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
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 str...
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| Format: | Article |
| Language: | English |
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Wiley
2017-01-01
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| Series: | Journal of Immunology Research |
| Online Access: | http://dx.doi.org/10.1155/2017/7407136 |
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| author | Anastasia Latanova Stefan Petkov Yulia Kuzmenko Athina Kilpeläinen Alexander Ivanov Olga Smirnova Olga Krotova Sergey Korolev Jorma Hinkula Vadim Karpov Maria Isaguliants Elizaveta Starodubova |
| author_facet | Anastasia Latanova Stefan Petkov Yulia Kuzmenko Athina Kilpeläinen Alexander Ivanov Olga Smirnova Olga Krotova Sergey Korolev Jorma Hinkula Vadim Karpov Maria Isaguliants Elizaveta Starodubova |
| author_sort | Anastasia Latanova |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-6e9b7040f8764a29b629bdc06de0204a |
| institution | Kabale University |
| issn | 2314-8861 2314-7156 |
| language | English |
| publishDate | 2017-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Immunology Research |
| spelling | doaj-art-6e9b7040f8764a29b629bdc06de0204a2025-02-03T01:21:47ZengWileyJournal of Immunology Research2314-88612314-71562017-01-01201710.1155/2017/74071367407136Fusion 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 MiceAnastasia Latanova0Stefan Petkov1Yulia Kuzmenko2Athina Kilpeläinen3Alexander Ivanov4Olga Smirnova5Olga Krotova6Sergey Korolev7Jorma Hinkula8Vadim Karpov9Maria Isaguliants10Elizaveta Starodubova11Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, RussiaDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, SwedenEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, RussiaDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, SwedenEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, RussiaEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, RussiaEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, RussiaChemistry Department, Belozersky Research Institute of Physico-Chemical Biology of Lomonosov Moscow State University, Moscow, RussiaLinköping University, Linköping, SwedenEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, RussiaGamaleja Research Center of Epidemiology and Microbiology, Moscow, RussiaEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, RussiaReverse 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.http://dx.doi.org/10.1155/2017/7407136 |
| spellingShingle | Anastasia Latanova Stefan Petkov Yulia Kuzmenko Athina Kilpeläinen Alexander Ivanov Olga Smirnova Olga Krotova Sergey Korolev Jorma Hinkula Vadim Karpov Maria Isaguliants Elizaveta Starodubova 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 Journal of Immunology Research |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| url | http://dx.doi.org/10.1155/2017/7407136 |
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