Creating an mRNA Vaccine for Tick-Borne Encephalitis by Choosing a Prototype Virus Strain

The mRNA-LNPs encoding TBEV prM/E genes were synthesized in vitro, purified, and encapsulated into lipid nanoparticles via microfluidic mixing. For mRNA production, linearized plasmid constructions based on the pBAC vector (Lucigen) were used to obtain mRNA containing the genes of the PrM/E TBEV. Plasmids preparations were performed as previously described [23,30,31], utilizing open reading frames of the target antigens (Table 1). The mRNA encapsulation efficiency into lipid particles and concentration were determined by SYBR Green dye (SYBR Green I, Lumiprobe, Moscow, Russia), followed by monitoring the change in fluorescence.

The following cell lines were used in this study: SPEV (BioLot, St Petersburg, Russia) for TBEV cultivation and SH-SY5Y (ATCC CRL-2266) for virus titration and assessment of virus-neutralization activity. Cells were cultivated at 37 °C and 5% CO₂ in DMEM culture medium (PanEco, Moscow, Russia) with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

A total of 10 virus strains were used: 3 of Far-Eastern subtype (TBEV-FE, Sofjin, Krasny-Yar-8 and 205 strains); 1 of European subtype (TBEV-Eu, Zmeinogorsk-1 strain); 4 of Siberian subtype (TBEV-Sib, Lesopark-11, Aina-1448, Vologda-2 and Buzuuchuk (Kyrgyz line) strains); 1 of Baikal subtype (TBEV-Baik) 258-83 and 1 of 4th subtype (prototype strain 178-79). Virus titration was carried out using the plaque assay on SPEV cell monolayers using an overlay of 1.5% carboxymethylcellulose (CMC). After 96 h of incubation, the number of plaques was counted and calculated with the titers in plaque-forming units per milliliter (PFU/mL) using a standard formula accounting for the dilution of the virus suspension. Moreover, for the determination of virus titers in TCID50 titrations were performed on SH-SY5Y cell line.

Animal sera were heat-inactivated at 56 °C for 30 min. Serial two-fold dilutions of the sera (starting from 1:10) were incubated with 100 TCID₅₀ of corresponding TBEV strain at 37 °C for 1 h. Subsequently, the mixture was transferred onto a monolayer of SH-SY5Y cells in 96-well plates. The plates were incubated for 96 h at 37 °C with 5% CO₂.

Each experiment included a virus control (without serum) and a cell control (without virus). The 50% neutralization titer (NT₅₀) was determined using an MTT assay. An MTT solution (3 mg/mL) was added to the wells, and the plates were incubated for 3 to 5 h at 37 °C. After incubation, the medium was removed from the wells and replaced with DMSO. The optical density (OD) was measured using a plate reader at 570 nm.

All procedures with animals were carried out according to current guidelines on the care and use of laboratory animals and approved by the Local Ethics Committee of N.F. Gamaleya National Research Center for Epidemiology and Microbiology (protocol No. 76 from 24 September 2024). Female mice from line BALB/c weighing 18–20 g (aged 6–8 weeks) were used in the study. Mice were acquired from Cyagen (China) animal nursery. Animals were kept in the vivarium at 22 ± 2 °C, relative humidity of 50 ± 10% and 12/12 h light/dark cycle. All mice were subjected to a 7-day quarantine prior to the experiment. All manipulations were conducted under biosafety level 3 (BSL-3) laboratory conditions in accordance with the 3R principles (Replacement, Reduction, Refinement). If an animal lost more than 20% of its initial body mass or demonstrated severe neurological impairments, it was humanely euthanized by cervical dislocation under deep anesthesia. Animals would be anesthetized by intramuscular injection of zoletil–xylazine (45 mg/kg and 7.5 mg/kg, respectively).

