Cross-reactive αβ T cell receptors (TCRs) recognizing multiple peptide variants can provide effective control of rapidly evolving viruses yet remain understudied. By screening 12 naturally occurring influenza-derived HLA-B*35:01–restricted nucleoprotein (NP) 418–426 epitopes (B*35:01-NP 418 ) that emerged since 1918 within influenza A viruses, including 2024 A/H5N1 viruses, we identified functional broadly cross-reactive T cells universally recognizing NP 418 variants. Binding studies demonstrated that TCR cross-reactivity was concomitant with diminished antigen sensitivity. Primary human B*35:01/NP 418 + CD8 + T cell lines displayed reduced cross-reactivity in the absence of CD8 coreceptor binding, validating the low avidity of cross-reactive B*35:01-NP 418 + CD8 + T cell responses. Six TCR–HLA-B*35:01/NP 418 crystal structures showed how cross-reactive TCRs recognized multiple B*35:01/NP 418 epitope variants. Specific TCR interactions were formed with invariant and conserved peptide-HLA features, thus remaining distal from highly varied positions of the NP 418 epitope. Our study defines molecular mechanisms associated with extensive TCR cross-reactivity toward naturally occurring viral variants highly relevant to universal protective immunity against influenza.

BACKGROUND:Coronavirus disease 2019 (COVID-19) frequently presents with neurological symptoms in human patients and leads to long-lasting brain pathology in a hamster model. There is no overt SARS-CoV-2 virus replication in central neurons. Whether viral proteins are sufficient to cause this pathology requires further investigations. The SARS-CoV-2 Spike-protein S1-subunit (S1-protein) has recently gained interest for causing neuroinflammation and accelerating aggregation of alpha-Synuclein (aSyn) in vitro. Here, we show the impact of S1-protein in a broad spectrum of brain regions after injection via three different application routes in C57/BL6 mice. METHODS:S1-protein was administered either intranasally, intravenously or intracerebrally. We quantified aSyn immunoreactivity and phosphorylated aSyn (pS129), microglia and astrocyte reactivity, ACE2/Neuropilin-1 receptor expression, and parvalbumin-positive interneurons in limbic system, basal ganglia, and cortical regions 14 days post-application. Plasma cytokine profiles were assessed 6 days post-injection. RESULTS:While intracerebral injection resulted in decreased aSyn immunoreactivity with increased pS129 in males, intravenous injection led to increased levels of aSyn immunoreactivity and microglia cell density, predominantly in brain regions associated with Parkinson's disease pathology. Intranasal application of S1-protein induced microgliosis in some brain regions but resulted in sex-dependent alterations of aSyn levels, with increases in females and decreases in males. All routes showed sex-dependent alterations in astrocytic reactivity, receptor expression, and parvalbumin-positive interneurons. CONCLUSION:Our results demonstrate that S1-protein itself leads to neuroinflammation, altered aSyn homeostasis, and disruption of inhibitory circuits in a route- and sex-dependent manner. These findings indicate the possibility of S1-protein being a crucial agent for both neuroinflammatory processes and altered protein regulation mechanisms. S1-protein trapped in tissue reservoirs could therefore explain symptoms occurring or persisting beyond viral clearance (Post COVID-19 condition).

Highly pathogenic avian influenza A viruses subtype H5N1 (HPAIV H5N1), subclade 2.3.4.4b infect multiple avian and mammalian species, posing a potential pandemic risk. Here we describe the outcomes of infection of ferrets with an HPAIV H5N1 virus, isolated from a European grey seal in 2023, compared with an older HPAIV H5N1 (A/Indonesia/05/2005). Overall, infection of ferrets with A/grey seal/Netherlands/302603/2023 caused more rapid mortality than infection of ferrets with A/Indonesia/05/2005. Animals developed severe pneumonia and irreversible hypothermia, associated with high levels of virus replication and histopathological changes in the respiratory tract and peripheral organs. As animal models for severe avian influenza virus infections in humans play a key role in the development of intervention strategies against these infections, these findings highlight the importance of using updated ferret models based on circulating virus strains.

