Ksenia Tuchynskaya1
Mikhail Vorovitch2,3
Yuriy Kruglov2Guzal Mostipanova2Ivan Kholodilov1Alla Ivanova2Victoria Kuchina1Anastasia Rogova1
Galina Karganova1,3*- 1Laboratory of Biology of Arbovirus, Federal State Autonomous Scientific Institution “Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences” (Institute of Poliomyelitis), Moscow, Russia
- 2Department of Encephalitis Vaccine, Federal State Autonomous Scientific Institution Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis), Moscow, Russia
- 3Institute of Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University, Moscow, Russia
West Nile virus (WNV) is widespread throughout the world. Occasionally, it causes outbreaks of the West Nile fever (WNF) disease, which can lead to severe CNS damage or death. At the same time, the virus’ expanding range is increasingly leading to the formation of mixed foci with other orthoflaviviruses, such as tick-borne encephalitis virus (TBEV). Based on long-term experience using inactivated vaccines to target tick-borne encephalitis (TBE), it seems sensible to create a complex inactivated vaccine targeting two antigens to protect the population against several orthoflaviviruses at once. The immunogenicity and efficacy of the mono-WNV and combined WNV/TBEV inactivated vaccines—based on WNV strain SHUA-3 and TBEV strain Sofjin against a wide range of WNV and TBEV strains—were compared in an in vitro neutralization assay, as well as in BALB/c mice in vivo. The mono vaccines showed a lack of cross-immunogenicity and protection, whereas the combined vaccine was immunogenic against five WNV strains of lineages 1 and 2 and five TBEV strains belonging to different virus subtypes. In the mouse model, the mono-WNV vaccine was effective against the three most pathogenic strains of WNV used in this work, while the combined WNV/TBEV vaccine was effective against both WNV and TBEV. Our work shows promise for the further development of a combined vaccine against WNF and TBE.
1 Introduction
West Nile virus (WNV) is an emerging worldwide neurotropic orthoflavivirus transmitted by mosquitoes. Other orthoflaviviruses that are pathogenic for humans include mosquito-borne viruses, such as Orthoflavivirus dengue (Dengue virus), Orthoflavivirus zikaense (Zika virus), Orthoflavivirus japonicum (Japanese encephalitis virus (JEV)), Orthoflavivirus louisense (St Louis virus), and tick-borne viruses, such as Orthoflavivirus encephalitidis (tick-borne encephalitis virus (TBEV)), Orthoflavivirus powassanense (Powassan virus (POWV)), and Orthoflavivirus omskense (Omsk haemorrhagic fever virus (OHFV)) (1, 2). The orthoflaviviruses genome is approximately 11,000 kb with one open reading frame, which encodes three structural (E, prM, and C) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). Furthermore, mature, enveloped orthoflavivirus particles are about 50 nm in size. The surface of orthoflaviviruses is covered with 90 anti-parallel homodimers of the E structural protein, which is the main target of the antibodies produced during infection (3).
WNV’s main vector is the Culex mosquito; however, as numerous studies have demonstrated, other genus can also serve this function (4, 5). The circulation of this virus is affecting multiple continents including Africa, Eurasia, the Americas, and Australia (6). Annual WNV-related morbidity depends on a combination of factors such as spring rainfall, summer temperatures, fires, etc. Of particular concern is the northwards spread of the primary vector of WNV as annual temperatures rise, a phenomenon that has the potential to precipitate new outbreaks (7).
There are currently nine genetic WNV lineages. Lineage 1 is divided into lineage 1a, which is pathogenic for humans and comprises isolates from Africa (8), Europe, the Middle East, Russia, and the Americas (6, 9); lineage 1b (Kunjin virus), which is distributed in Australia (10); and lineage 1c (now reassigned to Lin 5) isolates only found in India (11). Lineage 2, which is also pathogenic to humans, circulated in Africa until 2004. Thereafter, it spread to Eastern and Central Europe and Russia (12). Lineage 3 includes some isolates from Austria and the Czech Republic (13, 14), while lineage 4 has been isolated and reported from Russia (15). Lineage 6 is based on only a small gene fragment, has been described from Spain and lineages 7 (Koutango virus) and 8 have been isolated in the Somalia and Senegal respectively (16, 17). Putative lineage 9 (or line 4c) was identified in Uranotaenia unguiculata mosquitoes in Austria (18).
