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Progress and perspectives for DNA vaccines in Russia

Progress and perspectives for DNA vaccines in Russia B. Murashev, O. Nazarenko, E. Akulova, S. Verevochkin, M. Astanina, T. Krasnoselskih, D. Leoznov, E. Sokolovskiy, A. Kozlov. HIV-1 genome ( consensus sequence of the FSU subtype A HIV-1 variant ). GAG. POL. ENV. NEF. LTR. LTR.

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Progress and perspectives for DNA vaccines in Russia

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  1. Progress and perspectives for DNA vaccines in Russia B. Murashev, O. Nazarenko, E. Akulova, S. Verevochkin, M. Astanina, T. Krasnoselskih, D. Leoznov, E. Sokolovskiy, A. Kozlov

  2. HIV-1 genome (consensus sequence of the FSU subtype A HIV-1 variant) GAG POL ENV NEF LTR LTR accessory genes gag rt gp140 nef p7 p17 p24 prot RT int gp120 gp41 p15 vif vpr tat vpu p6 DNA-4 vaccine contained an equimolar mixture of four plasmids insaline solutionwith an overall concentration of 1 mg/mL Study Product

  3. Amino Acid Sequences of the Proteins Expressed by the Candidate DNA Vaccine 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.

  4. Plasmid DNA precipitation with isopropanol Alkaline lysis and KOAcprecipitation Protein precipitation with ammonium acetate Plasmid DNA precipitation with PEG 8000 Fermentation RNA Sephacryl S1000 А260 SC plasmid DNA DNA E.coli Quality control, immunization Large-scale Production of Plasmid DNA

  5. Plasmid DNA Fractions E.coli DNA 8% Concentration and diofiltration 3% Column purification Fraction Сhromatographic purificationpBMC Gag(A)-humplasmid 10% OC SC OC SC

  6. Quality Control of DNA Vaccines Purified by Chromatography

  7. Results of preclinical trials of the candidate DNA vaccine • DNA vaccine was not toxic for laboratory animals in acute toxicity experiments and corresponded to the 5th class of practically non-toxic substances. • Lethal doses LD50, LF16 and LD84 were impossible to estimate. The highest administered doses were 4 orders of magnitude higher than the proposed immunization dose. • DNA vaccine had no allergenicity. • DNA vaccine was not toxic after chronic administration in rats and dogs. • DNA vaccine had no pyrogenic effect.

  8. 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), % CD4 analysis CD4+, IFN+/IL4+ (minus control), % Time Time ( ( weeks weeks ) ) boost boost Immune Response Development After Three Serial i.m. Injections of the DNA Vaccine

  9. Antibody Titer Assessment to Antigen Р24 in Mice Sera after Three Serial Injections of the DNA Vaccine

  10. Summary • The candidate DNA vaccine consisting of four plasmids containing modified fragments of four subtype A HIV-1 genes (env, gag, pol, and nef) of was constructed. • Chromatographically purified DNA vaccine preparation meets all FDA и EMEA criteria for purity. • The preclinical studies of the candidate DNA vaccine preparations performed on laboratory animals demonstrated no toxicity and allergenicity. • Efficient stimulation of vaccine specific CTL, CD8+ IFNγ+ lymphocytes and induction of Th1 response were observed after the i.m. priming.

  11. Study Schema of Phase I trial of a prophylactic HIV-1 DNA-vaccine in St. Petersburg, Russia Demographics

  12. • • Biochemical blood examination Biochemical blood examination Blood Blood • • ELISA ELISA • • HIV HIV - - testing testing • • Blood processing and PBMC cryopreservation Blood processing and PBMC cryopreservation N N - - freezer frizer 2 2 g g g 1. IFN 1. IFN 1. IFN - - - ELISpot ELISpot ELISpot g g g a a a 2. ICS 2. ICS 2. ICS - - - IFN IFN IFN /TNF /TNF /TNF /IL /IL /IL - - - 2/CD3/CD8 2/CD3/CD8 2/CD3/CD8 Control K Control K Control K Control K - - - - flow cytometry (BC EPICS XL) flow cytometry (BC EPICS XL) flow cytometry (BC EPICS XL) 3 3 3 . CFSE . CFSE . CFSE - - - LPA LPA LPA CD8 CD8 CD8 &CD8 &CD8 &CD8 + + + - - - flow cytometry (BC EPICS XL) flow cytometry (BC EPICS XL) flow cytometry (BC EPICS XL) Nef Nef Nef Nef - - - - pool pool pool pool - - - - - - - - + + + + + + + + CD3 CD3 CD3 CD3 CD CD CD CD 8 8 8 8 CD3 CD3 CD3 CD3 CD CD CD CD 8 8 8 8 g g g + + + + - - - - CD3 CD3 CD3 CD3 CD CD CD CD 8 8 8 8 IFN IFN IFN Gag Gag Gag Gag - - - - pool pool pool pool RT RT RT RT - - - - pool pool pool pool a a a TNF TNF TNF Env Env Env Env - - - - pool pool pool pool CEF CEF CEF CEF - - - - pool pool pool pool IL IL IL - - - 2 2 2 PHA PHA PHA PHA Methods

