RESUMO
We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete "hot spots" with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.
Assuntos
Anticorpos Monoclonais Murinos/química , Bacteriófago lambda/genética , Epitopos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Biblioteca de Peptídeos , Animais , CamundongosRESUMO
A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by ß-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to ß-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble ß-1,3-glucan, but not by pustulan, a ß-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Candida albicans/efeitos dos fármacos , Candidíase/prevenção & controle , Vacinas Fúngicas/imunologia , Peptídeos/imunologia , Vacinação , Sequência de Aminoácidos , Animais , Anticorpos Anti-Idiotípicos/biossíntese , Anticorpos Anti-Idiotípicos/química , Candida albicans/imunologia , Candidíase/imunologia , Candidíase/microbiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/imunologia , Vacinas Fúngicas/administração & dosagem , Vacinas Fúngicas/química , Hemocianinas/química , Fatores Matadores de Levedura/química , Fatores Matadores de Levedura/imunologia , Camundongos , Mimetismo Molecular , Dados de Sequência Molecular , Micotoxinas/química , Micotoxinas/imunologia , Biblioteca de Peptídeos , Peptídeos/administração & dosagem , Peptídeos/química , Pichia/química , Pichia/metabolismo , Ratos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/imunologia , Vacinas de DNA , Vacinas de Subunidades Antigênicas , beta-Glucanas/química , beta-Glucanas/imunologiaRESUMO
A structural model of human ferroportin has been built using two Escherichia coli proteins belonging to the major facilitator superfamily of transporters. A potential iron binding site was identified in the inward-open conformation of the model, and its relevance was tested through measurement of iron export of HEK293T cells expressing wild-type or mutated ferroportin. Aspartates 39 and 181 were found to be essential for the transport ability of the protein. Noteworthy, the D181V mutation is naturally found in type 4 hemochromatosis with reticuloendothelial system iron retention phenotype. The outward-open conformation of ferroportin was also predicted, and showed that significant conformational changes must occur in the inward- to outward-open transition of ferroportin. In particular, putative iron ligands move several ångströms away from each other, leading to the logical conclusion that the iron binding site is not occupied by the metal in the outward-open conformation of ferroportin.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Ferro/metabolismo , Modelos Moleculares , Sítios de Ligação , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Escherichia coli/química , Células HEK293 , Hemocromatose/genética , Humanos , Ligantes , Mutação , Conformação ProteicaRESUMO
Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of â¼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Epitopos/imunologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Neisseria meningitidis/patogenicidade , Fatores de Virulência/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Técnicas de Visualização da Superfície Celular , Cristalografia por Raios X , Medição da Troca de Deutério , Mapeamento de Epitopos , Epitopos/química , Espectrometria de Massas , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/prevenção & controle , Modelos Moleculares , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica/imunologia , Ressonância de Plasmônio de Superfície , Fatores de Virulência/químicaRESUMO
There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunoprotection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine.