WNV lineage 1 and 2, which are responsible for recent outbreaks, are the most pathogenic for humans (19). However, strains within each lineage can be either highly or slightly pathogenic (20).
Repeated outbreaks of West Nile fever (WNF) caused by WNV occurred in Israel in the 1950s (21). Thereafter, sporadic human cases occurred in the Europe, Western Asia and the Middle East until the year 1990 (22). Nevertheless, in 1998, there was an increase in the number of WNF cases from domestic geese, and migratory and local bird populations were associated with reported outbreaks in Romania in 1996 and in southern Russia in 1999, and Israel, in 2000 (23). A major WNF outbreak occurred in the United States from 1999 to 2004. During this period, the virus spread rapidly across the country, resulting in more than 7,000 cases of neuroinvasive WNV disease caused by lineage 1 (24). At the same time, there was an outbreak in the Volgograd region of Russia, where about 1,000 cases were reported (25). In addition, a rise in disease incidence was observed in Russia in 2010, 2012 and 2019 (26). In European countries, outbreaks were observed in 2010 (in Greece) and 2018 (predominantly in Italy) (27, 28). It should be noted that, since 2004, both lineages 1 and 2 of WNV have co-circulated in Europe and Russia (26, 29, 30).
Approximately 80% of WNF cases are asymptomatic, while the remaining 20% are cases that often present with a febrile illness. However, disease develops in less than 1% of cases, with severe CNS lesions manifesting as multiple syndromes including meningitis, encephalitis and poliomyelitis (31, 32). At the highest risk of severe forms are elderly people, and those with comorbidities, those who have undergone immunosuppressive therapy, and those who have a single-nucleotide polymorphisms in several genes (33–36).
A separate problem in adequately estimating WNF case numbers is the presence of the serological cross-reactivity with other orthoflaviviruses, in particular with TBEV. Given the symptomatic similarity of these diseases, studies demonstrate that ELISA kits, which are most often used as the primary diagnostic tool for West Nile fever and tick-borne encephalitis (TBE), do not always facilitate correct diagnosis due to their low specificity (37–39). The co-circulation of these orthoflaviviruses in the same area, as well as active tourism, complicates the situation (40, 41). Aggregate epizootic and epidemiological studies show the sympatry zones of these two viruses in Europe, including Bulgaria (42), Spain (43), Slovakia (40), Germany (44), France (45), Poland (46), Hungary (47, 48), and Slovenia (49), as well as certain regions in Russia (50). However, unlike WNV, the inactivated TBE vaccines are licensed and widely used in Europe and Russia (51–55). They have shown high immunogenicity, efficacy and good protection against the various TBEV strains circulating in different territories (55–60). A new adjuvant-free inactivated TBE vaccine, cultivated in a Vero cell culture, was also developed (61).
There is currently no licensed emergency (62) or specific prophylaxis against WNF for humans (63, 64). Due to the fact that horses and some birds, like humans, are also a dead-end hosts for WNV, a few veterinary vaccines based on the whole inactivated virus and a live chimeric virus combining prM/E WNV and a canarypox main chain, have been used and show good protection for animals (65, 66). The development of a vaccine against WNF for humans is underway; developers are employing diverse platforms and are currently in phase 1 or 2 of clinical trials (63). We have recently developed an inactivated whole-virion vaccine against WNF (67). A significant challenge in developing orthoflavivirus vaccines is the potential for antibody-dependent enhancement (ADE) of infection, which is characterized by a more severe disease course in humans with pre-existing non-neutralizing antibodies or neutralizing antibodies in the sub-neutralizing concentrations. Nevertheless, the epidemiological and experimental evidences of ADE has only been demonstrated only for dengue and Zika viruses, predominantly against mosquito-borne orthoflaviviruses. For tick-borne orthoflaviviruses, there is no strong evidence in this regard (68, 69). The presence of two orthoflaviviruses in one area makes developing a combined vaccine against TBEV and WNV an urgent task.