The availability of influenza vaccines that can induce broadly protective immune responses is highly desirable and could also mitigate the impact of future influenza pandemics. Ideally, these vaccines also induce virus-specific CD8 + T cells, which have been identified as an independent correlate of protection. In the present study, we explored the use of an artificial immunogen that comprises of twenty highly conserved influenza virus CD8 + T cell epitopes with an HLA coverage of 99.5% of the world population. The highly attenuated viral vector Modified Vaccinia virus Ankara (MVA) was used to deliver the artificial poly-epitope sequence (rMVA-PE) and by using T cell lines raised against individual epitopes, we confirmed that the epitopes are liberated from the artificial immunogen. For efficient antigen processing and presentation, the epitopes were separated by spacer sequences. Stimulation of peripheral blood mononuclear cells of HLA-typed blood donors with rMVA-PE resulted in the activation of influenza virus-specific T cell responses. Furthermore, immunization of humanized HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice (HLA-A*02:01) with rMVA-PE induced influenza virus-specific CD8 + T cell responses. Thus, rMVA-PE proved to be immunogenic both in vitro and in vivo and constitutes a promising vaccine candidate for the induction of cross-reactive CD8 + T cell responses that could afford protection against antigenically distinct influenza A viruses (IAV) of various subtypes and species, and is currently considered for further clinical testing.

Tick-borne encephalitis virus (TBEV) vaccine breakthrough (VBT) infections are not uncommon in endemic areas. The clinical and immunological outcomes have been poorly investigated. We assessed the magnitude and specificity of virus-specific antibody and T cell responses after TBE in previously vaccinated subjects and compared the results with those of unvaccinated TBE patients and study subjects that received vaccination without VBT infection. Symptomatic TBEV infection of unvaccinated study subjects induced virus-specific antibody responses to the E protein and non-structural protein 1 (NS1) as well as T cell responses to structural and other non-structural (NS) proteins. After VBT infections, significantly impaired NS1-specific antibody responses were observed, while the virus-specific T cell responses to the NS proteins were relatively strong. VBT infection caused predominantly moderate to severe disease during hospitalization. The level of TBEV EDIII- and NS1-specific antibodies in unvaccinated convalescent patients inversely correlated with TBE severity and neurological symptoms early after infection.

Influenza viruses are notorious for their capacity to evade host immunity. Not only can they evade recognition by virus -neutralizing antibodies, there is also evidence that they accumulate mutations in epitopes recognized by virus -specific CD8+ T cells. In addition, we have shown previously that human influenza A viruses were less well recognized than avian influenza viruses by CD8+ T cells directed to the highly conserved, HLA-A*02:01 restricted M158-66 epitope located in the Matrix 1 (M1) protein. Amino acid differences at residues outside the epitope were responsible for the differential recognition, and it was hypothesized that this reflected immune adaptation of human influenza viruses to selective pressure exerted by M158-66 -specific CD8+ T cells in the human population. In the present study, we tested this hypothesis and investigated if selective pressure exerted by M158-66 epitopespecific CD8+ T cells could drive mutations at the extra-epitopic residues in vitro. To this end, isogenic influenza A viruses with the M1 gene of a human or an avian influenza virus were serially passaged in human lung epithelial A549 cells that transgenically express the HLA-A*02:01 molecule or not, in the presence or absence of M158-66 epitope-specific CD8+ T cells. Especially in the virus with the M1 gene of an avian influenza virus, variants emerged with mutations at the extra-epitopic residues associated with reduced recognition by M158-66 - specific T cells as detected by Next Generation Sequencing. Although the emergence of these variants was observed in the absence of selective pressure exerted by M158-66 epitope-specific CD8+ T cells, their proportion was much larger in the presence of this selective pressure.

Controlled human infection models (CHIMs) are a critical tool for the understanding of infectious disease progression, characterising immune responses to infection and rapid assessment of vaccines or drug treatments. There is increasing interest in using CHIMs for vaccine development and an obvious need for widely available and fit-for-purpose challenge agents. Inno4Vac is a large European consortium working towards accelerating and de-risking the development of new vaccines, including development of CHIMs for influenza, respiratory syncytial virus and Clostridium difficile. This report (in two parts) summarises a workshop held at the MHRA in 2021, focused on how to select CHIM candidate strains of influenza and respiratory syncytial virus (RSV) based on desirable virus characteristics and which immune assays would provide relevant information for assessing pre-existing and post-infection immune responses and defining correlates of protection. This manuscript (part 2) summarises presentations and discussions centred around RSV CHIMs and immune assays (an additional manuscript summarises influenza CHIM and immune assays: Inno4Vac workshop report Part 1: Controlled human influenza virus infection model (CHIVIM) strain selection and immune assays for CHIVIM studies, November 2021, MHRA, UK).

