RESUMO
Sepsis-leading to septic shock-is the leading cause of death in intensive care units. The systemic inflammatory response to infection, which is initiated by activated myeloid cells, plays a key role in the lethal outcome. Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory mediator, released by myeloid cells, that underlies a common genetic susceptibility to different infections and septic shock. Accordingly, strategies that are aimed at inhibiting the action of MIF have therapeutic potential. Here, we report the isolation and characterization of tailorable, small, affinity-matured nanobodies (Nbs; single-domain antigen-binding fragments derived from camelid heavy-chain Abs) directed against MIF. Of importance, these bioengineered Nbs bind both human and mouse MIFs with nanomolar affinity. NbE5 and NbE10 inhibit key MIF functions that can exacerbate septic shock, such as the tautomerase activity of MIF (by blocking catalytic pocket residues that are critical for MIF's conformation and receptor binding), the TNF-inducing potential, and the ability of MIF to antagonize glucocorticoid action. A lead NbE10, tailored to be a multivalent, half-life extended construct (NbE10-NbAlb8-NbE10), attenuated lethality in murine endotoxemia when administered via single injection, either prophylactically or therapeutically. Hence, Nbs, with their structural and pharmacologic advantages over currently available inhibitors, may be an effective, novel approach to interfere with the action of MIF in septic shock and other conditions of inflammatory end-organ damage.-Sparkes, A., De Baetselier, P., Brys, L., Cabrito, I., Sterckx, Y. G.-J., Schoonooghe, S., Muyldermans, S., Raes, G., Bucala, R., Vanlandschoot, P., Van Ginderachter, J. A., Stijlemans, B. Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.
Assuntos
Oxirredutases Intramoleculares/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Choque Séptico/tratamento farmacológico , Anticorpos de Domínio Único/farmacologia , Animais , Feminino , Meia-Vida , Humanos , Oxirredutases Intramoleculares/imunologia , Lipopolissacarídeos/toxicidade , Fatores Inibidores da Migração de Macrófagos/imunologia , Camundongos , Choque Séptico/induzido quimicamente , Choque Séptico/imunologia , Choque Séptico/patologia , Anticorpos de Domínio Único/imunologiaRESUMO
Transthyretin (TTR) is a transport protein of retinol and thyroxine in serum and CSF, which is mainly secreted by liver and choroid plexus, and in smaller amounts in other cells throughout the body. The exact role of TTR and its specific expression in Central Nervous System (CNS) remains understudied. We investigated TTR expression and metabolism in CNS, through the intranasal and intracerebroventricular delivery of a specific anti-TTR Nanobody to the brain, unveiling Nanobody pharmacokinetics to the CNS. In TTR deficient mice, we observed that anti-TTR Nanobody was successfully distributed throughout all brain areas, and also reaching the spinal cord. In wild-type mice, a similar distribution pattern was observed. However, in areas known to be rich in TTR, reduced levels of Nanobody were found, suggesting potential target-mediated effects. Indeed, in wild-type mice, the anti-TTR Nanobody was specifically internalized in a receptor-mediated process, by neuronal-like cells, which were identified as motor neurons. Whereas in KO TTR mice Nanobody was internalized by all cells, for late lysosomal degradation. Moreover, we demonstrate that in vivo motor neurons also actively synthesize TTR. Finally, in vitro cultured primary motor neurons were also found to synthesize and secrete TTR into culture media. Thus, through a novel intranasal CNS distribution study with an anti-TTR Nanobody, we disclose a new cell type capable of synthesizing TTR, which might be important for the understanding of the physiological role of TTR, as well as in pathological conditions where TTR levels are altered in CSF, such as amyotrophic lateral sclerosis.
Assuntos
Encéfalo/metabolismo , Neurônios Motores/metabolismo , Pré-Albumina/metabolismo , Medula Espinal/metabolismo , Administração Intranasal , Animais , Camundongos , Camundongos Knockout , Anticorpos de Domínio Único/administração & dosagemRESUMO
The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the "Nanobody platform" to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced ß-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.
Assuntos
Neoplasias de Cabeça e Pescoço/imunologia , Receptores CXCR/imunologia , Anticorpos de Domínio Único/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Arrestinas/imunologia , Arrestinas/metabolismo , Ligação Competitiva/imunologia , Camelídeos Americanos/imunologia , Linhagem Celular Tumoral , Quimiocina CXCL12/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/prevenção & controle , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Ensaio Radioligante , Receptores CXCR/genética , Receptores CXCR/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Anticorpos de Domínio Único/farmacologia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia , beta-ArrestinasRESUMO
The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.