Here, we report an in vivo study of the immunogenicity and protection efficacy of both WNV alone and combined TBEV/WNV vaccines, based on inactivated virions produced in Vero cell culture against TBEV and WNV strains. The mono-WNV vaccine candidate showed immunogenicity against a wide range of WNV strains belonging to 1 and 2 lineages, as well as high protection during experimental infection. The mixed TBEV/WNV vaccine showed similar immunogenicity results against different WNV and TBEV strains, whereas the monovaccines did not offer cross-protection. The findings of the combined vaccine’s in vivo protection assessment demonstrated favorable outcomes against both viruses.
2 Materials and methods
2.1 Animals
Inbred BALB/c mice (State Institution Scientific Center of Biotechnology, branch “Stolbovaya”, Moscow, Russia) with a weight of 10–12 grams were used in this study. The animals were kept and treated in accordance with the international recommendations for the treatment of laboratory animals (CIOMS recommendations, 1985, the Directive 2010/63/EU, and Appendix A to the European Convention ETS No. 123). The bioethics committee of Chumakov FSC R&D IBP RAS (Institute of Poliomyelitis) (protocol #13032023 from 15 March 2023) approved all experimental procedures performed on animals.
2.2 Cells and viruses
The Vero cell line was cultured in DMEM (Chumakov FSC R&D IBP RAS (Institute of Poliomyelitis), Moscow, Russia) supplemented with 10% FBS (FBS, Invitrogen, Waltham, MA, USA) and penicillin/streptomycin (Paneco, Russia) at 37°C with a 5% CO2 incubator. The porcine embryo kidney (PEK) cell line was maintained at 37°C in medium 199 with Hanks’ balanced salt solution and Earle’s balanced salt solutions (2:1, v:v, (Chumakov FSC R&D IBP RAS (Institute of Poliomyelitis), Moscow, Russia)), supplemented with 5% FBS (Paneco, Moscow, Russia).
The viruses used in these study and there passage history are described in Table 1. The viruses were stored as aliquots of culture fluid or 10% brain suspensions in saline solution (Chumakov FSC R&D IBP RAS (Institute of Poliomyelitis), Moscow, Russia) at -80°C until use. WNV strains SHUA-1 and SHUA-3 were isolated from the serum and saliva samples, respectively, of the patient diagnosed with WNF (67). No nucleotide substitutions were observed in the structural part of the genome of these strains. Strain SHUA-3 was utilized in the preparation of the vaccine, while strain SHUA-1 was employed in the virus challenge experiments.
Table 1. TBEV and WNV strains and other orthoflaviviruses used in the study.
2.3 Preparation of mono-WNV and combined WNV/TBEV vaccine candidates
The mono-WNV and combined WNV/TBEV vaccine candidates were prepared based on WNV SHUA-3 and TBEV Sofjin strains (Genbank KC806252), as described previously (61, 67). In brief, viruses were reproduced separately in a continuous Vero cell line. To inactivate the virus, 0.02% formaldehyde was added to the virus-containing cell culture fluid (VCF). The inactivated VCF (iWNV-VCF and iTBEV-VCF, respectively) was used to prepare mono-WNV and -TBEV concentrates. We performed clarification filtration to remove cell debris. Then iWNV and iTBEV concentrates were prepared by tangential ultrafiltration using a 300K-rated Pellicon 2 Biomax cassette membrane (Millipore, USA). The resulting concentrates were stored briefly at 2-8°C or long-term at -60 to -70°C. Both inactivated concentrates were further purified by gel filtration using Sepharose 6 FF as the sorbent (Cytiva, Marlborough, MA, USA).
Sample iWNV and iTBEV antigen concentration was estimated via a quantitative analysis of E protein content. The analysis was conducted using the ELISA Bioskin-WNV AG kit (Bioservice, Moscow, Russia) and ELISA VectoTBE-antigen kit (Vector-Best, Novosibirsk, Russia), respectively. The built-in of Thermo Scientific SkanIt PC software was used for calculations.
The final protein E concentration for the mono-WNV vaccine candidate was 3.6 µg per mL. Appropriate volumes of the both concentrates were mixed to achieve a final concentration of 3.6 μg per mL of protein E for each of iWNV and iTBEV components of the combined vaccine candidate.
