RESUMO
The NK-lysin antimicrobial peptide, first identified in mammals, possesses both antibacterial and cytotoxic activity against cancer cell lines. Homologue peptides isolated from different fish species have been examined for their functional characteristics in the last few years. In this study, a NK-lysin transcript was identified in silico from the head kidney transcriptome of the Antarctic teleost Trematomus bernacchii. The corresponding amino acid sequence, slightly longer than NK-lysins of other fish species, contains six cysteine residues that in mammalian counterparts form three disulphide bridges. Real time-PCR analysis indicated its predominant expression in T. bernacchii immune-related organs and tissues, with greatest mRNA abundance detected in gills and spleen. Instead of focusing on the full T. bernacchii derived NK-lysin mature molecule, we selected a 27 amino acid residue peptide (named NKL-WT), corresponding to the potent antibiotic NK-2 sequence found in human NK-lysin. Moreover, we designed a mutant peptide (named NKL-MUT) in which two alanine residues substitute the two cysteines found in the NKL-WT. The two peptides were obtained by solid phase organic synthesis to investigate their functional features. NKL-WT and NKL-MUT displayed antibacterial activity against the human pathogenic bacterium Enterococcus faecalis and the ESKAPE pathogen Acinetobacter baumannii, respectively. Moreover, at the determined Minimum Inhibitory Concentration and Minimum Bactericidal Concentration values against these pathogens, both peptides showed high selectivity as they did not exhibit any haemolytic activity on erythrocytes or cytotoxic activity against mammalian primary cell lines. Finally, the NKL-MUT selectively triggers the killing of the melanoma cell line B16F10 by means of a pro-apoptotic pathway at a concentration range in which no effects were found in normal mammalian cell lines. In conclusion, the two peptides could be considered as promising candidates in the fight against antibiotic resistance and tumour proliferative action, and also be used as innovative adjuvants, either to decrease chemotherapy side effects or to enhance anticancer drug activity.
Assuntos
Proteínas de Peixes , Perciformes , Humanos , Animais , Regiões Antárticas , Proteínas de Peixes/genética , Proteínas de Peixes/química , Peptídeos , Antibacterianos/farmacologia , Perciformes/genética , Perciformes/metabolismo , Proteolipídeos/genética , Proteolipídeos/química , Peixes/metabolismo , Mamíferos/metabolismoRESUMO
The increased incidence of invasive candidiasis and of patients at risk requires early diagnosis and treatment to improve prognosis and survival. The aim of this study was to set up a ten-protein array-based immunoassay to assess the IgG antibody responses against ten well-known immunogenic C. albicans proteins (Bgl2, Eno1, Pgk1, Pdc11, Fba1, Adh1, Als3, Hwp1, Hsp90 and Grp2) in 51 patients with invasive candidiasis (IC) and in 38 culture-negative controls (non-IC). Antibody levels were higher against Bgl2, Eno1, Pgk1, Als3, Hwp1 and Grp2, than against Adh1, Pdc11, Fba1 and Hsp90, irrespectively of the patient group considered. Moreover, the IgG levels against Bgl2, Eno1, Pgk1 and Grp2 were significantly higher in IC than in non-IC patients. Furthermore, the ROC curves generated by the analysis of the antibody responses against Bgl2, Grp2 and Pgk1 displayed AUC values above 0.7, thus discriminating IC and non-IC patients. According to these results, the employment of the microarray immunoassay (a rapid, sensitive and multiparametric system), in parallel with conventional diagnostics, can help to spot IC patients. This ultimately will allow to initiate an early, focused and optimized antifungal therapy.
