Institute of Microbiology and Virology Almaty, KAZAKHSTAN vladimirberezin@yahoo

1. Conference EastLink Klaipeda Science and Technology Park Klaipeda, 20-21 October 2010 Innovation biotechnologies for elaboration of antiviral preparations in Kazakhstan – the platform for cooperation Institute of Microbiology and Virology Almaty, KAZAKHSTAN vladimirberezin@yahoo.com

2. Biotechnology in global economy • Accordingly the UN expertise, biotechnology in XXI century will be one of main field of global industry connected with many directions - food production, pharmacology, ecology, power engineering etc. • Biotechnology on the base of modern achievements in microbiology, virology and immunology more and more useful in production of various biopreparations in many fields: - in medicine and veterinary (vaccines, diagnostic test-systems, antibiotics, antiviral preparations etc.) - in food industry (yeast, lactobacteria, enzymes etc.) - in alternative energy (biofuel), - in environmental control (utilization of waste materials, bioremediation of water and soil pollution etc.)

3. At present time annual increase of biotechnology market in the world is about 7%. It is one of highest index in global industry. • Total value of biotechnology market in 2010 estimated more than $2 trillions.

4. Biotechnology in prevention of infectious diseases • During last 30 years society had evidence of appearance of emerging and re-emerging infectious diseases (HIV, hepatitis C, hemorrhagic fevers, highly pathogenic avian influenza, antibiotic resistant microorganisms etc.). • Totally in the world epidemiology situation last years got worse and infectious diseases responsible for 15 millions deaths annually. • Production of pharmaceutical products for prevention of infectious diseases such as diagnostic test-systems, vaccines and therapeutic preparations is a large and very profitable sector of biotechnology.

5. Virology research in Kazakhstan Among total infectious diseases dangerous for human and animals about 70% related with viral infections. Therefore investigation in virology is important part of general biotechnology research. Virology research in Kazakhstan includes 3 main directions: • surveillance of viral diseases in Kazakhstan • development of vaccine preparations and improvement the efficacy of vaccines by elaboration of new adjuvant/delivery systems • elaboration of novel antiviral preparations of plant origin

6. Surveillance of avian-transmitted viral infections in Kazakhstan

7. Central Asia Regionand Kazakhstan Area: Kazakhstan is a biggest country in Central Asia region. This is 9th country in the world in size. Total area - 2,724,900 sq km. Kazakhstan located in the heart of Eurasia and extended from west to east from Caspian Sea and Volga to China (about 3,000 km) and from South to North from Tien Shan mountains to Siberia (about 1,600 km). Border: With Russia, China, Uzbekistan, Kyrgyzstan, Turkmenistan, Azerbaijan and Iran (through Caspian Sea).

8. Various Geographic Zones in Kazakhstan Natural water reservoirs in north, south-east and south-west Kazakhstan are very attractive for many species of waterfowl migratory birds

9. Major Flyways of Migratory birds in Eurasia • Kazakhstan located on the cross of flyways for many species of migratory birds. Geographic location and existence of many places in North and South Kazakhstan occupied migrating birds represent the reservoir for AI viruses spread and gene recombination.

10. Main flyways for penetration of avian-transmitted infections to the territory of Kazakhstan and boarding countries

11. Avian influenza viruses isolated in Kazakhstan in 2000-2010 • Viruses were isolated in 4 regions of Kazakhstan: - in north Kazakhstan near the border with Russia (wild birds and poultry birds) - in central Kazakhstan (poultry birds) - in south-east Kazakhstan on the cost of Caspian Sea (wild migratory birds) - in south-west Kazakhstan close to the border with China (wild migratory birds, poultry birds)

12. HP AIV H5N1 Outbreak in North Kazakhstan in 2005: Golubovka Settlement, Goose Farm • H5N1 outbreak was registered in July-August 2005 in Golubovka settlement and 3 villages located in north Kazakhstan close to the border with Russia. • In Golubovka settlement 6000 gooses on farm have infected and died during few days. Isolated virus was identified as HPAI H5N1. • Goose farm in Golubovka was burned and bulldozed under ground.

