RESUMO
Abs are glycoproteins that carry a conserved N-linked carbohydrate attached to the Fc whose presence and fine structure profoundly impacts on their in vivo immunogenicity, pharmacokinetics, and functional attributes. The host cell line used to produce IgG plays a major role in this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. In this study, we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the human endothelial kidney 293-F or Chinese hamster ovary-K1 systems. We show that the types of N-linked glycans between matched pairs of Fc mutants vary greatly and in particular, with respect, to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B-mediated agglutination of human erythrocytes when expressed in Chinese hamster ovary-K1, but not in human endothelial kidney 293-F cells.
Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Vírus da Influenza B/imunologia , Influenza Humana/tratamento farmacológico , Animais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Glicosilação , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/uso terapêutico , Influenza Humana/imunologia , Influenza Humana/virologiaRESUMO
Intravenous immunoglobulin (IVIG) is an established treatment for numerous autoimmune conditions. Although Fc fragments derived from IVIG have shown efficacy in controlling immune thrombocytopenia in children, the mechanisms of action are unclear and controversial. The aim of this study was to dissect IVIG effector mechanisms using further adapted Fc fragments on demyelination in an ex vivo model of the central nervous system-immune interface. Using organotypic cerebellar slice cultures (OSCs) from transgenic mice, we induced extensive immune-mediated demyelination and oligodendrocyte loss with an antibody specific for myelin oligodendrocyte glycoprotein (MOG) and complement. Protective effects of adapted Fc fragments were assessed by live imaging of green fluorescent protein expression, immunohistochemistry and confocal microscopy. Cysteine- and glycan-adapted Fc fragments protected OSC from demyelination in a dose-dependent manner where equimolar concentrations of either IVIG or control Fc were ineffective. The protective effects of the adapted Fc fragments are partly attributed to interference with complement-mediated oligodendroglia damage. Transcriptome analysis ruled out signatures associated with inflammatory or innate immune responses. Taken together, our findings show that recombinant biomimetics can be made that are at least two hundred-fold more effective than IVIG in controlling demyelination by anti-MOG antibodies.
Assuntos
Autoanticorpos/uso terapêutico , Cerebelo/patologia , Doenças Desmielinizantes/terapia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/uso terapêutico , Oligodendroglia/patologia , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Autoanticorpos/genética , Cerebelo/efeitos dos fármacos , Doenças Desmielinizantes/imunologia , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Imunoglobulinas Intravenosas/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Oligodendroglia/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Proteínas Recombinantes de Fusão/genéticaRESUMO
In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn297, either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein-Asn297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn221 sequon bound influenza virus hemagglutinin and disrupted influenza A-mediated agglutination of human erythrocytes.
Assuntos
Hemaglutinação/genética , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Orthomyxoviridae/genética , Polissacarídeos/genética , Receptores de IgG/genética , Glicosilação , Hemaglutinação/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Mutação , Orthomyxoviridae/imunologia , Polissacarídeos/imunologia , Receptores de IgG/imunologiaRESUMO
Multimeric fragment crystallizable (Fc) regions and Fc-fusion proteins are actively being explored as biomimetic replacements for IVIG therapy, which is deployed to manage many diseases and conditions but is expensive and not always efficient. The Fc region of human IgG1 (IgG1-Fc) can be engineered into multimeric structures (hexa-Fcs) that bind their cognate receptors with high avidity. The critical influence of the unique N-linked glycan attached at Asn-297 on the structure and function of IgG1-Fc is well documented; however, whether the N-linked glycan has a similarly critical role in multimeric, avidly binding Fcs, is unknown. Hexa-Fc contains two N-linked sites at Asn-77 (equivalent to Asn-297 in the Fc of IgG1) and Asn-236 (equivalent to Asn-563 in the tail piece of IgM). We report here that glycosylation at Asn-297 is critical for interactions with Fc receptors and complement and that glycosylation at Asn-563 is essential for controlling multimerization. We also found that introduction of an additional fully occupied N-linked glycosylation site at the N terminus at position 1 (equivalent to Asp-221 in the Fc of IgG1) dramatically enhances overall sialic acid content of the Fc multimers. Furthermore, replacement of Cys-575 in the IgM tail piece of multimers resulted in monomers with enhanced sialic acid content and differential receptor-binding profiles. Thus insertion of additional N-linked glycans into either the hinge or tail piece of monomers or multimers leads to molecules with enhanced sialylation that may be suitable for managing inflammation or blocking pathogen invasion.
