RESUMEN

Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.

Asunto(s)

Anticuerpos Antibacterianos , Staphylococcus aureus Resistente a Meticilina , Receptores de Superficie Celular , Anticuerpos de Dominio Único , Humanos , Antibacterianos/farmacología , Hemo/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/uso terapéutico , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Antígenos Bacterianos/inmunología , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Camélidos del Nuevo Mundo , Animales , Unión Proteica/efectos de los fármacos , Modelos Moleculares , Simulación de Dinámica Molecular

RESUMEN

Salmonella enterica is a leading cause of food-borne diseases in humans worldwide, resulting in severe morbidity and mortality. They are carried asymptomatically in the intestine or gallbladder of livestock, and are transmitted predominantly from animals to humans via the fecal-oral route. Thus, the best preventive strategy is to preemptively prevent transmission to humans by vaccinating livestock. Live attenuated vaccines have been mostly favored because they elicit both cellular and humoral immunity and provide long-term protective immunity. However, developing these vaccines is a laborious and time-consuming process. Therefore, most live attenuated vaccines have been mainly used for phenotypic screening using the auxotrophic replica plate method, and new types of vaccines have not been sufficiently explored. In this study, we used Radiation-Mutation Enhancement Technology (R-MET) to introduce a wide variety of mutations and attenuate the virulence of Salmonella spp. to develop live vaccine strains. The Salmonella Typhimurium, ST454 strain (ST WT) was irradiated with Cobalt60 gamma-irradiator at 1.5 kGy for 1 h to maximize the mutation rate, and attenuated daughter colonies were screened using in vitro macrophage replication capacity and in vivo mouse infection assays. Among 30 candidates, ATOMSal-L6, with 9,961-fold lower virulence than the parent strain (ST454) in the mouse LD50 model, was chosen. This vaccine candidate was mutated at 71 sites, and in particular, lost one bacteriophage. As a vaccine, ATOMSal-L6 induced a Salmonella-specific IgG response to provide effective protective immunity upon intramuscular vaccination of mice. Furthermore, when mice and sows were orally immunized with ATOMSal-L6, we found a strong protective immune response, including multifunctional cellular immunity. These results indicate that ATOMSal-L6 is the first live vaccine candidate to be developed using R-MET, to the best of our knowledge. R-MET can be used as a fast and effective live vaccine development technology that can be used to develop vaccine strains against emerging or serotype-shifting pathogens.

Asunto(s)

Refuerzo Biomédico , Vacunas contra la Salmonella , Animales , Anticuerpos Antibacterianos/genética , Femenino , Humanos , Inmunoglobulina G/genética , Ratones , Mutación , Vacunas contra la Salmonella/genética , Salmonella typhimurium , Porcinos , Vacunas Atenuadas

RESUMEN

Gram-positive organisms with their thick envelope cannot be lysed by complement alone. Nonetheless, antibody-binding on the surface can recruit complement and mark these invaders for uptake and killing by phagocytes, a process known as opsonophagocytosis. The crystallizable fragment of immunoglobulins (Fcγ) is key for complement recruitment. The cell surface of S. aureus is coated with Staphylococcal protein A (SpA). SpA captures the Fcγ domain of IgG and interferes with opsonization by anti-S. aureus antibodies. In principle, the Fcγ domain of therapeutic antibodies could be engineered to avoid the inhibitory activity of SpA. However, the SpA-binding site on Fcγ overlaps with that of the neonatal Fc receptor (FcRn), an interaction that is critical for prolonging the half-life of serum IgG. This evolutionary adaptation poses a challenge for the exploration of Fcγ mutants that can both weaken SpA-IgG interactions and retain stability. Here, we use both wild-type and transgenic human FcRn mice to identify antibodies with enhanced half-life and increased opsonophagocytic killing in models of S. aureus infection and demonstrate that antibody-based immunotherapy can be improved by modifying Fcγ. Our experiments also show that by competing for FcRn-binding, staphylococci effectively reduce the half-life of antibodies during infection. These observations may have profound impact in treating cancer, autoimmune, and asthma patients colonized or infected with S. aureus and undergoing monoclonal antibody treatment.