13. Phylogenetic analysis of H5N1 viruses isolated in Kazakhstan in 2005-2006

14. Products elaborated for influenza and Newcastle disease viruses control on the base of local strains Immunoenzime test-systems for simplified identification of influenza virus and Newcastle disease virus were elaborated using local strains isolated in Kazakhstan. Experimental series of test-systems were produced. PCR-kits for diagnostics of influenza virus and Newcastle disease virus on the base of local viral strains were prepared. Inactivated vaccines against H5N1 (“avian”) influenza virus and H1N1 (“swine”) influenza virus on the base of local influenza virus strains isolated in Kazakhstan were elaborated and investigated in preclinical and clinical trials.

15. Conclusions • It is very important choose “right” viral strain for preparation of most efficient vaccines and diagnostics test-systems. • Use of vaccines and test-systems based on strains isolated in “non-correct” regions or use old (not-actual) viral strains may significantly decrease the efficacy of vaccines and diagnostic preparations. • Creation of virus strain collections and gene-banks for biotechnology purposes and exchange of these materials between east countries and EU countries may be one of important points for collaboration.

16. 2. Elaboration of natural compounds for improvement the efficacy of vaccine preparations • Successful vaccination is one of the most important way for prevention of viral infections. Vaccination should increase the resistance against virus and, in the case of virus introduction, reduce levels of viral shedding. • Efficacy of vaccination mostly depended of immunostimulating activity of vaccine formulations and possibility to induce broad spectrum of immune responses.

17. ISCOM nanoparticles as an efficient vaccine formulation One of the most attractive vaccine formulations is immunostimulating complex (ISCOM) – special cage-like nanoparticle about 40-60nm in size containing viral antigens, lipids and immunostimulating compounds (saponins of plant origin). ISCOM may induces broad spectrum of immune responses (humoral, cellular and mucosal) is due the combination of successful antigen presentation and the powerful immunomodulatory capability of plant compounds. Agraval A. et al. J. Immune Based Therap.Vaccs. 1, 121-129, 2003. Morein B., Abasugra, I. J. Adv. Drug Deliv. Rev. 56, 367-382, 2004.

18. Novel low toxic saponins for ISCOM formation • At present time only one kind of saponins (saponin Quil A) isolated from South-American tree Quillaja Saponaria Molina has used for ISCOM formation and production of several vaccine preparations. This saponin possess relatively highly toxic and restricted for medical use. • Low toxicity immunostimulating saponins GG-6 and AH-6 suitable for ISCOM formation have been isolated from G. glabra and A. hippocas-tanum plants using HPLC fractionation. Toxicity of GG-6 and AH-6 saponins examined in chickens, chicken embryos and mice: 1- saponin Quil A (Iscotec AB, Sweden); 2 – crude saponin GG-6; 3- purified saponin GG-6; 4 – crude saponin AH-6; 5 – purified saponin AH-6 HPLC fractionation of saponins

19. Preparation of influenza vaccine based ISCOMs 1 2 3 Centrifugation Treatment by non-ionic detergent PurifiedHA+NA Saponins + lipids in non-ionic detergent Dialysis Self-assembling of ISCOMs incorporatedHA+NA, saponinsand lipids Electron microscopy of nanoparticles containingHA+NA of influenza virus H5N1 (А/Astana/RG/6.2/2009) andvarious saponins: GL-6 (1), АH-6 (2) and Quil A (3)

20. Antibody titers in mice sera after influenza H5N1 ISCOMs intranasal and subcutaneous immunization IgM IgG Intranasal Subcutaneous 1 – whole virus inactivated H1N1 vaccine; 2 – subunit HA+NA vaccine; 3 – subunit HA+NA vaccine + alum hydroxide adjuvant; 4 – ISCOMs assembled HA+NA antigens and saponin Quil A; 5 – ISCOMs incorporated HA+NA antigens and saponin AH-6; 6 – ISCOMs containing HA+NA antigens and saponin GG-6