Assuntos
Desenho de Fármacos , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Modelos Moleculares , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/metabolismo , Substituição de Aminoácidos , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Asparagina/metabolismo , Células CHO , Cricetulus , Cistina/metabolismo , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/química , Imunoglobulina G/genética , Estrutura Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Mutação , Domínios e Motivos de Interação entre Proteínas , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Diversity at pathogen genetic loci can be driven by host adaptive immune selection pressure and may reveal proteins important for parasite biology. Population-based genome sequencing of Plasmodium falciparum, the parasite responsible for the most severe form of malaria, has highlighted two related polymorphic genes called dblmsp and dblmsp2, which encode Duffy binding-like (DBL) domain-containing proteins located on the merozoite surface but whose function remains unknown. Using recombinant proteins and transgenic parasites, we show that DBLMSP and DBLMSP2 directly and avidly bind human IgM via their DBL domains. We used whole genome sequence data from over 400 African and Asian P. falciparum isolates to show that dblmsp and dblmsp2 exhibit extreme protein polymorphism in their DBL domain, with multiple variants of two major allelic classes present in every population tested. Despite this variability, the IgM binding function was retained across diverse sequence representatives. Although this interaction did not seem to have an effect on the ability of the parasite to invade red blood cells, binding of DBLMSP and DBLMSP2 to IgM inhibited the overall immunoreactivity of these proteins to IgG from patients who had been exposed to the parasite. This suggests that IgM binding might mask these proteins from the host humoral immune system.
Assuntos
Antígenos de Protozoários/metabolismo , Imunoglobulina M/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Humanos , Ligação ProteicaRESUMO
Plasmodium falciparum malaria is a major cause of mortality and severe morbidity. Its virulence is related to the parasite's ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family is central to both. Here, we present evidence of a P. falciparum evasion mechanism not previously documented: the masking of PfEMP1-specific IgG epitopes by nonspecific IgM. Nonspecific IgM binding to erythrocytes infected by parasites expressing the PfEMP1 protein VAR2CSA (involved in placental malaria pathogenesis and protective immunity) blocked subsequent specific binding of human monoclonal IgG to the Duffy binding-like (DBL) domains DBL3X and DBL5ε of this PfEMP1 variant. Strikingly, a VAR2CSA-specific monoclonal antibody that binds outside these domains and can inhibit IE adhesion to the specific VAR2CSA receptor chondroitin sulfate A was unaffected. Nonspecific IgM binding protected the parasites from FcγR-dependent phagocytosis of VAR2CSA(+) IEs, but it did not affect IE adhesion to chondroitin sulfate A or lead to C1q deposition on IEs. Taken together, our results indicate that the VAR2CSA affinity for nonspecific IgM has evolved to allow placenta-sequestering P. falciparum to evade acquired protective immunity without compromising VAR2CSA function or increasing IE susceptibility to complement-mediated lysis. Furthermore, functionally important PfEMP1 epitopes not prone to IgM masking are likely to be particularly important targets of acquired protective immunity to P. falciparum malaria.
Assuntos
Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Anticorpos Antiprotozoários/metabolismo , Reações Antígeno-Anticorpo , Antígenos de Protozoários/metabolismo , Epitopos/metabolismo , Membrana Eritrocítica/imunologia , Membrana Eritrocítica/parasitologia , Feminino , Humanos , Tolerância Imunológica , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Técnicas In Vitro , Malária Falciparum/complicações , Malária Falciparum/parasitologia , Fagocitose , Placenta/imunologia , Placenta/parasitologia , Plasmodium falciparum/patogenicidade , Gravidez , Complicações Parasitárias na Gravidez/imunologia , Complicações Parasitárias na Gravidez/parasitologiaRESUMO
Chloroquine (CQ) has been a mainstay of antimalarial drug treatment for several decades. Additional therapeutic actions of CQ have been described, including some reports of fungal inhibition. Here we investigated the action of CQ in fungi, including the yeast model Saccharomyces cerevisiae. A genomewide yeast deletion strain collection was screened against CQ, revealing that bck1Δ and slt2Δ mutants of the cell wall integrity pathway are CQ hypersensitive. This phenotype was rescued with sorbitol, consistent with cell wall involvement. The cell wall-targeting agent caffeine caused hypersensitivity to CQ, as did cell wall perturbation by sonication. The phenotypes were not caused by CQ-induced changes to cell wall components. Instead, CQ accumulated to higher levels in cells with perturbed cell walls: CQ uptake was 2- to 3-fold greater in bck1Δ and slt2Δ mutants than in wild-type yeast. CQ toxicity was synergistic with that of the major cell wall-targeting antifungal drug, caspofungin. The MIC of caspofungin against the yeast pathogen Candida albicans was decreased 2-fold by 250 µM CQ and up to 8-fold at higher CQ concentrations. Similar effects were seen in Candida glabrata and Aspergillus fumigatus. The results show that the cell wall is critical for CQ resistance in fungi and suggest that combination treatments with cell wall-targeting drugs could have potential for antifungal treatment.