The soluble form of the Respiratory Syncytial Virus (RSV) G protein (sG) bears resemblance to the chemokine fractalkine (CX₃CL1). Both RSV sG and CX3CL1 possess a mucin-like domain and a CX3C motif, exist in membrane-associated and soluble forms, and bind to the CX₃CR1 receptor expressed on immune and epithelial cells. To explore the biological significance of RSV sG and CX₃CR1 interaction, we produced wild type (WT) and CX₃C motif-deficient (CX3CMut) RSV sG proteins and determined their effects on CX₃CR1 signaling in monocytic cells. Both CX3CMut- and WT RSV sG failed to activate CX₃CR1 signaling directly. However, WT sG competed with CX₃CL1 for CX₃CR1 binding and reduced CX3CL1-induced CX₃CR1-activation, monocyte migration, and adhesion. The CX₃C motif of sG was crucial for competitive blocking of CX3CL1-mediated activation, as CX₃CMut sG did not affect these CX₃CR1 functions significantly. Thus, blockade of CX₃CR1 signaling by sG may allow RSV to dampen host immune responses.

Tick-borne encephalitis virus (TBEV) infection may cause acute central nervous system inflammation varying in clinical manifestations and severity. A possible correlation of TBEV-specific antibody and cell-mediated immune responses, shortly after infection, with clinical manifestations, severity and long-term outcome has been poorly investigated. In a cohort of thirty early tick-borne encephalitis (TBE) patients, we assessed the magnitude, specificity and functional properties of TBEV-specific T-cell and antibody responses. These responses early during disease were assessed in view of clinical manifestations, severity and long-term outcome. TBEV-specific T-cell responses to C, E, NS1, and NS5 proteins were significantly lower in patients with severe acute illness than in patients with mild TBE. Lower T-cell responses to E, NS1, and NS5 proteins also correlated with the development of meningoencephalomyelitis. Virus-specific antibody titres early after infection did not correlate with disease severity, clinical manifestations, or long-term outcome in this study, possibly due to the small number of patients of which matching serum and peripheral blood mononuclear cells were available. The findings suggest that virus-specific T cells afford a certain degree of protection against the development of severe TBEV-induced disease.

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that has been associated with congenital neurological defects in fetuses born to infected mothers. At present, no vaccine or antiviral therapy is available to combat this devastating disease. Repurposing drugs that target essential host factors exploited by viruses is an attractive therapeutic approach. Here, we screened a panel of clinically approved small-molecule kinase inhibitors for their antiviral effects against a clinical isolate of ZIKV and thoroughly characterized their mechanisms of action. We found that the Raf kinase inhibitors Dabrafenib and Regorafenib potently impair the replication of ZIKV, but not that of its close relative dengue virus. Time-of-addition experiments showed that both inhibitors target ZIKV infection at post-entry steps. We found that Dabrafenib, but not Regorafenib, interfered with ZIKV genome replication by impairing both negative- and positive-strand RNA synthesis. Regorafenib, on the other hand, altered steady-state viral protein levels, viral egress, and blocked NS1 secretion. We also observed Regorafenib-induced ER fragmentation in ZIKV-infected cells, which might contribute to its antiviral effects. Because these inhibitors target different steps of the ZIKV infection cycle, their use in combination therapy may amplify their antiviral effects which could be further explored for future therapeutic strategies against ZIKV and possibly other flaviviruses. IMPORTANCE:There is an urgent need to develop effective therapeutics against re-emerging arboviruses associated with neurological disorders like Zika virus (ZIKV). We identified two FDA-approved kinase inhibitors, Dabrafenib and Regorafenib, as potent inhibitors of contemporary ZIKV strains at distinct stages of infection despite overlapping host targets. Both inhibitors reduced viral titers by ~1 to 2 log10 (~10-fold to 100-fold) with minimal cytotoxicity. Furthermore, we show that Dabrafenib inhibits ZIKV RNA replication whereas Regorafenib inhibits ZIKV translation and egress. Regorafenib has the added benefit of limiting NS1 secretion, which contributes to the pathogenesis and disease progression of several flaviviruses. Because these inhibitors affect distinct post-entry steps of ZIKV infection, their therapeutic potential may be amplified by combination therapy and likely does not require prophylactic administration. This study provides further insight into ZIKV-host interactions and has implications for the development of novel antivirals against ZIKV and possibly other flaviviruses.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by RVF virus (RVFV). RVFV infections in humans are usually asymptomatic or associated with mild febrile illness, although more severe cases of haemorrhagic disease and encephalitis with high mortality also occur. Currently, there are no licensed human vaccines available. The safety and efficacy of a genetically engineered four-segmented RVFV variant (hRVFV-4s) as a potential live-attenuated human vaccine has been tested successfully in mice, ruminants, and marmosets though the correlates of protection of this vaccine are still largely unknown. In the present study, we have assessed hRVFV-4s-induced humoral and cellular immunity in a mouse model of RVFV infection. Our results confirm that a single dose of hRVFV-4s is highly efficient in protecting naïve mice from developing severe disease following intraperitoneal challenge with a highly virulent RVFV strain and data show that virus neutralizing (VN) serum antibody titres in a prime-boost regimen are significantly higher compared to the single dose. Subsequently, VN antibodies from prime-boost-vaccinated recipients were shown to be protective when transferred to naïve mice. In addition, hRVFV-4s vaccination induced a significant virus-specific T cell response as shown by IFN-γ ELISpot assay, though these T cells did not provide significant protection upon passive transfer to naïve recipient mice. Collectively, this study highlights hRVFV-4s-induced VN antibodies as a major correlate of protection against lethal RVFV infection.