Assuntos
Anticorpos/farmacologia , Quimiotaxia/efeitos dos fármacos , HIV-1 , Receptores CXCR4/imunologia , Replicação Viral/efeitos dos fármacos , Animais , Anticorpos/isolamento & purificação , Antígenos CD34 , Benzilaminas , Sítios de Ligação/genética , Células COS , Chlorocebus aethiops , Ciclamos , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Células HEK293 , Mobilização de Células-Tronco Hematopoéticas , Compostos Heterocíclicos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Influenza A virus infections impose a recurrent and global disease burden. Current antivirals against influenza are not always effective. We assessed the protective potential of monovalent and bivalent Nanobodies (Ablynx) against challenge with this virus. These Nanobodies were derived from llamas and target H5N1 hemagglutinin. Intranasal administration of Nanobodies effectively controlled homologous influenza A virus replication. Administration of Nanobodies before challenge strongly reduced H5N1 virus replication in the lungs and protected mice from morbidity and mortality after a lethal challenge with H5N1 virus. The bivalent Nanobody was at least 60-fold more effective than the monovalent Nanobody in controlling virus replication. In addition, Nanobody therapy after challenge strongly reduced viral replication and significantly delayed time to death. Epitope mapping revealed that the VHH Nanobody binds to antigenic site B in H5 hemagglutinin. Because Nanobodies are small, stable, and simple to produce, they are a promising, novel therapeutic agent against influenza.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Especificidade de Anticorpos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Nanocápsulas , Conformação Proteica , Organismos Livres de Patógenos Específicos , Fatores de TempoRESUMO
Despite the medical importance of respiratory syncytial virus (RSV) infections, there is no vaccine or therapeutic agent available. Prophylactic administration of palivizumab, a humanized monoclonal RSV fusion (F) protein-specific antibody, can protect high-risk children. Previously, we have demonstrated that RSV can be neutralized by picomolar concentrations of a camelid immunoglobulin single-variable domain that binds the RSV protein F (F-VHHb nanobodies). Here, we investigated the mechanism by which these nanobodies neutralize RSV and tested their antiviral activity in vivo. We demonstrate that bivalent RSV F-specific nanobodies neutralize RSV infection by inhibiting fusion without affecting viral attachment. The ability of RSV F-specific nanobodies to protect against RSV infection was investigated in vivo. Intranasal administration of bivalent RSV F-specific nanobodies protected BALB/c mice from RSV infection, and associated pulmonary inflammation. Moreover, therapeutic treatment with these nanobodies after RSV infection could reduce viral replication and reduced pulmonary inflammation. Thus, nanobodies are promising therapeutic molecules for treatment of RSV.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sinciciais Respiratórios/imunologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antivirais/imunologia , Antivirais/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/imunologia , Fatores de Tempo , Proteínas Virais de Fusão/imunologia , Carga Viral/efeitos dos fármacosRESUMO
Hepatitis B virus (HBV) infections represent a global health problem, since these account for 350 million chronic infections worldwide that result in 500,000-700,000 deaths each year. Control of viral replication and HBV-related disease and mortality are of utmost importance. Because the currently available antiviral therapies all have major limitations, new strategies to treat chronic HBV infection are eagerly awaited. Six single-domain antibodies (VHHs) targeting the core antigen of HBV (HBcAg) have been generated and three of these bound strongly to HBcAg of both subtype ayw and adw. These three VHHs were studied as intrabodies directed towards the nucleus or the cytoplasm of a hepatoma cell line that was co-transfected with HBV. A speckled staining of HBcAg was observed in the cytoplasm of cells transfected with nucleotropic VHH intrabodies. Moreover, an increased intracellular accumulation of hepatitis B e antigen (HBeAg) and a complete disappearance of intracellular HBcAg signal were observed with nuclear targeted HBcAg-specific VHHs. These results suggest that HBcAg-specific VHHs targeted to the nucleus affect HBcAg and HBeAg expression and trafficking in HBV-transfected hepatocytes.
Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Anticorpos Anti-Hepatite B/isolamento & purificação , Anticorpos Anti-Hepatite B/farmacologia , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Núcleo Celular/química , Núcleo Celular/virologia , Anticorpos Anti-Hepatite B/genética , Anticorpos Anti-Hepatite B/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Hepatócitos/virologia , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Replicação Viral/efeitos dos fármacosRESUMO
UNLABELLED: Hepatitis B virus (HBV) infections cause 500,000 to 700,000 deaths per year as a consequence of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Efficient and safe antivirals to treat chronically infected patients and consequently to prevent development of hepatocellular carcinoma are still awaited. We isolated five single-domain antibodies (VHHs) that recognize the most abundant envelope protein (S) of HBV. VHHs, when expressed and retained in the endoplasmic reticulum as intrabodies, reduced levels of secreted hepatitis B surface antigen (HBsAg) particles in a cellular HBV model. In a hydrodynamics-based HBV mouse model, these intrabodies caused a marked reduction in HBsAg concentrations and a 10- to >100-fold reduction in the concentration of HBV virions in plasma. CONCLUSION: VHHs potently inhibited secretion of HBV virions in vivo, showing that this approach might be useful in the treatment of HBV. To our knowledge, this is the first report of intrabody-mediated inhibition of viral secretion in mammals.
Assuntos
Anticorpos/farmacologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/imunologia , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Camelídeos Americanos/imunologia , Carcinoma Hepatocelular/imunologia , Linhagem Celular Tumoral , Antígenos de Superfície da Hepatite B/efeitos dos fármacos , Antígenos E da Hepatite B/efeitos dos fármacos , Antígenos E da Hepatite B/metabolismo , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Transfecção , Proteínas do Envelope Viral/imunologia , Vírion , Replicação Viral/efeitos dos fármacosRESUMO
Transthyretin (TTR) has intrinsic neurotrophic physiological activities independent from its thyroxine ligands, which involve activation of signaling pathways through interaction with megalin. Still, the megalin binding motif on TTR is unknown. Nanobodies (Nb) have the ability to bind "hard to reach" epitopes being useful tools for protein/structure function. In this work, we characterize two anti-TTR Nanobodies, with similar mouse TTR binding affinities, although only one is able to block its neuritogenic activity (169F7_Nb). Through epitope mapping, we identified amino acids 14-18, at the entrance of the TTR central channel, to be important for interaction with megalin, and a stable TTR K15N mutant in that region was constructed. The TTR K15N mutant lacks neuritogenic activity, indicating that K15 is critical for TTR neuritogenic activity. Thus, we identify the putative binding site for megalin and describe two Nanobodies that will allow research and clarification of TTR physiological properties, regarding its neurotrophic effects.
Assuntos
Sítios de Ligação/efeitos dos fármacos , Epitopos/efeitos dos fármacos , Pré-Albumina/farmacologia , Anticorpos de Domínio Único/farmacologia , Animais , Humanos , Ligantes , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/efeitos dos fármacos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacosRESUMO
The intrasplenic injection of human peripheral blood mononuclear cells (PBMCs) into severely immune deficient NOD/SCID mice, causes a massive and transient dominant expansion of human B cells in the spleen. This permits the easy isolation of human monoclonal antibodies specific for different antigens by a Kohler and Milstein-based method. Here we studied the human HIV-specific antibody response in the circulation of mice after intrasplenic transfer of PBMC from untreated HIV-infected patients with detectable to high viral load as well as from HAART-treated and from untreated patients, who kept an undetectable viral load (the latter referred to as "natural suppressors"). Excellent B cell expansion was obtained for all PBMC. High level replication of virus was observed after transfer of PBMC of untreated viremic patients only. A strong and multispecific HIV-specific antibody response was observed after transfer of PBMC of untreated viremic patients and natural suppressors. In contrast, only a weak and pauci-specific antibody response was detected in mice reconstituted with PBMC from successfully treated patients. Based on these observations we conclude that the use of the intraspleen mouse model confirmed a) the presence of HIV-specific circulating memory B cells in untreated patients and natural suppressors; b) the nearly complete absence of circulating memory B cells in patients receiving highly active antiretroviral therapy. Using the intraspleen model we generated large numbers of immortalized B cells and isolated two anti-p24 human monoclonal antibodies. We further conclude that the intraspleen huPBL NOD/SCID model is a small animal model useful for the analysis of the antibody response against HIV found in patients.
