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Immune responses in mice induced by multi-epitope DNA vaccine and protein vaccine of Crimean-Congo Hemorrhagic Fever Virus

Crimean-Congo Hemorrhagic Fever (CCHF), caused by the CCHF virus (CCHFV), is a severe tick borne zoonosis widely distributed in over 30 countries and regions. Currently, there is no licensed vaccine available for CCHF in China. To evaluate the cellular and humoral immune responses induced by multi-epitope DNA and protein vaccine of CCHF in BALB/c mice, a multi-epitope gene (MEPX) segment with tandem including six highly conservative and immunedominant B cell epitopes was designed based on the analysis of hydrophilicity and antigenic determinant sites in amino acid sequences of nucleoprotein and glycoprotein from CCHFV strain YL04057. The single and double-copy multi-epitope gene (MEPX and MEPX2) were respectively cloned into the eukaryotic expression vector pVAX I to construct the recombinant (r) plasmid pVAX-MEPX and pVAX-MEPX2 as DNA vaccines. The results of immunofluorescence in vitro showed that the pVAX-MEPX and pVAX-MEPX2 could be expressed in 293T cells. The recombinant prokaryotic plasmid pET-32a-MEPX and pET-32a-MEPX2 constructed previously were transformed them into E. coli BL21 (DE3), and recombinant multi-epitope proteins (rMEPX and rMEPX2) were obtained and purificated by Nickel affinity chromatography. Western blot results showed that rMEPX and rMEPX2 had good antigenicity. BALB/c mice were immunized with DNA vaccine alone, protein vaccine alone, and DNA prime followed by recombinant protein boost immunization strategy, respectively. After three immunizations, MTT assay, cytokine content assay, and ELISA assay for antibody titers were used to evaluate the immune response. The proliferation of mouse specific T lymphocytes in the enhanced by pVAX-MEPX2 combined with rMEPX2 boosting group was significant, and the expression levels of serum IFN-γ and IL-4 in mice were as high as 118.67 pg/mL and 135.33 pg/mL with significant difference compared to the control group (p<0.01), and serum antibody titer could reach up to 4.1×105. Double-copy multi-epitope vaccines groups (pVAX-MEPX2+ rMEPX2) generated better cellular and humoral immune responses by DNA prime-protein vaccine boost combinatorial immunization. This result could lay the foundation for the development of CCHFV multi-epitope vaccine candidates.


Introduction
Crimean-Congo Hemorrhagic Fever (CCHF) is an acute tick-borne zoonosis, which is caused by the Crimean-Congo Hemorrhagic Fever Virus (CCHFV). This virus is a member of the genus Nairovirus in the family Bunyaviridae [1]. It has been reported that 5 -40% of patients show severe bleeding, resulting in shock and even death [1]. In China, the first case of CCHF was reported in 1965. Thus, this disease is also known as Xinjiang Hemorrhagic Fever (XHF). At present, the virus strains have been isolated from patients in the Bachu region of Xinjiang, where it is considered to be the region with the highest incidence of CCHF in China [1][2][3]. Because of the high virulence and high pathogenicity of CCHFV, this research needs to be conducted in the high-level laboratories. As a result, the suitable animal model was lacked, which limits the in-depth study of effective diagnostic, prevention, and therapeutic methods for CCHF [4]. So far, there is no licensed vaccine available for CCHF in China [5].
New generation vaccines, such as recombinant subunit proteins and DNA vaccines, are gradually replacing the traditional vaccines [6,7]. For multi-epitope-based vaccines, the conserved immunedominant and neutralizing epitopes are typically selected for tandem expression, thereby reducing the interference and inhibition of extraneous sequences [8,9]. Because of its low cost, easy operation, high safety, and highly effective protection, it has attracted wide attention [9]. It has also been reported that the vaccine that is made by a multi-epitope concatenated tandem of multiple epitope constituent is more effective than a single epitope vaccine [10,11]. Therefore, the related study on multi-epitope gene tandem and its expression activity is of great significance for the development of novel epitope vaccines for viruses.
Our group previously investigated the antigenicity of the double-copy Multi-Epitope Peptide (MEPX 2 ) formed in tandem by 6 linear B-cell epitopes on the CCHFV nucleoprotein NP and glycoproteins GP (Gn and Gc) [12,13]. In order to further understand whether the multi-epitope gene is immunogenic, in this work, we constructed a multi-epitope gene eukaryotic expression plasmid and identified its expression in vitro.
We also used DNA vaccine alone, protein vaccine alone, and DNA prime followed by recombinant protein boost immunization strategy to immunize the BALB/c mice. Their immune response was evaluated. Our results could provide the basis to develop a novel multi-epitope vaccine for CCHFV in the future.

