Comparison of candidate vaccines against Tick-borne encephalitis based on mRNA, simian adenovirus serotype 25 and a chimeric replication-competent yellow fever vaccine strain

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Introduction

According to the World Health Organization, approximately 10,000–12,000 cases of tick-borne encephalitis (TBE)¹ are reported worldwide each year. The disease is caused by the tick-borne encephalitis virus (TBEV), which belongs to the family Flaviviridae and the genus Orthoflavivirus. In addition to TBEV, the family Flaviviridae includes other arboviruses that are pathogenic to humans, such as yellow fever virus (YFV), Japanese encephalitis virus, dengue fever virus, Zika virus, and West Nile virus [1]. Based on the results of phylogenetic analysis, five subtypes of TBEV have been identified: European, Siberian, Far Eastern, Baikal, and a new subtype, Himalayan [2, 3]. TBEV affects the central nervous system, leading to neurological complications of varying severity depending on the TBEV subtype [1, 4, 5].

To date, vaccination is the only way to prevent the development of the disease, as there is no effective treatment. All currently licensed vaccines are inactivated and based on European (Neudoerfl and K23 [6, 7]) or Far Eastern (Sofjin, 205 [8], and Senzhang [9]) strains. Although these vaccines are quite effective and safe, they have some limitations, such as vaccination schedules that include several doses of primary immunization with the need for subsequent regular revaccinations to maintain a protective level of immune response. Furthermore, traditional inactivated vaccines do not always provide complete protection, especially in older individuals. Thus, the development of new vaccines may overcome the shortcomings of existing inactivated vaccines against TBEV [10].

Approaches to vaccine development and production have evolved over many years, and today several technologies and platforms have been developed for their creation, including inactivated, toxoid, live attenuated vaccines, vaccines based on virus-like particles, synthetic peptides, polysaccharides and polysaccharide conjugates, viral vector-based vaccines, nucleic acid-based vaccines (DNA and messenger RNA (mRNA)) [11, 12]. Next-generation platforms, such as mRNA, pave the way for the development of low-cost, highly safe and effective vaccines with the potential for rapid mass vaccination. The main components of vaccines are antigens, which are obtained directly from the pathogen or by biotechnological means [11].

The structure of the virion of all flaviviruses, including TBEV, is similar and includes an envelope (formed by membrane (M) and envelope (E) proteins), a nucleocapsid (formed by the capsid (C) protein), and an RNA genome [13–16]. In addition to structural proteins, viral RNA encodes seven non-structural proteins [15, 17]. The E protein of TBEV is a surface glycoprotein that interacts with attachment factors and/or receptors on the plasma membrane of target cells and promotes the fusion of viral and endosomal membranes, as well as inducing the production of neutralizing antibodies [13, 15]. The precursor M protein (PrM) regulates the rearrangement of E proteins on the virus surface, leading to the transition from immature to mature virions [13, 17]. Thus, PrM and E proteins are considered the main preferred antigens for the development of vaccines not only against TBEV but also against other flaviviruses.

In this study, we selected three approaches for delivering antigens encoding the PrM and E regions of the viral glycoprotein and creating vaccine candidates: based on the live attenuated strain YFV 17DD-UN (as a genetic basis for the development of vaccines against other viruses), recombinant adenovirus (rAd) vectors (a popular tool for delivering genes into mammalian cells, especially in the development of vaccine candidates), and mRNA encapsulated in lipid nanoparticles (LNPs) (a promising platform for the development of mRNA vaccines). During our research, we created three candidate vaccines against TBEV based on the Sofjin-Chumakov strain (Far Eastern subtype). We used three approaches to deliver polyprotein fragments corresponding to the PrM and E proteins (the main TBEV antigen) into mammalian cells: as mRNA-LNP, as recombinant simian adenovirus serotype 25, and as a chimeric YFV 17DD-UN with TBEV gene insertions. We then evaluated the immunogenicity and protective efficacy of the resulting TBEV candidate vaccines in mice.

The aim of the study was to investigate the efficacy of candidate vaccines based on chimeric YFV, simian adenovirus serotype 25, and mRNA encoding a fragment of the viral polyprotein TBEV, which corresponds to the PrM and E regions.

Materials and methods

Scheme for creating three candidate vaccines

The Far Eastern subtype "Sofjin-Chumakov" strain of TBEV (GenBank accession ID: KC806252.1) was used to develop three candidate vaccines. Viral RNA was used as a template for obtaining complementary DNA (cDNA) using the High-Capacity RNA-to-cDNA kit (Thermo Fisher Scientific, USA). The oligonucleotide primer for PrM/E cDNA synthesis had the following composition: cDNA-TBEV 5'-GTG TCC ACA GCA CAG CCA ACA TC-3'. The resulting cDNA was then used to amplify a fragment (1.9 kb in size) by polymerase chain reaction (PCR) using the 2× Platinum SuperFi Green MasterMix kit (Thermo Fisher Scientific, USA). Primers with the following structure were selected for PCR: PrME-For 5'-TGC TGG TTG TTG TCC TGT TGG GA-3' and PrME-Rev 5'-ACC GCC AAG AAC TGT GTG CAG CG-3'. Amplification was performed according to the manufacturer's instructions. Oligonucleotide primers for cDNA synthesis, PCR fragment amplification, and plasmid construction were selected using the SnapGene program (version 7.2). The identity of the coding sequences was confirmed by Sanger sequencing. Depending on the viral vector, after one or more passages, the production of recombinant viruses was confirmed by high-throughput sequencing. Fig. 1 shows a diagram of the production of chimeric replication-competent attenuated YFV 17DD-UN/TBE(FE), recombinant replication-deficient simian adenovirus SAd25-TBEV(FE), and mRNA-TBEV(FE)-LNP.