21. Efficacy of ISCOM vaccinepreparations in chicken vaccination/challenge experiments 1 2 1 – titers of antibody in chicken sera 2 – protection against infection of highly pathogenic avian influenza virus • 1 – without vaccination • 2 – traditional vaccine without immunostimulator • 3 – ISCOM vaccine containing AH-6 • 4 – ISCOM vaccine containing GG-6 • 5 – ISCOM vaccine containing commercial saponin Quil A • 6 – vaccine mixed with traditional adjuvant alum hydroxide

22. Experimental preparation of immunostimulating saponin GG-6 (Asgipan-immuno+) for ISCOM formation

23. Conclusions • Novel low toxicity and highly immunostimulating active preparations GG-6 and AH-6 have been isolated from plants indigenous to Kazakhstan. • Use of such immunostimulating compounds in vaccine formulations may significantly increase the efficacy of vaccine preparations. • Development of modern more efficient and safe vaccine formulations against viral diseases, elaboration of novel biologically active compounds isolated from unique local plants and use that for improvement of vaccine preparations are attractive tasks for collaboration between east countries and EU countries.

24. 3. Elaboration of novel antivirus preparations based of natural plant compounds • Novel antiviral preparations with broad spectrum of antivirus activity based on natural compounds isolated from plants indigenous to Kazakhstan were elaborated. • It was established that number of Kazakhstanian plants contain high levels of compounds with antiviral activity. These compounds were isolated and characterized relatively spectrum of antiviral activity and toxicity. • Number of preparations were chosen for preclinical study.

25. Screening of plant preparations for antiviral activity Depression of infectious activity of avian influenza virus H7N1, strain A/FPV/Rostock/34 (in lg) after treatment of various preparations isolated from plants: 1 – AK01, 2 – AK11, 3 – AK17, 4 – AK27, 5 – MD6, 6 – SO12, 7 – GP12, 8 – KB8, 9 – virus without treatment Plant preparations were isolated from:Sedum (S.aizoon, S.Alberti, S.Ewersii, S.hybridum, S.mugodsharicum, S.pentapetalum, S.purpureum, S.telephium, S.tetramerum, S.acre, S.kamtczaticum) and Pseudosedum (P.bucharicum, P.karatavicum, P.ferganense, P.Lievenii, P.longidentatum)

26. 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Antiviral activity of plant preparations in comparison with commercial antiviral preparations Tamiflu and Amizon(% of inhibition) A/turkey/England/50/92 (H5N1) A/tern/South Africa/1/62 (H5N3) Antiviral preparations: 1 – AK27 2 – SO12 3 – KB8 4 – MD6 5 – GP12 6 – Tamiflu 7 – Amizon А/swine/Iowa/30 (H1N1)

27. Conclusions • Number of preparations isolated from plants indigenous to Kazakhstan have shown high levels of antiviral activity. • Three preparations: КВ-8, АК27 andSO12 actively depressed infection activity of influenza virus and Newcastle disease virus were elaborated. • КВ-8, АК27 andSO12 demonstrated higher levels of virus-inhibition activity in comparison with well-known antiviral preparations Oseltamivir (Tamiflu), Amizon and Remantadin. • Joint research/development of novel more effective antiviral preparations isolated from unique natural plant sources in Central Asia region could be a good point for collaboration between EU and east countries.

28. Summary • Biotechnology sector is one of the fastest growing field of global industry and play important role in economy of many counties. • Infectious diseases still have significant influence for social situation and economy in all countries and successful prevention of infectious diseases is directly related with stability of society and quality of life in the world. Therefore market of pharmaceutical products for prevention of infectious diseases is very important part of world economy. • Many viral infections are common for European countries and Central Asia region and may be quickly transmitted from one country to other because number of reasons (increased movements of people, expansion of international trade in foodstuffs and medicinal biological products, social and environmental changes etc.). Joint efforts in prevention of infectious diseases may provide more possibilities for success. • Number of products elaborated in Kazakhstan for viral infections control may be interesting for further joint research and promotion of pharmacology and veterinary products. • Cooperation in research /development and commercialization of successful biotechnology products may be a good platform for improvement of relations between EU and east countries.

29. Thank you for attention!