Ethics statement
The study was approved by the Committee on the Ethics of Animal Experiments of Xinjiang Key Laboratory of Biological Resources and Genetic Engineering (BRGE-AE001), Xinjiang University.

Analysis of the structure and antigen epitope of MEPX 2
SignalP4.1 and TMHHMM online analysis software were used to analyze signal peptides and transmembrane regions of the amino acid sequence of MEPX 2 . SignalP 4.1 server predicts the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms. The method incorporates a prediction of cleavage sites and a signal peptide/non-signal peptide prediction based on a combination of several artificial neural networks. DNAStar-Protean software was used to predict the possible BCEs on MEPX 2 . The prediction of secondary structure was based on Garnier and Robson [14], Chou and Fasman. [15] The hydrophilic scheme, flexible regimen, surface accessibility regimen and antigenicity index were analyzed and predicted using methods of the Kyte-Doolittle [16], Karplus-Schulz [17], Emini and Jameson-Wolf [18], respectively. Based on the results obtained from these analyses, peptides with good hydrophilicity, high accessibility, high flexibility and strong antigenicity were selected as epitope candidates.

Plasmids and antibodies
pVAX-Gc-NP2, pET-32a-MEPX, pET-32a-MEPX 2 previously constructed were stored by this research group [13,19]. Both healthy and CCHFV-infected sheep serum samples were provided by Professor Yujiang Zhang of Xinjiang Centers for Disease Control and Prevention. Serum samples of sheep infected with CCHFV were previously identified using indirect immunofluorescent assays (IFAs) and reverse transcription polymerase chain reactions (RT-PCRs) [20].

Construction of eukaryotic expression plasmids
In our previous study, a multi-epitope gene (MEPX) segment with tandem including six highly conservative and dominant B cell epitopes (NP 245-259 , NP 265-273 , Gn 39-58 , Gn 119-154 , Gc 226-245 , Gc 503-530 ) was designed based on the analysis of hydrophilicity and antigenic determinant sites in amino acid sequences of nucleoprotein and glycoprotein from CCHFV strain YL04057 [13]. The epitopes in MEPX were separated from each other by a flexible peptide Gly-Gly [13]. Based on the single-copy poly-epitope gene sequence designed by Yu Huan in our group [13], we have designed a double-copy multiepitope gene sequence optimized by E. coli preference codon (Fig 1(A)). The single-copy gene sequence MEPX and the double-copy gene sequence MEPX 2 was synthesized by Jierui Bioengineering Co. Ltd. in Shanghai, and inserted into the expression plasmid pVAXI by restriction endonuclease digestion with BamHI and XhoI, respectively. After being transformed into competent Escherichia coli DH5α cells, the recombinant plasmid pVAX-MEPX, pVAX-MEPX 2 was identified using restricted enzyme analysis and sequencing (Sangon Biotech, Shanghai, China). was used as the primary antibody, and horseradish peroxidase (HRP)-conjugated goat anti-mouse/ mouse anti-goat IgG (TransGen Biotech, Beijing, China) was used as the secondary antibody. The bound antibodies were observed through incubating membranes using ECL chmilumiescence reagents, and then were exposed in the LAS 4000 Ultrasensitive Chemiluminescence.

