Transcript Document

Workshop on Biodiversity and Climate Change
April 15-17, 2015
Astana, Republic of Kazakhstan
Use of triterpene saponins isolated from plants
indigenous to Kazakhstan as a novel adjuvants for for
mucosal immunization
Vladimir Berezin
Institute of Microbiology and Virology
Ministry of Education and Science of Kazakhstan
Mucosal (nasal) immunization as an efficient way to protect
against of respiratory infections
 Mucosal (nasal) immunization against mass respiratory infections such as
influenza is very attractive approach due a number of reasons: it needlefree, more safe, low cast, non-traumatic and can provide first barrier for
protection in primary infection gates.
 A major problem for the nasal vaccine has been the adjuvant, which is
required for efficient induction of immunity. Most vaccines today are
injectable and use aluminum salts as their adjuvant component. These
vaccines are unable to induce good immune response when delivered at
mucosal sites.
 In order to make mucosal vaccines more immunogenic, strong mucosal
adjuvants and/or novel antigens delivery systems are required.
Plant saponins as adjuvants
 Triterpen saponins of plant origin are one of the best candidates for incorporation to
mucosal vaccines as adjuvants. Saponins are natural compounds found in many wild
and cultivated plants, many of them already uses in traditional medicine and in food
industry.
 The molecular of triterpene saponins contain triterpenoid hydrophobic aglycone and
hydrophilic oligosaccharide chains. Due the combination of hydrophobic and
hydrophilic domains the molecular of saponin may interact with lipids and various
antigens and induce formation of immunostimulation nanocomplexes 60-100 nm in
size.
 Saponin-based adjuvants have the ability modulate cell mediated immune system as
well as to enhance local immune response and general antibody production.
Structure of triterpen saponin
ISCOMs contained viral
antigens, lipids and
triterpen saponin
Oda K. et al. Carbohydrate Research, Biol
Chem. 2000; 381(1):67–74
Kensil C.R. Crit Rev Ther Drug Carrier
Syst. 1996;13(1-2):1–55
Morein B., Abasugra, I. J. Adv. Drug Deliv.
Rev., 2004, 56:367-382
Drane D. et al. Expert review of vaccines
2007, 6: 761–772
Immunostimulatory saponins isolated from various plants
 Saponin Quil A isolated from the bark of
South-America tree Quillaya saponaria
Molina, (Dalsgaard K., 1978).
 Immunostimulation saponin QS21 with
lower toxicity obtained from Quil A by HPLC
fractionation, (Kensil C.R. et al., 1991).
 Immunostimulation saponins similar to Quil
A isolated from Polygala senega, plant
indigenous to Canada (Estrada et al., 2000)
and from Astragalus species indigenous to
Turkey (Yesilada et al., 2005).
Quillaya saponaria
Polygala senega
 Saponins with immunostimulatory activity
and low toxicity were isolated from number
of plants indigenous to Kazakhstan
(Saponaria officinalis, Glycyrrhiza glabra,
Aesculus hippocastanum etc.) (Berezin et
al., 2008, 2010).
Dalsgaard K. Acta Vet.Scand.Suppl., 1978, 69:1-40
Kensil C.et al., Immunol. 1991;146(2):431–437
Estrada A. et al. Comp.Immun.Microb.Inf.Dis. 2000; 23(1);27-43
Yesilada E. et al. J Ethnopharmacol. 2005;96(1-2):71–77
Berezin V. et al. J. Parasitology. J.Parasitology, 2008, 94 (2):381-385
Berezin V. et al., Veterinary Parasitology, 2010, 167: 28-35
Astragalus species
Saponaria officinalis
Aesculus hippocastanum Glycyrrhiza glabra
Purified imunostimulatory saponins AsgipanTM and GlabiloxTM
isolated from plants indigenous to Kazakhstan
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Purified triterpen saponins AsgipanTM and GlabiloxTM with high immunostimulatory activity
and low toxicity were isolated from Aesculus hippocastanum and Glycyrrhiza glabra plants
using HPLC fractionation.
Toxicity of isoalted saponins was investigated in doses exceeded doses need for
immunostimulation effect up to 1000 times (15,0 mg per animal). It was shown that
toxicity of saponins GlabiloxTM and AsgipanTM is significantly lower in comparison with
toxicity of commercial preparations of immunostimulatory saponin Quil A isolated from
Quillaya saponaria plant.
