Mass Production of Virus-Like Particles Using Chloroplast Genetic Engineering for Highly Immunogenic Oral Vaccine Against Fish Disease

Abstract

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis (VNN), which is one of the most serious fish diseases leading to mass mortality in a wide range of fish species worldwide. Although a few injectable inactivated vaccines are commercially available, there is a need for more labor-saving, cost-effective, and fish-friendly immunization methods. The use of transgenic plants expressing pathogen-derived recombinant antigens as edible vaccines is an ideal way to meet these requirements. In this study, chloroplast genetic engineering was successfully utilized to overexpress the red-spotted grouper NNV capsid protein (RGNNV-CP). The RGNNV-CP accumulated at high levels in all young, mature, and old senescent leaves of transplastomic tobacco plants (averaging approximately 3 mg/g leaf fresh weight). The RGNNV-CP efficiently self-assembled into virus-like particles (RGNNV-VLPs) in the chloroplast stroma of the transgenic lines, which could be readily observed by in situ transmission electron microscopy. Furthermore, intraperitoneal injection and oral administration of the crudely purified protein extract containing chloroplast-derived RGNNV-VLPs provided the sevenband grouper fish with sufficient protection against RGNNV challenge, and its immunogenicity was comparable to that of a commercial injectable vaccine. These findings indicate that chloroplast-derived VLP vaccines may play a promising role in the prevention of various diseases, not only in fish but also in other animals, including humans.

Keywords: aquaculture; chloroplast genetic engineering; oral vaccine; red-spotted grouper nervous necrosis virus (RGNNV); virus-like particles (VLPs).

Figure 1

Generation of transplastomic tobacco plants expressing RGNNV capsid protein (RGNNV-CP). (A) Physical map of the target region in the plastid genome (WT ptDNA) and schematic diagram of the chloroplast transformation vector, pRGNNV1 (construct not drawn to scale). Synthetic DNA covering the codon-optimized RGNNV-CP (RGNNV-CPpt) and the subsequent 3' non-cooding regioon (3' NCR) derived from the RNA2 genome of red-spotted grouper NNV (RGNNV) is driven by the tobacco plastid psbA promoter and its 5'-UTR (PpsbA), and the transcript is designed to be stabilized by the 3'-UTR of tobacco plastid psbA (TpsbA). The selectable marker gene aadA is under the control of the tobacco plastid ribosomal RNA operon promoter (Prrn). The transgenes are targeted to the intergenic spacer region between trnI and trnA in the tobacco plastid genome. The location of the probe used in Southern blot analysis is shown as a black bar. The wild-type and the transgenic chloroplast genomes give rise to hybridization signals corresponding to 2.3 kb and 5.3 kb XmnI-XmnI DNA fragments, respectively. (B) Southern blot analysis of two independent transgenic lines (RGNNV1-1 and RGNNV1-2). Total cellular DNA was digested with XmnI and subjected to hybridization analysis with a DIG-labeled probe shown in (A).

Figure 2

Transplastomic plants expressing RGNNV-CP grown under photoheterotrophic conditions. (A) Phenotype of wild-type (WT) and transplastomic (RGNNV1-1 and RGNNV1-2) plants grown on MS agar medium containing 3% (w/v) sucrose as carbon source. Bar = 3 cm. (B) Detection of RGNNV-CP accumulated in transplastomic plants (RGNNV1-1 and RGNNV1-2) by Coomassie Brilliant Blue (CBB) staining. Total, soluble, and insoluble protein extracts from 1.5 mg of fresh leaf were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and stained with CBB. The 37-kDa and 55-kDa bands correspond to RGNNV-CP and large subunit of Rubisco (RbcL), respectively.

Figure 3

Transplastomic plants expressing RGNNV-CP grown under photoautotrophic conditions. (A) Soil-grown wild-type (WT) and transplastomic (RGNNV1-1 and RGNNV1-2) plants were cultivated in a phytotron using sunlight and natural daylength, with a temperature cycle of 12 h at 26°C and 12 h at 23°C. Bar = 30 cm. (B) Mature leaves of WT and RGNNV1-2 plants. Bar = 5 cm. (C) Detection of RGNNV-CP accumulated in transplastomic plants (RGNNV1-1 and RGNNV1-2) by CBB staining. Total soluble protein (TSP) extracted from 1.5 mg of fresh leaf was separated by 13% SDS-PAGE gel and stained with CBB.

Figure 4

High accumulation of RGNNV-CP in all leaves of the transgenic tobacco plant. (A) TSPs extracted from 1.5 mg of different leaves of a transplastomic plant (RGNNV1-2) were separated by 13% SDS-PAGE and stained with CBB. TSP extracted from a mature leaf of wild-type (WT) plant was loaded as a control. (B) The RGNNV1-2 plant used for the analysis shown in (A). The leaves were numbered from top to bottom. (C) Western blot analysis to detect RGNNV-CP accumulated in the transplastomic plant. TSPs (500 ng) extracted from young (Y), mature (M), and old senescent (O) leaves of RGNNV1-2 correspond to leaf nos. 2, 10, and 20 in (B), respectively, were analyzed. TSP (5,000 ng) extracted from a mature leaf of wild-type (WT) was also loaded as a control. Blots were detected using an antiserum against a mixture of synthetic peptides derived from RGNNV-CP. A dilution series (50, 100, and 200 ng) of purified 6×His-tagged RGNNV-CP (RGNNV-CP-His) expressed in Escherichia coli was analyzed as a standard.

Figure 5

Purification of RGNNV virus-like particles (RGNNV-VLPs) from transplastomic tobacco plants. (A) TSP (T) extracted from mature leaves of transplastomic plants (RGNNV1-2) was analyzed by sucrose gradient sedimentation, and RGNNV-CP in each fraction was detected by CBB staining. Fraction 1 was obtained from the top of the tube. (B) Electron micrograph of the RGNNV-CP-enriched fractions recovered by the sucrose gradient sedimentation analysis shown in (A). RGNNV-VLPs derived from the transplastomic tobacco plants (Tobacco) and E. coli were visualized by negative staining with 2% (w/v) uranyl acetate and transmission electron microscopy (TEM). Bar = 100 nm.

Figure 6

Electron micrographs of RGNNV-VLPs present in high density in chloroplast stroma of transplastomic tobacco plants. In situ TEM images of chloroplasts from mature leaves of wild-type [WT; (A–C)] and the transplastomic [RGNNV1-2; (D–F)] plants. (B), (C), (E), and (F) are magnified views of the box regions in (A), (B), (D), and (E), respectively. Chl, chloroplast; S, starch granule; V, vacuole; CW, cell wall; and PG, plastoglobule.

Figure 7

Cumulative mortality after RGNNV challenge in sevenband grouper immunized with chloroplast-derived RGNNV-VLPs. Fish were vaccinated with PBS as control, crudely purified protein extracts from mature leaves of wild-type (WT) or the transplastomic (RGNNV1) tobacco plants, or a commercial inactivated vaccine (Inactivated) by intraperitoneal injection (IN) or oral administration (OR). On day 21 after immunization, the fish were challenged by intramuscular injection with RGNNV at a dose of 10^3.3 TCID₅₀ per fish and observed for an additional 14 days.