Mouse immunization
Mice were randomly divided into 8 groups (Table 1) Freund's complete adjuvant FCA was used for primary immunization and Freund's incomplete adjuvant FIA was used for booster immunization. Group PBS were immunized i.m with an equal volume of filter-sterilized PBS as negative control group. In the group pVAX-MEPX+rMEPX, pVAX-MEPX 2 +rMEPX 2 as the DNA prime-protein vaccine boost combinatorial immunization was administered: the primary immunization with plasmid DNA was performed, and then 14 d and 28 d after the first immunization, the recombinant protein were immunized i.m as the boosting immunization, respectively.
The serum samples were collected by the mice eyelids bleeding before the each administration and 14 d after the last immunization, followed by storage at -20 °C for antibody detection by ELISA. Splenocytes were harvested for splenocyte proliferation and cytokine assay on 14 d after final immunization. The specific immunization groups and strategy was shown in the table 1 below.

Statistical analysis
Statistical analyses were performed using GraphPad Prism version 5.0. The data was expressed as means and standard deviation. The absorbance at 450 nm of the mouse serum antibody, the cytokine IFN-γ and IL-4 levels of cell culture supernatant from the mouse spleen were analyzed using one-way ANOVA. The t-test was used to compare the mean valuesbetween groups. Differences between groups were considered significant at P < 0.05.

Structure analysis of MEPX 2 fragment and prediction of B cell antigen epitopes
Accessibility, variability, fragment mobility, charge distribution and hydrophilicity are important features of antigenic epitopes. The presence of flexible regions, such as coil and turn regions provides further evidence for epitope identification. In this study, the secondary structure of MEPX 2 was predicted using the methods of Garnier and Robson [14] and Chou and Fasman [15] based on the Gn gene sequence of CCHFV YL04057 strain ( Fig. 1(A)). The prediction results show that there is no transmembrane domain and signal peptide sequence in MEPX 2 . A hydrophilicity plot, flexibility plot, surface probability plot and antigenic index for the truncated protein were obtained using the methods of Kyte and Doolittle [16], Karplus and Schulz [17], Emini [18] and Jameson-Wolf [22], respectively. The analysis showed that the multi-epitope peptide is beneficial to the chimeric of the antibody, and contains the B-cell dominant epitope; the multiepitope peptide has strong hydrophilicity and antigenicity, and the flexible region is distributed more and can be seen in the figure. A flexible region was formed at the flexible peptide added during gene construction, indicating that the multi-epitope peptide construction strategy is correct.

Identification of recombinant plasmids pVAX-MEPX and pVAX-MEPX 2
The recombinant plasmids pVAX-MEPX and pVAX-MEPX 2 were digested by BamHI and XhoI in 1% agarose gel electrophoresis. The fulllength sequence of the MEPX gene was 400 bp, the fulllength sequence of the MEPX 2 gene was 800 bp (Fig 2(A)), consistent with the expected size. DNA sequencing confirmed that the nucleotide sequences of MEPX and MEPX 2 in the recombinant plasmid were accurate.

Expression and purification and Western blot identification of recombinant polyepitope protein
SDS-PAGE results showed that rMEPX and rMEPX 2 can be expressed in the supernatant (Fig 2 (B)), and can also be effectively purified using gradient elutes with imidazole (Fig 4). The protein purity can reach above 95%. Bands at approximately 34 kD and 49 kD clearly present in the Western Blot (Fig 4), indicating that the expressed rMEPX and rMEPX 2 could specifically bind to the anti-His mouse monoclonal antibody.
This result suggests that the recombinant protein rMEPX and rMEPX 2 was expressed correctly, and recognized specifically by positive sheep serum against CCHFV antibodies, indicating that rMEPX and rMEPX 2 have strong antigenicity.

Indirect ELISA detection of serum antibody levels in mice in different immunization groups
Indirect ELISA results showed that the IgG levels in the experimental groups were significantly higher than those in the control groups (Fig 5). ELISA data also showed that the experimental groups can effectively induce the humoral immune response after 14, 28, and 42 days. While the IgG levels in groups pVAX-MEPX and pVAX-MEPX 2 were lower than those in groups rMEPX and rMEPX 2 within 42 days, there is no significant difference in IgG levels among these groups after immunization of 42 days. The IgG level induced in group pVAX-MEPXrMEPX showed the highest level was reached at 42 days after immunization, and significantly higher than those of separate groups of DNA alone and protein alone (P < 0.01). After 42 days of immunization, the antibody titers in the mice serum immunized with pVAX-MEPX, pVAX-MEPX 2 , pVAXI, rMEPX, rMEPX 2 , PBS and co-immunization pVAX-MEPX 2 +rMEPX 2 were 1:1600, 1:3200, 1:102400, 1:204800, 1: 204800 and 1:409600, respectively. The antibody titer of the rMEPX and rMEPX 2 groups was 64 fold higher than that of the pVAX-MEPX and pVAX-MEPX 2 groups.