Assuntos
Proteínas de Bactérias/imunologia , Biblioteca de Peptídeos , Infecções Pneumocócicas/prevenção & controle , Streptococcus pneumoniae/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Peptídeos/imunologia , Infecções Pneumocócicas/mortalidade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Sorotipagem , Streptococcus pneumoniae/classificaçãoRESUMO
Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The ability of this bacterium to adhere to epithelial cells is considered as an essential early step in colonization and infection. By screening a whole genome phage display library with sera from infected patients, we previously identified three antigenic fragments matching open reading frame spr0075 of the strain R6 genome. This locus encodes for an approximately 120-kDa protein, herein referred to as plasminogen- and fibronectin-binding protein B (PfbB), which displays an LPXTG cell wall anchoring motif and six repetitive domains. In this study, by using isogenic pfbB-deleted mutants of the encapsulated D39 and of the unencapsulated DP1004 type 2 pneumococcal strains, we show that PfbB is involved in S. pneumoniae adherence to various epithelial respiratory tract cell lines. Our data suggest that PfbB directly mediates bacterial adhesion, because fluorescent beads coated with the recombinant PfbB sp17 fragment (encompassing one of the six repetitive domains and the C-terminal region) efficiently bound to epithelial cells. Mutants lacking PfbB bound to fibronectin and plasminogen considerably less efficiently than wild type bacteria, whereas sp17-coated beads specifically bound to both of these substrates. Taken together, our data suggest that, by directly interacting with fibronectin, PfbB significantly increases the ability of S. pneumoniae to adhere to human epithelial cells.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Células Epiteliais/microbiologia , Fibronectinas/metabolismo , Plasminogênio/metabolismo , Streptococcus pneumoniae/metabolismo , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Epiteliais/citologia , Humanos , Camundongos , Microesferas , Dados de Sequência Molecular , Fases de Leitura Aberta , Infecções Pneumocócicas/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Brucella melitensis and Brucella abortus are responsible for brucellosis in bovine and ovine species and for Malta fever in humans. The lipopolysaccharide (LPS) of Brucella is an important virulence factor and can elicit protective antibodies. Because of their potential importance in vaccine design and in serological diagnosis, we developed peptides mimicking the antigenic properties of distinctive antigenic determinants of Brucella LPS. These peptides were selected from several phage display random peptide libraries for their ability to bind monoclonal antibodies directed against the A- or C-type epitopes of Brucella LPS. Plasmids encoding for two of the isolated peptides induced, after DNA immunization, LPS-specific antibody responses. Although these responses were only moderate in extent, these data further suggest the feasibility of using peptide mimics of carbohydrate epitopes as immunogens, a property which may be useful in the design of novel anti-Brucella vaccines.
Assuntos
Antígenos de Bactérias/imunologia , Brucella , Epitopos/imunologia , Lipopolissacarídeos/imunologia , Mimetismo Molecular , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/genética , Brucella/química , Brucella/imunologia , Vacina contra Brucelose/genética , Vacina contra Brucelose/imunologia , Bovinos , Epitopos/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/imunologia , OvinosRESUMO
Anti-polysaccharide immunity is a key facet of protection against several bacterial pathogens. Problems exist with current polysaccharide vaccines and alternative strategies that deliver a protective response are needed. We have identified immunological peptide mimics of type 6B and 9V pneumococcal capsular polysaccharides that could be used as vaccine antigens. Peptides mimicking antigenic properties of serotype 6B capsular polysaccharide were obtained from a phage-displayed peptide library expressing dodecameric peptides, using a human monoclonal antibody (Db3G9). A murine monoclonal antibody (206, F-5) against the serotype 9V capsular polysaccharide identified three peptide mimotopes from the dodecameric peptide library and one from a random pentadecameric peptide library. In ELISA, binding of 206, F-5 and Db3G9 to phage displaying the selected mimotopes was significantly inhibited by type-specific pneumococcal polysaccharide. Peptides were conjugated to keyhole limpet haemocyanin and were used to immunise mice. Two peptides, MP13 and MP7, induced specific anti-6B and 9V polysaccharide antibodies, respectively. Mice immunised with MP7-keyhole limpet hemocyanin or MP13-keyhole limpet hemocyanin conjugate were significantly and specifically protected against a lethal challenge with pneumococci of the appropriate serotype. This study provides strong in vivo evidence that peptide mimics are alternatives to polysaccharide vaccines.
Assuntos
Cápsulas Bacterianas/imunologia , Mimetismo Molecular/imunologia , Peptídeos/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Polissacarídeos Bacterianos/imunologia , Streptococcus pneumoniae/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo/imunologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/administração & dosagem , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/administração & dosagem , VacinaçãoRESUMO
The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.
