To Örebro University

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.

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.

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.

PLNC8 alpha beta is a cationic antimicrobial peptide that previously has been reported to express both antibacterial and antiviral properties. This study aimed to further elucidate the antiviral effects of PLNC8 alpha beta and its impact on virus-induced cytotoxicity and inflammatory signaling in human alveolar epithelial cells (A549) infected with the flavivirus Kunjin. Complementary in silico analyses using molecular dynamics (MD) simulation were conducted to investigate the mechanism of action of PLNC8 alpha beta by studying the interaction of PLNC8 alpha and beta with models of a flavivirus membrane and a eukaryotic plasma membrane, respectively. Our findings demonstrated that PLNC8 alpha beta significantly reduces both extracellular and intracellular viral loads, as confirmed by plaque reduction assays and RT-PCR. The peptide also mitigated virus-induced cytotoxicity and inflammation. Notably, PLNC8 alpha beta modulated the virus-induced dysregulation of key signaling and inflammatory genes, such as TLR9, TLR3, NOD2, FOS, JUN, IL6, and CXCL8. MD simulation revealed that PLNC8 alpha beta exhibits higher binding affinity for a flavivirus membrane model compared to a model of the plasma membrane, likely due to stronger electrostatic interactions with anionic phospholipids. This selective interaction possibly accounts for a potent antiviral activity of PLNC8 alpha beta combined with a minimal cytotoxicity toward human cells. Overall, PLNC8 alpha beta shows significant promise as an antiviral agent against flavivirus infections and warrants further exploration for peptide-based antiviral therapies.

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.

Background. Tick-borne encephalitis (TBE) is one of the most important tick-transmitted diseases in Europe and Asia. Most infections with the TBE virus (TBEV) are asymptomatic or cause mild flu-like symptoms, but they may induce severe neurological disorders with permanent sequelae. The incidence of TBE cases showed a remarkable elevation in recent years probably due to the geographic expansion of TBEV and its vectors, which is concerning in the absence of a specific antiviral treatment. Vaccination remains the best protective measure against TBE. However, currently available vaccines have a burdensome immunization schedule, and poor immunogenicity in the elderly, which may contribute to observed vaccine failures, i.e., TBE occurrence in vaccinated people. One aim within the Developvaccines@oru project is to develop a novel TBE vaccine that could provide improved immunogenicity using fewer doses. Our strategy is to induce an immune response at possible sites of virus infection by a modified live attenuated vaccine based on Langat virus. 

Methods. Infectious clones of Langat virus (LGTV) based on the strain available in our laboratory are created followed by the generation of modified LGTV infectious clone as potential attenuated virus. Then, we compare them with the ¨original¨ LGTV strain using cell based and animal models.

Preliminary results. We successfully created LGTV infectious clones. In order to establish a baseline for animal experiments with our vaccine candidates, we planned a pilot study using the “original” Langat virus. First, we conducted a pre-pilot experiment to optimize the study design and evaluation methods. Preliminary data on the establishment process of vaccine candidates in vitro as well as cellular and humoral immune response in mice in response to the LGTV infectious clone are presented.

Conclusion. Further investigation of modified LGTV clone seems interesting in the development approach of new TBEV vaccine candidate.

Tick-borne encephalitis (TBE) is one of the most important tick-transmitted diseases in Europe and Asia. The incidence of TBE cases showed a remarkable elevation in recent years probably due to the geographic expansion of TBEV and its vectors, which is concerning in the absence of a specific antiviral treatment. Vaccination remains the best protective measure against TBE. However, currently available vaccines have a burdensome immunization schedule, and poor immunogenicity in the elderly, which may contribute to observed vaccine failures, i.e., TBE occurrence in vaccinated people. One aim within the Developvaccines@oru project is to develop a novel TBE vaccine that could provide improved immunogenicity using fewer doses. Our strategy is to induce an immune response at possible sites of virus infection by a modified live attenuated vaccine based on Langat virus (LGTV). Infectious clones of Langat virus (LGTV IC) based on the strain available in our laboratory are created followed by the generation of modified LGTV IC as potential attenuated virus. Then, we compare them with the ¨original¨ LGTV strain using cell based and animal models. In our laboratory, we f successfully created LGTV IC. In order to establish a baseline for animal experiments with our vaccine candidates, we planned a pilot study using the “original” LGTV and LGTV IC strains. First, we conducted a pre-pilot experiment to optimize the study design and evaluation methods. Our preliminary data shows that intramuscular administration of both strains was well tolerated in mice.  In contrast to the original LGTV, LGTV IC was found to cause a transient, but significant reduction in body weight. ELISA results showed that mice antibodies after LGTV IC infection cross reacted with TBEV antigens. The T lymphocytes, isolated from these mice spleens, showed Interferon gamma secretion when stimulated with both LGTV and TBEV peptides. However, this cellular response revealed higher in original LGTV infected mice. Moreover, we confirm that LGTV IC show lower viraemia peak than original LGTV, both occurring at 2 days post infection. Besides, we have successfully rescued modified LGTV infectious clones with individual or combined mutations in genomic regions coding for NS3, NS5 and/or 3’non-coding region of LGTV genome. Further in-vitro and in-vivo investigation of the modified and potentially attenuated LGTV clone, seems interesting in the development approach of new TBEV vaccine candidate. 

