To Örebro University

OBJECTIVES: We aimed to 1) detect tick-borne encephalitis virus (TBEV) RNA in clinical samples from patients with TBE, 2) characterise the detected RNA using Sanger sequencing, and 3) examine whether RNA detection was associated with disease severity.

METHODS: We studied 137 patients infected and diagnosed with TBE between 2016 and 2021 in Region Örebro County and Region Värmland. Biobanked serum (n = 129) and cerebrospinal fluid (CSF; n = 110) samples were analysed. Serum was tested for TBEV-specific antibodies, and both serum and CSF for TBEV RNA using PCR. Following nested PCR, the 5' non-coding region (5'NCR) of five samples underwent Sanger sequencing. Disease severity was assessed based on intensive care unit (ICU) admission, duration of ICU stay and need for mechanical ventilation.

RESULTS: TBEV RNA was detected in 5 serum samples (3.9 %) and 7 CSF samples (6.4 %), representing 10 patients (7.3 %). Patients with detectable RNA were older, more frequently admitted to an ICU (p = 0.04), and more often required mechanical ventilation (p = 0.01) compared to those without detectable TBEV RNA. Sequencing of the 5'NCR in four patients revealed differences from the 5 ´NCR of the Swedish reference strain Torö-2003. The Örebro sequences were identical but differed from the Värmland sequences at two nucleotide positions.

CONCLUSIONS: TBEV RNA was detectable in both serum and CSF of TBE patients, and its presence was associated with more frequent ICU admission and need for mechanical ventilation. Sequencing of the 5'NCR revealed genetic variation between TBEV sequences from patients in Örebro and Värmland.

Background: Tick-borne encephalitis (TBE) is a growing public health concern in Europe and Asia, driven by the increasing spread of the TBE virus (TBEV) and its tick vectors. Although current vaccines provide protection, their multi-dose schedule and reduced efficacy in the elderly contribute to occasional vaccine failures. This study aims to develop a novel TBE vaccine offering enhanced protection with fewer doses, focusing on mucosal immunization.Methods: Infectious clone of Langat virus (LGTV IC) was designed and rescued- in our laboratory. We assessed the safety and immunogenicity of the LGTV IC as a live-attenuated TBE vaccine platform in a murine model. Mice were vaccinated with LGTV IC via intranasal or intramuscular routes at low or high doses. We evaluated viremia,viral presence in cerebrospinal fluid, general health, and immune responses.Results: Intranasal immunization with LGTV IC induced strong immune responses. It elicited robust anti-TBEVIgG responses and strong TBEV NS3-specific IFN-γ and IL-2 production. Notably, low-dose intranasal immunization outperformed higher doses of both routes, inducing a more balanced and effective immune response. Low-dose intranasal administration was well tolerated, with no clinical signs, weight loss, or viral presence in the central nervous system. In contrast, intranasal immunization caused potential adverse effects atelevated doses.Conclusion: These findings support LGTV IC as a promising vaccine platform for TBE, with intranasal administration emerging as a putative safe, well-tolerated, and effective needle-free alternative to intramuscular injection when given at a low dose. Ongoing efforts are focused on further attenuating LGTV IC to enhance its safety profile for future applications.

 Tick-borne encephalitis (TBE) is one of the most important tick-transmitted diseases in Europe and Asia. TBE virus (TBEV) infections lead to a diversity of disease outcomes ranging from mild flu-like symptoms to severe neurological disorders. With no specific antiviral treatment available, vaccination remains the most effective protective strategy for TBE. Currently, only inactivated TBE vaccines are available on the market, which require repeated booster doses to sustain immunity. In addition, vaccine breakthroughs are reported in some patients, especially in the elderly. In contrary to inactivated vaccines, live-attenuated viral vaccines could provide long-term, or even lifelong immunity after a single dose. Langat virus (LGTV) is a naturally attenuated strain of TBEV, which makes it a potential candidate for a live-attenuated TBE vaccine. In this study, we engineered and rescued four infectious clones (ICs) of LGTV using RNA- and DNA-based reverse genetics methods. Next generation sequencing of the rescued ICs showed that the viruses rescued by DNA-based methods were more similar to the parental LGTV sequence and showed higher genetic stability after passaging in cell culture. One of the DNA-launched LGTV IC was further evaluated in vitro and in vivo which exhibited growth kinetics and immune profile comparable to the LGTV strain in our laboratory. This reverse genetics platform will be utilized to introduce targeted mutations within the LGTV genome to develop a live-attenuated TBE vaccine.

Tick-borne encephalitis (TBE) is one of the most important tick-transmitted diseases in Europe and Asia. With no specific antiviral treatment available, vaccination remains the most effective protective strategy for TBE. Unlike currently available inactivated TBE vaccines that require repeated boosters, live-attenuated vaccines could offer lifelong immunity with a single dose. Langat virus (LGTV) is a naturally attenuated strain of TBE virus (TBEV). In this study, we engineered and rescued four infectious clones (ICs) of LGTV using RNA- and DNA-based reverse genetics methods. The ICs rescued by DNA-based method showed higher genetic stability in cell culture. One of the ICs rescued by DNA-based method was further evaluated in vitro and in vivo, which exhibited growth kinetics and immune profile comparable to the LGTV strain in our laboratory. This reverse genetics platform will be utilized to introduce targeted mutations within the LGTV genome to develop a live-attenuated TBE vaccine.