Assuntos
Anticorpos , Mimetismo Molecular , Antígenos O , Peptídeos , Shigella flexneri/imunologia , Animais , Anticorpos/química , Anticorpos/imunologia , Sequência de Carboidratos , Cristalografia por Raios X , Camundongos , Conformação Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Antígenos O/química , Antígenos O/imunologia , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , TermodinâmicaRESUMO
BACKGROUND: The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. RESULTS: An antigenic sequence corresponding to amino acids 420-457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. CONCLUSION: The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Epitopos de Linfócito B/imunologia , Epitopos Imunodominantes/imunologia , Serina Endopeptidases/imunologia , Streptococcus pneumoniae/imunologia , Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos , Epitopos de Linfócito B/isolamento & purificação , Humanos , Epitopos Imunodominantes/isolamento & purificação , Imunoglobulina A/metabolismo , Biblioteca de Peptídeos , Infecções Pneumocócicas/imunologia , Streptococcus/enzimologia , Streptococcus pneumoniae/enzimologiaRESUMO
Neisseria meningitidis serogroup B (MenB) is a leading cause of sepsis and meningitis in children. No vaccine is available for the prevention of these infections because the group B capsular polysaccharide (CP) (MenB CP) is unable to stimulate an immune response, due to its similarity with human polysialic acid. Because the MenB CP bears both human cross-reactive and non-cross-reactive determinants, we developed immunogenic peptide mimics of the latter epitopes. Peptides were selected from phage display libraries for their ability to bind to a protective anti-MenB CP mAb. One of these peptides (designated 9M) induced marked elevations in serum bactericidal activity, but not polysialic acid cross-reacting Abs, after gene priming followed by carrier-conjugate boosting. Moreover, the occurrence of bacteremia was prevented in infant rats by administration of immune sera before MenB challenge. 9M is a promising lead candidate for the development of an effective and affordable anti-MenB vaccine.
Assuntos
Bacteriemia/prevenção & controle , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/imunologia , Mimetismo Molecular , Neisseria meningitidis Sorogrupo B , Peptídeos/imunologia , Polissacarídeos Bacterianos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Monoclonais/imunologia , Cápsulas Bacterianas , DNA/genética , Soros Imunes/imunologia , Imunização , Imunização Passiva , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/farmacologia , Plasmídeos/genética , Polissacarídeos Bacterianos/química , Ratos , Ratos Endogâmicos BBRESUMO
To improve tumor targeting in a subset of patients, where tumor cells do not express the well-known tumor antigens widely used in immunotherapy, we have developed a novel biotechnological tool. It is useful for tumors of various origins for the identification of tumor-associated proteins, which are differentially expressed in tumor cells with respect to normal tissue, and exposed on the cell surface. For this purpose, a combination of techniques, such as "suppression subtractive hybridization" and "transmembrane trapping," was employed. In applying this novel approach to breast cancer, we identified a large panel of cDNA fragments encoding for the well-known tumor-associated surface antigens, such as erb-B2, erbB3 and the urokinase receptor and, more importantly, for several clones overexpressed in breast cancer, whose cDNA fragments match the sequences of hypothetical transmembrane proteins with unknown function. The latter may represent novel tumor-specific targets.
Assuntos
Antígenos de Neoplasias/isolamento & purificação , Biotecnologia/métodos , Proteínas de Neoplasias/isolamento & purificação , Neoplasias/química , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/genética , Neoplasias da Mama/química , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Expressão Gênica , Biblioteca Gênica , Humanos , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/metabolismo , Hibridização de Ácido Nucleico/métodosRESUMO
OBJECTIVE: In this article we provide evidence of a significant spontaneous humoral response in cancer patients. METHODS: A panel of tumor-associated antigens, previously identified through serological screening of phage-displayed cDNA libraries from solid human tumors, breast carcinoma cell lines and human testis by employing breast cancer patient sera, was used in this study to survey sera from 182 patients with known disease histories and clinical stages. RESULTS: This analysis reveals a statistically significant association between tumor disease and presence in peripheral blood of IgG antibodies against four autoantigens. One of these antigens (D7-1) is particularly interesting in that the antibody response against it grows with cancer progression from stages I through IV, with an incidence of 13.2, 13.5, 18.2 and 27%, respectively. The significance of this stage-dependent increase in the incidence is confirmed by the Mantel-Haenszel Chi-squared test (P=0.001). CONCLUSIONS: Our data confirm association between breast cancer diagnosis of patients and presence in their peripheral blood of antibodies against several autoantigens identified by phage display.