 

Fig. 1. Schematic representation of the design of chimeric virus YFV 17DD-UN/TBEV(FE), a recombinant SAd25-TBEV(FE) and mRNA-TBEV(FE)-LNP used in this study. a – schematic representation of TBEV(FE) genome; b – schematic representation of YFV 17DD-UN/TBEV(FE) genome; c – schematic representation of SAd25-TBEV(FE) genome; d – schematic representation of mRNA-TBEV(FE)-LNP.

Рис. 1. Схематическое изображение конструкции химерного вируса желтой лихорадки (ВЖЛ) 17DD-UN/КЭ(FE), рекомбинантного SAd25-КЭ(FE) и мРНК-КЭ(FE)-ЛНП. а – схематическое изображение генома КЭ(FE); б – схематическое изображение генома ВЖЛ 17DD-UN/КЭ(FE); в – схематическое изображение генома SAd25-КЭ(FE); г – схема мРНК-КЭ(FE)-ЛНП.

 

Obtaining a chimeric replication-competent attenuated yellow fever virus 17DD-UN/TBE(FE)

To obtain chimeric YFV 17DD-UN/TBE(FE) based on the YFV 17DD-UN strain (ID GenBank OP796361.1) with inserts of nucleotide sequences encoding the PrM and E regions of TBEV, the pSMART BAC plasmid from the CopyRight v. 2.0 Bac Cloning kit (Lucigen, USA) was used. All molecular cloning work was performed using the Escherichia coli Top10 strain. Plasmid construction and assembly were performed using the Gibson Assembly Ultra MasterMix kit (Codex DNA, USA) according to the manufacturer's instructions.

To assemble the hybrid construct, Vero E6 cells were transfected. For this purpose, cells were seeded in 48-well plates (2 × 10⁵ cells per well) one day before the experiment. Then, cells were transfected in Opti-MEM medium (Gibco, USA) using Lipofectamine 2000 (LF2000; ThermoFisher, USA). Virus replication was confirmed by one-step reverse transcription PCR (RT-PCR) followed by observation of the development of the virus-induced cytopathic effect (CPE).

Amplification of the chimeric YFV 17DD-UN/TBE(FE) was performed using a quantitative one-step RT-PCR, as described previously [18]. The oligonucleotide primers and probes for the NS5 non-structural protein gene of YFV 17DD-UN (backbone) and the E gene of TBEV (insert) were as follows: forward primer YFV NS5F 5'-GCG GTA TCT TGA GTT TGA GG-3', reverse primer NS5R 5'-AGG TCT CTG ATC ACA TAT CCT AG-3', probe NS5TM 5'-FAM-AGC CAA TGC CTTC CAC TCC TCC TC-BHQ1-3' (20) and forward primer TBEV(FE) ChTBE-Fe-for 5'-gTC AAA gTA gAg CCg CAT AC-3', reverse primer ChTBE-Fe-rev 5'-T TCT CCg Agg AAg CCg TgA A-3', probe ChTBE-Fe-Z 5'-R6G-CgT CgC TgC TAA TgA gAC TCA CAg Tgg-BHQ1-3' (this study). To confirm the production of PreM and E, the virus-containing TBEV fluid was examined using enzyme-linked immunosorbent assay (ELISA).

To determine the virus titer, Vero E6 cells were seeded in 24-well plates (10⁵ cells per well) one day before the experiment. Various virus dilutions were then added to the cell monolayer. After virus adsorption, the medium was removed and the cells were covered with 0.7% carboxymethylcellulose (Sigma, USA) in DMEM medium. The plates were incubated for 4–5 days at 37°C and 5% CO₂. After that, the cells were fixed with a 5% paraformaldehyde solution and stained with a 1% crystal violet solution. The virus titer was expressed in PFU/mL (plaque-forming units) [19].

Obtaining recombinant replication-deficient simian adenovirus SAd25-TBE(FE)

The recombinant replication-deficient vector based on simian adenovirus serotype 25 (SAd25), containing the PrM and E TBEV nucleotide sequences, was obtained by homologous recombination in E. coli BJ5183 cells. Bacterial cells were transformed by electroporation according to the manufacturer's instructions using a MicroPulser device (Bio-Rad, Hercules, USA). The resulting plasmid (pSAd25-TBE(FE)) was analyzed by PCR, restriction mapping, and high-throughput sequencing according to standard protocols [18, 20]. In pSAd25-TBE(FE), the deleted region E1 of the adenovirus genome was replaced with a cassette containing the target gene (CMV promoter, nucleotide sequence of the PrM/E polyprotein of the same strain used to create the chimeric YFV 17DD-UN/TBE(FE), and a polyadenylation signal). To increase the packaging capacity of the vector, the E3 region of the adenovirus genome was also deleted.

Plasmid pSAd25-TBE(FE) was used to generate recombinant adenovirus SAd25-TBE(FE). HEK293 cells were seeded in 24-well culture plates and incubated overnight until 80% confluence was reached. To remove the bacterial component, plasmid DNA was hydrolyzed with PacI and SspI restriction enzymes and then transfected into HEK293 cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. After visual detection of cytopathic effect (CPE) using an inverted microscope CKX41 (Olympus, Japan), the cells in the culture medium were subjected to three freeze-thaw cycles.

Recombinant simian adenovirus was accumulated in HEK293 cell culture. HEK293 cells were seeded in 10 cm diameter Petri dishes at a density of 15–17 × 10⁶ cells per dish. The next day, 65–75% confluent monolayer cells were infected with recombinant adenovirus SAd25-TBE(FE) at a dose of 10⁷ TCID₅₀ per dish. After 2 days, upon reaching 90–100% CPE, the infected cells were collected, concentrated by low-speed centrifugation, resuspended in buffer (0.01 M Tris-HCl pH 8.0, 0.01 M NaCl, 5 mM EDTA) and subjected to three freeze-thaw cycles to destroy the cell and nuclear membranes and release the virus from the cells. Cell lysates were centrifuged at 5000 rpm for 10 min at room temperature, after which the precipitate was removed. The recombinant adenovirus was then purified by double ultracentrifugation in a CsCl gradient (both stepwise and equilibrium) on an Optima XPN-90 ultracentrifuge (Beckman Coulter Inc., USA).

