On the vaccination of shrimp against white spot syndrome virus

More than a decade after its discovery inSouth-East Asia, White Spot Syndrome Virus (WSSV) is still the most important (viral) pathogen in the shrimp culture industry. Despite the shift from culturingPenaeusmonodon towards the presumed less susceptibleLitopenaeusvannamei , the use of specific pathogen free shrimp and the development of more advanced shrimp culturing techniques, WSSV continues to scourge shrimp farms. Therefore there is an urgent need for effective intervention strategies. Vaccination is the generally used method to prevent viral infections in vertebrates. The success of this method depends on the immunological memory generated by the adaptive immune system. Unfortunately, shrimps, as any other arthropod, do not have such an adaptive immune system implying that vaccination would never work. However, some phenomenological observations have been made, indicating that there might be an analogous defense system present in shrimp. With this in mind experiments in this thesis are presented to determine if and how shrimp can be protected against WSSV via vaccination.At the start of this research project several studies were available describing various major structural proteins present in the WSSVvirionincluding the majorvirionenvelope proteins VP28 and VP19 andvirionstructural proteins VP26, VP24 and VP15 (see thesis vanHulten, 2001). In this research a number of these proteins were investigated in more detail as potential vaccine candidates. For one of these, the majornucleocapsidprotein VP15, it was determined that it was probably (one of) the DNA-binding protein(s) of WSSV (Chapter 2). Experiments revealed that VP15 binds non-specifically to double-stranded DNA, but has a strong preference tosupercoiledDNA, suggesting a possible role in the packaging of the WSSV genome. Furthermore, VP15 formshomomultimersbut does not interact with any of the other major WSSV structural proteins and unlike other basic DNA-binding proteins VP15 was notphosphorylated.The next structural protein studied in more detail was VP28 which, because of its abundance and location in the envelope of the WSSVvirion, was another potential candidate for use as a WSSV vaccine. The involvement of the VP28 protein in the infection process of WSSV was studied in virus neutralization experiments using polyclonal antibodies generated against the VP28 protein in rabbit (Chapter 3). The antiserum neutralized WSSV infection of P.monodon in a dose-dependent manner, whereas the pre-immune rabbit serum did not. These results suggested that VP28 is located on the surface of the virus particle and is likely to play a key role in the initial steps of the systemic WSSV infection in shrimp. Although the results from the neutralization experiments seemed conclusive, further research revealed that the observed neutralization is probably notIgG-based. Experiments showed that some rabbit pre-immune sera are already able to neutralize WSSV and furthermore, purifiedIgGfrom sera that neutralized WSSV was not able to neutralize the virus (Chapter 4). Therefore, it could be concluded that in most cases the neutralization is not antibody based, but caused by unidentified serum components.VP28 and the other major envelope protein VP19 were tested in vaccination and challenge experiments. The first experiments were performed via injection of the antigens and virus as this guaranteed a controlled and reproducible application. Injection with recombinant MBP-VP19 or a mixture of MBP-VP19 and His ₆ -VP28 significantly reduced and delayed mortality upon WSSV challenge, suggesting a specific role of VP19 in the systemic defense response of shrimp (Chapter 5). To study the onset and duration of the vaccination, groups of shrimp were challenged two or twenty-five days after vaccination. After the challenge, VP19-vaccinated shrimp showed a significant better survival compared to the controls with a Relative Percent Survival (RPS) of 33% and 57% at two and 25 days after vaccination, respectively. Also the groups vaccinated with VP28 and a mixture of VP19 and VP28 showed a significantly better survival challenged two days after vaccination (RPS of 44% and 33% respectively), but no longer after twenty-five days (Chapter 6).Although these injection experiments clearly showed that shrimp are indeed capable of specifically recognizing foreign proteins and exhibit a kind of adaptive memory, the injection vaccination technique is far from suited for use under shrimp farming conditions. Therefore the potential of oral vaccination of shrimp using the same viral envelope proteins was investigated (Chapter 7). In this setup P.monodon shrimp were fed commercial food pellets coated with inactivated bacteria thatoverexpressedboth envelope proteins VP19 and VP28. In order to approach the natural route of WSSV infection and subject the virus to the full array of immunological responses of the shrimp, the challenge was performed via immersion of the shrimp in WSSV containing seawater. When the challenge was performed three days after a seven-day vaccination period, VP28 vaccinated shrimp showed a significant lower cumulative mortality compared to shrimp vaccinated with bacteria containing empty vectors (RPS of 61%), while vaccination with VP19 provided no protection. To determine the onset and duration of protection of VP28, challenges were performed three, seven and twenty-one days after the seven-day vaccination period. A significantly higher survival was observed both three and seven days post vaccination (RPS of 64% and 77%, respectively), but the protection was reduced twenty-one days after the vaccination (RPS of 29%). These results strongly suggest that a specific immune response and ultimately protection can be generated in an invertebrate species like shrimp.

In an effort to investigate whether the oral vaccination effects were limited to P.monodon or based on more universal mechanisms, the vaccination experiments were applied to an alternative host for WSSV, the Pacific White shrimpLitopenaeusvannamei(Chapter 8). Also this species showed a significantly lower cumulative mortality upon VP28 vaccination compared to the control groups. This outcome points to a shared and therefore universal adaptive response mechanism present in crustaceans. It is still not clear whether this response is WSSV specific or more generally directed against viruses.

In recent years more evidence has become available suggesting the presence of a specific immune response and adaptive memory in invertebrates. The results presented in this thesis support this view by showing that the shrimp's immune system is able to specifically recognize and react upon WSSV structural proteins or more in general, proteins lacking known pathogen associated molecular patterns. Furthermore, the studies described in this thesis have shown that vaccination of shrimp against WSSV can be successful, which opens the way to the design of new strategies to control WSSV and other invertebrate pathogens.