Lymphocyte proliferation reaction
In order to analyze the proliferation of mouse spleen T cells stimulated by each immunization group, the spleen was aseptically taken after 7 days of the third immunization. The proliferation of splenic T lymphocytes was analyzed using MTT assay. Fig. 6 exhibited that the stimulation index of DNA prime-protein vaccine boost combinatorial immunization group (pVAX-MEPX+rMEPX and pVAX-MEPX 2 +rMEPX 2 ) was significantly higher than protein immunization alone, and the index is slightly higher than DNA immunization. The difference between pVAX-MEPX+rMEPX and pVAX-MEPX 2 +rMEPX 2 group was extremely significant (P < 0.01). The stimulation index of DNA immunization group (pVAX-MEPX and pVAX-MEPX 2 group) was significantly higher than that of protein immunization group ( rMEPX and rMEPX 2 group) (P < 0.01). In contrast, there is no significant difference in stimulation index between pVAX-MEPX group and rMEPX 2 group.

The detection of IL-4 and IFN-γ cytokine levels
After 7 days of the last immunization, the expression of IFN-γ and IL-4 in the supernatant of the mouse spleen T lymphocyte culture was examined. The difference between the treatment groups is statistically significant (P < 0.05). The IFN-γ level in the DNA alone group (pVAX-MEPX and pVAX-MEPX 2 group) was higher than that in the protein alone group (rMEPX and rMEPX 2 group), while the IFN-γ level in the pVAX-MEPX 2 group combined with the rMEPX 2 group was the highest (119.375±10.284 pg/mL), which was slightly higher than the pVAX-MEPX combined rMEPX group (102.708 ± 6.437 pg/mL).
In terms of IL-4 levels, the protein alone immunization group (rMEPX and rMEPX 2 group) was higher than the DNA alone immunization group (pVAX-MEPX and pVAX-MEPX 2 group), and the DNA-protein combination group (pVAX-MEPX+rMEPX and pVAX-MEPX 2 +rMEPX 2 group) was higher than the protein alone immunization group (rMEPX and rMEPX 2 group). The IL-4 levels in the pVAX-MEPX 2 group combined with the rMEPX 2 group were significantly different from those in the rMEPX and rMEPX 2 groups (P<0.05). The highest expression IL-4 level in pVAX-MEPX 2 combined with rMEPX 2 group was 135.187±9.805 pg/mL, which is slightly higher than that of pVAX-MEPX combined with rMEPX group (119.035±7.768 pg/mL).