To determine the LD₅₀ five virus dilutions (10⁵, 10³, 10¹, 5 and 2 PFU/animal) were prepared in sterile phosphate-buffered saline (PBS). Seven virus strains were used: Sofjin, Krasny Yar-8, 205, Zmeinogorsk-1, Lesopark-11, 178-79 and 258-83. Each virus dilution was administered intraperitoneally (i.p.) to a group of 6 mice in a volume of 100 μL. The control group (n = 6) received an equivalent volume of PBS. The general condition, behavior, and clinical symptoms of the animals were monitored for 12 days. The first death occurred on day 7 post infection. Body weight was measured once every 7 days until the first deaths occurred, and then daily thereafter.

To evaluate the cross-reactive immune response, 6–8-week-old female BALB/c mice were immunized with one of ten different mRNA-LNP formulations (10 groups, n = 6) (Table 1). Each animal received two 50 μL intramuscularly injections containing 5 µg of mRNA, administered 14 days apart. The mRNA dose was selected based on our previous study [30]. Control mice received an equivalent volume of PBS via the same route. Two weeks after the booster immunization, blood samples were collected via cardiac puncture into clot activator tubes (VACUETTE^®, Greiner Bio-One, Vienna, Austria) for serum isolation. The obtained serum was used to assess virus-neutralizing activity.

To evaluate the protective efficacy, female BALB/c mice aged 6–8 weeks were immunized twice with a 14-day interval. Five mRNA-PrM/E-LNP vaccine variants were used: Sofjin_FE, Krasny Yar_FE, 205_FE, 178-79_4 and a mixture of Sofjin_FE + Krasny Yar_FE; for each vaccine variant, a group of 52 mice was immunized (5 groups, n = 52). Before the challenge infection, the immunized mice from each vaccine group were redistributed. Therefore, 52 immunized mice were divided into eight subgroups (n = 6): seven groups for infection with different TBEV strains (one group = one virus) and one control group of immunization. Additionally, four mice were sacrificed prior to infection to collect serum for analysis. In parallel, control groups (injected with PBS) were formed for their consequent infection with seven TBEV strains: Sofjin_FE, Krasny Yar-8_FE, 205_FE, Zmeinogorsk-1_Eu, Lesopark-11_Sib, 178-79_4, 258-83_Baik (7 groups, n = 6). In 14 days after the second immunization, the animals were infected with TBEV at doses of 100 LD₅₀. LD₅₀ was calculated by the Kerber method based on the previously determined titer of each strain. After infection, animals were weighed daily and their clinical condition was evaluated, documenting the following parameters: body mass, motor activity, presence of neurological symptoms (pareses, paralyses, seizure activity) and survival rate. The animals were monitored for 21 days.

Viral load and virus replication were assessed using quantitative reverse transcription PCR (RT-qPCR) with hybridization-fluorescence detection.

Organ samples (brain, heart, liver, spleen) were homogenized in 1 mL of PBS with protease inhibitors (cOmplete ULTRA Tablets—Mini, EDTA-free, EASYpacks) using aTissueLyser II (50 Hz, 5 min). The homogenates were centrifuged for 20 min at 12,000× g and at 4 °C. The supernatants were collected and stored at −80 °C.

Total RNA was extracted from 100 μL of tissue lysate using ExtractRNA reagent (Eurogen, Moscow, Russia) according to the manufacturer's guide. Reverse transcription and qPCR were conducted using a commercial kit for TBEV RNA detection AmpliSense^® TBEV, B. burgdorferi sl, A. phagocytophillum, E. chaffeensis/E. muris-FL (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) according to the manufacturer's guide. The amplification conditions were as follows: 50 °C for 15 min and 95 °C for 5 min, followed 5 cycles of 95 °C—10 s, 60 °C—35 s and 72 °C—15 s, followed 45 cycles 95 °C—10 s, 56 °C—35 s and 72 °C—30 s.

Data analysis was performed with the CFX 96 Real-Time PCR Detection System software (CFX Manager 3.1, Bio-Rad, Hercules, CA, USA), monitoring the accumulation of the FAM fluorescent signal, which corresponds to the accumulation of TBEV cDNA.