Background

Chronic liver disease caused by Hepatitis C virus (HCV), contributes to a major health burden worldwide. Although chronic infections with the HCV can be effectively cured by antivirals, but the cured patients can be re-infected as they lack protective immune responses. In addition, the relatively high cost of the HCV treatment brings concerns about the accessibility, especially in the developing countries. Hence, there exists a need for cost effect vaccines with high efficiency to control and possibly eradicate HCV globally.

Methods

We have developed and utilized flavivirus replicons as delivery system to prime hepatitis-specific immune responses. We generated subgenomic replicons of Tick-borne encephalitis virus, Langat virus, West-Nile virus (WNV), and Kunjin virus (KUNV) expressing a fusion protein between the HCV NS3/4A and core protein of stork hepatitis B virus. Antigen expression and immunogenicity of these flavivirus replicons was evaluated in vitro and in vitro, respectively.

Results

Transfection experiments showed that the antigen expression by KUNV and WNV replicons was several folds higher than the antigen expression of control DNA plasmid with CMV promoter. In a murine model, KUNV replicon triggered a potent cellular immune response with respect to priming of HCV NS3/4A-specific T cells as determined by ELISpot, and polyfunctionality assays. In addition, vaccination with KUNV replicon also induced HCV NS3/4A-specific humoral immune response.

Conclusion

These findings indicate that the KUNV replicon-based HCV vaccine can induce both the neutralizing antibodies and protective T cell responses. Thus, the newly developed KUNV replicon-based vaccine is an attractive candidate to provide protection against HCV.

Flaviviruses are usually transmitted to humans via mosquito or tick bites, whose infections may lead to severe diseases and fatality. During intracellular infection, they remodel the endoplasmic reticulum (ER) membrane to generate compartments scaffolding the replication complex (RC) where replication of the viral genome takes place. In this study, we purified the ER membrane fraction of virus infected cells to identify the proteins that were enriched during flavivirus infection. We found that tripartite motif-containing proteins (TRIMs) including TRIM38, TRIM21, and TRIM14 were significantly enriched during infection with mosquito-borne (West Nile virus strain Kunjin and Zika virus (ZIKV)) and tick-borne (Langat virus (LGTV)) flaviviruses. Further characterizations showed that TRIM21 and TRIM14 act as restriction factors against ZIKV and LGTV, while TRIM38 hinders ZIKV infection. These TRIMs worked as interferon-stimulated genes to mediate IFN-I response against LGTV and ZIKV infections. Restriction of ZIKV by TRIM14 and TRIM38 coincides with their colocalization with ZIKV NS3. TRIM14-mediated LGTV restriction coincides with its colocalization with LGTV NS3 and NS5 proteins. However, TRIM21 did not colocalize with ZIKV and LGTV NS3 or NS5 protein suggesting its antiviral activity is not dependent on direct targeting the viral enzyme. Finally, we demonstrated that overexpression of TRIM21 and TRIM14 restricted LGTV replication.

The annual number of tick-borne encephalitis (TBE) cases in Norway has increased dramatically from 1 case in 1998 to 113 in 2023. Characterization of TBE virus (TBEV) genomes from both clinical samples and tick vectors is necessary to understand disease severity and transmission dynamics. However, clinical samples with intact virus are rare because TBE is usually diagnosed by serology in the post-viremic phase, when the viral load is low and undetectable by molecular methods such as polymerase chain reaction (PCR). To date, Mandal-2009 is the only TBEV sequence from Norway with complete virus genome, sequenced directly from the tick vector. We used a combined approach with newly designed overlapping primer pairs and nanopore sequencing together with Sanger sequencing to obtain nearly complete TBEV genomes from both patient and tick samples from Norway. The patient had severe TBE complicated with hemophagocytic lymphohistiocytosis (HLH). The patient and tick samples were collected 16 km apart, from Telemark and Vestfold Counties, respectively. Pairwise genomic comparison showed 99.7 % identity, and phylogenetic analysis revealed that these sequences were closely related to the TBEV strain from Kumlinge in Åland, Finland, rather than to the previously published Norwegian variant Mandal-2009. These findings confirm the existence of novel TBEV variants in the endemic areas of Telemark and Vestfold Counties of Norway. Our findings highlight the need for continuous monitoring and characterization of novel TBEV genomes in Norway and Europe.

