Immune protection induced by E2 recombinant glycoprotein of bovine viral diarrhea virus in a murine model

Abstract

Bovine viral diarrhea virus (BVDV) is considered the most important viral pathogen in ruminants worldwide due to the broad range of clinical manifestations displayed by infected animals. Therefore, infection with BVDV leads to severe economic losses in several countries' beef and dairy industries. Vaccination prevents reproductive failure and gastrointestinal and respiratory disorders caused by BVDV infection. However, considering their limitations, conventional vaccines such as live, attenuated, and killed viruses have been applied. Hence, different studies have described subunit vaccines as an effective and safe alternative for BVDV protection. Therefore, in this study, the ectodomain of E2 (E2e) glycoprotein from NADL BVDV strain was expressed in mammalian cells and used in two vaccine formulations to evaluate immunogenicity and protection against BVDV conferred in a murine model. Formulations consisted of solo E2e glycoprotein and E2e glycoprotein emulsified in adjuvant ISA 61 VG. Five groups of 6 mice of 6-to-8-week-old were immunized thrice on days 1, 15, and 30 by intraperitoneal injection with the mentioned formulations and controls. To evaluate the conferred protection against BVDV, mice were challenged six weeks after the third immunization. In addition, the humoral immune response was evaluated after vaccination and challenge. Mice groups inoculated with solo E2e and the E2e + ISA 61 VG displayed neutralizing titers; however, the E2 antibody titers in the E2e + ISA 61 VG group were significantly higher than the mice group immunized with the solo E2e glycoprotein. In addition, immunization using E2e + ISA 61 VG prevents animals from developing severe lesions in surveyed tissues. Moreover, this group acquired protection against the BVDV challenge, evidenced by a significant reduction of positive staining for BVDV antigen in the lungs, liver, and brain between the experimental groups. Our findings demonstrated that using E2e + ISA 61 VG induces greater BVDV protection by an early humoral response and reduced histopathological lesions and BVDV antigen detection in affected organs, indicating that E2e + ISA 61 VG subunit formulation can be considered as a putative vaccine candidate against BVDV. The efficacy and safety of this vaccine candidate in cattle requires further investigation.

Keywords: E2 recombinant glycoprotein; adjuvant; bovine viral diarrhea virus; murine model; virus neutralization.

Figure 1

Experimental timeline. A schematic diagram of immunization protocol. The numbers indicate the days during the trial. Arrows indicate the trial procedure.

Figure 2

Expression of BVDV E2 recombinant protein in SDS-PAGE and Western blot. (A) Lane 1: Molecular weight marker. Lane 2: Expression of E2e recombinant protein recovered and purified from the supernatant of HEK-293T transfected cells. (B) Detection of E2e recombinant protein by Western blot using monoclonal anti-His tag antibodies diluted 1:1,000. (C) Detection of E2e recombinant protein by Western blot using anti-BVDV antibodies diluted 1:1,000. (D) Detection of E2e recombinant protein by Western blot using monoclonal anti-myc tag antibodies diluted 1:1,000.

Figure 3

Two-way hierarchical cluster analysis based on positive RT-PCR results of BVDV detection. (A) Dendrogram shows the grouping pattern of BVDV RNA-positive detection in the lungs, brain, liver, spleen, stomach, intestine, and kidneys from experimental groups of mice used in this study. (B) Spectrum of colors between green and red shows the correlation intensity among the treatment of each experimental group and BVDV-positive percentage values obtained from mice tissues evaluated using RT-PCR results.

Figure 4

Neutralizing antibody titer. Comparison of viral neutralization titer of each experimental group. Neutralizing antibodies were detected by VNT. Titers are expressed as the reciprocal of the highest dilution that neutralized viral infectivity. ***p < 0.001, **p < 0.01.

Figure 5

Representative histopathological features of the lung, liver, spleen, intestine, kidney, brain, and stomach sections of immunized and challenged mice from experimental groups 1–3. Group 5 (PSS) was included as a negative control and Group 3 as a positive control. Compared to the negative control group 5, mice immunized with solo E2e formulation and BVDV NADL strain showed coagulative necrosis and lymphocytic infiltration in the liver (squares and black arrows in liver tissue, respectively). Similarly, mice from groups 1 and 3 exhibited mild-to-moderate glomerulitis and tubular necrosis (black arrows and squares in kidney tissues, respectively).

Figure 6

Immunohistochemical staining of BVDV in tissues from immunized and challenged mice. BVDV antigen was detected in the lungs, liver, spleen, intestine, kidneys, brain, and stomach of mice with variable immunopositivity in the experimental groups 1–3, whereas group 5 (PSS) was included as a negative control.