The Envelope Protein of Tick-Borne Encephalitis Virus Influence Neuron Entry, Pathogenicity and Vaccine Protection

doi:10.21203/rs.3.rs-24796/v1

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The Envelope Protein of Tick-Borne Encephalitis Virus Influence Neuron Entry, Pathogenicity and Vaccine Protection

https://doi.org/10.21203/rs.3.rs-24796/v1

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Journal Publication

published 28 Sep, 2020

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Background: Tick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection ranges from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known, however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection.

Method: Mice were infected with different TBEV strains, and high virulent and low virulent TBEV were chosen. Sequence alignment were used to identify differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes and MEFs. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated.

Results: We identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild type (WT) Torö and this correlated with enhanced pathogenicity in vivo. The E protein is also the major target of neutralizing antibodies, thus genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we choose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains.

Conclusions: Our findings show that two amino acid substitutions of the E protein found in 93/783, A83T and A463S, enhanced Torö infection of neurons as well as pathogenesis in vivo, furthermore we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.

Neurobiology of Disease

Tick-borne encephalitis virus

strain

European subtype

envelope protein

pathogenesis

neurovirulence

Tick-borne encephalitis virus (TBEV) is positive-sense, single-stranded RNA virus belonging to the family of Flaviviridae and genus Flavivirus. In Europe, TBEV is considered to be the most important arthropod-borne viral infection, and causes about 10,000–13,000 cases worldwide every year [1, 2]. TBEV is most commonly transmitted by the bite of an infected tick, but transmission by consumption of unpasteurized dairy products [3, 4] as well as via solid organ transplantation may occur [5]. The symptoms of a TBEV infection ranges from a subclinical form to a mild febrile illness, to meningitis and encephalitis. The exact determinants of the disease severity are not known, however, infectious dose, age, genotype and immune status of the host as well as the virulence of the virus strain are thought to play a role [6–8].

Based on phylogenetic differences TBEV is divided into three different subtypes, European, Siberian and Far Eastern [9]. At least two more subtypes, the Baikalian and the Himalayan subtype are proposed [10]. The genetic difference on the amino acid level is up to 2% within a subtype and 5–6% in between subtypes [11]. In general, the European subtype is associated with milder disease and lower case fatality rates (0.5-2%) compared to the Siberian and Far Eastern subtypes (fatality rates between 5–20%) [12, 13]. However, both mild and severe cases have been observed with all three subtypes [14].

Once infected there is no specific antiviral therapy against TBEV so treatment is restricted to supportive care and symptomatic therapy. However, there are two commercially available vaccines in Europe, FSME-IMMUN® and Encepur®, which are considered to be safe and efficacious [15, 16]. Although the vaccine is considered to be very efficient, vaccine breakthroughs have occurred despite complete vaccination [17–20]. Previous studies have shown that serum from vaccine breakthrough patients contained high titers of antibodies able to neutralize the vaccine strain Neudörfl (FSME-IMMUN®) and the closely related strain Hypr [17, 21]. Furthermore, it was shown that neutralizing titers decreased with increased genetic diversity from the Hypr strain [21], thus genetic variance in natural isolates might contribute to vaccine breakthroughs.

Not much is known about the viral determinants responsible for disease severity in TBEV infection, since the virus is often undetectable in TBE patients, thus making the correlation between virus strain and disease severity difficult. Previous studies have shown that cell culture adaptations of TBEV have led to amino acid changes in the envelope (E) protein resulting in stronger viral interactions with glycosaminoglycans, which was accompanied with attenuated neuroinvasiveness [22, 23]. But so far, no amino acid substitutions in the E protein have been found to increase TBEV infection of the main target cell in the brain, the neurons.

Here we wanted to identify novel virulence determinants in TBEV infection. We infected mice with nine different TBEV strains. We identified 93/783 (European subtype) as a highly neurovirulent and pathogenic TBEV strain compared to strains from the European, Siberian and Far Eastern subtypes. After sequence alignment we found two unique amino acid residues in the E protein of 93/783 that was cloned into the infectious clone of Torö (based on the Torö-2003 strain, European subtype). We found that transfer of the E protein of 93/783 into Torö enhanced the infection rate of neurons and also enhanced the pathogenesis in mice. Compared to the vaccine strains of TBEV, these strains differ in regions of the E-protein known to be involved in neutralization. Therefore, we investigated the neutralizing capacity of sera from people vaccinated with FMSE-IMMUN®, Encepur® and a combination of both vaccines against these viruses. In general, we found lower neutralizing antibody titers against all tested strains in the Encepur® group compared to the other two groups. Furthermore, FSME-IMMUNE® and the combined vaccine groups showed higher titers against Neudörfl compared to 93/783 and Torö strains.