BACKGROUND:Rift Valley fever virus, a pathogen to ruminants, camelids, and humans, is an emerging mosquito-borne bunyavirus currently endemic to Africa and the Arabian Peninsula. Although animals are primarily infected via mosquito bites, humans mainly become infected following contact with infected tissues or fluids of infected animals. There is an urgent need for adequate countermeasures, especially for humans, because effective therapeutics or vaccines are not yet available. Here we assessed the safety, tolerability, and immunogenicity of a next-generation, four-segmented, live-attenuated vaccine candidate, referred to as hRVFV-4s, in humans. METHODS:A first-in-human, single-centre, randomised, double-blind, placebo-controlled trial was done in Belgium in which a single dose of hRVFV-4s was administered to healthy volunteers aged 18-45 years. Participants were randomly assigned using an interactive web response system. The study population encompassed 75 participants naive to Rift Valley fever virus infection, divided over three dosage groups (cohorts) of 25 participants each. All participants were followed up until 6 months. Using a staggered dose escalating approach, 20 individuals of each cohort were injected in the deltoid muscle of the non-dominant arm with either 104 (low dose), 105 (medium dose), or 106 (high dose) of 50% tissue culture infectious dose of hRVFV-4s as based on animal data, and five individuals per cohort received formulation buffer as a placebo. Primary outcome measures in the intention-to-treat population were adverse events and tolerability. Secondary outcome measures were vaccine-induced viraemia, vaccine virus shedding, Rift Valley fever virus nucleocapsid antibody responses (with ELISA), and neutralising antibody titres. Furthermore, exploratory objectives included the assessment of cellular immune responses by ELISpot. The trial was registered with the EU Clinical Trials Register, 2022-501460-17-00. FINDINGS:Between August and December, 2022, all 75 participants were vaccinated. No serious adverse events or vaccine-related severe adverse events were reported. Pain at the injection site (51 [85%] of 60 participants) was most frequently reported as solicited local adverse event, and headache (28 [47%] of 60) and fatigue (28 [47%] of 60) as solicited systemic adverse events in the active group. No vaccine virus RNA was detected in any of the blood, saliva, urine, or semen samples. Rift Valley fever virus nucleocapsid antibody responses were detected in most participants who were vaccinated with hRVFV-4s (43 [72%] of 60 on day 14) irrespective of the administered dose. In contrast, a clear dose-response relationship was observed for neutralising antibodies on day 28 with four (20%) of 20 participants responding in the low-dose group, 13 (65%) of 20 responding in the medium-dose group, and all participants (20 [100%] of 20) responding in the high-dose group. Consistent with the antibody responses, cellular immune responses against the nucleocapsid protein were detected in all dose groups, whereas a more dose-dependent response was observed for the Gn and Gc surface glycoproteins. Neutralising antibody titres declined over time, whereas nucleocapsid antibody responses remained relatively stable for at least 6 months. INTERPRETATION:The hRVFV-4s vaccine showed a high safety profile and excellent tolerability across all tested dose regimens, eliciting robust immune responses, particularly with the high-dose administration. The findings strongly support further clinical development of this candidate vaccine for human use. FUNDING:The Coalition for Epidemic Preparedness Innovations with support from the EU Horizon 2020 programme.