  13. IFNγ-ELISpot CFSE LPA ICS PBMCs proliferation, % % of T cells producing IFNg and/or TNFa and/or IL-2 SFU/106PBMC CD3+CD8+ p>0.07 CD8+ p>0.45 CD3+CD8- p>0.08 CD8- p>0.91 p>0.13 Dependence on groups/doses IFNγ-ELISPOT (square), ICS (circle) and CFSE LPA (triangle) results dependence on groups/doses. CD3+CD8+ (black circle), CD3+CD8- (open circle), CD8+ (black triangle) and CD8- (open triangle), medians (red dotted line) with interquartile range (black line). p - Kruskal-Wallis test (ANOVA). SFU – Spot Forming Units, PBMC- Peripheral Blood Mononuclear Cells

  14. IFNγ-ELISpot ICS CFSE LPA 150 100 PBMCs proliferation, % SFU/106PBMC % of T cells producing IFNg and/or TNFa and/or IL-2 50 0 14 25 40 60 Days CD8- p>0.45 CD8+ p>0.70 CD3+CD8+ p>0.06 CD3+CD8- p>0.18 p>0.48 Dependence on visits IFNγ-ELISPOT (square), ICS (circle) and CFSE LPA (triangle) results dependence on visits. CD3+CD8+ (black circle), CD3+CD8- (open circle), CD8+ (black triangle) and CD8- (open triangle), medians (red dotted line) with interquartile range (black line). p - Kruskal-Wallis test (ANOVA). SFU – Spot Forming Units, PBMC- Peripheral Blood Mononuclear Cells.

  15. IFNγ-ELISpot ICS CFSE LPA SFU/106PBMC PBMCs proliferation, % % of T cells producing IFNg and/or TNFa and/or IL-2 p>0.08 CD3+CD8+ p>0.44 CD3+CD8- p>0.45 CD8+ p>0.66 CD8- p>0.65 Dependence on antigens IFNγ-ELISPOT (square), ICS (circle) and CFSE LPA (triangle) results dependence on antigens (peptide pools). CD3+CD8+ (black circle), CD3+CD8- (open circle), CD8+ (black triangle) and CD8- (open triangle), medians (red dotted line) with interquartile range (black line). p - Kruskal-Wallis test (ANOVA). SFU – Spot Forming Units, PBMC- Peripheral Blood Mononuclear Cells. Nef, Gag, RT and Gp140 – peptide pools, spanning HIV-1 subtype A-EE proteins, consisted of 15-mer peptides overlapping by 11 aa. Peptides were synthesized in State Research Institute of Highly Pure Biopreparations (St. Petersburg, Russia).

  16. p**<0.002 p**<0.0001 % of T cells producing two or three cytokines % of T cells producing IFNg or TNFa or IL-2 IL2 IL2 TNFa TNFa IFNg IFNg IFNg/IL2 IFNg/IL2 TNFa/IL2 TNFa/IL2 IFNg/TNFa IFNg/TNFa IFNg/TNFa/IL2 CD3+CD8- IFNg/TNFa/IL2 CD3+CD8+ CD3+CD8- p*<0.02 CD3+CD8+ p*<0.006 CD3+CD8- p*<0.01 CD3+CD8+ p*>0.17 ICS cytokine expression profile results CD3+CD8+ (black circle), CD3+CD8- (open circle), medians (red dotted line) with interquartile range (black line). Left axis - monocytokines, right axis - two or three cytokines. p* - the comparisons across groups were performed using the Kruskal-Wallis analysis of variance (ANOVA) test. p** - Mann-Whitney test.

  17. Summary results

  18. Conclusions • DNA-4 vaccine is well tolerated. • Vaccination with DNA-4 regimen elicited moderate CD4+ and CD8+ T-cell responses, but modest humoral responses. • This regimen should be considered as part of future vaccine strategies.

  19. Cloning and analysis of full-length genomes of HIV-1 Biomedical Center, Research Institute of Pure Biochemicals, St.Petersburg, Russia: Masharsky A., Murashev B., Klimov N. Research Institute for Epidemiology and Microbiology, Minsk, Belarus: Eremin V. Institute of Epidemiology and Infectious Diseases, Kiev, Ukraine: Kravchenko O., Shcherbinskaya A. Clinical trials Pavlov Medicine University, St.Petersburg, Russia: Sokolovsky E., Krasnoselskih T., Lioznov D. Biomedical Center, Research Institute of Pure Biochemicals, St.Petersburg, Russia: Murashev B., Nazarenko O., Akulova E., Verevochkin S., Astanina M. Design and purification of DNA vaccines against HIV-1 Biomedical Center, Research Institute of Pure Biochemicals, St.Petersburg, Russia: Murashev, B., Kazenova E., Romanovich A., Murasheva I., Pavlova M., Kreslavskiy T., Dukhovlinova E., Dorofeyeva E., Galachyants Y., Klimov N. Pre-clinical trials of HIV vaccines Biomedical Center, Research Institute of Pure Biochemicals, Research Institute for Toxicology St.Petersburg, Russia:Murashev B., Klimov N., Dukhovlinova E., Dukhovlinov I., Murashova I., Smirnova I., Kobatov A., Nikonov B., Kolbasov S. Sechenov Medical Academy, Moscow, Russia: Boichenko M., Vorobiev A Dr. A.P. Kozlov - The Biomedical Center and St. Petersburg State University, St. Petersburg, Russia Collaborators:

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