Assuntos
Anticorpos Antifúngicos/sangue , Candidíase Invasiva/diagnóstico , Análise Serial de Proteínas/métodos , Proteínas Fúngicas/imunologia , Humanos , Imunoensaio , Imunoglobulina G/sangue , Proteínas Recombinantes/imunologiaRESUMO
Candida auris is a multidrug-resistant fungus known to be a global public health problem. The skin-based transmission, together with the marked resistance to drugs, resulted in its rapid spread to all continents. The aim of this study was to identify an essential oil (EO) active in the fight against C. auris. A total of 15 EOs were tested against 10 clinical strains of C. auris. Cinnamomum zeylanicum EO (CZ-EO) was the most effective (MIC90 and MFC90 equal to 0.06% vol/vol). Three fractions obtained from CZ-EO, and the cinnamaldehyde (CIN), the major chemical compound, were tested to identify the principal compound effectives against C. auris. All CIN-containing samples showed anti-fungal activity. To study the synergy with fluconazole, CZ-EO, its active fraction (FR2), and CIN were tested in checkerboard tests. Results show that CZ-EO and FR2, but not CIN, synergize with fluconazole. Furthermore, only the copresence of CZ-EO or FR2 synergize with fluconazole at therapeutic concentrations of the drug (0.45 ± 0.32 µg/mL and 0.64 ± 0.67 µg/mL, respectively), while CIN only shows additive activity. In vivo studies conducted on Galleria mellonella larvae show the absence of toxicity of CZ-EO up to concentrations of 16% vol/vol, and the ability of CZ-EO to reactivate the efficacy of fluconazole when formulated at synergic concentrations. Finally, biochemical tests were made to study the mechanism of action of CZ-EO. These studies show that in the presence of both fluconazole and CZ-EO, the activity of fungal ATPases decreases and, at the same time, the amount of intracellular drug increases. IMPORTANCE This study highlights how small doses of CZ-EO are able to inhibit the secretion of fluconazole and promote its accumulation in the fungal cell. In this manner, the drug is able to exert its pharmacological effects bypassing the resistance of the yeast. If further studies will confirm this synergy, it will be possible to develop new therapeutic formulations active in the fight against C. auris resistances.
RESUMO
Aspergillus fumigatus has become a leading cause of fungal morbidity and mortality, especially in immunocompromised patients. This fungus is able to grow as a multicellular community and produce a hydrophobic extracellular matrix (ECM), mainly composed of galactomannan and α-1,3 glucans, to protect itself from host defenses and antimicrobial drugs. This matrix envelops the fungus hyphae, binding them into a contiguous sheath on the colony surface, forming a biofilm and increasing the fungal resistance to adverse environmental factors. Adherence to host cells and resistance to physical removal play a key role in fungal colonization and invasion of the host and in a wide range of infections. Here we show that, by using atomic force spectroscopy, it is possible to exploit the peculiar hydrophobicity of the biofilm components (i.e., cell walls, ECM) to detect the biofilm spread, its growth, and lysis on rough surfaces. By means of this approach, we demonstrate that alginate lyase, an enzyme known to reduce negatively charged alginate levels in microbial biofilms, reduces the biofilm adhesion forces suggesting a loss of ECM from the biofilm, which could be used to enhance pharmacological treatments.
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/crescimento & desenvolvimento , Biofilmes , Proteínas Fúngicas/química , Polissacarídeo-Liases/química , Aspergillus fumigatus/química , Aspergillus fumigatus/citologia , Biofilmes/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Polissacarídeo-Liases/metabolismoRESUMO
Intestinal parasites account for the majority of parasitic diseases, particularly in endemic areas. Most are transmitted via contaminated food. Because of increased immigration and travel, enteric parasitoses are now distributed worldwide. Between May 2006 and December 2008, we examined stool specimens from 5,351 patients (4,695 Italians, 656 non-Italians) for ova and parasites using microscopy, culture techniques, and molecular methods. Stools from 594 patients (11.1%) were contaminated and for all patients samples combined, a total of 700 intestinal parasites were counted. Ninety of the 594 infected patients had more than one parasite in their stools. Parasites causing intestinal disease occurred in 8.8% of patients. The prevalence was over twice as high among non-Italians (26.8% vs 8.9% in Italians, p<0.001) and higher in males (13.0% vs 9.5% in females, p=0.003). Most isolates were pathogenic protozoa, including in decreasing order of frequency: Blastocystis hominis, Giardia intestinalis, Entamoeba histolytica, and Cyclospora cayetanensis. The latter two species tended to be more common in Italians, although not at significant level (3.6% (15/418) vs 1.7% (3/176) in non-Italians, OR: 2.15; 95%CI: 0.6011.70, p=0.22). Helminthes were found in 28 patients, mainly non-Italians (5.7% (10/176) vs 4.3% (18/418), OR: 1.34; 95%CI: 0.543.13, p=0.47). Ascaris lumbricoides and Hymenolepis nana were the most common. Strongyloides stercoralis, Enterobius vermicularis, Taenia spp. and Trichuris trichiura were also found. Intestinal parasites are a serious problem in developing countries, but should not be underestimated in industrialised countries.
