Vladimir Berezin Institute of Microbiology and Virology

1. 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 BerezinInstitute of Microbiology and Virology Ministry of Education and Science of Kazakhstan

2. 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 needle-free, 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.

3. 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. • 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 ISCOMs contained viral antigens, lipids and triterpen saponin Structure of triterpen saponin

4. 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). • 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). Quillaya saponaria Polygala senega Astragalus species Saponaria officinalis • 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 Aesculus hippocastanum Glycyrrhiza glabra

5. Purified imunostimulatory saponins AsgipanTM andGlabiloxTM isolated from plants indigenous to Kazakhstan • Purified triterpen saponins AsgipanTM and GlabiloxTM with high immunostimulatory activity and low toxicity were isolated from Aesculus hippocastanum and Glycyrrhiza glabra plantsusing 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). Toxicity of various saponin preparations: 1. Quil A; 2. G. glabra extract; 3. Glabilox; 4. A. hippocastanum extract; 5. Asgipan HPLC fractionation of plant extracts derived from A.hippocastanum (A) and G.glabra (B) plants

6. Levels of IgM/IgG antibody and cytokines in mice sera after single intranasal immunization of ISCOMs H1N1 subunit vaccine IgG IgM 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 incorporatedHA+NA antigens and saponin AsgipanTM 6 – ISCOMs incorporatedHA+NA antigens and saponin GlabiloxTM

7. Protection against influenza H1N1 lethal infection after single intranasal immunization of H1N1 subunit vaccine based ISCOMs On axis abscissa – groups ofmice 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 incorporatedHA+NA antigens and saponin AsgipanTM 5 – ISCOMs incorporatedHA+NA antigens and saponin GlabiloxTM

8. Levels of IgM/IgG antibody and cytokines in mice sera after single intranasal immunization of whole virus influenza vaccine mixed with SAPOMAX adjuvant IgM IgG IL-2 IFN-γ 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

9. Protection against H1N1 influenza virus infection after single intranasal immunization of H1N1 whole virus inactivated vaccine mixed with SAPOMAX adjuvant On axis abscissa – groups ofmice 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/St-Petersburg/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

10. Summary • 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.