Assuntos
Antimaláricos/farmacologia , Parede Celular/efeitos dos fármacos , Cloroquina/farmacologia , Farmacorresistência Fúngica , Saccharomyces cerevisiae/efeitos dos fármacos , Aspergillus fumigatus/efeitos dos fármacos , Transporte Biológico , Candida albicans/efeitos dos fármacos , Candida glabrata/efeitos dos fármacos , Caspofungina , Sinergismo Farmacológico , Equinocandinas/farmacologia , Lipopeptídeos , Testes de Sensibilidade Microbiana , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sorbitol/farmacologiaRESUMO
Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study, we report on the properties of a novel human IgA1, constructed using a single-chain variable fragment clone (2E9), selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial α-crystallin Ag and for the human FcαRI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-γ significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls, suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-γ in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of FcαRI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoglobulina A/uso terapêutico , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD/uso terapêutico , Sítios de Ligação de Anticorpos/imunologia , Células CHO , Cricetinae , Cricetulus , Humanos , Imunização Passiva/métodos , Imunoglobulina A/administração & dosagem , Imunoglobulina A/metabolismo , Camundongos , Camundongos Transgênicos , Mycobacterium bovis/imunologia , Receptores Fc/genética , Receptores Fc/metabolismo , Receptores Fc/uso terapêutico , alfa-Cristalinas/imunologiaRESUMO
α-Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex. Aberrant glycosylation of the protein has been linked to various forms of congenital muscular dystrophy. Unusually α-DG has previously been demonstrated to be modified with both O-N-acetylgalactosamine and O-mannose initiated glycans. In the present study, Fc-tagged recombinant mouse α-DG was expressed and purified from human embryonic kidney 293T cells. α-DG glycopeptides were characterized by glycoproteomic strategies using both nano-liquid chromatography matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry. A total of 14 different peptide sequences and 38 glycopeptides were identified which displayed heterogeneous O-glycosylation. These data provide new insights into the complex domain-specific O-glycosylation of α-DG.
Assuntos
Distroglicanas/química , Proteômica , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia Líquida , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
OBJECTIVES: Recent work with the yeast model revealed that the antiprotozoal drug quinine competes with tryptophan for uptake via a common transport protein, causing cellular tryptophan starvation. In the present work, it was hypothesized that similar interactions may occur in malaria patients receiving quinine therapy. PATIENTS AND METHODS: A direct observational study was conducted in which plasma levels of drug and amino acids (tryptophan, tyrosine and phenylalanine) were monitored during quinine treatment of malaria patients with Plasmodium falciparum infections. RESULTS: Consistent with competition for uptake from plasma into cells, plasma tryptophan and tyrosine levels increased ≥2-fold during quinine therapy. Plasma quinine levels in individual plasma samples were significantly and positively correlated with tryptophan and tyrosine in the same samples. Control studies indicated no effect on phenylalanine. Chloroquine treatment of Plasmodium vivax-infected patients did not affect plasma tryptophan or tyrosine. During quinine treatment, plasma tryptophan was significantly lower (and quinine significantly higher) in patients experiencing adverse drug reactions. CONCLUSIONS: Plasma quinine levels during therapy are related to patient tryptophan and tyrosine levels, and these interactions can determine patient responses to quinine. The study also highlights the potential for extrapolating insights directly from the yeast model to human malaria patients.
