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.

Keywords: CCHFV; CD24; Cytokines; IFNAR−/− mice; Lethal Challenge; nucleocapsid.

Figure 1

Immunization scheme.

Figure 2

(A,B) Propagation of Ank-2 strain of Crimean Congo hemorrhagic fever virus (CCHFV) in Scott and White No. 13 (SW-13) cells on day five (A: cell control, B: virus infected cells). Magnification ×400. (C) Plasmid construct used for DNA immunization in this study. In vitro homologous recombination between the empty vector and amplified nucleocapsid, flanked by 50 bp homologous arms to the EcoRI recognition sequence of the vector multiple cloning site, occurs in the presence of the Seamless Ligation Cloning Extract (SLiCE) lysate from PPY bacteria, adenosine triphosphate (ATP), 1,4-Dithiothreitol (DTT), and MgCl₂ at 37 °C. (D–G) N protein expression in the cells transfected by pV-N13 via indirect immunofluorescence assay (IIFA) 72 hours post DNA delivery (D: phase contrast; E: fluorescent contrast). CD24 protein expression in the cells transfected by pCD24 via IIFA after 72 h (F: phase contrast; G: fluorescent contrast). (H) Western blot analysis of the BHK21-C13 cells transfected with pV-N13. The expected protein (~52 kDa) was detected after 72 h (lane 1). We included pVAX-1 transfected cells (lane 2) and cell control (lane 3) in the assay.

Figure 3

Serological assays (A) IgG1 response. (B) IgG2a response. (C) IgG2b response. (D) IgG3 response: In all of the mentioned assays, the pV-N13 plus pCD24 and pCD24 groups are dominant. (E) IgM response: pV-N13, pV-N13 plus pCD24, and pCD24 groups are a stimulator of the IgM response. (F) Comparison of IgG1, IgG2a, and IgG2a/IgG1 ratio responses. * p < 0.05; ** p < 0.01; and *** p < 0.001 versus the pVAX-1 group. All of the data are shown as mean ± standard deviation (SD). (G–H) Detection of the N specific antibodies present in the serum samples in the Baby Hamster Kidney (BHK)-N cells (G: pV-N13 immunized mice serum samples, H: pV-N13 plus pCD24 immunized mice serum samples).

Figure 3

Serological assays (A) IgG1 response. (B) IgG2a response. (C) IgG2b response. (D) IgG3 response: In all of the mentioned assays, the pV-N13 plus pCD24 and pCD24 groups are dominant. (E) IgM response: pV-N13, pV-N13 plus pCD24, and pCD24 groups are a stimulator of the IgM response. (F) Comparison of IgG1, IgG2a, and IgG2a/IgG1 ratio responses. * p < 0.05; ** p < 0.01; and *** p < 0.001 versus the pVAX-1 group. All of the data are shown as mean ± standard deviation (SD). (G–H) Detection of the N specific antibodies present in the serum samples in the Baby Hamster Kidney (BHK)-N cells (G: pV-N13 immunized mice serum samples, H: pV-N13 plus pCD24 immunized mice serum samples).

Figure 4

Cytokine responses in the supernatant of CCHFV stimulated splenocytes of the immunized BALB/c mice. (A) Interferon (IFN)-gamma response: As demonstrated here, the pV-N13 plus pCD24 group’s result is higher in comparison with the other groups. The pCD24 stimulation level is also significant. (B) IL-2 response: IL-2 response is predominant in the pV-N13 plus pCD24 and pCD24 groups. (C) IL-4 response: As shown, the pV-N13 plus pCD24 and pV-N13 immunized BALB/c mice demonstrated the highest amount. (D) IL-5 response: the IL-5 response is predominant in the pV-N13 plus pCD24 and pCD24 groups. (E) IL-6 response: pV-N13 plus pCD24 and pV-N13 groups are higher in comparison with the other groups. (F) IL-13 response: All of the immunized mice showed almost identical levels of IL-13 secretion. (G) IL-10 response: When comparing the N expressing groups to the empty vectors, the pV-N13 plus pCD24 group elicited a pronounced IL-10 response in the BALB/c mice model. Also, pCD24 has the potential to induce IL-10 responses. (H) TNF-alpha response: As shown, the pV-N13 plus pCD24 group stimulated the highest amount. * p < 0.05; ** p < 0.01, and *** p < 0.001 versus the pVAX-1 group. The ‘ns’ in the graphs indicates non-significant data. All of the data are shown as mean ± SD.

