Comparison of in vivo Adjuvanticity of Liposomal PO CpG ODN with Liposomal PS CpG ODN: Soluble Leishmania Antigens as a Model

Document Type: Original Article

Authors

1 Nanotechnology Research Centre, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Biotechnology Research Centre, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Centre for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran

Abstract

Objective(s)
CpG oligodeoxynucleotides (CpG ODNs) have been shown to have potent immunostimulatory adjuvant activity for a wide range of antigens. Due to susceptibility of phosphodiester CpG ODNs (PO CpG) to nuclease degradation, nuclease-resistant phosphorothioate CpG ODNs (PS CpG) were currently utilized in an in vivo model. In this study, according to some recently reported drawbacks with PS CpG, the adjuvant potential of liposomal PO CpG as a substitute for PS CpG was evaluated.
Materials and Methods
Soluble Leishmania antigens (SLA) as a model antigen and distearoylphosphatidylcoline (DSPC) as a neutral lipid were employed to prepare liposomes. Susceptible BALB/c mice received buffer, SLA, Lip-SLA, Lip-SLA-PS CpG, Lip-SLA-PO CpG, SLA+PS CpG, or SLA+PO CpG subcutaneously 3 times with 3 weeks intervals and then were challenged with Leishmania major’s live promastigotes. Blood and spleen samples were analyzed to determine the level and type of antibodies and cytokines. The number of live parasites in the spleen of immunized mice was determined. Moreover, the lesion size progress was assessed weekly by footpad swelling measurement.
Results
The results showed that mice immunized with Lip-SLA-PS CpG or Lip-SLA-PO CpG developed a significantly smaller footpad swelling, higher level of anti SLA IgG antibodies before and after challenge, and lower spleen parasite burden compared with the control groups. However, there was no significant difference between mice received Lip-SLA-PS CpG and those received Lip-SLA-PO CpG.
Conclusion
The results demonstrated that liposomal PO CpG ODN could be used instead of PS CpG ODN to overcome the possible drawbacks.

Keywords


1. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Ann Rev Immunol 2002; 20:709-760.

2. Klinman DM, Currie D, Gursel I, Verthelyi D. Use of CpG oligodeoxynucleotides as immune adjuvants. Immunol Rev 2004; 199:201-216.

3. Jaafari MR, Badiee A, Khamesipour A, Samiei A, Soroush D, Kheiri MT, et al. The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome. Vaccine 2007; 25:6107-6117.

4. Krieg AM, Efler SM, Wittpoth M, Al Adhami MJ, Davis HL. Induction of systemic TH1-like innate immunity in normal volunteers following subcutaneous but not intravenous administration of CPG 7909, a synthetic B-class CpG oligodeoxynucleotide TLR9 agonist. J Immunother 2004; 27:460-471.

5. Cooper CL, Davis HL, Angel JB, Morris ML, Elfer SM, Seguin I, et al. CpG 7909 adjuvant improves hepatitis B virus vaccine seroprotection in antiretroviral-treated HIV-infected adults. Aids 2005; 19:1473-1479.

6. Brown DA, Kang SH, Gryaznov SM, DeDionisio L, Heidenreich O, Sullivan S, et al. Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J Biol Chem 1994; 269:26801-26805.

7. Levin AA. A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides. Biochim Biophys Acta 1999; 1489:69-84.

8. Agrawal S, Temsamani J, Galbraith W, Tang J. Pharmacokinetics of antisense oligonucleotides. Clin Pharmacokinet 1995; 28:7-16.

9. Sands H, Gorey-Feret LJ, Cocuzza AJ, Hobbs FW, Chidester D, Trainor GL. Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate. Mol Pharmacol 1994; 45:932-943.

10. Zhao Q, Matson S, Herrera CJ, Fisher E, Yu H, Krieg AM. Comparison of cellular binding and uptake of antisense phosphodiester, phosphorothioate, and mixed phosphorothioate and methylphosphonate oligonucleotides. Antisense Res Dev 1993; 3:53-66.

11. Krieg AM, Stein CA. Phosphorothioate oligodeoxynucleotides: antisense or anti-protein? Antisense Res Dev 1995; 5:241.