Ethic statement

Infection experiments with animal were performed in compliance with the German animal welfare law (TierSchG BGBl. S. 1105; 25.05.1998) at the Helmholtz Center for Infection Research in Braunschweig. The mice were housed and handled in accordance with good animal practice as defined by FELASA. All animal experiments were approved by the Lower Saxony State Office of Consumer Protection and Food Safety under permit number AZ 33.19-42502-04-15/1895. For primary cell isolation, mice were maintained under specific pathogen-free conditions and experiments were approved and carried out according to the guidelines set out by the regional animal ethical committee (Umeå, Sweden) under approval number A77-14. For the serological analysis: The research was carried out in line with “The Code of Ethics of the World Medical Association” (Declaration of Helsinki) and according to good clinical practice guidelines. In accordance with local legislation, no formal approval by a research ethics committee was required, because either anonymous samples or sera for research purposes were used.

Viruses and cells

VeroB4 cells were cultured in medium 199/EBSS (Hyclone) supplemented with 10% FBS (Hyclone), 100 U/mL of penicillin and 100 µg/mL streptomycin (Gibco). Astrocytes and neurons were isolated and cultured as previously described [24, 25]. In brief, astrocytes were isolated from cerebral cortices between postnatal day 1 and 4, seeded on poly-D-lysine (Sigma) coated T75 flasks and cultured in DMEM (Sigma) supplemented with 10% heat-inactivated FBS (Hyclone), 100 U/mL of penicillin and 100 µg/mL streptomycin (Gibco) and 2 mM L-glutamine (Gibco). Neurons were isolated at embryonic day 17 and seeded on poly-D-lysine coated plates and cultured in neurobasal medium supplemented with B27 (Gibco), 100 U/mL of penicillin and 100 µg/mL streptomycin (Gibco) and 2 mM L-glutamine (Gibco). 93/783 virus strain was isolated from the serum of a 56 year old male in Sweden. Serum samples were taken 5 days post onset of symptoms and the patient later developed a biphasic course of disease [26], the virus was a kind gift of S. Vene (Folkhälsoinstitutet, Stockholm, Sweden), Torö-2003 (from now on denoted as Torö) [27], M14/10, HM467/09, Neudörfl, Hypr 71 (from now on denoted Hypr), AS33 [28] (Table 1). All virus stocks were grown and titrated on VeroB4 cells.

Virus strain	TBEV subtype	Source of isolation	Year of Isolation	Country of isolation	Passage history	Reference
Torö-2003	European	I ricinus	2003	Sweden	Infectious clone[27], 2 passages in VeroB4 cells	[27, 29] GenBank Accession no. DQ401140.3
HM467/09	European	I ricinus	2009	Germany	3 passages in VeroB4 cells
Neudörfl	European	I ricinus	1970	Austria	Multiple lab passages in mouse brain and call culture
Hypr71	European	Human blood	1953	Czechoslovakia	Multiple lab passages in mouse brain and cell culture
AS33	European	I ricinus	2005	Germany	7 passages in Vero B4 cells	[28] GenBank Accession no GQ266392.1
93/783	European	Serum of a 56 year old male		Sweden	Isolation in suckling mouse brain, 4 passages in Vero B4 cells.	[26]
M14/10	Siberian	I persulcatus	2010	Mongolia	7 passages in Vero B4 cells	[30]
Aina	Siberian	Human blood	1963	Russia		[31]
Sofjin	Far Eastern	Human brain		Russia	Multiple lab passages in mouse brain and cell culture	[32]

Viral infection of mice

C57BL/6 mice were bought from Harlan laboratories/ENVIGO. Six- to ten-week-old mice were intraperitoneally infected with the 10⁴ focus forming units (FFU) of the indicated strain of TBEV in 100-µl phosphate buffered saline (PBS). For intracranial infections, mice were anesthetized by intraperitoneal injection with a mixture of ketamine (100 µg/g body weight) and xylazine (5 µg/g body weight). Mice were then injected with 10² FFU of the indicated strain of TBEV in 20 µl PBS. Mice that lost more than 20% of their body weight were sacrificed. For organ harvesting, mice were perfused with 20 ml of PBS and organs were harvested in Trizol reagent for RNA extraction. Experiments were performed in the biosafety level 3 (BSL3) facility at the Helmholtz Center for Infection Research.

