Bovine Herpesvirus Type 4 (BoHV-4) Vector Delivering Nucleocapsid Protein of Crimean-Congo Hemorrhagic Fever Virus Induces Comparable Protective Immunity against Lethal Challenge in IFNα/β/γR−/− Mice Models

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of a tick-borne infection with a significant mortality rate of up to 40% in endemic areas, with evidence of geographical expansion. Due to a lack of effective therapeutics and control measures, the development of a protective CCHFV vaccine remains a crucial public health task. This paper describes, for the first time, a Bovine herpesvirus type 4 (BoHV-4)-based viral vector (BoHV4-∆TK-CCHFV-N) and its immunogenicity in BALB/c and protection potential in IFNα/β/γR−/− mice models in comparison with two routinely used vaccine platforms, namely, Adenovirus type 5 and a DNA vector (pCDNA3.1 myc/His A), expressing the same antigen. All vaccine constructs successfully elicited significantly elevated cytokine levels and specific antibody responses in immunized BALB/c and IFNα/β/γR−/− mice. However, despite highly specific antibody responses in both animal models, the antibodies produced were unable to neutralize the virus in vitro. In the challenge experiment, only the BoHV4-∆TK-CCHFV-N and Ad5-N constructs produced 100% protection against lethal doses of the CCHFV Ank-2 strain in IFNα/β/γR−/− mice. The delivery platforms could not be compared due to similar protection rates in IFNα/β/γR−/− mice. However, during the challenge experiment in the T cell and passive antibody transfer assay, BoHV4-∆TK-CCHFV-N was dominant, with a protection rate of 75% compared to others. In conclusion, vector-based CCHFV N protein expression constitutes an effective approach for vaccine development and BoHV-4 emerged as a strong alternative to previously used viral vectors.

Animal Models for Crimean-Congo Hemorrhagic Fever Human Disease

“…Additionally, CCHFV produces severe disease in STAT-1 −/− mice and mice lacking both the IFN-I receptor and IFN-gamma receptor (IFNAGR −/− ). These animals have deficiencies in both IFN-I and type II interferon (IFN-γ) signaling [78,79]. We recently developed a novel murine system by exploiting an antibody against IFN-I receptor A (MAR1-5A3) that was previously shown to produce severe disease models with other unrelated viruses [80,81].…”

“…Protective efficacy of various CCHFV vaccines including DNA, MVA-vectored and adenovirus-vector vaccines have been reported in IFNAR −/− mice, including those on the 129 and C57BL/6 backgrounds [82,107,112,113]. Another group reported protective efficacy of a vaccine expressing N on a Bovine herpesvirus type 4 vector in mice lacking both type I and type II interferon receptors [79]. Rodriguez, et al reported the protective efficacy of a recombinant a vesicular stomatitis virus (rVSV) expressing the glycoproteins in STAT-1 −/− mice from lethal challenge [86].…”

Immunological Analysis of a CCHFV mRNA Vaccine Candidate in Mouse Models

“…Additionally, it has been documented that antibodies produced against this protein have no neutralizing effect on the virus. However, despite the lack of neutralizing antibodies, vaccination using different expression platforms delivering the S segment sufficiently stimulated the immune response to protect the challenged mice models [5,6,7,8]. To clarify this phenomenon, one study documented that vaccines based on this gene elicited balanced Th1 and Th2 responses, which resulted in protection in genetically modified mice models against lethal doses [9].…”

Differential Growth Characteristics of Crimean-Congo Hemorrhagic Fever Virus in Kidney Cells of Human and Bovine Origin

“…This procedure was repeated two more times. The virus was titrated between each passage using a standard TCID50 assay in SW-13 cells [28]. RNA extraction from the virus-infected aqueous phase of the cell cultures was performed by QIAamp Viral RNA Mini Kit (QIAGEN, Germantown, MD, USA) according to the manufacturer's instructions.…”

“…The cycling conditions were as follows: cDNA synthesis at 50 • C for 10 min, initial denaturation at 95 • C for 2 min, and 40 cycles of denaturation at 95 • C for 5 s, and annealing/extension at 60 • C for 30 s. The primers and probe were targeting the nucleoprotein gene (small segment) as previously described [27]: qPCR-F (5 -GGACATAGGTTTCCGTGTCA-3 ), qPCR-R (5 -TCCTTCTAATCATGTCTGACAGC-3 ) and qPCR-probe (5 -FAM-AGAACAACTTGCCAATTACCAACAGGC-BHQ1-3 ). Standard linear plasmids were prepared by 10-fold dilutions equivalent to 2 × 10 3 to 2 × 10 8 copies/reaction mixture using pCD-N1 as the template [28]. The reactions were carried out in one step by a Rotor-Gene Q instrument (QIAGEN, Germantown, MD, USA) and all calculations were then transformed to log-based viral loads (copies/mL).…”