RNA Nanovaccine Protects against White Spot Syndrome Virus in Shrimp

Abstract

In the last 15 years, crustacean fisheries have experienced billions of dollars in economic losses, primarily due to viral diseases caused by such pathogens as white spot syndrome virus (WSSV) in the Pacific white shrimp Litopenaeus vannamei and Asian tiger shrimp Penaeus monodon. To date, no effective measures are available to prevent or control disease outbreaks in these animals, despite their economic importance. Recently, double-stranded RNA-based vaccines have been shown to provide specific and robust protection against WSSV infection in cultured shrimp. However, the limited stability of double-stranded RNA is the most significant hurdle for the field application of these vaccines with respect to delivery within an aquatic system. Polyanhydride nanoparticles have been successfully used for the encapsulation and release of vaccine antigens. We have developed a double-stranded RNA-based nanovaccine for use in shrimp disease control with emphasis on the Pacific white shrimp L. vannamei. Nanoparticles based on copolymers of sebacic acid, 1,6-bis(p-carboxyphenoxy)hexane, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane exhibited excellent safety profiles, as measured by shrimp survival and histological evaluation. Furthermore, the nanoparticles localized to tissue target replication sites for WSSV and persisted through 28 days postadministration. Finally, the nanovaccine provided ~80% protection in a lethal WSSV challenge model. This study demonstrates the exciting potential of a safe, effective, and field-applicable RNA nanovaccine that can be rationally designed against infectious diseases affecting aquaculture.

Keywords: WSSV; dsRNA; nanovaccine; polyanhydride; shrimp.

Figure 1

In vitro release kinetics of dsRNA-loaded nanoaparticles. Nanoparticles loaded with 11% WSSV 477 dsRNA were incubated in saline for approximately 1 month and the release kinetics were measured. Data are presented as the mean ± standard error of the mean (SEM) and are representative of 3 replicates per formulation.

Figure 2

Stability of released dsRNA from nanoparticles. Released dsRNA from nanoparticles was evaluated for using gel electrophoresis.

Figure 3

Safety profile of nanoparticles in shrimp. Shrimp were injected with 500 mg of either 20:80 CPTEG:CPH or 20:80 CPH:SA nanoparticles by reverse gavage (RG). (A) Shrimp survival was monitored through 60 days postinjection. Data are expressed as the mean ± SEM of three independent experiments. No statistical difference between groups at day 60. (B) Blinded histopathological analysis of tissue was performed by fixing live shrimp in Davidson’s fixative. Scale bar = 20 mm.

Figure 4

Biodistribution of 1% rhodamine-loaded nanoparticles in L. vannamei post-RG administration. Shrimp were injected with 250 μg of either 20:80 CPTEG:CPH or 20:80 CPH:SA rhodamine-loaded nanoparticles by reverse gavage (RG). At indicated time intervals, animals were dissected into cephalothorax, hepatopancreas, gut, gills, and stomach. (A) Qualitative image analysis. Tissue samples were placed on an imaging tray and rhodamine fluorescence was captured using Carestream Multispectral Imager. Images shown are representative of results obtained from two independent experiments. (B) In vivo fluorescence quantification of rhodamine. Data are expressed as the mean fold change over controls ± SEM of two independent experiments. Statistical differences (p < 0.05) of either nanoparticle treatment in comparison to the control are indicated by *, while differences between the two nanoparticle formulations are indicated by #.

Figure 5

Shrimp survival against WSSV challenge. (A) Shrimp were injected by reverse gavage (RG) with 11 μg of either soluble WSSV 477 dsRNA or 11 μg WSSV 477 dsRNA encapsulated in 100 μg 20:80 CPTEG:CPH or 20:80 CPH:SA nanoparticles. Three days postvaccination animals were challenged with a lethal dose of WSSV and survival was monitored through day 12. Sham controls were injected with water prior to challenge. Soluble _eGFP was used as non-shrimp specific vaccination control. The negative controls did not receive any viral dose. n = 60 (two independent experiments with 30 animals/treatment) except the negative control group (n = 40). (B) Photomicrographs of shrimp tissue postchallenge. Scale bar = 20 μm. (C) Viral load measured using WSSV-specific qRT-PCR.

Figure 6

Principal component analysis of viral load, survival, and histopathology. Principal component analysis was used to determine the effects of nanovaccines on key vaccine efficacy parameters: survival, viral load, and histopathology postchallenge. The results were plotted three dimensionally and the similarity among formulations is defined through the comparison of Euclidean distances, where formulations in close proximity to one another represent greater similarity.