The purity and identity of SAd25-TBE(FE) were confirmed by PCR and whole-genome sequencing. The titer of the purified virus was determined by TCID₅₀ analysis on HEK293 cells [21]. The results were recorded on days 10–12 after cell transduction. The number of viral particles (vp) was determined using reagents from the Pico488 double-stranded DNA quantification kit (Lumiprobe, USA) in accordance with the manufacturer's instructions.

SDS-PAGE was performed in a 12% polyacrylamide gel using the Lammli method [22]. Precision Plus Protein was used as a molecular weight standard. Adenovirus preparations were diluted to a concentration of 10¹⁰ vp/mL in filtered (PES filter, pore size 0.22 μm) milliQ water. Then, 1 mL of the preparation was transferred to a 10 × 10 × 45 mm cuvette and measured three times. Particle size was determined using Zetasizer software.

For expression analysis, SAd25-TBE(FE) virus and control SAd25-EGFP virus (expressing the EGFP reporter protein gene) were inoculated into 3 cm diameter Petri dishes with 80–90% monolayer of Vero E6 cells. After 72 hours, the cells and medium were collected and analyzed by ELISA.

Obtaining mRNA-TBEV(FE)-LNP

The development of an mRNA platform for efficient and sustained expression of the gene of interest, including DNA cloning procedures, plasmid DNA isolation and purification, in vitro transcription (IVT), and mRNA packaging into LNP, has been described previously [25]. A linear bacterial plasmid based on pJAZZ-OK (Lucigen, USA) with PrM and E coding regions was used as a template for mRNA production. pDNA for IVT was isolated from electrocompetent E. coli BigEasy-TSA cells (Lucigen, USA) using the Plasmid Maxi Kit (QIAGEN). pDNA was cleaved with BsmBI-v2 restriction endonuclease (NEB), followed by purification of the product by phenol-chloroform extraction and ethanol precipitation. IVT was performed as described previously [26]. A 100 μL reaction mixture was prepared containing 3 μg of DNA template, 3 μL of T7 RNA polymerase (Biolabmix) and 10X T7 buffer (TriLink), 4 mM trinucleotide cap 1 analogue (3'-OMe-m7G)-5'-ppp-5'-(2'-OMeA)pG (Biolabmix), 5 mM m1ΨTP (Biolabmix) replacing UTP, and 5 mM GTP, ATP, and CTP. After 2 h of incubation at 37 °C, 6 μL of DNase I (Thermo Fisher Scientific, USA) was added for another 15 min, after which the mRNA was precipitated with 2M LiCl (incubation for 1 hour on ice followed by centrifugation for 30 minutes at 14,000g, 4 °C) and thoroughly washed with 80% ethanol. RNA integrity was assessed by electrophoresis in 8% denaturing polyacrylamide gel.

The assembly of LNP has also been described previously [23]. All lipid components were dissolved in ethanol in a molar ratio of 46.3 : 9.0 : 42.7 : 1.6 (ionizable lipid : DSPC : cholesterol : PEG lipid). The ionizable lipid Acuitas (ALC-0315) and PEG lipid (1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000) were purchased from Cayman Chemical Company; DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol were purchased from AvantiResearch and Merck, respectively. The lipid mixture was mixed with a 10 mM sodium citrate acidifying buffer (pH 3.0) containing mRNA (0.2 mg/mL) in a volume ratio of 3 : 1 (aqueous solution:ethanol) using a NanoAssemblr Ignite device (Precision NanoSystems, Canada). The ratio of ionizable nitrogen atoms in the ionizable lipid to the number of phosphate groups in mRNA (N : P ratio) was set to 6 for each formulation. The preparations were dialyzed against PBS (pH 7.2) in Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific, USA) for at least 24 hours. The preparations were passed through a 0.22 μm pore size filter and stored at 4 °C (PBS) until use. The diameter and size distribution, as well as the zeta potential of mRNA-LNP, were measured using a Zetasizer Nano ZS (Malvern Panalytical, UK) according to the operating instructions.

The efficiency of encapsulation and mRNA concentration were determined using SYBR Green dye (SYBR Green I, Lumiprobe), as described previously [23, 24]. Transfection of HEK293 cells with mRNA encoding PreM/E was performed as previously described [23, 25]. To confirm the production of PreM and E antigens, cell culture fluid was examined by ELISA.

Efficiency of mRNA encapsulation

The efficiency of encapsulation and mRNA concentration were determined using SYBR Green dye (SYBR Green for PCR, Lumiprobe, Moscow, Russia) followed by fluorescence measurement. mRNA-LNP samples were diluted with TE buffer (pH 8.0) in the absence or presence of 2% Triton-X-100 in a black 96-well plate. Standard mRNA was serially diluted with TE buffer in the absence or presence of 2% Triton-X-100 to construct calibration curves. The plate was then incubated for 10 min, after which SYBR Green dye (diluted 100-fold) was added to each well for RNA binding. Fluorescence was measured at 497 nm excitation and 522 nm emission using a Varioscan LUX (Thermo Fisher Scientific Inc., USA). The mRNA concentrations after LNP destruction with Triton X-100 (with total mRNA) and before LNP destruction (with external mRNA) were determined using the corresponding standard curves. The concentration of mRNA loaded into the LNP was determined as the difference between the two concentrations multiplied by the dilution factor of the initial sample. The encapsulation efficiency was calculated using the formula: (E%) = (C total mRNA) – (C external mRNA) / (C total mRNA) × 100%.

