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THE PILOT PRODUCTION AND QUALITY CONTROL OF HIV DNA VACCINE FOR THE FIRST PHASE OF  CLINICAL TRIAL

THE PILOT PRODUCTION AND QUALITY CONTROL OF HIV DNA VACCINE FOR THE FIRST PHASE OF  CLINICAL TRIAL Dobrynin P. Biomedical Center St. Petersburg, Russia. HIV-1 genome ( consensus sequence of the FSU subtype A HIV-1 variant ). GAG. POL. ENV. NEF. LTR. LTR. acces s ory genes.

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THE PILOT PRODUCTION AND QUALITY CONTROL OF HIV DNA VACCINE FOR THE FIRST PHASE OF  CLINICAL TRIAL

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  1. THE PILOT PRODUCTION AND QUALITY CONTROL OF HIV DNA VACCINE FOR THE FIRST PHASE OF THE PILOT PRODUCTION AND QUALITY CONTROL OF HIV DNA VACCINE FOR THE FIRST PHASE OF  CLINICAL TRIAL Dobrynin P. Biomedical Center St. Petersburg, Russia

  2. HIV-1 genome (consensus sequence of the FSU subtype A HIV-1 variant) GAG POL ENV NEF LTR LTR accessory genes Equal mixture p7 p17 p24 prot RT int gp120 gp41 p15 vif vpr tat vpu p6 1mg/ml plasmid DNA solution gag rt gp140 nef pBMCgag pBMCrt pBMCenv pBMCnef i.m. immunization The candidate DNA vaccine, "DNA-4"

  3. The amino acid sequences of 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.

  4. Chromatographic purification and obtaining the final form product Preparation of inoculate culture Fermentation and lysis of the biomass Bacterial museum Alkaline lysis and plasmid precipitation Quality control Structure of pilot "DNA4" manufacturing plant

  5. Plasmid DNA precipitation with isopropanol Concentration, diafiltration biomass Alkaline lysis, KOAcprecipitation, filtration Bacterial museum Sephacryl S1000 RNA Plasmid DNA precipitation with PEG 8000 Fermentation А260 Plasmid DNA DNA E.coli Plasmids quality control Plasmids concentration, diafiltration, sterilization Mixing, sterilization, ampouling Quality control Scheme of production and purification of "DNA-4” vaccine

  6. Analysis of the fractions Genomic DNA of E.coli plasmid DNA 6% Concentration anddiafiltration 2% Column purification and regeneration fractions Purification of plasmidpBMC RT(A)-hum 10% OC SC open circular supercoiled

  7. Analysis of the fractions Genomic DNA of E.coli plasmid DNA 8% Concentration anddiafiltration 3,5% Column purification and regeneration fractions Purification of plasmid pBMC Nef(A)-hum 10% OC SC open circular supercoiled

  8. Purification of plasmid pBMC Gp140(A)-hum plasmid DNA Genomic DNA of E.coli Analysis of the fractions 8% 10% OC SC Concentration anddiafiltration 3% open circular supercoiled Column purification and regeneration fractions

  9. Genomic DNA of E.coli Analysis of the fractions plasmid DNA 8% Concentration anddiafiltration 3% Column purification and regeneration fractions Purification of plasmid pBMC Gag(A)-hum 10% OC SC open circular supercoiled

  10. Quality control of drug-DNA vaccine, "DNA-4”

  11. Plasmid identity. Polymerase chain reaction (PCR) followed by agarose gel electrophoresis Electropherograms of PCR productsfor the first three batches of the drug "DNA-4" along the paths : 1, 3, 5, 7 – PCR to detect fragments of genes nef, rt, gag, and gp140, respectively; 2, 4, 6, 8 – corresponding negative controls.

  12. К- К+ К+ DNA contamination. The content of the genomic DNA of host cells. PCR and Southern hybridization.  1ng/1mg Radiographs of the analysis of genomic DNA content of cells of the host in two batches of drugs "DNA-4”(A and B, respectively). As a specific gene fragment probe used CutE E.coli marked with -dTsTF along the paths : 1-6 - Sample calibration curve E.coli genomic DNA with a titer of 1 ng, 500 pg, 50 pg, 10pg, 1 pg and 0.1 pg, respectively; 7-9 - the investigated samples containing 2, 1 and 0.5 mkg of total recombinant DNA, respectively.

  13. М 1 2 3 4 5 6 7 8 М 1 2 3 4 5 6 7 8 Biological activity. Transformation of cell lines 293T (human), RNA isolation, reverse transcription (RT) and PCR followed by electrophoresis. Electropherograms of the analysis of biological activity of the two drugs "DNA-4“ along the paths : 1 – RT-PCR for gene nef; 2 - RT-PCR for gene rt; 3 - RT-PCR for gene gag; 4 - RT-PCR for genegp140. 5, 6, 7, 8 - negative controls for genes nef, rt, gag, gp140, respectively,

  14. Analysis of the distribution of DNA vaccines by intramuscular injection in the bodies of mice Electrophoregram analysis of plasmid DNA in the lung. Electrophoregram analysis of plasmid DNA in the spleen. Electrophoregram analysis of plasmid DNA in muscle  (injection site). Electrophoregram analysis of plasmid DNA in the brain. Electrophoregram analysis of plasmid DNA in the fractions of blood. Electrophoregram analysis of plasmid DNA in the heart.

  15. Distribution and lifetime of the vaccine, "DNA-4” in mice when administered intramuscularly n / a - the point was not analyzed

  16. Analysis of the nef gene expression in various organs after DNA immunization of mice n / a - the point was not analyzed

  17. CD4+lymphocytes CD8+lymphocytes 0.02 % 0.87 % 5.00 % 18.7 % IFNg "DNA-4 " Stimulation control control "DNA-4 " Stimulation IL4 CD69 Stimulation of DNA-vaccine "DNA-4” expression of IFN  and / or IL4 in CD8 + and CD4 + splenocytes in vitro mouse

  18. 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. Results Of Preclinical Trials Of The Candidate DNA Vaccine

  19. 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 ) ) im boost boost The development of T-cell immune response when mice were immunized three times by intramuscular vaccine, "DNA-4"

  20. Analysis of specific antibodies to p24 protein in mice immunized three times, "DNA-4"  

  21. 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. • Increase of HIV-1 proteins expression in eucaryotic cells 293T after gene modification was achieved. • 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. immunization.

  22. Acknowledgments Biomedical Center, Research Institute of Pure Biochemicals, St.Petersburg, Russia: Masharsky A., Murashev B., Murasheva I., Nazarenko O., Smirnova G., Ryzhova T., Sergeeva E., Shevchenko A., Kazennova E., Akulova E., Kolbasov S. Dukhovlinov I., Dorofeyeva E., Galachyants Y.,, Zerov Y., Klimov N., Selezneva L., Kozlov A.

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