Rescue and cloning of chimeric virus

Generation of a chimeric clone containing E gene of 93/783 in the Torö background. The infectious clone was constructed using two plasmids: pTBEV-CME and pTBEV-luc-rep for Torö[29, 33]. pTBEV-CME comprises the 5´NCR, the structural genes, and 124 bp of NS1 of Torö-2003 cloned into the pcDNA3.1 vector. The E gene in pTBEV-CME was replaced with the E gene of 93/783 using BbvCI and ApaI restriction sites to generate pTBEV-CME^93/783. The cloning work was performed using standard molecular biology procedures, and verified by sequencing (Eurofins MWG Operon, Ebersberg, Germany). Two overlapping PCR fragments comprising 5´NCR-NS1 and NS1-3´NCR of TBEV were amplified from pTBEV-CME^93/783 and pTBEV-luc-rep, respectively. SP6 promoter sequence was introduced upstream 5´NCR in the first PCR fragment. Both PCR fragments were mixed by 1:1 Molar ratio and 12 cycles of denaturation, annealing and elongation were performed to generate the full length TBEV DNA with SP6 promotor sequence. The overlapping sequence in both fragments facilitated to anneal the fragments with one another, followed by elongation with KOD KOD Hot Start DNA polymerase (Novagen®). Capped RNA transcripts of complete TBEV genome were synthesized using MEGAscript® SP6 in vitro transcription kit (Ambion) as per the manufacturer’s instructions. A mixed (1:1) population of HEK293 and VeroB4 cells was transfected with in vitro-transcribed RNA to rescue the infectious virus as previously described [27].

RNA isolation and qPCR

RNA was isolated from cells and organs as previously described [24, 34]. Total RNA (500 ng for tissue samples and 200 ng for cultured cells) was used to synthesize cDNA using Quantitect reverse transcription kit (Qiagen) according to the manufacturer’s instructions. GAPDH transcripts were detected by Quantitect primer assay (Qiagen) and the KAPA SYBR FAST qPCR kit (KAPA Biosystems), TBEV transcripts were detected using previously described primers and probes [35], qPCR was run using a StepOnePlus fast real-time PCR system (Applied Biosystems).

Viral infections, virus initial infection and entry assay

TBEV was titrated using focusing forming assay as previously described [36]. In brief, viruses were serial diluted in 10-fold dilutions, followed by 1 h of inoculation at 37°C and 5% CO₂ on VeroB4 cells. After 48 h of infection cells were fixed with 4% formaldehyde, permeabilized in PBS containing 0.5% trition-X-100 and 20 mM glycine. Virus was detected using primary monoclonal mouse antibodies directed against TBEV E (1:1000, 1786,3 [37]) and secondary anti-mouse HRP conjugated antibodies (1:2000, Thermo Fisher Scientific). Viral foci were then revealed by incubation with TrueBlue peroxidase substrate (KPL, Gaithersburg, MD). TROPHOS assay was performed as previously described [24, 25].

The initial infection events including binding and entry was analyzed by infecting cells with an MOI of 1 for 1 h at 37°C 5% CO₂, followed by three washes with PBS and before cell lysis. To analyze entry of virions into cells fresh medium was added after 1 h infection and cells were incubated for another 2 h at 37°C and 5% CO₂ before being washed with PBS-EDTA, trypsinized for 10 minutes, washed 3 times in PBS and then lysed. Viral burdens were measured using qPCR and presented in relation to 93/783.

Flow cytometry

Neurons were isolated from C57BL/6 mice at embryonic day 17 and cultured for 7 days before infection with 0.1 MOI of 93/783, Torö93E or Torö at 37°C 5% CO₂ for 1 h. After 12 h of infection cells were treated with 5 µg/mL of brefeldin A. 24 hours post infection (h.p.i) cells were fixed in 4% formaldehyde. Then permeabilized in PBS 20 mM EDTA, 2% FBS, 0.02% NaN₃ and 0.2% Tween-20, stained with primary antibodies monoclonal mouse antibodies directed against TBEV E (1:500, 1786) and polyclonal rabbit antibodies directed against tubulin β3 (1:500 Biolegend PRB-435P) for 60 minutes and secondary antibodies, donkey anti-rabbit Alexa Fluor 488 (1:2000, A21206 Thermo Fisher Scientific) and donkey anti-mouse Alexa Fluor 647 (1:2000, A31571 Thermo Fisher Scientific) for 60 minutes. After one wash cells were taken up in PBS 2% FBS and analyzed with flow cytometry using a FACSLSRII instrument (Becton Dickinson). Results were analyzed using FACSDiva software (Becton Dickinson). Tubulin β3 was used to gate for neurons and the percentage of infected cells was determined within this population.

