No detection of tick-borne encephalitis virus RNA in blood, urine or saliva of hospitalised immunocompetent tick-borne encephalitis patients.

Tick-borne encephalitis (TBE) is usually diagnosed based on the presence of TBE virus (TBEV)-specific IgM and IgG antibodies in serum. However, antibodies induced by vaccination or cross-reactivity to previous flavivirus infections may result in false positive TBEV serology. Detection of TBEV RNA may be an alternative diagnostic approach to detect viral presence and circumvent the diagnostic difficulties present when using serology. Viral RNA in blood is commonly detectable only in the first viremic phase usually lasting up to two weeks, and not in the second neurologic phase, when the patients contact the health care system and undergo diagnostic work-up. TBEV RNA has previously been detected in urine in a few retrospective TBE cases in the neurologic phase, and furthermore RNA of other flaviviruses has been detected in patient saliva. In this study, blood, saliva and urine were collected from 31 hospitalised immunocompetent patients with pleocytosis and symptoms of aseptic meningitis and/or encephalitis, suspected to have TBE. We wanted to pursue if molecular testing of TBEV RNA in these patient materials may be useful in the diagnostics. Eleven of the 31 study patients were diagnosed with TBE based on ELISA detection of TBEV specific IgG and IgM antibodies. None of the study patients had TBEV RNA detectable in any of the collected patient material.

Influence of adjuvant type and route of administration on the immunogenicity of Leishmania-derived tick-borne encephalitis virus-like particles - A recombinant vaccine candidate.

Tick-borne encephalitis virus (TBEV) is a tick-borne flavivirus that induces severe central nervous system disorders. It has recently raised concerns due to an expanding geographical range and increasing infection rates. Existing vaccines, though effective, face low coverage rates in numerous TBEV endemic regions. Our previous work demonstrated the immunogenicity and full protection afforded by a TBEV vaccine based on virus-like particles (VLPs) produced in Leishmania tarentolae cells in immunization studies in a mouse model. In the present study, we explored the impact of adjuvants (AddaS03™, Alhydrogel®+MPLA) and administration routes (subcutaneous, intramuscular) on the immune response. Adjuvanted groups exhibited significantly enhanced antibody responses, higher avidity, and more balanced Th1/Th2 response. IFN-γ responses depended on the adjuvant type, while antibody levels were influenced by both adjuvant and administration routes. The combination of Leishmania-derived TBEV VLPs with Alhydrogel® and MPLA via intramuscular administration emerged as a highly promising prophylactic vaccine candidate, eliciting a robust, balanced immune response with substantial neutralization potential.

Exposure of cattle to tick-borne encephalitis virus in the historical endemic zone in north-eastern France.

BACKGROUND: Tick-borne encephalitis (TBE) is a severe human neuroinfection caused by TBE virus (TBEV). TBEV is transmitted by tick bites and by the consumption of unpasteurized dairy products from infected asymptomatic ruminants. In France, several food-borne transmission events have been reported since 2020, raising the question of the level of exposure of domestic ungulates to TBEV. In this study, our objectives were (i) to estimate TBEV seroprevalence and quantify antibodies titres in cattle in the historical endemic area of TBEV in France using the micro virus neutralisation test (MNT) and (ii) to compare the performance of two veterinary cELISA kits with MNT for detecting anti-TBEV antibodies in cattle in various epidemiological contexts. A total of 344 cattle sera from four grid cells of 100 km² in Alsace-Lorraine (endemic region) and 84 from western France, assumed to be TBEV-free, were investigated. RESULTS: In Alsace-Lorraine, cattle were exposed to the virus with an overall estimated seroprevalence of 57.6% (95% CI: 52.1-62.8%, n = 344), varying locally from 29.9% (95% CI: 21.0-40.0%) to 92.1% (95% CI: 84.5-96.8%). Seroprevalence did not increase with age, with one- to three-year-old cattle being as highly exposed as older ones, suggesting a short-life duration of antibodies. The proportion of sera with MNT titres lower than 1:40 per grid cell decreased with increased seroprevalence. Both cELISA kits showed high specificity (> 90%) and low sensitivity (less than 78.1%) compared with MNT. Sensitivity was lower for sera with neutralising antibodies titres below 1:40, suggesting that sensitivity of these tests varied with local virus circulation intensity. CONCLUSIONS: Our results highlight that cattle were highly exposed to TBEV. Screening strategy and serological tests should be carefully chosen according to the purpose of the serological study and with regard to the limitations of each method.

