogênio A/isolamento & purificação , Pepsinogênio C/genética , Pepsinogênio C/isolamento & purificação , Encaminhamento e Consulta , Estômago/microbiologia , Estômago/patologia , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Adulto Jovem
3.
PLoS Negl Trop Dis ; 14(9): e0008557, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976512

RESUMO

Novel diagnostic tools are a major challenge for brucellosis research, especially in developing countries. Herein, we established a handheld quantum dot (QD) immunochromatographic device for the fast detection of brucellosis antibodies in the field. Total bacterial protein extracted from Brucella 104M served as labelling and coating antigen. QD labelling and immunochromatography methods were used to optimise reaction conditions, labelling conditions, reaction temperature and storage temperature. QD test strips were employed to test brucellosis serum to determine their sensitivity, specificity and stability. Test strips were compared with Rose Bengal test, standard agglutination test and colloidal gold immunochromatographic assay. Labelled Brucella total protein displayed good specificity and no cross-reactivity. The concentration of labelled total bacterial protein was 3.9 mg/ml, the coating concentration was 2.0 mg/ ml, and the serum titre with the lowest detection sensitivity was 1:25. The optimal reaction temperature for the test strip was 25-30°C. The test strip was stable after storage at room temperature and the repeatability was high, with a coefficient of variation of 4.0%. After testing 199 serum samples, the sensitivity of the QD test strip was 98.53%, the specificity was 93.57%, and the coincidence rate with the standard agglutination test was 96.98%. The developed QD immunochromatographic method can be used for rapid detection and preliminary screening of brucellosis in the field.


Assuntos
Brucella/imunologia , Brucelose/diagnóstico , Cromatografia de Afinidade/métodos , Pontos Quânticos , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/análise , Brucelose/imunologia , Humanos , Fitas Reagentes/análise , Sensibilidade e Especificidade
4.
PLoS Pathog ; 16(8): e1008733, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817694

RESUMO

Staphylococcus aureus (S. aureus) is one of the most common bacterial infections worldwide, and antibiotic resistant strains such as Methicillin-Resistant S. aureus (MRSA) are a major threat and burden to public health. MRSA not only infects immunocompromised patients but also healthy individuals and has rapidly spread from the healthcare setting to the outside community. However, all vaccines tested in clinical trials to date have failed. Immunocompromised individuals such as patients with HIV or decreased levels of CD4+ T cells are highly susceptible to S. aureus infections, and they are also at increased risk of developing fungal infections. We therefore wondered whether stimulation of antifungal immunity might promote the type of immune responses needed for effective host defense against S. aureus. Here we show that vaccination of mice with a fungal ß-glucan particle (GP) loaded with S. aureus antigens provides protective immunity to S. aureus. We generated glucan particles loaded with the four S. aureus proteins ClfA, IsdA, MntC, and SdrE, creating the 4X-SA-GP vaccine. Vaccination of mice with three doses of 4X-SA-GP promoted protection in a systemic model of S. aureus infection with a significant reduction in the bacterial burden in the spleen and kidneys. 4X-SA-GP vaccination induced antigen-specific Th1 and Th17 CD4+ T cell and antibody responses and provided long-term protection. This work suggests that the GP vaccine system has potential as a novel approach to developing vaccines for S. aureus.


Assuntos
Saccharomyces cerevisiae/imunologia , Infecções Estafilocócicas/imunologia , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Coagulase/administração & dosagem , Coagulase/genética , Coagulase/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Saccharomyces cerevisiae/química , Infecções Estafilocócicas/microbiologia , Vacinas Antiestafilocócicas/administração & dosagem , Vacinas Antiestafilocócicas/genética , Staphylococcus aureus/genética , Células Th1/imunologia , Células Th17/imunologia , Vacinação , beta-Glucanas/administração & dosagem , beta-Glucanas/imunologia
5.
PLoS One ; 15(8): e0237394, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822419

