Long-Term Protection and Serologic Response of European Sea Bass Vaccinated with a Betanodavirus Virus-Like Particle Produced in Pichia pastoris

Abstract

Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass (Dicentrarchus labrax) in the Mediterranean. In the current studies, we tested a novel Pichia-produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus "Red-Spotted Grouper Nervous Necrosis Virus" (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.

Keywords: VLP vaccine; betanodavirus; correlate of protection; efficacy; recombinant vaccine.

Figure 1

Setup during challenge in Study 2 (DTU). The fish were kept in cylindrical 180 L tanks with aerated saltwater (12‰) at 26 °C (±1 °C). A bio-filter pump was installed in each tank and the wastewater was UV and heat treated.

Figure 2

Visualization of RGNNV VLPs by cryo-TEM.

Figure 3

Western blot stained with rabbit-anti-nodavirus. (1) RGNNV (strain 283.2009), (2) RGNNV VLP, (3) MilliQ H₂O. The RGNNV sample was concentrated virus obtained by ultracentrifugation of cell culture supernatant and was not quantified since impurities from the cell culture medium would contribute to total protein. The molecular weight indicated by the marker must be interpreted with caution since the marker was pre-stained (as described in Section 2.3). Uncropped photos are shown in the supplementary material (Figure S2).

Figure 4

Prevalence of -specific IgM in ELISA (Study 1). Serum from vaccinated fish was sampled 27 days (“1 m.p.v.”, n = 5) and 57 days (“2 m.p.v.”, n = 10) after vaccination.

Figure 5

Prevalence of RGNNV-specific IgM in ELISA (Study 2). Plasma from sea bass 1 (n = 3–5) or 10 (n = 10) months post-vaccination (m.p.v).

Figure 6

Geometric mean neutralizing titer in vaccinated fish on Day 27 (n = 5) or 57 (n = 10) post-vaccination, Study 1. VLP high = 20 µg/fish, medium = 5 µg/fish or low = 1.25 µg/fish. Bars indicate geometric standard deviation. The lowest serum dilution analyzed was 1:20, which is why a negative sample was <1:20. When calculating the geometric mean titer, these were set to a value of 10, except in the PBS group where all were <1:20, and set as 0.

Figure 7

Kaplan–Mayer survival curve of the three experimental challenges with intramuscular injection of RGNNV.

Figure 8

Data on individual fish. (A) Neutralizing titer before challenge and (B) RGNNV virus titer (TCID50/g) isolated from brain tissue from dead/moribund fish as a function of the day the fish was terminated/diseased. The titers were log transformed to obtain linearity. The colors indicate the treatment (20, 5 or 1.25 µg VLP, commercial vaccine or PBS).

Figure 9

Immunohistochemistry of the encephalon from moribund sea bass (PBS, negative control group). Bright red coloring indicates the presence of RGNNV antigens. (A–C) Cerebellum (*) with intense immunostaining at different magnifications; the cortical of the optic lobes appears scarcely affected. Cerebellar Purkinje cell soma (arrows) appears strongly IHC positive, while their dendritic process (arrowheads) is also highlighted in the molecular layer. (D) Diencephalon showing intense immunostaining.

Figure 10

Immunohistochemistry of brain from vaccinated surviving sea bass (VLP 20 µg/fish). Bright red immunostaining indicates the presence of RGNNV antigens. (A,B) Cerebellum at different magnifications showing mild coloring (arrows). (C,D) Mesencephalon with scattered staining (arrows). (D) Higher magnification of the squared section in (C).