Cellular Immunogenicity of Novel Gene Immunogens in Mice Monitored by in Vivo Imaging

Starodubova, Elizaveta; Krotova, Olga; Holzmann, David; Kuzmenko, Yulia; Engström, Gunnel; Legzdina, Diana; Latyshev, Oleg; Eliseeva, Olesja; Maltais, Anna Karin; Tunitskaya, V. L.; Карпов, В.Л.; Bråve, Andreas; Isaguliants, Maria

doi:10.2310/7290.2012.00011

Cited by 8 publications

(17 citation statements)

References 66 publications

(127 reference statements)

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“…We have earlier shown that diminishment of bioluminescence induced by the expression of reporter gene coinjected with DNA immunogen correlates with the induction of polyfunctional cellular responses against the immunogen [8, 47] specifically with IFN- γ /Il-2 responses of CD4 T cells [8]. To monitor this effect, mice were coinjected with RT gene variants and a reporter gene encoding firefly luciferase (Luc) and assessed for the levels of bioluminescence on days 1, 3, 6, 9, 15, and 22 postimmunization.…”

Section: Resultsmentioning

confidence: 99%

Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice

Latanova

Petkov

Kuzmenko

et al. 2017

Journal of Immunology Research

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Reverse transcriptase (RT) is a key enzyme in viral replication and susceptibility to ART and a crucial target of immunotherapy against drug-resistant HIV-1. RT induces oxidative stress which undermines the attempts to make it immunogenic. We hypothesized that artificial secretion may reduce the stress and make RT more immunogenic. Inactivated multidrug-resistant RT (RT1.14opt-in) was N-terminally fused to the signal providing secretion of NS1 protein of TBEV (Ld) generating optimized inactivated Ld-carrying enzyme RT1.14oil. Promotion of secretion prohibited proteasomal degradation increasing the half-life and content of RT1.14oil in cells and cell culture medium, drastically reduced the residual polymerase activity, and downmodulated oxidative stress. BALB/c mice were DNA-immunized with RT1.14opt-in or parental RT1.14oil by intradermal injections with electroporation. Fluorospot and ELISA tests revealed that RT1.14opt-in and RT1.14oil induced IFN-γ/IL-2, RT1.14opt-in induced granzyme B, and RT1.14oil induced perforin production. Perforin secretion correlated with coproduction of IFN-γ and IL-2 (R = 0,97). Both DNA immunogens induced strong anti-RT antibody response. Ld peptide was not immunogenic. Thus, Ld-driven secretion inferred little change to RT performance in DNA immunization. Positive outcome was the abrogation of polymerase activity increasing safety of RT-based DNA vaccines. Identification of the molecular determinants of low cellular immunogenicity of RT requires further studies.

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Section: Resultsmentioning

confidence: 99%

Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice

Latanova

Petkov

Kuzmenko

et al. 2017

Journal of Immunology Research

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“…This was done by assessing the level of expression in the injection sites of the reporter gene of firefly luciferase, co-delivered with the IN gene variants [83]. As we have recently shown, co-injection of Luc reporter gene with a potent gene immunogen results in a rapid loss of the in vivo reporter activity (decrease of bioluminescence) [83]. Here, co-delivery of Luc and IN genes led to a significant, 10- to 15-fold decrease in the total photon flux from the site of immunization three weeks post immunization.…”

Section: Discussionmentioning

confidence: 99%

Consensus HIV-1 FSU-A Integrase Gene Variants Electroporated into Mice Induce Polyfunctional Antigen-Specific CD4+ and CD8+ T Cells

et al. 2013

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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.

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“…[8][9][10][11] With this in mind, we have undertaken a study of the oxidative stress and oxidative stress response on a panel of HIV-1 reverse transcriptase (RT) genes of high to low immunogenicity. [34][35][36] All RT genes were found to induce oxidative stress in the transfected human embryonic kidney cells. The capacity of an RT gene to induce ROS correlated with its capacity to induce RT-specific IFN-γ responses in mice.…”

Section: Introductionmentioning

confidence: 99%

Oxidative stress induced by HIV-1 reverse transcriptase modulates the enzyme’s performance in gene immunization

Isaguliants

Smirnova

Ivanov

et al. 2013

Human Vaccines & Immunotherapeutics

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quinone oxidoreductase (Nqo1) and heme oxygenase 1 (HO-1), indicating the induction of oxidative stress response. The capacity to induce oxidative stress and stress response appeared to be an intrinsic property of a vast variety of RTs: enzymatically active and inactivated, bearing mutations of drug resistance, following different routes of processing and presentation, expressed from viral or synthetic expression-optimized genes. The total ROS production induced by RT genes of the viral origin was found to be lower than that induced by the synthetic/expression-optimized or chimeric RT genes. However, the viral RT genes induced higher levels of ROS production and higher levels of HO-1 mRNA than the synthetic genes per unit of protein in the expressing cell. The capacity of RT genes to induce the oxidative stress and stress response was then correlated with their immunogenic performance. For this, RT genes were administered into BALB/c mice by intradermal injections followed by electroporation. Splenocytes of immunized mice were stimulated with the RT-derived and control antigens and antigen-specific proliferation was assessed by IFN-γ/IL-2 Fluorospot. RT variants generating high total ROS levels induced significantly stronger IFN-γ responses than the variants inducing lower total ROS, while high levels of ROS normalized per unit of protein in expressing cell were associated with a weak IFN-γ response. Poor gene immunogenicity was also associated with a high (per unit of protein) transcription of antioxidant response element (ARE) dependent phase II detoxifying enzyme genes, specifically HO-1. Thus, we have revealed a direct link between the propensity of the microbial proteins to induce oxidative stress and their immunogenicity.

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Cellular Immunogenicity of Novel Gene Immunogens in Mice Monitored by in Vivo Imaging

Cited by 8 publications

References 66 publications

Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice

Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice

Consensus HIV-1 FSU-A Integrase Gene Variants Electroporated into Mice Induce Polyfunctional Antigen-Specific CD4+ and CD8+ T Cells

Oxidative stress induced by HIV-1 reverse transcriptase modulates the enzyme’s performance in gene immunization

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