An Oral Microencapsulated Vaccine Loaded by Sodium Alginate Effectively Enhances Protection Against GCRV Infection in Grass Carp (Ctenopharyngodon idella)

Abstract

Grass carp reovirus (GCRV) is highly infectious and lethal to grass carp, causing huge economic losses to the aquaculture industry annually. Currently, vaccination is the most effective method against viral infections. Among the various vaccination methods, the oral vaccination is an ideal way in aquaculture. However, low protective efficiency is the major problem for oral vaccination owing to some reasons, such as antigen degradation and low immunogenicity. In our study, we screened the antigenic epitopes of GCRV-II and prepared an oral microencapsulated vaccine using sodium alginate (SA) as a carrier and flagellin B (FlaB) as an adjuvant, and evaluated its protective effects against GCRV-II infection in grass carp. The full length and three potential antigenic epitope regions of GCRV-II VP56 gene were expressed in Escherichia coli and purified by glutathione affinity column respectively. The optimal antigen (VP56-3) was screened by enzyme-linked immunosorbent assay (ELISA). Adjuvant FlaB was also expressed in E. coli and purified by Ni²⁺ affinity column. Subsequently, we prepared the oral vaccines using sodium alginate as a carrier. The vaccine (SA-VP56-3/FlaB) forms microsphere (1.24 ± 0.22 μm), examined by transmission electron microscopy, scanning electron microscopy, and dynamic light scattering assay. SA-VP56-3/FlaB vaccine has excellent stability, slow-release, and low toxicity by dynamic light scattering assay, release dynamic assay, in vivo fluorescence imaging system, hemolytic activity and cytotoxicity. Then we vaccinated grass carp orally with SA-VP56-3/FlaB and measured immune-related parameters (serum neutralizing antibody titer, serum enzyme activity (TSOD, LZM, C3), immune-related genes ((IgM, IFN1, MHC-II, CD8 in head kidney and spleen), IgZ in hindgut)). The results showed that SA-VP56-3/FlaB significantly induced strong immune responses, compared to other groups. The highest survival rate achieved in SA-VP56-3/FlaB microencapsulated vaccine (56%) in 2 weeks post GCRV challenge, while 10% for the control group. Meanwhile, the tissue virus load in survival grass carp is lowest in SA-VP56-3/FlaB group. These results indicated that SA-VP56-3/FlaB could be a candidate oral vaccine against GCRV-II infection in aquaculture.

Keywords: flagellin (FlaB); grass carp; grass carp reovirus (GCRV); oral microencapsulated vaccine; sodium alginate (SA).

Figure 1

Recombinant expression and screening of VP56 full-length and fragment protein. (A) SDS-PAGE analyses of VP56 full-length and fragment protein. Lane M: protein marker; Lane 1: Purified VP56-1; Lane 2: Purified VP56-2; Lane 3: Purified VP56-3. Lane 4: Purified VP56. (B) ELISA assays were used to screen full-length and fragment proteins to identify those with the highest binding capacity to anti GCRV-II serum. (C) Verification of the inhibitory ability of each protein serum against GCRV-II by neutralizing antibody assay. Data are expressed as mean ± SD (n = 4). Different lowercase letters in each group (a, b and c) denote significant variations suggested by the Kruskal-Wallis statistics followed by the Dunn's multiple comparison (p < 0.05).

