RESUMO
Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human gamma 1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (K(d) of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Vaccinia virus/imunologia , Vacínia/prevenção & controle , Vírus da Varíola/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Peso Corporal , Modelos Animais de Doenças , Mapeamento de Epitopos , Humanos , Regiões Constantes de Imunoglobulina/genética , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Glicoproteínas de Membrana/imunologia , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Pan troglodytes , Vacínia/imunologia , Proteínas do Envelope Viral/imunologiaRESUMO
Four single-chain variable fragments (scFvs) against protective antigen (PA) and 2 scFvs against lethal factor (LF) of anthrax were isolated from a phage display library generated from immunized chimpanzees. Only 2 scFvs recognizing PA (W1 and W2) neutralized the cytotoxicity of lethal toxin in a macrophage lysis assay. Full-length immunoglobulin G (IgG) of W1 and W2 efficiently protected rats from anthrax toxin challenge. The epitope recognized by W1 and W2 was conformational and was formed by C-terminal amino acids 614-735 of PA. W1 and W2 each bound to PA with an equilibrium dissociation constant of 4x10-11 mol/L to 5x10(-11) mol/L, which is an affinity that is 20-100-fold higher than that for the interaction of the receptor and PA. W1 and W2 inhibited the binding of PA to the receptor, suggesting that this was the mechanism of protection. These data suggest that W1 and W2 chimpanzee monoclonal antibodies may serve as PA entry inhibitors for use in the emergency prophylaxis against and treatment of anthrax.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Antígenos de Bactérias/química , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Mapeamento de Epitopos , Epitopos/imunologia , Feminino , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/isolamento & purificação , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/isolamento & purificação , Macrófagos/imunologia , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Pan troglodytes , Biblioteca de Peptídeos , Ratos , Ratos Endogâmicos F344 , Receptores de Peptídeos/metabolismoRESUMO
Chimpanzee Fabs against the B5 envelope glycoprotein of vaccinia virus were isolated and converted into complete mAbs with human gamma 1 heavy chain constant regions. The two mAbs (8AH8AL and 8AH7AL) displayed high binding affinities to B5 (Kd of 0.2 and 0.7 nM). The mAb 8AH8AL inhibited the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro, protected mice from subsequent intranasal challenge with virulent vaccinia virus, protected mice when administered 2 days after challenge, and provided significantly greater protection than that afforded by a previously isolated rat anti-B5 mAb (19C2) or by vaccinia immune globulin. The mAb bound to a conformational epitope between amino acids 20 and 130 of B5. These chimpanzee/human anti-B5 mAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox.
