RESUMO
Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.
Assuntos
Interleucina-2 , Transplante de Órgãos , Camundongos , Humanos , Animais , Linfócitos T Reguladores , Sobrevivência de Enxerto , Transplante HomólogoRESUMO
The increase in emerging drug resistant Gram-negative bacterial infections is a global concern. In addition, there is growing recognition that compromising the microbiota through the use of broad-spectrum antibiotics can impact long term patient outcomes. Therefore, there is the need to develop new bactericidal strategies to combat Gram-negative infections that would address these specific issues. In this study, we report and characterize one such approach, an antibody-drug conjugate (ADC) that combines (i) targeting the surface of a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of an antimicrobial peptide. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance: Pseudomonas aeruginosa. To target this organism, we designed an ADC by fusing an antimicrobial peptide to the C-terminal end of the VH and/or VL-chain of a monoclonal antibody, VSX, that targets the core of P. aeruginosa lipopolysaccharide. This ADC demonstrates appropriately minimal levels of toxicity against mammalian cells, rapidly kills P. aeruginosa strains, and protects mice from P. aeruginosa lung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study might result in a broadly useful strategy for targeting specific pathogenic microorganisms without further augmenting antibiotic resistance.
Assuntos
Infecções Bacterianas , Imunoconjugados , Animais , Camundongos , Pseudomonas aeruginosa , Anticorpos Monoclonais/farmacologia , Antibacterianos/farmacologia , Peptídeos Antimicrobianos , MamíferosRESUMO
SARS coronavirus 1 (SARS-CoV-1) causes a respiratory infection that can lead to acute respiratory distress characterized by inflammation and high levels of cytokines in the lung tissue. In this study we constructed a herpes simplex virus 1 replication-defective mutant vector expressing SARS-CoV-1 spike protein as a potential vaccine vector and to probe the effects of spike protein on host cells. The spike protein expressed from this vector is functional in that it localizes to the surface of infected cells and induces fusion of ACE2-expressing cells. In immunized mice, the recombinant vector induced antibodies that bind to spike protein in an ELISA assay and that show neutralizing activity. The spike protein expressed from this vector can induce the expression of cytokines in an ACE2-independent, MyD88-dependent process. These results argue that the SARS-CoV-1 spike protein intrinsically activates signaling pathways that induce cytokines and contribute directly to the inflammatory process of SARS.
Assuntos
Anticorpos Neutralizantes/imunologia , Herpesvirus Humano 1/genética , Imunidade Inata , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Fusão Celular , Linhagem Celular , Citocinas/imunologia , Vetores Genéticos , Humanos , Camundongos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologiaRESUMO
IgA nephropathy (IgAN) is the most prevalent cause of primary glomerular disease worldwide, and the cytokine A PRoliferation-Inducing Ligand (APRIL) is emerging as a key player in IgAN pathogenesis and disease progression. For a panel of anti-human APRIL antibodies with known antagonistic activity, we sought to define their structural mode of engagement to understand molecular mechanisms of action and aid rational antibody engineering. Reliable computational prediction of antibody-antigen complexes remains challenging, and experimental methods such as X-ray co-crystallography and cryoEM have considerable technical, resource, and throughput barriers. To overcome these limitations, we implemented an integrated and accessible experimental-computational workflow to more accurately predict structures of antibody-APRIL complexes. Specifically, a yeast surface display library encoding site-saturation mutagenized surface positions of APRIL was screened against a panel of anti-APRIL antibodies to rapidly obtain a comprehensive biochemical profile of mutational impact on binding for each antibody. The experimentally derived mutational profile data were used as quantitative constraints in a computational docking workflow optimized for antibodies, resulting in robust structural models of antibody-antigen complexes. The model results were confirmed by solving the cocrystal structure of one antibody-APRIL complex, which revealed strong agreement with our model. The models were used to rationally select and engineer one antibody for cross-species APRIL binding, which quite often aids further testing in relevant animal models. Collectively, we demonstrate a rapid, integrated computational-experimental approach to robustly predict antibody-antigen structures information, which can aid rational antibody engineering and provide insights into molecular mechanisms of action.
Assuntos
Complexo Antígeno-Anticorpo/química , Mutação , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/química , Biblioteca Gênica , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/química , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Traditionally, such approaches have largely relied on random mutagenesis and display formats, which fail to address related critical attributes of the antibody, such as effector functions or biophysical stability. We have developed a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. Using this framework, we identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, we engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors. An antibody harboring representative Fc designs demonstrates a half-life improvement of > 9 fold in transgenic mice and > 3.5 fold in cynomolgus monkeys, and maintains robust effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.