RESUMO

Bordetella pertussis vaccine escape mutants that lack expression of the pertussis antigen pertactin (Prn) have emerged in vaccinated populations in the last 10-20 years. Additionally, clinical isolates lacking another acellular pertussis (aP) vaccine component, filamentous hemagglutinin (FHA), have been found sporadically. Here, we show that both whole-cell pertussis (wP) and aP vaccines induced protection in the lungs of mice, but that the wP vaccine was more effective in nasal clearance. Importantly, bacterial populations isolated from the lungs shifted to an FHA-negative phenotype due to frameshift mutations in the fhaB gene. Loss of FHA expression was strongly selected for in Prn-deficient strains in the lungs following aP but not wP vaccination. The combined loss of Prn and FHA led to complete abrogation of bacterial surface binding by aP-induced serum antibodies. This study demonstrates vaccine- and anatomical site-dependent adaptation of B. pertussis and has major implications for the design of improved pertussis vaccines.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Bordetella pertussis/fisiologia , Vacinas contra Difteria, Tétano e Coqueluche Acelular/imunologia , Hemaglutininas/metabolismo , Fatores de Virulência de Bordetella/metabolismo , Animais , Anticorpos Antibacterianos/imunologia , Bordetella pertussis/imunologia , Regulação da Expressão Gênica , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Vacinação , Coqueluche/metabolismo , Coqueluche/patologia , Coqueluche/prevenção & controle
6.
Proc Natl Acad Sci U S A ; 117(37): 22992-23000, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32855300

RESUMO

Antibodies may bind to bacterial pathogens or their toxins to control infections, and their effector activity is mediated through the recruitment of complement component C1q or the engagement with Fcγ receptors (FcγRs). For bacterial pathogens that rely on a single toxin to cause disease, immunity correlates with toxin neutralization. Most other bacterial pathogens, including Staphylococcus aureus, secrete numerous toxins and evolved multiple mechanisms to escape opsonization and complement killing. Several vaccine candidates targeting defined surface antigens of S. aureus have failed to meet clinical endpoints. It is unclear that such failures can be solely attributed to the poor selection of antibody targets. Thus far, studies to delineate antibody-mediated uptake and killing of Gram-positive pathogens remain extremely limited. Here, we exploit 3F6-hIgG1, a human monoclonal antibody that binds and neutralizes the abundant surface-exposed Staphylococcal protein A (SpA). We find that galactosylation of 3F6-hIgG1 that favors C1q recruitment is indispensable for opsonophagocytic killing of staphylococci and for protection against bloodstream infection in animals. However, the simple removal of fucosyl residues, which results in reduced C1q binding and increased engagement with FcγR, maintains the opsonophagocytic killing and protective attributes of the antibody. We confirm these results by engineering 3F6-hIgG1 variants with biased binding toward C1q or FcγRs. While the therapeutic benefit of monoclonal antibodies against infectious disease agents may be debatable, the functional characterization of such antibodies represents a powerful tool for the development of correlates of protection that may guide future vaccine trials.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Fagocitose/imunologia , Proteína Estafilocócica A/imunologia , Animais , Linhagem Celular , Glicosilação , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia
7.
PLoS One ; 15(8): e0237940, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32853255

RESUMO

Acidovorax citrulli, a seedborne bacterium and quarantine pest, causes the devastating bacterial fruit blotch disease in cucurbit plants. Immunological assays such as ELISA are widely used in routine field inspections for this bacterium. However, to the best of our knowledge, none of the currently available monoclonal antibodies (MAbs) can detect all common A. citrulli strains. We therefore aimed to produce a panel of MAbs and to develop an ELISA-based method capable of detecting all A. citrulli strains. We used a high-throughput bead array technique to screen and characterize A. citrulli-specific MAbs produced from hybridoma clones. The hybridoma library was simultaneously screened against five A. citrulli strains (PSA, KK9, SQA, SQB and P) and the closely related bacterium, Delftia acidovorans. Three MAbs exhibiting different binding patterns to A. citrulli were used to develop an ELISA-based method called "double antibody pairs sandwich ELISA" (DAPS-ELISA). DAPS-ELISA employing mixtures of MAbs was able to specifically detect all 16 A. citrulli strains tested without cross-reactivity with other bacteria. By contrast, our previously developed MAb capture-sandwich ELISA (MC-sELISA) and a commercial test kit detected only 15 and 14 of 16 strains, respectively. The sensitivity of the DAPS-ELISA ranged from 5×105 to 1×106 CFU/mL, while those of the MC-sELISA and the commercial test kit ranged from 5×104 to 1×107 CFU/mL and 5×104 to 5×105 CFU/mL, respectively. DAPS-ELISA thus represents an alternative method enabling rapid, accurate, and inexpensive detection of all A. citrulli strains. The method can be applied to seed testing prior to planting as well as to routine field inspections.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Comamonadaceae/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Sorogrupo , Hibridomas , Limite de Detecção
8.
Arch Dis Child ; 105(9): 825-829, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32601082

