Gut mucosal immune responses and protective efficacy of oral yeast Cyprinid herpesvirus 2 (CyHV-2) vaccine in Carassius auratus gibelio

Abstract

Cyprinid herpesvirus 2 (CyHV-2) causes herpesviral hematopoietic necrosis (HVHN) disease outbreaks in farmed Cyprinid fish, which leads to serious economic losses worldwide. Although oral vaccination is considered the most suitable strategy for preventing infectious diseases in farmed fish, so far there is no commercial oral vaccine available for controlling HVNN in gibel carp (C. auratus gibelio). In the present study, we developed for the first time an oral vaccine against CyHV-2 by using yeast cell surface display technology and then investigated the effect of this vaccine in gibel carp. Furthermore, the protective efficacy was evaluated by comparing the immune response of a single vaccination with that of a booster vaccination (booster-vaccinated once 2 weeks after the initial vaccination). Critically, the activities of immune-related enzymes and genes expression in vaccine group, especially in the booster vaccine group, were higher than those in the control group. Moreover, strong innate and adaptive immune responses could be elicited in both mucosal and systemic tissues after receipt of the oral yeast vaccine. To further understand the protective efficacy of this vaccine in gibel carp, we successfully developed the challenge model with CyHV-2. Our results showed the relative percent survival was 66.7% in the booster vaccine group, indicating this oral yeast vaccine is a promising vaccine for controlling CyHV-2 disease in gibel carp aquaculture.

Keywords: CyHV-2; gibel carp; mucosal immune response; oral vaccination; protective efficacy; yeast surface display.

Figure 1

The sampling position of the gibel carp’s gut. Black line with double arrowheads demonstrates the exact location of foregut, midgut, and hindgut sampling in this study. Yellow triangles indicate the dividing line of the foregut, midgut, and hindgut.

Figure 2

Schematic diagram of the yeast vaccine and experimental strategy. (A) pYD1-ORF25 recombinant plasmids displayed on the yeast cell surface through attachment of Aga2 to Aga1 via disulfide bonds. (B) Positive clones of pYD1-ORF25 identification by agarose gel electrophoresis. (C) Immunofluorescence assay of EBY100/pYD1-ORF25 fusion protein Expression. Scale bars, 20 μm. (D) Strategy to vaccinate gibel carp. Briefly, vaccine and bo-vaccine fish were orally administered with CyHV-2 yeast vaccine at 1, 2, and 3 days (vaccine) and 1, 2, 3, 18, 19, and 20 days (booster vaccine), respectively. Take samples at the 10, 17, 27, and 34 days to test the safety of the yeast vaccine (cumulative survival after vaccination and morphological changes of gut), body fluid biochemistry indices, and immune-related gene expression. Challenge with CyHV-2 at the 41 days to test intestinal pathological changes, viral load, and relative percent survival.

Figure 3

Safety evaluation of oral administration of CyHV-2 yeast vaccine. (A, C, E) Histological examination by H&E staining of foregut (A), midgut (C), and hindgut (E) from Control fish and Vaccine fish and Bo-vaccine fish (n = 6 fish per group). Scale bars, 50 μm. Con, control; Va, vaccine; Bo-va, booster vaccine. (B, D, F) The length-width ratio of foregut (B), midgut (D), and hindgut (F) intestinal folds in control fish, vaccine fish, and booster vaccine fish (n = 6 fish per group). Control vs. Vaccine and Bo-vaccine, ns, not significant, unpaired Student’s t-test (at 10dpv and 17dpv), one-way ANOVA with Bonferroni correction (at 27dpv and 34dpv). Data are representative of three different independent experiments (mean ± SEM).

Figure 4

Effects of vaccination on levels of body fluid biochemical parameters of gibel carp. Complement C3, TSOD and LZM enzyme activities in foregut, midgut, and hindgut mucus, as well as serum were detected by using commercial kits (n = 6 fish per group). Data are representative of three different independent experiments (mean ± SEM).

Figure 5

Immune responses in gibel carp vaccinated with oral administration of CyHV-2 yeast vaccine. Heat map illustrates expression profiles of immune genes in gibel carp foregut (A), midgut (C), hindgut (E), spleen (G), and head kidney (I) of vaccine and bo-vaccine vs. control fish measured at 10,17, 27, and 34 dpv (n = 6 fish per group). Color value: log₂ (fold change). (B, D, F, H, J) Fold changes of igt, igm and igd in foregut, midgut, hindgut, spleen, and head kidney was detected at 34 dpv (n = 6 fish per group). Control vs. Vaccine and Bo-vaccine: ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Bonferroni correction. Data are representative of three different independent experiments (mean ± SEM).

Figure 6

Pathological changes of gibel carp after challenge with CyHV-2 among the control group, vaccine group, and booster vaccine group. (A) Strategy to challenge with CyHV-2. Briefly, at 41 days, control, vaccinated, and booster-vaccinated fish were challenged by injection with CyHV-2, and at 4 dpc, the six groups of fish treatments (including: Con [control fish + PBS], Va [vaccinated fish +PBS], Bo-va [booster-vaccinated fish + PBS], Con+Cha [control fish + CyHV-2], Va+Cha [vaccinated fish + CyHV-2], Bo-va+Cha [booster-vaccinated fish + CyHV-2]) were analyzed for pathological changes of foregut, midgut, and hindgut, respectively. (B) The clinical observation following challenge with CyHV-2. (C–E) Histological examination by H&E staining of foregut, midgut, and hindgut from Con, Va, Bo-va, Con+Cha, Va+Cha, and Bo-va+Cha groups fish (n = 6 fish per group). Black triangle indicates epithelial cell sloughing and necrosis. Blue triangle indicates the goblet cells. Scale bars, 50 μm. (F–H) The length-width ratio of foregut (C), midgut (D), and hindgut (E) intestinal folds in these groups fish from C-E (n = 6 fish per group). Different superscript letters in each group (a, b, c) denote significant variations suggested by the Kruskal–Wallis statistics at 95% of significance, followed by the Dunn test with Bonferroni adjustment as the post hoc test (p < 0.05). Data are representative of three different independent experiments (mean ± SEM).

Figure 7

The viral load and survival rate of gibel carp after challenge with CyHV-2. (A) The histogram showed loads of CyHV-2 in different tissues at 4 dpc (n = 6 fish per group). ns, not significant, *P < 0.05, ***P < 0.001, one-way ANOVA with Bonferroni correction. Data are representative of three different independent experiments (mean ± SEM). (B) The cumulative mortalities of gibel carp challenged with CyHV-2. Statistical differences were evaluated by log-rank (Mantel-Cox) test. (C) Efficacy of yeast vaccine in gibel carp under laboratory conditions. RPS, Relative survival rate = (1 - the ratio of mortality percent in the vaccine group or booster vaccine group to in the control group) × 100%.