Dynamic light scattering

Particle size was determined using a Zetasizer Nano ZS (Malvern, UK) nano-sizer by dynamic light scattering (DLS). The DLS method determines the diffusion coefficient of dispersed particles in a liquid after analyzing the correlation function of scattered light intensity fluctuations. The hydrodynamic diameter of the particle is then calculated from the diffusion coefficient obtained.

For the Zetasizer Nano ZS, the SAd25-TBE(FE) sample was diluted to a concentration of 7.3 × 10⁹/mL in filtered (PES filter, pore size 0.22 μm) milliQ water. Then, 1 mL of the diluted sample was transferred to a measuring cuvette (10 × 10 × 45 mm, polystyrene/polystyrene material, Sarstedt, Germany). After that, the particle size was measured three times.

The mRNA-LNP suspension was diluted 20-fold with water and loaded into the cuvette for measurement at 25 °C. LNPs in PBS were read using the solvent parameter "water". One measurement consisted of three readings, each obtained from 13 measurements. The presented DRS data for the mRNA-LNP preparation represent the average value of these three readings.

The results were calculated using Zetasizer software (Malvern, UK).

Enzyme-linked immunosorbent assay (ELISA) for the detection of PrM and E glycoproteins

The medium or virus-containing fluid was collected and analyzed using a set of reagents for determining the TBEV antigen, "VectorTBEV-antigen" (Vector-Best, Russia).

The ELISA results were evaluated using a µ-Quant device (Bio-Tek Instruments, USA) at a wavelength of 450 nm with a reference wavelength of 620 nm. The ELISA threshold value was calculated in accordance with the manufacturer's instructions using the following formula:

Threshold value = average optical density (OD) of three negative controls + 0.25 (SD).

Animal studies

All procedures involving animals were performed in accordance with current guidelines for the care and use of laboratory animals and were approved by the Local Ethics Committee of the N.F. Gamaleya National Research Center for Epidemiology and Microbiology (protocol No. 63 dated October 5, 2023). In accordance with institutional and national guidelines, all experiments were performed in ABSL/BSL-2 and -3 facilities.

The mice were purchased from the Stolbovaya Laboratory Animal Breeding Facility (Russia). All animals were kept in separate cages (10 mice per cage) with controlled temperature (20–24 °C) and humidity (45–65%). Throughout the study, the mice had free access to food and water.

Immunization

To evaluate the immunogenicity and protective efficacy of the recombinant viruses and mRNA-LNP obtained, female BALB/c mice (aged 6–8 weeks) were divided into groups (10 animals in each). Depending on the vaccine platform used to deliver PrM and E glycoproteins (viruses or mRNA-LNP), mice were immunized with one or two doses of the experimental vaccine subcutaneously or intramuscularly. The control group consisted of mice that were injected with phosphate-buffered saline (PBS) (unvaccinated group, 10 individuals). The health and behavior of the mice were monitored once a day until the end of the study. When neurological symptoms such as paresis appeared, the mice were euthanized. After 28 days of observation, blood samples were taken from the tail vein. Blood serum was taken to determine the level of neutralizing antibodies.

Challenge with tick-borne encephalitis virus

To analyze the protective efficacy of recombinant viruses and mRNA-LNP, immunized mice were infected with the Sofjin-Chumakov strain of TBEV. The TBEV strain was obtained from the State Virus Collection (SVC No. 1/2). The viral stock for the experiments was a clarified brain homogenate obtained by intracerebral infection of newborn BALB/c mice. The virus titer was determined using SPEV cells (Biolot, Russia). After immunization, animals in all groups were intraperitoneally infected with TBEV at a dose of 100 LD₅₀. LD₅₀ was calculated using the Reed and Mench method [26]. The health and behavior of the mice were monitored once a day for a week after TBEV infection, and then twice a day until the end of the study. After 20 days of observation, the surviving animals were euthanized.

Assessment of serum neutralizing activity after immunization

Different dilutions of serum obtained from immunized animals were mixed in equal proportions with 100 TCID₅₀ of the Sofjin-Chumakov strain of TBEV and incubated for 1 hour at 37 °C. After incubation, the mixture of serum and virus was transferred to a 96-well plate containing a monolayer of porcine embryonic kidney (SPEV) cells. The plates were incubated at 37°C and 5% CO₂ for 4 days. The last serum dilution demonstrating complete protection against virus-induced CPE was taken as the virus neutralizing titer.

Statistical analysis

All differences were considered statistically significant at p ≤ 0.05. The log-rank test (Mantel-Cox test) was used to interpret the Kaplan-Meier survival analysis. The Kruskal–Wallis test was used to compare virus-neutralizing antibody levels, followed by Dunn's post hoc multiple comparison test. All data were obtained using GraphPad software, 8th edition (GraphPad Software, USA).

Results

Generation of a chimeric replicative-competent attenuated yellow fever virus 17DD-UN/TBE(FE)

In our previous study [20], we obtained a chimeric YFV 17DD-UN/TBE(Eu) using the infectious subgenomic amplicon (ISA) method. In this study, we obtained a chimeric YFV 17DD-UN/TBE(FE) using bacterial artificial chromosomes (Fig. 1 b). For this purpose, the infectious cDNA backbone of YFV 17DD-UN was cloned into the pSMART BAC plasmid, then cells were transfected, followed by evaluation of virus-induced CPE and confirmation of virus replication by real-time RT-PCR. CPE and virus replication were observed in Vero E6 cells both after transfection and after virus passages. After several passages in Vero E6 cells, the presence of the complete YFV 17DD-UN genome was confirmed by high-throughput sequencing. Comparative analysis of the complete genomes of YFV 17DD-UN (GenBank ID OP796361.1) (generated by ISA) and YFV 17DD-UN (generated using artificial chromosomes) in the study did not reveal any nucleotide substitutions.

