Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: The present invention provides compositions useful in and methods for producing populations of infectious, replication-defective alphavirus replicon particles that contain no replication competent alphavirus particles, as determined by passage on cells in culture. The compositions include helper and replicon nucleic acid molecules that can further reduce the predicted frequency for formation of replication-competent virus and can optimize manufacturing strategies and costs.

Abstract: Viral replicon selected nucleic acid expression libraries are useful for analyzing multiple antigens associated with a parasite, pathogen or neoplasia or for preparing immunogenic compositions for generating immune responses specific for the parasite, pathogen or neoplasia. Alphavirus replicon particles representative of the nucleic acid expression library are preferred. The nucleic acid library can be a random library, or it can be prepared after a selection step, for example, by differential hybridization prior to cloning into the replicon vector.

Abstract: The present invention provides methods and compositions comprising a population of alphavirus replicon particles comprising alphavirus replicon RNAs, wherein a first replicon RNA comprises nucleic acid encoding cytomegalovirus pp65 and IE1 protein or immunogenic fragments thereof, and a second replicon RNA comprises nucleic acid encoding cytomegalovirus gB protein or an immunogenic fragment thereof, and wherein each of the two replicon RNAs is contained within a separate alphavirus replicon particle.

Abstract: The present invention provides compositions useful in and methods for producing populations of infectious, replication-defective alphavirus replicon particles that contain no replication competent alphavirus particles, as determined by passage on cells in culture. The compositions include helper and replicon nucleic acid molecules that can further reduce the predicted frequency for formation of replication-competent virus and can optimize manufacturing strategies and costs.

Abstract: Provided herein are methods for producing alphavirus replicon particles in high yield; replicon RNAs are electroporated into permissive cells, where the cells are at a relatively high density, together with at least one helper nucleic acid providing the necessary functions for packaging. After a growth period in appropriate medium, alphavirus replicon particles are harvested from the surfaces of the cells in which they were produced using a salt wash in which the salt concentration is from about 0.2 to about 5 M sodium chloride, calcium chloride, magnesium chloride, potassium chloride, ammonium acetate, ammonium bicarbonate, among others. After dilution, if necessary, the particles can be purified by a suitable chromatographic technique.

Abstract: The present invention provides compositions useful in and methods for producing populations of infectious, replication-defective alphavirus replicon particles that contain no replication-competent alphavirus particles, as determined by passage on cells in culture. The compositions include helper and replicon nucleic acid molecules that can further reduce the predicted frequency for formation of replication-competent virus and can optimize manufacturing strategies and costs.

Abstract: The present invention provides methods and compositions comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit protease, integrase, RNase H and/or reverse transcriptase activity, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle.

Abstract: The present invention provides methods and compositions comprising a population of alphavirus replicon particles comprising alphavirus replicon RNAs, wherein a first replicon RNA comprises nucleic acid encoding cytomegalovirus pp65 and IE1 protein or immunogenic fragments thereof, and a second replicon RNA comprises nucleic acid encoding cytomegalovirus gB protein or an immunogenic fragment thereof, and wherein each of the two replicon RNAs is contained within a separate alphavirus replicon particle.

Abstract: Viral replicon selected nucleic acid expression libraries are useful for analyzing multiple antigens associated with a parasite, pathogen or neoplasia or for preparing immunogenic compositions for generating immune responses specific for the parasite, pathogen or neoplasia. Alphavirus replicon particles representative of the nucleic acid expression library are preferred. The nucleic acid library can be a random library, or it can be prepared after a selection step, for example, by differential hybridization prior to cloning into the replicon vector.

Abstract: The present invention provides methods and compositions comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a g&agr;g gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the g&agr;g gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the g&agr;g gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit protease, integrase, RNase H and/or reverse transcriptase activity, and wherein the nucleic acids are each contained within a separate alphavirus replicon

Abstract: Provided herein are methods for producing alphavirus replicon particles in high yield; replicon RNAs are electroporated into permissive cells, where the cells are at a relatively high density, together with at least one helper nucleic acid providing the necessary functions for packaging. After a growth period in appropriate medium, alphavirus replicon particles are harvested from the surfaces of the cells in which they were produced using a salt wash in which the salt concentration is from about 0.2 to about 5 M sodium chloride, calcium chloride, magnesium chloride, potassium chloride, ammonium acetate, ammonium bicarbonate, among others. After dilution, if necessary, the particles can be purified by a suitable chromatographic technique.

Abstract: The present invention provides compositions useful in and methods for producing populations of infectious, replication-defective alphavirus replicon particles that contain no replication-competent alphavirus particles, as determined by passage on cells in culture. The compositions include helper and replicon nucleic acid molecules that can further reduce the predicted frequency for formation of replication-competent virus and can optimize manufacturing strategies and costs.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: The present invention provides methods and compositions comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit protease, integrase, RNase H and/or reverse transcriptase activity, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as is compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.