Assuntos
Especificidade de Anticorpos , Linfócitos B/imunologia , Vias de Administração de Medicamentos , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Modelos Animais , Animais , Linfócitos B/fisiologia , Linfócitos B/virologia , Anticorpos Anti-HIV/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Baço/imunologia , Carga ViralRESUMO
Although a limited number of HIV-infected patients have broadly neutralizing antibodies, it has not been examined whether these antibodies can protect against infection with primary virus in vivo. Here we screened the plasma of 23 HIV-1-infected patients for broadly neutralizing antibodies. Purified antibodies from subjects with broad and more narrow responses were administered to huPBL-NOD/Scid mice that were subsequently challenged with primary viruses of clade A, B and CRF01_AE. Although we observed a lack of correlation between the data from the in vitro neutralization assay and the results from the passive immunization experiments, we report for the first time that antibodies from HIV-infected persons can inhibit replication of primary virus isolates in an animal model.
Assuntos
Anticorpos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Replicação Viral/imunologia , Adulto , Idoso , Animais , Anticorpos/isolamento & purificação , Anticorpos/uso terapêutico , Contagem de Linfócito CD4 , Modelos Animais de Doenças , Feminino , Infecções por HIV/prevenção & controle , Infecções por HIV/terapia , HIV-1/isolamento & purificação , Humanos , Imunização Passiva/métodos , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/uso terapêutico , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Testes de Neutralização , Carga ViralRESUMO
BACKGROUND: Chronic hepatitis B (HBV) and hepatitis C (HCV) patients have elevated plasma levels of soluble CD14 (sCD14). We examined whether human hepatocytes produce sCD14 in vivo, and whether HBV or HCV infections influence this chimeric production. METHODS: uPA-SCID mice were transplanted with primary human hepatocytes and some animals were subsequently infected with HBV or HCV. Plasma from these mice was analyzed for the presence of human sCD14. The liver was examined via immunohistochemistry. RESULTS: A soluble form of human CD14 could be detected in the plasma from successfully transplanted mice, while it was completely absent in non-transplanted control animals. The isoform of this human sCD14 corresponded with the most abundant isoform found in human plasma. CD14 levels in circulation were not significantly different between non-infected, HBV infected and HCV infected animals. CONCLUSIONS: Our data indicate that human hepatocytes produce sCD14 in vivo, and that liver cells might be the major source of sCD14 in normal human plasma. In addition we demonstrate that HBV and HCV infections have no direct influence on the production of sCD14 by human hepatocytes in this chimeric model.
Assuntos
Hepatite B/fisiopatologia , Hepatite C/fisiopatologia , Hepatócitos/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Animais , Western Blotting , Transplante de Células , DNA Viral/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Hepatite B/sangue , Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite C/sangue , Hepatite C/metabolismo , Hepatócitos/transplante , Humanos , Endogamia , Receptores de Lipopolissacarídeos/sangue , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Endogâmicos , Camundongos SCID , Camundongos Transgênicos , Solubilidade , Transplante Heterólogo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The hepatitis B virus nucleocapsid or core antigen is extremely immunogenic during infection and after immunization. This review summarizes several features of the nucleocapsid which explain this exceptionally high immunogenicity: a unique three-dimensional folding, the presence of a region that interacts with immunoglobulins outside the classical antibody-binding site, the presence of many CD4+ T cell epitopes, and the presence of encapsidated nucleic acids. Because of these features, nucleocapsids efficiently interact and activate antigen presenting cells, especially nai;ve B cells. This leads to the generation of a dominant Th1 immunity phenotype and the secretion of high levels of IgM and IgG anti-nucleocapsid antibodies.
Assuntos
Anticorpos Anti-Hepatite B/biossíntese , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Nucleocapsídeo/imunologia , Animais , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Anticorpos Anti-Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/química , Vírus da Hepatite B/ultraestrutura , Humanos , Camundongos , Modelos Moleculares , Nucleocapsídeo/química , Dobramento de Proteína , Estrutura Terciária de Proteína , RNA/imunologiaRESUMO
Rabies virus causes lethal brain infection in about 61000 people per year. Each year, tens of thousands of people receive anti-rabies prophylaxis with plasma-derived immunoglobulins and vaccine soon after exposure. Anti-rabies immunoglobulins are however expensive and have limited availability. VHH are the smallest antigen-binding functional fragments of camelid heavy chain antibodies, also called Nanobodies. The therapeutic potential of anti-rabies VHH was examined in a mouse model using intranasal challenge with a lethal dose of rabies virus. Anti-rabies VHH were administered directly into the brain or systemically, by intraperitoneal injection, 24 hours after virus challenge. Anti-rabies VHH were able to significantly prolong survival or even completely rescue mice from disease. The therapeutic effect depended on the dose, affinity and brain and plasma half-life of the VHH construct. Increasing the affinity by combining two VHH with a glycine-serine linker into bivalent or biparatopic constructs, increased the neutralizing potency to the picomolar range. Upon direct intracerebral administration, a dose as low as 33 µg of the biparatopic Rab-E8/H7 was still able to establish an anti-rabies effect. The effect of systemic treatment was significantly improved by increasing the half-life of Rab-E8/H7 through linkage with a third VHH targeted against albumin. Intraperitoneal treatment with 1.5 mg (2505 IU, 1 ml) of anti-albumin Rab-E8/H7 prolonged the median survival time from 9 to 15 days and completely rescued 43% of mice. For comparison, intraperitoneal treatment with the highest available dose of human anti-rabies immunoglobulins (65 mg, 111 IU, 1 ml) only prolonged survival by 2 days, without rescue. Overall, the therapeutic benefit seemed well correlated with the time of brain exposure and the plasma half-life of the used VHH construct. These results, together with the ease-of-production and superior thermal stability, render anti-rabies VHH into valuable candidates for development of alternative post exposure treatment drugs against rabies.