An artificial chromosome containing the complete genome of strain YFV 17DD-UN was then used as a molecular clone to restore a viable chimeric virus. The nucleotide sequences of the viral glycoproteins PrM and E of the YFV 17DD-UN backbone were replaced with the same glycoproteins of TBEV. The plasmid construct was assembled using the Gibson method (Gibson assembly). Sanger sequencing confirmed the identity of PrM and E of TBEV(FE) in YFV 17DD-UN backbone. Subsequent transfection and passages in Vero E6 cells demonstrated the successful construction of the chimeric YFV 17DD-UN/TBE(FE), which was confirmed by real-time RT-PCR and CPE assessment. The virus-containing medium collected from Vero E6 cells was also examined by ELISA, which confirmed the expression of PreM and E TBEV antigen proteins by the chimeric virus (Table).

 

Table. Production of PreM and E TBEV antigen evaluated by ELISA

Таблица. Анализ экспрессии гликопротеинов PreM и E ВКЭ методом ИФА

Sample type Образец	Optical density (mean)* Оптическая плотность (среднее значение)*
Negative control (buffer for dilution) Отрицательный контроль (буфер для разведения)	0.053
Cut-off value Cut-off значение	0.30
YFV 17DD-UN/TBEV(FE) (culture medium) YFV 17DD-UN/КЭ(FE) (культуральная среда)	2.609
YFV 17DD-UN/TBEV(FE) (cell lysate) YFV 17DD-UN/КЭ(FE) (клеточный лизат)	3.908
SAd25-TBEV(FE) (culture medium) SAd25-КЭ(FE) (культуральная среда)	3.598
SAd25-TBEV(FE) (cell lysate) SAd25-КЭ(FE) (клеточный лизат)	4.000
mRNA-PrM/E-TBEV(FE) (culture medium) mRNA-PrM/E-КЭ(FE) (культуральная среда)	3.653
mRNA-PrM/E-TBEV(FE) (cell lysate) mRNA-PrM/E-КЭ(FE) (клеточный лизат)	4.000
HEK293 (culture medium) HEK293 (культуральная среда)	0.049
HEK293 (cell lysate) HEK293 (клеточный лизат)	0.060
Vero E6 (culture medium) Vero E6 (культуральная среда)	0.055
Vero E6 (cell lysate) Vero E6 (клеточный лизат)	0.056
Positive control Положительный контроль	3.106

Note. * – an optical density reading > 2.5 units is considered an indicator of high absorption, not a quantitative parameter. Measurements in this range are semi-quantitative.

Примечание. * – показания оптической плотности > 2,5 единиц рассматривается как индикатор высокой степени поглощения, а не как количественный параметр. В данном диапазоне измерения носят полуколичественный анализ.

 

Generation of recombinant replication-deficient simian adenovirus SAd25-TBEV(FE)

We previously developed a technological platform based on SAd25 [20]. In this study, we used the same approach to obtain a recombinant simian adenovirus containing nucleotide sequences that encode the TBEV PrM and E regions. One of the important aspects of successful complementation of adenoviruses with E1 gene deletion is the functional interaction of the E1B 55K protein (produced by the transcomplementing cell line) with the E4 34K protein in the viral genome. However, the development of complementary cell lines for different types of replication-deficient vectors is a laborious and complex process. Thus, the availability of adenoviruses that are non-pathogenic to humans and capable of replicating in cells such as HEK293 is a significant advantage. To obtain the SAd25-TBE(FE) virus, HEK293 cells were transfected with the pSAd25-TBE(FE) plasmid construct (Fig. 1b). The similarity of the E1B 55K Ad5 and SAd25 protein sequences is approximately 56%. However, we were able to successfully revive replication-deficient SAd25 in HEK293 cells.

After amplifying and purifying the virus, we measured the particle size and analyzed the protein spectrum. The size of the SAd25-TBE(FE) virion was 108.23 ± 26.64 nm (Fig. 2a). Analysis of the protein spectrum of the recombinant adenovirus SAd25-TBE(FE) indicates the presence of major capsid proteins – hexon (II), penton base (III) and fiber (IV), as well as minor proteins (V, VI). The molecular weight of SAd25-TBE(FE) polypeptides was approximately as follows: hexon (II) – 120–170 kDa; penton base (III) – 51–65 kDa; pIIIa and fibrin – 60–65 kDa; minor protein pV ~ 48 kDa; protein associated with hexon pVI – 22–24 kDa (Fig. 2c). The expression of the PreM and E genes of the corresponding proteins was determined by ELISA (table).

 

Fig. 2. Characteristics of the recombinant simian adenovirus SAd25-TBEV(FE). a – results of the analysis of the homogeneity of the distribution of recombinant adenoviral particles by size; b – SDS-page of SAd25-TBEV(FE).

Рис. 2. Характеристика рекомбинантного аденовируса обезьян SAd25-КЭ(FE). а – результаты анализа однородности распределения рекомбинантных аденовирусных частиц по размерам; б – SDS-PAGE SAd25-КЭ(FE).

 

Generation of mRNA-TBEV(FE)-LNP

To obtain mRNA-TBEV(FE)-LNP, nucleotide sequences of the PrM/E polyprotein of TBEV were synthesized and cloned into the linear bacterial plasmid pJAZZ-OK, as described previously [23, 24] (Fig. 1d). The in vitro synthesized mRNAs included a cap-1 structure at the 5'-end; a poly(A) tail 100 nucleotides long at the 3'-end; and the 5'- and 3'-untranslated regions of human hemoglobin alpha subunit (HBA₁) mRNA.

N1-methylpseudouridines (m1Ψ) were co-transcriptionally incorporated into mRNA instead of 100% uridines (U). mRNA-LNP preparations were prepared using a NanoAssemblr Ignite microfluidic device. The encapsulation efficiency was 90% (SD 1.2%) with a particle size of 88.64 ± 32.4 nm (Fig. 3).

