Institute of Microbiology and Virology Almaty, KAZAKHSTAN

vladimirberezin@yahoo.com

Conference EastLinkKlaipeda Science and Technology ParkKlaipeda, 20-21 October 2010

Innovation biotechnologies for elaboration of antiviral preparations

in Kazakhstan – the platform for cooperation

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.)

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.

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.

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

1. Surveillance of avian-transmitted viral infections in Kazakhstan

Central Asia Region and 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).

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

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.

Main flyways for penetration of avian-transmitted infections to the territory of Kazakhstan and

boarding countries

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)

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.

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

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.

Amplification Plots

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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.

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.

ISCOM nanoparticles as an efficient vaccine formulation

One of the most attractive vaccine formulations is One of the most attractive vaccine formulations is immunostimulating complex (ISCOM) – special cage-immunostimulating complex (ISCOM) – special cage-like nanoparticle about 40-60nm in size containing like nanoparticle about 40-60nm in size containing viral antigens, lipids and immunostimulating viral antigens, lipids and immunostimulating compounds (saponins of plant origin). compounds (saponins of plant origin).

ISCOM may induces broad spectrum of immune ISCOM may induces broad spectrum of immune responses (humoral, cellular and mucosal) is due the responses (humoral, cellular and mucosal) is due the combination of successful antigen presentation and combination of successful antigen presentation and the powerful immunomodulatory capability of plant the powerful immunomodulatory capability of plant compounds.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.

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.

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HPLC fractionation of saponins

Novel low toxic saponins for ISCOM formation

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Toxicity of GG-6 and AH-6 saponins examined in chickens, Toxicity of GG-6 and AH-6 saponins examined in chickens, chicken embryos and mice:chicken embryos and mice:1- saponin Quil A (Iscotec AB, Sweden); 2 – crude saponin GG-6;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-63- purified saponin GG-6; 4 – crude saponin AH-6; 5 – purified saponin AH-6

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Preparation of influenza vaccine based ISCOMs

Electron microscopy of nanoparticles Electron microscopy of nanoparticles containingcontaining HA+NA of influenza virus H5N1 HA+NA of influenza virus H5N1 ((А/А/AstanaAstana//RGRG/6.2/2009/6.2/2009) and) and various saponinsvarious saponins: : GL-6 (GL-6 (11), ), ААH-6 (H-6 (22) and Quil A () and Quil A (33))

Treatment by non-ionicdetergent

Centrifugation

Saponins + lipidsin non-ionic detergent

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Self-assembling of ISCOMs incorporated HA+NA, saponins and lipids

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Antibody titers in mice sera after influenza H5N1 ISCOMs intranasal and subcutaneous immunization

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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

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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

Experimental preparation of immunostimulating saponin GG-6 (Asgipan-

immuno+) for ISCOM formation

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.

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.

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Screening of plant preparations for antiviral activity

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)

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

Antiviral activity of plant preparations in comparison with commercial antiviral preparations Tamiflu and

Amizon(% of inhibition)

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A/tern/South Africa/1/62 (H5N3) A/turkey/England/50/92 (H5N1)

А/swine/Iowa/30 (H1N1)

Antiviral preparations: 1 – AK27 2 – SO12 3 – KB8 4 – MD6 5 – GP12 6 – Tamiflu 7 – Amizon

Conclusions

Number of preparations isolated from plants indigenous to Kazakhstan have shown high levels of antiviral activity.

Three preparations: КВ-8, АК27 and SO12 actively depressed infection activity of influenza virus and Newcastle disease virus were elaborated.

КВ-8, АК27 and SO12 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.

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.

Thank you for attention!