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Towards An HIV-1 Vaccine A.P. Kozlov The Biomedical Center Russian-German Cooperation Forum “Biotechnology – Life Sciences” St. Petersburg, Russia September 26, 2006 Confirmation of the first cases of HIV infection in St.Petersburg in 1987 Kozlov et al. 1987 AMPLIFICATION AND CLONING OF FULL-LENGTH HIV-1 GENOME vpu 5'LTR vpr vif pol gag 3'LTR rev env nef tat 0 1000 I 2000 3000 III II 4000 5000 PCR 6000 7000 VIIa V IV 9000 п.н. 8000 VI VIIb VII Cloning THE FULL-LENGTH GENOME HIV-1 PHYLOGENY F K CRF03-AB 98BY10443 03-AB.KAL153 03-AB.98RU001 C B H D J A2 G 98UA0116 A.97BL006 A1 A-FSU HIV-1 SUBTYPES IN THE FORMER SOVIET UNION Other B subtypes A/B 10% 5% 5% REFERENCES •Bobkov et al., 1997; 1998; 2000. •Liitsola et al., 1998; 2000. •Lukashov et al., 1998; 1999. •Masharsky et al., 1999. A 80% •Nabatov et al., 1997; 1998. •Novitsky et al., 1998. HIV DNA-vaccine concept - DNA-vaccine inducing strong CD8+, CD4+ T-cell response - Based on HIV subtype А - Contains 4 HIV genes: gag, pol, nef and gp120 - For strengthening the expression all four genes must be modified: codon optimization, RRE and INS destruction - Two forms of DNA-vaccine: DNA solution for injections and tablets containing live attenuated Salmonella thyphimurium cells containing the same DNAs - The combination of these two vaccine forms in primeboost vaccination procedure is hoped to induce both CD8+, CD4+ T-cell response and antibody B-cell anti-HIV response The pBMC plasmid used as a vector for the production of DNA vaccines against HIV-1 T7 Bgl II pCMV Amp R pBMC 3265 bp Nhe I Apa I Xba I Xho I Not I EcoR V EcoR I BamH I Kpn I Hind III BGH polyA BGH Pvu II ColE1 Ori HIV-1 genome (GenBank #AF413987) GAG LTR p17 p24 POL p7 p6 prot RT accesory genes p15 int vif vpr tat vpu ENV gp120 NEF LTR gp41 gag rt gp160 nef pBMCgag pBMCrt pBMCenv pBMCnef Two forms of vaccine preparations production pBMCgag pBMCrt pBMCenv pBMCnef pBMCgag pBMCrt pBMCgag pBMCrt 1mg/ml plasmid DNA vaccine pBMCnef Transformation Equimolar mixing pBMCgag pBMCenv pBMCrt pBMCenv pBMCnef Salmonella typhimurium-T10 pBMCenv pBMCnef Cultivation i.m. immunization p.o. immunization Lyophilized tablet 1,21010 CFU/tablet Purification of DNA vaccines Fermentation Alkaline lysis and KOAc precipitation Plasmid DNA precipitation with isopropanol RNA First purification Plasmid DNA Plasmid DNA precipitation with PEG 8000 OC DNA E.coli Sephacryl S1000 pBMCrt pBMCgag Final plasmid forms pBMCenv 0 pBMCnef А26 Second purification А260 CC Protein precipitation with ammonium acetate Quality control, immunization The quality of DNA vaccines purified by chromatography Parameter Method DNA vaccines Supercoiled DNA Agarose gel electrophoresis > 90% RNA contaminant Agarose gel electrophoresis Not detected E.coli DNA Southern blot <10 ng/mg Purity Spectrophotometric scanning =210-300 nm А260/А280 >1,75 А260/А230 >1,9 Protein BCA-assay < 10 ng/mg pDNA Endotoxin Endotoxin < 0,05 ng/mg pDNA Plasmid identity Restriction analysis, PCR, sequencing + Flow diagram of experimental immunization of mice with a DNA vaccine and analysis of immune responses in the mice Cytokine boosting DNA vaccine Production of new lines of target cells Cytotoxicity test CD8+ TCR MHC I Target cell effector cell % of specific lysis CD8 peptide spleen blood Time course of the response Response Recombinant proteins of HIV-1 Stimulation of cytokine secretion (IFN) ELISA time Blood and washout Ig levels Vaccine trials in Russian Federation World Health Organization Recommendations National regulations for conducting pre-clinical and clinical trials The goals of pre-clinical trials are to evaluate: Stability (shelf life time) of new vaccine at various temperatures of storage Immunogenicity of the new vaccine Toxicity of the new vaccine using three species of animals, including: 1. Acute toxicity trials: several groups of animals are administered once with different vaccine doses followed by daily examination during 14 days. After euthanasia on day 15 histological examination of internal organs is conducted. Lethal doses LD50, LD16 and LD84 are estimated. 