Serum samples

A total of 36 anonymous serum samples from TBE vaccine recipients was included from the German National Consultant Laboratory for TBEV in our study. Sera were stored at -80° C until use. Sera from TBE vaccinated individuals (FSME Immun, Pfizer (no. 11); Encepur, GSK (no. 12); or a combination of FSME Immun and Encepur (no. 13)) who, according to anamnestic information, had never suffered from TBE. All individuals had a complete basic immunization and the last vaccine shot was more than three months before blood sampling.

Focusing forming reduction neutralization test

Sera were diluted 1:5 in DMEM supplemented with 100 U/mL of penicillin and 100 µg/mL streptomycin (Gibco) and inactivated at 56°C for 30 minutes and were further diluted in two-fold serial dilutions in DMEM. Diluted sera were mixed with 100 FFU of 93/783, Torö or Neudörfl and incubated at 37°C 5% CO₂ for 30 minutes and were then added to VeroB4 cells. Viral foci were revealed by focusing forming assay. Neutralizing antibody titers were calculated as the reciprocal of serum dilution that resulted in 80% reduction of foci compared to virus control. Sera from an unvaccinated subject was used as negative control.

Statistical analysis

Statistical analysis were performed using GraphPad Prism Software. Unpaired t test was used to analyze all cell culture data, Mann-Whitney test was used to analyze difference of viral burdens in infected mice, log-rank (Mantel-Cox) test was used to analyze significance in survival experiments. Multiple comparison Friedman Dunn's multiple comparisons test was used to analyze the different vaccines ability to neutralize the different TBEV strains and multiple comparison Kruskal Wallis multiple comparison Dunn's multiple comparisons test was used to analyze the effects of different vaccines on the Swedish strains. The specific tests used are indicated in the figure legends.

93/783 is a highly pathogenic and neurovirulent TBEV strain

In order to identify TBEV virulence determinants, C57BL/6 mice were intraperitoneally infected with 10⁴ focus forming units (FFU) of 6 different TBEV strains beloning to the European subtype (Fig. 1A). Mice were highly susceptible to all TBEV strains, and all mice died within 11 days, except for AS33 infected mice that showed 80% mortality at 13 days post infection. Mice infected with 93/783 died signficantly earlier compared to mice infected with the other 5 European strains (p < 0.001 vs Torö, Hypr and AS33, and p < 0.01 vs HM467/09 and Neudörfl, significance were calculated using Log-rank (Mantel-Cox)). To further investigate the pathogenicity of 93/783 we decided to compare it to Torö, which is an infectious cDNA clone that can be genetically manipulated. Torö has a similar pathogenicity profile as other European strains (Hypr and Neudörfl) which makes it a suitable model to investigate virulence determinants. The Siberian and Far Eastern subtypes of TBEV have been associated with more severe disease compared to the European subtypes [12, 13]. To compare the pathogenicity of 93/783 and Torö to Siberian and Far Eastern TBEV strains we infected mice intraperitoneally with 93/783 and Torö as well as two Siberian strains, M14/10 and Aina, and one Far Eastern strain Sofjin (Fig. 1B). Mice infected with 93/783 died significantly earlier compared to mice infected with all other strains (Sofjin p < 0.001, Aina and M14/10 p < 0.01 Log-rank (Mantel-Cox) test) while Torö infected mice survived longer compared to mice infected with the Siberian strains. Thus 93/783 was shown to be more pathogenic than all tested TBEV strains, even those beloning to other subtypes.

The pathogenecity of a neurotropic virus is largely dependent on its neuroinvasiveness (ability to enter the CNS) and its neurovirulence (ability to cause disease within the CNS). To investigate the neurovirulence of the two different TBEV strains, mice were infected intracranially with 10² FFU of 93/783 or Torö (Fig. 1C). Both strains were highly neurovirulent, and all mice died within 8 days of infection. However, mice infected with 93/783 died significantly earlier than Torö infected mice (p < 0.001) indicating that 93/783 is more neurovirulent. Taken together we concluded that 93/783 is a highly pathogenic and neurovirulent TBEV strain.