Asunto(s)

Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Opsonización/inmunología , Ingeniería de Proteínas , Secuencia de Aminoácidos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Activación de Complemento , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta Inmunológica , Humanos , Fagocitosis/inmunología , Unión Proteica , Ingeniería de Proteínas/métodos , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/inmunología , Receptores Fc/genética , Proteína Estafilocócica A/inmunología , Staphylococcus aureus/inmunología

RESUMEN

Staphylococcal enterotoxin B (SEB) simultaneously crosslinks MHC class II antigen and TCR, promoting proliferation of T cells and releasing a large number of toxic cytokines. In this report, we computationally examined the possibility of using a single-chain biparatopic bispecific antibody to target SEB and prevent TCR binding. The design was inspired by the observation that mixing two anti-SEB antibodies 14G8 and 6D3 can block SEB-TCR activation, and we used 14G8-6D3-SEB tertiary crystal structure as a template. Twelve simulation systems were constructed to systematically examine the effects of the designed bispecific scFV MB102a, including isolated SEB, MB102a with different linkers, MB102a-SEB complex, MB102a-SEB-TCRß complex, MB102a-SEB-TCR-MHC II complex, and MB102a-SEB-MHC II. Our all atom molecular dynamics simulations (total 18,900 ns) confirmed that the designed single-chain bispecific antibody may allosterically prevent SEB-TCRß chain binding and inhibit SEB-TCR-MHC II formation. Subsequent analysis indicated that the binding of scFV to SEB correlates with SEB-TCR binding site motion and weakens SEB-TCR interactions.

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/inmunología , Enterotoxinas/inmunología , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/genética , Anticuerpos Biespecíficos/genética , Sitios de Unión de Anticuerpos , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Enterotoxinas/química , Humanos , Ratones , Simulación de Dinámica Molecular , Conformación Proteica , Ingeniería de Proteínas , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Staphylococcus aureus/inmunología , Linfocitos T/inmunología , Linfocitos T/microbiología

RESUMEN

Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.

Asunto(s)

Anticuerpos Antibacterianos , Anticuerpos Monoclonales/farmacología , Borrelia burgdorferi , Enfermedad de Lyme , Sustitución de Aminoácidos , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/farmacología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Borrelia burgdorferi/genética , Borrelia burgdorferi/inmunología , Modelos Animales de Enfermedad , Humanos , Lipoproteínas/genética , Lipoproteínas/inmunología , Enfermedad de Lyme/tratamiento farmacológico , Enfermedad de Lyme/genética , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/transmisión , Macaca fascicularis , Macaca mulatta , Masculino , Ratones , Ratones Transgénicos , Mutación Missense , Garrapatas/inmunología , Garrapatas/microbiología

RESUMEN

Vibrio cholerae causes the severe diarrheal disease cholera. Clinical disease and current oral cholera vaccines generate antibody responses associated with protection. Immunity is thought to be largely mediated by lipopolysaccharide (LPS)-specific antibodies, primarily targeting the O-antigen. However, the properties and protective mechanism of functionally relevant antibodies have not been well defined. We previously reported on the early B cell response to cholera in a cohort of Bangladeshi patients, from which we characterized a panel of human monoclonal antibodies (MAbs) isolated from acutely induced plasmablasts. All antibodies in that previous study were expressed in an IgG1 backbone irrespective of their original isotype. To clearly determine the impact of affinity, immunoglobulin isotype and subclass on the functional properties of these MAbs, we re-engineered a subset of low- and high-affinity antibodies in different isotype and subclass immunoglobulin backbones and characterized the impact of these changes on binding, vibriocidal, agglutination, and motility inhibition activity. While the high-affinity antibodies bound similarly to O-antigen, irrespective of isotype, the low-affinity antibodies displayed significant avidity differences. Interestingly, despite exhibiting lower binding properties, variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies, suggesting that how the MAb binds to the O-antigen may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.IMPORTANCE Immunity to the severe diarrheal disease cholera is largely mediated by lipopolysaccharide (LPS)-specific antibodies. However, the properties and protective mechanisms of functionally relevant antibodies have not been well defined. Here, we have engineered low and high-affinity LPS-specific antibodies in different immunoglobulin backbones in order to assess the impact of affinity, immunoglobulin isotype, and subclass on binding, vibriocidal, agglutination, and motility inhibition functional properties. Importantly, we found that affinity did not directly dictate functional potency since variants derived from the low-affinity MAbs had comparable agglutination and motility inhibition properties to the potently binding antibodies. This suggests that how the antibody binds sterically may be critical to function. In addition, not only pentameric IgM and dimeric IgA, but also monomeric IgA, was remarkably more potent than their IgG counterparts at inhibiting motility. Finally, analyzing highly purified F(ab) versions of these antibodies, we show that LPS cross-linking is essential for motility inhibition.

