Co-Delivery Effect of CD24 on the Immunogenicity and Lethal Challenge Protection of a DNA Vector Expressing Nucleocapsid Protein of Crimean Congo Hemorrhagic Fever Virus

Abstract

Crimean Congo hemorrhagic fever virus (CCHFV) is the causative agent of a globally-spread tick-borne zoonotic infection, with an eminent risk of fatal human disease. The imminent public health threat posed by the disseminated virus activity and lack of an approved therapeutic make CCHFV an urgent target for vaccine development. We described the construction of a DNA vector expressing a nucleocapsid protein (N) of CCHFV (pV-N13), and investigated its potential to stimulate the cytokine and total/specific antibody responses in BALB/c and a challenge experiment in IFNAR−/− mice. Because of a lack of sufficient antibody stimulation towards the N protein, we have selected cluster of differentiation 24 (CD24) protein as a potential adjuvant, which has a proliferative effect on B and T cells. Overall, our N expressing construct, when administered solely or in combination with the pCD24 vector, elicited significant cellular and humoral responses in BALB/c, despite variations in the particular cytokines and total antibodies. However, the stimulated antibodies produced as a result of the N protein expression have shown no neutralizing ability in the virus neutralization assay. Furthermore, the challenge experiments revealed the protection potential of the N expressing construct in an IFNAR −/− mice model. The cytokine analysis in the IFNAR−/− mice showed an elevation in the IL-6 and TNF-alpha levels. In conclusion, we have shown that targeting the S segment of CCHFV can be considered for a practical way to develop a vaccine against this virus, because of its ability to induce an immune response, which leads to protection in the challenge assays in the interferon (IFN)-gamma defective mice models. Moreover, CD24 has a prominent immunologic effect when it co-delivers with a suitable foreign gene expressing vector.

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

“…Five microliters of template RNA (500 ng) was added to complete the master mix to 20 μl. 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 ].…”

“…To investigate viral load and replication pattern in the different human and bovine kidney cell lines, a local CCHFV strain Ank-2 (GenBank Accession number: MK309333) [27] was used in the study. CCHFV strain Ank-2 was isolated in SW-13 cells and after two rounds of virus propagation, virus replication was verified by real-time PCR targeting the S segment and follow-up sequencing.…”

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

“…To confirm the in vitro expression of N proteins from the pCD-N1 and pDC516-N constructs, an indirect immunofluorescence assay (IIFA) was performed in BHK21-C13 cells, as previously described [23]. Briefly, BHK21-C13 cells were cultivated in 24-well plates for 24 h before pCD-N1 transfection by Lipofectamine3000.…”

“…The CCHFV Ank-2 strain, which was previously demonstrated to be lethal for IFNα/β/γR−/− mice, was used in the intra-peritoneal challenge experiment [23]. The immunization schedule (vaccine constructs and their respective backbones) of IFNα/β/γR−/− mice (8–12-week-old female) was similar to BALB/c mice.…”

Crimean-Congo Hemorrhagic Fever Mouse Model Recapitulating Human Convalescence

“…Although the glycoproteins of CCHFV exhibit the greatest genetic diversity, we detected sustained CCHFV-specific T-cell responses against the more-conserved CCHFV nucleoprotein. Vaccine-induced responses solely against the nucleoprotein can be protective (24,25), and it is therefore likely that immune responses against the more-conserved CCHFV nucleoprotein could provide cross protection. Studies with monoclonal antibodies against the glycoproteins have shown that although there exist antigenic differences among diverse strains of CCHFV, there are neutralizing epitopes conserved among divergent strains (26,27).…”