93/783 replicates better within the brain

As 93/783 was found to be more neurovirulent compared to Torö, we wanted to investigate if this correlated with increased viral replication within a certain brain region. Mice were intracranially infected with 10² FFU of Torö or 93/783, and different brain regions were isolated at 1, 2 and 5 days post infection (d.p.i.) (Fig. 2). Viral replication was detected with both strains in all brain regions, olfactory bulb, cerebrum, cerebellum and brainstem. Higher levels of viral replication in 93/783 infected mice were found in olfactory bulb (Fig. 2A), cerebrum (Fig. 2B) and brainstem (Fig. 2D) at 2 d.p.i and in all four different brain regions at 5 d.p.i. Interestingly viral load of 93/783 increased immediately between day 1 to day 2 in olfactory bulb, cerebrum and brainstem whereas no signficant increase was found in Torö infected mice. Thus higher neurovirulence of 93/783 correlated with increased viral replication at early timepoints and higher viral burdens at 5 d.p.i within all parts of the brain.

93/783 possess two unique aminoacid substitutions in the envelope protein which are important for neuron infection

Sequence alignements of all investigated strains was performed to identify unique amino acid substitutions in 93/783 that could contribute to the increased pathogenicity. In the E protein of 93/783, we found two unique amino acids which were not found in any of the other strains used for the survival experiments (Fig. 1). The alanine at residue 83 (Torö) in domain II of the E protein was a threonine in 93/783 and the alanine at residue 463 (Torö) in the stem anchor region was a serine in 93/783 (Fig. 3A-B). Residue 83 in domain II was found to be located on the surface of the virion particle whereas residue 463 is burried in the membrane. The E protein is involved in receptor binding, membran fusion and also constitute the main target for neutralizing antibodies [38, 39], thus the E protein plays an important role in pathogenesis. To determine the function of these amino acid substitutions, a chimeric Torö virus was generated replacing the E protein with the E protein of 93/783, denoted Torö93E (Fig. 3C).

A previous study has shown that reduced TBEV growth in neuroblastoma cells correlated with attenuation and reduced neuroinvasiveness in mice [40]. Since we observed that 93/783 was more neurovirulent and replicated better within the mouse brain compared to Torö, we investigated the replication ability of the different strains in primary astrocytes and neurons which are the most abundant cell types in the brain. Mouse embryonic fibroblast (MEF) were used as control to monitor viral replicataion in a non-CNS cell type. Cells were infected with 93/783, Torö93E and Torö and viral replication was determined at different time points by qPCR (Fig. 3D-F). The 3 h.p.i time point was used as an input control and RNA levels were normalized against the input using the ∆∆CT method. The percentage infected cells was analyzed by immunoflourescent staining of the TBEV E protein (Fig. 3G-I). In MEFs no significant difference between the different TBEV strains was found (3D and G). In astrocytes 93/783 replicated better compared to Torö and Torö93E at both 24 and 48 h.p.i (Fig. 3E), however, this was not due to more cells being infected (Fig. 3H). Torö and Torö93E showed very similar replication and infection efficiency, indicating that the E protein is not influencing the replication efficiency in astrocytes (Fig. 3E and H). The TBEV replication was similar for the three strains in neurons at later time points (Fig. 3F), however, we observed more neurons infected with 93/783 and Torö93E compared to Torö at 48 h.p.i (Fig. 3I), thus the E protein of 93/783 might influence infection efficiency in neurons. Taken together we found that the E protein of 93/783 enhanced Torö viral spread in neurons specifically, which could be of importance for pathogenesis.

The E protein of 93/783 enhances binding, entry and infection of neurons

Since the E protein of TBEV is involved in receptor binding, membrane fusion and entry we wanted to investigate the mechanism of enhanced initial infection of 93/783. To analyse the early initial events during infection we blocked the secretory pathway by treating the cells with 5 µg/mL brefeldin A (BFA) at 12 h.p.i before the release of new virions have started (Fig. 4A-D). Cells were fixed 24 h.p.i and stained with antibodies against the TBEV E protein. We found no significant difference in the infection rate between the different strains in MEFs or astrocytes (Fig. 4A and B). However, 93/783 as well as Torö93E infected neurons much more efficently compared to Torö (Fig. 4C and D). Since the E protein mediates receptor binding and membrane fusion we wanted to charcterize the initial binding and entry events under physiological conditions, at 37°C. Cells were infected for 1 h and lysed for RNA extraction (Fig. 4E) or infected for 3 h followed by trypsinization (Fig. 4F), to remove bound viruses which did not enter the cell before RNA extraction. The viral burdens were measured by qPCR and were presented in relation to the 93/783 strain. The 93/783 strain showed much more efficient receptor binding and entry into neurons compared to the Torö strain. This was mediated by the E-protein of 93/783 strain, because the chimeric virus showed better infection at 1 and 3 h.p.i compared to the Torö strain (Fig. 4E and F). Taken together, 93/783 was found to be a highly pathogenic TBEV strain in mice and by introducing the E protein of 93/783 into Torö the initial infection and entry into neurons was enhanced, indicating that the amino acid substitions A83T and A463S might be important virulence determinants by increasing neuronal infection.

