Recombinant Subunit Vaccine Candidate against the Bovine Viral Diarrhea Virus

Abstract

Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.

Keywords: BVDV subgenotypes; E2 glycoprotein; bovine viral diarrhea virus; subunit vaccine.

Figure 1

Individual clones expressing different E2 proteins. Bright fields, fluorescences, and flow cytometry histograms from clones expressing (A) 1aE2, (B) 1bE2, (C) 1cE2, (D) 1dE2, and (E) 1eE2. The P3 region of histograms gathers cells with the highest fluorescence intensity.

Figure 2

Immunoidentification of the five E2 proteins. Western blot assay of purified E2 proteins using specific primary antibodies against (A) c-Myc tag, (B) HA tag, (C) VSV tag, (D) V5 tag, and (E) E tag. Anti-6xHis antibody was also used. 1—Molecular Weight Marker AccuRuler RGB Plus prestained protein ladder (Maestrogen, Taiwan), 2—Anti-specific tag, 3—anti-6xHis tag, 4—Merge of anti-specific tag and anti-6xHis tag, meaning they identified the same E2 protein.

Figure 3

Vaccine candidate’s immunogenicity in sheep. (A) Immunization protocol. (B) Time-course of humoral immune response in sheep, i.m. immunized on days 0 and 21 with 50 µg, 100 µg, and 150 µg of the vaccine candidate. Data represented means ± SD analyzed by the Friedman test, followed by Dunn’s multiple comparison test, n = 8. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.

Figure 4

Vaccine candidate’s immunogenicity in bovines. (A) Immunization protocol. (B) Time course of humoral immune response in cattle immunized with 50 µg of the vaccine candidate. The commercial vaccine CattleMaster^MR Gold FP 5 was used as the positive control. Data represent means ± SD analyzed by the Friedman test and Dunn’s multiple comparisons test. The significance between groups at each time was analyzed by the Kruskal–Wallis test and Dunn’s multiple comparisons test. (C) Antibody titers in the sera of immunized animals at day 56. Data were analyzed by the Mann–Whitney test. (D) The individual contribution of every E2 antigen to the humoral immune response. Data were analyzed by the Friedman test and Dunn’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.

Figure 5

Cellular immune response by measuring cytokine transcripts or protein levels. Relative transcript expression from (A) IFN-γ, (B) IL-12, and (C) IL-4 was measured using the 2^−ΔΔCt method [39]. The GAPDH gene was used as housekeeping. (D) IFN-γ in vitro determination using unstimulated and stimulated PBMC from day 56. The mean ± SD for each group (n = 8 animals) is represented. Data were analyzed by the Friedman test, followed by Dunn’s multiple comparisons test. Statistical significance between groups at each time was determined by the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. * p ≤ 0.05 and ** p ≤ 0.01.

Figure 6

In vitro neutralization assay. Primary bovine embryo kidney cells were treated with a serum-antigen mixture using sera from days 35 and 56 after the first immunization and the reference antigen BVDV1a. Values represent the mean ± SD of 8 animals. Statistics were determined using the Wilcoxon matched-pairs signed rank test for different times. The Kruskal–Wallis test, followed by Dunn’s multiple comparison tests was used to measure the significance between groups at each time. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

Figure 7

Scheme of the expression vectors coding the five E2 proteins from different BVDV subgenotypes. Bicistronic transcriptional units designed to produce E2 proteins also contain the GFP molecule as a fluorescent marker linked by an IRES sequence. Transcriptional units were controlled using the CMV promoter/enhancer and the SV40 cleavage and polyadenylation sequence. Every E2 protein has a distinct tag (c-Myc, HA, VSV, V5, and E) for immunoidentification. A second tag (6xHis tag) was also included for purification purposes.