Figure 2

Recombinant expression of VP56-3 and FlaB, preparation and biosafety assay of the oral microencapsulated vaccine. (A) SDS-PAGE and WB analyses of VP56-3 and FlaB. Lane M: protein marker; Lane 1: Purified FlaB; Lane 2: Purified VP56-3; Lane 3: FlaB was confirmed by WB with His-tag mAb; Lane 4: VP56-3 was confirmed by WB with GST-tag mAb. (B) Image of SA under TEM (left); Image of SA-VP56-3/FlaB under TEM (right). (C) Image of SA under SEM (left); Image of SA-VP56-3/FlaB under SEM (right). (D) Nanoparticle size analysis. (E) PDI distribution coefficient. (F) Zeta potential analysis. (G) Hemolysis study of SA, VP56-3/FlaB, and SA-VP56-3/FlaB. Hemolytic activity of each group was detected with 2% grass carp erythrocytes for 1 h at 37 °C. Data are expressed as mean ± SD (n = 4). (H) Toxicity effect of SA, VP56-3/FlaB, and SA-VP56-3/FlaB on CIK cells. MTT method was used for determination. Data are expressed as mean ± SD (n = 4). Different lowercase letters in each group (a and b) denote significant variations suggested by the Kruskal-Wallis statistics followed by the Dunn’s multiple comparison (p < 0.05).

Figure 3

In vitro release rate and intestinal imaging of gavage SA (FITC-VP56-3/FlaB). (A) Release rate of SA-VP56-3/FlaB in PBS (PH = 7.4) or grass carp intestinal fluid over 36h. (B) Fluorescence of control in the intestine of grass carp with 0 h, 6 h, 12 h (left); Fluorescence of FITC-VP56-3/FlaB in the intestine of grass carp with 0 h, 6 h, 12 h (mid); Fluorescence of SA (FITC-VP56-3/FlaB) in the intestine of grass carp with 0 h, 6 h, 12 h (right).

Figure 4

Protection rate of SA-VP56-3/FlaB against GCRV-II (10⁷TCID₅₀/mL) infection and tissues viral load. (A) Schematic of the oral immunization, GCRV-II challenge, and sampling. (B) Survival of grass carp (n = 50) was monitored and calculated within 14 days after the viral challenge. (C) Head kidney viral load of surviving grass carp on D42. (D) Spleen viral load of surviving grass carp on D42. (E) Hindgut viral load of surviving grass carp on D42. ΔCT indicates the difference between CT_vp56 and CT_{18S rRNA}. Data are expressed as mean ± SD (n = 5). Different lowercase letters in each group (a, b, c and d) denote significant variations suggested by the Kruskal-Wallis statistics followed by the Dunn’s multiple comparison (p < 0.05).

Figure 5

Effect of SA-VP56-3/FlaB on nonspecific immune parameters. (A) Serum TSOD activity. (B) Serum LZM activity. (C) Serum C3 activity, determined on D0, D28, D40. Data are expressed as mean ± SD (n = 4). Different lowercase letters in each group (a, b and c) denote significant variations suggested by the Kruskal-Wallis statistics followed by the Dunn's multiple comparison (p < 0.05).

Figure 6

Transcriptional response of immune-related genes in head kidney, spleen, and hindgut of grass carp after oral immunization and viral challenge. Total RNAs were extracted from the head kidney, spleens, and hindgut on D0, D28, D40, and the transcription levels of immune-related genes. (A–C) The expression levels of immune-related genes (IFN1, MHC-II, CD8) mRNA in head kidney. (D–F) The expression levels of immune-related genes (IFN1, MHC-II, CD8) mRNA in spleen. (G) The expression level of IgZ mRNA in hindgut. The 18S rRNA gene was used as the control gene. Data are expressed as mean ± SD (n = 4). Different lowercase letters in each group (a, b, c and d) denote significant variations suggested by the Kruskal-Wallis statistics followed by the Dunn’s multiple comparison (p < 0.05).

Figure 7

Expression levels of IgM mRNA in head kidney and spleen of grass carp, serum neutralizing antibody levels, and antigen binding capacity. (A) IgM mRNA expression level in head kidney on D0, D28, D42. (B) IgM mRNA expression level in spleen on D0, D28, D40. (C) ELISA assay to determine the binding ability of each group of serum to specific antigens on D28. (D) Neutralizing antibody assay to determine the inhibitory ability of each group of serum against GCRV-II on D28. Data are expressed as mean ± SD (n = 4). Different lowercase letters in each group (a, b, c and d) denote significant variations suggested by the Kruskal-Wallis statistics followed by the Dunn’s multiple comparison (p < 0.05).