RESUMEN
Previously, a mucosal model of immunization against human immunodeficiency virus type 1 (HIV-1) was established by using influenza virus as a vector for the neutralizing gp41 epitope ELDKWA. Whether replication of this chimeric influenza virus in the upper respiratory tract of mice is sufficient for inducing mucosal immune responses in the genital tract was investigated. An immunization strategy was established that permits the virus to replicate in the murine upper respiratory tracts but not in the lungs. Intranasal application of the chimeric virus induced HIV-1-specific antibodies in sera and genital tract. In addition, chimeric virus-specific antibody-secreting cells were detected in lymphocyte populations obtained from lungs, spleens, and urogenital tracts. These results indicate that replication of the chimeric influenza/ELDKWA virus in the upper respiratory tract is sufficient to induce systemic immune responses as well as local immune responses in the genital tract.
Asunto(s)
Anticuerpos Anti-VIH/biosíntesis , VIH-1/inmunología , Virus de la Influenza A/inmunología , Pulmón/inmunología , Mucosa Nasal/inmunología , Virus Reordenados/inmunología , Vagina/inmunología , Vacunas contra el SIDA/inmunología , Administración Intranasal , Animales , Anticuerpos Monoclonales/metabolismo , Quimera , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/genética , Humanos , Inmunidad Mucosa , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Membrana Mucosa/inmunología , Membrana Mucosa/virología , Mucosa Nasal/virología , Fragmentos de Péptidos/inmunología , Virus Reordenados/genética , Vagina/metabolismo , Vagina/virología , Replicación ViralRESUMEN
We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40 degreesC selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.
Asunto(s)
Virus de la Influenza A/crecimiento & desarrollo , Proteínas no Estructurales Virales/fisiología , Animales , Línea Celular , Embrión de Pollo , Chlorocebus aethiops , Perros , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Virus de la Influenza A/fisiología , Ratones , Ratones Endogámicos BALB C , Nucleocápside/biosíntesis , Biosíntesis de Péptidos , Eliminación de Secuencia , Transfección , Células Vero , Proteínas de la Matriz Viral/biosíntesis , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
During the 1996 influenza epidemic in Vienna we obtained influenza A virus specimens (Vienna/47/96, Vienna/81/96) which grow efficiently in African green monkey kidney (Vero) cells but not in embryonated chicken eggs. Amplification of the specimens in Vero cells resulted in progeny that agglutinated human but not chicken erythrocytes. Reassortment analysis suggested that the haemagglutinin (HA) might be responsible for the host restriction. Vero cells were infected with the Vienna/47/96 virus and then transfected with reconstituted ribonucleoprotein complexes containing HA genes from egg-adapted strains. Subsequent selective passages in embryonated chicken eggs resulted in selection of transfectant viruses, growing in eggs and containing the transfected HAs. The results demonstrate that host restriction of the Vero-adapted Vienna/47/96 virus is due to its HA. Moreover, the experiments showed that the Vienna/47/96 strain can be used as helper virus for reverse genetics experiments.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Virus de la Influenza A/genética , Transfección , Animales , Secuencia de Bases , Línea Celular , Embrión de Pollo , Chlorocebus aethiops , ADN Viral , Perros , Glicoproteínas Hemaglutininas del Virus de la Influenza/fisiología , Humanos , Virus de la Influenza A/patogenicidad , Datos de Secuencia Molecular , Células VeroRESUMEN
The baculovirus expression system was utilized to serve as a tool for ligand selection, demonstrating the applicability of the system to the generation and screening of eukaryotic expression libraries. The HIV-1-gp41 epitope 'ELDKWA', specific for the neutralizing human mAb 2F5, was inserted into the antigenic site B of influenza virus hemagglutinin and expressed on the surface of baculovirus infected insect cells. In order to improve the antigenicity of the epitope within the hemagglutinin, and therefore enhance the specific binding of 2F5, we inserted three additional, random amino acids adjacent to the epitope. This pool of hemagglutinin genes was directly cloned into the baculovirus Ac-omega. To identify distinct proteins displayed on the cellular surface, we developed a screening protocol to select for specific binding capacity of individual viral clones. Using fluorescence activated cell sorting (FACS) we isolated a baculovirus clone displaying the epitope with markedly increased binding capacity out of a pool of 8000 variants in only one sorting step. Binding properties of the identified ligand were examined by FACS performing a competition assay.
Asunto(s)
Baculoviridae/genética , Epítopos/biosíntesis , Biblioteca de Péptidos , Transfección/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Baculoviridae/metabolismo , Secuencia de Bases , Línea Celular , Epítopos/química , Citometría de Flujo , Proteína gp41 de Envoltorio del VIH/biosíntesis , Proteína gp41 de Envoltorio del VIH/química , VIH-1/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/biosíntesis , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Ligandos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Alineación de Secuencia , Spodoptera , beta-Galactosidasa/biosíntesisRESUMEN
OBJECTIVE: To investigate whether variations of the conserved gp41 amino-acid sequence ELDKWA affect its binding or neutralization by monoclonal antibody (MAb) 2F5. DESIGN AND METHODS: Neutralization assays were performed with primary isolates from different HIV-1 subtypes and the sequences corresponding to the 2F5 epitope region were analysed. Studies of MAb 2F5 peptide reactivity were performed by spot analysis, using peptides immobilized on cellulose. The frequency of emergence of neutralization-resistant virus variants was determined by immune selection experiments in the presence of MAb 2F5. RESULTS: Primary isolates from clades A, B and E were neutralized by MAb 2F5. Neutralization sensitivity correlated with the presence of the LDKW motif. A K-to-N change in the core sequence was identified in a neutralization-resistant patient isolate. Neutralization resistant virus variants that were selected in the presence of MAb 2F5 were found to contain D-to-N, D-to-E, or K-to-N changes within the LDKW sequence. Neither in natural isolates nor in variants obtained under immune selection conditions in the laboratory were changes in the L and W positions observed. Studies of MAb 2F5 binding to variations of the ELDKWA peptide confirmed that the changes at the first and last positions did not significantly reduce binding capacity, whereas amino-acid changes from D to N, D to E, and K to N almost completely abrogated binding of MAb 2F5. CONCLUSION: Sequence analysis of a variety of primary isolates suggests that the major determinant of MAb 2F5 binding corresponds to the amino-acid sequence LDKW. Naturally occurring and in vitro selected neutralization-resistant viruses contained changes in the D and K positions of the ELDKWA motif.
Asunto(s)
Variación Antigénica , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Anticuerpos Monoclonales/inmunología , Humanos , Análisis de SecuenciaRESUMEN
Previously, we constructed a chimeric influenza virus that expresses the highly conserved amino acid sequence ELDKWA of gp41 of human immunodeficiency virus type 1 (HIV-1). Antisera elicited in mice by infection with this chimeric virus showed neutralizing activity against distantly related HIV-1 isolates (T. Muster, R. Guinea, A. Trkola, M. Purtscher, A. Klima, F. Steindl, P. Palese, and H. Katinger, J. Virol. 68:4031-4034, 1994). In the present study, we demonstrated that intranasal immunizations with this chimeric virus are also able to induce a humoral immune response at the mucosal level. The immunized mice had ELDKWA-specific immunoglobulins A in respiratory, intestinal, and vaginal secretions. Sustained levels of these secretory immunoglobulins A were detectable for more than 1 year after immunization. The results show that influenza virus can be used to efficiently induce secretory antibodies against antigens from foreign pathogens. Since long-lasting mucosal immunity in the genital and intestinal tracts might be essential for protective immunity against HIV-1, influenza virus appears to be a promising vector for HIV-1-derived immunogens.