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Isotipos de Inmunoglobulinas/metabolismo , Antígenos O/inmunología , Vibrio cholerae O1/inmunología , Anticuerpos Antibacterianos/genética , Anticuerpos Monoclonales/clasificación , Anticuerpos Monoclonales/genética , Sitios de Unión de Anticuerpos , Inmunoglobulina A/genética , Inmunoglobulina A/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas/clasificación , Isotipos de Inmunoglobulinas/genética , Isotipos de Inmunoglobulinas/inmunología , Vibrio cholerae O1/química

RESUMEN

Monoclonal antibody (MAb) 2C7 recognizes a lipooligosaccharide epitope expressed by most clinical Neisseria gonorrhoeae isolates and mediates complement-dependent bactericidal activity. We recently showed that a recombinant human IgG1 chimeric variant of MAb 2C7 containing an E430G Fc modification (2C7_E430G), which enhances complement activation, outperformed the parental MAb 2C7 (2C7_WT) in vivo Because natural infection with N. gonorrhoeae often does not elicit protective immunity and reinfections are common, approaches that prolong bacterial control in vivo are of great interest. Advances in DNA-based approaches have demonstrated the combined benefit of genetic engineering, formulation optimizations, and facilitated delivery via CELLECTRA-EP technology, which can induce robust in vivo expression of protective DNA-encoded monoclonal antibodies (DMAbs) with durable serum activity relative to traditional recombinant MAb therapies. Here, we created optimized 2C7-derived DMAbs encoding the parental Fc (2C7_WT) or complement-enhancing Fc variants (2C7_E430G and 2C7_E345K). 2C7 DMAbs were rapidly generated and detected throughout the 4-month study. While all complement-engaging 2C7 variants facilitated rapid clearance following primary N. gonorrhoeae challenge (day 8 after DMAb administration), the complement-enhancing 2C7_E430G variant demonstrated significantly higher potency against mice rechallenged 65 days after DMAb administration. Passive intravenous transfer of in vivo-produced, purified 2C7 DMAbs confirmed the increased potency of the complement-enhancing variants. This study highlights the ability of the DMAb platform to launch the in vivo production of antibodies engineered to promote and optimize downstream innate effector mechanisms such as complement-mediated killing, leading to hastened bacterial elimination.IMPORTANCENeisseria gonorrhoeae has become resistant to most antibiotics in clinical use. Currently, there is no safe and effective vaccine against gonorrhea. Measures to prevent the spread of gonorrhea are a global health priority. A monoclonal antibody (MAb) called 2C7, directed against a lipooligosaccharide glycan epitope expressed by most clinical isolates, displays complement-dependent bactericidal activity and hastens clearance of gonococcal vaginal colonization in mice. Fc mutations in a human IgG1 chimeric version of MAb 2C7 further enhance complement activation, and the resulting MAb displays greater activity than wild-type MAb 2C7 in vivo Here, we utilized a DNA-encoded MAb (DMAb) construct designed to launch production and assembly of "complement-enhanced" chimeric MAb 2C7 in vivo The ensuing rapid and sustained MAb 2C7 expression attenuated gonococcal colonization in mice at 8 days as well as 65 days postadministration. The DMAb system may provide an effective, economical platform to deliver MAbs for durable protection against gonorrhea.