Two kinds of influenza vaccine preparations contained saponins as immunostimulation
component were prepared: (1) subunit vaccine on the base of ISCOMs (nanoparticles
contained HA+NA antigens, saponins and lipids); (2) whole virus inactivated vaccine
mixed with saponin/lipid particulate adjuvant (SAPOMAX).
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Chickens
Chiken embryos
Mice
1
2
3
4
5
Saponins
HPLC fractionation of plant extracts derived from
A.hippocastanum (A) and G.glabra (B) plants
Toxicity of various saponin preparations:
1. Quil A; 2. G. glabra extract; 3. Glabilox;
4. A. hippocastanum extract; 5. Asgipan
Levels of IgM/IgG antibody and cytokines in mice sera after single
intranasal immunization of ISCOMs H1N1 subunit vaccine
14000
25000
12000
20000
10000
15000
8000
IgM
6000
IgG
10000
4000
5000
2000
0
0
1
2
3
4
5
6
1
2
3
4
5
6
IFN-γ
IL-2
IL-4
IL-10
1 – placebo
2 – subunit HA+NA vaccine
3 – subunit HA+NA vaccine + alum hydroxide adjuvant
4 – ISCOMs incorporated HA+NA antigens and saponin Quil A
5 – ISCOMs incorporated HA+NA antigens and saponin AsgipanTM
6 – ISCOMs incorporated HA+NA antigens and saponin GlabiloxTM
Protection against influenza H1N1 lethal infection after single
intranasal immunization of H1N1 subunit vaccine based ISCOMs
On axis abscissa – groups of mice immunized vaccine in various doses: 1,0; 3,0 and 5,0 ug/animal.
On axis ordinate – % of protection from H1N1 influenza virus, strain A/St-Petersburg/5/09 (H1N1)
influenza virus in dose 100 EID50
1 – placebo
2 – subunit HA+NA vaccine + alum hydroxide adjuvant
3 – ISCOMs incorporated HA+NA antigens and saponin Quil A
4 – ISCOMs incorporated HA+NA antigens and saponin AsgipanTM
5 – ISCOMs incorporated HA+NA antigens and saponin GlabiloxTM
Levels of IgM/IgG antibody and cytokines in mice sera after single
intranasal immunization of whole virus influenza vaccine mixed with
SAPOMAX adjuvant
12000
25000
10000
20000
8000
15000
6000
IgM
10000
IgG
4000
5000
2000
0
0
1
2
3
4
5
1
2
3
4
5
IFN-γ
IL-2
IL-4
IL-10
1 – placebo
2 – whole virus inactivated vaccine without adjuvant;
3 – whole virus inactivated vaccine + alum hydroxide adjuvant;
4 – Whole virus inactivated vaccine + SAPOMAX-AG adjuvant
5 – Whole virus inactivated vaccine + SAPOMAX-GL adjuvant
Protection against H1N1 influenza virus infection after single
intranasal immunization of H1N1 whole virus inactivated vaccine
mixed with SAPOMAX adjuvant
100
90
80
70
60
50
40
30
20
10
0
1
2
3
4
5
On axis abscissa – groups of mice immunized whole virus inactivated
vaccine in doses 3,0 ug and 5,0 ug per animal.
On axis ordinate – % of protection from H1N1 influenza virus, strain A/StPetersburg/5/09 in dose 100 EID50
1 – placebo
2 – whole virus inactivated vaccine without adjuvant;
3 – whole virus inactivated vaccine + alum hydroxide adjuvant;
4 – Whole virus inactivated vaccine + SAPOMAX-AG adjuvant
5 – Whole virus inactivated vaccine + SAPOMAX-GL adjuvant
Summary
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Purified triterpen saponins AsgipanTM and GlabiloxTM with high
immunostimulatory activity and low toxicity have been isolated from KZ
plants Aesculus hippocastanum and Glycyrrhiza glabra by HPLC
fractionation. These saponins were used for creation of subunit influenza
vaccine based nanoparticles incorporated viral antigens, lipids and purified
saponins and also for preparation of saponin/lipid particulate adjuvants.
Intranasal immunization with H1N1 subunit influenza contained purified
HA+NA antigens, lipids and saponins induced high levels of Th1 and Th2
immune responses and protected against experimental influenza infection.
Intranasal immunization of whole virus inactivated H1N1 influenza vaccine
mixed with saponin/lipid particulate adjuvant stimulated high levels of
humoral and cellular immune responses and induced protection against
lethal influenza infection.
The results of study have shown that purified triterpen saponins isolated
from plants indigenous to Kazakhstan may be used as efficient adjuvants
for creation of influenza vaccine preparations for mucosal (intranasal)
immunization.