Human antibodies in Mexico and Brazil neutralizing tick-borne flaviviruses.

Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are spread by mosquitoes and cause human disease and mortality in tropical areas. In contrast, Powassan virus (POWV), which causes severe neurologic illness, is a flavivirus transmitted by ticks in temperate regions of the Northern hemisphere. We find serologic neutralizing activity against POWV in individuals living in Mexico and Brazil. Monoclonal antibodies P002 and P003, which were derived from a resident of Mexico (where POWV is not reported), neutralize POWV lineage I by recognizing an epitope on the virus envelope domain III (EDIII) that is shared with a broad range of tick- and mosquito-borne flaviviruses. Our findings raise the possibility that POWV, or a flavivirus closely related to it, infects humans in the tropics.

A combined cross-sectional analysis and case-control study evaluating tick-borne encephalitis vaccination coverage, disease and vaccine effectiveness in children and adolescents, Switzerland, 2005 to 2022.

BackgroundTick-borne encephalitis (TBE) is a severe, vaccine-preventable viral infection of the central nervous system. Symptoms are generally milder in children and adolescents than in adults, though severe disease does occur. A better understanding of the disease burden and duration of vaccine-mediated protection is important for vaccination recommendations.AimTo estimate TBE vaccination coverage, disease severity and vaccine effectiveness (VE) among individuals aged 0-17 years in Switzerland.MethodsVaccination coverage between 2005 and 2022 was estimated using the Swiss National Vaccination Coverage Survey (SNVCS), a nationwide, repeated cross-sectional study assessing vaccine uptake. Incidence and severity of TBE between 2005 and 2022 were determined using data from the Swiss disease surveillance system and VE was calculated using a case-control analysis, matching TBE cases with SNVCS controls.ResultsOver the study period, vaccination coverage increased substantially, from 4.8% (95% confidence interval (CI): 4.1-5.5%) to 50.1% (95% CI: 48.3-52.0%). Reported clinical symptoms in TBE cases were similar irrespective of age. Neurological involvement was less likely in incompletely (1-2 doses) and completely (≥ 3 doses) vaccinated cases compared with unvaccinated ones. For incomplete vaccination, VE was 66.2% (95% CI: 42.3-80.2), whereas VE for complete vaccination was 90.8% (95% CI: 87.7-96.4). Vaccine effectiveness remained high, 83.9% (95% CI: 69.0-91.7) up to 10 years since last vaccination.ConclusionsEven children younger than 5 years can experience severe TBE. Incomplete and complete vaccination protect against neurological manifestations of the disease. Complete vaccination offers durable protection up to 10 years against TBE.

Severe Tick-Borne Encephalitis (TBE) in a Patient with X-Linked Agammaglobulinemia; Treatment with TBE Virus IgG Positive Plasma, Clinical Outcome and T Cell Responses.