ABSTRACTThe current outbreak of HPAI H5N1 virus infections in dairy cattle in the USA underscores the need for easily accessible methods to rapidly assess host susceptibility for infection with known and emerging influenza viruses. Here we show that ex vivo lung slice cultures from calves provide a useful method to rapidly screen host susceptibility to a range of influenza A viruses.

Tick-borne encephalitis (TBE) is a serious neurological disease caused by TBE virus (TBEV). Because antiviral treatment options are not available, vaccination is the key prophylactic measure against TBEV infections. Despite the availability of effective vaccines, cases of vaccination breakthrough infections have been reported. The multienzymatic non-structural protein 3 (NS3) of orthoflaviviruses plays an important role in polyprotein processing and virus replication. In the present study, we evaluated NS3 of TBEV as a potential vaccine target for the induction of protective immunity. To this end, a recombinant modified vaccinia virus Ankara that drives the expression of the TBEV NS3 gene (MVA-NS3) was constructed. MVA-NS3 was used to immunize C57BL/6 mice. It induced NS3-specific immune responses, in particular T cell responses, especially against the helicase domain of NS3. However, MVA-NS3-immunized mice were not protected from subsequent challenge infection with a lethal dose of the TBEV strain Neudoerfl, indicating that in contrast to immunity to prME and NS1, NS3-specific immunity is not an independent correlate of protection against TBEV in this mouse model.

The four serotypes of dengue virus (DENV1–4) continue to pose a major public health threat. The first licenced dengue vaccine, which expresses the surface proteins of DENV1–4, has performed poorly in immunologically naïve individuals, sensitising them to antibody-enhanced dengue disease. DENV non-structural protein 1 (NS1) can directly induce vascular leakage, the hallmark of severe dengue disease, which is blocked by NS1-specific antibodies, making it an attractive target for vaccine development. However, the intrinsic ability of NS1 to trigger vascular leakage is a potential drawback of its use as a vaccine antigen. Here, we modified DENV2 NS1 by mutating an N-linked glycosylation site associated with NS1-induced endothelial hyperpermeability and used modified vaccinia virus Ankara (MVA) as a vector for its delivery. The resulting construct, rMVA-D2-NS1-N207Q, displayed high genetic stability and drove efficient secretion of NS1-N207Q from infected cells. Secreted NS1-N207Q was composed of dimers and lacked N-linked glycosylation at position 207. Prime–boost immunisation of C57BL/6J mice induced high levels of NS1-specific antibodies binding various conformations of NS1 and elicited NS1-specific CD4+ T-cell responses. Our findings support rMVA-D2-NS1-N207Q as a promising and potentially safer alternative to existing NS1-based vaccine candidates, warranting further pre-clinical testing in a relevant mouse model of DENV infection.

Introduction Tick-borne encephalitis virus (TBEV) is one of the most relevant tick-transmitted neurotropic arboviruses in Europe and Asia and the causative agent of tick-borne encephalitis (TBE). Annually more than 10,000 TBE cases are reported despite having vaccines available. In Europe, the vaccines FSME-IMMUN® and Encepur® based on formaldehyde-inactivated whole viruses are licensed. However, demanding vaccination schedules contribute to sub-optimal vaccination uptake and breakthrough infections have been reported repeatedly. Due to its immunogenic properties as well as its role in viral replication and disease pathogenesis, the non-structural protein 1 (NS1) of flaviviruses has become of interest for non-virion based flavivirus vaccine candidates in recent years. Methods Therefore, immunogenicity and protective efficacy of TBEV NS1 expressed by neuraminidase (NA)-deficient Influenza A virus (IAV) or Modified Vaccinia virus Ankara (MVA) vectors were investigated in this study. Results With these recombinant viral vectors TBEV NS1-specific antibody and T cell responses were induced. Upon heterologous prime/boost regimens partial protection against lethal TBEV challenge infection was afforded in mice. Discussion This supports the inclusion of NS1 as a vaccine component in next generation TBEV vaccines.