Tick-borne encephalitis (TBE) is a significant disease in Europe and Asia, with a risingincidence due to the spread of the TBE virus (TBEV) and its vectors. Current TBE vaccinesprovide good protection, but they have a complex immunization schedule and lower efficacy inthe elderly, leading to occasional vaccine failures. We aim to develop a novel TBE vaccine toprovide better protection with fewer doses through mucosal immunization. The current workcovers a pilot study that evaluates live-attenuated TBE vaccine in vivo, using Langat virus(LGTV) platform that we developed based on a rescued LGTV infectious clone (LGTV IC).In the current mouse study, LGTV IC was administered via intranasal and intramuscular routesat two doses (10³ and 10⁵ PFU). The study assessed tolerability, viremia profile, and inducedimmunogenicity.As a result, we show that intranasal immunization with LGTV IC induced strong immuneresponses and revealed a favorable safety profile in a dose-dependent manner. Low-doseintranasal administration was well tolerated, with no clinical signs, weight loss, or viral presencein the central nervous system. It elicited robust anti-TBEV IgG antibodies that successfullyneutralized both LGTV and TBEV and induced strong cellular immunity, characterized byTBEV NS3-specific IFNγ and IL-2 secreting cells. Notably, low-dose mucosal immunizationoutperformed both high-dose intranasal and intramuscular administration in generating abalanced immune response. In contrast, high-dose intranasal immunization caused significantweight loss and minimal viral detection in CSF, indicating potential adverse effects at elevateddoses.These findings support the potential of low-dose mucosal immunization with LGTV IC as a safeand effective TBE vaccination strategy. Further attenuation of LGTV IC is underway to enhancesafety for future development.

Chronic liver disease, resulting from Hepatitis B virus (HBV), Hepatitis D virus (HDV), or Hepatitis C virus (HCV) infections, contributes to a major health burden worldwide. Chronic infections with the hepatitis C virus (HCV) can be effectively cured by antivirals. However, the cured patients can be re-infected as they lack protective immune responses. In addition, the relatively high cost of the HCV treatment brings concerns about the accessibility, especially in the developing countries. Hence, there exists a need for cost effect vaccines with high efficiency to control and possibly eradicate Hepatitis viruses globally. The vaccine should induce either, or both, neutralizing antibodies and protective T cell responses. We have developed and utilized flavivirus replicons as delivery system to prime hepatitis-specific T cell responses. We generated subgenomic replicons of Tick-borne encephalitis virus (TBEV), Langat virus (LGTV), West-Nile virus (WNV), and Kunjin virus (KUNV) expressing either a fusion protein between the HCV NS3/4A and a stork hepatitis B virus core or a vaccine candidate gene of HB/DV. Transfection experiments showed that the antigen expression by KUNV and WNV replicons was several folds higher than the antigen expression of control DNA plasmid with CMV promoter. The immunogenicity of these flavivirus replicons was evaluated in mice. The KUNV replicon triggered a potent cellular immune response with respect to priming of HCV NS3/4A-specific T cells as determined by ELISpot, and polyfunctionality. In short, the newly developed KUNV replicon- based vaccine is an attractive candidate to provide protection against hepatitis viruses.

Background. Tick-borne encephalitis (TBE) is a significant disease in Europe and Asia, witha rising incidence due to the spread of the TBE virus (TBEV) and its vectors. Currentvaccines, although essential, have a complex immunization schedule and lower effectiveness in the elderly, leading to occasional vaccine failures. We aim to create a new TBE vaccine that provides better protection with fewer doses.

Aim. We aim to develop an effective vaccine that can be delivered via the mucosal route. The current study covers a pilot study that evaluates live-attenuated vaccine using LGTV IC ascandidate for TBE vaccine.

Methods. We developed a genetically modified Langat virus (LGTV) so-called LGTV infectious clone (LGTV IC). In-vivo evaluation was done on mice model to assess tolerabilityand immunogenicity of LGTV IC. LGTV IC was delivered to mice via two different routes,mucosal and intramuscular, in two different doses. The study assessed the peak and kinetics ofviremia, as well as the elicited humoral, cellular, and mucosal immune responses.

Results. The mucosal administration of LGTV IC intranasally, at 1 x 103 PFU, was well tolerated in mice, as evidenced by the absence of clinical signs post-administration and no observed body weight loss. This immunization regimen consistently elicited anti-TBEV IgG antibodies in serum, irrespective of the dosage. Furthermore, a robust cellular immune response targeting TBEV non-structural protein 3 antigens (NS3), as well as capsid (C) and envelope (E) antigens, was observed. Intriguingly, lower dosage mucosal immunizationproved more effective in eliciting both humoral and cellular immune responses compared tohigher dosage mucosal administration and intramuscular immunization. Evidence of mucosal immunity was detected in select mucosal samples from the low dosage mucosal immunizationgroup.

Conclusion and perspective. Administration of LGTV IC via mucosal route seems promising suggesting that LGTV IC can be considered as a candidate for a live TBE vaccine.Ongoing efforts involve further attenuation of LGTV IC, with successful attenuated variants undergoing in-vitro evaluation. Subsequently, these variants will be assessed in vivo as potential live-attenuated vaccine candidates.