Asunto(s)

Anticuerpos Antibacterianos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Vacunas Bacterianas/inmunología , Epítopos/inmunología , Gonorrea/prevención & control , Inmunización Pasiva , Inmunoglobulina G/administración & dosificación , Neisseria gonorrhoeae/inmunología , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/administración & dosificación , Activación de Complemento , Femenino , Gonorrea/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C

RESUMEN

Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68-81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68-61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Escherichia coli Enterotoxigénica/inmunología , Inmunoglobulina A Secretora/inmunología , Nicotiana/metabolismo , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/metabolismo , Anticuerpos Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Afinidad de Anticuerpos , Adhesión Bacteriana/efectos de los fármacos , Células CACO-2 , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/terapia , Ácido Gástrico/metabolismo , Glicosilación , Humanos , Inmunoglobulina A Secretora/genética , Inmunoglobulina A Secretora/metabolismo , Inmunoglobulina A Secretora/uso terapéutico , Inmunoterapia , Ratones , Plantas Modificadas Genéticamente , Nicotiana/genética

RESUMEN

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.

Asunto(s)

Anticuerpos Antibacterianos/aislamiento & purificación , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/aislamiento & purificación , Inmunoglobulina G/aislamiento & purificación , Adolescente , Adulto , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Biopsia , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Gastrinas/genética , Gastrinas/aislamiento & purificación , Gastritis Atrófica/diagnóstico , Gastritis Atrófica/genética , Gastritis Atrófica/microbiología , Gastritis Atrófica/patología , Gastroscopía/métodos , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Humanos , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Pepsinógeno A/genética , Pepsinógeno A/aislamiento & purificación , Pepsinógeno C/genética , Pepsinógeno C/aislamiento & purificación , Derivación y Consulta , Estómago/microbiología , Estómago/patología , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patología , Adulto Joven

RESUMEN

The type VI secretion system (T6SS) is a nanomachine capable of killing adjacent microbial cells in a contact-dependent manner. Due to limited studies, relatively little is known about the range of marine bacteria that are susceptible to T6SS attack. Here, 15 diverse marine bacterial isolates from the phyla Bacteroidetes and Ɣ-Proteobacteria were challenged against the marine bacterium and human pathogen, Vibrio cholerae, which has a well described T6SS. V. cholerae killed several of the tested Ɣ-Proteobacteria, including members of the orders Vibrionales, Alteromonadales, Oceanospirillales, and Pseudomonadales. In contrast, V. cholerae co-existed with multiple Bacteroidetes and Ɣ-Proteobacteria isolates, but was killed by Vibrio coralliilyticus. Follow-up experiments revealed that five V. coralliilyticus strains, including known coral and shellfish pathogens survived the T6SS challenge and killed V. cholerae. By using predicted protein comparisons and mutagenesis, we conclude that V. coralliilyticus protected itself in the challenge by using its own T6SS to kill V. cholerae. This study provides valuable insight into the resilience and susceptibility of marine bacteria to the V. cholerae T6SS, and provides the first evidence for a functional T6SS in V. coralliilyticus, both of which have implications for human and ocean health.

Asunto(s)

Sistemas de Secreción Tipo VI/genética , Vibrio cholerae/patogenicidad , Vibrio/genética , Animales , Antozoos/microbiología , Anticuerpos Antibacterianos/genética , Proteínas Bacterianas/genética , Bacteroidetes/genética , Regulación Bacteriana de la Expresión Génica/genética , Proteobacteria/genética , Mariscos/microbiología , Vibrio cholerae/genética

RESUMEN

Single-chain antibodies from camelids have served as powerful tools ranging from diagnostics and therapeutics to crystallization chaperones meant to study protein structure and function. In this study, we isolated a single-chain antibody from an Indian dromedary camel (ICab) immunized against a bacterial 14TM helix transporter, NorC, from Staphylococcus aureus We identified this antibody in a yeast display screen built from mononuclear cells isolated from the immunized camel and purified the antibody from Escherichia coli after refolding it from inclusion bodies. The X-ray structure of the antibody at 2.15 Å resolution revealed a unique feature within its CDR3 loop, which harbors a Zn2+-binding site that substitutes for a loop-stabilizing disulfide bond. We performed mutagenesis to compromise the Zn2+-binding site and observed that this change severely hampered antibody stability and its ability to interact with the antigen. The lack of bound Zn2+ also made the CDR3 loop highly flexible, as observed in all-atom simulations. Using confocal imaging of NorC-expressing E. coli spheroplasts, we found that the ICab interacts with the extracellular surface of NorC. This suggests that the ICab could be a valuable tool for detecting methicillin-resistant S. aureus strains that express efflux transporters such as NorC in hospital and community settings.