Assuntos
Linfócitos B/imunologia , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Regulação Alostérica/genética , Animais , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular , Redes Reguladoras de Genes , Meia-Vida , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Mutação/genética , Ligação Proteica/genética , Engenharia de Proteínas , Estabilidade Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Dengue virus (DENV) infection imposes enormous health and economic burden worldwide with no approved treatment. Several small molecules, including lovastatin, celgosivir, balapiravir and chloroquine have been tested for potential anti-dengue activity in clinical trials; none of these have demonstrated a protective effect. Recently, based on identification and characterization of cross-serotype neutralizing antibodies, there is increasing attention on the potential for dengue immunotherapy. Here, we tested the ability of VIS513, an engineered cross-neutralizing humanized antibody targeting the DENV E protein domain III, to overcome antibody-enhanced infection and high but brief viremia, which are commonly encountered in dengue patients, in various in vitro and in vivo models. We observed that VIS513 efficiently neutralizes DENV at clinically relevant viral loads or in the presence of enhancing levels of DENV immune sera. Single therapeutic administration of VIS513 in mouse models of primary infection or lethal secondary antibody-enhanced infection, reduces DENV titers and protects from lethal infection. Finally, VIS513 administration does not readily lead to resistance, either in cell culture systems or in animal models of dengue infection. The findings suggest that rapid viral reduction during acute DENV infection with a monoclonal antibody is feasible.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Antígenos Virais/química , Antígenos Virais/genética , Linhagem Celular , Chlorocebus aethiops , Reações Cruzadas/imunologia , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Modelos Animais de Doenças , Epitopos , Feminino , Humanos , Soros Imunes , Imunoterapia , Técnicas In Vitro , Camundongos , Modelos Estruturais , Mutação , Testes de Neutralização , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sorogrupo , Células THP-1 , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Ensaio de Placa ViralRESUMO
Despite useful in vivo activity, no therapeutic against dengue virus (DENV) has demonstrated efficacy in clinical trials. Herein, we explored dosing and virological endpoints to guide the design of human trials of VIS513, a pan-serotype anti-DENV IgG1 antibody, in non-human primates (NHPs). Dosing VIS513 pre- or post-peak viremia in NHPs neutralized infectious DENV although RNAemia remained detectable post-treatment; differential interaction of human IgGs with macaque Fc-gamma receptors may delay clearance of neutralized DENV. Our findings suggest useful antiviral utility of VIS513 and highlight an important consideration when evaluating virological endpoints of trials for anti-DENV biologics.
Assuntos
Anticorpos Antivirais/administração & dosagem , Antivirais/administração & dosagem , Vírus da Dengue/imunologia , Dengue/terapia , Fatores Imunológicos/administração & dosagem , Imunoterapia/métodos , Animais , Avaliação Pré-Clínica de Medicamentos , Macaca , Resultado do TratamentoRESUMO
Broadly neutralizing monoclonal antibodies (bNAbs) for viral infections, such as HIV, respiratory syncytial virus (RSV), and influenza, are increasingly entering clinical development. For influenza, most neutralizing antibodies target influenza virus hemagglutinin. These bNAbs represent an emerging, promising modality for treatment and prophylaxis of influenza due to their multiple mechanisms of antiviral action and generally safe profile. Preclinical work in other viral diseases, such as dengue, has demonstrated the potential for antibody-based therapies to enhance viral uptake, leading to enhanced viremia and worsening of disease. This phenomenon is referred to as antibody-dependent enhancement (ADE). In the context of influenza, ADE has been used to explain several preclinical and clinical phenomena. Using structural and viral kinetics modeling, we assess the role of ADE in the treatment of influenza with a bNAb.
Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Facilitadores , Influenza Humana/imunologia , Influenza Humana/terapia , Modelos Biológicos , Modelos Moleculares , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Dengue/imunologia , Epitopos/imunologia , Humanos , Influenza Humana/virologia , Viremia/imunologia , Viroses/imunologiaRESUMO
Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (-24 h) or double-dose (-12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (-12 h) combined with oseltamivir at 50 mg/kg (-12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Anticorpos Amplamente Neutralizantes , Humanos , Influenza Humana/terapia , Camundongos , Camundongos EndogâmicosRESUMO
Dengue is the most common vector-borne viral disease, causing nearly 400 million infections yearly. Currently there are no approved therapies. Antibody epitopes that elicit weak humoral responses may not be accessible by conventional B cell panning methods. To demonstrate an alternative strategy to generating a therapeutic antibody, we employed a non-immunodominant, but functionally relevant, epitope in domain III of the E protein, and engineered by structure-guided methods an antibody directed to it. The resulting antibody, Ab513, exhibits high-affinity binding to, and broadly neutralizes, multiple genotypes within all four serotypes. To assess therapeutic relevance of Ab513, activity against important human clinical features of dengue was investigated. Ab513 mitigates thrombocytopenia in a humanized mouse model, resolves vascular leakage, reduces viremia to nearly undetectable levels, and protects mice in a maternal transfer model of lethal antibody-mediated enhancement. The results demonstrate that Ab513 may reduce the public health burden from dengue.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Vírus da Dengue/fisiologia , Dengue/terapia , Epitopos Imunodominantes/química , Sequência de Aminoácidos , Animais , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/imunologia , Modelos Animais de Doenças , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fagocitose , Engenharia de Proteínas , Receptores Fc/imunologia , Alinhamento de SequênciaRESUMO
Identification of epitopes on viral proteins for the design/identification of broadly-neutralizing monoclonal antibodies (bnAbs) or specific immunogens for vaccine development is hampered by target amino acid diversity. Recently, bnAbs have been isolated for variable viruses by screening B cells from infected individuals for neutralization breadth. Epitope mapping and structural analysis of bnAbs revealed, while some of these bnAbs target glycan moieties, most target protein regions that are conserved in sequence and/or structure. However, almost universally viruses develop mutations that allow escape from neutralization suggesting protein function may not be dependent on the observed conservation. An alternative method for identification of conserved amino acid sequences utilizes an amino acid network-based approach. Calculation of a significant interaction network (SIN) score allows for selection of amino acids that are conserved and constrained within the protein system. Amino acids with high SIN scores are predicted to mutate at lower frequency due to the impact mutation has on the structure/function of a protein. By ascertaining regions of high SIN score, therapeutics can be appropriately designed to target these regions of low mutability. Further, the use of atomic interaction networks to examine protein structure and protein-protein interfaces can complement existing structure-based computational approaches for therapeutic engineering.
Assuntos
Aminoácidos/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antivirais/farmacologia , Sequência Conservada , Epitopos/imunologia , Vírus/imunologia , Aminoácidos/genética , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Antivirais/isolamento & purificação , Descoberta de Drogas/métodos , Mapeamento de Epitopos , Epitopos/genética , Conformação Proteica , Vírus/genéticaRESUMO
Chronic hepatitis C virus (HCV) infection is the most common cause of end-stage liver disease, often leading to liver transplantation, in which case circulating virions typically infect the transplanted liver within hours and viral concentrations can quickly exceed pre-transplant levels. MBL-HCV1 is a fully human monoclonal antibody recognizing a linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423). The ability of MBL-HCV1 to prevent HCV recurrence after liver transplantation was investigated in a phase 2 randomized clinical trial evaluating six MBL-HCV1-treated subjects and five placebo-treated subjects. MBL-HCV1 treatment significantly delayed time to viral rebound compared with placebo treatment. Here we report results from high-throughput sequencing on the serum of each of the eleven enrolled subjects prior to liver transplantation and after viral rebound. We further sequenced the sera of the MBL-HCV1-treated subjects at various interim time points to study the evolution of antibody-resistant viral variants. We detected mutations at one of two positions within the antibody epitope--mutations of N at position 415 to D, K or S, or mutation of N at position 417 to S. It has been previously reported that N415 is not glycosylated in the wild-type E2 protein, but N417S can lead to glycosylation at position 415. Thus N415 is a key position for antibody recognition and the only routes we identified for viral escape, within the constraints of HCV fitness in vivo, involve mutating or glycosylating this position. Evaluation of mutations along the entire E1 and E2 proteins revealed additional positions that changed moderately before and after MBL-HCV1 treatment for subsets of the six subjects, yet underscored the relative importance of position 415 in MBL-HCV1 resistance.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Evolução Biológica , Biomarcadores/metabolismo , Hepatite C Crônica/terapia , Transplante de Fígado , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais/imunologia , Método Duplo-Cego , Seguimentos , Glicosilação , Hepatite C Crônica/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Dados de Sequência Molecular , Prognóstico , RNA Viral/sangue , RNA Viral/genética , Recidiva , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/antagonistas & inibidoresRESUMO
Size exclusion high performance liquid chromatography analysis of a human monoclonal antibody (mAb) showed the presence of a new species that eluted with a retention time between the dimeric and monomeric species of the antibody. Extensive characterization of this species, referred to as "shoulder," indicated that it was a mAb containing an extra light chain and had a molecular weight of approximately 175 kDa. The extra light chain was found to be non-covalently associated with the Fab portion of the protein. The relative amount of shoulder (typically 1-3% of the total mAb present) varied with the Chinese hamster ovary cell line producing the mAb and was not influenced by the growth conditions. Our three-step mAb purification platform using protein A, anion exchange, and cation exchange process steps was successful at removing dimer and higher and lower molecular weight species, but not the shoulder impurity. It was found that hydrophobic interaction chromatography could be used in place of cation exchange to exploit the subtle differences in hydrophobicity between monomer and shoulder. We developed an antibody polishing process using Butyl Sepharose HP resin that is capable of removing the majority of high and low molecular weight impurities yielding 99% pure mAb monomer, virtually devoid of the shoulder species, with a step recovery of about 80%.
