RESUMEN
The neutralization of tumor necrosis factor alpha (TNFα) with biopharmaceuticals is a successful therapy for inflammatory diseases. Currently, one of the main TNFα-antagonists is Etanercept, a dimeric TNF-R2 ectodomain. Considering that TNFα and its receptors are homotrimers, we proposed that a trimeric TNF-R2 ectodomain could be an innovative TNFα-antagonist. Here, the 3cTNFR2 protein was designed by the fusion of the TNF-R2 ectodomain with the collagen XV trimerization domain. 3cTNFR2 was produced in HEK293 cells and purified by immobilized metal affinity chromatography. Monomers, dimers, and trimers of 3cTNFR2 were detected. The interaction 3cTNFR2-TNFα was assessed. By microscale thermophoresis, the KD value for the interaction was 4.17 ± 0.88 nM, and complexes with different molecular weights were detected by size exclusion chromatography-high performance liquid chromatography. Moreover, 3cTNFR2 neutralized the TNFα-induced cytotoxicity totally in vitro. Although more studies are required to evaluate the anti-inflammatory effect, the results suggest that 3cTNFR2 could be a TNFα-antagonist agent.
Asunto(s)
Antiinflamatorios/farmacología , Colágeno/genética , Endotoxinas/antagonistas & inhibidores , Etanercept/farmacología , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Supervivencia Celular/efectos de los fármacos , Colágeno/metabolismo , Endotoxinas/metabolismo , Endotoxinas/toxicidad , Etanercept/química , Etanercept/metabolismo , Expresión Génica , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Ingeniería de Proteínas/métodos , Multimerización de Proteína , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS-PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (<10-9â M and >10-8â M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.
Asunto(s)
Adalimumab/química , Etanercept/química , Factor de Necrosis Tumoral alfa , Humanos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/químicaRESUMEN
Antimicrobial peptides (AMPs) are amphipathic peptides, which play an important role in innate defence. These peptides are gene-encoded and either constitutively expressed and/or upregulated during an infection. NK-lysins are AMPs with a three-dimensional globular structure. They are larger molecules, which comprise 74-78 amino acid residues and six conserved cysteine residues forming three disulphide bonds. Cathelicidins are a family of antimicrobial peptides that act as important components of the innate immune system with a broad spectrum of antimicrobial activity and immunomodulatory properties. Although they are widely studied in mammals, little is known about their immunomodulatory function. In the present study, we identified and characterized for the first time four NK-lysin-like transcripts from Atlantic salmon (Salmo salar) based on EST reported sequences. In vitro, NK-lysin derived peptides were able to induce the expression of IL-1ß and IL-8 in Salmo salar head kidney leukocytes. We also tested Salmo salar cathelicidin 1 derived peptide in a similar assay, showing its ability to induce the expression of IFN-γ. These results indicate that NK-lysin and cathelicidin 1 derived peptides are able to modulated immune response, suggesting their potential use to enhance immune response in fish.
Asunto(s)
Catelicidinas/genética , Proteínas de Peces/inmunología , Factores Inmunológicos/inmunología , Proteolípidos/genética , Salmo salar/inmunología , Animales , Catelicidinas/inmunología , Enfermedades de los Peces/inmunología , Riñón Cefálico/citología , Riñón Cefálico/inmunología , Inmunidad Innata , Interferón gamma/inmunología , Leucocitos/inmunología , Proteolípidos/inmunologíaRESUMEN
A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10(7) M(-1). The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.