Assuntos
Blastocystis hominis/isolamento & purificação , Entamoeba histolytica/isolamento & purificação , Giardia lamblia/isolamento & purificação , Hospitais de Ensino , Enteropatias Parasitárias/diagnóstico , Enteropatias Parasitárias/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Blastocystis/epidemiologia , Criança , Pré-Escolar , Entamebíase/epidemiologia , Feminino , Giardíase/epidemiologia , Humanos , Lactente , Recém-Nascido , Enteropatias Parasitárias/parasitologia , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for serious hospital infections worldwide and represents a global public health problem. Curcumin, the major constituent of turmeric, is effective against MRSA but only at cytotoxic concentrations or in combination with antibiotics. The major issue in curcumin-based therapies is the poor solubility of this hydrophobic compound and the cytotoxicity at high doses. In this paper, we describe the efficacy of a composite nanoparticle made of curcumin (CU) and graphene oxide (GO), hereafter GOCU, in MRSA infection treatment. GO is a nanomaterial with a large surface area and high drug-loading capacity. GO has also antibacterial properties due mainly to a mechanical cutting of the bacterial membranes. For this physical mechanism of action, microorganisms are unlikely to develop resistance against this nanomaterial. In this work, we report the capacity of GO to support and stabilize curcumin molecules in a water environment and we demonstrate the efficacy of GOCU against MRSA at a concentration below 2 µg ml-1. Further, GOCU displays low toxicity on fibroblasts cells and avoids haemolysis of red blood cells. Our results indicate that GOCU is a promising nanomaterial against antibiotic-resistant MRSA.
RESUMO
Today, liposomes are an advanced technology of drug carriers with a dozen drugs in clinical practice and many more in clinical trials. A bottleneck associated with the clinical translation of liposomes has long been 'opsonization', i.e. the adsorption of plasma proteins at the liposome surface resulting in their rapid clearance from circulation. For decades, the most popular way to avoid opsonization has been grafting polyethylene glycol (PEG) onto the liposome surface. Recent studies have clarified that grafting PEG onto the liposome surface reduces, but does not completely prevent protein binding. In this work, we employed dynamic light scattering, zeta-potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), semi-quantitative densitometry and cell imaging to explore the bio-nano-interactions between human plasma (HP) and Onivyde, a PEGylated liposomal drug that has recently been approved by the Food and Drug Administration (FDA) for the treatment of metastatic pancreatic ductal adenocarcinoma (PDAC). To properly evaluate the role of PEGylation, an unPEGylated variant of Onivyde was used as a reference. Collectively, our findings suggest that: (i) although PEGylated, Onivyde is not "stealth" in HP; (ii) surface chemistry is more important than PEGylation in controlling the bio-nano-interactions between Onivyde and plasma components. Of note is that the PC was found to boost the cellular uptake of Onivyde in the pancreas ductal adenocarcinoma cell line (PANC-1) thus suggesting its prominent role in its indication for PDAC treatment. Relevant implications for drug delivery and drug design are discussed.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Polietilenoglicóis/química , Coroa de Proteína , Linhagem Celular Tumoral , Humanos , LipossomosRESUMO
It is now established that the gastric pathogen Helicobacter pylori has the ability to form biofilms in vitro as well as on the human gastric mucosa. The aim of this study is to evaluate the antimicrobial effects of Clarithromycin on H. pylori biofilm and to enhance the effects of this antibiotic by combining it with Alginate Lyase, an enzyme degrading the polysaccharides present in the extracellular polymeric matrix forming the biofilm. We evaluated the Clarithromycin minimum inhibition concentration (MIC) on in vitro preformed biofilm of a H. pylori. Then the synergic effect of Clarithromycin and Alginate Lyase treatment has been quantified by using the Fractional Inhibitory Concentration index, measured by checkerboard microdilution assay. To clarify the mechanisms behind the effectiveness of this antibiofilm therapeutic combination, we used Atomic Force Microscopy to analyze modifications of bacterial morphology, percentage of bacillary or coccoid shaped bacteria cells and to quantify biofilm properties. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1584-1591, 2016.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Claritromicina/farmacologia , Helicobacter pylori/efeitos dos fármacos , Polissacarídeo-Liases/metabolismo , Antibacterianos/química , Claritromicina/química , Relação Dose-Resposta a Droga , Helicobacter pylori/metabolismo , Testes de Sensibilidade Microbiana , Relação Estrutura-AtividadeRESUMO
The availability of genes coding for monoclonal Fab fragments of a desired specificity permits their expression in bacteria and provides a simple method for the generation of good quality reagents. In this paper we describe a new phagemid vector for the production of recombinant Fabs from genes obtained from phage display combinatorial libraries. The phagemid features an antibiotic resistance cassette which, once inserted between the heavy chain fragment and the light chain genes, avoids unwanted recombination and preserves useful restriction sites not affecting the Fab production rate.
