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V la dimir Berezin Institute of Microbiology and Virology A lmaty, Kazakhstan

Triterpen saponins isolated from plants indigenous to Kazakhstan as efficient adjuvants for mucosal immunization. 5 th World Congress on Virology December 07-09, 2015, Atlanta, USA. V la dimir Berezin Institute of Microbiology and Virology A lmaty, Kazakhstan.

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V la dimir Berezin Institute of Microbiology and Virology A lmaty, Kazakhstan

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  1. Triterpen saponins isolated from plants indigenous to Kazakhstan as efficient adjuvants for mucosal immunization 5th World Congress on Virology December 07-09, 2015, Atlanta, USA Vladimir Berezin Institute of Microbiology and VirologyAlmaty, Kazakhstan

  2. Mucosal surfaces as a route for immunization Mucosal surfaces are the gates for many infectious agents and mucosal immune response serve as a first line of defense. Mucosal immunization can provide strong protective immunity by induction of specific neutralizing antibodies at local mucosal surfaces and by induction of general serum and cellular immunity. The main routes for mucosal immunization are: ocular, nasal, oral, pulmonary, vaginal and rectal. Main routes for mucosal immunization: • Ocular immunization can be carried using eye drops. • Nasal immunization is carried using sprays or drops. • Oral immunization. Vaccines can be delivered orally in the form of liquids or pills. • Pulmonary immunization can be carried by aerosols or powders. • Vaginal and rectal immunization. Vaccines can be delivered to the vaginal or rectal mucosal membrane, using creams or suppositories. S. Mitragotri Nature Reviews Immunology, 2005,5: 905-916

  3. Nasal immunization as an efficient way to protect against of respiratory infections • Nasal immunization against mass respiratory infections such as influenza is attractive due a number of reasons: it needle-free, safe, low cast, non-traumatic and can provide first barrier for protection in primary infection gates. • A major problem for nasal vaccines 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.

  4. Most common adjuvants for vaccine use • Adjuvants can generally be divided into two classes: vehicleswhich present vaccine antigens to the immune system in a more efficient way and immunostimulatorswhich affect the immune system. • Hundreds of compounds and molecules have been extensively evaluated as adjuvants but only few currently uses in vaccine preparations. Vaccine adjuvant systems: enhancing the efficacy of subunit protein antigens. P. Mohammed et al. Int. J. Pharm. 2008, 364(2), 272-280 Vaccine adjuvants: Current state and future trends. N. Petrovsky and J.C.Aguilar, Immunology and Cell Biology. 2004, 82, 488-496 Current adjuvants and new perspectives in vaccine formulation. E. Montomoli et al., Expert Rev. Vaccines. 2011, 10(7), 1053-1061

  5. Plant saponins as adjuvants Triterpen saponins of plant origin are one of the best candidates for incorporation to mucosal vaccines as adjuvants. These natural compounds found in many wild and cultivated plants, many of them already used in traditional medicine and for food production. 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 another amphipatic molecules and induce formation of micellar nanocomplexes. 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 Jacobsen N.E. et al. Carbohydrate Research, 1996, v.280, p.1-14. Structure of triterpen saponins

  6. Immunostimulation saponins as a natural component of 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, Gpsóphila paniculáta, Allokhruza gypsophiloides, Aesculus hippocastanum etc.) (Berezin et al., 2002, Alexuk et al., 2005). Quillaya saponaria Polygala senega Saponaria officinalis Astragalus species 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., “The World of Microbes”, Paris, 2002, p. 271-272 Alexuk P. et al. Cytokines and inflammation, 2005, 4 (2):75-76 Gpsóphila paniculáta Allokhruza gypsophiloides