Asunto(s)
Anticuerpos Monoclonales/química , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/química , Neisseria meningitidis/metabolismo , Polisacáridos/química , Animales , Calibración , Ensayo de Inmunoadsorción Enzimática , Límite de Detección , Infecciones Meningocócicas/microbiología , Ratones , Ratones Endogámicos BALB C , Serogrupo , Especificidad de la EspecieRESUMEN
Background: Gout, the most common inflammatory arthritis disease, and hyperuricaemia onset are influenced by environmental and genetic factors. We sought to investigate these factors in an Indigenous community in Guam. Methods: In this cross-sectional study, the University of Guam led the qualitative inquiry with the native community, training (pre-screening of participants, data collection methods, and biospecimen handling), study implementation (outreach and recruitment, data collection, and DNA extraction and quantification), and qualitative and epidemiologic data analyses. Recruitment targets were based on demographic representation in current census data. The University of Otago collaborated on ethics guidance, working with Indigenous communities, and led the genetic sequencing and genetic data analysis. Participants were recruited in Guam from Fall 2019 to Spring 2022. Results: Of the 359 participants, most self-identified as Native CHamorus (61.6%) followed by Other Micronesians (22.0%), and Filipinos (15.6%). The prevalence of metabolic conditions from highest to lowest were obesity (55.6%), hyperuricaemia (36.0%), hypertension (27.8%), gout (23.0%), diabetes (14.9%), cardiovascular disease (8.4%), kidney disease (7.3%), and liver disease (3.4%). Compared to Filipinos and Other Micronesians, significantly more CHamorus had hyperuricaemia (42.1% versus 26.8% in Filipinos and 25.3% in Other Micronesians), gout (28.5% versus 21.4% and 8.9%), diabetes (19.5% versus 8.9% and 6.3%), and hypertension (33.9% versus 19.6% and 16.5%). Conclusions: We estimated the prevalence of metabolic conditions, especially gout and hyperuricaemia, and found statistical differences among major ethnic groups in Guam, all while obtaining the Indigenous community's feedback on the genetic study and building gout research capacity. The results of ongoing genetic sequencing will be used to understand molecular causes of gout in Guam.
RESUMEN
The development of molecules specific for M. tuberculosis-infected cells has important implications, as these tools may facilitate understanding of the mechanisms regulating host pathogen interactions in vivo. In addition, development of new tools capable to targeting M. tuberculosis-infected cells may have potential applications to diagnosis, treatment, and prevention of tuberculosis (TB). Due to the lack of CD1b polymorphism, M. tuberculosis lipid-CD1b complexes could be considered as universal tuberculosis infection markers. The aim of the present study was to display on the PIII surface protein of m13 phage, a human αß single-chain T-cell receptor molecule specific for CD1b:2-stearoyl-3-hydroxyphthioceranoyl-2´-sulfate-α-α´-D-trehalose (Ac2SGL) which is a complex presented by human cells infected with M. tuberculosis. The results showed the pIII fusion particle was successfully displayed on the phage surface. The study of the recognition of the recombinant phage in ELISA and immunohistochemistry showed the recognition of CD1b:Ac2SGL complexes and cells in human lung tissue from a tuberculosis patient respectively, suggesting the specific recognition of the lipid-CD1b complex.
Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos CD1/inmunología , Técnicas de Visualización de Superficie Celular , Glucolípidos/inmunología , Mycobacterium tuberculosis/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Tuberculosis/inmunología , Bacteriófago M13 , Línea Celular , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Humanos , Pulmón/inmunología , Pulmón/microbiología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas ViralesRESUMEN
BACKGROUND: Immunoglobulin A is the most abundant isotype in secretions from mucosal surfaces of the gastrointestinal, respiratory and genitourinary tracts and in external secretions such as colostrum, breast milk, tears and saliva. The high concentration of human secretory IgA (hsIgA) in human colostrum strongly suggests that it should play an important role in the passive immune protection against gastrointestinal and respiratory infections. MATERIALS AND METHODS: Human secretory IgA was purified from colostrum. The reactivity of hsIgA against mycobacterial antigens and its protective capacity against mycobacterial infection was evaluated. RESULTS: The passive administration of hsIgA reduces the pneumonic area before challenge with M. tuberculosis. The intratracheal administration of M. tuberculosis preincubated with hsIgA to mice greatly reduced the bacterial load in the lungs and diminished lung tissue injury. CONCLUSIONS: HsIgA purified from colostrum protects against M. tuberculosis infection in an experimental mouse model.
Asunto(s)
Calostro/inmunología , Inmunización Pasiva , Inmunoglobulina A Secretora/administración & dosificación , Inmunoglobulina A Secretora/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/prevención & control , Animales , Antígenos Bacterianos/inmunología , Carga Bacteriana , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/fisiología , Tuberculosis Pulmonar/inmunologíaRESUMEN
Murine hybridoma monoclonal antibodies (MAbs) were produced against the capsular polysaccharide (CPs) of serogroups A, C, W135 and Y meningococci (MenA, MenC, MenW, MenY) in order to develop immunological reagents for the identification of meningococcal polysaccharides. Each serogroup-specific MAb reacted with the CPs from its homologous serogroup only and did not react with CPs from the other three serogroups. The affinity constant (Ka) of the four MAbs measured by non-competitive ELISA was 6.62 × 10(9), 2.76 × 10(9), 1.48 × 10(9) and 3.8 × 10(9) M(-1) for MenA, MenC, MenW and MenY MAbs respectively. The application of these MAbs for identity tests was demonstrated by their abilities to correctly identify the CPs from serogroups A, C, W135 and Y in meningococcal CPs-based vaccines through ELISA. The MAbs obtained in this work are a very valuable set of tools for study meningococcal polysaccharides vaccines.