Assuntos
Antimaláricos/administração & dosagem , Interações Medicamentosas , Malária Falciparum/tratamento farmacológico , Quinina/administração & dosagem , Triptofano/antagonistas & inibidores , Tirosina/antagonistas & inibidores , Adulto , Idoso , Antimaláricos/farmacologia , Feminino , Humanos , Malária Vivax/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Plasma/química , Quinina/farmacologia , Triptofano/metabolismo , Tirosina/metabolismo , Adulto JovemRESUMO
IgM is an ancestral Ab class found in all jawed vertebrates, from sharks to mammals. This ancient ancestry is shared by malaria parasites (genus Plasmodium) that infect all classes of terrestrial vertebrates with whom they coevolved. IgM, the least studied and most enigmatic of the vertebrate Igs, was recently shown to form an intimate relationship with the malaria parasite Plasmodium falciparum. In this article, we discuss how this association might have come about, building on the recently determined structure of the human IgM pentamer, and how this interaction could affect parasite survival, particularly in light of the just-discovered Fc mu R localized to B and T cell surfaces. Because this parasite may exploit an interaction with IgM to limit immune detection, as well as to manipulate the immune response when detected, a better understanding of this association may prove critical for the development of improved vaccines or vaccination strategies.
Assuntos
Interações Hospedeiro-Parasita/imunologia , Evasão da Resposta Imune/imunologia , Imunoglobulina M/metabolismo , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Plasmodium falciparum/imunologia , Receptores Fc/metabolismo , Animais , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Feminino , Variação Genética/imunologia , Interações Hospedeiro-Parasita/genética , Humanos , Evasão da Resposta Imune/genética , Imunoglobulina M/sangue , Imunoglobulina M/deficiência , Malária Falciparum/prevenção & controle , Masculino , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores Fc/sangue , Receptores Fc/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Acanthamoeba granulomatous encephalitis (AGE), caused by Acanthamoeba castellanii, is a fatal infection of immunocompromised individuals. The pathogenesis of blood-brain barrier (BBB) breach remains unknown. Using a novel in vitro BBB infection model under flow conditions, demonstrates that increases in flow rates lead to decreased binding of A. castellanii to host cells. This is a distinct departure from previous findings under static conditions. However, similarly to static conditions binding of A. castellanii to host cells is host mannose dependent. Disruption of the host cell monolayer was independent of amoeba binding, but dependent on secreted serine proteases. For the first time we report the binding dynamics of A. castellanii under physiological conditions, showing that BBB disruption is not directly linked to binding, instead it is reliant on secreted proteases. Our results offer a platform on which therapies designed at modulating physiological parameters can improve the outcome of infection with A. castellanii.
Assuntos
Acanthamoeba castellanii/fisiologia , Amebíase/parasitologia , Barreira Hematoencefálica/parasitologia , Encefalite/parasitologia , Serina Proteases/metabolismo , Encéfalo/citologia , Encéfalo/parasitologia , Células Cultivadas , Meios de Cultivo Condicionados , Células Endoteliais/parasitologia , Endotélio Vascular/citologia , Endotélio Vascular/parasitologia , Humanos , Hidrodinâmica , Lectina de Ligação a Manose/metabolismo , Microvasos/metabolismo , Microvasos/parasitologiaRESUMO
Murine immunoglobulin G (IgG) plays an important role in mediating protective immune responses to malaria. We still know relatively little about which IgG subclasses protect against this disease in mouse models, although IgG2a and IgG2b are considered to be the most potent and dominate in successful passive transfer experiments in rodent malarias. To explore the mechanism(s) by which the different mouse IgG subclasses may mediate a protective effect, we generated mouse IgG1, IgG2a, IgG2b and IgG3 specific for the C-terminal 19-kDa region of Plasmodium falciparum merozoite surface protein 1 (PfMSP1(19)), and to the homologous antigen from Plasmodium yoelii (P. yoelii), both major targets of protective immune responses. This panel of eight IgGs bound antigen with an affinity comparable to that seen for their epitope-matched parental monoclonal antibodies (mAbs) from which they were derived, although for reasons of yield, we were only able to explore the function of mouse IgG1 recognizing PfMSP1(19) in detail, both in vitro and in vivo. Murine IgG1 was as effective as the parental human IgG from which it was derived at inducing NADPH-mediated oxidative bursts and degranulation from neutrophils. Despite showing efficacy in in vitro functional assays with neutrophils, the mouse IgG1 failed to protect against parasite challenge in vivo. The lack of protection afforded by MSP1(19)-specific IgG1 against parasite challenge in wild type mice suggests that this Ab class does not play a major role in the control of infection with mouse malaria in the Plasmodium berghei transgenic model.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Especificidade de Anticorpos , Imunoglobulina G/biossíntese , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium falciparum/imunologia , Plasmodium yoelii/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Imunofluorescência , Imunização Passiva , Imunoglobulina G/classificação , Imunoglobulina G/imunologia , Malária/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmodium berghei/imunologia , Alinhamento de Sequência , Organismos Livres de Patógenos EspecíficosRESUMO
