Humoral Immune Response in Immunized Sheep with Bovine Coronavirus Glycoproteins Delivered via an Adenoviral Vector

Abstract

Bovine coronavirus (BCoV) is distributed globally and mainly causes different clinical manifestations: enteric diarrhea in calves, winter dysentery in adults, and respiratory symptoms in cattle of all ages. Low mortality and high morbidity are the hallmarks of BCoV infection, usually associated with substantial economic losses for the livestock industry. Vaccination, combined with the implementation of biosecurity measures, is the key strategy for the prevention of infections. This pilot study evaluates the immunogenicity of a recombinant vaccine containing two BCoV antigens (S and M) in sheep, compared to vaccines containing only the M or S protein. Three groups of sheep were inoculated intramuscularly at day 0 and day 21 with recombinant adenoviruses expressing BCoV S protein (AdV-BCoV-S), BCoV M protein (AdV-BCoV-M), or both proteins (AdV-BCoV-S + M). Serum antibodies were evaluated using immunofluorescence (IF) and serum neutralization (SN) tests. Moderate seroconversion was observed by day 21, but serum antibodies detected via SN increased from 1:27.5 (day 21) to 1:90 (day 28) in sheep inoculated with the recombinant AdV expressing both the S- and M-BCoV proteins. Based on the SN results, a repeated-measures ANOVA test indicated a more significant difference in immune response between the three groups (F = 20.47; p < 0.001). The experimental investigation produced satisfactory results, highlighting that the S + M recombinant vaccine was immunogenic, stimulating a valid immune response. Despite some inherent limitations, including a small sample size and the absence of challenge tests, the study demonstrated the efficacy of the immune response induced via the recombinant vaccine containing both S and M proteins compared to that induced via the individual proteins S or M.

Keywords: bovine coronavirus; immune response; vaccine.

Figure 1

Construction of an assessment of recombinant adenoviruses expressing BCoV S and M glycoproteins. (A) Diagram (not to scale) of Ad5-bocS-ΔRS-HA-GFP (Ad5-S) and Ad5-bovM-HA-GFP (Ad5-M) genomic structure composed of different elements: 5′ and 3′ inverted terminal repeat (ITR; gray), adenoviral packaging signal (Ψ; dark blue), human cytomegalovirus immediate early enhancer/promoter (CMV; light blue), open reading frame coding for S or M glycoprotein (S or M; yellow), bovine growth hormone polyadenylation signal (pA; dark green), human phosphoglycerate kinase 1 promoter (PGK; light blue), open reading frame coding for green fluorescent protein (GFP; green), Herpes Simplex Virus thymidine kinase polyadenylation signal (pA; dark green), and E1A/B and E3 deleted human adenovirus type 5 genome backbone (ΔE1-E3Ad5 backbone; orange). Phase contrast and fluorescence images (scale bar corresponds to 50 µm) of Ad5-S (B) and Ad5-M (D) infected HEK293T cells at 24 and 48 h (hs) post-infection (P.I.). Ad5-S and Ad5-M viral titer, (C) and (E), respectively, was measured and expressed as log10 per ml of transducing units (T.U.) of viral particles released at 24 and 48 h P.I. when HEK293T cells were infected with an M.O.I. Values are the means ± standard errors of three independent experiments. (F) Immunofluorescent staining of Ad5-S or Ad5-M transduced primary cultures of bone marrow-derived ovine mesenchyme stem/stromal cells and the untransduced control (UT control). Since S and M were HA-tagged, immunostaining was performed with an anti-HA mAb. GFP and anti-HA were colocalized by merging the images (merge). (G) Western immunoblotting of Ad5-S or Ad5-M transduced primary cultures of bone marrow-derived ovine mesenchyme stem/stromal cells. Two different number of cell protein extracts were loaded (1 = 10 µg, and 2 = 30 µg), the negative control (−) was made with 30 µg of protein extract that came from untransduced primary cultures of bone marrow-derived ovine mesenchyme stem/stromal cells, and L is the protein-size ladder lane (kDa).

Figure 2

Assessment of HEK293T growth in OvS. MTT assay comparing HEK293T growth with medium containing 10% of FBS and the same number of HEK293T growth with medium containing 10% of OvS at different time points (24, 48, 72, and 96 h); absolute values are expressed as optical density (O.D.) (A) The data presented are the means ± standard errors of triplicate measurements (p > 0.05, as measured with Student’s t test). (B,C) Ad5-S and Ad5-M titer was measured and compared between cells’ growth with FBS or OvS at 24 and 48 h post-infection and expressed as log₁₀ T.U. per mL of viral particles released. Values are the means ± standard errors of three independent experiments.

Figure 3

Monitoring of immune response in the AdV-BCoV-S protein group, AdV-BCoV-M protein group, and AdV-BCoV-S + M proteins group using IF test and SN test. In the plots, the x-axis represents the time point (days) in which the antibody titer was evaluated, while the y-axis represents the antibody titer expressed as an absolute value. (A): The antibody titer detected in each animal of the three groups included in the experiment using IF and SN at four checkpoints (day 0, day 21, day 28, and day 72). (B): Timeline of the procedure conducted during the experiment.