Assuntos
Anticorpos Monoclonais/biossíntese , Regulação da Expressão Gênica , Vetores Genéticos , Fragmentos Fab das Imunoglobulinas/biossíntese , Anticorpos Monoclonais/genética , Resistência ao Cloranfenicol/genética , Desoxirribonucleases de Sítio Específico do Tipo II , Escherichia coli , Vetores Genéticos/genética , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Proteínas Recombinantes de Fusão/biossínteseRESUMO
A cDNA encoding a Schistosoma mansoni cyclophilin (SmCyP) has been cloned by polymerase chain reaction amplification using degenerate oligonucleotides based on known conserved cyclophilin (CyP) sequences and by screening an expression cDNA library. The cDNA sequence encodes a 21.5-kDa protein, which shares 59% sequence identity with human CyP B. The SmCyP protein was expressed in Escherichia coli with a hexahistidine affinity tag at its amino terminus and antibodies to the purified (His6)-SmCyP fusion protein were raised in a rabbit. Fractionation of parasite material followed by immunoblot analysis revealed that schistosome CyP is a soluble protein. The N-terminus of the predicted protein contains a hydrophobic region, suggestive of a signal sequence. Accordingly, a recombinant SmCyP protein, lacking the first 23 amino acids was found to share the same gel electrophoretic mobility as the parasite-derived CyP protein, suggesting cleavage of a leader sequence. Hybridization of genomic DNA to a full-length cDNA probe indicates that the SmCyP gene is present as a single copy. Immunohistological experiments in conjunction with confocal scanning laser microscopy and immune electron microscopy show that SmCyP is present in abundance in the adult worm as well as in the schistosomula. The function of CyP in the schistosome is presently unclear, but since its ligand, cyclosporin A, has antischistosomal activity, its function is expected to be a vital one.
Assuntos
Isomerases de Aminoácido/biossíntese , Proteínas de Transporte/biossíntese , Proteínas de Helminto/biossíntese , Schistosoma mansoni/metabolismo , Isomerases de Aminoácido/análise , Isomerases de Aminoácido/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Clonagem Molecular , Códon , Sequência Conservada , Primers do DNA , DNA Complementar , DNA de Helmintos/química , DNA de Helmintos/metabolismo , Escherichia coli , Genes de Helmintos , Proteínas de Helminto/genética , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Fases de Leitura Aberta , Peptidilprolil Isomerase , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Schistosoma mansoni/genética , Schistosoma mansoni/ultraestrutura , Homologia de Sequência de AminoácidosRESUMO
Experimental evidence indicates Epstein Barr virus (EBV) envelope glycoprotein gp350/220 elicits a potent virus neutralizing response in the infected human host that may play an important role in restricting viral pathogenesis. In this study, we report the molecular cloning in combinatorial phage display vectors, of the IgG1 repertoire of an individual naturally infected with EBV, and describe the recovery and characterization of a monoclonal antibody recognizing gp350/220. A detailed understanding of the human antibody response in EBV infection will identify antibodies of potential use in anti-viral prophylaxis and will advance the production of more effective vaccine candidates.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Antígenos Virais/imunologia , Herpesvirus Humano 4/imunologia , Fragmentos Fab das Imunoglobulinas/genética , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Sequência de Bases , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Dados de Sequência MolecularRESUMO
Labeling of recombinant Fab molecules is an important yet cumbersome and time-consuming procedure that is needed in many immunological experimental designs. This work describes the development of a novel expression vector fusing to the carboxyterminal of the Fab heavy chain fragments a tag peptide (FLAG) that is consistently recognized by a mouse monoclonal antibody. The presence of the FLAG peptide does not alter the binding characteristics of the unmodified Fab molecule, as demonstrated by relative affinity determinations and competition experiments. This new method is suitable for extensive utilization in immunological experimental work using recombinant Fabs.