Respiratory syncytial virus (RSV) infections are a constant public health problem, especially in infants and older adults. Virtually all children will have been infected with RSV by the age of two, and reinfections are common throughout life. Since antigenic variation, which is frequently observed among other respiratory viruses such as SARS-CoV-2 or influenza viruses, can only be observed for RSV to a limited extent, reinfections may result from short-term or incomplete immunity. After decades of research, two RSV vaccines were approved to prevent lower respiratory tract infections in older adults. Recently, the FDA approved a vaccine for active vaccination of pregnant women to prevent severe RSV disease in infants during their first RSV season. This review focuses on the host response to RSV infections mediated by epithelial cells as the first physical barrier, followed by responses of the innate and adaptive immune systems. We address possible RSV-mediated immunomodulatory and pathogenic mechanisms during infections and discuss the current vaccine candidates and alternative treatment options.

Introduction:Tick-borne encephalitis virus (TBEV) is an important human pathogen that can cause a serious disease involving the central nervous system (tick-borne encephalitis, TBE). Although approved inactivated vaccines are available, the number of TBE cases is rising, and breakthrough infections in fully vaccinated subjects have been reported in recent years.Methods:In the present study, we generated and characterized a recombinant Modified Vaccinia virus Ankara (MVA) for the delivery of the pre-membrane (prM) and envelope (E) proteins of TBEV (MVA-prME).Results:MVA-prME was tested in mice in comparison with a licensed vaccine FSME-IMMUN® and proved to be highly immunogenic and afforded full protection against challenge infection with TBEV.Discussion:Our data indicate that MVA-prME holds promise as an improved next-generation vaccine for the prevention of TBE.

Influenza viruses (IVs) cause substantial global morbidity and mortality. Given the limited range of licensed antiviral drugs and their reduced efficacy due to resistance mutations, repurposing FDA-approved kinase inhibitors as fast-tracked host-targeted antivirals is an attractive strategy. We identified six FDA-approved non-receptor tyrosine kinase-inhibitors (NRTKIs) as potent inhibitors of viral replication of pandemic and seasonal IVs in vitro. We validated their efficacy in a biologically and clinically relevant ex vivo model of human precision-cut lung slices. We identified steps of the virus infection cycle affected by these inhibitors and assessed their effect(s) on host responses. Their overlapping targets suggest crosstalk between Abl, EGFR, and PDGFR pathways during IAV infection. Our data and established safety profiles of these NRTKIs provide compelling evidence for further clinical investigations and repurposing as host-targeted influenza antivirals. Moreover, these NRTKIs have broad-spectrum antiviral potential given that their kinase/pathway targets are critical for the replication of many viruses.

Introduction:Naturally attenuated Langat virus (LGTV) and highly pathogenic tick-borne encephalitis virus (TBEV) share antigenically similar viral proteins and are grouped together in the same flavivirus serocomplex. In the early 1970s, this has encouraged the usage of LGTV as a potential live attenuated vaccine against tick-borne encephalitis (TBE) until cases of encephalitis were reported among vaccinees. Previously, we have shown in a mouse model that immunity induced against LGTV protects mice against lethal TBEV challenge infection. However, the immune correlates of this protection have not been studied.Methods:We used the strategy of adoptive transfer of either serum or T cells from LGTV infected mice into naïve recipient mice and challenged them with lethal dose of TBEV.Results:We show that mouse infection with LGTV induced both cross-reactive antibodies and T cells against TBEV. To identify correlates of protection, Monitoring the disease progression in these mice for 16 days post infection, showed that serum from LGTV infected mice efficiently protected from developing severe disease. On the other hand, adoptive transfer of T cells from LGTV infected mice failed to provide protection. Histopathological investigation of infected brains suggested a possible role of microglia and T cells in inflammatory processes within the brain.Discussion:Our data provide key information regarding the immune correlates of protection induced by LGTV infection of mice which may help design better vaccines against TBEV.