1 / 37

Development of HIV vaccine in Russia

Development of HIV vaccine in Russia. A.P.Kozlov The Biomedical Center, Research Institute of Pure Biochemicals and St.Petersburg University St. Petersburg, Russia. The History of Russian HIV Vaccine Project.

hayden
Download Presentation

Development of HIV vaccine in Russia

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. Development of HIV vaccine in Russia A.P.Kozlov The Biomedical Center, Research Institute of Pure Biochemicals and St.Petersburg University St. Petersburg, Russia

  2. The History of Russian HIV Vaccine Project 1994 –conference «Concept of National AIDS Policy» (St.Petersburg). Suggestion to organize national HIV vaccine project is included into the documents of the conference 1996 – failure to incorporate HIV vaccine project into the Federal Budget 1997 – program «Vaccines of new generation…» is incorporated into Federal Budget under the Ministry of Science and Technologies. Russian HIV vaccine project is a part of the program 1998-2005 – realization of the project 2006 – decision to prolong the term of the program is made 2008-2010 – realization of the second phase of the program May 1997–The US PresidentClintonannounced HIV vaccinedevelopment program June 1997 –G8 Summit in Denver (USA) decided to provide the resources necessary to accelerate AIDS vaccine research, and enhance international scientific cooperation and collaboration 2006 –G8 Summit in St.Petersburg (Russia) confirmed the commitment to HIV vaccine development

  3. HIV Vaccine research in Russia St.Petersburg: Biomedical Center and Research Institute of Highly Pure Biochemicals, SPb State University – DNA Vaccine Moscow: Institute of Immunology – Recombinant proteins "HIVrepol" (p24/gp41) Novosibirsk: Vector Laboratories – virus-like particle polyepitope DNA/protein vaccine All candidate vaccines passed phase I clinical trials

  4. 1995 (5%) Others (1%) (1%) 2009 A (93%) HIV subtypes in Russia (1995-2009) Bobkova et al., 2010

  5. Subtypes A and B introduction to IDUs in the FSU in the early 90ies A A/B A A B A A

  6. Subtypes A and B introduction to IDUs in the FSU in the early 90ies A/B A A/B A A/B A/B A A/B A A/B A A/B B A A A

  7. THE SCHEME OF AMPLIFICATION AND CLONING OF FULL-LENGTH HIV-1 GENOME vpu 5'LTR 3'LTR rev vpr pol gag vif env nef 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 bp tat VIIa III V I II IV VI VIIb VII

  8. THE FULL-LENGTH GENOME HIV-1 PHYLOGENY CRF03-AB F 98BY10443 K 03-AB.KAL153 03-AB.98RU001 C B H D J A2 98UA0116 G A.97BL006 A-FSU A1

  9. HIV-1 subtype A genetic diversity in the FSU

  10. 0,3 0,29 0,28 0,27 0,26 0,25 0,24 0,23 0,22 0,21 0,2 0,19 0,18 0,17 0,16 0,15 0,14 0,13 0,12 0,11 Pairwise distance 0,1 0,09 0,08 0,07 0,06 0,05 0,04 0,03 0,02 0,01 200 400 600 800 1 000 1 200 1 400 1 600 1 800 2 000 2 200 2 400 2 600 2 800 3 000 3 200 3 400 3 600 3 800 4 000 4 200 4 400 4 600 4 800 5 000 5 200 5 400 5 600 5 800 6 000 6 200 Window: 400 bp, Step: 10 bp, GapStrip: On, J-C Correction: On Position (bp) Genetic distance scanning analysis of the FSU subtype A HIV-1 strains (20 full-length genomes from the FSU) SimPlot

  11. 0,3 0,29 0,28 0,27 0,26 0,25 0,24 0,23 0,22 0,21 0,2 0,19 0,18 0,17 0,16 0,15 0,14 0,13 0,12 Pairwise distance 0,11 0,1 0,09 0,08 0,07 0,06 0,05 0,04 0,03 0,02 0,01 200 400 600 800 1 000 1 200 1 400 1 600 1 800 2 000 2 200 2 400 2 600 2 800 3 000 3 200 3 400 3 600 3 800 4 000 4 200 4 400 4 600 4 800 5 000 5 200 5 400 5 600 5 800 6 000 6 200 Position (bp) Window: 400 bp, Step: 10 bp, GapStrip: On, J-C Correction: On Genetic distance scanning analysis of the FSU subtype A HIV-1 strains (20 full-length genomes from the FSU) SimPlot pairwise genetic distance < 8.6%

  12. env, 7044-7259 bp of HIV-1HXB2 genome 9,0 8,0 7,0 6,0 5,0 Distance to consensus, % 4,0 3,0 2,0 1,0 0,0 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005- 2007- 2006 2009 n = 64471818 41100 111 12 23 27 48 median = 0,50,51,41,21,41,41,91,94,43,4 3,7 max =3,4 2,9 3,4 4,9 8,1 7,4 5,4 3,4 8,6 7,4 8,8 509subtype A (A-FSU) HIV-1 strains: Russia - 263, Ukraine - 53, Belarus - 44, Kazakhstan - 16, Uzbekistan - 25, Moldova - 6, Latvia - 100, Lithuania -1, Georgia -1. maximum upper quartile median lower quartile minimum Genetic diversity of HIV-1 strains collected in the FSU countries in 1996-2009 Dukhovlinova et al., 2009