 

Fig. 3. Results of the analysis of the homogeneity of the distribution of mRNA-TBEV(FE)-LNP particles by size.

Рис. 3. Результаты анализа однородности распределения частиц мРНК-ВКЭ(FE)-ЛНП.

 

Evaluation of PrM and E glycoprotein production

The expression of PrM and E proteins synthesized in vitro by mRNA-TBEV(FE)-LNP using recombinant replication-deficient adenovirus SAd25-TBEV(FE) and replication-competent attenuated virus was confirmed by transfection of HEK293 and Vero E6 cell cultures. The culture medium and/or cell lysate were then examined by ELISA (Table 1). The results confirmed the translational activity of synthetic mRNA and the presence of antigen in the obtained YFV 17DD-UN/TBE(FE) and SAd25-TBE(FE).

Evaluation of the immunogenicity of the chimeric replication-competent attenuated yellow fever virus 17DD-UN/TBE(FE), recombinant replication-deficient adenovirus SAd25-TBE(FE), and mRNA-TBE(FE)-LNP

To evaluate the immunogenicity of the recombinant YFV 17DD-UN/TBE(FE) and SAd25-TBE(FE) obtained, as well as mRNA-TBE(FE)-LNP, BALB/c mice were subcutaneously injected with YFV 17DD-UN/TBE(FE) at a dose of 10⁴ BOE per mouse (YFV 17DD-UN/TBE(FE) group, 10 individuals per group), intramuscularly SAd25-TBE(FE) at a dose of 5 × 10¹⁰ IU per mouse (SAd25-TBE(FE) 2 groups, 10 individuals per group) and intramuscularly mRNA-TBE(FE)-LNP at a dose of 5 μg per mouse (mRNA-TBE(FE)-LNP group, 10 individuals). The doses were selected during preliminary experiments. Animals that received PBS (unvaccinated group, 10 individuals) were used as control groups. Three groups of BALB/c mice received one dose of YFV 17DD-UN/TBE(FE), SAd25-TBE(FE), and PBS, while the other two groups received two doses of SAd25-TBE(FE) and mRNA-TBE(FE)-LNP (Fig. 4).

 

Fig. 4. Experimental design.

Рис. 4. Экспериментальный дизайн исследования.

 

After immunization, the animals were observed for 4 weeks.

To evaluate the neutralizing activity of sera against the homologous TBEV strain "Sofjin-Chumakov", blood samples were collected 28 days after immunization. It was found that immunization of mice with a dose of 10⁴ PFU YFV 17DD-UN/TBE(FE) did not lead to the formation of neutralizing antibodies. Neutralizing antibodies were detected in half of the animals that received a single dose of SAd25 TBE(FE). Immunization with two doses of SAd25 TBE(FE) and mRNA-TBE(FE)-LNP led to the formation of antibodies in all animals in the groups (Fig. 5).

 

Fig. 5. Titers of neutralizing antibodies in serum samples from mice immunized with YFV 17DD-UN/TBEV(FE), SAd25-TBEV(FE) and mRNA-TBEV(FE)-LNP. Individual values of neutralization antibody titer for each sample are shown. Data are presented as geometric mean with 95% CI. Significance is based on one-way ANOVA with Dunn's multiple comparisons test (*p ≤ 0.01, **p ≤ 0.001, ***p ≤ 0.0001).

Рис. 5. Титры нейтрализующих антител в сыворотке мышей, иммунизированных ВЖЛ 17DD-UN/КЭ(FE), SAd25-КЭ(FE) и мРНК-КЭ(FE)-ЛНП. Точками показаны индивидуальные значения для каждого образца. Данные представлены как среднее геометрическое с 95% доверительным интервалом (ДИ). Значимость данных оценивали на основе однофакторного дисперсионного анализа (ANOVA) с критерием множественных сравнений Данна (*p ˂ 0,05, **p ≤ 0,01, ***p ≤ 0,0001).

 

Analysis of the titer of virus-neutralizing antibodies in blood sera revealed a statistically significant difference between all groups. In the mRNA group, the antibody titer was higher than in other groups.

To evaluate the ability of the chimeric replication-competent attenuated YFV 17DD-UN/TBE(FE), recombinant replication-deficient adenovirus and SAd25-TBE(FE), as well as mRNA-TBE(FE)-LNP to protect animals from lethal infection caused by TBEV, immunized animals were infected with TBEV (Sofjin-Chumakov strain). Experimental infection of animals at a dose of 100 LD₅₀ resulted in 100% survival in the mRNA-TBE(FE)-LNP groups and in both SAd25-TBE(FE) groups. Infection with the Sofjin-Chumakov strain of TBEV at a dose of 100 LD₅₀ resulted in 85.7% and 0% survival in the YFV 17DD-UN/TBE(FE) and placebo groups, respectively. Significant differences between survival curves (p < 0.0001) were detected using the log-rank test (Mantel-Cox criterion) (Fig. 6).

 

Fig. 6. Assessment of protective efficacy of YFV 17DD-UN/TBEV(FE), SAd25-TBEV(FE) and mRNA-TBEV(FE)-LNP. The survival of mice was monitored for 20 days. Kaplan–Meier survival analysis immunized mice infected with TBEV strain Sofjin at 100 LD₅₀.

Рис. 6. Оценка протективности ВЖЛ 17DD-UN/КЭ(FE), SAd25-КЭ(FE) и мРНК-КЭ(FE)-ЛНП. За выживаемостью мышей наблюдали в течение 20 сут. Анализ выживаемости по Каплану–Майеру для иммунизированных мышей, инфицированных штаммом вируса клещевого энцефалита «Софьин-Чумаков» в дозе 100 LD₅₀.

