RESUMEN
The hepatitis B core antigen (HBcAg) was used to present the HIV epitopes and mimics selected by phage display. The HIV epitopes were inserted into the el loop of HBcAg. The influence of insertions on the ability of chimeric HBcAg to assemble itself was studied. Special soft was made use of to detect the regularities between certain physical-and-chemical properties of amine-acid residua (belonging to an inserted alien peptide) and the presence or loss of the ability of HBcAg to assemble itself. Recommendations are provided of how to overcome difficulties related with the presentation of alien epitopes.
Asunto(s)
Epítopos/inmunología , VIH-1/inmunología , VIH-2/inmunología , Antígenos del Núcleo de la Hepatitis B/química , Antígenos del Núcleo de la Hepatitis B/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Anticuerpos Antivirales/biosíntesis , Sistemas de Liberación de Medicamentos , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Antígenos del Núcleo de la Hepatitis B/metabolismo , Humanos , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína , Vacunas Sintéticas , Vacunas ViralesRESUMEN
Bacteriophages bearing peptides reacting with antihemagglutinating monoclonal antibodies (MAb) 10H10 to tick-borne encephalitis (TBE) virus protein E were selected from a phage-display peptide library by affinity selection and enzyme immunoassay. The library contained randomized peptides that are 6 amino acids long, fused with protein pIII and exposed on the surface of the bacteriophage. No significant homology between the sequences of selected peptides and TBE virus protein E was detected. Computer software was created to locate the conformation epitopes on the surface of protein E. Amino acids R73, C74, T76, M77, N103, C105, L107, and S112 were found to form a discontinuous epitope recognized by MAb 10H10. These amino acids are remote in the protein sequence but close in the tertiary structure and form a whole epitope located in the structural domain II of protein E. Presumably the localized amino acids bind to the cellular receptor for TBE virus.
Asunto(s)
Antígenos Virales/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Hemaglutininas Virales/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Bacteriófagos/genética , Epítopos/análisis , Epítopos/genética , Epítopos/inmunología , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/análisis , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Alineación de Secuencia , Proteínas del Envoltorio Viral/químicaRESUMEN
Recombinant strains producing hepatitis B virus (HBcAg) core protein and chimeric core protein exposing on its surface the major immunogenic epitope of HBsAg (HBcAg-HBs) were constructed on the base of attenuated S. typhimurium SL 7202 strain. The resultant Salmonella strains produced proteins which were capable of self-assembly into virus-like particles and showed antigenic properties of both core and surface hepatitis B proteins. A single rectal immunization with recombinant S. typhimurium induced humoral and cellular immune response to HBcAg and HBsAg. Specific anti-HBcAg were detected in animal sera and intestinal tissues, which indicated the formation of specific mucosal immunity.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/genética , Salmonella typhimurium/genética , Animales , Epítopos/inmunología , Antígenos del Núcleo de la Hepatitis B/biosíntesis , Antígenos de Superficie de la Hepatitis B/biosíntesis , Inmunidad Celular , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Recombinación Genética , Salmonella typhimurium/inmunologíaRESUMEN
Three new approaches to design effective immunogens are considered. At first, we derived an expression vector from bacteriophage M13 allowing the exposure of short peptides on the virion surface. EIA demonstrates that antibodies against a recombinant phage carrying the antigenic determinant of the HIV-1 gag protein reacted with the 17-kDa core protein of the virus and also with its polyprotein precursor p55 in immunoblotting. In another approach, we chose the hepatitis B core antigen (HBcAg) particle as a vehicle for the presentation of foreign antigenic determinants to the immune system. Chimerical particles of HBcAg containing epitope of the VEE virus were obtained. A vector system for insertion of foreign antigenic determinants and production of both hybrid and wild HBcAg proteins were also obtained. The third approach relies on construction of immunogens from different T- and B-cell epitopes of the HIV-1. We suggested to construct HIV-1 vaccines in a form of the TBI (T- and B-cell epitopes containing Immunogen) with a predetermined tertiary structure, namely, a four-alpha-helix bundle. The gene of the TBI protein consisting of nine HIV-1 epitopes was synthesized and expressed in Escherichia coli cells. Mice immunized with TBI showed humoral and cellular immune responses to HIV-1. Anti-TBI antibodies displayed HIV-1 neutralizing activity. These new approaches offer promise in the development of new effective vaccines.
Asunto(s)
Vacunas contra el SIDA , Antígenos Virales/inmunología , Vacunas Sintéticas , Vacunas Virales , Secuencia de Aminoácidos , Animales , Antígenos Virales/química , Antígenos Virales/genética , Bacteriófago M13 , Secuencia de Bases , Cartilla de ADN , Diseño de Fármacos , Virus de la Encefalitis Equina del Este/genética , Virus de la Encefalitis Equina del Este/inmunología , Virus de la Encefalitis Equina Venezolana/genética , Epítopos/química , Epítopos/inmunología , Escherichia coli , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Genes gag , VIH-1/inmunología , Antígenos del Núcleo de la Hepatitis B/biosíntesis , Antígenos del Núcleo de la Hepatitis B/inmunología , Caballos , Humanos , Ratones , Modelos Estructurales , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Linfocitos T/inmunologíaAsunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Viral de la Expresión Génica , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Recombinante , Virus de la Encefalitis Equina Venezolana/genética , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Plásmidos , Proteínas Virales , alfa 1-Antitripsina/genética , beta-Galactosidasa/genéticaRESUMEN
A hybrid beta-lactamase gene with a synthetic tuftsin-coding DNA fragment inserted at the Pst I-site of pBR322 plasmid has been obtained and its expression has been studied. Radioactive amino acids have been used to show that in E. coli chi 925 minicells up to 30% of newly synthesized chimeric protein is secreted into periplasm providing the tuftsin transport. After hybrid protein cleavage with CNBr, tuftsin has been isolated using ion-exchange and thin-layer chromatography.