Assuntos
Anticorpos Monoclonais/química , Eletroforese em Gel de Poliacrilamida , Cadeias Leves de Imunoglobulina/química , Animais , Células CHO , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peso MolecularRESUMO
Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 µg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Corynebacterium diphtheriae/imunologia , Antitoxina Diftérica/isolamento & purificação , Antitoxina Diftérica/uso terapêutico , Difteria/prevenção & controle , Imunoterapia/métodos , Animais , Linhagem Celular , Toxina Diftérica/antagonistas & inibidores , Modelos Animais de Doenças , Mapeamento de Epitopos , Cobaias , Voluntários Saudáveis , Humanos , Testes de Neutralização , Ligação Proteica , Análise de SobrevidaRESUMO
Mutations in the gene encoding human SOD1 (hSOD1) can cause amyotrophic lateral sclerosis (ALS) yet the mechanism by which mutant SOD1 can induce ALS is not fully understood. There is currently no cure for ALS or treatment that significantly reduces symptoms or progression. To develop tools to understand the protein conformations present in mutant SOD1-induced ALS and as possible immunotherapy, we isolated and characterized eleven unique human monoclonal antibodies specific for hSOD1. Among these, five recognized distinct linear epitopes on hSOD1 that were not available in the properly-folded protein but were available on forms of protein with some degree of misfolding. The other six antibodies recognized conformation-dependent epitopes that were present in the properly-folded protein with two different recognition profiles: three could bind hSOD1 dimer or monomer and the other three were specific for hSOD1 dimer only. Antibodies with the capacity to bind hSOD1 monomer were able to prevent increased hydrophobicity when mutant hSOD1 was exposed to increased temperature and EDTA, suggesting that the antibodies stabilized the native structure of hSOD1. Two antibodies were tested in a G93A mutant hSOD1 transgenic mouse model of ALS but did not yield a statistically significant increase in overall survival. It may be that the two antibodies selected for testing in the mouse model were not effective for therapy or that the model and/or route of administration were not optimal to produce a therapeutic effect. Therefore, additional testing will be required to determine therapeutic potential for SOD1 mutant ALS and potentially some subset of sporadic ALS.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Epitopos/imunologia , Superóxido Dismutase/imunologia , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/enzimologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Epitopos/química , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoprecipitação , Camundongos , Camundongos Transgênicos , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Superóxido Dismutase/químicaRESUMO
UNLABELLED: Recognition of hepatitis C virus (HCV)-infected hepatocyes and interferon (IFN) induction are critical in antiviral immune response. We hypothesized that cell-cell contact between plasmacytoid dendritic cells (pDCs) and HCV-infected cells was required for IFN-α induction through the involvement of cell-surface molecules. Coculture of human peripheral blood mononuclear cells (PBMCs) with genotype 1a full-length (FL) HCV genomic replicon cells or genotype 2a Japanese fulminant hepatitis type 1 (JFH-1) virus-infected hepatoma cells (JFH-1), and not with uninfected hepatoma cells (Huh7.5), induced IFN-α production. Depletion of pDCs from PBMCs attenuated IFN-α release, and purified pDCs produced high levels of IFN-α after coculture with FL replicons or JFH-1-infected cells. IFN-α induction by HCV-containing hepatoma cells required viral replication, direct cell-cell contact with pDCs, and receptor-mediated endocytosis. We determined that the tetraspanin proteins, CD81 and CD9, and not other HCV entry receptors, were required for IFN-α induction in pDCs by HCV-infected hepatoma cells. Disruption of cholesterol-rich membrane microdomains, the localization site of CD81, or inhibition of the CD81 downstream molecule, Rac GTPase, inhibited IFN-α production. IFN-α induction involved HCV RNA and Toll-like receptor (TLR) 7. IFN-α production by HCV-infected hepatoma cells was decreased in pDCs from HCV-infected patients, compared to healthy controls. We found that preexposure of healthy PBMCs to HCV viral particles attenuated IFN-α induction by HCV-infected hepatoma cells or TLR ligands, and this inhibitory effect could be prevented by an anti-HCV envelope glycoprotein 2-blocking antibody. CONCLUSION: Our novel data show that recognition of HCV-infected hepatoma cells by pDCs involves CD81- and CD9-associated membrane microdomains and induces potent IFN-α production.
