Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: An uptake and processing study in the midgut of Penaeus monodon A. Kulkarni a , J.H.W.M. Rombout a, b, * , I.S.B. Singh c , N.S. Sudheer c , J.M. Vlak b , C.M.A. Caipang a , M.F. Brinchmann a , V. Kiron a a Faculty of Biosciences and Aquaculture, University of Nordland, Bodø, Norway b Wageningen University, Wageningen, The Netherlands c National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Kochi, India article info Article history: Received 10 July 2012 Received in revised form 19 September 2012 Accepted 14 October 2012 Available online 26 October 2012 Keywords: White spot syndrome virus Recombinant VP28 Antigen processing Penaeus monodon Immunohistochemistry abstract Several 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. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction White spot syndrome virus (WSSV), the causative agent of white spot disease in shrimps, has been described as the most calamitous virus ever since its first appearance in Northeast Asia [1]. Therefore, there is a worldwide effort to control or manage this disease. The morphology, molecular characterization, morphogenesis and pathogenesis of WSSV have been extensively studied [2]. The virus most likely enters per os when healthy shrimps scavenge on diseased individuals although entry via the gills cannot be excluded. The primary target of the ingested WSSV is the stomach, and the interaction of WSSV with the epithelial cells of the gut has been demonstrated [3,4]. Based on the viral accommodation concept suggesting the existence of immunological memory it was hypothesized that shrimps could be vaccinated against viral disease [5]. Several attempts were made to immunize shrimps against WSSV, through intramuscular injections and oral feeding of formalin-inactivated virus and/or recombinant viral envelope proteins [6e9]. VP28 is a major envelope protein of WSSV that takes part in the systemic infection of the shrimp through its attachment and entry into host cells [10,11]. Most of the vaccination trials were aimed to enhance survival, to extend the duration of protection and to increase the efficacy of vaccine delivery. However, there is a dearth of information on how the animal processes and utilizes the vaccine components. The present study describes the uptake and processing of two ‘vaccine’ candidates e formalin-inactivated virus preparation (IVP) and recombinant VP28 (rec-VP28) e within the gut of the shrimp after oral intubation. For comparison the oral infection process of live WSSV was studied. In addition, haemocyte aggregation and * Corresponding author. Faculty of Biosciences and Aquaculture , University of Nordland, 8049 Bodø, Norway. Tel.: þ47 755 17399; fax: þ47 755 17457. E-mail address: jan.rombout@wur.nl (J.H.W.M. Rombout). Contents lists available at SciVerse ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2012.10.028 Fish & Shellfish Immunology 34 (2013) 159e166