E protein of 93/783 enhances pathogenicity of Torö

As we found that Torö93E infect neurons more efficiently compared to Torö, we set out to investigate if this was important for pathogenesis in vivo. Mice were infected intraperitoneally with 93/783, Torö or Torö93E and survival was monitored over time (Fig. 5). The medium suvival time of 93/783 infected mice was 7.5 days. Torö infected mice survived longer with a medium survial time of 11.5 days. Mice infected with Torö93E died slightly earlier compared to mice infected with Torö with a medium survival time of 10.4 days (P = 0,0847 Log-rank (Mantel-Cox) test), thus the E protein of 93/783 may contribute to the pathogenicity.

TBEV vaccine neutralizes vaccine strain more efficently compared to natural isolates

Over the years there has been an increase in TBE vaccine breakthroughs in Sweden [14, 18], but the underlying mechanisms are not fully known. Neutralizing antibodies against the E protein play an important role in controling TBEV infections. We hypothesized that naturally circulating Swedish TBEV strains may not be neutralized equally efficient by vaccine derived antibodies due to the detected amino acid differences in exposed regions of the E protein know to interact with neutralizing antibodies.

Sequence alignment of the E protein was performed, comparing amino acid differences among the vaccine strains Neudörfl (FSME IMMUNE®) and K23 (Encepur®) and the Swedish strains Torö and 93/783 (Fig. 6A). We found 5 amino acid in the E protein which differed between the strains. The amino acid substutition A83T, that was unique in 93/783 among the strains used in our survival analysis, was also found in the vaccine strain K23. K23 also harboured two unique amino acid substitutions N52K and K136R which were not found in Torö, Neudörfl or 93/783, whereas Neudörfl possesed one unique amino acid substition V167I compared to the other strains. The A463S amino acid substitution was only found in 93/783 (data not shown). One of the amino acid substitions in 93/783, A83T was found close to the interface between E-protein and neutralizing antibodies (Fig. 6F and H) [37, 39] thus this substition might be important for neutralization.

To test our hypothesis we performed focusing forming neutralization test on sera from 36 vaccinated individuals. The induviduals were vaccinateds either by FSME-IMMUN® (based on Neudörfl strain, N = 11), Encepur® (based on K23 strain, N = 12) or a combination of both vaccines (N = 13). The neutralization efficiency was tested against the two Swedish strains 93/783 and Torö as well as the vaccine strain Neudörfl.

The human sera from individuals vaccinated by Neudörfl-based FSME-IMMUN® or a combination of both vaccines (Fig. 6B and D) neutralized Neudörfl significantly better compared to the Swedish isolates Torö and 93/783. Since V167I is the only difference between Neudörfl and the Swedish strains in the E protein, this indicates that this residue might contribute to the difference in neutralizing antibody titers (Fig. 6F-G). We also found that FMSE-IMMUN® or a combination of both vaccines neutralized the Swedish strains Torö and 93/783 more efficiently compared to Encepur® (Fig. 6E). This might be due to the fact that the E protein of the Swedish strains is more similar to Neudörfl compared to K23 (Fig. 6A). Despite this, both FSME-IMMUN® and the combination of both vaccines resulted in sera with neutralizing activity above the treshold for what is considered neutralizing titers. Interestingly, the sera from K23-based Encepur® vaccine showed overall lower neutralizing titers to all three strains tested (Fig. 6B-E), with some sera not reaching to neutalizing titers, but there were no significant difference between the strains (Fig. 6C). This indicates that the residue N52K is important for generating a neutralizing response against several different viruses. This amino acid has been shown previously to be involved in the antibody epitope (Fig. 6F-G) in other studies [39], thus genetic divergence resulted in reduced neutralizing antibody titers and might be a contributing factor to vaccine breakthroughs.

