Protection of Cultured Shrimp from White Spot Syndrome Virus (WSSV) by Recombinant Proteins, rVP28 and rVP19-28

Abstract

흰반점증후군바이러스 (White Spot Syndrome Virus, WSSV) 는 1992년 Taiwan에서 처음 발견 되었으며 봉입체의 타원형, 이중가닥 DNA 바이러스로 주로 갑각류에 감염된다. 특히, WSSV는 새우 백점병의 원인 바이러스로 양식 새우에서 대량폐사를 일으켜 경제적으로 큰 손실을 입히고 있으나 이를 효과적으로 제어할 수 있는 방법이 아직 보고되어 있지 않다. 따라서 본 연구에서는 재조합 WSSV envelop protein을 이용하여 WSSV를 제어하기 위한 유사백신 개발을 목표로 연구를 수행하였다. 현재까지 알려진 WSSV의 주요 단백질로는 VP19, 26, 28, 281, 466을 포함하여 약 40여종이 있으며, 주로 VP19와 28을 이용한 연구결과들이 많이 보고되어 있다. 한편, 최근 재조합 단백질을 이용한 바이러스 백신 연구에 있어 다양한 항원의 조합이 가능한 fusion protein이 많이 이용되고 있다. 그러므로 본 연구에서는 WSSV의 주요 구조단백질인 VP19와 VP28을 모두 포함하는 VP19-28 fusion protein을 제조한 다음 VP28과 함께 재조합 백신으로서의 효능을 평가하기 위해 연구를 수행하였다. 우선 감염된 새우로부터 WSSV를 분리한 후 단백질을 coding하는 VP19와 VP28 DNA fragment를 정제된 바이러스로부터 PCR을 통해 증폭하였다. 이것을 동일한 제한효소인 Bam HⅠ으로 처리한 뒤 T4 ligase로 연결하여 fusion gene을 제작하였다. 그런 다음 pET-28a(+) vector에 클로닝하고 E. coli BL21에 형질전환시킨 후 발현과정을 거쳐 재조합 단백질을 확보하였다. 다양한 배양 시간과 IPTG 농도 조건으로 test해 본 결과, pET-VP19-28은 37℃에서 0.5 mM IPTG로 induction한 다음 2시간 이후부터 재조합 fusion protein (rVP19-28) 을 확인할 수 있었다. 단백질 농도는 Bradford-assay을 이용하여 평균 1.0 mg/ml의 단백질 농도를 확인 할 수 있었으며 이를 SDS-PAGE에서 확인하였다. 또한 pET-VP19-28에는 His-tag이 포함되어 있어 이를 affinity column chromatography로 정제 하였고 anti-His antibody를 이용하여 western blot으로 확인 하였다. 한편, rVP19-28의 WSSV에 대한 면역효과를 검증하기 위하여 중화효과 (neutralization effect) 와 백신효과 (vaccination effect) 에 대해 rVP28과 비교하여 test를 수행 하였다. 먼저 각각의 정제된 rVP19-28과 rVP28를 토끼에 접종하여 항체를 생산하였고 이를 ELISA로 확인 하였다. 또한 실험에 사용할 바이러스 적정 농도를 구하기 위해 in vivo titration test를 수행 하였다. 중화효과 실험에 사용된 새우는 평균 5 g 정도의 Penaeus chinensis (대하) 이며 중화반응은 항체와 바이러스 반응액을 혼합한 후 28℃에서 1시간 incubation 시킨 다음 실험용 새우에 접종 하였다. 약 2주 경과 후 positive control과 비교 하였을 때 rVP19-28과 rVP28에 의해 생성된 항체에 의해 WSSV가 효과적으로 불활성화 됨을 알 수 있었다. 백신효과 실험은 rVP19-28과 rVP28이 포함된 각각의 백신사료를 준비하고 이를 2주일간 지속적으로 투여한 다음, titration test를 통해 확인된 적정농도인 10² 으로 희석된 WSSV 액을 이용하여 공격실험을 수행하였다. 그 결과 positive control은 접종 후 8일째 100% mortality를 보인데 비해 rVP19-28과 rVP28의 경우는 접종 후 21일까지 각각 40%와 30%의 누적폐사율을 보여 백신으로서의 효과를 확인할 수 있었다. 따라서 VP28 뿐만 아니라 fusion protein인 VP19-28도 백신으로써 효과가 있음을 확인 하였으므로 추후 WSSV 유사 백신 개발에 적용 될 수 있을 것이다.

