title: Consensus HIV-1 FSU-A integrase gene variants electroporated into mice induce polyfunctional antigen-specific CD4+ and CD8+ T cells.
abstract: Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN) of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V) with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids) were highly expressed in human and murine cell lines (>0.7 ng/cell). Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus-infected cells via both MHC class I and II pathways.
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Olga
authors.initials: O
authors.lastname: Krotova
authors.forename: Elizaveta
authors.initials: E
authors.lastname: Starodubova
authors.forename: Stefan
authors.initials: S
authors.lastname: Petkov
authors.forename: Linda
authors.initials: L
authors.lastname: Kostic
authors.forename: Julia
authors.initials: J
authors.lastname: Agapkina
authors.forename: David
authors.initials: D
authors.lastname: Hallengärd
authors.forename: Alecia
authors.initials: A
authors.lastname: Viklund
authors.forename: Oleg
authors.initials: O
authors.lastname: Latyshev
authors.forename: Eva
authors.initials: E
authors.lastname: Gelius
authors.forename: Tomas
authors.initials: T
authors.lastname: Dillenbeck
authors.forename: Vadim
authors.initials: V
authors.lastname: Karpov
authors.forename: Marina
authors.initials: M
authors.lastname: Gottikh
authors.forename: Igor M
authors.initials: IM
authors.lastname: Belyakov
authors.forename: Vladimir
authors.initials: V
authors.lastname: Lukashov
authors.forename: Maria G
authors.initials: MG
authors.lastname: Isaguliants
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D024882:Drug Resistance, Viral / Q000235:genetics* / Q000276:immunology; D018274:Electroporation; D004926:Escherichia coli; D005434:Flow Cytometry; D019427:HIV Integrase / Q000096:biosynthesis / Q000235:genetics*; D019428:HIV Integrase Inhibitors / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000276:immunology; D006801:Humans; D008156:Luciferases; D008213:Lymphocyte Activation / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D015363:Quinolones
chemical_list: D019428:HIV Integrase Inhibitors; D015363:Quinolones; C509700:elvitegravir; D008156:Luciferases; D019427:HIV Integrase