 

Discussion

All currently available inactivated vaccines against TBE do not provide 100% protection against infection and disease development, and cases of TBE are reported annually among vaccinated individuals² [27–28]. The reasons for the limited effectiveness of vaccines against TBE are being actively studied, which contributes to a deeper understanding of the mechanisms of the protective immune response [29, 30]. In this context, the development of improved vaccines and new vaccination strategies is considered a promising approach to improving the effectiveness of prevention of this disease [10].

The aim of this study was to compare the immunogenicity and protective efficacy of the same TBEV antigenic component presented in different vaccine platforms that differ fundamentally in their delivery mechanism, expression pattern, and immune response formation. For this purpose, a nucleotide sequence encoding a fragment of the viral polyprotein corresponding to the PrM and E regions was used and implemented in a live chimeric vaccine, a recombinant adenovirus vector, and an mRNA platform.

Although the vaccine against YFV was developed more than 80 years ago, it remains one of the most effective live attenuated vaccines, providing long-lasting, and in some cases lifelong, immunity after a single dose. This has led to sustained interest in using the genome of the 17D vaccine strain as a genetic basis for the development of chimeric vaccines against other flaviviruses, including TBEV.

Since 2012, two YFV-based vaccines have been approved for the prevention of Japanese encephalitis and dengue fever [31, 32]. It has previously been shown that chimeric replication-competent vaccine candidates against TBEV, including Langat/DENV-4, TBEV/DENV-4, and TBEV/JEV (ChinTBEV), had lower neurovirulence in mice and non-human primates compared to the original viruses [33–35]. In our previous work [18], we created and characterized a chimeric virus of the European (EU) subtype of TBEV and the YFV 17DD-UN strain, in which the PrM and E regions of YFV 17DD-UN were replaced with fragments of PrM and E of the European (EU) subtype of TBEV. The chimeric virus was obtained using the ISA method. In this study, a chimeric replication-competent YFV 17DD-UN/TBE(FE) was obtained, for which a safe immunizing dose was selected during preliminary experiments. It should be noted that the live chimeric vaccine differs fundamentally from other platforms studied in its ability to replicate in vivo and potentially to elicit a longer-lasting immune response after a single immunization.

Recombinant adenovirus vectors are widely used in the development of vaccines against various infectious diseases due to their favorable safety profile and ability to induce both humoral and cellular immune responses [36–38]. With regard to flaviviruses, it has previously been shown that adenoviral vectors expressing structural proteins are capable of eliciting neutralizing antibodies and a T-cell response [39, 40]. However, data on the use of adenoviral vectors expressing the PrM and E regions of the TBEV viral polyprotein remain limited.

Although adenovirus vectors are a popular platform for developing vaccines against various flavivirus infections, including diseases caused by Zika, dengue, and West Nile viruses, research on TBE is very limited. To date, only two studies have been published on adenoviruses as vaccine candidates against TBE [41–43]. These studies investigated a human adenovirus type 5-based vector expressing the TBEV nonstructural protein NS1. Experiments in mice demonstrated good immunogenicity and protective efficacy; however, no further studies have been conducted. Thus, the recombinant simian adenovirus serotype 25 expressing the TBEV prM/E polyprotein, which we have developed and characterized, is a promising candidate for the development of a vaccine against TBEV.

Recently, there has been growing interest in the development of mRNA-based vaccines; such mRNAs do not integrate into the genome, are characterized by short-term antigen expression, are not capable of pathogenicity reversal, do not induce vector-specific humoral immune responses, and, moreover, mRNA does not need to cross the nuclear barrier for protein expression, which allows the transfection of non-dividing or slowly dividing cells, such as dendritic cells [44, 45]. To protect against degradation by host nucleases, mRNA is encapsulated in LNPs. Over the past 10 years, several effective mRNA-LNP vaccines have been developed against many viral infections, including influenza [46], HIV [47], rabies [48], chikungunya [49], human cytomegalovirus infection [50], flaviviruses DENV [51], ZIKV [52], Powassan [53], and SARS-CoV-2 [54]. Furthermore, we have previously demonstrated the efficacy of multivalent mRNA vaccines against seasonal influenza [24] and a combined mRNA vaccine against influenza and SARS-CoV-2 [25]. With regard to flaviviruses, it has been shown that transfection of cells with mRNA-LNP encoding prM/E structural proteins leads to the secretion of subviral particles with antigenic properties similar to those of viral virions and stimulates the formation of neutralizing antibodies [55]. Thus, the mRNA-LNP platform is a promising approach to creating a vaccine against TBE, allowing for rapid updating and combination of antigen composition as needed.

Analysis of our experimental data allows us to identify a number of features of the vaccine platforms studied. Thus, the mRNA platform was characterized by the induction of the highest titers of virus-neutralizing antibodies, which is probably due to the effective expression of the encoded fragment of the viral polyprotein and the two-dose immunization regimen. A high level of humoral immune response correlated with 100% survival of animals after infection with a lethal dose of TBEV. The vaccine based on a recombinant adenovirus vector also provided complete protection of animals from lethal infection, despite lower neutralizing antibody titers compared to the mRNA platform. This may indicate the contribution of additional immune defense mechanisms, including the cellular immune response characteristic of virus vector platforms. The chimeric replication-competent virus demonstrated a protective effect at a relatively low immunizing dose and a single administration, but was accompanied by the formation of low or undetectable neutralizing antibody titers. The data obtained suggest that the protective effect in this case may be due to the peculiarities of vaccine virus replication in vivo and the involvement of immune mechanisms that are not limited to the level of circulating antibodies. Thus, differences in virus-neutralizing antibody titers and survival rates emphasize that the effectiveness of vaccine platforms cannot be assessed solely on the basis of a single immunological parameter, but must be considered in the context of their biological and technological characteristics.