Asunto(s)
Anticuerpos Antibacterianos , Anticuerpos Monoclonales de Origen Murino , Cápsulas Bacterianas , Neisseria meningitidis Serogrupo A , Neisseria meningitidis Serogrupo C , Neisseria meningitidis Serogrupo W-135 , Neisseria meningitidis Serogrupo Y , Polisacáridos Bacterianos , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Monoclonales de Origen Murino/inmunología , Especificidad de Anticuerpos , Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Ratones , Neisseria meningitidis Serogrupo A/química , Neisseria meningitidis Serogrupo A/inmunología , Neisseria meningitidis Serogrupo C/química , Neisseria meningitidis Serogrupo C/inmunología , Neisseria meningitidis Serogrupo W-135/química , Neisseria meningitidis Serogrupo W-135/inmunología , Neisseria meningitidis Serogrupo Y/química , Neisseria meningitidis Serogrupo Y/inmunología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunologíaRESUMEN
BACKGROUND: A monoclonal antibody (mAb) of the IgA isotype, designated TBA61, is specific for the Acr protein of Mycobacterium tuberculosis (MTB). TBA61 has been used in studies exploring protection against tuberculosis (TB), and its efficacy has been proven using different challenge models. To purify the mouse IgA isotype, a combination of methods, such as globulin precipitation, ion exchange, and gel filtration, is usually required to achieve a satisfactory degree of purity. METHODS: To minimise the number of chromatographic steps, we proposed to employ immunoaffinity chromatography using the Acr protein of MTB as a specific ligand for this mAb. For this purpose, the HspX gene was cloned and expressed in Escherichia coli, and recombinant Acr (rAcr) was coupled to a cyanogen bromide-activated Sepharose 4B matrix, which was used to purify TBA61 mAb from ascites produced in mice in a single step. RESULTS: The recovery from the purification procedure was 1.46 mg per mL of ascites. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot showed a high purity. The purified mAb retained its reactivity against the Acr protein based on enzyme-linked immunosorbent assay (ELISA) and western blot. CONCLUSION: The purification method used is rapid, simple, and specific and can be easily scaled up.
RESUMEN
Micronesia began to be peopled earlier than other parts of Remote Oceania, but the origins of its inhabitants remain unclear. We generated genome-wide data from 164 ancient and 112 modern individuals. Analysis reveals five migratory streams into Micronesia. Three are East Asian related, one is Polynesian, and a fifth is a Papuan source related to mainland New Guineans that is different from the New Britain-related Papuan source for southwest Pacific populations but is similarly derived from male migrants ~2500 to 2000 years ago. People of the Mariana Archipelago may derive all of their precolonial ancestry from East Asian sources, making them the only Remote Oceanians without Papuan ancestry. Female-inherited mitochondrial DNA was highly differentiated across early Remote Oceanian communities but homogeneous within, implying matrilocal practices whereby women almost never raised their children in communities different from the ones in which they grew up.