RESUMO

OBJECTIVE: Despite substantial variation of streptococcal antibody titres among global populations, there is no data on normal values in sub-Saharan Africa. The objective of this study was to establish normal values for antistreptolysin O (ASO) and antideoxyribonuclease B (ADB) antibodies in Uganda. DESIGN: This was an observational cross-sectional study. SETTING: This study was conducted at Mulago National Referral Hospital, which is located in the capital city, Kampala, and includes the Uganda Heart Institute. PATIENTS: Participants (aged 0-50 years) were recruited. Of 428 participants, 22 were excluded from analysis, and 183 (44.4%) of the remaining were children aged 5-15 years. MAIN OUTCOME MEASURES: ASO was measured in-country by nephelometric technique. ADB samples were sent to Australia (PathWest) for analysis by enzyme inhibition assay: 80% upper limit values were established. RESULTS: The median ASO titre in this age group was 220 IU/mL, with the 80th percentile value of 389 IU/mL. The median ADB titre in this age group was 375 IU/mL, with the 80th percentile value of 568 IU/mL. CONCLUSIONS: The estimated Ugandan paediatric population standardised 80% upper-limit-of-normal ASO and ADB titres is higher than many global populations. Appropriateness of using population-specific antibody cutoffs is yet to be determined and has important implications for the sensitivity and specificity of rheumatic fever diagnosis.


Assuntos
Anticorpos Antibacterianos/sangue , Streptococcus pyogenes/imunologia , Adolescente , Adulto , Fatores Etários , Anticorpos Antibacterianos/imunologia , Antiestreptolisina/imunologia , Criança , Pré-Escolar , Estudos Transversais , Desoxirribonucleases/imunologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Valores de Referência , Infecções Estreptocócicas/sangue , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/imunologia , Uganda/epidemiologia , Adulto Jovem
9.
Appl Environ Microbiol ; 86(17)2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32591386

RESUMO

Staphylococcus aureus and other coagulase-positive Staphylococcus spp. bind the Fc region of IgG antibodies through expression of protein A (SpA). These species have consequently been a source of false-positive signals in antibody-based assays designed to detect other target bacteria. Here, flow cytometry was used to study the influence of a number of factors on the SpA-mediated binding of single cells to an anti-human IgG antibody, including strain, heat killing, overnight storage, growth phase, cell physiology, surface adhesion, and growth in model food systems. Through the costaining of antibody-stained cells with the permeability dye propidium iodide and calcein violet AM, the cell physiological status was related to SpA-mediated antibody binding. Generally, permeabilized cells lacking esterase activity did not strongly bind antibody. The binding of a number of commercially available polyclonal IgG antibodies to non-Staphylococcus spp. was also characterized. Not all SpA-expressing species showed strong binding of mouse IgG, and one species not known to express SpA showed strong binding. Most SpA-expressing strains bound rabbit IgG antibodies to some extent, whereas only one strain bound goat IgG. To reduce or eliminate SpA-mediated IgG binding, the following products were evaluated as blocking reagents and applied prior to staining with primary or secondary antibody: normal rabbit serum, mouse IgG isotype control, goat IgG, and a commercial FcR blocking reagent. Only the FcR blocking reagent consistently reduced SpA-mediated binding of Staphylococcus spp. to antibodies against other species and could be recommended as a blocking reagent in immunoassays designed to detect non-Staphylococcus species.IMPORTANCE This study characterizes a widespread but little-studied problem associated with the antibody-based detection of microbes-the Staphylococcus protein A (SpA)-mediated binding of IgG antibodies-and offers a solution: the use of commercial FcR blocking reagent. A common source of false-positive signals in the detection of microbes in clinical, food, or environmental samples can be eliminated by applying this study's findings. Using flow cytometry, the authors demonstrate the extent of heterogeneity in a culture's SpA-mediated binding of antibodies and that the degree of SpA-mediated antibody binding is strain, growth phase, and food matrix dependent and influenced by simulated food processing treatments and cell adherence. In addition, our studies of SpA-mediated binding of Staphylococcus spp. to antibodies against other bacterial species produced a very nuanced picture, leading us to recommend testing against multiple strains of S. aureus and S. hyicus of all antibodies to be incorporated into any immunoassay designed to detect a non-Staphylococcus spp.


