BEI Inactivated Vaccine Induces Innate and Adaptive Responses and Elicits Partial Protection upon Reassortant Betanodavirus Infection in Senegalese Sole

Abstract

Nervous necrosis virus (NNV), the causative agent of viral encephalopathy and retinopathy (VER), is one of the most threatening viruses affecting marine and freshwater fish species worldwide. Senegalese sole is a promising fish species in Mediterranean aquaculture but also highly susceptible to NNV and VER outbreaks, that puts its farming at risk. The development of vaccines for aquaculture is one of best tools to prevent viral spread and sudden outbreaks, and virus inactivation is the simplest and most cost-effective method available. In this work, we have designed two inactivated vaccines based on the use of formalin or binary ethylenimine (BEI) to inactivate a reassortant NNV strain. After vaccination, the BEI-inactivated vaccine triggered the production of specific IgM-NNV antibodies and stimulated innate and adaptive immune responses at transcriptional level (rtp3, mx, mhcii and tcrb coding genes). Moreover, it partially improved survival after an NNV in vivo challenge, reducing the mid-term viral load and avoiding the down-regulation of immune response post-challenge. On the other hand, the formalin-inactivated vaccine improved the survival of fish upon infection without inducing the production of IgM-NNV antibodies and only stimulating the expression of herc4 and mhcii genes (in head-kidney and brain, respectively) during the vaccination period; this suggests that other immune-related pathways may be involved in the partial protection provoked. Although these vaccines against NNV showed encouraging results, further studies are needed to improve sole protection and to fully understand the underlying immune mechanism.

Keywords: BEI; Senegalese sole; antibodies; formalin; gene expression; immune response; inactivated vaccine; nervous necrosis virus.

Figure 1

BEI-iSs160 vaccine induces specific humoral immunity. Specific anti-NNV IgM levels in the serum of Senegalese sole specimens 7-, 15- and 30-days after intraperitoneal vaccination with low and high dosages (10⁵ and 10⁷ TCID₅₀/mL, iSs160L and iSs160H, respectively) of BEI-iSs160 vaccine (inactivated with BEI) and form-iSs160 (formalin inactivated) or PBS (Control). Data represent the mean ± standard error of the mean (SEM; n = 6 fish/group and time point). Lower letters denote statistical differences between groups at a same time-point (p < 0.05), whilst asterisks indicate those between time-points in the same group (* p < 0.05; **** p < 0.0001).

Figure 2

Vaccination produces the increment of immune response at transcriptional level in head-kidney. Expression of immune-related genes (A) rtp3, (B) herc4, (C) mx, (D) mhcii and (E) tcrb in the head-kidney of Senegalese sole specimens 7-, 15- and 3-days after intraperitoneal vaccination with low and high dosages (10⁵ and 10⁷ TCID₅₀/mL, iSs160L and iSs160H, respectively) of BEI-iSs160 vaccine (inactivated with BEI) and form-iSs160 (formalin inactivated) or PBS (Control). Data represent the mean ± standard error of the mean (SEM; n = 6 fish/group and time). Lower letters denote statistical differences between groups at a same time-point (p < 0.05), whilst asterisks indicate those between time-points in the same group (* p < 0.05; **** p < 0.0001).

Figure 3

Vaccination barely alters the immune response at transcriptional level in brain. Expression of immune-related genes (A) rtp3, (B) herc4, (C) mx, (D) mhcii and (E) tcrb in the brain of Senegalese sole specimens 7-, 15- and 30-days after intraperitoneal vaccination with low and high dosages (10⁵ and10⁷ TCID₅₀/mL, iSs160L and iSs160H, respectively) of BEI-iSs160 vaccine (inactivated with BEI) and form-iSs160 (formalin inactivated) or PBS (Control). Data represent the mean ± standard error of the mean (SEM; n = 6 fish/group and time). Lower letters denote statistical differences between groups at a same time-point (p < 0.05), whilst asterisks indicate those between time-points in the same group (* p < 0.05; ** p < 0.01).

Figure 4

All vaccines induce partial protection and BEI-iSs160 decreases the viral load after challenge. (A) viral load (n = 3 fish/group and time) in brain and (B) Percent of survival during 45 days of in vivo infection in intraperitoneally vaccinated Senegalese sole specimens vaccinated with low and high dosages (10⁵ and 10⁷ TCID₅₀/mL, iSs160L and iSs160H, respectively) of BEI-iSs160 vaccine (inactivated with BEI) and form-iSs160 (formalin inactivated) or PBS (Control). Infection was performed by 3 h of immersion with 10⁷ TCID₅₀/mL. Survival rates were compared between groups using Kaplan Meier test. Lower letters denote statistical differences between groups of the same time point according to the two-way ANOVA test (p < 0.05).

Figure 5

Vaccination does not alter specific humoral immunity upon NNV infection. Specific anti-NNV IgM levels in the serum of Senegalese sole specimens 45 days after 3 h of immersion with 10⁵ TCID₅₀/mL of Ss160 in previously intraperitoneally vaccinated Senegalese sole specimens vaccinated with low and high dosages (10⁵ and 10⁷ TCID₅₀/mL, iSs160L and iSs160H, respectively) of BEI-iSs160 vaccine (inactivated with BEI) and form-iSs160 (formalin inactivated) or PBS (Control). Infection was performed by 3 h of immersion with 10⁷ TCID₅₀/mL. Data represent the mean ± standard error of the mean (SEM; n = 6 fish/group and time). Statistical assay was performed by the two-way ANOVA test (p < 0.05).

Figure 6

Vaccination mainly decreases the immune response upon NNV infection at a transcriptional level in head-kidney but not in brain. Expression of immune-related genes in (A) head-kidney and (B) brain of Senegalese sole specimens 45 days after 3 h of immersion with 10⁵ TCID₅₀/mL of Ss160 in fish previously vaccinated with PBS (Control) or with low and high dosages (10⁵ and 10⁷ TCID₅₀/mL, iSs160L and iSs160H, respectively) of BEI-iSs160 vaccine (inactivated with BEI) and form-iSs160 (formalin inactivated) or PBS (Control). Data represent the mean ± standard error of the mean (SEM; n = 6 fish/group and time). Lower letters denote statistical differences between groups according to the two-way ANOVA test (p < 0.05).