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beefzoologistBiotechnology

Feb 21, 2013 (4 years and 5 months ago)

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Institute of Microbiology and Virology

Almaty, KAZAKHSTAN

vladimirberezin@yahoo.com

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

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 9
th

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

H
5
N
1

(“avian”)

influenza

virus

and

H
1
N
1

(“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
-0,08
0,02
0,12
0,22
0,32
0,42
0,52
0,62
0,72
0,82
0,92
15
17
19
21
23
25
27
29
Cycles
Fluorescence (dRn)
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

immunostimulating

complex

(ISCOM)



special

cage
-
like

nanoparticle

about

40
-
60
nm

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.


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.


Time (min)
0
5
10
15
20
25
30
35
40
Absorbance
0.0
0.1
0.2
0.3
0.4
G. glabra
HPLC fractionation of saponins

Novel low toxic saponins for ISCOM formation

0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1
2
3
4
5
Saponins
Chickens
Chiken embryos
Mice
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

Time (min)
0
5
10
15
20
25
30
35
40
Absorbance
0.0
0.1
0.2
0.3
0.4
A. hippocanastanum
Preparation of influenza vaccine based ISCOMs



Electron microscopy of nanoparticles

containing

HA+NA of influenza virus H5N1
(
А/
Astana
/
RG
/6.2/2009
) and

various saponins
:
GL
-
6 (
1
),
А
H
-
6 (
2
) and Quil A (
3
)

Treatment by

non
-
ionic

detergent

Centrifugation

Saponins

+
lipids

in non
-
ionic detergent

Dialysis

Self
-
assembling of ISCOMs incorporated

HA+NA
,
saponins

and lipids



1

2

3

Purified

HA+NA


Antibody titers in mice sera after influenza H5N1

ISCOMs intranasal and subcutaneous immunization

0
500
1000
1500
2000
2500
3000
1
2
3
4
5
6
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

0
1000
2000
3000
4000
5000
6000
1
2
3
4
5
6
0
5000
10000
15000
20000
25000
1
2
3
4
5
6
0
5000
10000
15000
20000
25000
1
2
3
4
5
6
Efficacy of ISCOM vaccine

preparations in chicken
vaccination/challenge experiments


0
2000
4000
6000
8000
10000
12000
14000
16000
18000
1
2
3
4
5
6
1


titers of antibody in chicken sera

2


protection against infection of highly pathogenic avian influenza virus


1

2

0
10
20
30
40
50
60
70
80
90
100
1
2
3
4
5
6
7

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.


O
O
H
O
O
O
H
O
O
C
H
3
O
O
H
O
H
O
H
H
3
C
O
O
H
H
O
O
H
3
C
O
O
O
H
O
H
O
C
H
3
O
H
3
C
O
O
O
H
H
O
O
O
H
O
H
O
O
H
O
H
O
O
H
O
O
O
H
O
H
O
H
O
O
H
O
H
H
O
O
O
H
O
O
H
H
O
O
O
H
O
O
H
H
O
O
O
H
O
O
C
H
3
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
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)

0
20
40
60
80
100
АК27
MD
SO12
ГДА
ГДГ
тамифлю
амиксин
0,5
5
50

1 2 3 4 5 6 7

0
20
40
60
80
100
АК27
MD
SO12
ГДА
ГДГ
тамифлю
амиксин
0,5
5
50

1 2 3 4 5 6 7

0
20
40
60
80
100
АК27
MD
SO12
ГДА
ГДГ
тамифлю
амиксин
0,5
5
50

1 2 3 4 5 6 7

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

SO
12
actively depressed
infection activity of influenza virus and Newcastle disease virus
were elaborated.



КВ
-
8, АК27
and

SO
12
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 (i
ncreased
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!