Cross-Reactive anti-Nucleocapsid Protein Immunity against Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus in Multiple Species

Abstract

The World Health Organization estimates that there may be three billion people at risk of infection by Crimean-Congo Hemorrhagic Fever Virus (CCHFV), a highly lethal, emerging orthonairovirus carried by ticks. On the other hand, the closely related Hazara virus (HAZV), a member of the same serogroup, has not been reported as a pathogen for humans. Given the structural and phylogenetic similarities between these two viruses, we evaluated the immunological similarities of the nucleocapsid protein (NP) of these two viruses in multiple species. Strong antigenic similarities were demonstrated in anti-NP humoral immune responses against HAZV and CCHFV in multiple species using convalescent human CCHF sera, rabbit and mouse polyclonal antiserum raised against CCHFV, and mouse polyclonal antiserum against CCHFV-NP in enzyme immunoassays. We also report a convincing cross-reactivity between NPs in Western blots using HAZV-infected cell lysate as antigen and inactivated CCHFV and CCHFV-NP-immunized mice sera. These results suggest that NPs of HAZV and CCHFV share significant similarities in humoral responses across species and underline the potential utility of HAZV as a surrogate model for CCHFV.IMPORTANCE CCHFV and HAZV, members of the Nairoviridae family, are transmitted to mammals by tick bites. CCHFV is considered to be a severe threat to public health and causes hemorrhagic diseases with a high mortality rate, and there are neither preventative nor therapeutic medications against CCHFV disease. HAZV, on the other hand, is not a pathogen to humans and can be studied under BSL-2 conditions. The antigenic relationship between these viruses is of interest for vaccines and for preventative investigations. Here, we demonstrate cross-reactivity in anti-NP humoral immune response between NPs of HAZV and CCHFV in multiple species. These results underline the utility of HAZV as a surrogate model to study CCHFV infection.

FIG 1

Purification of HAZV rNP by Ni-nitrilotriacetic acid (NTA) affinity chromatography. (A) Chromatogram of 6×His-tagged HAZV rNP monitored by UV 280-nm absorbance. (B) Analysis of elution fractions with Coomassie-stained 12% Tris-glycine SDS-PAGE gel. M, marker; 1 to 5, elution fractions.

FIG 2

Detection of cross-reaction by Western blotting. (A) Purified HAZV rNP was run on 12% SDS-PAGE gels and transferred to nitrocellulose membranes. CCHFV-immunized/infected mouse and human sera, CCHFV-NP-immunized mouse sera, and anti-His tag antibodies were used. +, CCHFV infected or immunized; −, healthy or naive. (B) HAZV-infected and uninfected cell lysates were separated at equal amounts on 12% polyacrylamide gels by SDS-PAGE and transferred to nitrocellulose membranes. CCHFV-immunized mouse and rabbit sera were used as primary antibodies. M, marker; +, HAZV infected; −, HAZV uninfected cell lysate.

FIG 3

Detection of cross-reaction by EIA by using CCHF convalescent-phase human sera. EIA plates were coated with 0.1 μg/well ultrapure HAZV rNP and, as a positive control, with 0.1 μg/well ultrapure CCHFV rNP and analyzed using CCHF convalescent-phase and healthy human sera. Twenty CCHFV-positive and 20 CCHFV-negative serum samples were used for both EIAs. Standard deviation (SD) was calculated based on the OD values of 18 true-negative samples, and the cutoff value was calculated with the formula 2 × SD plus mean value. (A) Results from 16 of 20 patients remained higher than the cutoff value for HAZV rNP EIA. The sensitivity and specificity of HAZV rNP EIA is 80% and 90%, respectively. (B) Results from 18 of 20 patients remained higher than the cutoff value for CCHFV rNP EIA. The sensitivity and specificity of CCHFV rNP EIA is 90%. Each data point was calculated by averaging the optical density values obtained from three independent experiments performed in duplicates at an absorbance value of 450 nm. EIA data are compared by Mann-Whitney U test (P < 0.0001).

FIG 4

Detection of cross-reaction in cross-species by EIA. (A) Plates were coated with 10 μg/well ultrapure HAZV rNP and analyzed against 200-fold-diluted CCHFV-immunized rabbit sera. (B) Plates were coated with 1 μg/well and analyzed against 100-fold-diluted CCHFV-immunized mouse sera. Unprimed rabbit and mouse sera were used at the same dilutions as for the positive controls. Each data point was calculated by averaging the optical density values obtained from three independent experiments performed in duplicates at an absorbance value of 450 nm. Standard deviation (SD) was calculated based on the OD values of negative samples, and the cutoff value was calculated with formula 2 × SD plus mean value. OD values of positive samples remained higher than the cutoff value. The cutoff values were 0.176 and 0.131 for inactive-CCHFV-immunized rabbit and mouse EIA, respectively. EIA data were compared with two-tailed unpaired t test and found to be significant.

FIG 5

Titration curve EIA using CCHFV-rNP-immunized mouse sera. (A) Plates were coated with 1 μg/well ultrapure HAZV rNP, with 1 μg/well CCHFV rNP as a positive control. (B) EIA was performed using CCHFV-rNP-immunized mouse sera. Positive and negative serum samples were serially diluted 2-fold with the initial dilution of 1/1,000, and HAZV rNP and CCHFV rNP were able to capture antibodies in sera even at a dilution of 1/32,000. Each data point was calculated by averaging the optical density values obtained from three independent experiments performed in duplicates at an absorbance value of 450 nm. Standard deviation (SD) was calculated based on the OD values of negative samples, and the cutoff value was calculated with formula 2 × SD plus mean value. OD values of positive samples in each dilution remained higher than the cutoff value. The cutoff values were 0.069 and 0.053 for HAZV rNP and CCHFV rNP EIA, respectively. EIA data were compared with two-tailed unpaired t test and found to be significant.