Assuntos
Antígenos de Neoplasias/biossíntese , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Neoplasias/sangue , Adulto , Formação de Anticorpos , Antígenos de Neoplasias/metabolismo , Autoantígenos/química , Neoplasias da Mama/imunologia , Clonagem Molecular , DNA Complementar/metabolismo , Feminino , Humanos , Imunoglobulina G/química , Masculino , Pessoa de Meia-Idade , Biblioteca de Peptídeos , Plasmídeos/metabolismoRESUMO
BACKGROUND: CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. METHODS: The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. RESULTS: The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. CONCLUSION: The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance.
Assuntos
Antígeno Carcinoembrionário/química , Região Variável de Imunoglobulina/química , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Antineoplásicos/farmacologia , Bactérias/metabolismo , Biotinilação , Western Blotting , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , DNA/química , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Citometria de Fluxo , Biblioteca Gênica , Humanos , Fragmentos de Imunoglobulinas/química , Imuno-Histoquímica , Cinética , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese , Metástase Neoplásica , Biblioteca de Peptídeos , Estrutura Terciária de Proteína , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície , TransfecçãoRESUMO
In the present work we demonstrate the efficient display of functional scFv antibodies on the bacteriophage lambda capsid. A single-chain (scFv) anti-CEA antibody gene was cloned in two different vectors to obtain fusion of the scFv antibody to the N- or C-terminus of the bacteriophage lambda capsid protein D (gpD). Lambda bacteriophage assembly occurs in the reducing environment of the cytoplasm; despite this the lambda-displayed anti-CEA antibody fragments retain the capacity to recognize the antigen, indicating correct single-chain antibody folding. Efficient production of functional scFv exposed on lambda capsid with viable antigen binding specificity allowed us to study and compare the capacity of display, the stability of recombinant antibody expression and the assembly efficiency of bacteriophage particles decorated with recombinant antibody fused to the amino- or carboxy-terminus of lambda D protein.
Assuntos
Bacteriófago lambda/imunologia , Fragmentos de Imunoglobulinas/imunologia , Western Blotting , Proteínas do Capsídeo/imunologia , Antígeno Carcinoembrionário/imunologia , Cromatografia de Afinidade , Humanos , Região Variável de Imunoglobulina/imunologia , Oligopeptídeos , Biblioteca de Peptídeos , Peptídeos/imunologia , Proteínas Recombinantes/imunologiaRESUMO
Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.
Assuntos
Marcadores de Afinidade/química , Anticorpos Monoclonais/imunologia , Bacteriófagos/química , Peptídeos/química , Tenascina/imunologia , Marcadores de Afinidade/isolamento & purificação , Sequência de Aminoácidos , Anticorpos Monoclonais/isolamento & purificação , Western Blotting , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Dados de Sequência Molecular , Peptídeos/imunologia , Peptídeos/isolamento & purificação , Ressonância de Plasmônio de SuperfícieRESUMO
Vibrio cholerae is the etiological agent of cholera. V. cholerae serogroup O1 had been, until 1992, the only serogroup responsible for large epidemics and pandemics of cholera. In 1992, a new serotype of V. cholerae emerged in South-East Asia that caused a massive outbreak of cholera in India and neighboring countries. The new serotype was named V. cholerae O139. The main differences between V. cholerae O139 and O1 are that the former possesses a capsular polysaccharide and different lipopolysaccharide. Capsular polysaccharides are, in general, T-independent antigens giving rise to poor immune responses lacking immunological memory. In order to overcome this, monoclonal antibodies against the capsular polysaccharide of V. cholerae O139 were used to screen different phage-displayed random peptide libraries. Eight different phage clones were selected and characterized using enzyme immunoassay with the monoclonal antibodies, and then tested for specificity by competition with V. cholerae O139 capsular polysaccharide. Selected peptides were sequenced, synthesized and conjugated to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). The conjugated peptides were used to immunize mice. It is evident that the anti-peptide mouse antibodies bind to the V. cholerae O139 capsular polysaccharide. In addition, the anti-peptide antibodies are protective in a suckling mouse model. The protective efficacy is both specific and dose-dependent. A PCT (PCT/IT2003/000489) with the publication number WO 2004/056851 has been filed for the sequences of the eight peptides.