  7. Saponins-based adjuvants/delivery systems Immunostimulating complexes (ISCOMs) - highly immunogenic antigen formulation contains saponinQuil A or QS21, lipids and antigens of different origin: viral, bacterial, parasite etc. (Morein et al.,1984). ISCOMs initiated high levels of antigen-specific immune responses including humoral and CD4/CD8 cell-mediated responses, stimulation of IL and INF-gamma production, and elicitation of mucosal immune response through various routes of immunization: subcutaneous, intranasal and oral (Morein, Abasugra, 2004). ISCOMATRIX™ adjuvant contains QS21 saponin, cholesterol and phospholipids - saponin-basedparticulateadjuvantcapableofinducingsignificantantibody andcellularimmuneresponses when mixed with various antigens (Drain et al., 2007) ISCOMATRIX™ Morein B. et al., Nature, 1984;308(5958):457–460 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

  8. Use of saponins in vaccine preparations • Saponins have used as integral part of vaccine preparations is about 60 years. Crude saponin mixture was applied as adjuvant for veterinary vaccines (Espinet, 1951). Foot-and-mouth disease vaccine Partially purified immunostimulatory saponins were isolated from the bark of Quillaya saponaria, South-American tree (Dalsgaard, 1974). Foot-and-mouth disease vaccine Veterinary vaccines: equine influenza virus; bovine diarrhea virus; feline leukemia, canine Lyme disease - licensed ISCOMs vaccines Saponin Quil A, saponin QS-21, ISCOMs (Kensilet al., 1995; Morein, Abasugra, 2004) Human vaccines: Flu, HIV-1, HIV-2, HBV, HCV, vaccine against cancer - preclinical and clinical trials ISCOMs, ISCOMATRIX™ (Morein, Abasugra, 2004; Drane et al., 2007)

  9. Triterpen saponins “Glabilox” and “Asgipan” isolated from plants indigenous to Kazakhstan • Purified saponins “Glabilox” and “Asgipan” were isolated by HPLC fractionation from plants Aesculus hippocastanum and Glycyrrhiza glabra indigenous to Kazakhstan. • Both plants are widely used for production of medical preparations and in food industry. Aesculus hippocastanum Berezin et al., J.Parasitology, 2008, 94 (2):381-385 Berezin et al., Veterinary Parasitology, 20010, 167: 28-35 Glycyrrhiza glabra

  10. Study of the toxicity of saponins Glabilox and Asgipan • Toxicity of initial plant extracts and purified triterpen saponins Glabilox and Asgipan was investigated on various animal models: mice, rats, chickens, chicken embryos, rabbits and guinea pigs. • Toxicity of saponins was studied in doses exceeded doses need for immunostimulation effect up to 1000 times (15,0 mg per animal). • Toxicity of saponins Glabilox and Asgipanwas significantly lower in comparison with toxicity of commercial preparations of saponin Quil A. A B On axis abscissa – tested preparations; on axis ordinate – level of toxic reactions. A – guinea pigs; B – chickens, chicken embryos, mice. 1 (a) – Quil A; 2 (b) – G. glabra crude extract; 3 (c,d,e,f,g) – Glabilox; 4 (h) – A. hippocastanum crude extract; 5 (I,j,k,l,m) - Asgipan

  11. Adjuvants/delivery systems prepared on the base of triterpen saponins Glabilox and Asgipan Immunostimulating complexes (ISCOMs) incorporated triterpen saponins Glabilox (1) or Asgipan (2), lipids and purified influenza virus external antigens (HA+NA) 1 2 • Particulate adjuvants “Glabilox-MX” (1) and “Asgipan-MX” (2) on the base of nanoparticles contained lipids and triterpen saponins Glabilox or Asgipan 2 1

  12. Experimental influenza vaccine preparations for intranasal immunization contained saponins Glabilox and Asgipan as adjuvant component Subunit influenza vaccine on the base of immunostimulating complexes (ISCOMs) contained purified influenza virus external antigens hemagglutinin (HA) and neuraminidase (NA), triterpensaponinsGlabilox or Asgipan and lipids Whole virus inactivated influenza vaccine mixed with particulate adjuvants Glabilox-MXand Asgipan-MX

