Development of vaccines against scuticociliatosis caused by Philasterides dicentrarchi

Abstract

스쿠티카증은 섬모충의 일종인 스쿠티카충이 어류 등에 감염되어 심각한 폐사를 일으키는 질병으로서 이를 제어하기 위한 여러가지 화학요법제를 사용한 시도가 있었지만, 아직까지 효과적인 치료제가 개발된 바가 없으며, 따라서 스쿠티카증을 제어하기 위해서는 백신 개발을 통한 예방이 최선책이라 할 수 있다. 본 연구는 스쿠티카증의 주요 원인체인 Philasterides dicentrarchi 에 대한 백신 개발을 위한 연구에 중점을 두었다. 1. Immobilization antigen independent protection of fish against scuticociliatosis 섬모충의 주요 항원인 immobilization antigens (i-antigen)이 스쿠티카증에 대한 항원후보로서 적합한지 조사하기 위해, CHSE-214 세포를 이용하여 배양한 P. dicentrarchi lysate를 넙치에 주사하여 만든 항혈청으로 배양충 및 감염충에 대해 in vitro killing, agglutination, ADCK assay를 수행하였다. 배양충은 immune sera 내에서 강하게 agglutination 되었지만, agglutination 되었던 충들이 몇시간 안에 다시 활성을 되찾는 것을 관찰하였다. 또한 매우 낮은 혈청 농도에서까지 ADCK에 의해 killing되었으나, agglutination이 강하게 일어난 고농도 혈청에서는 오히려 ADCK에 의한 killing 이 완벽하게 충을 죽이지는 못하였다. 감염충은 고농도의 항혈청에서 agglutination은 되지 않았지만, ADCK 에 의해서는 배양충보다 쉽게 killing 되었다. 이러한 결과로 스쿠티카충의 i-antigen 은 항체가 존재하면 그 type을 바꾸어 ADCK에 의한 killing 을 피할 수 있게 되므로, i-antigen은 백신의 후보 항원으로 적합하지 않음을 알 수 있었다. 또한 넙치 치어에 감염충과 배양충의 lysate를 주사한 후 감염실험시, 두 경우 모두 높은 방어효과를 나타내었다. 따라서 본 연구에서는, 비록 i-antigen type은 다르지만 배양충을 백신에 사용하였을 때 감염에 방어효과를 나타내었다는 것은 i-antigen과는 별개의 방어기작이 스쿠티카충 lysates에 의해 유도되며, cell line을 이용하여 배양한 스쿠티카충은 백신의 antigen으로 사용할 수 있음을 제안하였다. 2. CpG ODN에 의한 어류의 스쿠티카충에 대한 저항성 증진 넙치 치어에, immunostimulatory CpG ODN 1668을 주사하여, 혈청의 살충능과 공격실험에서의 생존능을 조사하였다. 그 결과, 실험구의 혈청은 대조구에 비해 높은 스쿠티카충 살충능을 보였고, 감염실험에서도 높은 생존율을 나타내었다. 따라서 본 연구에서는, CpG ODN 이 넙치에서 보체의 대체경로를 활성화 시키고, 이러한 보체에 의한 살충능은 P. dicentrarchi 감염에 대한 선천면역의 중요한 요소이며, 따라서, CpG ODN의 스쿠티카증에 의한 피해를 감소시키기 위한 면역자극물로서 사용가능성을 제시하였다. 3. 스쿠티카충 백신 개발을 위한 실험 모델로서 zebrafish 의 응용 본연구에서는, 스쿠티카충 백신 시스템의 개발과 함께, 이후의 백신 실험의 효과를 검증하기 위한 시스템으로서 zebrafish의 이용가능성에 대해 연구하였다. 이를 위해, 감염실험을 먼저 수행한 결과 zebrafish는 전형적인 스쿠티카증의 증상들을 나타내며 다량 폐사하는 것을 관찰하였다. 이후에 스쿠티카충 lysate를 zebrafish에 vaccination한 후 감염실험 하여 방어효과를 조사하였다. 본 연구에서는, 또한 Quillaja saponin, Montanide, and Freund？s incomplete adjuvant 와 같은 여러가지 adjuvants 의 효과도 함께 조사하였다. 그 결과, 스쿠티카충 lysate만을 주사한 그룹에서도 비교적 높은 생존율을 나타내었고, 각각의 adjuvant를 함께 투여하였을 때 그 효과가 증가하였다. 4. 감염형 P. dicentrarchi 에서 특이적으로 발현되는 유전자의 동정 재조합 단위 백신의 개발을 위한 후보 항원의 검색을 위해, subtraction subtractive hybridization (SSH) 법을 수행하여 subtracted cDNA library를 구축하였고, 이를 분석하여 어체에 감염된 상태의 P. dicentrarchi에서 특이적으로 발현되는 유전자들을 확보하였으며, 그 발현은 semi-quantitative RT-PCR을 수행하여 검증하였다. 5. 스쿠티카충 항원의 재조합 단백질의 투여로 유도되는 방어 효과 이전의 연구와 본 연구에서 확보한 P. dicentrarchi의 유전자 중에서 재조합 단위 백신의 개발을 위한 6가지 항원후보 유전자-β-tubulin (BTU), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), acidic ribosomal PO protein (PO), plasma membrane Ca^(2+)ATPase (PMCA), leucine rich repeat containing protein (LRR), surfaceprotein (SP)-를 선정하였다. 이들 유전자의 전체 ORF 또는 부분 ORF sequence를 클로닝하여, E. coli 에서 재조합단백질을 제조하기 위해, site-derected mutagenesis를 수행한 후 각각의 재조합 단백질을 발현, 정제하여 이들 단백질의 방어효과를 zebrafish 에 immunization 하여 조사하였다. 주사와 경구 투여법으로 immunization 한 결과 LRR 과 sufrace protein이 높은 방어효과를 나타내었다. 또한 경구투여시 GAPDH, LRR, SP를 혼합하였을 때, 방어효과가 단독으로 투여하였을 때보다 증가하였다. 6. 스쿠티카충 항원 표면발현 E. tarda ghost (ETG)의 방어 효과 새로운 단위 백신의 delivery system의 개발을 위해, 위 여섯 가지 항원후보 단백질을 세포 표면에 발현시킨 Edwardsiella tarda ghost (ETG)를 제작하여 zebrafish 에 투여한 후 그 방어 효과를 확인하였다. 주사 및 경구 투여법으로 immunization 한 결과surface protein이 가장 높은 방어 효과를 나타내었으며, 재조합 단백질을 투여하였을 때와 마찬가지로, GAPDH, LRR, SP를 혼합하여 투여하였을 때 방어효과가 증가하는 것을 확인하였다. 본 연구에서는, 어류의 스쿠티카증을 예방하기 위한 일련의 연구들을 통해 긍정적인 결과들을 얻었다. 이러한 결과들을 토대로, 본 연구에서 개발된 백신 시스템은 추후 양식현장 적용을 통해 넙치 양식에 있어 치명적인 스쿠티카증을 제어함으로써 양식어류의 생산성 향상에 기여할 수 있을 것이다.