Asunto(s)

Anticuerpos Antibacterianos/química , Proteínas Bacterianas/inmunología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/inmunología , Anticuerpos de Dominio Único/química , Zinc/metabolismo , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Afinidad de Anticuerpos , Sitios de Unión , Camelus , Staphylococcus aureus Resistente a Meticilina/inmunología , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Estabilidad Proteica , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Zinc/química

RESUMEN

Background & objectives: : Shiga toxin (Stx) is produced by Shigella dysenteriae, a Gram-negative, facultative anaerobic bacillus that causes shigellosis, haemolytic uraemic syndrome (HUS) and Reiter's syndrome. The detection methods for shiga toxin needs to be rapid, accurate, reliable and must be extensively evaluated under field conditions. The aim of this study was to develop rapid, sensitive and specific detection method for Stx. Methods: : Mice and rabbits were immunized with purified recombinant Shiga toxin B (rStxB). Using these antibodies dot ELISA, sandwich ELISA and flow through assay were developed. Results: : The high-titre antibodies specifically reacted with purified rStxB. Dot-ELISA, sandwich ELISA and flow-through assay were developed and standardized that could detect StxB with limit of detection (LOD) of 9.75, 9.7 ng/ml and 0.46 µg/cassette, respectively. Interpretation & conclusions: : The rStxB was used to produce antibodies to avoid handling of pathogen. The Flow through assay 'developed was specific, rapid and field amenable.

Asunto(s)

Disentería Bacilar/diagnóstico , Síndrome Hemolítico-Urémico/diagnóstico , Toxina Shiga/aislamiento & purificación , Shigella dysenteriae/genética , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Artritis Reactiva/diagnóstico , Artritis Reactiva/genética , Artritis Reactiva/microbiología , Disentería Bacilar/genética , Disentería Bacilar/microbiología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Síndrome Hemolítico-Urémico/genética , Síndrome Hemolítico-Urémico/microbiología , Humanos , Ratones , Toxina Shiga/genética , Shigella dysenteriae/patogenicidad

RESUMEN

Host immunogenetic factors can affect late complications of urogenital infections with Chlamydia trachomatis. These findings are creating new avenues for updating existing risk prediction models for C. trachomatis-associated tubal factor infertility (TFI). Research into host factors and its utilization may therefore have future implications for diagnosing C. trachomatis-induced infertility. We outline the epidemiological situation regarding C. trachomatis and TFI in high-income countries. Thereupon, we review the main characteristics of the population undergoing fertility work-up and identify screening and diagnostic strategies for TFI currently in place. The Netherlands is an exemplary model for the state of the art in high-income countries. Within the framework of existing clinical approaches, we propose a scenario for the translation of relevant genome-based information into triage of infertile women, with the objective of implementing genetic profiling in the routine investigation of TFI. Furthermore, we describe the state of the art in relevant gene- and single nucleotide polymorphism (SNP) based clinical prediction models and place our perspectives in the context of these applications. We conclude that the introduction of a genetic test of proven validity into the assessment of TFI should help reduce patient burden from invasive and costly examinations by achieving a more precise risk stratification.

Asunto(s)

Infecciones por Chlamydia/genética , Chlamydia trachomatis/genética , Pruebas Genéticas , Infertilidad Femenina/genética , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Infecciones por Chlamydia/epidemiología , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/aislamiento & purificación , Chlamydia trachomatis/patogenicidad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Infertilidad Femenina/diagnóstico , Infertilidad Femenina/epidemiología , Infertilidad Femenina/microbiología , Países Bajos/epidemiología , Polimorfismo de Nucleótido Simple/genética

