RESUMEN
Cytotoxic T lymphocytes (CTLs) expressing the CD8 surface marker recognize peptides in association with major histocompatibility complex (MHC) class I molecules. Although most peptides expressed on MHC class I molecules are derived from self- or virally encoded proteins, delivery of exogenous proteins to the cytosol can result in their being processed for presentation to CTLs on MHC class I molecules. We describe two fusion proteins (PEMa and PENP), consisting of the binding and translocating domains of Pseudomonas exotoxin A (PE), fused to peptide epitopes from influenza A matrix protein and nucleoprotein, respectively. These fusion proteins were internalized and processed by MHC class I-positive target cells, resulting in sensitization of target cells for lysis by peptide-specific CTLs. A point mutation known to interfere with intoxication by wild-type PE also reduced the ability of PEMa to sensitize target cells. Fusion of peptide or polypeptide epitopes with PE provides a potential means of eliciting CTLs without the use of self-replicating agents, as well as a useful probe for studying MHC class I-restricted antigen processing.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Epítopos/metabolismo , Exotoxinas/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/metabolismo , Factores de Virulencia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Células Cultivadas , Codón/genética , Citotoxicidad Inmunológica , ADN Viral/genética , Epítopos/genética , Epítopos/inmunología , Exotoxinas/genética , Exotoxinas/inmunología , Glutatión Transferasa/genética , Glutatión Transferasa/aislamiento & purificación , Glutatión Transferasa/metabolismo , Humanos , Virus de la Influenza A/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Osteosarcoma , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Bazo/inmunología , Células Tumorales Cultivadas , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Protein carriers vary in their ability to increase the immunogenicity of poorly immunogenic or T-lymphocyte-independent antigens. We examined one such carrier, the outer membrane protein complex derived from Neisseria meningitidis serogroup B strain B11, in an attempt to determine why this outer membrane protein complex was more immunogenic in young infants and in relevant animal models than two other carriers used in conjugates made with Haemophilus influenzae type b polysaccharide, a T-cell-independent antigen. A single protein of the outer membrane protein complex, the class 2 porin protein, was purified and shown to function as a T-helper lymphocyte carrier protein. Unexpectedly, it was also found to have mitogenic activity for lymphocytes that was not due to lipopolysaccharide. This mitogenic activity appears to date to be unique to this carrier protein of the carrier proteins tested and may contribute to the ability of the H. influenzae type b conjugate vaccine made with the outer membrane protein complex to generate IgG anti-polysaccharide antibody responses in mice and infant monkeys and protective immune responses in infants less than 6 months of age.