  13. Preparation of subunit influenza vaccine on the base of nanoparticles contained HA+NA flu antigens, lipids and saponins Treatment by non-ionic detergent Centrifugation PurifiedHA+NA i HA+NA +saponins + lipids in non-ionic detergent Dialysis Self-assembling of ISCOMs nanoparticles containingHA+NA, saponinsand lipids Electron microscopy of ISCOMs incorporatedinfluenza virus antigens andplant saponins

  14. Immunization protocol Virus: A/H1N1 influenza virus: strainA/St-Petersburg/5/09 (H1N1) pdm09 Vaccine preparations: - subunit influenza vaccine without adjuvant - subunit influenza vaccine plus alum hydroxide adjuvant - subunit influenza vaccine on the base of ISCOMs containing purified HA+NA lipids and purified saponins Glabilox or Asgipan Animals: mice Doses: 1.0 - 5.0 µg of HA+NA per animal Route of immunization: intranasal Sera investigation: 21 days following single immunization Challenge: 21 days following single immunization using 100 lethal doses of A/St-Petersburg/5/09 (H1N1) influenza virus

  15. Influenza subunit vaccine: titers of IgM and IgG antibody in mice sera IgM IgG On axis abscissa – groups ofmice immunized vaccine preparations in doses 3,0 and 5,0 ug/animal On axis ordinate – titers of IgM and IgG antibody 1 – placebo 2 – subunit HA+NA vaccine 3 – subunit HA+NA vaccine + alum hydroxide adjuvant 4 – ISCOMs incorporatedHA+NA antigens lipids and saponin Asgipan 5 – ISCOMs incorporatedHA+NA antigens lipids and saponin Glabilox

  16. Influenza subunit vaccine: IgG subclasses in mice sera IgG1 IgG2a IgG2b On axis abscissa – groups ofmice immunized vaccine preparations in doses 3,0 and 5,0 ug/animal On axis ordinate – titers of IgG1, IgG2a and IgG2b antibody 1 – placebo 2 – subunit HA+NA vaccine 3 – subunit HA+NA vaccine + alum hydroxide adjuvant 4 – ISCOMs incorporatedHA+NA antigens, lipids and saponin Asgipan 5 – ISCOMs incorporatedHA+NA antigens, lipids and saponin Glabilox

  17. Influenza subunit vaccine: levels of IFN-γ and IL-2, IL-4 and IL-10in mice sera IL-2 IFN-γ IL-10 IL-4 On axis abscissa – groups ofmice immunized vaccine preparations in doses 3,0 and 5,0 ug/animal On axis ordinate – levels of cytokines in mice sera 1 – placebo 2 – subunit HA+NA vaccine 3 – subunit HA+NA vaccine + alum hydroxide adjuvant 4 – ISCOMs incorporatedHA+NA antigens, lipids and saponinAsgipan 5 – ISCOMs incorporatedHA+NA antigens, lipids and saponinGlabilox

  18. Influenza subunit vaccine: protection against H1N1 influenza virus infection after single intranasal (1) and s/c (2) immunization 1 2 1 1 1 2 2 2 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 incorporatedHA+NA antigens, lipids and saponin Asgipan 4 – ISCOMs incorporatedHA+NA antigens, lipids and saponin Glabilox

  19. Particulate adjuvant contained saponin/lipid nanoparticles Mixture of saponins, lipids and non-ionic detergent Dialysis Particulate adjuvant contained saponin/lipid nanoparticles Electron microscopy of saponin/lipid particulate adjuvant

  20. Immunization protocol Virus: A/H1N1 influenza virus: strainA/St-Petersburg/5/09 (H1N1) pdm09 Vaccine preparations: - whole virus inactivated vaccine without adjuvant - whole virus inactivated vaccine contained alum hydroxide adjuvant - whole virus inactivated vaccine contained Glabilox-MX or Asgipan-MX adjuvants Animals: mice Doses: 1.0 - 5.0 µg of HA+NA per animal Route of immunization: intranasal Sera investigation: 21 days following single immunization Challenge: 21 days following single immunization using 100 lethal doses of A/St-Petersburg/5/09 (H1N1) influenza virus