RESUMEN

Brucellosis is caused by the genus Brucella. Brucella is widely distributed in cattle, swine, sheep, goat and other mammals including human. Animal brucellosis causes severe economic losses and affects related international transportation and trade. Human brucellosis causes both acute and chronic symptoms of multi-organ dysfunction. Brucella type IV secretion system (T4SS) VirB5 was required for macrophages infection and essential for virulence in mice. VirB5 is located on the cell surface and serves as a specific adhesin targeting host cell receptors. The aim of this study was to isolate and characterize a specific human domain antibody against Brucella abortus (B. abortus) VirB5 from human single domain antibody (sdAb or VHH) phage display library. Following five rounds of screening, an sdAb named as BaV5VH4 showed the highest affinity by enzyme-linked immunosorbent assay (ELISA). Its interaction with B. abortus VirB5 was verified by binding assay, dot blot and molecular docking. These findings in this paper could greatly help elucidate the molecular mechanisms of Brucella infection, and accelerate the development of sdAbs-based vaccines and neutralizing therapeutics of brucellosis.

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Brucella abortus/inmunología , Brucelosis/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/aislamiento & purificación , Bacteriófagos/inmunología , Secuencia de Bases , Brucella abortus/genética , Brucella abortus/aislamiento & purificación , Brucelosis/economía , Bovinos , Ensayo de Inmunoadsorción Enzimática , Cabras , Humanos , Immunoblotting , Ratones , Simulación del Acoplamiento Molecular , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Ovinos , Porcinos , Virulencia , Factores de Virulencia/metabolismo , Zoonosis/prevención & control

RESUMEN

To understand how the interaction between an intracellular bacterium and the host immune system contributes to outcome at the site of infection, we studied leprosy, a disease that forms a clinical spectrum, in which progressive infection by the intracellular bacterium Mycobacterium leprae is characterized by the production of type I IFNs and antibody production. Dual RNA-seq on patient lesions identifies two independent molecular measures of M. leprae, each of which correlates with distinct aspects of the host immune response. The fraction of bacterial transcripts, reflecting bacterial burden, correlates with a host type I IFN gene signature, known to inhibit antimicrobial responses. Second, the bacterial mRNA:rRNA ratio, reflecting bacterial viability, links bacterial heat shock proteins with the BAFF-BCMA host antibody response pathway. Our findings provide a platform for the interrogation of host and pathogen transcriptomes at the site of infection, allowing insight into mechanisms of inflammation in human disease.

Asunto(s)

Lepra/inmunología , Lepra/microbiología , Mycobacterium leprae/genética , ARN Bacteriano , RNA-Seq , Adulto , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Factor Activador de Células B/inmunología , Femenino , Interacciones Huésped-Patógeno , Humanos , Inmunidad Humoral/genética , Interferón Tipo I/metabolismo , Lepra/patología , Masculino , Mycobacterium leprae/inmunología , Células Plasmáticas/inmunología , ARN Mensajero , ARN Ribosómico , Transcriptoma

RESUMEN

Campylobacter jejuni (C. jejuni) is a foodborne intestinal pathogen and major cause of gastroenteritis worldwide. C. jejuni proteins that are immunogenic have been sought for their potential use in the development of biomarkers, diagnostic assays, or subunit vaccines for humans or livestock. To identify new immunogenic C. jejuni proteins, we used a native protein microarray approach. A protein chip, with over 1400 individually purified GST-tagged C. jejuni proteins, representing over 86% of the proteome, was constructed to screen for antibody titers present in test sera raised against whole C. jejuni cells. Dual detection of GST signals was incorporated as a way of normalizing the variation of protein concentrations contributing to the antibody staining intensities. We detected strong signals to 102 C. jejuni antigens. In addition to antigens recognized by antiserum raised against C. jejuni, parallel experiments were conducted to identify antigens cross-reactive to antiserum raised against various serotypes of E. coli or Salmonella or to healthy human sera. This led to the identification of 34 antigens specifically recognized by the C. jejuni antiserum, only four of which were previously known. The chip approach also allowed identification of conformational antigens. We demonstrate in the case of Cj1621 that antigen signals are lost to denaturing conditions commonly used in other approaches to identify immunogens. Antigens identified in this study include those possessing sequence features indicative of cell surface localization, as well as those that do not. Together, our results indicate that the unbiased chip-based screen can help reveal the full repertoire of host antibodies against microbial proteomes.