  13. High HIV-1 incidence and low genetic diversity of subtype A FSU provide a window of opportunity for HIV vaccine efficacy trials in Russia

  14. Development of DNA vaccine technology Plasmid DNA injection induced in mice antibodies against protein coded by plasmid DNA Intramuscular injection of plasmid DNA coding influenza antigens induced protective immune response in mice Advantages of DNA vaccines: • DNA vaccine does not replicate in organism and does not revert to pathogetic form. • DNA vaccine is not immunogenic and allergenic by itself. • The same technology allows to produce DNA vaccines against different pathogens. • DNA vaccines are simple in development and production, stable during the storage. • DNA immunization allows to induce either cellular or humoral immune responses or their combination.

  15. Induction of cellular and humoral immune responses after DNA vaccination Kutzler et al., 2008

  16. The share of DNA platform in clinical trials in 2007 Kutzler et al., 2008

  17. DNA vaccine against HIV «DNA-4» HIV-1 genome (consensus sequence of the FSU subtype A HIV-1 variant) GAG POL ENV NEF LTR LTR accessory genes p7 p17 p24 prot RT int gp120 gp41 p15 vif vpr tat vpu p6 gag rt gp140 nef Equimolar mixture of 4 plasmids 1 mg/ml plasmid DNA in saline pBMCgag i.m. immunization pBMCrt pBMCenv pBMCnef

  18. Amino acid sequences of HIV proteins expressed by DNA vaccine «DNA-4» Gag MGARASVLSGGKLDAWEKIRLRPGGKKKYRIKHLVWASRELERFALNPSLLETSEGCQQILEQLQPTLKTGSEEVKSLYNTVATLYCVHQRIEIKD TKEALDKIEEIQNENKQKTQQATGTGSSSKVSQNYPIVQNAQGQMTHQSMSPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDSNMMLNIVGG HQAAMQMLKDTINEEAAEWDRLHPAQAGPFPPGQMREPRGSDIAGTTSTLQEQIGWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPVSILDIRQGP KEPFRDYVDRFFKTLRAEQATQEVKNWMTETLLVQNADPDCKAILRALGPGATLEEMMTACQGVGGPGHKARVLAEAMSQVQNANIMMQKSNFRGP KRIKCFNCGKEGHLARNCRAPRKKGCWKCGKEGHQMKNCTERQANFLGRIWPSSKGRPGNFPQSRPEPSAPPAEDFGRGEEITPPLKQEQKDREQH PPSISLKSLFGNDPLSQ RT MPISPIETVPVTLKPGMDGPKVKQWPLTEEKIKALTDICKEMEKEGKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIP HPAGLKKKKSVTVLHVGDAYFSVPLDESFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPSIFQSSMTKILEPFRLKNPEIVIYQYMHHLYVGS DLEIGQHRTKIEELRAHLLSWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIMLPDKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAK ALTDIVTLTEEAELELAENREILKEPVHGVYYDPSKDLVAEIQKQGQDQWTYQIYQEPFKNLKTGKYAKKGSAHTNDVKQLTAVVQKVATEGIIIW GKTPKFRLPIQKETWEAWWMEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETF Nef M—KWSKSSIVGWPQVRERIRRAPAPAARGVGPVSQDLDKHGAVTSSNTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGAFDLSHFLKEKGG LDGLIYSKKRQEILDLWVYHTQGYFPDWQNYTPGPGIRFPLTFGWCYKLVPVDPAEVEEATEGENNSLLHPICQHGMDDEEKEVLMWKFDSRLALT HRARELHPEFYKDC gp140 MDAMKRGLCCVLLLCGAVFVSPSKAAENLWVTVYYGVPVWRDAETTLFCASDAKAYDKEVHNVWATHACVPTDPDPQEIALENVTEKFDMWKNNMV EQMQTDIISLWDQSLKPCVKLTPLCVTLNCAEPSSTSSNNSSVNSNSSDGLFEEMKNCSFNMTTELRDKRKTVHSLFYKLDIVSTGSNGSGQYRLI NCNTSAMTQACPKVTFEPIPIYYCAPAGFAILKCKDTNFTGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEKEVMIRSENITDNGKIIIVQLTE PVNITCIRPGNNTRTSIRIGPGQTFYATGDVIGDIRKAYCNVSRAAWNSTLQKISTQLRKYFNNKTIIFKNSPGGDLEVTTHSFNCGGEFFYCNTT DLFNSTWDENGTVTNSTKANGTITLPCRIKQIINMWQRVGQAMYAPPIKGSIRCESNITGLLLTRDGGGGTNSSNETFRPIGGNMRDNWRSELYKY KVVKIEPIGVAPTRA-(cleavage site and fusion peptide, 500-534aa)-TVQARQLLSGIVQQQSNLLRAIEAQQHLLKLTV WGIKQLQARVLAVERYLKDQQ-(region between two heptad repeats, 589-618aa)-IWDNMTWMQWDREVINYTDIIYDLIE KSQNQQEKNEQDLLALDKWASLWSWFDISNW-(transmembrane and cytoplasmic region, 676-860aa) All amino acid sequences correspond to consensus sequence of the FSU subtype A HIV-1 variant.