Assuntos
Vetores Genéticos , Fragmentos Fab das Imunoglobulinas/biossíntese , Peptídeos , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Animais , Bactérias/genética , Sequência de Bases , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Dados de Sequência Molecular , OligopeptídeosAssuntos
Isomerases de Aminoácido/genética , Proteínas de Transporte/genética , Schistosoma japonicum/genética , Schistosoma mansoni/genética , Isomerases de Aminoácido/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Clonagem Molecular , Sequência Conservada , Ciclosporina/metabolismo , Ciclosporina/farmacologia , DNA Complementar/genética , Dados de Sequência Molecular , Peptidilprolil Isomerase , Schistosoma japonicum/efeitos dos fármacos , Schistosoma japonicum/metabolismo , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/metabolismo , Esquistossomicidas/metabolismo , Esquistossomicidas/farmacologia , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Recombinant Schistosoma mansoni cyclophilin proteins of the A and the B subtypes (SmCYP A and B) were expressed in bacterial cells as histidine- and maltose-binding fusion proteins and also as nonfused proteins. In addition, S. mansoni CYPs were produced in Sf9 insect cells in their natural forms. Purified recombinant SmCYP B was found to possess a peptidyl-prolyl cis-trans isomerase (PPIase) activity, with a kcat/Km value of 8.2 x 10(5) M-1 s-1. The SmCYP B isoform is approximately two to three times more active than SmCYP A. SmCYP B-specific RNA appears to be more abundant in adult schistosomes than SmCYP A RNA in Northern blots. These results support the conclusion that SmCYP B represents the major schistosomal CYP. The PPIase-associated activity of both CYPs was inhibitable by the immunosuppressive drug cyclosporin A (CsA). We attempt to explain differences in PPIase activities and in CsA inhibition by examining models of the two CYPs complexed to CsA.
Assuntos
Peptidilprolil Isomerase/genética , Proteínas Recombinantes de Fusão/genética , Schistosoma mansoni/enzimologia , Animais , Sítios de Ligação , Western Blotting , Linhagem Celular , Clonagem Molecular , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Histidina/química , Soros Imunes/imunologia , Maltose/química , Modelos Moleculares , Peptidilprolil Isomerase/antagonistas & inibidores , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/isolamento & purificação , Peptidilprolil Isomerase/metabolismo , Conformação Proteica , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
It is well known that immunoglobulins with no identifiable immunogenic origin, called natural antibodies, are present in the sera of healthy individuals and their role as a defence against important pathogens has been proposed. Unfortunately, the studies are hampered by the fact that these immunoglobulins seem to have low affinity and to be polyreactive, and are commonly available in polyclonal preparations. Lately, new technologies for the production of monoclonal antibodies became available, and in particular the cloning of genes coding for antibody fragments in combinatorial phage display vectors provided a handy tool for the selection of human monoclonal antibodies. In this work, we describe the successful development of a technology for the molecular cloning of combinatorial phage display libraries containing genes coding exclusively for antibody fragment of the IgM or IgD phenotype. These libraries can be useful for molecular cloning of monoclonal antibodies of the IgM and IgD phenotype and can help elucidate the role played by natural antibodies in defence against infectious agents.
