Immune responses in multiple hosts to Nucleocapsid Protein (NP) of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)

Abstract

In 2019, the World Health Organization declared 3 billion to be at risk of developing Crimean Congo Hemorrhagic Fever (CCHF). The causative agent of this deadly infection is CCHFV. The data related to the biology and immunology of CCHFV are rather scarce. Due to its indispensable roles in the viral life cycle, NP becomes a logical target for detailed viral immunology studies. In this study, humoral immunity to NP was investigated in CCHF survivors, as well as in immunized mice and rabbits. Abundant antibody response against NP was demonstrated both during natural infection in humans and following experimental immunizations in mice and rabbits. Also, cellular immune responses to recombinant NP (rNP) was detected in multispecies. This study represents the most comprehensive investigation on NP as an inducer of both humoral and cellular immunity in multiple hosts and proves that rNP is an excellent candidate warranting further immunological studies specifically on vaccine investigations.

Fig 1. Expression and purification of CCHFV rNP by Ni-NTA affinity chromatography.

(A) Analysis of the rNP solubility in fractions of cell extracts on Coomassie stained 12% Tris-Glycine SDS-PAGE gel after expression in BL21 (DE3) with 1 mM IPTG induction for three hours. M: marker (Cat. #24052, Intron Biotechnology), P: pellet, S: supernatant samples obtained after lysis of bacteria under native conditions. (B) Chromatogram of 6xHis-tagged CCHFV rNP monitored by UV 280 nm absorbance with ÄKTA pure chromatography through HisTrap Excel columns. (C) Analysis of elution fractions obtained from after Ni-NTA Column Chromatography on Coomassie stained 12% Tris-Glycine SDS-PAGE gel. M: marker (Cat. #24052, Intron Biotechnology), 1–8: Elution fractions. D) Western blot analysis of purified rNP using goat polyclonal anti His tag-HRP at 1:1000 dilution.

Fig 2. Analysis of the authenticity of rNP.

Purified rNP ran on 12% polyacrylamide gel by SDS-PAGE and transferred on nitrocellulose membranes. Western blot analysis performed with serum samples from different species that have anti-CCHFV antibodies (CCHFV survivors and iCCHFV immunized rabbits) or anti-NP antibodies (rNP immunized mice samples and anti-rNP mAb; 2D3). Except for rabbit serum (1:10), all primary antibodies were prepared at 1:1.000 final dilution in blocking buffer. (+): CCHFV infected or immunized/rNP immunized; (-): mock immunized rabbit or mice, or healthy human sera.

Fig 3. Analysis of antigenic fidelity of recombinant NP by EIA.

EIA plates were coated either with rNP (1μg/well) or whole viral antigen (1μg/well) and analyzed using sera, collected from a mouse pre- and post-immunization with inactive CCHFV at 1:1000 final dilution. The NP specific antibodies were detected with HRP-conjugated anti-mouse IgG antibody. Each sample was tested in duplicate and measured at the absorbance value of 450 nm, and the bars represent the mean value of samples. Each data represents mean ± SEM. Pre- and post- immunization data for each group was statistically analyzed separately by Welch’s t-test. **** p< 0.0001.

Fig 4. Evaluation of NP-specific antibody response using CCHF convalescent sera.

A serum panel consisting of 28 CCHFV-IgG positive and 23 CCHFV-IgG-negative serum samples were analyzed by in-house CCHFV rNP based IgG EIA. The plates were coated with rNP (1μg/well), serum samples were used at 1:100 final dilution and detected with HRP-conjugated anti-mouse IgG antibody. The mean optical density obtained from anti-CCHFV IgG negative human sera samples was 0.070 and standard deviation was 0.030. The difference of optical densities between anti-CCHFV IgG antibody positive and negative sera groups was analyzed by Mann-Whitney U test and found statistically significant. (p< 0,0001). The cut-off value was calculated as follows: 2SD + mean absorbance of each CCHFV negative samples. Each data represents mean ± SEM. Each data was calculated by averaging the optical density values obtained from three independent experiments performed in duplicates at an absorbance value of 450 nm. Gray bars represent CCHFV antibody negative, black bars represent CCHFV positive human sera.

Fig 5. Detection of NP-specific IgG antibodies in multiple hosts.

The endpoint NP-specific IgG titers in sera samples from different species were determined with EIA in which rNP (1μg/well) was utilized as a solid phase antigen. Serum samples were serially diluted in a range of 1:100 to 1:32000 for mice sera, 1:100 to 1:16000 for human sera, and 1:10 to 1:6400 for rabbit sera and incubated with the relevant HRP-conjugated secondary antibodies. To detect the endpoint IgG titers for each species, cut-off values were calculated by using the corresponding dilution of the related negative sample. The cut-off values were determined as follows: 2SD + mean absorbance of each negative sample. Each data represents mean ± SEM, calculated by averaging the optical density values measured from three independent experiments performed in duplicates at an absorbance value of 415 nm. iCCHFV: Inactivated CCHFV.

Fig 6. rNP-elicited footpad swelling in mice.

Mice were immunized with rNP as test antigen or with BSA and PBS as positive and negative controls, respectively as described. One week after last immunization, DTH reactions in the footpad were induced by a subcutaneous rNP (50 μg in final), BSA (50 μg in final) or PBS challenge. The thickness of footpads was measured before and 24, 48 and 72 hours after challenge by electronic caliper gauge. Each bar represents mean values ± SEM of each immunization group (The data were from 5 mice per group). PBS and BSA were utilized as a negative and positive control, respectively. The differences of mean footpad thickness of mice compared to corresponding negative controls were statistically significant (**, p < 0.01; ***, p< 0.001; ****, p< 0.0001; and ns, not significant). Two-way ANOVA and Bonferroni’s multiple comparison tests were used for statistical analysis between all groups at the same time point. ΔFootpad Swelling: The difference in the thickness of antigen injected and PBS injected footpad, BSA: Bovine serum albumin, NP: nucleoprotein, PBS: Phospate buffered saline.

Fig 7. Measurement of cytokines released into CCHFV rNP stimulated mice lymphocytes medium.

The media of combined lymphocyte and splenocytes cultures of mice containing secreted cytokines were used to detect different cytokine levels. Levels of cytokines were quantified by multi-analyte ELISA. The absorption was measured at 450 nm by the spectrophotometer. Absorbance values more than two times the corresponding negative control absorbance values are interpreted as positive samples according to manufacturers’ instructions and indicated with an asterisks (*). rNP: mice immunized with rNP, NC: mice immunized with PBS as a negative control group.