Asunto(s)

Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Campylobacter jejuni/inmunología , Campylobacter jejuni/metabolismo , Proteoma/inmunología , Proteoma/metabolismo , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/genética , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Infecciones por Campylobacter/inmunología , Campylobacter jejuni/genética , Reacciones Cruzadas , Humanos , Ratones , Análisis por Matrices de Proteínas/métodos , Conformación Proteica , Proteoma/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína

RESUMEN

BACKGROUND: Congenital toxoplasmosis is an important disease that occurs when pregnant women become infected with Toxoplasma gondii during pregnancy. The aim of this study was to investigate the presence of T. gondii B1 gene in placental tissues of IgM seronegative women. MATERIALS AND METHODS: In this research, chronic toxoplasmosis was identified through examination of blood samples in a group of pregnant women by anti-Toxoplasma IgG and IgM ELISA and nested-PCR techniques. IgG avidity test was used to estimate the onset of infection in some pregnant women with chronic infection. After delivery, some umbilical cord and neonatal blood were tested by anti-Toxoplasma IgM ELISA, and also the B1 gene of T. gondii was investigated in their placental tissue by nested-PCR. Some factors such as blood sampling time and some clinical symptoms experienced during pregnancy were recorded. RESULTS: One hundred and sixty seven out of 653 (25.6%) pregnant women were positive for anti-Toxoplasma IgG. Of them, 165 (98.8%) were negative for anti-T. gondii IgM. Six out of 10 (60%) placental tissues from IgG seropositive, IgM seronegative women were positive for T. gondii B1 gene, while anti-Toxoplasma IgM was negative in the umbilical cord and neonatal blood samples. The results of IgG avidity test showed low avidity in one and high avidity in two women's samples. The B1 gene was not found in the blood samples of any of the six mothers. The most symptoms experienced during pregnancy were headache and nausea. CONCLUSION: The detection of B1 gene in placental tissues of the healthy newborn infants reiterates that presence of T. gondii in the placenta does not always result in congenital toxoplasmosis.

Asunto(s)

Genes Protozoarios , Técnicas de Diagnóstico Molecular , Placenta/química , Toxoplasma/genética , Toxoplasmosis Congénita/diagnóstico , Toxoplasmosis Congénita/genética , Adulto , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/genética , Afinidad de Anticuerpos , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ADN Protozoario , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Inmunoglobulina M/análisis , Inmunoglobulina M/inmunología , Recién Nacido , Reacción en Cadena de la Polimerasa , Embarazo , Reproducibilidad de los Resultados

RESUMEN

Tissue factor pathway inhibitor-2 (TFPI-2) has previously been characterized as an endogenous anticoagulant. TFPI-2 is expressed in the vast majority of cells, mainly secreted into the extracellular matrix. Recently we reported that EDC34, a C-terminal peptide derived from TFPI-2, exerts a broad antimicrobial activity. In the present study, we describe a previously unknown antimicrobial mode of action for the human TFPI-2 C-terminal peptide EDC34, mediated via binding to immunoglobulins of the classes IgG, IgA, IgE, and IgM. In particular the interaction of EDC34 with the Fc part of IgG is of importance since this boosts interaction between the immunoglobulin and complement factor C1q. Moreover, we find that the binding increases the C1q engagement of the antigen-antibody interaction, leading to enhanced activation of the classical complement pathway during bacterial infection. In experimental murine models of infection and endotoxin challenge, we show that TFPI-2 is up-regulated in several organs, including the lung. Correspondingly, TFPI-2-/- mice are more susceptible to pulmonary Pseudomonas aeruginosa bacterial infection. No anti-coagulant role of TFPI-2 was observed in these models in vivo. Furthermore, in vivo, the mouse TFPI-2-derived C-terminal peptide VKG24, a homolog to human EDC34 is protective against systemic Escherichia coli bacterial infection. Moreover, in sputum from cystic fibrosis patients TFPI-2 C-terminal fragments are generated and found associated with immunoglobulins. Together our data describe a previously unknown host defense mechanism and therapeutic importance of TFPI-2 against invading Gram-negative bacterial pathogens.