Here we identified novel virulence determinants contributing to disease severity in TBEV infection. By screening different TBEV strains in mice we identified one highly pathogenic European strain, 93/783. 93/783 is a low passage strain isolated from the sera of a human TBE patient in Sweden, who later developed encephalitis [26]. The human isolate 93/783 has been under selection pressure of a mammalian immune response whereas the other strains are either tick isolates (Torö, M14/10, HM467/11) or references strain with high passage number in cell culture (Hypr, Neudörfl, Aina Sofjin). This might be one factor contributing to the high virulence of 93/783, as passaging of TBEV in cell culture has previously been reported to result in attenuation in vivo [22, 41]. However, when we analyzed the prevalence of the T83 residue in other TBEV strains deposited in GenBank (using: www.viprbrc.org) we found only 7 TBEV sequences out of more than 1000, indicating that it is a quite rare amino acid at this position. Furthermore, the viruses carrying this rare amino acid in position 83 are isolates from both ticks and patients [42, 43] and belong to different subtypes, suggesting that it is neither a common mammalian adaptation of the virus, nor a specific trait of a certain subtype.

We found 93/783 to be more neurovirulent than Torö. An immediate increase in viral replication was observed in brains intracranially infected with 93/783 mice, whereas viral replication was only detectable 5 d.p.i in Torö infected mice. Faster spread and higher viral burdens might be due to more infected cells in the brain. This correlated with faster disease development and could be important determinants of pathogenesis. The E protein is involved in receptor binding and membrane fusion and could be of great importance for fast spread and infection within the brain. Apart from its role in receptor binding and membrane fusion, the E protein also constitutes the most important antigenic structure [38]. Previous studies have indicated that the E protein is important for pathogenesis, amino acid changes in the E protein which resulted in increased net positive charge showed lower pathogenicity and neuroinvasiveness in vivo [22, 41, 44]. The increased in positive charge results in stronger binding to glycosaminoglycans which is an advantage in vitro, but reduces the neuroinvasiveness of the virus in vivo by impeding the virus ability to spread from cell to cell [44]. Similar findings have been observed for mosquito-borne flaviviruses [45]. Furthermore, an antibody escape mutant, containing a Y384H mutation in the E protein, also resulted in reduced neuroinvasivness [46] demonstrating the diverse roles of the E protein in TBEV pathogenesis. To test the importance of A83T and A463S amino acids a chimeric Torö93E virus was generated, by transferring the E gene from 93/783 into Torö. We found that the E protein of 93/783 mediated better binding and infection into neurons, and also contributed to the pathogenicity in mice. Since the A83T amino acid substitution is located on the surface it is more likely that this amino acid contributes to increased infection of neurons.

Interestingly, we saw no difference between the strains during the infection in MEFs, indicating that the effect of A83T residue on virus entry and infection is cell-type-specific for neurons. In astrocytes, no difference in number of infected cells were detected between the strains, and this might be due to low number of infected cells and the rare event of TBEV infected astrocytes [34, 47, 48]. The low infection rate could be explained by the fact that the fast type I interferon response of astrocytes prevents the infection of the neighboring cells [25]. However, 93/783 replicated more efficiently in astrocytes compared to Torö, whereas the chimera virus behaved like Torö. This could be explained by amino acid differences in other parts of the virus, both in other structural as well as non-structural (NS) proteins e.g. NS3 and NS5. Which could contribute to pathogenesis. These amino acid differences might explain the moderate change in pathogenicity of the chimeric virus in mice. These differences will be further investigated.

There are two commercially available vaccines against TBEV in Europe. These are considered to be safe and efficacious, however, vaccine breakthroughs occur [14]. In a retrospective study concerning reported TBEV cases in the Stockholm County in Sweden during 2006 to 2015, it was found that 81% of vaccine failure patients were over 50 years old, 26% of the patients had diseases which involved the immune system, the mortality rate was 6% [49]. The exact mechanisms behind the vaccine breakthroughs are not known, but the immune response of the patient as well as the genetics of the viral strain might contribute.

Here we wanted to investigate if natural variation of in the E protein could affect the efficiency of neutralization by the TBEV vaccines. When we compared the amino acid sequence of the E protein of Torö, 93/783 and the two vaccine strains Neudörfl and K23, we found that the amino acid differences among the strains were in previously identified neutralizing epitopes and neighboring residues had been found to affect neutralization [39]. To investigate the role of these amino acid differences for neutralizing antibody titers we tested the sera from individuals vaccinated with either FSME-IMMUN®, Encepur or a combination of both vaccines. We found that sera from FSME-IMMUN® vaccinated patients possessed higher neutralizing titers against the vaccine strain Neudörfl compared to the natural isolates Torö and 93/783. Neutralizing antibodies have been shown to be induced by both FSME-IMMUN® and Encepur®, and vaccine-induced protection against TBEV has been shown to be mediated by antibodies against the E protein [50–52]. The only difference in amino acid sequence of the E protein of Torö and Neudörfl is the I167V which could be important for neutralization and explain the higher neutralizing titers against Neudörfl in FSME-IMMUN® vaccinated patients.