Assuntos
Cápsulas Bacterianas/imunologia , Cólera/prevenção & controle , Mimetismo Molecular , Peptídeos/administração & dosagem , Peptídeos/imunologia , Vibrio cholerae O139/imunologia , Animais , Anticorpos Antibacterianos/sangue , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Hemocianinas/química , Hemocianinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Biblioteca de Peptídeos , Soroalbumina Bovina/química , Soroalbumina Bovina/imunologiaRESUMO
Infection with Toxoplasma gondii causes morbidity and mortality in congenitally infected and immunocompromised individuals. Both humoral and cell-mediated immunity are involved in host resistance to invasion of the parasite. Among putative vaccine candidates, the T. gondii microneme proteins appear to be promising, because they are responsible for the invasion process. The present work focused on studying the immunogenicity of microneme proteins in infected individuals and in a mouse model of chronic toxoplasmosis. We identified 5 distinct antigenic regions within MIC2, MIC4, MIC2-associated protein, and apical membrane antigen 1 gene products, which were recognized by (1) T cells from both adults with acquired infection and children with congenital infection and (2) antibodies from all patients. Finally, we demonstrated that DNA immunization with microneme fragments elicited effective protection in mice (84% reduction in brain-cyst burden), suggesting that a combination of these antigenic regions should be considered in the design of potential vaccines against toxoplasmosis.
Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Toxoplasmose/prevenção & controle , Vacinas de DNA/imunologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Encéfalo/parasitologia , Moléculas de Adesão Celular/imunologia , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/sangue , Lactente , Ativação Linfocitária , Masculino , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Complicações Parasitárias na Gravidez/imunologia , Proteínas de Protozoários/imunologia , Linfócitos T/imunologia , Toxoplasmose Congênita/imunologiaRESUMO
BACKGROUND: Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy. Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX). METHODS: Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed. The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D. Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer. RESULTS: A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified. Three of these antigens (T7-1, T11-3 and T11-9) were found to be overexpressed in tumors as compared to normal breast. A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones (T6-2, T6-7, T7-1, T9-21 and T9-27). CONCLUSIONS: Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease. The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis.
Assuntos
Antígenos Glicosídicos Associados a Tumores/genética , Bacteriófago lambda/genética , Neoplasias da Mama/genética , Biblioteca Gênica , Neoplasias/genética , Testículo/química , Testículo/metabolismo , Sequência de Aminoácidos , Antígenos Glicosídicos Associados a Tumores/sangue , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/imunologia , Neoplasias da Mama/sangue , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Vetores Genéticos/genética , Humanos , Masculino , Análise de Sequência de DNA/métodos , Testes Sorológicos/métodosRESUMO
Synthetic peptides containing a phosphorylation site for protein kinase CK2 were used to investigate their binding properties to other peptides/proteins. The aim of this work was to find an efficient procedure to search for these peptide/protein ligands. The goal was successfully achieved through screening of random peptide libraries displayed on phage. Peptides corresponding to the amino terminal region of topoisomerase I were synthesized in both phosphorylated and unphosphorylated form and used to screen the libraries. Four of the selected sequences were also tested for their reactivity with synthetic peptides corresponding to the carboxy terminal region of the largest subunit of RNA polymerase II. The positive reaction detected supports the hypothesis that the isolated sequences may represent mimics of ligands of proteins phosphorylated by protein kinase CK2.