PURPOSE: A patient with X-linked agammaglobulinemia (XLA) and severe tick-borne encephalitis (TBE) was treated with TBE virus (TBEV) IgG positive plasma. The patient's clinical response, humoral and cellular immune responses were characterized pre- and post-infection. METHODS: ELISA and neutralisation assays were performed on sera and TBEV PCR assay on sera and cerebrospinal fluid. T cell assays were conducted on peripheral blood the patient and five healthy vaccinated controls. RESULTS: The patient was admitted to the hospital with headache and fever. He was not vaccinated against TBE but receiving subcutaneous IgG-replacement therapy (IGRT). TBEV IgG antibodies were low-level positive (due to scIGRT), but the TBEV IgM and TBEV neutralisation tests were negative. During hospitalisation his clinical condition deteriorated (Glasgow coma scale 3/15) and he was treated in the ICU with corticosteroids and external ventricular drainage. He was then treated with plasma containing TBEV IgG without apparent side effects. His symptoms improved within a few days and the TBEV neutralisation test converted to positive. Robust CD8+ T cell responses were observed at three and 18-months post-infection, in the absence of B cells. This was confirmed by tetramers specific for TBEV. CONCLUSION: TBEV IgG-positive plasma given to an XLA patient with TBE without evident adverse reactions may have contributed to a positive clinical outcome. Similar approaches could offer a promising foundation for researching therapeutic options for patients with humoral immunodeficiencies. Importantly, a robust CD8+ T cell response was observed after infection despite the lack of B cells and indicates that these patients can clear acute viral infections and could benefit from future vaccination programs.

Phase 3 immunogenicity and safety study of a tick-borne encephalitis vaccine in healthy Japanese participants 1 year of age and older.

BACKGROUND: Tick-borne encephalitis (TBE) virus infects the central nervous system and may lead to severe neurological complications or death. This study assessed immunogenicity, safety, and tolerability of TBE vaccine in Japanese participants 1 year of age and older. METHODS: This phase 3, multicenter, single-arm, open-label study was conducted in Japanese adult (≥ 16 years) and pediatric (1-< 16 years) populations. Participants received a single 0.5-mL (adult) or 0.25-mL (pediatric) dose of TBE vaccine at each of 3 visits. The primary endpoint was the proportion of participants who were seropositive (neutralization test [NT] titer ≥ 1:10) 4 weeks after Dose 3. Secondary and exploratory endpoints included NT seropositivity rates 4 weeks after Dose 2, immunoglobulin G (IgG) seropositivity 4 weeks after Doses 2 and 3, NT geometric mean titers (GMTs), IgG geometric mean concentrations (GMCs), and geometric mean fold rises. Primary safety endpoints were frequencies of local reactions, systemic events, adverse events (AEs), and serious AEs. RESULTS: Among 100 adult and 65 pediatric participants, 99.0 % and 100.0 % completed the study, respectively. NT seropositivity was achieved in 98.0 % adult and 100.0 % pediatric participants after Dose 3; seropositivity after Dose 2 was 93.0 % and 92.3 %, respectively. In both age groups, IgG seropositivity was ≥ 90.0 % and ≥ 96.0 % after Doses 2 and 3, respectively; GMTs and GMCs were highest 4 weeks after Dose 3. Reactogenicity events were generally mild to moderate in severity and short-lived. AEs were reported by 15.0 % (adult) and 43.1 % (pediatric) of participants. No life-threatening AEs, AEs leading to discontinuation, immediate AEs, related AEs, or deaths were reported. No serious AEs were considered related to TBE vaccine. CONCLUSIONS: TBE vaccine elicited robust immune responses in Japanese participants 1 year of age and older. The 3-dose regimen was safe and well tolerated, and findings were consistent with the known safety profile of this TBE vaccine. CLINICALTRIALS: gov: NCT04648241.

Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Identification of Host Factors Differentially Induced by Clinically Diverse Strains of Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) is an important human arthropod-borne virus that causes tick-borne encephalitis (TBE) in humans. TBEV acutely infects the central nervous system (CNS), leading to neurological symptoms of various severity. No therapeutics are currently available for TBEV-associated disease. Virus strains of various pathogenicity have been described, although the basis of their diverse clinical outcome remains undefined. Work with infectious TBEV requires high-level biocontainment, meaning model systems that can recapitulate the virus life cycle are highly sought. Here, we report the generation of a self-replicating, noninfectious TBEV replicon used to study properties of high (Hypr) and low (Vs) pathogenic TBEV isolates. Using a Spinach2 RNA aptamer and luciferase reporter system, we perform the first direct comparison of Hypr and Vs in cell culture. Infectious wild-type (WT) viruses and chimeras of the nonstructural proteins 3 (NS3) and 5 (NS5) were investigated in parallel to validate the replicon data. We show that Hypr replicates to higher levels than Vs in mammalian cells, but not in arthropod cells, and that the basis of these differences map to the NS5 region, encoding the methyltransferase and RNA polymerase. For both Hypr and Vs strains, NS5 and the viral genome localized to intracellular structures typical of positive-strand RNA viruses. Hypr was associated with significant activation of IRF-3, caspase-3, and caspase-8, while Vs activated Akt, affording protection against caspase-mediated apoptosis. Higher activation of stress-granule proteins TIAR and G3BPI were an additional early feature of Vs but not for Hypr. These findings highlight novel host cell responses driven by NS5 that may dictate the differential clinical characteristics of TBEV strains. This highlights the utility of the TBEV replicons for further virological characterization and antiviral drug screening. IMPORTANCE Tick-borne encephalitis virus (TBEV) is an emerging virus of the flavivirus family that is spread by ticks and causes neurological disease of various severity. No specific therapeutic treatments are available for TBE, and control in areas of endemicity is limited to vaccination. The pathology of TBEV ranges from mild to fatal, depending on the virus genotype. Characterization of TBEV isolates is challenging due to the requirement for high-containment facilities. Here, we described the construction of novel TBEV replicons that permit a molecular comparison of TBEV isolates of high and low pathogenicity.

Balanced T and B cell responses are required for immune protection against Powassan virus in virus-like particle vaccination.

Powassan virus (POWV) is a tick-borne pathogen for which humans are an incidental host. POWV infection can be fatal or result in long-term neurological sequelae; however, there are no approved vaccinations for POWV. Integral to efficacious vaccine development is the identification of correlates of protection, which we accomplished in this study by utilizing a murine model of POWV infection. Using POWV lethal and sub-lethal challenge models, we show that (1) robust B and T cell responses are necessary for immune protection, (2) POWV lethality can be attributed to both viral- and host-mediated drivers of disease, and (3) knowledge of the immune correlates of protection against POWV can be applied in a virus-like particle (VLP)-based vaccination approach that provides protection from lethal POWV challenge. Identification of these immune protection factors is significant as it will aid in the rational design of POWV vaccines.

Tick-borne encephalitis nonstructural protein NS1 expressed in E. coli retains immunological properties of the native protein.

There is evidence that flaviviral NS1 glycoprotein plays an important role in the pathology of tick-borne encephalitis (TBE) and NS1-specific antibodies are detected in the blood of patients with TBE. This makes NS1 a good target for the development of therapeutic inhibitors and NS1 could be an important biomarker for the early diagnosis of TBE in vaccinated individuals. Eukaryotic expression systems are mainly used to produce recombinant tick-borne encephalitis virus (TBEV) NS1. The expression of TBEV NS1 proteins in eukaryotic cells was successful, but there were some limitations. Several attempts have also been made to obtain the NS1 protein in Escherichia coli cells; however, they were unsuccessful due to the low solubility of the recombinant protein and improper folding. In this study, using Trx-tag as a fusion partner, soluble Trx-fused TBEV NS1 protein was first produced in the E. coli BL21 strain. In addition, insoluble Trx-fused TBEV NS1 protein was obtained when cultivation conditions were changed to increase the productivity. The insoluble TBEV NS1 obtained from inclusion bodies was solubilized using chaotropic reagents and successfully refolded using dialysis. Both soluble variant and successfully refolded from inclusion bodies variant showed immunological properties similar to the native TBEV NS1 protein and were recognized by specific monoclonal antibodies (mAbs), immune ascetic fluid in ELISA, western blot, and competitive analysis.

