Enfeksiyöz Nekrotik Hepatitis Aşısının Üretiminde Poli (D, L-Laktik-Ko-Glikolik Asit) (PLGA) Biyopolimerinin Adjuvant Etkisinin Araştırılması

Bu çalışmada, koyunların enfeksiyöz nekrotik hepatitis hastalığına karşı alüminyum hidroksit ve poli (D, L-Laktik-Ko-Glikolik Asit) (PLGA) adjuvantları kullanılarak üretilen aşıların kobaylarda oluşturduğu bağışıklık düzeyleri karşılaştırıldı. Clostridium novyi tip A kültürünün formol ile inaktivasyonundan sonra, alüminyum hidrokside adsorbe edilmiş ve çift emülsiyon çözücü buharlaştırma yöntemi uygulanarak PLGA (laktid/glikolid oranı: (50/50), moleküler ağırlık: 30.000-60.000 dalton) ile enkapsüle edilmiş antjen içeren iki farklı toksoid aşı hazırlandı. Aşıların oluşturduğu bağışıklık düzeyini belirlemek için 5-6 aylık ve ağırlıkları 400-500 gram olan erkek kobaylar kullanıldı. Her biri 10 kobaydan oluşan 3 grup oluşturuldu. Birinci gruptakilere 21 gün arayla çift doz (2 ml+2ml) alüminyum hidroksitli aşı, ikinci gruptakilere PLGA mikrosferli aşı tek doz (2 ml) ve üçüncü gruptakilere PLGA mikrosferli aşı yarım doz (1ml) derialtı yolla verildi. Birinci gruptaki kobaylardan rapel aşılamadan, ikinci ve üçüncü gruptaki kobaylardan tek doz aşılamadan sonraki 15, 30 ve 45. günlerde kalpten kan örnekleri alınarak havuzlanmış serum örnekleri elde edildi. Kan serumlarındaki antikor düzeyi fare Toksin Nötralizasyon Test (TNT) ile belirlendi. Tek doz mikrosferli aşı ile çift doz alüminyum hidroksitli aşı uygulamalarından sonraki 30. ve 45. günlerde aynı düzeyde antikor (8 IU/ml) saptandı. Ancak 15. günde çift doz alüminyum hidroksitli aşının antikor düzeyi 4 IU/ml iken, tek doz PLGA mikrosferli aşının antitor düzeyi 2 IU/ml olarak bulundu. Yarım doz PLGA mikrosferli aşı verilen kobaylarda yeterli düzeyde (antikor titresi˂2.5 IU/ml) bağışıklık elde edilemedi. Sonuç olarak, tek dozlu veteriner aşıların geliştirilmesi amacıyla farklı polimer tipi ve enkapsulasyon yöntemleri kullanılarak daha geniş kapsamlı çalışmaların yapılması gerekmektedir.

Anahtar Kelimeler:

C. novyi, aşı, adjuvant, PLGA

PDF

___

1. Akagi T, Baba M, Akashi M. (2012). Biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine. Adv Polym Sci. 247, 31-64.
2. Amitoto K, Sasaki O, Isogai M, et al. (1998). The protective effect of Clostridium novyi type B alpha-toxoid against challenge with spores in guinea pigs. J Vet Med Sci. 60, 681-685.
3. Arda M, Minbay A, Leloğlu N, ve ark. (1997). Özel Mikrobiyoloji, Medisan Yayın Serisi No:13, Medisan Yayınevi, Ankara.
4. Ardehali M, Darakhshan H, Moosawi M. (1986). Mass production and standardization of Clostridium oedematiens vaccine against black disease (infectious necrotic hepatitis) of sheep. Dev Biol Stand. 64, 137-140.
5. Arnon R, Yedidia TB. (2003). Old and new vaccine approaches. Int Immunopharmacol. 3, 1195-1204.
6. Baş T, Alp R. (2005). Clostridial aşıların kombine hazırlanması. Pendik Vet Mikrobiyol Derg. 36, 35-45.
7. Blood DC, Radostits OM. (1989). Veterinary Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses, 7 th., London.
8. Büyüktanır Ö. (2010). Günümüzde biyoteknolojik bakteriyel aşılar. Atatürk Üniversitesi Vet Bil Derg. 5, 97-105.
9. Cleland JL. (1995). Design and production of single-immunization vaccines using polylactide polyglycolide microsphere systems. in: Powell M. F., Newman M. J. (Eds.) Vaccine design: The subunit and adjuvant approach. Plenum Press, New York.
10. Clem AS. (2011). Fundamentals of vaccine immunology. J Glob Infect Dis. 3, 73-78.
11. Eldridge JH, Staas, JK, Meulbroek JA, et al. (1991). Biodegradable and biocompatible poly(dl-lactideco-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxin-neutralizing antibodies. Infect Immun. 59, 2978–2986.
12. Feng G, Jiang Q, Xia M, et al. (2013). Enhanced immune response and protective effects of nano-chitosan-based DNA vaccine encoding T cell epitopes of Esat-6 and FL against Mycobacterium tuberculosis infection. PLoS One. 8, 4.
13. Gupta A, Chaphalkar SR. (2015). Vaccine adjuvants: the current necessity of life. Shiraz E-Med J. 16, 1-11.
14. Gutierro I, Hernandez RM, Igartua M, et al. (2002). Size dependent immune response after subcutaneous, oral and intranasal administration of BSA loaded nanospheres. Vaccine. 21, 67-77.
15. Isobe M, Yamazaki Y, Oida SI, et al. (1996). Bone morphogenetic protein encapsulated with a biodegradable and biocompatible polymer. J Biomed Mater Res. 32, 433–438.
16. Jaganathan KS, Singh P, Prabakaran D, et al. (2004). Development of a single-dose stabilized poly(D, L-lactic-co-glycolic acid) microspheres-based vaccine against hepatitis B. J Pharm Pharmacol. 56, 1243-1250.
17. Jaganathan KS, Rao YUB, Singh P, et al. (2005). Development of a single dose tetanus toxoid formulation based on polymeric microspheres: a comparative study of poly(D, L-lactic-co-glycolic acid) versus chitosan microspheres. Int J Pharm. 294, 23–32.
18. Johansen P, Linda M, Tamber H, et al. (2000). Immunogenicity of single-dose diphtheria vaccines based on PLA/PLGA microspheres in guinea pigs. Vaccine. 18, 209-215.
19. Johansen P, Martinez-Gomez JM, Gander B. (2007). Development of synthetic biodegradable microparticulate vaccines: a roller coaster story. Exp Rev Vac. 6, 471-474.
20. Kerimoğlu O, Alarçin, E. (2002). Poly(lactic-co-glycolic acid) based drug delivery devices for tissue engineering and regenerative medicine. ANKEM Derg. 26, 86-98.
21. Khademi F, Sahebkar A, Fasihi-Ramand M, et al. (2018). Induction of strong immune response against a multicomponent antigen of Mycobacterium tuberculosis in BALB/c mice using PLGA and DOTAP adjuvant. APMIS. 126, 509–514.
22. Kılıç A, Kılınç Ü. (2005). Enfeksiyöz nekrozan hepatit aşısı uygulanan koyunlarda aşının oluşturduğu bağışıklık düzeyinin araştırılması. FÜ Sağ Bil Derg.19, 145-149.