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Infecciones por Escherichia coli/inmunología , Escherichia coli/inmunología , Glicoproteínas/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Anticuerpos Antibacterianos/genética , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/patología , Glicoproteínas/genética , Humanos , Ratones , Ratones Noqueados , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/patología , Regulación hacia Arriba/inmunología

RESUMEN

Interleukin 6 (IL-6), a pleiotropic cytokine, plays an important role in humoral immune response, not only inducing the differentiation of B cells into plasma cells, but also promoting antibody-secreting cells (ASCs) to produce antibodies. In this study, Nile tilapia (Oreochromis niloticus) IL-6 (OnIL-6) was identified and characterized at expression level in response to bacterial infection and promotion of antibody production. The open reading frame of OnIL-6 ORF is consisted of 663 bp encoding a polypeptide of 220 amino acids. The deduced OnIL-6 protein contained an IL-6/G-CSF family signature, two conserved cysteine, and four α-helix bundles, which was highly homologous to other species. Spatial mRNA expression analysis revealed that the highest expression of OnIL-6 was observed in the thymus. After in vivo challenges of lipopolysaccharide (LPS) and Streptococcus agalactia (S. agalactiae), OnIL-6 expressions were significantly up-regulated in head kidney and spleen. The similar up-regulation of OnIL-6 was observed in the head kidney and spleen leukocytes in vitro stimulation with LPS and S. agalactiae. In addition, inducement with the recombinant OnIL-6 ((r)OnIL-6) in vitro caused significant increases in expressions of both sIgM and mIgM. Moreover, the (r)OnIL-6 stimulation enhanced the secretion of sIgM (more especially in P50 plasma-like B cells) and the production of mIgM in P60 and P70 B cell subsets (resting B cells, activated B cells and plasmablast-like B cells) in vitro. Taken together, this study indicated that OnIL-6 might be involved in host defense against bacterial infection and promote the production of antibody in Nile tilapia.

Asunto(s)

Cíclidos/inmunología , Proteínas de Peces/inmunología , Interleucina-6/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/genética , Subgrupos de Linfocitos B/inmunología , Cíclidos/genética , Cíclidos/microbiología , Enfermedades de los Peces/inmunología , Proteínas de Peces/biosíntesis , Proteínas de Peces/genética , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/genética , Interleucina-6/química , Interleucina-6/genética , Modelos Moleculares , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/veterinaria , Streptococcus agalactiae/inmunología , Streptococcus agalactiae/patogenicidad

RESUMEN

Infection with Helicobacter pylori may result in the emergence of gastric adenocarcinoma. Among various toxins assisting pathogenesis of H. pylori, the vacuolating cytotoxin A (VacA) is one of the most potent toxins known as the major cause of the peptic ulcer and gastric adenocarcinoma. To isolate single-chain variable fragments (scFvs) against two conserved regions of VacA, we capitalized on the phage display technology and a solution-phase biopanning (SPB). Characterization of scFvs was carried out by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and surface plasmon resonance (SPR). Bioinformatics analyses were also performed in order to characterize the structural and functional properties of the isolated scFvs and the interaction(s) between the isolated antibodies (Ab)-antigen (Ag). After four rounds of biopanning, the positive colonies detected by scFv ELISA were harvested to extract the plasmids and perform sequencing. Of several colonies, three colonies showed high affinity to the VacA1 and two colonies for the VacA2. Further complementary examinations (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot, SPR, and flow cytometry) displayed the high affinity and specificity of the isolated scFvs to the VacA. Docking results revealed the interaction of the complementarity-determining regions (CDRs) with the VacA peptide. In conclusion, for the first time, we report on the isolation of several scFvs against conserved residues of VacA toxin with high affinity and specificity, which may be used as novel diagnostic/therapeutic tool in the H. pylori infection.

Asunto(s)

Anticuerpos Antibacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Anticuerpos Antibacterianos/genética , Western Blotting , Técnicas de Visualización de Superficie Celular , Secuencia Conservada/genética , Ensayo de Inmunoadsorción Enzimática , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/química , Helicobacter pylori/genética