Discussion
The method of vaccination prevention has been successfully applied to control a variety of viral diseases, such as avian influenza, Newcastle disease and Hepatitis B [23][24][25]. However, because CCHFV is a biological safety class IV pathogen, it requests a high level of laboratory and instrument, but suitable experimental animal models were lacked.
As a result, there is currently no effective CCHF prevention, treatment, and commercially available vaccines. The multi-epitope gene-based vaccines can induce favorable and specific immune responses, and effectively avoid the side effects of other adverse epitopes in intact antigens [26]. Meanwhile, there is no risk of gene integration into host cells, largely enhancing safety [8]. Reasonable epitope design can improve immune efficacy and have the capability to focus the immune response on conserved epitopes [27]. In addition, the use of bioinformatics methods to obtain candidate epitope peptides, combined with epitope mapping to identify the fine epitopes, can facilitate the development of viral multi-epitope vaccines [12]. The use of reverse genetics technology to prepare multi-epitope gene-based vaccines provides a potential candidate tool for the CCHF prevention..
Nucleoproteins and glycoproteins are two major structural proteins of CCHFV, and play a key role in the viral infection process. Nucleoprotein NP is the main antigen that induces humoral and cellular immunity in the body [28]. The mature glycoprotein GP is very important in the process of virus infection and pathogenicity, and can induce neutralizing antibodies. Based on this, the multi-epitope vaccines design of current study includes 2 epitopes on NP2 segment, which was previously identified CCHFV YL04057 strains [19], and 4 immuno-dominant, which was highly conserved linear B-cell epitopes on glycoproteins (Gn and Gc) predicted by bioinformatics software [13]. These epitopes were used to construct the multi-epitope DNA vaccines and protein vaccines. Previous reports suggest the effect of DNA vaccine immunization alone could be limited due to the host resistance, low levels of expression, and inappropriate transfection in vivo [29,30].
The delivery of DNA vaccines by electroporation in vivo can enhance T cell responses by increasing the efficiency of antigen-specific T cells and the ability of T cells to secrete more IFN-γ [30]. In this study, the in vivo electroporation method was used as a DNA vaccine delivery method. This study was designed in view of the DNA primed protein boost protocol and Freund's adjuvant as a highly effective adjuvant to receive a strong immune response. Different experimental groups of DNA, recombinant protein alone, and DNA prime-protein vaccine boost combinatorial immunization were designed and performed. The results showed that the constructed multi-epitope DNA vaccines (pVAX-MEPX and pVAX-MEPX 2 ) generate a higher level of IFN-γ than the control group pVAXI, indicating a significant Th1-type immune response was produced [31]. The multi-epitope protein vaccines (rMEPX and rMEPX 2 ) stimulated higher levels of IL-4 compared to the PBS control group, suggesting a significant Th2-type immune response [32]. However, the DNA prime-protein vaccine boost combinatorial immunization group stimulated Th1 and Th2-type immune responses, were higher than the DNA and protein alone group, which is in good agreement with the results of Memarnejadian A et al. [33].
From the perspective of elicited antibody level, pVAX-MEPX combined with rMEPX and pVAX-MEPX 2 combined with rMEPX 2 immunized mice produced antibody titers up to 2.05×105 and 4.1×105, respectively. This value was much higher than recombinant immunized DNA alone, and was 2-fold higher than that of recombinant protein alone group. This finding confirms that the DNA prime-protein boost strategy can enhance antibody levels, which is consistent with Liang's study result [34]. The mechanism may be enhanced by the stimulation of antigen-specific memory B cells, followed by the protein immune coordination through the process of DNA primary immunization [35].
Meanwhile, the prime-boost immunization strategy amplifies selective B or T cells with greater affinity, increasing the number of memory cells specific for common antigens [36]. We found that the levels of both cellular and humoral immune responses produced by mice immunized with rMEPX 2 and pVAX-MEPX 2 combined with double-copy multiepitope recombinant DNA (pVAX-MEPX 2 ) and pVAX-MEPX 2 were significantly higher than those of pVAX-MEPX, rMEPX and pVAX-MEPX combined with rMEPX. This result also revealed that high epitope densities can induce the production of high-affinity antibodies. Yan Xuhua et al. [37] found that the increase in the number of epitope repeats can enhance the recognition and interaction of B-cell surface antibody with multi-epitope peptides. Our previous study also confirmed that the double-copy multi-epitope peptide has better antigenicity [13]. However, since CCHFV is a highly pathogenic virus and requires a high level of safety laboratory, there is no cell model and animal model to detect CCHFV in China. Therefore, this study did not further detect whether the antibodies induced by immunized mice have neutralization and protection effects.
In conclusion, our results revealed that the multi-epitope DNA vaccine and recombinant multi-epitope protein vaccine could stimulate mice to produce specific humoral and cellular immune responses. Double-copy multi-epitope vaccines generated better cellular and humoral immune responses by DNA prime-protein vaccine boost combinatorial immunization strategy. The pVAX-MEPX 2 gene prime followed by boosting recombinant protein rMEPX 2 immunization induced the strongest antigenspecific immune effect, would be expected to serve as vaccine candidates for the prevention and control of CCHFV. Further investigation by establishing the CCHFV animal model is needed to clarify whether the antibodies have neutralizing activity and neutralization protection.