23. Kim MG, Park JY, Shon Y, et al. (2014). Nanotechnology and vaccine development. Asian J Pharm Sci. 9, 227-235.
24. Kumar M. (2000). Nano ve microparticles as controlled drug delivery devices. J Pharm Sci. 3, 234-258.
25. Lee NH, Lee JA, Park SY, et al. (2012). A review of vaccine development and research for industry animals in Korea. Clin Exp Vaccine Res. 1, 18-34.
26. Lee S, Nguyen MT. (2015). Recent advances of vaccine adjuvants for infectious diseases. Immune Network. 15, 51-57.
27. Li P, Luo Z, Liu P, et al. (2013). Bioreducible alginate-poly (ethylenimine) nanogels as an antigen-delivery system robustly enhance vaccine-elicited humoral and cellular immune responses. J Control Release. 168, 271–279.
28. Li P, Asokanathan C, Liu F, et al. (2016). PLGA nano/micro particles encapsulated with pertussis toxoid (PTd) enhances Th1/Th17 immune response in a murine model. Int J Pharm. 513, 183-190.
29. Lin CY, Lin SJ, Yang YC, et al. (2015). Biodegradable polymeric microsphere-based vaccines and their applications in infectious diseases. Hum Vaccin Immunother. 11, 650-656.
30. Lowry OH, Rosbrough NJ, Farr AL, et al. (1951). Protein measurement with the folin phenol reagent. J Biol Chem. 193, 265-275.
31. Peek LJ, Middaugh CR, Berkland C. (2008). Nanotechnology in vaccine delivery. Adv Drug Delier Rev. 60, 915–928.
32. Quintilio W, Takata CS, Sant’Anna OA, et al. (2009). Evaluation of a diphtheria and tetanus PLGA microencapsuled vaccine formulation without stabilizers. Curr Drug Deliv. 6, 297-304.
33. Rosas JE, Pedraz JL, Hernandez RM, et al. (2002). Remarkably high antibody levels and protection against P. falciparum malaria in Aotus monkeys after a single immunisation of SPf66 encapsulated in PLGA microspheres. Vaccine. 20, 1707-1710.
34. Sanghi DK, Tiwle R. (2014). A detail comprehensive review on vaccines. Int J Res Dev Pharm L Sci. 3, 887-895.
35. Shakya AK, Nandakumar KS. (2013). Applications of polymeric adjuvants in studying autoimmune responses and vaccination against infectious diseases. J R Soc Interface. 10, 1-6.
36. Shi L, Caulfield MJ, Chern RT, et al. (2002). Pharmaceutical and immunological evaluation of a single-shot hepatitis B vaccine formulated with PLGA microspheres. J Pharmacet Sci. 91, 1019-1035.
37. Singh SM, Alkie TN, Nagy E, et al. (2016). Delivery of an inactivated avian influenza virus vaccine adjuvanted with poly(D,L-lactic-co-glycolic acid) encapsulated CpG ODN induces protective immune responses in chickens. Vaccine. 34, 4807-4813.
38. Souza CD, Bannantine JP, Brown WC, et al. (2017). Nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp. paratuberculosis peptides stimulate a pro-immune proﬁle in bovine macrophages. J Appl Microbiol. 123, 54—65.
39. Spickler AR, Roth JA, (2003). Adjuvants in veterinary vaccines: Modes of action and adverse effects. J Vet Intern Med. 17, 273–281.
40. Taha-Abdelaziz K, Hodgins DC, Alkie TN, et al. (2018). Oral administration of PLGA-encapsulated CpG ODN and Campylobacter jejuni lysate reduces cecal colonization by Campylobacter jejuni in chickens. Vaccine. 36, 388-394.
41. Thomas C, Gupta V, Ahsan F. (2010). Particle size influences the immune response produced by hepatitis B vaccine formulated in inhalable particles. Pharm Res. 27, 905-919.
42. Webster A, Frank CL. (1985). Comparison immune response stimulated in sheep, rabbits and guinea pigs by the administration of multicomponent clostridial vaccines. Australian Vet J. 62, 112-114.
43. Woo HS, Kim SR, Yoon M, et al. (2018). Combined poly(lactide-co-glycolide) microspheres containing diphtheria toxoid for a single-shot immunization. AAPS Pharm Sci Tech. 19, 1160-1167.