BACKGROUND: Human immunoglobulin G (IgG) plays an important role in mediating protective immune responses to malaria. Although human serum immunoglobulin A (IgA) is the second most abundant class of antibody in the circulation, its contribution, if any, to protective responses against malaria is not clear. RESULTS: To explore the mechanism(s) by which IgA may mediate a protective effect, we generated fully human IgA specific for the C-terminal 19-kDa region of Plasmodium falciparum merozoite surface protein 1 (PfMSP1 19), a major target of protective immune responses. This novel human IgA bound antigen with an affinity comparable to that seen for an epitope-matched protective human IgG1. Furthermore, the human IgA induced significantly higher NADPH-mediated oxidative bursts and degranulation from human neutrophils than the epitope-matched human IgG1 from which it was derived. Despite showing efficacy in in vitro functional assays, the human IgA failed to protect against parasite challenge in vivo in mice transgenic for the human Fcα receptor (FcαRI/CD89). A minority of the animals treated with IgA, irrespective of FcαRI expression, showed elevated serum TNF-α levels and concomitant mouse anti-human antibody (MAHA) responses. CONCLUSIONS: The lack of protection afforded by MSP1 19-specific IgA against parasite challenge in mice transgenic for human FcαRI suggests that this antibody class does not play a major role in control of infection. However, we cannot exclude the possibility that protective capacity may have been compromised in this model due to rapid clearance and inappropriate bio-distribution of IgA, and differences in FcαRI expression profile between humans and transgenic mice.
Assuntos
Anticorpos Antiprotozoários/imunologia , Imunoglobulina A/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium falciparum/imunologia , Proteínas Recombinantes/imunologia , Animais , Antígenos CD/genética , Modelos Animais de Doenças , Humanos , Imunização Passiva , Imunoglobulina G/metabolismo , Malária/imunologia , Malária/prevenção & controle , Camundongos , Camundongos Transgênicos , NADPH Oxidases/metabolismo , Plasmodium berghei , Receptores Fc/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pathogens frequently use multivalent binding to sialic acid to infect cells or to modulate immunity through interactions with human sialic acid-binding immunoglobulin-type lectins (Siglecs). Molecules that interfere with these interactions could be of interest as diagnostics, anti-infectives or as immune modulators. This review describes the development of molecular scaffolds based on the crystallizable fragment (Fc) region of immunoglobulin (Ig) G that deliver high-avidity binding to innate immune receptors, including sialic acid-dependent receptors. The ways in which the sialylated Fc may be engineered as immune modulators that mimic the anti-inflammatory properties of intravenous polyclonal Ig or as blockers of sialic-acid-dependent infectivity by viruses are also discussed.
Assuntos
Fragmentos Fc das Imunoglobulinas , Imunoglobulina G , Ácido N-Acetilneuramínico , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Animais , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Ácido N-Acetilneuramínico/imunologia , Ácido N-Acetilneuramínico/uso terapêuticoRESUMO
Quinine is a major drug of choice for the treatment of malaria. However, the primary mode of quinine action is unclear, and its efficacy is marred by adverse reactions among patients. To help address these issues, a genome-wide screen for quinine sensitivity was carried out using the yeast deletion strain collection. Quinine-sensitive mutants identified in the screen included several that were defective for tryptophan biosynthesis (trp strains). This sensitivity was confirmed in independent assays and was suppressible with exogenous Trp, suggesting that quinine caused Trp starvation. Accordingly, quinine was found to inhibit [(3)H]Trp uptake by cells, and the quinine sensitivity of a trp1Delta mutant could be rescued by overexpression of Trp permeases, encoded by TAT1 and TAT2. The site of quinine action was identified specifically as the high affinity Trp/Tyr permease, Tat2p, with which quinine associated in a Trp-suppressible manner. A resultant action also on Tyr levels was reflected by the Tyr-suppressible quinine hypersensitivity of an aro7Delta deletion strain, which is auxotrophic for Tyr (and Phe). The present genome-wide dataset provides an important resource for discovering modes of quinine toxicity. That potential was validated with our demonstration that Trp and Tyr uptake via Tat2p is a major target of cellular quinine toxicity. The results also suggest that dietary tryptophan supplements could help to avert the toxic effects of quinine.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Antimaláricos/farmacologia , Quinina/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Triptofano/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Antimaláricos/química , Transporte Biológico/efeitos dos fármacos , Desenho de Fármacos , Farmacorresistência Fúngica/genética , Mutação , Quinina/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Trítio , Triptofano/biossíntese , Triptofano/químicaRESUMO
Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fcalpha/mu receptor (hFcalpha/muR). Ligand polymerization status was crucial for the interaction, because hFcalpha/muR binding did not occur with monomeric Ab of either class. hFcalpha/muR bound IgM with an affinity in the nanomolar range, whereas the affinity for dimeric IgA (dIgA) was tenfold lower. Panels of mutant IgM and dIgA were used to identify regions critical for hFcalpha/muR binding. IgM binding required contributions from both Cmu3 and Cmu4 Fc domains, whereas for dIgA, an exposed loop in the Calpha3 domain was crucial. This loop, comprising residues Pro440-Phe443, lies at the Fc domain interface and has been implicated in the binding of host receptors FcalphaRI and polymeric Ig receptor (pIgR), as well as IgA-binding proteins produced by certain pathogenic bacteria. Substitutions within the Pro440-Phe443 loop resulted in loss of hFcalpha/muR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA-binding proteins were shown to inhibit the dIgA-hFcalpha/muR interaction. Therefore, we have identified a motif in the IgA-Fc inter-domain region critical for hFcalpha/muR interaction, and highlighted the multi-functional nature of a key site for protein-protein interaction at the IgA Fc domain interface.
Assuntos
Afinidade de Anticorpos , Imunoglobulina A/química , Imunoglobulina M/química , Receptores Fc/imunologia , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Células COS , Chlorocebus aethiops , Humanos , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Proteínas Mutantes/imunologia , Mutação , Domínios e Motivos de Interação entre Proteínas/imunologia , Multimerização Proteica , Estrutura Terciária de Proteína , Receptores Fc/genéticaRESUMO
The binding of nonspecific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Herein we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4beta domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H chain and requires the IgM Cmu4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4beta domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cmu4 domain are equivalent to those regions of IgG and IgA recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.
Assuntos
Imunoglobulina M/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Eritrócitos/imunologia , Eritrócitos/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina M/química , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
Antigen-specific antibodies (Abs) to the 19-kDa carboxy-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)) play an important role in protective immunity to malaria. Mouse monoclonal Abs (MAbs) 12.10 and 12.8 recognizing MSP1(19) can inhibit red cell invasion by interfering with MSP1 processing on the merozoite surface. We show here that this ability is dependent on the intact Ab since Fab and F(ab')(2) fragments derived from MAb 12.10, although capable of binding MSP1 with high affinity and competing with the intact antibody for binding to MSP1, were unable to inhibit erythrocyte invasion or MSP1 processing. The DNA sequences of the variable (V) regions of both MAbs 12.8 and 12.10 were obtained, and partial amino acid sequences of the same regions were confirmed by mass spectrometry. Human chimeric Abs constructed by using these sequences, which combine the original mouse V regions with human gamma1 and gamma3 constant regions, retain the ability to bind to both parasites and recombinant MSP1(19), and both chimeric human immunoglobulin G1s (IgG1s) were at least as good at inhibiting erythrocyte invasion as the parental murine MAbs 12.8 and 12.10. Furthermore, the human chimeric Abs of the IgG1 class (but not the corresponding human IgG3), induced significant NADPH-mediated oxidative bursts and degranulation from human neutrophils. These chimeric human Abs will enable investigators to examine the role of human Fcgamma receptors in immunity to malaria using a transgenic parasite and mouse model and may prove useful in humans for neutralizing parasites as an adjunct to antimalarial drug therapy.
Assuntos
Anticorpos Antiprotozoários/imunologia , Eritrócitos/parasitologia , Imunoglobulina G/imunologia , Proteína 1 de Superfície de Merozoito/antagonistas & inibidores , Plasmodium falciparum/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antiprotozoários/metabolismo , Sequência de Bases , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Proteína 1 de Superfície de Merozoito/imunologia , Proteína 1 de Superfície de Merozoito/metabolismo , Camundongos , Dados de Sequência Molecular , Neutrófilos/imunologia , Ligação Proteica , RNA , Explosão RespiratóriaRESUMO
The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcgammaRI. This important finding documents the capacity of FcgammaRI to mediate potent antimalaria immunity and supports the development of FcgammaRI-directed therapy for human malaria.