Assuntos
Anticorpos Antibacterianos/imunologia , Receptores Fc/metabolismo , Proteína Estafilocócica A/imunologia , Staphylococcus/metabolismo , Anticorpos Antibacterianos/metabolismo , Citometria de Fluxo , Ligação Proteica , Proteína Estafilocócica A/metabolismo
10.
Am J Pathol ; 190(10): 2095-2110, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32598882

RESUMO

Inhalational anthrax, a disease caused by inhaling Bacillus anthracis spores, leads to respiratory distress, vascular leakage, high-level bacteremia, and often death within days. Anthrax lethal toxin and edema toxin, which are composed of protective antigen (PA) plus either lethal factor (LF) or edema factor (EF), respectively, play an important yet incompletely defined role in the pulmonary pathophysiology. To better understand their contribution, we examined the structural integrity of the alveolar-capillary barrier in archival formalin-fixed lungs of cynomolgus monkeys challenged with the fully virulent B. anthracis Ames wild-type strain or the isogenic toxin-deficient mutants ΔEF, ΔLF, and ΔPA. Pulmonary spore challenge with the wild-type strain caused high mortality, intra-alveolar hemorrhages, extensive alveolar septal sequestration of bacteria and neutrophils, diffuse destabilization of epithelial and endothelial junctions, increased markers of coagulation and complement activation (including tissue factor and C5a), and multifocal intra-alveolar fibrin deposition. ΔEF challenge was lethal and showed similar alveolar-capillary alterations; however, intra-alveolar hemorrhages, bacterial deposition, and markers of coagulation or complement were absent or markedly lower. In contrast, ΔLF or ΔPA challenges were nonlethal and showed no signs of alveolar bacterial deposition or alveolar-capillary changes. These findings provide evidence that lethal toxin plays a determinative role in bacterial dissemination and alveolar-capillary barrier dysfunction, and edema toxin may significantly exacerbate pulmonary pathologies in a systemic infection.


Assuntos
Antraz/patologia , Bacillus anthracis/patogenicidade , Bacteriemia/patologia , Pulmão/patologia , Infecções Respiratórias/patologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Pulmão/efeitos dos fármacos , Macaca fascicularis/imunologia , Neutrófilos/imunologia , Esporos Bacterianos/imunologia , Esporos Bacterianos/patogenicidade , Virulência/imunologia
11.
Ann Hematol ; 99(8): 1895-1906, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32519092