2. Chronic toxicity trials: everyday administration of vaccine during 30-days period, examination, analysis of clinical, biochemical and haematological parameters during 37-days period, pathological and histological examination of internal organs after 37-day period. Allergenicity: evaluation of anaphylactic ability, development of immune complexes in the skin, evaluation of mast cells degranulation and evaluation of allergenicity by conjuctive probe. Pyrogenicity (for injectable forms): direct measurement of rectal temperature in rabbits All trials must be done using three pilot lots of vaccine. DNA-vaccine DNA-vaccine: - is not toxic for laboratory animals in acute experiments, belongs 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; - has no allergenicity; - is no toxic after chronic administration in rats and dogs; - has no pyrogenic effect. Oral attenuated Salmonella-based DNA-vaccine Salmonella-based vaccine: - is not toxic for laboratory animals in acute experiments, LD50, LD16 and LD84 are 2 orders of magnitude higher than the proposed immunization dose for humans; - is not toxic after chronic administration in rats and dogs as shown in 1-month trial using the everyday doses which are 10and 100-fold higher than supposed immunization dose; - has no allerginicity, - has no local irritative activity, - has no immunotoxic effect. HIV incidence in St.Petersburg IDU Cohort (PTN 033 study) # HIV seroconversions 20/520 (8 (42%) at 6 month during FUP: visit) Estimate of HIV 4.5 per 100 p-y Incidence: (95% CI.2.7, 7.0) Factors significantly -Drug Injection of associated with psychostimulants Incidence: (ephidrine based and amphetamines) -≥ 3 or more sexual partners in last 6 months (univariate analysis) Source: Kozlov et al., 2006 Conclusion: We are ready for clinical trials of HIV vaccines New HIV-Vaccine Initiative - St. Petersburg State University - The Biomedical Center - Research Institute of Pure Biochemicals Molecular epidemiology Masharsky A., Verevochkin S., Nabatov A., Biomedical Center, Research Institute of Pure Biochemicals, St.Petersburg, Russia Cloning and analysis of full-length genomes of HIV-1 Masharsky A., Verevochkin S., Nabatov A., Murashev B., Klimov N., Eremin V., Kravchenko O., Shcherbinskaya A. Biomedical Center, Research Institute of Pure Biochemicals, St.Petersburg, Russia, Research Institute for Epidemiology and Microbiology, Minsk, Belarus, Institute of Epidemiology and Infectious Diseases, Kiev, Ukraine Design, purification, and immunological testing of DNA vaccines against HIV-1 Murashev, B., Romanovich A., Murasheva I., Pavlova M., Kreslavskiy T., Dukhovlinova Y., Dorofeyeva Y., Galachyants Y., Klimov N. Biomedical Center, Research Institute of Pure Biochemicals Pre-clinical trials of new HIV vaccines Klimov N., Duhovlinova E., Murashev B., Duhovlinov I., Murashova I., Smirnova I., Kobatov A., Nikonov B., Kolbasov S., Boichenko M., Vorobiev A. Biomedical Center, Research Institute of Pure Biochemicals, Research Institute for Toxicology, St.Petersburg, Sechenov Medical Academy, Moscow PTN/Cohort building Ryder R., Shaboltas A., Hoffman I. University of Northern Carolina, USA, St.Petersburg State Universuty, Biomedical Center Sponsors: - Russian Program “New Generation of Vaccines and Medical Diagnostic Systems of the Future” 1997-2005 - BTEP DHHS USA 2002-2005 - NIH PTN 2000-2006