1 / 19

Towards An HIV-1 Vaccine

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. AMPLIFICATION AND

abiba
Télécharger la présentation

Towards An HIV-1 Vaccine

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. Towards An HIV-1 Vaccine A.P. Kozlov The Biomedical Center Russian-German Cooperation Forum “Biotechnology – Life Sciences” St. Petersburg, Russia September 26, 2006

  2. Confirmation of the first cases of HIV infection in St.Petersburg in 1987

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

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

  5. HIV-1 SUBTYPES IN THE FORMER SOVIET UNION Other subtypes 5% B 5% A/B 10% • REFERENCES • Bobkov et al., 1997; 1998; 2000. • Liitsola et al., 1998; 2000. • Lukashov et al., 1998; 1999. • Masharsky et al., 1999. • Nabatov et al., 1997; 1998. • Novitsky et al., 1998. A 80%

  6. 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 andgp120 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 prime-boost vaccination procedure is hoped to induce both CD8+,CD4+ T-cell response and antibody B-cell anti-HIV response

  7. The pBMC plasmid used as a vector for the production of DNA vaccines against HIV-1 HIV-1 genome (GenBank #AF413987) GAG POL ENV NEF LTR LTR accesory genes p7 p17 p24 prot RT int gp120 gp41 p15 vif vpr tat vpu p6 gag rt gp160 nef pBMCgag pBMCrt pBMCenv pBMCnef

  8. Two forms of vaccine preparations production pBMCgag pBMCrt pBMCenv pBMCnef Transformation Equimolarmixing pBMCgag pBMCrt pBMCenv pBMCnef 1mg/ml plasmid DNA vaccine Salmonella typhimurium-T10 Cultivation i.m. immunization pBMCgag pBMCgag pBMCrt pBMCrt pBMCenv pBMCenv pBMCnef pBMCnef p.o. immunization Lyophilized tablet 1,21010CFU/tablet

  9. Purification of DNA vaccines Fermentation Plasmid DNA precipitation with isopropanol Alkaline lysis and KOAcprecipitation Protein precipitation with ammonium acetate RNA Plasmid DNA precipitation with PEG 8000 First purification Second purification А260 А260 CC Plasmid DNA Sephacryl S1000 OC DNA E.coli Final plasmid forms Quality control, immunization pBMCrt pBMCnef pBMCenv pBMCgag

  10. The quality of DNA vaccines purified by chromatography

  11. Production of new lines of target cells Cytotoxicity test Cytokine boosting % of specific lysis DNA vaccine CD8+ effector cell MHC I TCR Target cell spleen CD8 peptide Stimulation of cytokine secretion (IFN) Time course of the response Recombinant proteins of HIV-1 Response blood ELISA time Blood and washout Ig levels Flow diagram of experimental immunization of mice with a DNA vaccine and analysis of immune responses in the mice

  12. Vaccine trials in Russian Federation World Health OrganizationRecommendations 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 measurementof rectal temperature in rabbits All trials must be done using three pilot lots of vaccine.

  13. 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.

  14. 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 10- and 100-fold higher than supposed immunization dose; - has no allerginicity, - has no local irritative activity, - has no immunotoxic effect.

  15. HIV incidence in St.Petersburg IDUCohort (PTN 033 study) Source: Kozlov et al., 2006

  16. Conclusion:We are ready for clinical trials of HIV vaccines

  17. New HIV-Vaccine Initiative- St. Petersburg State University- The Biomedical Center- Research Institute of Pure Biochemicals

  18. Molecular epidemiology Masharsky A., Verevochkin S., Nabatov A., Biomedical Center, Research Institute of Pure Biochemicals, St.Petersburg, Russia 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, SechenovMedical Academy, Moscow 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 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 PTN/Cohort building Ryder R., Shaboltas A., Hoffman I. University of Northern Carolina, USA, St.Petersburg State Universuty, Biomedical Center

  19. 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

More Related