Asunto(s)
ADN Antiguo , ADN Mitocondrial , Migración Humana , Pueblo Asiatico/genética , Niño , ADN Mitocondrial/genética , Femenino , Historia Antigua , Migración Humana/historia , Humanos , Masculino , Micronesia , OceaníaRESUMEN
Complex recombinant glycoproteins produced as potential biopharmaceuticals in goat's milk have an aberrant pattern of N-glycosylation due to the lack of multi-antennary structures. Overexpression of glycosyltransferases may increase oligosaccharide branching of the desired glycoproteins. Here, human erythropoietin fused to human IgG Fc (EPO-Fc) was co-expressed with N-acetyl-glucosaminyltransferase-IVa (GnT-IVa) by adenoviral transduction in goat mammary gland to evaluate the in vivo modification of N-glycosylation pattern in this tissue. Adenoviral vectors, containing the EPO-Fc and GnT-IVa sequences were assembled for in vitro and in vivo expression in mammalian cell culture or in goat mammary gland. Protein detection was assessed by gel electrophoresis and western blot, and N-glycans were identified by HPLC and mass spectrometry. GnT-IVa overexpression and its colocalization with EPO-Fc in the Golgi apparatus of SiHa cells were demonstrated. N-glycan analysis of in vitro and in vivo expression of EPO-Fc modified by GnT-IVa (EPO-Fc/GnT-IVa) showed an increase in high molecular weight structures, which corresponded to tri- and tetra-antennary N-glycans in SiHa cells and mostly tri-antennary N-glycans in goat's milk from transformed mammary tissue. The results confirmed that successful modification of the goat mammary gland secretion pathway could be achieved by co-expressing glycoenzymes together with the glycoprotein of interest. This is the first report of modification of the N-glycosylation pattern in the goat mammary gland in vivo, and constitutes a step forward for improving the use of the mammary gland as a bioreactor for the production of complex recombinant proteins.
Asunto(s)
Glicoproteínas/metabolismo , Glándulas Mamarias Animales/metabolismo , Animales , Células Cultivadas , Eritropoyetina , Femenino , Glicosilación , Cabras , Humanos , N-Acetilglucosaminiltransferasas , Transducción GenéticaRESUMEN
Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, we developed a new strategy based on the sequential insertion of transgenes for generating stable clones producing high levels of a chimeric human follicle-stimulating hormone (hscFSH). Gene insertion was done by transducing HEK-293 cells with a lentiviral vector containing a bicistronic transcriptional unit for expressing hscFSH and GFP genes. Clone selection was performed by flow cytometry coupled to cell sorting, and the GFP gene was further removed by CRE-mediated site-specific recombination. High-producing clones of hscFSH were obtained after three rounds of lentiviral transduction. Expression levels increased in a step-wise manner from 7 to 23 pg/cell/day, with a relatively constant rate of 7 pg/cell/day in each round of transduction. The GFP gene was successfully removed from the cell genome without disturbing the hscFSH gene expression. Clones generated using this approach showed stable expression levels for more than two years. This is the first report describing the sequential insertion of transgenes as an alternative for increasing the expression levels of transformed cell lines. The methodology described here could notably impact on biotechnological industry by improving the capacity of mammalian cells to produce biopharmaceuticals.
Asunto(s)
Hormona Folículo Estimulante/biosíntesis , Mutagénesis Insercional/métodos , Transgenes/genética , Biotecnología/métodos , Células Clonales , Citometría de Flujo/métodos , Hormona Folículo Estimulante/genética , Expresión Génica , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Lentivirus/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transducción GenéticaRESUMEN
Lipids, glycolipids and lipopeptides derived from Mycobacterium tuberculosis (Mtb) are presented to T cells by monomorphic molecules known as CD1. This is the case of the Mtb-specific sulfoglycolipid Ac2SGL, which is presented by CD1b molecules and is recognized by T cells found in tuberculosis (TB) patients and in individuals with latent infections. Our group, using filamentous phage display technology, obtained two specific ligands against the CD1b-Ac2SGL complex: (i) a single chain T cell receptor (scTCR) from a human T cell clone recognizing the CD1b-AcSGL complex; and (ii) a light chain domain antibody (dAbκ11). Both ligands showed lower reactivity to a synthetic analog of Ac2SGL (SGL12), having a shorter acyl chain as compared to the natural antigen. Here we put forward the hypothesis that the CD1b endogenous spacer lipid (EnSpacer) plays an important role in the recognition of the CD1b-Ac2SGL complex by specific T cells. To support this hypothesis we combined: (a) molecular binding assays for both the scTCR and the dAbκ11 antibody domain against a small panel of synthetic Ac2SGL analogs having different acyl chains, (b) molecular modeling of the CD1b-Ac2SGL/EnSpacer complex, and (c) modeling of the interactions of this complex with the scTCR. Our results contribute to understand the mechanisms of lipid presentation by CD1b molecules and their interactions with T-cell receptors and other specific ligands, which may help to develop specific tools targeting Mtb infected cells for therapeutic and diagnostic applications.
