A Novel Quadruple Gene-Deleted BoHV-1-Vectored RVFV Subunit Vaccine Induces Humoral and Cell-Mediated Immune Response against Rift Valley Fever in Calves

Abstract

Rift Valley fever virus (RVFV) is considered to be a high biodefense priority based on its threat to livestock and its ability to cause human hemorrhagic fever. RVFV-infected livestock are also a significant risk factor for human infection by direct contact with contaminated blood, tissues, and aborted fetal materials. Therefore, livestock vaccination in the affected regions has the direct dual benefit and one-health approach of protecting the lives of millions of animals and eliminating the risk of severe and sometimes lethal human Rift Valley fever (RVF) disease. Recently, we have developed a bovine herpesvirus type 1 (BoHV-1) quadruple gene mutant virus (BoHV-1qmv) vector that lacks virulence and immunosuppressive properties due to the deletion of envelope proteins UL49.5, glycoprotein G (gG), gE cytoplasmic tail, and US9 coding sequences. In the current study, we engineered the BoHV-1qmv further by incorporating a chimeric gene sequence to express a proteolytically cleavable polyprotein: RVFV envelope proteins Gn ectodomain sequence fused with bovine granulocyte-macrophage colony-stimulating factor (GMCSF) and Gc, resulting in a live BoHV-1qmv-vectored subunit vaccine against RVFV for livestock. In vitro, the resulting recombinant virus, BoHV-1qmv Sub-RVFV, was replicated in cell culture with high titers. The chimeric Gn-GMCSF and Gc proteins expressed by the vaccine virus formed the Gn-Gc complex. In calves, the BoHV-1qmv Sub-RVFV vaccination was safe and induced moderate levels of the RVFV vaccine strain, MP12-specific neutralizing antibody titers. Additionally, the peripheral blood mononuclear cells from the vaccinated calves had six-fold increased levels of interferon-gamma transcription compared with that of the BoHV-1qmv (vector)-vaccinated calves when stimulated with heat-inactivated MP12 antigen in vitro. Based on these findings, we believe that a single dose of BoHV-1qmv Sub-RVFV vaccine generated a protective RVFV-MP12-specific humoral and cellular immune response. Therefore, the BoHV-1qmv sub-RVFV can potentially be a protective subunit vaccine for cattle against RVFV.

Keywords: BoHV-1 mutant; BoHV-1 vector; Gn and Gc; RVFV; cattle; immunogenicity; subunit-vaccine.

Figure 1

Schematic of bovine herpesvirus virus type 1 (BoHV-1) genome showing the UL49.5, glycoprotein G (gG), gE cytoplasmic tail (gECT), and US9-deletion loci and pBoHV-1gECT-∆Us9∆, pBoHV-1gG∆, and pBoHV-1UL49.5 (30-32-CT) ∆ used to construct the BoHV-1qmv vector. Also shown is the chimeric RVFV glycoprotein Gn-GMCSF fusion (Gn+), p2A, and Gc sequences insertion into the gG-deletion locus of the BoHV-1qmv genome, resulting in the BoHV-1qmv Sub-RVFV vaccine.

Figure 2

Schematic showing the immunization and sample collection scheme for the calf experiment. Intranasal—intranasal inoculation; Subcut.—Subcutaneous injection; PFU—plaque forming units.

Figure 3

Immunoblot analysis of the BoHV-1qmv Sub-RVFV expressing chimeric RVFV Gn-GMCSF and RVFV Gc proteins using rabbit anti-RVFV Gn (left panel), anti-FLAG monoclonal antibody (mAbs) (middle left panel), rabbit anti-RVFV Gc (middle right panel), and anti-V5 mAbs (right panel), respectively. The RVFV Gn- and Gc-specific protein bands were absent in the mock-infected KOP-R cell lysates.

Figure 4

Immunoblot analysis of the BoHV-1qmv Sub-RVFV-expressed chimeric Gn-GMCSF and Gc proteins for glycosylation. KOP-R cells were infected with the BoHV-1qmv Sub-RVFV. Infected cell lysates were immunoprecipitated with either (A) RVFV Gn- or (B) RVFV Gc-specific antibodies. Immunoprecipitated proteins were either untreated (−) or treated with PNGase F (+) or Endo H (+) and subjected to SDS-PAGE and immunoblotting with FLAG tag (Gn-specific) or V5 tag (Gc-specific) antibodies.

Figure 5

The BoHV-1qmv Sub-RVFV vaccine virus-expressed Gn-GMCSF and Gc proteins form the Gn–Gc complex in the infected cells in vitro. KOP-R cells were infected with the BoHV-1qmv Sub-RVFV. Infected cell lysates were immunoprecipitated with (A) FLAG tag- or V5 tag-specific antibodies followed by immunoblotting with RVFV Gc- or Gn-specific rabbit polyclonal antibodies to identify the co-immunoprecipitated proteins. (B) Similarly, the RVFV Gn-immunoprecipitated proteins were immunoblotted with FLAG tag- or V5 tag-specific antibodies to recognize the co-immunoprecipitated proteins.

Figure 6

In vitro characterization of BoHV–1qmv Sub-RVFV. (A,B) Plaque size analysis of BoHV-1qmv Sub-RVFV compared to that of BoHV–1 wt. Shown are the pictures of areas containing representative plaques of each virus. The bar graph shows the average plaque size of at least 50 plaques with SD (*** p < 0.001). (C) One–step growth analysis of BoHV-1qmv Sub-RVFV compared with the BoHV–1 wt.

Figure 7

Indirect immunofluorescence assay (IIFA) to determine the stability of BoHV-1qmv Sub-RVFV. MDBK cells were infected with BoHV-1qmv Sub-RVFV from either passage 1 or passage 10. At 24 h post-infection, cells were fixed and IIFA were performed using rabbit anti-FLAG or mouse anti-V5 as primary antibodies and donkey anti-rabbit IgG Alexa Fluor 647 or donkey anti-mouse IgG Alexa fluor 488 as secondary antibodies. Chimeric protein expressions were indicated by bright far-red fluorescent signals for Gn-GMCSF-FLAG and bright apple-green fluorescent signals for Gc-V5 (Magnifications 200×).

Figure 8

Nasal shedding of both BoHV-1qmv vector (▲) and BoHV-1qmv Sub-RVFV () in immunized calves assessed by qPCR and virus isolation. (A) After the inoculation into the KOP-R cells, the virus was isolated from each animal’s nasal swab and titrated in confluent KOP-R cells by plaque assay. Shown are the virus titers in the plaque-forming unit/mL of the nasal swab. (B) DNA was isolated from nasal swabs following inoculation and BoHV-1-qPCR was performed. The mean copy numbers of the BoHV-1 genome are shown in 100 ng of total DNA. PFU/mL; dpv—days post-vaccination.

Figure 9

BoHV-1 and RVFV-specific neutralizing antibody titer in serum. BoHV-1- and RVFV-specific serum-neutralizing (SN) antibody titer developed in calves after BoHV-1qmv vector and BoHV-1qmv Sub-RVFV vaccination. (A) BoHV-1-specific SN titers. The data represent the mean + standard deviation. (B) RVFV-specific SN antibody titer following BoHV-1qmv vector and BoHV-1qmv Sub-RVFV immunization. The dot plot graph shows each animal’s mean values and individual titer with standard deviation.

Figure 10

Fold-change in pre-vaccination and post-vaccination RVFV-specific interferon-gamma (IFN–γ) mRNA transcript expression upon in vitro stimulation by RVFV MP12 strain antigen.