  19. control control w.t. nef w.t. gag syn. gag syn. nef 1/1 syn. nef 1/50 syn. nef 1/10 33kDa 72kDa 54kDa 24kDa Nef Gag Expression of native and synthetic HIV-1 genes in 293T cells in vitro

  20. CD4+ cells CD8+ cells IL4 0.02 % 0.87 % IFN 5.00 % 18.7 % «DNA-4» stimulation Control Control «DNA-4» stimulation CD69 Unspecific stimulation of IFN and/orIL4expression in CD8+ иCD4+ mice splenocytes by DNA vaccine in vitro

  21. Examination of the HIV-1 DNA vaccine toxicity in rodents, dogs and rabbits DNA vaccine is not toxic for laboratory animals, belongs to the 5th class of practically nontoxic substances. Lethal doses LD50, LD16 and LD84 were impossible to be estimated. The highest administered doses were 4 orders of magnitude higher the proposed immunization dose. Has no allergic effect. The vaccine is not toxic after chronic administration in rats and dogs. Has no pyrogenic effect in rabbits. The vaccine may be recommended for conducting phase I of clinical trails.

  22. CTL analysis 8 E:T 10:1 lysis E:T 20:1 6 E:T 50:1 (minus control), % 4 Specific 2 0 0 1 2 3 4 5 6 7 8 9 10 11 12 Time ( weeks ) CD8 analysis CD8+, IFN+ (minus control), % Time ( weeks ) CD4 analysis CD4+, IFN+/IL4+ (minus control), % Time ( weeks ) imm boost boost Cellular immune response development after thrice-repeated i.m. immunization with HIV DNA vaccine in mice

  23. Analysis of antibodies against HIV-1 p24 after thrice-repeated i.m. immunization with HIV DNA vaccine in mice  

  24. Scheme of large-scale production of HIV DNA vaccine «DNA-4» Ethanol precipitation of plasmid DNA Diafiltration of biomass Lysis of biomass and lysate filtration E. coli museum Filtration of DNA precipitate Fermentation Sephacryl S1000 Quality control of individual plasmids Concentration and diafiltration of plasmid DNA RNA А260 Plasmid DNA E.coli DNA Mixing, sterilization, ampouling, marking Final quality control

  25. Purification of plasmid pBMC RT(A)-hum Analysis of fractions Genomic DNAE.coli Plasmid DNA 6% 10% Relaxed SC Concentration and diafiltration 2% Relaxed SC Washing and regeneration of column Fractions

  26. Lots of HIV DNA vaccine "DNA-4" for clinical trials

  27. Scheme of phase I clinical trials of vaccine "DNA-4" 4 раза 4 раза 4 раза

  28. Analysis of immune response on HIV DNA vaccine "DNA-4" in clinical trials

  29. Pool of peptides overlapping HIV-1 subtype A proteins Length of peptides - 15 aa Overlapping - 11 aa

  30. control PHA Sample 1 Sample 2 Sample 3 Sample 4 ELISPOT analysis

  31. Proliferation analysis of CFSE labelled T cells by cytofluorimeter

  32. Single cytokine Two cytokines Three cytokines CD3-CD8- CD3+CD8+ CD3+CD8- IL-2 TNF CD3/CD4 IFN TNF IFN CD3/CD8 IL-2 CD3/CD4 CD3-CD4- CD3+CD4+ CD3+CD4- TNF IFN IL-2 CD3/CD4 IL-2 TNF CD3/CD4 Intracellular cytokine staining: IFN/TNF/IL-2/CD3/CD4&8

  33. Conclusions • The candidate DNA vaccine “DNA-4” is safe and immunogenic • in animals • 2. The Phase 1 clinical trial of the candidate DNA vaccine “DNA-4” have • shown that it is safe in humans. Immunogenicity of this vaccine • in trial participants is being studied

  34. Acknowledgements The Biomedical Center and State Research Institute of Highly Pure Biopreparations, St. Petersburg, Russia: Masharsky A., Verevochkin S., Murashev B., Dukhovlinova E., Shaboltas A., Toussova O., Skochilov R., Krasnoselskikh T., Nabatov A., Klimov N., Dukhovlinov I., Murasheva I., Gagarina S., Timofeeva M., Levchenko Yu., Nazarenko O., Akulova E., Ryzhova T., Al-Shekhadat R., Shevchenko A. et al.

More Related