Assuntos
Anticorpos Antivirais/genética , Antígenos Virais/imunologia , Clonagem Molecular , Genes de Imunoglobulinas , Imunoglobulina D/genética , Imunoglobulina M/genética , Anticorpos Monoclonais/genética , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Bacteriófagos/genética , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Antígenos HIV/imunologia , Antígenos da Hepatite C/imunologia , Humanos , Imunidade Inata , Imunoglobulina D/sangue , Imunoglobulina D/imunologia , Fragmentos de Imunoglobulinas/genética , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Biblioteca de Peptídeos , Plasmídeos/genéticaRESUMO
The preparation of random combinatorial libraries exposed on the surface of phage provides a route for the selection of diverse high affinity human monoclonal antibodies. However, in particular settings, the isolation of genes coding for a rare antibody can be elusive because some epitopes are predominant and because, in the case of impure antigens, the protein or any compound of interest can be present in relatively minimal amount. In this paper, we describe the successful utilization of a new strategy of "preadsorption" panning that allowed us to clone a rare human monoclonal antibody fragment and to access a different antibody repertoire. The procedure is easy, fast, inexpensive, can be used together with other panning techniques and can be particularly useful in cloning antibodies against rare or unknown determinants.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Clonagem Molecular , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Anticorpos Monoclonais/genética , Antígenos Virais/imunologia , Bacteriófagos , Sequência de Bases , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Herpes Simples/virologia , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Pessoa de Meia-Idade , Dados de Sequência Molecular , Biblioteca de PeptídeosRESUMO
Evidence from clinical and experimental studies indicates that hepatitis C virus E2 (HCV/E2) glycoprotein is the major target of a putatively protective immune response. However, even in the presence of a vigorous production of anti-HCV/E2 antibodies, reinfection can occur. Dissection of the human immune response against HCV/E2 indicated that blocking of binding of HCV/E2 to target cells [neutralization of binding (NOB) activity] varies widely among antibody clones. Moreover, in vivo, simultaneous binding of antibodies to distinct epitopes can induce conformational changes and synergies that may be relevant to understanding the anti-HCV immune response. In this study, human recombinant Fabs were generated by affinity-selecting a phage display repertoire library with antibody-coated HCV/E2. These Fabs, which share the same complementarity-determining region DNA sequences, had higher affinity than other anti-HCV/E2 Fabs but showed no NOB activity even at the highest concentrations. Binding of Fabs to HCV/E2 caused conformational changes modifying Fab-binding patterns and reducing, with a negative synergistic effect, Fab-mediated NOB activity. These data suggest that some antibody clones have the potential to modify HCV/E2 conformation and that, in this state, binding of this glycoprotein to its cellular target is less prone to inhibition by some antibody clones. This can explain why high anti-HCV/E2 antibody titers do not directly correlate with protection from infection. Information on the interactions among different antibody clones can contribute to understanding virus-host interplay and developing more effective vaccines.
Assuntos
Anticorpos Antivirais/imunologia , Hepacivirus/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais/química , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/imunologia , Dados de Sequência Molecular , Testes de Neutralização , Biblioteca de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
Demonstration of antibodies inhibiting key viral functions is the basis for the design of an effective vaccine. Dissection of the human antibody response by repertoire cloning may be a powerful means to address this issue. In this study, a panel of human monoclonal recombinant Fab fragments specific for hepatitis C virus (HCV) E2 envelope protein was generated. The selection procedure was designed to select for cross-genotype reactive antibodies. Sequences coding five different human recombinant Fabs specific for the HCV/E2 protein were cloned and characterized. The ability of the cloned antibody fragments to inhibit adhesion of recombinant envelope E2 protein to target cells was assayed. While affinity of the different antibody fragments appeared similar, activity in inhibiting E2 binding to target cells varied considerably from one Fab fragment to another. Two Fabs were not able to inhibit E2 binding at high concentration (40 microg/mL), while three other Fab clones were active in neutralizing 50% of the E2 binding at concentrations ranging from 3 to 0.35 microg/mL.
Assuntos
Anticorpos Anti-Hepatite C/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos , Feminino , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Vacinas Virais/imunologiaRESUMO
Clinical and experimental evidence indicates that the hepatitis C virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate for the development of an effective anti-HCV vaccine. Identification of the human epitopes that are conserved among isolates and are able to elicit protective antibodies would constitute a significant step forward. This work describes the mapping of the B-cell epitopes present on the surface of HCV/E2, as recognized by the immune system during infection, by the analysis of the reciprocal interactions of a panel of human recombinant Fabs derived from an HCV-infected patient. Three unrelated epitopes recognized by antibodies with no neutralization-of-binding (NOB) activity were identified; a fourth, major epitope was defined as a clustering of minor epitopes recognized by Fabs endowed with strong NOB activity.