No neutralizing effect of pre-existing tick-borne encephalitis virus antibodies against severe acute respiratory syndrome coronavirus-2: a prospective healthcare worker study.

Certain immunizations including vaccination against tick-borne encephalitis virus (TBEV) have been suggested to confer cross-protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Within a prospective healthcare worker (HCW) cohort, we assessed the potentially protective role of anti-TBEV antibodies against SARS-CoV-2 infection. Among 3352 HCW, those with ≥ 1 previous TBEV vaccination (n = 2018, 60%) showed a reduced risk of SARS-CoV-2 seroconversion (adjusted odds ratio: 0.8, 95% CI: 0.7-1.0, P = 0.02). However, laboratory testing of a subgroup of 26 baseline and follow-up samples did not demonstrate any neutralizing effect of anti-TBEV antibodies against SARS-CoV-2 in live-virus neutralization assay. However, we observed significantly higher anti-TBEV antibody titers in follow-up samples of participants with previous TBEV vaccination compared to baseline, both TBEV neutralizing (p = 0.001) and total IgG (P < 0.0001), irrespective of SARS-CoV-2 serostatus. Based on these data, we conclude that the observed association of previous TBEV vaccination and reduced risk of SARS-CoV-2 infection is likely due to residual confounding factors. The increase in TBEV follow-up antibody titers can be explained by natural TBEV exposure or potential non-specific immune activation upon exposure to various pathogens, including SARS-CoV-2. We believe that these findings, although negative, contribute to the current knowledge on potential cross-immunity against SARS-CoV-2 from previous immunizations.

A pigtailed macaque model of Kyasanur Forest disease virus and Alkhurma hemorrhagic disease virus pathogenesis.

Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.

West Nile Virus and Tick-Borne Encephalitis Virus Are Endemic in Equids in Eastern Austria.

The emergence of West Nile virus (WNV) and Usutu virus (USUV) in addition to the autochthonous tick-borne encephalitis virus (TBEV) in Europe causes rising concern for public and animal health. The first equine case of West Nile neuroinvasive disease in Austria was diagnosed in 2016. As a consequence, a cross-sectional seroprevalence study was conducted in 2017, including 348 equids from eastern Austria. Serum samples reactive by ELISA for either flavivirus immunoglobulin G or M were further analyzed with the plaque reduction neutralization test (PRNT-80) to identify the specific etiologic agent. Neutralizing antibody prevalences excluding vaccinated equids were found to be 5.3% for WNV, 15.5% for TBEV, 0% for USUV, and 1.2% for WNV from autochthonous origin. Additionally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect WNV nucleic acid in horse sera and was found to be negative in all cases. Risk factor analysis did not identify any factors significantly associated with seropositivity.

Seroepidemiologic survey of emerging vector-borne infections in South Korean forest/field workers.

With global warming and lush forest change, vector-borne infections are expected to increase in the number and diversity of agents. Since the first report of severe fever with thrombocytopenia syndrome (SFTS) in 2013, the number of reported cases has increased annually in South Korea. However, although tick-borne encephalitis virus (TBEV) was detected from ticks and wild rodents, there is no human TBE case report in South Korea. This study aimed to determine the seroprevalence of TBEV and SFTS virus (SFTSV) among forest and field workers in South Korea. From January 2017 to August 2018, a total 583 sera were obtained from the forest and field workers in South Korea. IgG enzyme-linked immunosorbent assay (ELISA) and neutralization assay were conducted for TBEV, and indirect immunofluorescence assay (IFA) and neutralization assay were performed for SFTSV. Seroprevalence of TBEV was 0.9% (5/583) by IgG ELISA, and 0.3% (2/583) by neutralization assay. Neutralizing antibody against TBEV was detected in a forest worker in Jeju (1:113) and Hongcheon (1:10). Only 1 (0.2%) forest worker in Yeongju was seropositive for SFTSV by IFA (1:2,048) and neutralizing antibody was detected also. In conclusion, this study shows that it is necessary to raise the awareness of physicians about TBEV infection and to make efforts to survey and diagnose vector-borne diseases in South Korea.

