Development and validation of subunit vaccine to prevent porcine epidemic diarrhea via oral vaccination : 돼지 유행성 설사 예방을 위한 아단위 경구 백신의 개발 및 효과 검정

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농업생명과학대학 농생명공학부
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서울대학교 대학원
PEDsubunit vaccineinclusion bodysoluble antigenneutralizing antibodysoluble aP2HPMCPoral vaccinechallenge assay
학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부 동물생명공학과, 2016. 8. 최윤재.
Porcine epidemic diarrhea (PED) is a highly contagious enteric disease of swine. Recent outbreaks of PED in Asia, North America and Europe, which caused by highly pathogenic variant strains, have greatly affected global swine industry in economic perspectives. Therefore, development of more effective control measurement such as vaccines, are urgently needed to combat newly emerged PEDV variants. In Korea, most of the commercial PED vaccines are made by Vero cell-attenuated live or killed PED virus, which used for vaccinating sows through oral or intramuscular route. Although those commercial PED vaccines have been proved to be effective, safety concerns are steadily raised about their application in the field. As an alternative vaccine candidate, recombinant subunit vaccines have been considered as a promising vaccine candidate for the prevention of PED, because it is safer, easier to produce and cost-effective than attenuated live or killed vaccines.
The present study was aimed to develop a recombinant subunit antigen-based oral vaccine system for the effective prevention of PED. The study is divided into two parts: (i) Production of soluble recombinant spike protein of porcine epidemic diarrhea virus in E. coli for the development of effective subunit vaccine, (ii) Evaluation of subunit vaccine loaded hydroxypropyl methylcellulose phthalate microspheres and RANKL secreting L. lactis as an effective oral vaccine candidate in pregnant sow.
In study 1, we aimed to produce recombinant spike protein of PEDV in E. coli as a subunit antigen. Initially, the neutralizing epitope regions of PEDV S protein such as COE (aa 499-636), S1D (aa 637-789) and SP1 (aa 499-789), were chosen and expressed as glutathione S-transferase (GST) fusion proteins in E. coli. However, unlike previous report, expression of GST-COE, GST-S1D and GST-SP1 fusion proteins in E. coli invariably resulted in inclusion bodies (IBs). Besides, to produce those proteins in soluble form, we adopted chaperone co-expression and alkaline pH solubilization strategies. We found that co-expression of TF with recombinant proteins at 15 C was most useful in soluble production of GST-COE and GST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble GST-COE (P1) and GST-S1D (P2) were purified by GST-tag affinity chromatography with yields of 5-7.5 mg per 1 liter E. coli culture. Purified soluble proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. Furthermore, large amount (180-250 mg/l culture) of soluble GST-COE (aP1) and GST-S1D (aP2) were recovered from IBs by alkaline pH solubilization strategy. In in vivo immunization study, considering cost-effectiveness of the subunit vaccine for industrial application, we firstly tested whether IBs could be used as subunit vaccine antigen. However, intramuscular immunization of GST-SP1 IBs (SP1 is the COE and S1D combined region) did not induce serum neutralizing antibodies in pigs. On the other hand, systemic injection of mice and pigs through subcutaneous or intramuscular route with both soluble GST-COE and GST-S1D (P1, P2, aP1 and aP2) elicited highly potent serum IgG and neutralizing antibodies, suggesting that solubility of the antigen may play important role in the generation of serum neutralizing antibodies against PEDV. Even tough, since production yields of soluble aP1 and aP2 produced by alkaline pH solubilization method were much higher than P1 and P2, and therefore, those two antigens may have potential to be used for the development of recombinant subunit antigen-based oral vaccine strategy.
In study 2, soluble aP2 antigen was selected and used for the development of an effective oral vaccination strategy for the prevention of PED. For the effective delivery of aP2 antigen through oral route, we used hydroxypropyl methylcellulose phthalate (HPMCP (aP2)) as a carrier system. The HPMCP (aP2) microspheres were prepared by W1/O/W2 double emulsion method. The size of the microsphere was 1-10 µm. The microspheres showed spherical morphology under a scanning electron microscope. The loading content and efficiency were 6.3% and 42.5%, respectively. Moreover, in vitro release test revealed that 70% of aP2 were efficiently released from microspheres at pH 7.0 within 1 h, while only 20% of aP2 were released at pH 2.0. As an oral vaccine adjuvant system, we chose RANKL secreting L. lactis (LL RANKL) to enhance the efficacy of mucosal immune response. To evaluate whether oral vaccination of pregnant sows with HPMCP (aP2) microspheres and LL RANKL could elicit potent lactogenic immunity, and thus protect suckling piglets from virulent PEDV challenging. Sows were vaccinated through oral route at 4 weeks before fallowing for two times with 2-weeks interval. As a result, neutralizing antibody titers of serum, colostrum and whey of sows received oral vaccines were increased after 4 weeks first vaccination. Furthermore, piglets delivered from sows vaccinated with HPMCP (aP2) microspheres and LL RANKL showed lower overall mortality rate (25% and 75%) than piglets from sows vaccinated with commercial PED vaccine (88% and 50%). Therefore, our current oral vaccine strategy based on recombinant subunit antigen could be a promising vaccination strategy for the prevention of PED.

Key words: PED, subunit vaccine, inclusion body, soluble antigen, neutralizing antibody, soluble aP2, HPMCP, oral vaccine, challenge assay

Student number: 2010-31338
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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