RESUMO

After allogeneic hematopoietic stem cell transplantation (HSCT), patients are repetitively vaccinated to reduce the risk of infection caused by the immune deficiency following allogeneic HSCT. By the vaccination of transplanted patients, the humoral memory function can be restored in the majority of cases. It is unknown, however, to what extent memory B cells derived from the donor contribute to the mobilization of antibody-secreting cells and long-term humoral memory in patients after allogeneic HSCT. We therefore analyzed patients after allogeneic HSCT for memory B cell responses 7 days after single vaccination against tetanus toxoid (TT), diphtheria toxoid (DT), pertussis toxoid (PT), Haemophilus influenzae type b (Hib), and poliovirus. Patients showed an insufficient mobilization of plasmablasts (PB) after vaccination, whereas healthy subjects (HD, n = 13) exhibited a significant increase of PB in the peripheral blood. Regarding vaccine-specific antibody-secreting PB, all HD responded against all vaccine antigens, as expected. However, only 65% of the patients responded with a measurable increase in IgG-secreting PB against TT, 65% against DT, 33% against PT, and 53% against poliovirus. Correspondingly, the antibody titers on day 7 after vaccination did not increase in patients. A significant increase of serum titers for the vaccine antigens was detectable in the majority of patients only after repetitive vaccinations. In contrast to the low mobilization of vaccine-specific PB after vaccination, a high number of PB before vaccination was detectable in patients following allogeneic HSCT. High frequencies of circulating PB correlated with the incidence of moderate/severe chronic GVHD. In summary, patients showed a weak mobilization of antigen-specific PB and an inadequate increase in antibody titers 7 days after the first vaccination. Patients with moderate or severe chronic GVHD in their history had a significantly higher percentage of IgG-secreting PB prior to vaccination. The antigen specificity of these IgG-secreting PB is currently unknown.


Assuntos
Linfócitos B/imunologia , Vacina contra Difteria, Tétano e Coqueluche/administração & dosagem , Vacinas Anti-Haemophilus/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Memória Imunológica , Vacinas Pneumocócicas/administração & dosagem , Vacina Antipólio de Vírus Inativado/administração & dosagem , Vacinação , Adolescente , Adulto , Idoso , Aloenxertos , Anticorpos Antibacterianos/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Vacinas Combinadas/administração & dosagem
12.
Food Chem ; 329: 127224, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32516716

RESUMO

To overcome the drawbacks of antibody labeling dependence and single-readout system in the conventional lateral flow immunoassays (LFIAs) as well as the non-targeted combination of new capture agents reported recently for pathogen detection, in this work, a multi-readout and label-free LFIA was proposed for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7) based on a nanozyme-bacteria-antibody sandwich pattern. A type of functional nanozyme-mannose modified Prussian blue (man-PB), was introduced as the recognition agent as well as signal indicator. Apart from original signal intensity on the T-line, the peroxidase-like catalytic activity-driven generation of colorimetric signal could be used as another format of quantitation. Importantly, such LFIA could exhibit excellent performance for target pathogens detection separately with a quantitative range of 102-108 cfu·mL-1 and a low detection limit of 102 cfu·mL-1 based on different readout formats, indicating the application potential of the proposed LFIA in real samples.


Assuntos
Escherichia coli O157/isolamento & purificação , Imunoensaio , Nanopartículas/química , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Catálise , Colorimetria , Escherichia coli O157/imunologia , Ferrocianetos/química , Microbiologia de Alimentos , Limite de Detecção , Manose/química , Nanopartículas/metabolismo
13.
J Vis Exp ; (159)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32510489

RESUMO

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to opsono-phagocytic killing assays. An advantage of the opsono-adherence assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/imunologia , Anticorpos Antibacterianos/imunologia , Bacillus anthracis/imunologia , Aderência Bacteriana , Proteínas Opsonizantes/imunologia , Animais , Antraz/microbiologia , Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Fluoresceína-5-Isotiocianato/metabolismo , Fluorescência , Humanos , Macrófagos/imunologia , Camundongos , Primatas/imunologia , Primatas/microbiologia , Células RAW 264.7
14.
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
15.
Can J Microbiol ; 66(9): 529-534, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32396022

RESUMO

Fusobacterium nucleatum is becoming increasingly recognised as an emerging pathogen, gaining attention as a potential factor for exacerbating colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism; thus, we have initiated studies to examine the bacterium's surface glycochemistry. In an effort to characterise the surface carbohydrates of F. nucleatum, the aims of this study were to investigate the structure of the lipopolysaccharide (LPS) O-antigen of the cancer-associated isolate F. nucleatum strain CC 7/3 JVN3 C1 (hereafter C1) and to develop monoclonal antibodies (mAbs) to the LPS O-antigen that may be beneficial to the growing field of F. nucleatum research. In this study, we combined several technologies, including nuclear magnetic resonance (NMR) spectroscopy, to elucidate the structure of the LPS O-antigen repeat unit as -[-4-ß-Gal-3-α-FucNAc4N-4-α-NeuNAc-]-. We have previously identified this structure as the LPS O-antigen repeat unit from strain 10953. In this present study, we developed a mAb to the C1 LPS O-antigen and confirmed the mAbs cross-reactivity to the 10953 strain, thus confirming the structural identity.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Fusobacterium nucleatum/imunologia , Antígenos O/química , Antígenos O/imunologia , Animais , Antígenos de Bactérias/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Sorotipagem , Fatores de Virulência
16.
Am J Cardiol ; 125(11): 1651-1654, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32279835