Assuntos
Vírus da Raiva/imunologia , Raiva/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Meia-Vida , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Raiva/prevenção & controle , Raiva/virologia , Vacina Antirrábica/imunologia , Vírus da Raiva/genética , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/genética , Distribuição Tecidual , Carga ViralRESUMO
FcRn is a key player in several immunological and non-immunological processes, as it mediates maternal-fetal transfer of IgG, regulates the serum persistence of IgG and albumin, and transports both ligands between different cellular compartments. In addition, FcRn enhances antigen presentation. Thus, there is an intense interest in studies of how FcRn binds and transports its cargo within and across several types of cells, and FcRn detection reagents are in high demand. Here we report on phage display-selected Nanobodies that target human FcRn. The Nanobodies were obtained from a variable-domain repertoire library isolated from a llama immunized with recombinant human FcRn. One candidate, Nb218-H4, was shown to bind FcRn with high affinity at both acidic and neutral pH, without competing ligand binding and interfering with FcRn functions, such as transcytosis of IgG. Thus, Nb218-H4 can be used as a detection probe and as a tracker for visualization of FcRn-mediated cellular transport.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores Fc/imunologia , Receptores Fc/metabolismo , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Animais , Transporte Biológico , Camelídeos Americanos/imunologia , Camelídeos Americanos/metabolismo , Células HEK293 , Haplorrinos , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/imunologia , Ligantes , Camundongos , Ratos , Suínos , Transcitose/imunologiaRESUMO
In 1989, a new type of antibody was identified, first in the sera of dromedaries and later also in all other species of the Camelidae family. These antibodies do not contain a light chain and also lack the first constant heavy domain. Today it is still unclear what the evolutionary advantage of such heavy chain-only antibodies could be. In sharp contrast, the broad applicability of the isolated variable antigen-binding domains (VHH) was rapidly recognized, especially for the development of therapeutic proteins, called Nanobodies(®). Here we summarize first some of the unique characteristics and features of VHHs. These will next be described in the context of different experimental therapeutic applications of Nanobodies against different viruses: HIV, Hepatitis B virus, influenza virus, Respiratory Syncytial virus, Rabies virus, FMDV, Poliovirus, Rotavirus, and PERVs. Next, the diagnostic application of VHHs (Vaccinia virus, Marburg virus and plant Tulip virus X), as well as an industrial application (lytic lactococcal 936 phage) will be described. In addition, the described data show that monovalent Nanobodies can possess unique characteristics not observed with conventional antibodies. The straightforward formatting into bivalent, multivalent, and/or multispecific Nanobodies allowed tailoring molecules for potency and cross-reactivity against viral targets with high sequence diversity.