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Antígenos Bacterianos/inmunología , Antígenos CD1/inmunología , Modelos Moleculares , Mycobacterium tuberculosis/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Antígenos CD1/genética , Humanos , Proteínas Recombinantes/inmunologíaRESUMEN
Vaccination has reduced morbidity and mortality of many diseases that previously caused devastating epidemics and deaths globally. Vaccines as a biological product may contain microorganisms or their derivatives. This aspect together with the fact that they are administered to healthy individuals (mainly children) means that approximately 70% of vaccines development time is dedicated to quality control. Monoclonal antibodies (MAbs) have become essential analytical tools for application in ELISAs, Western and Dot blotting, immunoprecipitation, and flow cytometric assays that ensure the quality control of vaccines. The aim of this work is to present a review of the methods used to obtain a platform of MAbs against Neisseria meningitidis polysaccharide antigens to use as an analytical tool for quality control of anti-meningococcal polysaccharide (Ps) vaccines. The MAbs obtained are used in five sandwich ELISAs developed for Ps quantification. The assays showed good reproducibility and repeatability, with quantitation and detection limits below 1 ng/mL. Dot Blot, as the Identity test of the Ps vaccine, was carried out to positively identify licensed and experimental vaccines. All assays described are suitable for the screening of multiple vaccine samples and could be useful for monitoring lot-to-lot consistency and stability.
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Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Infecciones Meningocócicas/inmunología , Vacunas Meningococicas/normas , Neisseria meningitidis/inmunología , Polisacáridos Bacterianos/inmunología , Control de Calidad , Humanos , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/administración & dosificación , Polisacáridos Bacterianos/clasificaciónRESUMEN
Tuberculosis (TB) is the main cause of mortality among all infectious diseases. The presentation of lipids by CD1b molecules and the interactions of the CD1b-lipid complexes with the immune receptors are important for the understanding of the immune response to Mycobacterium tuberculosis (Mtb), and to develop TB control methods. A specific domain antibody (dAbk11) recognizing the complex of CD1b with Mtb sulphoglycolipid (Ac2SGL) had been previously developed. In order to study the interactions of dAbk11 with Ac2SGL:CD1b, the conformation of Ac2SGL within CD1b was first modelled. The orientation of dAbκ11 with Ac2SGL:CD1b was then predicted by a docking experiment and the complex was sampled using molecular dynamics simulation. Data showed that dAbκ11 Tyr32 OH plays a decisive role in interacting with Ac2SGL alkyl tail HO17. The binding free energy calculation showed that Ac2SGL establish strong hydrophobic interactions with dAbκ11. The model also predicted a higher affinity for the natural sulfoglycolipid (Ac2SGL) than the synthetic analogue (SGL12), which was supported by the ELISA data. These results shed light on the likely mechanism of interactions between Ac2SGL:CD1b and dAbκ11, thus making possible to envision the strategies for dAbκ11 optimization for possible future applications.
Asunto(s)
Antígenos CD1/inmunología , Tuberculosis/inmunología , Anticuerpos Antibacterianos/inmunología , Presentación de Antígeno/inmunología , Glucolípidos/metabolismo , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular/métodos , Mycobacterium tuberculosis/inmunologíaRESUMEN
A phage-displayed peptide approach was used to identify ligands mimicking antigenic determinants of hepatitis A virus (HAV) for the first time. Bacteriophages displaying HAV mimotopes were isolated from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Selected phage-peptides were screened for reactivity with sera from HAV infected patients and healthy controls. Four cloned peptides with different sequences were identified as mimotopes of HAV; three of them showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV. One clone was recognised by 92% of the positive sera. The phagotopes competed effectively with HAV for absorption of anti-HAV-specific antibodies in human sera, as determined by ELISA. The four phage clones induced neutralising anti-HAV antibodies in immunised mice. These results demonstrate the potential of this method to elucidate the disease related epitopes of HAV and to use these mimotopes in diagnostic applications or in the development of a mimotope-based hepatitis A vaccine without the necessity of manipulation of the virus.