12. Deng GM, Nilsson IM, Verdrengh M, Collins LV, Tarkowski A. Intra-articularly localized bacterial DNA containing CpG motifs induces arthritis. Nat Med 1999; 5:702-705.

13. Sparwasser T, Hultner L, Koch ES, Luz A, Lipford GB, Wagner H. Immunostimulatory CpG-oligodeoxynucleotides cause extramedullary murine hemopoiesis. J Immunol 1999; 162:2368-2374.

14. Heikenwalder M, Polymenidou M, Junt T, Sigurdson C, Wagner H, Akira S, et al. Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration. Nat Med 2004; 10:187-92.

15. Kim D, Rhee JW, Kwon S, Sohn WJ, Lee Y, Kim DW, et al. Immunostimulation and anti-DNA antibody production by backbone modified CpG-DNA. Biochem Biophys Res Commun 2009; 379:362-367.

16. Kindrachuk J, Potter JE, Brownlie R, Ficzycz AD, Griebel PJ, Mookherjee N, et al. Nucleic acids exert a sequence-independent cooperative effect on sequence-dependent activation of Toll-like receptor 9. J Biol Chem 2007; 282:13944-13953.

17. Haas T, Metzger J, Schmitz F, Heit A, Muller T, Latz E, et al. The DNA sugar backbone 2' deoxyribose determines toll-like receptor 9 activation. Immunity 2008; 28:315-323.

18. Yasuda K, Rutz M, Schlatter B, Metzger J, Luppa PB, Schmitz F, et al. CpG motif-independent activation of TLR9 upon endosomal translocation of "natural" phosphodiester DNA. Eur J Immunol 2006; 36:431-436.

19. Klinman DM. Use of CpG oligodeoxynucleotides as immunoprotective agents. Expert Opin Biol Ther 2004; 4:937-946.

20. Semple SC, Klimuk SK, Harasym TO, Hope MJ. Lipid-based formulations of antisense oligonucleotides for systemic delivery applications. Methods Enzymol 2000; 313:322-341.

21. Wilson KD, de Jong SD, Tam YK. Lipid-based delivery of CpG oligonucleotides enhances immunotherapeutic efficacy. Adv Drug Deliv Rev 2009; 61:233-242.

22. Chikh GG, Kong S, Bally MB, Meunier JC, Schutze-Redelmeier MP. Efficient delivery of Antennapedia homeodomain fused to CTL epitope with liposomes into dendritic cells results in the activation of CD8+ T cells. J Immunol 2001; 167:6462-6470.

23. Alving CR. Liposomal vaccines: clinical status and immunological presentation for humoral and cellular immunity. Ann N Y Acad Sci 1995; 754:143-152.

24. O'Hagan DT, Singh M. Microparticles as vaccine adjuvants and delivery systems. Expert Rev Vaccines 2003; 2:269-283.

25. Copland MJ, Rades T, Davies NM, Baird MA. Lipid based particulate formulations for the delivery of antigen. Immunol Cell Biol 2005; 83:97-105.

26. Tsujimura H, Tamura T, Kong HJ, Nishiyama A, Ishii KJ, Klinman DM, et al. Toll-like receptor 9 signaling activates NF-kappaB through IFN regulatory factor-8/IFN consensus sequence binding protein in dendritic cells. J Immunol 2004; 172:6820-6827.

27. Belkaid Y, Von Stebut E, Mendez S, Lira R, Caler E, Bertholet S, et al. CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major. J Immunol 2002; 168:3992-4000.

28. Muller I, Kropf P, Louis JA, Milon G. Expansion of gamma interferon-producing CD8+ T cells following secondary infection of mice immune to Leishmania major. Infect Immun 1994; 62:2575-2581.

29. Sharifi I, FeKri AR, Aflatonian MR, Khamesipour A, Nadim A, Mousavi MR, et al. Randomised vaccine trial of single dose of killed Leishmania major plus BCG against anthroponotic cutaneous leishmaniasis in Bam, Iran. Lancet 1998; 351:1540-1543.