Indirect enzyme-linked immunosorbent assay (ELISA) was used to evaluate the titers of specific anti-TBEV antibodies in serum of immunized mice. Recombinant E proteins were used as the antigen. E proteins obtained from various TBEV strains were used as the antigen: 205_FE), Lesopark-11_Sib, Vologda-2_Sib and 886/84_Baik. A total of 0.5 μg of antigen in purified water was added into the wells of a 96-well immunoassay plate (NEST, Beijing, China). The plate was incubated at 4 °C for 16 h, followed by quintuple washing with PBST (0.01 M PBS with 0.05% Tween-20). Nonspecific binding was blocked with 1% solution of bovine serum albumin (BSA) in PBS (BSA-PBS) at 37 °C for 1 h, followed by subsequent washing with PBST. Consequently, 0.1 mL of investigated serum samples were added in different dilutions in 1% BSA-PBS and incubated at 37 °C for 1 h, once again followed by subsequent washing with PBST. After that, 0.1 mL of HRP-conjugated anti-mouse IgG in BSA-PBS were added and incubated at 25 °C for 45 min. Finally, after another washing with PBST, 0.1 mL of TMB were added and incubated for 10 min at room temperature, then stopped with 0.1 mL of 1 M sulfuric acid.

Endpoint antibody titers (EAT) were determined in serially diluted serum samples. Intact mouse serum (PBS group) was used as a negative control (K-) at dilutions ranging from 1:100 to 1:100,000 with a 10-fold step in PBST–BSA.

Results were interpreted based on the signal-to-noise ratio (S/N), where S represents the optical density (OD) value of the sample and N is the mean OD value of the negative control. Samples with value of S/N > 3 were considered positive.

Statistical analysis, as well as plotting heat maps and graphs, was performed using GraphPad Prism 6.0. Virus-neutralizing antibody levels were compared using the Kruskal–Wallis test and Dunn's multiple comparison post hoc test. Data are expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical significance was set at p < 0.05. ELISA data are presented as geometric means from at least three independent experiments.

We designed and synthesized a panel of ten mRNA-LNP constructs encoding the PrM/E proteins of distinct TBEV strains. The panel was designed to encompass the primary genetic diversity of the virus, including next representatives: three related to the Far-Eastern subtype (Sofjin, Krasny Yar-8, 205), one to the European subtype (Zmeinogorsk-1), four to the Siberian subtype (Lesopark-11, Aina-1448, Vologda-2, Buzuuchuk) and one each to the fourth subtypes (178-79) and Baikal subtype (258-83). For the selection of TBEV strains for vaccine mRNA candidate development, we relied on virus-neutralization data published previously [32], as well as the spectrum of currently dominant subtypes. Thus, according to earlier experiments, immunization with the Krasny Yar variant of TBEV, which belongs to subtype 1, provided the highest virus-neutralizing activity. The main variants used in Russian vaccines are strains belonging to subtype 1 (Sofjin and 205), while, among currently circulating strains, representatives of the third subtype are increasingly gaining dominance [15,32].

To obtain the mRNA-PrM/E-TBEV-LNP in this work, linear plasmid constructions pBAC were used as described previously [30]. These constructions contained open reading frames for the investigated TBEV antigens. To ensure stability of the poly(A) tract during vector propagation in bacterial culture, the tract was divided into segments. The first 30 adenines were divided with the next 70 by a 10-nucleotide spacer. All plasmid constructions were verified by Sanger sequencing.

Immunization with the ten mRNA-PrM/E-TBEV candidates elicited formation neutralizing antibodies in mice. Sera from immunized animals neutralized both homologous and heterologous TBEV strains. Average 50% neutralizing serum titer (NT₅₀) ranged from 1:16.9 to 1:84.4, signifying presence of neutralizing antibodies (Figure 1b, Table 2). The highest efficacy in regard to cross-neutralizing activity was exhibited by mRNA-PrM/E-Krasny Yar-8_FE, while mRNA-PrM/E-Zmeinogorsk_Eu and mRNA-PrM/E-178-79_4 have shown the lowest efficacy (Figure 1, Table 2). A notable observation was that for several candidates, neutralization titers against heterologous viral subtypes were higher than those against the homologous subtype used for immunogen design. This pattern aligns with prior clinical and experimental observations in TBEV immunology, where immunization with one subtype (e.g., European) can induce a serum response with superior activity against a heterologous subtype (e.g., Far-Eastern) [4].