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Bacteriófagos/inmunología , Antígenos de Hepatitis A/inmunología , Virus de la Hepatitis A Humana/inmunología , Imitación Molecular/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Bacteriófagos/genética , Ensayo de Inmunoadsorción Enzimática , Antígenos de Hepatitis A/química , Virus de la Hepatitis A Humana/genética , Humanos , Sueros Inmunes/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de Neutralización , Péptidos/inmunología , Péptidos/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
OBJECTIVE/BACKGROUND: The development of new tools capable of targeting Mycobacterium tuberculosis (Mtb)-infected cells have potential applications in diagnosis, treatment, and prevention of tuberculosis. In Mtb-infected cells, CD1b molecules present Mtb lipids to the immune system (Mtb lipid-CD1b complexes). Because of the lack of CD1b polymorphism, specific Mtb lipid-CD1b complexes could be considered as universal Mtb infection markers. 2-Stearoyl-3-hydroxyphthioceranoyl-2'-sulfate-α-α'-d-trehalose (Ac2SGL) is specific for Mtb, and is not present in other mycobacterial species. The CD1b-Ac2SGL complexes are expressed on the surface of human cells infected with Mtb. The aim of this study was to generate ligands capable of binding these CD1b-Ac2SGL complexes. METHODS: A synthetic human scFv phage antibody library was used to select phage-displayed antibody fragments that recognized CD1b-Ac2SGL using CD1b-transfected THP-1 cells loaded with Ac2SGL. RESULTS: One clone, D11-a single, light-variable domain (kappa) antibody (dAbκ11)-showed high relative binding to the Ac2SGL-CD1b complex. CONCLUSION: A ligand recognizing the Ac2SGL-CD1b complex was obtained, which is a potential candidate to be further tested for diagnostic and therapeutic applications.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Antígenos CD1/inmunología , Glucolípidos/inmunología , Mycobacterium tuberculosis/inmunología , Anticuerpos de Cadena Única/genética , Tuberculosis/inmunología , Anticuerpos Antibacterianos/genética , Antígenos CD1/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Expresión Génica , Humanos , Mycobacterium tuberculosis/genética , Anticuerpos de Cadena Única/inmunología , Tuberculosis/microbiologíaRESUMEN
Tuberculosis (TB) remains an important cause of mortality and morbidity. The TB vaccine, BCG, is not fully protective against the adult form of the disease and is unable to prevent its transmission although it is still useful against severe childhood TB. Hence, the search for new vaccines is of great interest. In a previous study, we have shown that proteoliposomes obtained from Mycobacterium smegmatis (PLMs) induced cross reactive humoral and cellular response against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLMs, a murine model of progressive pulmonary TB was used. Animals immunized with PLMs with and without alum (PLMs/PLMsAL respectively) showed protection compared to non-immunized animals. Mice immunized with PLMsAL induced similar protection as that of BCG. Animals immunized with BCG, PLMs and PLMsAL showed a significant decrease in tissue damage (percentage of pneumonic area/lung) compared to non-immunized animals, with a more prominent effect in BCG vaccinated mice. The protective effect of the administration of PLMs in mice supports its future evaluation as experimental vaccine candidate against Mtb.
Asunto(s)
Mycobacterium smegmatis/inmunología , Proteolípidos/inmunología , Vacunas contra la Tuberculosis , Tuberculosis Pulmonar/prevención & control , Adyuvantes Inmunológicos , Compuestos de Alumbre , Animales , Vacuna BCG , Carga Bacteriana , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Masculino , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/aislamiento & purificación , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/patología , Neumonía Bacteriana/prevención & control , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patologíaRESUMEN
Tuberculosis (TB) is one of the most important causes of mortality and morbidity due to infectious diseases. BCG, the vaccine in use, is not fully protective against TB. In a previous study, we have shown that proteoliposomes (outer membrane extracts), obtained from BCG (PLBCG) were able to induce humoral immune responses against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLBCG alone or as a booster with BCG, a murine model of progressive pulmonary TB was used. Animals immunized with PLBCG adjuvanted with alum (PLBCG-Al) showed similar protection to that conferred by BCG. The group immunized with PLBCG-Al as a booster to BCG gave superior protection than BCG as evidenced by a reduction of bacterial load in lungs 2 months after infection with Mtb. Animals immunized with BCG, PLBCG-Al and this formulation as a booster of BCG, showed a significant decrease of tissue damage (percentage of pneumonic area/lung) compared with non-immunized animals. These results demonstrate that immunization with PLBCG-Al alone or as a booster to BCG induce appropriate protection against challenge with Mtb in mice and support the future evaluation of PLBCG as a promising vaccine candidate against Mtb.