30. Momeni AZ, Jalayer T, Emamjomeh M, Khamesipour A, Zicker F, Ghassemi RL, et al. A randomised, double-blind, controlled trial of a killed Leishmania major vaccine plus BCG against zoonotic cutaneous leishmaniasis in Iran. Vaccine 1999; 17:466-472.

31. Khalil EA, El Hassan AM, Zijlstra EE, Mukhtar MM, Ghalib HW, Musa B, et al. Autoclaved Leishmania major vaccine for prevention of visceral leishmaniasis: a randomised, double-blind, BCG-controlled trial in Sudan. Lancet 2000; 356:1565-1569.

32. Khamesipour A, Rafati S, Davoudi N, Maboudi F, Modabber F. Leishmaniasis vaccine candidates for development: a global overview. Indian J Med Res 2006; 123:423-438.

33. Noazin S, Khamesipour A, Moulton LH, Tanner M, Nasseri K, Modabber F, et al. Efficacy of killed whole-parasite vaccines in the prevention of leishmaniasis: a meta-analysis. Vaccine 2009; 27:4747-5473.

34. Afrin F, Rajesh R, Anam K, Gopinath M, Pal S, Ali N. Characterization of Leishmania donovani antigens encapsulated in liposomes that induce protective immunity in BALB/c mice. Infect Immun 2002; 70:6697-6706.

35. Manuel S, Laura R, rez, Miguel AP, Victor MG, lez, et al. Searching genes encoding Leishmania antigens for diagnosis and protection. Scholarly Research Exchange; 2009.

36. Badiee A, Jaafari MR, Samiei A, Soroush D, Khamesipour A. Coencapsulation of CpG oligodeoxynucleotides with recombinant Leishmania major stress-inducible protein 1 in liposome enhances immune response and protection against leishmaniasis in immunized BALB/c mice. Clin Vaccine Immunol 2008; 15:668-674.

37. Noazin S, Modabber F, Khamesipour A, Smith PG, Moulton LH, Nasseri K, et al. First generation leishmaniasis vaccines: a review of field efficacy trials. Vaccine; 26:6759-6767.

38. Noazin S, Khamesipour A, Moulton LH, Tanner M, Nasseri K, Modabber F, et al. Efficacy of killed whole-parasite vaccines in the prevention of leishmaniasis-A meta-analysis. Vaccine 2009; 27:4747-4753.

39. Scott P, Pearce E, Natovitz P, Sher A. Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes. J Immunol 1987; 139:221-227.

40. Chu RS, Targoni OS, Krieg AM, Lehmann PV, Harding CV. CpG Oligodeoxynucleotides Act as Adjuvants that Switch on T Helper 1 (Th1) Immunity. J Exp Med 1997; 186:1623-31.

41. Weeratna RD, Brazolot Millan CL, McCluskie MJ, Davis HL. CpG ODN can re-direct the Th bias of established Th2 immune responses in adult and young mice. FEMS Immunol Medical Microbiol 2001; 32:65-71.

42. Badiee A, Jaafari MR, Khamesipour A. Leishmania major: immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes. Exp Parasitol 2007; 115:127-134.

43. Taswell C. Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. J Immunol 1981; 126:1614-1619.

44. Roman M, Martin-Orozco E, Goodman JS, Nguyen MD, Sato Y, Ronaghy A, et al. Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat Med 1997; 3:849-854.

45. Stacey KJ, Sweet MJ, Hume DA. Macrophages ingest and are activated by bacterial DNA. J Immunol 1996; 157:2116-22.

46. Sun S, Zhang X, Tough DF, Sprent J. Type I interferon-mediated stimulation of T cells by CpG DNA. J Exp Med 1998; 188:2335-2342.

47. Halpern MD, Kurlander RJ, Pisetsky DS. Bacterial DNA induces murine interferon-gamma production by stimulation of interleukin-12 and tumor necrosis factor-alpha. Cell Immunol 1996 10; 167:72-78.

48. Yi AK, Chace JH, Cowdery JS, Krieg AM. IFN-gamma promotes IL-6 and IgM secretion in response to CpG motifs in bacterial DNA and oligodeoxynucleotides. J Immunol 1996; 156:558-564.