The results obtained show that the choice of vaccine platform should take into account not only the level of induced antibodies, but also factors such as the frequency of immunization, the dose used, the ability to replicate in vivo, and the potential contribution of cellular immunity. It should be noted that direct comparison of the immunogenicity of different vaccine platforms is limited by differences in doses, frequency of administration, and mechanisms of immune response formation. Each of the platforms studied has its own advantages and limitations related to safety and duration of immunity. Thus, this study highlights the promise of further research into various vaccine platforms against TBEV and their possible adaptation to specific objectives for further optimization of vaccine prevention strategies for this disease.

Conclusion

Overall, the presented study demonstrates the practical feasibility of using different vaccine platforms to deliver the same antigenic component of the TBEV and highlights their fundamental differences in terms of the immune response they elicit and their protective efficacy. The experimental data obtained confirm the promise of the mRNA platform as the most powerful approach for inducing humoral immunity, and also indicate the significant potential of adenoviral and chimeric replication-competent vectors capable of providing protection through alternative immunological mechanisms. Taken together, the data obtained expand our understanding of possible strategies for vaccine prevention of TBE and form the scientific basis for the further development and optimization of new-generation vaccines, taking into account requirements for efficacy, safety and practical applicability.

¹ Tick-borne encephalitis. Available at: https://www.who.int/health-topics/tick-borne-encephalitis#tab=tab_1.

² Tick-borne encephalitis ‒ Annual Epidemiological Report for 2020. Available at: https://www.ecdc.europa.eu/en/publications-data/tick-borne-encephalitis-annual-epidemiological-report-2020.

About the authors

Andrei E. Siniavin

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Author for correspondence.
Email: andreysi93@ya.ru
ORCID iD: 0000-0001-7576-2059

PhD, Research Fellow

Russian Federation, Moscow; Moscow

Vladimir A. Gushchin

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Lomonosov Moscow State University

Email: wowaniada@gmail.com
ORCID iD: 0000-0002-9397-3762

Doctor of Biological Sciences, Professor, Head of the Department of Epidemiology, Head of the Laboratory of Mechanisms of Population Variability of Pathogenic Microorganisms

Russian Federation, Moscow; Moscow; Moscow

Nadezhda A. Kuznetsova

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: nadyakuznetsova0@yandex.ru
ORCID iD: 0000-0002-7399-7628

 PhD, Senior Research Fellow

Russian Federation, Moscow

Leonid I. Russu

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: plano77@bk.ru
ORCID iD: 0000-0001-6353-9917

Research Fellow

Russian Federation, Moscow

Elizaveta V. Marchuk

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: elizaveta.divisenko@yandex.ru
ORCID iD: 0009-0000-5470-4917

Research Fellow

Russian Federation, Moscow

Denis A. Kleymenov

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: mne10000let@yandex.ru
ORCID iD: 0000-0001-9422-7238

PhD, Senior Research Fellow, Head of the Laboratory of Translational Biomedicine

Russian Federation, Moscow

Maria A. Nikiforova

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: marianikiforova@inbox.ru
ORCID iD: 0000-0001-5823-6508

Research Fellow

Russian Federation, Moscow

Olga V. Zubkova

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation; P. Hertsen Moscow Oncology Research Institute – Branch of the National Medical Research Radiology Center, Ministry of Health of the Russian Federation

Email: olga-zubkova@yandex.ru
ORCID iD: 0000-0001-7893-8419

Doctor of Biological Sciences, Senior Research Fellow

Russian Federation, Moscow; Moscow

Olga D. Popova

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: olga.popova31@yandex.ru
ORCID iD: 0000-0003-3248-1227

Research Fellow

Russian Federation, Moscow

Irina V. Vavilova

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: vavilovairinav@yandex.ru
ORCID iD: 0009-0008-6272-0368

Junior Research Fellow

Russian Federation, Moscow

Tatiana A. Ozharovskaia

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: o.tatiana09@yahoo.com
ORCID iD: 0000-0001-7147-1553

PhD, Research Fellow

Russian Federation, Moscow

Elena P. Mazunina

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: lenok27microb@gmail.com
ORCID iD: 0000-0003-0701-5152

Research Fellow

Russian Federation, Moscow

Evgeniia N. Bykonia

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: evgeniya_bikonya@mail.ru
ORCID iD: 0000-0002-2269-1469

Research Fellow

Russian Federation, Moscow

Elena V. Shidlovskaya

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: lenitsa@gmail.com
ORCID iD: 0000-0001-8199-6417

Research Fellow

Russian Federation, Moscow

Evgeny V. Usachev

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: evgenyvusachev@gmail.com
ORCID iD: 0000-0001-5197-2465

PhD, Senior Research Fellow

Russian Federation, Moscow

Olga V. Usacheva

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: lyapina@rambler.ru
ORCID iD: 0009-0000-8133-1036

Research Fellow

Russian Federation, Moscow

Andrey A. Pochtovyi

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation; I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University)

Email: a.pochtovyi@yandex.ru
ORCID iD: 0000-0003-1107-9351

PhD, Senior Research Fellow, Head of the Biotechnology Laboratory

Russian Federation, Moscow; Moscow

Vladimir I. Zlobin

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: vizlobin@mail.ru
ORCID iD: 0000-0002-0164-5113

Doctor of Medical Sciences, Academician of the Russian Academy of Sciences, Leading Researcher

Russian Federation, Moscow

Denis Y. Logunov

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: logunov@gamaleya.org
ORCID iD: 0000-0003-4035-6581

Doctor of Biological Sciences, Academician of the Russian Academy of Sciences, Director

Russian Federation, Moscow

Alexander L. Gintsburg

National Research Centre for Epidemiology and Microbiology Named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation; I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University)

Email: gintsburg@gamaleya.org
ORCID iD: 0000-0003-1769-5059

Academician of the Russian Academy of Sciences, Scientific Director

Russian Federation, Moscow; Moscow

References

Supplementary files