  21. Titers of HI and VN antibody in mice sera after single intranasal immunization with H1N1 whole virus inactivated vaccine HI-antibody VN-antibody On axis abscissa – groups ofmice immunized vaccine preparations in doses 3,0 and 5,0 ug/animal On axis ordinate – titers of hemagglutinin inhibition (HI) and virus neutralizing (VN) antibody 1 – Placebo 2 – Whole virus inactivated vaccine without adjuvant 3 – Whole virus inactivated vaccine + alum hydroxide adjuvant 4 – Whole virus inactivated vaccine + Asgipan-MX adjuvant 5 – Whole virus inactivated vaccine + Glabilox-MX adjuvant

  22. Titers ofIgG andIgM antibody in mice sera after single intranasal immunization with H1N1 whole virus inactivated influenza vaccine IgM IgG On axis abscissa – groups ofmice immunized vaccine preparations in doses 3,0 and 5,0 ug/animal On axis ordinate – titers of IgM and IgG antibody 1 – Placebo 2 – Whole virus inactivated vaccine without adjuvant 3 – Whole virus inactivated vaccine + alum hydroxide adjuvant 4 – Whole virus inactivated vaccine + Asgipan-MX adjuvant 5 – Whole virus inactivated vaccine + Glabilox-MX adjuvant

  23. IgG subclasses in mice sera after single intranasal immunization with H1N1 whole virus inactivated influenza vaccine IgG1 IgG2a IgG2b 1 – placebo 2 – whole virus inactivated vaccine without adjuvant 3 – whole virus inactivated vaccine + alum hydroxide adjuvant 4 – whole virus inactivated vaccine + Asgipan-MX adjuvant 5 – whole virus inactivated vaccine + Glabilox-MX adjuvant

  24. IFN-γ (A), IL-2 (B), IL-4 (C) and IL-10 (D) in mice sera after single intranasal immunization with H1N1 whole virus inactivated influenza vaccine IFN-γ IL-2 IL-10 IL-4 1 – placebo 2 – whole virus inactivated vaccine without adjuvant; 3 – whole virus inactivated vaccine + alum hydroxide adjuvant; 4 – whole virus inactivated vaccine + Asgipan-MX adjuvant 5 – whole virus inactivated vaccine + Glabilox-MX adjuvant

  25. Protection against lethal H1N1 influenza virus infection after single intranasal (1) and s/c (2) immunization of H1N1 whole virus inactivated influenza vaccine 1 2 1 1 1 2 2 2 On axis abscissa – groups ofmice immunized whole virus inactivated vaccine in 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 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 + Asgipan-MX adjuvant 5 – whole virus inactivated vaccine + Glabilox-MX adjuvant

  26. Summary Low toxicity triterpen saponins Glabilox and Asgipan isolated from A.hippocastanum and G.glabra plants indigenous to Kazakhstan may be used for assembling of immunostimulating nanoparticles incorporated viral antigens and for preparation of saponin/lipids particulate adjuvants. Intranasal immunization of H1N1 subunit influenza vaccine based immunostimulating nanocomplexes contained purified virus glycoprotein antigens, lipids and triterpen saponins induced high levels of Th1 and Th2 immune responses and protection against lethal experimental H1N1 influenza infection. Intranasal immunization of H1N1 whole virus inactivated influenza vaccine mixed with particulate saponin/lipid adjuvants also stimulated high levels of antibody and cellular immune responses and protection against lethal influenza infection. The results of study have shown good ability of triterpen saponins isolated from plants indigenous to Kazakhstan as efficient and non-toxic adjuvants for creation of influenza vaccine preparations for mucosal/intranasal immunization.

  27. Acknowledgements Institute of Microbiology and Virology of the Ministry of Education and Science Almaty, Kazakhstan grant MESRK #0366GF4; grant MESRK #0359GF4

  28. Thank you for attention!

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