Combining segments 9 and 10 in DNA and recombinant protein vaccines conferred superior protection against tilapia lake virus in hybrid red tilapia (oreochromis sp.) compared to single segment vaccines

Abstract

Tilapia lake virus (TiLV) now affects Nile tilapia culture worldwide, with no available commercial vaccine for disease prevention. DNA and recombinant protein-based vaccines were developed and tested following viral isolation and characterization. The viral strain isolated from diseased hybrid red tilapia (Oreochromis sp.) shared high levels of morphological and genomic similarity (95.49-99.52%) with other TiLV isolates in the GenBank database. TiLV segment 9 (Tis9) and segment 10 (Tis10) DNA vaccines (pcDNA-Tis9 and pcDNA-Tis10) and recombinant protein vaccines (Tis9 and Tis10) were prepared and tested for their efficacy in juvenile hybrid red tilapia. Fish were immunized with either single vaccines (pcDNA-Tis9, pcDNA-Tis10, Tis9 and Tis10) or combined vaccines (pcDNA-Tis9 + pcDNA-Tis10 and Tis9 + Tis10) by intramuscular injection and intraperitoneal injection for DNA and protein vaccines, respectively. Negative controls were injected with PBS or a naked pcDNA3.1 vector in the same manner. An experimental challenge with TiLV was carried out at 4 weeks post-vaccination (wpv) by intraperitoneal injection with a dose of 1 × 10⁵ TCID₅₀ per fish. Relative percent survival (RPS) ranged from 16.67 ± 00.00 to 61.11 ± 9.62%. The Tis10 and pcDNA-Tis10 vaccines conferred better protection compared to Tis9 and pcDNA-Tis9. Highest levels of protection were observed in pcDNA-Tis9 + pcDNA-Tis10 (61.11 ± 9.62%) and Tis9 + Tis10 (55.56 ± 9.62%) groups. Specific antibody was detected in all vaccinated groups at 1-4 wpv by Dot Blot method, with the highest integrated density at 2 and 3 wpv. In silico analysis of Tis9 and Tis10 revealed a number of B-cell epitopes in their coil structure, possibly reflecting their immunogenicity. Findings suggested that the combination of Tis9 and Tis10 in DNA and recombinant protein vaccine showed high efficacy for the prevention of TiLV disease in hybrid red tilapia.

Keywords: DNA vaccine; TiLV; TiLV ORF10; TiLV ORF9; recombinant protein vaccine; tilapia (fish).

Figure 1

TiLV detection of infected tilapia organs, infected cell cultures and transmission electron microscope (TEM) analyses. (A) Clinical signs of infected fish include skin redness and congestion around the eye and head (asterisk), severe skin hemorrhage (black arrow), skin erosion (blue arrow) and fin rot (red arrow). (B) Infected fish were screened for TiLV infection from different organs using RT-PCR and specific PCR products approximately 500 bps were observed. Lane M: DNA marker; Lane P: positive control; Lane N: negative control; Lanes 1-3: brain, liver and spleen tissues from infected fish, respectively. (C) E-11 infected cells revealed CPEs with plaque formation (asterisk) and syncytial formation (black arrow) at 7 days post infection (dpi). (D) Uninfected E-11 cells. (E) TK-1 infected cells revealed CPEs with cell swelling, vacuolated appearance (red arrow) followed by cell detachment at 7 dpi. (F) Uninfected TK-1 cells. (G) Detection of TiLV from E-11 and TK-1 infected cells by RT-PCR. Lane M: DNA marker; Lane P: positive control; Lane N: negative control; Lanes 1-2: uninfected E-11 and TK-1, respectively; Lanes 3-4: infected E-11 and TK-1 cells, respectively. (H, I) TEM micrograph of the purified TiLV from infected TK-1 culture supernatant that showed isolated virion (H), and aggregated virions in small group (I).

Figure 2

Expression and purification of Tis9 and Tis10 proteins. (A) Western blot analysis of both proteins from the E. coli expression system. Tis9 and Tis10 proteins expressed approximately 32 kDa and 37 kDa, respectively (Lane M: prestained protein marker). (B, C) SDS-PAGE analysis of (B) Tis9 and (C) Tis10 proteins purification with coomassie blue staining. (Lane M: prestained protein marker; Lane S: supernatant; Lane P: precipitation of the E. coli was induced; Lane F: unbound flowthrough; Lane W: wash fraction; Lanes: E1-E2 = elute fractions.

Figure 3

Cumulative mortality curves of the recombinant protein vaccines (Tis9, Tis10 and Tis9+Tis10 groups) and DNA vaccines (pcDNA-Tis9, pcDNA-Tis10 and pcDNA-Tis9+Tis10 groups) after challenge with 1 x 10⁵ TCID50 of TiLV. Differences among the groups were analyzed using one-way ANOVA and Tukey’s post hoc tests. Statistical significance is indicated by single asterisk (*, P < 0.05) or double asterisks (**, P < 0.01) or ns (not significant) compared to the PBS or pcDNA3.1 (negative control) groups at the termination day (day 28).

Figure 4

Dot blot analysis for identification of antibody production from vaccinated fish. Integrated densities from dot blot results were converted to values and normalized to the PBS or pcDNA3.1 negative control group using Image Lab software (Bio-Rad). Data are represented as means ± SD (n = 3). Different letters above the bars indicate significant differences within treatments (P < 0.05).

Figure 5

3D structure of Tis9 and Tis10 proteins showing their surface polarity, linear and conformational B cell epitope regions and relative proportions of amino acid residues in conformational epitopes. (A) Tis9 and (B) Tis10 models are shown in surface presentation, nonpolar residues (blue) buried in the core, whereas polar residues (red) tend to occur at the surface of proteins. (C and D) Predicted linear epitope and conformational epitope from the ElliPro results. Polar surface regions in (A) and (B) displayed linear epitopes (represented as red ribbon) and conformational epitopes (represented as red CPK along with surface structure on the Tis9 (C) and Tis10 (D) models. Their N-terminus and C-terminus are indicated by arrows.