Scuticociliatosis, caused by a ciliated protozoan parasite, is a well-known culprit of massive death of cultured fish. Although several attempts have been conducted to treat the disease by various chemotherapeutics, there is at present no effective in vivo chemotherapeutics especially for internal infections. Thus, prevention by effective vaccine development has been considered as the best way to control scuticociliatosis. This study, therefore, focused on the development of vaccines against fish scuticociliatosis caused by Philasterides dicentrarchi which is the most suspicious species for scuticociliatosis occurred in farmed olive flounder Paralichthys olivaceus in Korea. 1. Immobilization antigen-independent protection of fish against scuticociliatosis In order to test whether immobilization antigens (i-antigens) of P. dicentrarchi could be suitable antigenic targets against scuticociliatosis, polyclonal olive flounder sera were raised against P. dicentrarchi by immunization with lysates of ciliates grown using CHSE-214 cells. Although the immune sera showed clear agglutination activity against the CHSE-cultured ciliates, the agglutinated ciliates were not killed but escaped from the agglutinated mass within a few hours. Whereas ciliates isolated from fish artificially infected with the same population of CHSE-cultured ciliates were not agglutinated by the immune sera. In ADCK assays, the immune sera completely killed the CHSE-cultured ciliates at relatively higher serum dilutions (showing low or no agglutination activity). However, CHSE-cultured ciliates were not killed completely at lower immune serum dilutions (showing high agglutination activity). In contrast to CHSE-cultured ciliates, the ciliates isolated from infected fish were killed at lower dilutions of the immune sera in spite of no agglutination response. Considering the presence of various i-antigen types, ability to change i-antigen type in response to corresponding antibody and relatively low ADCK activity at high agglutination titer, i-antigens of P. dicentrarchi may not be good targets for subunit vaccine development. To determine whether olive flounder immunized with lysate of P. dicentrarchi could be protected immobilization antigen (i-antigen) type independently, fish were immunized with lysate of ciliates obtained from in vitro cultures or ciliates obtained from infected fish. Fish immunized with ciliates obtained from infected fish were completely protected against artificial infection with in vitro cultured P. dicentrarchi, in spite of a weak or no serum agglutination activity against in vitro cultured ciliates. The present results indicate that i-antigen-independent protection was elicited by immunization of fish with the ciliates lysate, and the cultured ciliates would provide a good source for preparation of vaccines, which are cross-protective against various i-antigen types of P. dicentrarchi. 2. Effects of CpG ODN on the resistance of fish against scuticociliatosis The direct effects of CpG-ODN 1668 on the resistance of olive flounder against scuticociliatosis were investigated by analyzing the serum scuticocidal activity and the survival rate of fish against P. dicentrarchi challenge. Fish injected with CpG-ODN 1668 showed significantly higher serum scuticocidal activity than control fish injected with PBS alone. This result suggests that CpG-ODN might activate an alternative pathway of complement of olive flounder, and complement-mediated killing might be an important innate immune factor in the resistance against P. dicentrarchi infection and CpG ODNs may be used as potential immunostimulants to lessen cultured fish loss caused by scuticociliates. 3. Application of zebrafish as an experimental model for scuticociliatosis Zebrafish was firstly applied to an experimental model for scuticociliatosis caused by P. dicentrarchi. The susceptibility of zebrafish to infection of P. dicentrarchi was assessed by intraperitoneal injection of the ciliates, which produced typical symptoms of scuticociliatosis and significant mortality. The potential use of zebrafish as a model to evaluate the vaccine efficacy against scuticociliatosis was analyzed by immunization of zebrafish with the ciliates lysate. Furthermore, the effect of different adjuvants, such as Quillaja saponin, Montanide, and Freund？