49. Kurreck J. Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem 2003; 270:1628-1644.

50. Shi F, Hoekstra D. Effective intracellular delivery of oligonucleotides in order to make sense of antisense. J Control Release 2004; 97:189-209.

51. Rao M, Alving CR. Delivery of lipids and liposomal proteins to the cytoplasm and golgi of antigen-presenting cells. Adv Drug Deliv Rev 2000; 41:171-188.

52. Klinman DM, Currie D, Gursel I, Verthelyi D. Use of CpG oligodeoxynucleotides as immune adjuvants. Immunol Rev 2004; 199:201-216.

53. Jiao X, Wang RY-H, Qiu Q, Alter HJ, Shih JW-K. Enhanced hepatitis C virus NS3 specific Th1 immune responses induced by co-delivery of protein antigen and CpG with cationic liposomes. J Gen Virol 2004; 85:1545-53.

54. Suzuki Y, Wakita D, Chamoto K, Narita Y, Tsuji T, Takeshima T, et al. Liposome-encapsulated CpG oligodeoxynucleotides as a potent adjuvant for inducing type 1 innate immunity. Cancer Res 2004; 64:8754-8760.

55. Rhee EG, Mendez S, Shah JA, Wu CY, Kirman JR, Turon TN, et al. Vaccination with heat-killed Leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against Leishmania major infection. J Exp Med 2002; 195:1565-1573.

56. Zimmermann S, Egeter O, Hausmann S, Lipford GB, Rocken M, Wagner H, et al. CpG oligodeoxynucleotides trigger protective and curative Th1 responses in lethal murine leishmaniasis. J Immunol 1998; 160:3627-3630.

57. Walker PS, Scharton-Kersten T, Krieg AM, Love-Homan L, Rowton ED, Udey MC, et al. Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFN-gamma-dependent mechanisms. Proc Natl Acad Sci U S A 1999; 96:6970-6975.

58. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408:740-745.

59. Rhee EG, Mendez S, Shah JA, Wu C-y, Kirman JR, Turon TN, et al. Vaccination with heat-killed Leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+and CD8+T cell responses and protection against Leishmania major infection. J Exp Med 2002; 195:1565-1573.

60. Badiee A, Jaafari MR, Khamesipour A, Samiei A, Soroush D, Kheiri MT, et al. The role of liposome charge on immune response generated in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63). Exp Parasitol 2009; 121:362-369.

61. Mazumdar T, Anam K, Ali N. Influence of phospholipid composition on the adjuvanticity and protective efficacy of liposome-encapsulated Leishmania donovani antigens. J Parasitol 2005; 91:269-274.

62. Kuwana M, Matsuura E, Kobayashi K, Okazaki Y, Kaburaki J, Ikeda Y, et al. Binding of beta 2-glycoprotein I to anionic phospholipids facilitates processing and presentation of a cryptic epitope that activates pathogenic autoreactive T cells. Blood 2005; 105:1552-1557.

63. Foged C, Arigita C, Sundblad A, Jiskoot W, Storm G, Frokjaer S. Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition. Vaccine 2004; 22:1903-1913.

64. Jiao X, Wang RYH, Feng Z, Alter HJ, Shih JWK. Modulation of cellular immune response against hepatitis C virus nonstructural protein 3 by cationic liposome encapsulated DNA immunization. Hepatology 2003; 37:452-460.

65. Heravi Shargh V, Jaafari MR, Khamesipour A, Jalali SA, Firouzmand H, Abbasi A, et al. Cationic liposomes containing soluble Leishmania antigens (SLA) plus CpG ODNs induce protection against murine model of leishmaniasis. Parasitol Res 2012; 111:105-114.

66. McConville MJ, Thomas-Oates JE, Ferguson MA, Homans SW. Structure of the lipophosphoglycan from Leishmania major. J Biol Chem 1990; 265:19611-19623.

67. Souto-Padron T. The surface charge of trypanosomatids. An Acad Bras Cienc 2002; 74:649-75.