Assuntos
Células Dendríticas/patologia , Hepacivirus/isolamento & purificação , Hepatócitos/metabolismo , Hepatócitos/patologia , Interferon-alfa/metabolismo , Tetraspanina 28/fisiologia , Tetraspanina 29/fisiologia , Tetraspaninas/fisiologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Estudos de Casos e Controles , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Dendríticas/fisiologia , Endocitose/fisiologia , Hepatite C/metabolismo , Hepatite C/patologia , Hepatite C/virologia , Hepatócitos/virologia , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Microdomínios da Membrana/fisiologia , Replicação Viral/fisiologiaRESUMO
Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5-1.0 log(10) reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/uso terapêutico , Hepatite C Crônica/terapia , Hepatite C/prevenção & controle , Sequência de Aminoácidos , Animais , Linhagem Celular , Modelos Animais de Doenças , Hepatite C/imunologia , Hepatite C/virologia , Hepatite C Crônica/imunologia , Humanos , Transplante de Fígado , Mutação , Testes de Neutralização , Pan troglodytes , RNA Viral/sangue , Tetraspanina 28/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Carga ViralRESUMO
Replacement of polyclonal anti-rabies immunoglobulin (RIG) used in rabies post-exposure prophylaxis (PEP) with a monoclonal antibody will eliminate cost and availability constraints that currently exist using RIG in the developing world. The human monoclonal antibody RAB1 has been shown to neutralize all rabies street isolates tested; however for the laboratory-adapted fixed strain, CVS-11, mutation in the G glycoprotein of amino acid 336 from asparagine (N) to aspartic acid (D) resulted in resistance to neutralization. Interestingly, this same mutation in the G glycoprotein of a second laboratory-adapted fixed strain (ERA) did not confer resistance to RAB1 neutralization. Using cell surface staining and lentivirus pseudotyped with rabies virus G glycoprotein (RABVpp), we identified an amino acid alteration in CVS-11 (K346), not present in ERA (R346), which was required in combination with D336 to confer resistance to RAB1. A complete analysis of G glycoprotein sequences from GenBank demonstrated that no identified rabies isolates contain the necessary combination of G glycoprotein mutations for resistance to RAB1 neutralization, consistent with the broad neutralization of RAB1 observed in direct viral neutralization experiments with street isolates. All combinations of amino acids 336 and 346 reported in the sequence database were engineered into the ERA G glycoprotein and RAB1 was able to neutralize RABVpp bearing ERA G glycoprotein containing all known combinations at these critical residues. These data demonstrate that RAB1 has the capacity to neutralize all identified rabies isolates and a minimum of two distinct mutations in the G glycoprotein are required for abrogation of RAB1 neutralization.
Assuntos
Substituição de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Glicoproteínas/imunologia , Vírus da Raiva/imunologia , Proteínas do Envelope Viral/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/metabolismo , Asparagina/metabolismo , Sítios de Ligação de Anticorpos , Clonagem Molecular , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida/métodos , Testes de Neutralização , Mutação Puntual , Vírus da Raiva/genética , Vírus da Raiva/metabolismo , Análise de Sequência de Proteína , Transfecção , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Anti-Hepatite C/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/isolamento & purificação , Sequência Conservada , Mapeamento de Epitopos , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/isolamento & purificação , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Testes de NeutralizaçãoRESUMO
Rabies is a zoonosis that results in millions of human exposures worldwide each year. Human monoclonal antibodies (HuMAbs) that neutralize rabies virus may represent one viable strategy for post-exposure prophylaxis in humans, and have many advantages over current human or equine rabies immune globulin. Transgenic mice carrying human immunoglobulin genes were used to isolate human monoclonal antibodies that neutralized rabies virus. Several HuMAbs were identified that neutralized rabies virus variants from a broad panel of isolates of public health significance. HuMAb 17C7 was the most promising antibody identified because it neutralized all rabies virus isolates tested. HuMAb 17C7 recognizes a conformational epitope on the rabies virus glycoprotein which includes antigenic site III. HuMAb 17C7 protected hamsters from a lethal dose of rabies virus in a well-established in vivo model of post-exposure prophylaxis.