s incomplete adjuvant on the protective efficacy of the vaccine was investigated. The considerably high survival in the fish immunized with the antigen alone and no or low mortalities in the groups of fish immunized with the antigen plus adjuvants suggests that the ciliate lysate itself is highly immunogenic to zebrafish and the adaptive immune responses of zebrafish might be accelerated by the adjuvants or the protective potential of the antigen and adjuvants might synergistically interact. In spite of several shortcomings such as difficulties in sampling of serum and leucocytes enough to routine immunological analyses, zebrafsih might be the most convenient experimental animal for scuticociliatosis. 4. Identification of preferentially expressed genes in fish-infected P. dicentrarchi To identify a putative set of genes expressed in infection state of P. dicentrarchi, a subtracted cDNA library representative mRNAs specific to fish-infected P. dicentrarchi was generated using SSH for the first time, and genes associated with signal transduction, cell proliferation, membrane transporter, protein translocation, and transcription regulation were identified. The differential gene expression patterns were confirmed by semi-quantitative RT-PCR. These differential expressions may be needed for the ciliates to survive in the host fish, and part of the genes might be used as antigen candidates for development of scuticociliatosis vaccines. 5. Protection of fish from scuticociliatosis by immunization with recombinantly produced antigens Six genes, β-tubulin (BTU), glyceraldehyde 3-phosphate dehydrogenase (PdGAPDH), acidic ribosomal PO protein (PdPO), plasma membrane Ca^(2+) ATPase (PdPMCA), leucine rich repeat containing protein (PdLRR) and surface protein (PdSP) were tested to investigate the possibility as target antigens in vaccine development by immunization of each recombinant proteins using zebrafish. Among these antigen candidates, LRR and SP were effective in both injection and oral administration. This study suggested that recombinant subunit vaccines can induce protective immune responses against scuticociliatosis in fish. 6. Protection efficacy of ScuAg-surface displayed Edwardsiella tarda ghost (ETG) vaccine To develop more advanced vaccine systems, the combination vaccine system which scuticociliate antigens were expressed on the E. tarda ghosts was developed and the protection efficacy was investigated by immunization of prepared ETGs using zebrafish. As a result, among tested antigens, SP group showed the highest protection followed by LRR in the injection experiment. Moreover, in oral immunization, SP group showed the highest survival rate (RPS 23%) and Mix group of GAPDH, LRR and SP showed increasing survival rate. Although partial protection against challenge infection in both injection and oral immunization was demonstrated, survival rates were relatively lower than that of purified recombinant protein immunization, because the expression levels of P. dicentrarchi antigens on the surface of ETGs were relatively lower than expected and purified proteins. Therefore, this ETG vaccine system can be applied as a convenient delivery system of subunit vaccines against scuticociliatosis after more studies for increasing levels of protein expression. In this study, several positive results and information were obtained by studies on the development of vaccines to prevent scuticociliatosis although some questions to be solved with more integrated and repeated studies. Effective vaccines in aquaculture industry can not only contribute to increase productivity of aquaculture by preventing diseases but also decrease environmental risks which could be caused by using chemotherapeutics. Because treatment of fish using drugs has been the only way to control diseases by parasites in the past and the development of parasite vaccines in fish is very valuable and has merits itself scientifically, this study will be useful for future studies to investigate fish parasitic diseases providing new approach methods and views. Moreover, the systems used in this study may be applicable for development vaccines against fish bacterial and viral vaccines as well as fish parasites, thus further studies should be preformed continually. If these vaccine systems can be applied successfully in the field, it is certain that this study will contribute to increase productivity of aquaculture.