Citation: Kim, M.-J.; Kim, S.-H.; Kim, J.-O.; Lee, T.-K.; Jang, I.-K.; Choi, T.-J. Efficacy of White Spot Syndrome Virus Protein VP28-Expressing Chlorella vulgaris as an Oral Vaccine for Shrimp. Viruses 2023, 15, 2010. https://doi.org/10.3390/v15102010 Academic Editor: Daniel M. P. Ardisson-Araújo Received: 6 September 2023 Revised: 18 September 2023 Accepted: 26 September 2023 Published: 27 September 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). viruses Article Efficacy of White Spot Syndrome Virus Protein VP28-Expressing Chlorella vulgaris as an Oral Vaccine for Shrimp Min-Jeong Kim 1 , Su-Hyun Kim 1 , Jong-Oh Kim 1 , Taek-Kyun Lee 2 , In-Kwon Jang 3 and Tae-Jin Choi 1, * 1 Department of Microbiology, School of Marine and Fisheries Sciences, Pukyong National University, Busan 48513, Republic of Korea; rnfma00082@naver.com (M.-J.K.); olem1140@gmail.com (S.-H.K.); jokim@pknu.ac.kr (J.-O.K.) 2 South Sea Environment Research Division, Korea Institute of Ocean Science & Technology, Geoje-si 53201, Republic of Korea; tklee@kiost.ac.kr 3 Junggyeom Co., Ltd., Goyang-si 10223, Republic of Korea; jangik2001@gmail.com * Correspondence: choitj@pknu.ac.kr; Tel.: +82-51-620-6367; Fax: +82-51-611-6358 Abstract: The white spot syndrome virus (WSSV) is the causative agent of white spot disease, which kills shrimp within a few days of infection. Although WSSV has a mortality rate of almost 100% and poses a serious threat to the shrimp farming industry, strategies for its prevention and treatment are extremely limited. In this study, we examined the efficacy of VP28, a recombinant WSSV protein expressed in Chlorella vulgaris (C. vulgaris), as an oral shrimp vaccine. When compared with the control group, in which WSSV had a cumulative mortality of 100%, shrimp treated with 5% VP28-expressing C. vulgaris in their feed only had a 20% cumulative mortality rate 12 days after the WSSV challenge. When compared with the nonvaccinated group, the transcription of anti-lipopolysaccharide factor, C-type lectin, and prophenoloxidase genes, which are involved in shrimp defense against WSSV infection, was upregulated 29.6 fold, 15.4 fold, and 11.5 fold, respectively. These findings highlight C. vulgaris as a potential host for industrial shrimp vaccine production. Keywords: WSSV; VP28; vaccine; Chlorella vulgaris; immunization 1. Introduction Global shrimp farming is an important source of seafood, with the rise in demand every year leading to the production of about 5.51 million tons of shrimp in 2019 [1]. Whiteleg shrimp (Litopenaeus vannamei), the most farmed species of penaeid shrimp worldwide [1,2], is mainly produced in Thailand, Vietnam, and China. However, shrimp farming faces significant risks because of the threat of bacterial, fungal, protozoan, and viral infections [3], with viruses posing a particularly high risk. Currently, the main viruses of concern are the white spot syndrome virus (WSSV) and the yellow-head virus [4]. WSSV, which was first discovered in 1992 on shrimp farms in Asia, causes white spot disease (WSD), the most common disease in shrimp farms worldwide and a major cause of high economic losses [5]. WSSV-infected shrimp typically die within seven days, at a mortality rate of almost 100% [6]. The genome of WSSV, a circular, double-stranded DNA virus of the genus Whispovirus (family Nimaviridae)[7], is about 300 kilobase pairs long, but the genome size varies across isolates. WSSV is a bacilliform, nonoccluded, enveloped virus made up of a tail and a virion (210–380 nm long and 70–167 nm wide). Of the more than 40 characterized WSSV proteins, 21 are found in the envelope, 5 in the tegument, and 10 in the nucleocapsid [8]. WSSV’s viral proteins (VP) 15, VP19, VP24, VP26, and VP28 [8] play critical roles during infection. To date, there are no effective ways of treating or preventing the highly infectious WSSV, probably because shrimp lack adaptive immunity [9]. Nevertheless, protection Viruses 2023, 15, 2010. https://doi.org/10.3390/v15102010 https://www.mdpi.com/journal/viruses