Clinical Presentation and Laboratory Diagnostic Work-Up of a Horse with Tick-Borne Encephalitis in Switzerland.

Tick-borne encephalitis is an important viral tick-borne zoonosis in Europe and Asia. The disease is induced by tick-borne encephalitis virus (TBEV). This report describes a 16-year-old Warmblood gelding presenting with sudden onset of lethargy, ataxia, and muscle fasciculations on the nostrils, the lips, and the eye lids as the most important clinical findings. The horse further had a mild facial nerve paralysis with drooping of the right upper and lower lip. Diagnosis was based on paired serum samples using TBEV-ELISAs revealing high serum IgM in the first sample with normal IgM in the second sample and an increase in serum IgG and neutralizing antibodies, indicating acute and recent infection. TBEV was confirmed by a virus-neutralization test, revealing a fivefold increase in antibodies 32 days after of the onset of clinical signs. Although the specific PCR on cerebrospinal fluid (CSF) was negative, TBEV-specific IgG and IgM were identified in the CSF of the horse. Treatment consisted of anti-inflammatory and anti-oxidative treatment and the horse recovered with a mild drooping of the right nostril as the only remaining clinical sign. TBEV infection is a potential differential diagnosis of neurological disease in horses living in endemic areas and this is the first report to describe the diagnostic criteria in a horse as recommended in humans with suspected TBEV infection.

Computational and Rational Design of Single-Chain Antibody against Tick-Borne Encephalitis Virus for Modifying Its Specificity.

Tick-borne encephalitis virus (TBEV) causes 5-7 thousand cases of human meningitis and encephalitis annually. The neutralizing and protective antibody ch14D5 is a potential therapeutic agent. This antibody exhibits a high affinity for binding with the D3 domain of the glycoprotein E of the Far Eastern subtype of the virus, but a lower affinity for the D3 domains of the Siberian and European subtypes. In this study, a 2.2-fold increase in the affinity of single-chain antibody sc14D5 to D3 proteins of the Siberian and European subtypes of the virus was achieved using rational design and computational modeling. This improvement can be further enhanced in the case of the bivalent binding of the full-length chimeric antibody containing the identified mutation.

In-silico design of envelope based multi-epitope vaccine candidate against Kyasanur forest disease virus.

Kyasanur forest disease virus (KFDV) causing tick-borne hemorrhagic fever which was earlier endemic to western Ghats, southern India, it is now encroaching into new geographic regions, but there is no approved medicine or effective vaccine against this deadly disease. In this study, we did in-silico design of multi-epitope subunit vaccine for KFDV. B-cell and T-cell epitopes were predicted from conserved regions of KFDV envelope protein and two vaccine candidates (VC1 and VC2) were constructed, those were found to be non-allergic and possess good antigenic properties, also gives cross-protection against Alkhurma hemorrhagic fever virus. The 3D structures of vaccine candidates were built and validated. Docking analysis of vaccine candidates with toll-like receptor-2 (TLR-2) by Cluspro and PatchDock revealed strong affinity between VC1 and TLR2. Ligplot tool was identified the intermolecular hydrogen bonds between vaccine candidates and TLR-2, iMOD server confirmed the stability of the docking complexes. JCAT sever ensured cloning efficiency of both vaccine constructs and in-silico cloning into pET30a (+) vector by SnapGene showed successful translation of epitope region. IMMSIM server was identified increased immunological responses. Finally, multi-epitope vaccine candidates were designed and validated their efficiency, it may pave the way for up-coming vaccine and diagnostic kit development.