1 / 15

DNA-IMMUNOGEN BASED ON A CONSENSUS INTEGRASE OF HIV-1 SUBTYPE A

DNA-IMMUNOGEN BASED ON A CONSENSUS INTEGRASE OF HIV-1 SUBTYPE A. Krotova Olga 1,2,3. 1 Engelhardt Institute of Molecular Biology, Moscow (Russia); 2 MTC, Karolinska Institutet, Stockholm (Sweden); 3 Ivanovsky Institute of Virology, Moscow (Russia ). St.-Petersburg , 201 2 г. HIV EPIDEMICS.

petra
Download Presentation

DNA-IMMUNOGEN BASED ON A CONSENSUS INTEGRASE OF HIV-1 SUBTYPE A

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. DNA-IMMUNOGEN BASED ON A CONSENSUS INTEGRASE OF HIV-1 SUBTYPE A Krotova Olga1,2,3 1Engelhardt Institute of Molecular Biology, Moscow (Russia); 2MTC, Karolinska Institutet, Stockholm (Sweden); 3Ivanovsky Institute of Virology, Moscow (Russia). St.-Petersburg, 2012 г.

  2. HIV EPIDEMICS

  3. CONSENSUS INTEGRASE 34 full-length amino acid integrase sequences from treatment naïve patients isolated on the territory of the former Soviet Union (Belarus, Estonia, Georgia, Russia, Ukraine, and Uzbekistan) BioEdit IN_a

  4. 1. IN_a 2. IN_a_e3 + H51Y, E92Q, S147G, E157Q, K160Q 64 64 64 64 D V V D 3.IN_in 1 50 212 288 288 288 288 1 1 1 50 50 50 212 212 212 4. IN_in_e3 + H51Y, E92Q, S147G, E157Q, K160Q pET15b (prokaryotic) pVAX1 (eukaryotic) INTEGRASE DESIGN AMINO ACID SEQ NUCLEOTIDE SEQ Optimization of IN nucleotide sequences for expression: • codon usage (OPTIMIZER) • secondary mRNA structure (UNAFold)

  5. PROKARYOTIC EXPRESSION WB analysis anti-IN • Purification by Ni-NTA agarose chromatography • SDS-PAGE analysis with subsequent staining by Coomassie blue • Western blotting analysis using ployclonal anti-IN antibodies

  6. In vitro INTEGRASE ACTIVITY TESTS U5 = 32P-U5B + U5A U5-2 = 32P-U52 + U5A 3’-processing Strand transfer Unspecific exo- activity

  7. EUKARYOTIC EXPRESSION HeLa cells • IN variants were also expressed in: • HEK293 • NIH3T3

  8. HALF-LIFE OF ACTIVE AND INACTIVE CONSENSUS IN(cycloheximide-chase) anti-IN anti-Actin 11,3 h 4,7 h

  9. PROTEASOMAL DEGRADATION PATHWAY anti-IN anti-Actin

  10. IFNγ FLUOROSPOT experiments IL2 IFNγ/IL2

  11. Intracellular cytokine staining (ICCS) • CD4+ • CD8+ • IFNγ • IL2 • IL4 • TNFα

  12. CONCLUSIONS • Consensus INs whose genes were optimized for eukaryotic expression are expressed at high levels both in eukaryotic and prokaryotic cell lines; • Active consensus integrase is even more active (2-fold) than HXB2 integrase of clade B, while mutation of inactivation totally inhibits integrase activities; • Inactivation mutation was shown to enhance protein half-life 3-fold; • Active consensus integrases degrade via proteasomal pathway, while inactive forms do not; • All IN genes are immunogenicand revealed IFN-gamma/IL2/TNFaprofile of cytokine expression according to in vitro T-cell stimulation tests (Fluorospot and ICCS). RESUME • Inactive variants can be used as components of the multi-gene vaccine against HIV-1; • The consensus gene approach can be applied to other variable viruses, as HCV.

  13. ACKNOWLEDGEMENTS Engelhardt Institute of Molecular Biology, Moscow (Russia) Starodubova Elizaveta Karpov Vadim MTC, Karolinska Institutet, Stockholm (Sweden) Petkov Stefan Viklund Alecia Kostic Linda Hallengärd David Isaguliants Maria Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow (Russia) Agapkina Julia Gottikh Marina Ivanovsky Institute of Virology, Moscow (Russia) Latyshev Oleg Academic Medical Center of the University of Amsterdam, Department of Medical Microbiology, Amsterdam (Netherlands) Lukashov Vladimir

  14. In vivo assessment of immune response: IVIS

More Related