Figure 5

Cytokine responses in the serum samples of the immunized BALB/c mice. (A) IFN-gamma response: pCD24 vector (on day 28) stimulated a prominent production of IFN-gamma. pV-N13 plus pCD24 on day 28 is by far dominant when compared with other groups. (B) IL-2 response: pCD24 and pV-N13 plus pCD24 possessed the highest amount. Moreover, the IL-2 level in the pV-N13 group is also adequate. (C) IL-4 response: IL-4 stimulation in pCD24 and pV-N13 plus pCD24 is considerable when compared with the other groups. (D) IL-5 response: Interestingly, the pV-N13 plus pCD24 group has potential to stimulate this cytokine in immunized mice in a higher level compared to the remaining groups. The amount of IL-5 in the pCD24 immunized mice is also significantly elevated. (E) IL-6 response: pCD24 and pV-N13 plus pCD24 immunized mice possessed the highest level of IL-6 in the serum samples. (F) IL-10 response: The results indicated the predominance of the pV-N13 plus pCD24 group. (G) IL-13 response: The resu lts are comparable to those of IFN-gamma. (H) TNF-alpha response: pV-N13 plus pCD24 construct stimulated the highest levels of TNF-alpha. * p < 0.05; **p < 0.01, and *** p < 0.001 versus pVAX-1 group. All of the data are shown as mean ± SD.

Figure 6

Cytokine responses in the serum samples of the immunized IFNAR^−/− mice. (A) IFN-gamma response: pV-N13 and pV-N13 plus pCD24 showed relatively high levels of IFN-gamma on day 28 (before challenge). Despite pV-N13 plus pCD24, in the pV-N13 group, this cytokine was elevated on day 41 (13 days after challenge). (B) IL-2 response: The results of IL-2 are comparable to INF-gamma. (C) IL-4 response: pV-N13 and pV-N13 plus pCD24 demonstrated the potential to stimulate IL-4 before challenge on day 28 and 13 days after challenge on day 41. (D) IL-5 response: pV-N13 plus pCD24 is predominant compared with pV-N13. However, both groups demonstrated a decrease on day 41. (E) IL-6 response: pV-N13 plus pCD24 appeared more immunogenic than pV-N13 via the IL-6 levels. (F) TNF-alpha response: The results are comparable to IL-6 and both N expressing constructs had the potential to elicit adequate TNF-alpha responses before and after challenge. * p < 0.05; ** p < 0.01, and *** p < 0.001 versus pVAX-1 group. All of the data are shown as mean ± SD.

Figure 7

Challenge experiment. (A) Challenge assay to find the suitable strain of CCHFV: Four different isolates were assayed in IFNAR^−/− to identify the lethal strains. (B) Survival rate in the challenge assay with Ank-2 strain: the pV-N13 and pV-N13 plus pCD24 groups survived in the lethal dose challenge of the IFNAR^−/− mice. (C) Percentage of body weight: Despite the lethal challenge, the pV-N13 and pV-N13 plus pCD24 groups showed an almost stable body weight range. All of the data are shown as mean ± SD.

Figure 8

Viral loads (copy/µL of 100 ng RNA) in the tissues of the challenged IFNAR^−/− mice infected with 1000 TCID₅₀ of the Ank-2 strain. The virus copy numbers in the brain (A: all groups; B: all groups except the positive control), liver (C: all groups; D: all groups except the positive control), and spleen (E: all groups) are provided. The most significant virus clearance was observed in the pV-N13 and pV-N13 plus pCD24 groups. All of data are shown as mean ± SD.