Tick-borne encephalitis virus (TBEV) and Langat virus (LGTV) are both members of Flavivirus genus within the Flaviviridae family. TBEV is the main pathogenic arbovirus circulating in Europe, Russia, and China. Flaviviruses are characterized by a positive sense single-stranded RNA genome and an enveloped icosahedral virion structure. Previously, it has been observed that flavivirus envelope (E) protein and non-structural protein 1 (NS1) both play a critical role in the pathology of flavivirus. Therefore, in this study, we aim to investigate flavivirus E and NS1 protein as a good target for the development of a subunit vaccine with further potential as a putative diagnostic tool to distinguish between TBEV infected from TBEV vaccinated individuals. Thus, we have generated 4 different successful constructs with TBEV (E and NS1) and LGTV (E and NS1) in the pET SUMO vector. Restriction digestion and sequencing analysis confirmed successful clones of interest and their right orientation. Next, the right clones were transformed in BL21 (DE3) one shoot chemically competent E. coli and induce the expression with 0.5 mM IPTG in culture medium following 0-4h, and 24h incubation period. Next, bacterial cell pellets were collected and used for SDSPAGE/Western blot analysis. We used the champion™ pET SUMO expression system which may produce high levels of soluble protein in bacteria. It employs a small ubiquitin-related modifier (SUMO) fusion, belonging to the growing family of ubiquitin-related proteins, to enhance the solubility of expressed fusion proteins. We have stained with 6x-His tag antibody of interest (mouse monoclonal) for targeting both TBEV- E/NS1 and LGTV-E/NS1 proteins. Among them, the expression of TBEV-NS1 and LGTV-E proteins was verified and confirmed. Several attempts have also been made to obtain the TBEV-E and LGTV-NS1 protein in E. coli cells; however, these require further optimization with a suitable time and dose of IPTG induction. We have used the BL21(DE3) expression system, which could maximize the expression of soluble protein. After successful expression, the 13-kd SUMO moiety will be cleaved by the highly specific and active SUMO (ULP1) protease at the carboxyl terminal, producing a native protein. Furthermore, a protein purification assay (e-g., NI-NTA column/ÄKTA Protein Purification Systems) will be developed to obtain native recombinant protein. The purified proteins will be studied in combination with suitable adjuvants as putative TBE subunit vaccines. They will also be characterized with the potential to develop new tools for TBE diagnostics. 

Removal of genes coding for major parts of capsid (C), premembrane (prM), and envelope (E) proteins on the flavivirus genome aborts the production of infectious virus particles where the remaining genome forms a replicon that retains replicability in host cells. The C-prM-E proteins can also be expressed in trans with the flavivirus replicons to generate single-round infectious replicon virus-like particles (RVPs). In this study, we characterized the use of RVPs based on the Kunjin strain of WNV (WNV_KUN) as a putative WNV vaccine candidate. In addition, the WNV_KUN C-prM-E genes were substituted with the Crimean-Congo hemorrhagic fever virus (CCHFV) genes encoding the glycoproteins Gn and Gc to generate a WNV_KUN replicon expressing the CCHFV proteins. To generate RVPs, the WNV_KUN replicon was transfected into a cell line expressing the WNV_KUN C-prM-E. Using immunoblotting and immunofluorescence assays, we showed that the replicon can express the CCHFV Gn and Gc proteins and the RVPs can transduce cells to express WNV_KUN proteins and the CCHFV Gn and Gc proteins. Our study also revealed that these RVPs have potential as a vaccine platform with low risk of recombination as it infects cells only in one cycle. The immunization of mice with the RVPs resulted in high seroconversion to both WNV E and NS1 but limited seroconversion to CCHFV Gn and Gc proteins. Interestingly, we found that there was enhanced production of WNV E, NS1 antibodies, and neutralizing antibodies by the inclusion of CCHFV Gc and Gn into WNV_KUN RVPs. Thus, this study indicates a complementary effect of the CCHFV Gn and Gc proteins on the immunogenicity by WNV_KUN RVPs, which may be applied to develop a future vaccine against the WNV.

Potent broad-spectrum antiviral agents are urgently needed to combat existing and emerging viral infections. This is particularly important considering that vaccine development is a costly and time consuming process and that viruses constantly mutate and render the vaccine ineffective. Antimicrobial peptides (AMP), such as bacteriocins, are attractive candidates as antiviral agents against enveloped viruses. One of these bacteriocins is PLNC8 αβ, which consists of amphipathic peptides with positive net charges that display high affinity for negatively charged pathogen membrane structures, including phosphatidylserine rich lipid membranes of viral envelopes. Due to the morphological and physiological differences between viral envelopes and host cell plasma membranes, PLNC8 αβ is thought to have high safety profile by specifically targeting viral envelopes without effecting host cell membranes. In this study, we have tested the antiviral effects of PLNC8 αβ against the flaviviruses Langat and Kunjin, coronavirus SARS-CoV-2, influenza A virus (IAV), and human immunodeficiency virus-1 (HIV-1). The concentration of PLNC8 αβ that is required to eliminate all the infective virus particles is in the range of nanomolar (nM) to micromolar (μM), which is surprisingly efficient considering the high content of cholesterol (8–35%) in their lipid envelopes. We found that viruses replicating in the endoplasmic reticulum (ER)/Golgi complex, e.g. SARS-CoV-2 and flaviviruses, are considerably more susceptible to PLNC8 αβ, compared to viruses that acquire their lipid envelope from the plasma membrane, such as IAV and HIV-1. Development of novel broad-spectrum antiviral agents can significantly benefit human health by rapidly and efficiently eliminating infectious virions and thereby limit virus dissemination and spreading between individuals. PLNC8 αβ can potentially be developed into an effective and safe antiviral agent that targets the lipid compartments of viral envelopes of extracellular virions, more or less independent of virus antigenic mutations, which faces many antiviral drugs and vaccines.