title: Immunogenicity testing of a novel engineered HIV-1 envelope gp140 DNA vaccine construct.
abstract: DNA vaccines expressing the envelope (env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating strong immune responses. In this study, we described the development of a recombinant plasmid DNA (pEK2P-B) expressing an engineered codon-optimized envelope gp140 gene of primary (nonrecombinant) HIV-1 subtype B isolate 6101. Codon usage and RNA optimization of HIV-1 structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo. To further increase the expression, a synthetic IgE leader with kozak sequences were fused into the env gene. The cytoplasmic tail of the gene was also truncated to prevent recycling. The expression of env by the recombinant pEK2P-B was evaluated using T7 coupled transcription/translation. The construct demonstrated high expression of the HIV-1 env gene in eukaryotic cells as demonstrated in transfected 293-T and RD cells. Immunogenicity of pEK2P-B was evaluated in mice using IFN-gamma ELISpot assay, and the construct was found to be highly immunogenic and crossreactive with HIV-1 clade C env peptides. Three immunodominant peptides were also mapped out. Furthermore, by performing a CFSE flow cytometry-based proliferation assay, 2.4 and 1.5% proliferation was observed in CD4+, CD8+, and CCR+ memory T cells, respectively. Therefore, this engineered synthetic optimized env DNA vaccine may be useful in DNA vaccine and other studies of HIV-1 immunogenicity.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6100, USA.
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Kumar
authors.forename: Jian
authors.initials: J
authors.lastname: Yan
authors.forename: Karuppiah
authors.initials: K
authors.lastname: Muthumani
authors.forename: Mathura P
authors.initials: MP
authors.lastname: Ramanathan
authors.forename: Hanna
authors.initials: H
authors.lastname: Yoon
authors.forename: George N
authors.initials: GN
authors.lastname: Pavlakis
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Felber
authors.forename: Maninder
authors.initials: M
authors.lastname: Sidhu
authors.forename: Jean D
authors.initials: JD
authors.lastname: Boyer
authors.forename: David B
authors.initials: DB
authors.lastname: Weiner
mesh_terms: D016915:AIDS Vaccines / Q000235:genetics* / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003429:Cross Reactions; D004279:DNA, Viral / Q000235:genetics; D018604:Epitope Mapping; D005260:Female; D015870:Gene Expression; D015686:Gene Products, env / Q000235:genetics* / Q000276:immunology*; D015752:Genes, env; D005818:Genetic Engineering; D015488:HIV Antigens / Q000235:genetics / Q000276:immunology; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016176:T-Lymphocyte Subsets / Q000276:immunology; D014162:Transfection; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology; D054299:env Gene Products, Human Immunodeficiency Virus
chemical_list: D016915:AIDS Vaccines; D004279:DNA, Viral; D015686:Gene Products, env; D015488:HIV Antigens; D016056:Immunodominant Epitopes; D019444:Vaccines, DNA; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1