There was no significant difference in neutralizing titers against Torö, 93/783 or Neudörfl in the sera of Encepur® vaccinated individuals. Interestingly, FSME-IMMUN® was shown to the neutralize Swedish strains better compared to Encepur®, where some sera showed borderline neutralizing titers after Encepur vaccination and one serum did not neutralize 93/783 at all. A previous study found that the amino acid K52 is very important for efficient neutralization by Encepur®, this amino acid is found in the K23 vaccine strain [53], however, this is the only TBEV strain found in the gene bank database sequenced that contains this amino acid in that position (www.viprbrc.org). This could also explain why the Swedish isolates which lacks the K52 are not neutralized as efficiently by Encepur®. Thus, genetic divergence from the vaccine strain might reduce neutralizing antibody titers and might be a risk factor for vaccine breakthroughs.

In summary, we identified two unique amino acids in the E protein of a highly pathogenic and neurovirulent TBEV strain, which enhanced TBEV infection and entry of neurons. The A83T substitution does not change the charge of the E protein but specifically increase TBEV entry and infection of neurons, TBEVs primary target cell in vivo. Furthermore, this correlated with higher infection in the brain and increased pathogenicity in mice. In contrast, this amino acid is not directly involved in determining the neutralization capability of the vaccines FSME-IMMUN® and Encepur®. However, lower neutralizing titers against the Swedish strains was observed with the Encepur vaccinated sera, compared to FSME-IMMUNE, probably because of position 52 in the vaccine strain K23 (Encepur®). Sera from FSME-Immune and combined vaccination showed overall higher neutralizing titers against the Swedish strains. Since these observations are from a relatively small sample size, they need to be followed up thoroughly in order to give clear advice about vaccinations.

CNS

Central nervous system; hpi:hours post infection; d.p.i:days post infection; BFA:brefeldin A; E:envelope; MEF:Mouse embryonic fibroblast; qPCR:Quantitative reverse transcription PCR; PBS:phosphate buffered saline; FFU:Focus forming unit; WT:Wild-type; TBEV:Tick-borne encephalitis virus;

Ethics approval: Infection experiments with animal were performed in compliance with the German animal welfare law (TierSchG BGBl. S. 1105; 25.05.1998) at the Helmholtz Center for Infection Research in Braunschweig. The mice were housed and handled in accordance with good animal practice as defined by FELASA. All animal experiments were approved by the Lower Saxony State Office of Consumer Protection and Food Safety under permit number AZ 33.19-42502-04-15/1895. For primary cell isolation, mice were maintained under specific pathogen-free conditions and experiments were approved and carried out according to the guidelines set out by the regional animal ethical committee (Umeå, Sweden) under approval number A77-14. For the serological analysis: The research was carried out in line with “The Code of Ethics of the World Medical Association (Declaration of Helsinki)” and according to good clinical practice guidelines. In accordance with local legislation, no formal approval by a research ethics committee was required, because either anonymous samples or sera for research purposes were used.

Consent to participate: Not applicable.

Consent for publication: Not applicable

Availability of data and material: All data and material is available upon request.

Competing interests: The authors declare that they have no competing interests.

Author’s contributions: R.L. performed all primary cell culture experiments. E.W. and S.S. performed the in vivo infections. E.R. did the modelling of the E protein. E.R. and R.L. performed the sequence analysis. N.A. constructed the chimeric viruses. A.L. did the FACS analysis. G.D. and M.B. provided the human sera. R.L., A.K and A.K.Ö. designed the study and wrote the manuscript. All authors read and critical revised the final version of the manuscript.

Acknowledgement: The authors thank S. Vene for providing the 93/783 strain.

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The Envelope Protein of Tick-Borne Encephalitis Virus Influence Neuron Entry, Pathogenicity and Vaccine Protection

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Abstract

Figures

Background

Methods

Ethic statement

Viruses and cells

Viral infection of mice

Rescue and cloning of chimeric virus

RNA isolation and qPCR

Viral infections, virus initial infection and entry assay

Flow cytometry

Serum samples

Focusing forming reduction neutralization test

Statistical analysis

Results

93/783 is a highly pathogenic and neurovirulent TBEV strain

93/783 replicates better within the brain

The E protein of 93/783 enhances binding, entry and infection of neurons

E protein of 93/783 enhances pathogenicity of Torö

TBEV vaccine neutralizes vaccine strain more efficently compared to natural isolates

Discussion

Conclusions

Abbreviations

Declarations

References

Status:

Journal Publication

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