Assuntos
Anticorpos Antivirais/uso terapêutico , Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Região Variável de Imunoglobulina/uso terapêutico , Viroses/diagnóstico , Viroses/tratamento farmacológico , Vírus/imunologia , Animais , Anticorpos Antivirais/imunologia , Camelídeos Americanos/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Viroses/imunologiaRESUMO
The polymeric immunoglobulin receptor (pIgR) ensures the transport of dimeric immunoglobulin A (dIgA) and pentameric immunoglobulin M (pIgM) across epithelia to the mucosal layer of for example the intestines and the lungs via transcytosis. Per day the human pIgR mediates the excretion of 2 to 5 grams of dIgA into the mucosa of luminal organs. This system could prove useful for therapies aiming at excretion of compounds into the mucosa. Here we investigated the use of the variable domain of camelid derived heavy chain only antibodies, also known as VHHs or Nanobodies®, targeting the human pIgR, as a transport system across epithelial cells. We show that VHHs directed against the human pIgR are able to bind the receptor with high affinity (â¼1 nM) and that they compete with the natural ligand, dIgA. In a transcytosis assay both native and phage-bound VHH were only able to get across polarized MDCK cells that express the human pIgR gene in a basolateral to apical fashion. Indicating that the VHHs are able to translocate across epithelia and to take along large particles of cargo. Furthermore, by making multivalent VHHs we were able to enhance the transport of the compounds both in a MDCK-hpIgR and Caco-2 cell system, probably by inducing receptor clustering. These results show that VHHs can be used as a carrier system to exploit the human pIgR transcytotic system and that multivalent compounds are able to significantly enhance the transport across epithelial monolayers.
Assuntos
Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Receptores de Imunoglobulina Polimérica/metabolismo , Transcitose , Animais , Células CACO-2 , Camelídeos Americanos , Polaridade Celular , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Mapeamento de Epitopos , Humanos , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC(50) of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5.The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve "best-in-class" and broader neutralization capacity.
Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Camelídeos Americanos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/imunologia , Vírus/imunologia , Animais , Anticorpos Neutralizantes/isolamento & purificação , Especificidade de Anticorpos , Antivirais/imunologia , Antivirais/isolamento & purificação , Antivirais/metabolismo , Reações Cruzadas , Relação Dose-Resposta Imunológica , Epitopos/imunologia , Genótipo , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Virus da Influenza A Subtipo H5N1/imunologia , Lyssavirus/genética , Lyssavirus/imunologia , Vírus Sinciciais Respiratórios/imunologia , Proteínas Virais/imunologiaRESUMO
The hepatitis B virus core (HBc) virus-like particle (VLP) is known as one of the most immunogenic antigens and carrier vehicles in different immunization strategies. Recent findings are suggesting the potential of the HBc VLPs as an oral immunogen. Here, we focus on the induction of serum humoral responses by oral administration of HBc VLPs in preparations substantially free of lipopolysaccharide and immunomodulating encapsidated RNA. The full-length HBc antigen was used, because the C-terminal arginine-rich tail may contribute to the immunogenicity of the antigen as the region is involved in cell surface heparan sulfate binding and internalization of the protein. Serum antibody levels and isotypes were determined following oral administration of the HBc VLPs with the perspective of using the HBc VLP as an immunostimulatory and carrier molecule for epitopes of blood-borne diseases in oral immunization vaccination strategies. Following oral administration of the HBc VLP preparations to mice, a strong serum humoral response was induced with mainly immunoglobulin G2a (IgG2a) antibodies, pointing toward a Th1 response which is essential in the control of intracellular pathogens. Intraperitoneal immunization with the HBc VLP induced a stronger, mixed Th1/Th2 response. Finally, a comparison was made with the induced serum humoral response following oral administration of the recombinant cholera toxin B pentamer, a commonly used oral immunogen. These immunizations, in contrast, induced predominantly antibodies of the IgG1 isotype, indicative of a Th2 response. These data suggest that the HBc VLP can be an interesting carrier molecule in oral vaccine development.
Assuntos
Anticorpos Antibacterianos/imunologia , Toxina da Cólera/administração & dosagem , Anticorpos Anti-Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/administração & dosagem , Administração Oral , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/classificação , Antígenos Virais/imunologia , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Anticorpos Anti-Hepatite B/sangue , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The nucleocapsid or core particle of the hepatitis B virus has become one of the favourite recombinant vaccine carriers for foreign peptides, proteins and stimulatory oligonucleotides. The core protein consists of three regions: an N-terminal, a central and a C-terminal region that can accommodate the addition or insertion of the foreign sequences. The protamine-like C-terminal region that binds host RNA randomly during recombinant particle formation is often truncated. It is commonly thought that these truncations do not affect particle assembly. Recent studies have demonstrated that the C-terminal domains mediate a glycosaminoglycan-dependent attachment of nucleocapsids to the plasma membranes of host cells. This interaction might well contribute to the immunogenicity of nucleocapsids. Testing the hypothesis that full-length particles might be safer and superior for the induction of an immune response against the nucleocapsids and inserted sequences, requires the availability of purified particles. In this report, we detail a novel method for the synthesis and purification of full-length core particles essentially free of RNA from Escherichia coli.