Based on this data, the following mRNA-PrM/E-TBEV candidates were selected for further evaluation of protective efficacy: Sofjin_FE, Krasny Yar-8_FE and 205_FE (all belonging to the Far-Eastern subtype), as well as mRNA-PrM/E-TBEV-178-79_4. It is, therefore, anticipated that it will be confirmed that the vaccine mRNA variants based on TBEV strains of subtype 1 induce the highest immune response compared to the mRNA variant based on strain subtype 4.

To evaluate protective efficacy, groups of BALB/c mice were immunized by five different mRNA-PrM/E-LNP vaccines encoding the prM/E proteins of the following strains: Sofjin_FE, Krasny Yar-8_FE, 205_FE, 178-79_4, or a combination of Sofjin_FE and Krasny Yar-8_FE. Animals were immunized with mRNA (5 μg per animal) twice with a 14-day interval, while control animals received PBS. Two weeks after the booster immunization and prior to challenge, four mice from the vaccine group were euthanized for serum collection to assess anti-TBEV antibody titers and virus-neutralizing activity (VNA). The remaining mice were then challenged intraperitoneally with one of seven TBEV strains (Sofjin_FE, Krasny Yar-8_FE, 205_FE, Zmeinogorsk-1_Eu, Lesopark-11_Sib, 178-79_4 and 258-83_Baik) at a dose of 100LD₅₀. The experimental design is summarized in Figure 2a.

During the observation period, animal deaths were recorded only in the control groups (PBS). The onset of symptoms occurred between days 7 and 10; mortality was observed by day 15 post infection in all control groups. The survival rate of animals in the mRNA groups was 100% (Figure 2b).

VNA titer of sera of immunize mice was evaluated against eight different TBEV strains (Sofjin_FE, Krasny Yar-8_FE, 205_FE, Zmeinogorsk-1_Eu, Lesopark-11_Sib, 178-79_4, 258-83_Baik, Buzuuchuk_Sib); the results are shown in Figure 2c–e. As in the first experiment, analysis of serum samples from immunized animals has shown cross-VNA against eight TBEV strains (Figure 2c).

The viral load data, as determined by one-step RT-qPCR, correlated with the results of survival. Viral RNA was detected in homogenates of the brain, heart, and spleen of mice from the control (PBS) group, while viral RNA was not detected in any organs from mice immunized with the mRNA vaccine (Figure 3). As anticipated, the highest viral RNA load was observed in brain homogenates; however, no virus was detected in the liver samples.

Antibody levels in the serum of immunized mice were assessed by an in-house ELISA. Four different E proteins of TBEV were used in analysis—205_FE, Lesopark-11_Sib, Vologda-2_Sib, 886/84_Baik—to assess specific antibodies against these variants.

All serum samples tested positive (S/N > 3) against all four antigens (Figure 4a). The strongest immune response was observed when the E protein from the 205_FE strain was used as the antigen.

To determine the antibody titer, the geometric mean titer (GMT) was calculated from all serum dilutions that yielded a positive result (Figure 4b). The endpoint titers varied depending on the antigen used.

In summary, immunization with the mRNA candidates elicited specific antibodies against all target antigens.

The immunogenicity of the mRNA vaccine candidates was assessed by measuring antigen-specific antibody levels in mouse sera using ELISA. The candidate based on the Krasny Yar-8_FE TBEV strain demonstrates the highest antibody titers against a panel of heterologous antigens (Table 3), indicating its potential to elicit a broad cross-reactive immune response.

Comprehensive in vitro analysis of sera from immunized mice demonstrated the presence of cross-neutralizing activity against both homologous and heterologous TBEV subtypes. In the assessment of binding antibodies, cross-reactivity with all four antigen variants was also observed. Collectively, these data identify the mRNA-PrM/E-Krasny Yar_FE construct as the lead candidate, demonstrating superior performance across all evaluated parameters.