Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: An uptake and processing study in the midgut of Penaeus monodon

Abstract

a b s t r a c tSeveral oral vaccination studies have been undertaken to evoke a better protection against white spot syndrome virus (WSSV), a major shrimp pathogen. Formalin-inactivated virus and WSSV envelope protein VP28 were suggested as candidate vaccine components, but their uptake mechanism upon oral delivery was not elucidated. In this study the fate of these components and of live WSSV, orally intubated to black tiger shrimp (Penaeus monodon) was investigated by immunohistochemistry, employing antibodies specific for VP28 and haemocytes. The midgut has been identified as the most prominent site of WSSV uptake and processing. The truncated recombinant VP28 (rec-VP28), formalin-inactivated virus (IVP) and live WSSV follow an identical uptake route suggested as receptor-mediated endocytosis that starts with adherence of luminal antigens at the apical layers of gut epithelium. Processing of internalized antigens is performed in endo-lysosomal compartments leading to formation of supra-nuclear vacuoles. However, the majority of WSSV-antigens escape these compartments and are transported to the inter-cellular space via transcytosis. Accumulation of the transcytosed antigens in the connective tissue initiates aggregation and degranulation of haemocytes. Finally the antigens exiting the midgut seem to reach the haemolymph. The nearly identical uptake pattern of the different WSSV-antigens suggests that receptors on the apical membrane of shrimp enterocytes recognize rec-VP28 efficiently. Hence the truncated VP28 can be considered suitable for oral vaccination, when the digestion in the foregut can be bypassed.

Antisense RNA mediated protection from white spot syndrome virus (WSSV) infection in Pacific white shrimp Litopenaeus vannamei

“…Ahanger et al (2014) studied the effect of antisense constructs targeting VP28 gene of WSSV in P. monodon and reported 90% survival of the animals. The results are in agreement with previous studies wherein VP28 was observed to be highly immunogenic and hence a potential vaccine candidate against WSSV infection (Kulkarni et al, 2013;Yumiao et al, 2013). However, it should be noted that all shrimp exposed to WSSV eventually died in our experiments and the relative short time frame of the cited experiments above masks the eventual fate of their experimental animals perhaps leading to an over enthusiastic conclusion.…”

VP24 Is a Chitin-Binding Protein Involved in White Spot Syndrome Virus Infection

“…Numerous studies have explored different vaccination strategies to protect shrimp from WSSV infection, including inactivated WSSV vaccines (45), recombinant protein-based vaccines (46,47), DNA-based vaccines (48)(49)(50), and RNA-based vaccines (51)(52)(53)(54)(55)(56)(57). VP28, which is the most abundant WSSV envelope protein, is the major target for vaccine design, and oral vaccination is thought to be the most practical route for vaccine delivery (58).…”

Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV‐vaccines and live white spot syndrome virus

“…WSSV‐free (confirmed by PCR ) P. monodon (body weight 18–20 g), obtained from a local shrimp farm in Cochin (India) were reared as described in Kulkarni et al. .…”

“…The truncated form of VP28 (pET28a‐His6‐VP28) expressed in Escherichia coli BL 21 cells was obtained from the Virology group of Wageningen University, The Netherlands. Over‐expression and subsequent purification of rec‐VP28 was performed at UiN as described earlier . Briefly, the transformed E. coli BL 21 cells were cultured on Luria‐Bertani (Life Technologies, Paisley, UK)‐kanamycin (50 μg/mL; Life Technologies) medium added with 1 mM isopropyl β‐ D ‐1‐thiogalactopyranoside (IPTG, Life Technologies) at 37°C for 5 h. The harvested transformed cells were resuspended in 1 mL PBS having 100 μL lysis solution [0.2% SDS (Sigma, St. Louis, MO, USA), 1% Triton™ X‐100 (Sigma), 10 mM EDTA (Sigma), 0.2M NaCl (Sigma)].…”

“…In our previous study, we have reported that midgut is the main site of uptake and processing of oral WSSV‐antigens . In addition, cellular responses of shrimp to the antigens were described.…”