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Background: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies. Methods: We designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen. Results: The optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation. Conclusions: The purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen's suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment. [ABSTRACT FROM AUTHOR]

Crimean‐Congo hemorrhagic fever orthonairovirus (CCHFV) is listed as a priority pathogen by the World Health Organization due to the severity of disease, propensity for spread to nonendemic regions, and absence of a vaccine or specific treatment. The immune correlates of protection are not clearly defined and hence the importance of investigating host immune responses in survivors. We have previously shown that survivors generate memory T cell responses that are long‐lived and this study aimed to further define specific viral proteins targeted by the T cell response. The NSM, GP38, highly variable mucin‐like domain, and N‐terminus of GC regions in CCHFV are considered immunogenic regions and were investigated using peptide libraries representing regions of interest. An interferon gamma ELISpot assay was used to identify responses in peripheral blood mononuclear cells isolated from 12 survivors of laboratory confirmed CCHFV infections. IFN‐γ responses were detected from eight survivors, against nine peptides, including four peptides located in the NSM region and five peptides located in the GP38 protein. No response was detected against peptides representing the mucin‐like domain. In conclusion, the results suggest the presence of a long‐lasting T cell memory response upon stimulation with viral epitopes in survivors of infection. [ABSTRACT FROM AUTHOR]

Crimean-Congo hemorrhagic fever virus (CCHFV) causes severe acute human disease with lethal outcome. The knowledge about the immune response for this human health threat is highly limited. In this study, we have screened the glycoprotein of CCHFV for novel linear B-cell epitopic regions using a microarray approach. The peptide library consisted of 168 synthesized 20mer peptides with 10 amino acid overlap covering the entire glycoprotein. Using both pooled and individual human sera from survivors of CCHF disease in Turkey five peptide epitopes situated in the mucin-like region and GP 38 (G15-515) and GN G516-1037 region of the glycoprotein were identified as epitopes for a CCHF immune response. An epitope walk of the five peptides revealed a peptide sequence located in the GN region with high specificity and sensitivity. This peptide sequence, and a sequence downstream, reacted also against sera from survivors of CCHF disease in South Africa. The cross reactivity of these peptides with samples from a geographically distinct region where genetically diverse strains of the virus circulate, enabled the identification of unique peptide epitopes from the CCHF glycoprotein that could have application in development of diagnostic tools. In this study clinical samples from geographically distinct regions were used to identify conserved linear epitopic regions of the glycoprotein of CCHF. [ABSTRACT FROM AUTHOR]

Crimean–Congo hemorrhagic fever virus (CCHFV) infrequently causes hemorrhagic fever in humans with a case fatality rate of 30%. Currently, there is neither an internationally approved antiviral drug nor a vaccine against the virus. A replicon based on the Sindbis virus vector encoding the complete open reading frame of a CCHFV nucleoprotein from a South African isolate was prepared and investigated as a possible candidate vaccine. The transcription of CCHFV RNA and recombinant protein production by the replicon were characterized in transfected baby hamster kidney cells. A replicon encoding CCHFV nucleoprotein inserted in plasmid DNA, pSinCCHF-52S, directed transcription of CCHFV RNA in the transfected cells. NIH-III heterozygous mice immunized with pSinCCHF-52S generated CCHFV IgG specific antibodies with notably higher levels of IgG2a compared to IgG1. Splenocytes from mice immunized with pSinCCHF-52S secreted IFN-γ and IL-2, low levels of IL-6 or IL-10, and no IL-4. No specific cytokine production was registered in splenocytes of mock-immunized mice (p < 0.05). Thus, our study demonstrated the expression of CCHFV nucleoprotein by a Sindbis virus vector and its immunogenicity in mice. The spectrum of cytokine production and antibody profile indicated predominantly Th1-type of an anti-CCHFV immune response. Further studies in CCHFV-susceptible animals are necessary to determine whether the induced immune response is protective. [ABSTRACT FROM AUTHOR]