Invertebrate constitute 95% of all animal species and rely on defense mechanism primarily based on a broad range of cellular innate immune responses. Because of the lack of a known adaptive immune response, the potential for vaccination against viral pathogens is uncertain. However, a few reports suggested that the presence of such a response in crustaceans (Venegas et al., 2000; Wu et al., 2002) and this has opened up the possibility of vaccination as an intervention strategy to combat viral disease in shrimp. In the study present here we have analyzed if viral proteins can be used to elicit an immune response in shrimp leading to protection against WSSV. Because VP28 and VP19 protein are the most exposed proteins abundantly present in the WSSV envelop and react strongly with polyclonal antibodies generated against complete virions in rabbit (van Hulten et al., 2000a), so we selected VP28 and fusion protein VP(19+28) for use in the subunit vaccines. Three different trials (Neutralization test, vaccination test by injection and Vaccination by oral feeding) were carried out to examine the possibility of immunization of P. chinesis against WSSV. Neutralization of virus with antiserum have been performed to study the role of virion proteins or their domains in the infection process. However, standardized (primary) shrimp cell culture was not available and therefore an in vivo approach was followed. The in vivo neutralization test showed that WSSV infection was neutralized by the rVP28 polyclonal antiserum. The preimmune serum control resulted in a small positive effect on shrimp survival. This could be due to compounds present in the serum stimulating the shrimp defense system. It suggest that a small general immune response can be provided by injection of foreign proteins. A similar resulted was reported by Wistteveldt et al. (2004a, b). The result of neutralization indicated that WSSV could be neutralized by rVP28 polyclonal antiserum in a dose-dependent manner and VP28 plays an important role in the systemic infection of WSSV in shrimp. In vaccination experiment we chose bacteria for protein expression and as an antigen dilivery vehicle since the production for commecial application as well established and cheap. At first we carried out Vaccination test by intramuscular injection to ensure the application of a consistent amount of proteins per shrimp. Even though this technique is far from practical under shrimp farming conditions, it is suitable in determining the vaccinating potential of proteins. After 1 week WSSV challenge, all shrimps in positive control died whereas the mortality of shrimps injected with VP28 protein was reduced 40%. This result indicated that the resistance system of shrimp was enhanced to against WSSV using its structural proteins as a subunit vaccine. However since injection vaccinations in shrimp are not practically feasible in shrimp farming, so to this end we have used oral vaccination. The challenge with WSSV was performed using immersion, as the challenge pressure can be well controlled in contrary to challenge using infected tissue. In a natural situation shrimp become infected through both oral and water-borne route and the gills are thought to be a major oint of viral entry (Chang et al., 1996; Tan et al., 2001). This vaccination test by oral feeding showed a significantly low cumulative mortality in vaccinated shrimp compared to control group. The rVP28 gave a better protective effect than rVP(19+28), therefore it suggests that correctly folded VP28 protein provides better protective response in shrimp than VP(19+28), or maybe because molecule size of VP(19+28) protein was so big that the protein could not penetrate biological membrane in internal organs. As protection against WSSV is maintained up to three weeks after vaccination, it is unlikely that the presence of residual VP28 could block WSSV infection by blocking receptors needed by the virus to enter shrimp cells. The way in which the protection is obtained by the shrimp immune system remains to be resolved. Protection could for example be generated by prevention of entry of WSSV by secreted neutralizing substances or by blocking the virus spread entry. Oral vaccination with VP28 results in a reduction in mortality to a level unlike that observed in the injection vaccinations. Possibly the reverse situation for VP28 exists; if elicits a high reaction in the intestinal tract, but a much lower reation when injected. Another explanation could lie within the challenge methods: with the immersion challenge method WSSV is confronted with a different set of immunological defenses of the shrimp and may be neutralized by a process which is circumvented by using injection challenge. Overall, these oral vaccination experiments have shown that shrimp can specifically recognize and react on WSSV structural proteins. Altogether these results suggest that a specific memory exists in invertebrate or more specific in crustaceans as the data obtained are in line with the results found for the copepod, which is a minute crustacean (Kurtz and Franz, 2003). This study shows that the shrimp immune system is able to specifically recognize WSSV structural proteins and that vaccination of shrimp against WSSV might be possible and opens the way to the design of new strategies to control WSSV and other inveterbrate pathogens.