RESUMO

In many cases, atrial fibrillation (AF) is associated with a history of cardiac inflammation. One of the potential pathogens responsible for atrial inflammation might be Borrelia burgdorferi - a pathogen involved in Lyme carditis. This study aimed to assess whether the serological history of Borrelia infection was associated with the risk of AF. The study included 113 AF patients and 109 patients in sinus rhythm. All patients underwent a clinical evaluation, echocardiography and had their blood taken for the assessment of anti-Borrelia IgG antibodies. Patients with AF compared with the non-AF group had more often serological signs of Borrelia infection (34.5% vs 6.4%; p <0.0001). The multivariate analysis showed that positive results for anti-Borrelia IgG antibodies were a strong independent predictor of AF (odds ratio 8.21; 95% confidence interval 3.08 to 21.88; p < 0.0001). In conclusion, presented data show that exposure to Borrelia spp. infection is associated with an increased risk of AF. Whether the early treatment of Lyme disease lowers the risk of AF development remains to be explored.


Assuntos
Fibrilação Atrial/epidemiologia , Doença de Lyme/epidemiologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antibacterianos/imunologia , Grupo Borrelia Burgdorferi/imunologia , Ecocardiografia , Feminino , Humanos , Imunoglobulina G/imunologia , Doença de Lyme/imunologia , Masculino , Análise Multivariada , Fatores de Risco , Testes Sorológicos
17.
PLoS One ; 15(4): e0230782, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294093

RESUMO

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


Assuntos
Antraz/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Epitopos/imunologia , Dermatopatias Bacterianas/imunologia , Adulto , Vacinas contra Antraz/imunologia , Especificidade de Anticorpos/imunologia , Bacillus anthracis/imunologia , Feminino , Humanos , Imunidade Humoral/imunologia , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Testes de Neutralização/métodos , Turquia , Adulto Jovem
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.
Cancer Causes Control ; 31(6): 601-606, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32222845

RESUMO

PURPOSE: Helicobacter pylori (H. pylori) is the leading cause of gastric cancer. High antibody levels to H. pylori virulence factors Vacuolating cytotoxin A (VacA) and Cytotoxin-associated gene A (CagA) have been suggested as gastric cancer risk markers. In the USA, H. pylori sero-prevalence is twofold higher in African Americans compared to whites. We sought to assess whether African Americans also exhibit higher antibody levels to VacA and CagA. METHODS: Antibody responses to H. pylori proteins were measured by multiplex serology in 686 African Americans and whites of the Southern Community Cohort Study. Among VacA- and CagA-seropositives, we analyzed the association of race with antibody level using logistic regression models to produce odds ratios (OR) and 95% confidence intervals (CI). RESULTS: Sero-positive African Americans had significantly higher mean antibody levels to both VacA and CagA, which resulted in increased odds for the highest quartile of antibody levels compared to sero-positive whites (VacA, OR: 6.08; 95% CI 3.41, 10.86; CagA, OR: 3.77; 95% CI 1.61, 8.84). CONCLUSION: Our findings support future studies to assess the association of differential antibody responses by race with risk of gastric cancer in the USA, which could then aid in developing targeted H. pylori eradication strategies.


Assuntos
Afro-Americanos/estatística & dados numéricos , Anticorpos Antibacterianos , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Grupo com Ancestrais do Continente Europeu/estatística & dados numéricos , Helicobacter pylori , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Infecções por Helicobacter/epidemiologia , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Helicobacter pylori/patogenicidade , Humanos , Estudos Soroepidemiológicos , Sudeste dos Estados Unidos , Fatores de Virulência/imunologia
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