2.4 Vaccination and virus injection
In all vaccination experiments, 8-week-old BALB/c mice were used. Animals were immunized intramuscularly at two-week intervals with 50 μl per mouse (0.1 of the human dose as estimated for EverVac vaccine (61)) into the hind thigh muscle with the mono and combined WNV/TBEV vaccine candidates. In comparative protection experiments, aluminium hydroxide (Al(OH)3, SPI Pharma, France) was used as an adjuvant. Al(OH)3 and both concentrates were mixed to achieve a final concentration of 0.8 mg/ml Al(OH)3, with the final concentration of the iWNV and iTBEV components remaining the same as previously described. The protein E antigen concentration for the monovaccines against TBE and WNF was 180 ng per mouse; for the combined vaccine, this was 180 ng each of TBEV and WNV antigens. Blood for immunogenicity study of the developed vaccine was taken by decapitation a total of two weeks after the last immunization. A virus challenge was also carried out at this stage at s dose 100LD50 for all studied viruses. The number of mice is described in the Results section for each experiment separately.
2.5 Analyzing the presence of WNV and TBEV RNA in mouse brain by RT-PCR
RNA extraction was performed using TRI Reagent LS (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. M-MLV (Eurogen, Moscow, Russia) and WNRT-R (CGGTWYTGAGGGCTTACRTGG) for WNV or TBE/Pow3’ (5’-AGCGGGTGTTTTTCCGAGTC-3’) primers were used for the reverse transcription. PT-PCR was performed on a DNA Engine Analyzer (BioRad, Hercules, CA, USA) utilizing the RT-qPCR kit (Syntol, Russia), in accordance with the manufacturer’s instructions, using primers WNRT-R, WNRT-F (CGGAAGTYGRGTAKACGGTGCTG) and probe-WNV ((FAM)-WCCCCAGGWGGACTG-(BHQ1)) as a probe for WNV and F-TBE (5’-GGGCGGTTCTTGTTCTCC-3’), R-TBE (5’-ACACATCACCTCCTTGTCAGACT-3’) and TBE-probe ((FAM)- TGAGCCACCATCACCCAGACACA-(BHQ1)) as a probe for TBEV. The Sabin I poliovirus strain was used as the internal control, as described previously (70).
2.6 Virus titration in the cell cultures and 50% Plaque Reduction Neutralization Test (PRNT50)
As described earlier, TBEV strains virus titers were determined on the PEK cell culture in 24-well plates (71). Those of the WNV strains were established on the Vero cell culture in 24-well, in a process identical to that of the PEK cell methodology, using methylcellulose coating.
In 24-well plates, PRNT50 was performed on PEK cell monolayers for TBEV and on Vero cell monolayers for WNV. For the PRNT50 assay, mouse blood samples were collected by BALB/c mouse decapitation. Sera were obtained by centrifugation on 1000g for 30 min and stored in aliquots at −20°C. The details of the PRNT50 are described elsewhere (71).
Every experiment included controls, i.e., negative and positive murine sera with known antibody titers. The neutralization antibody (NAb) titers were calculated according to the modified Reed and Muench method (72).
2.7 Virus titration in mice
For the quantification of a 50% lethal dose (LD50) of TBEV and WNV, 8-week-old BALB/c mice in groups of five were injected intraperitoneally (i/p) with 300 μL of 10-fold dilutions of the virus in saline solution (Chumakov FSC R&D IBP RAS (Institute of Poliomyelitis), Moscow, Russia) and observed for clinical symptoms and mortality for 21 days (59). Ruffled fur, hunched posture, limb paresis and paralysis, and body weight loss were considered disease symptoms. The lethal dose of virus resulting in 50% mortality (expressed in log LD50/ml) was calculated according to the Kerber method (73).
2.8 WNV strains genome sequencing
To obtain the complete genome of WNV strains (Asio_otus/RUS/14/2021, HP-90), we used the specific primers described previously (67). The PCR products obtained were analyzed in an agarose gel, and bands of target length were extracted. The bands were purified using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany) and sequenced on the Applied Biosystems 3500 Genetic Analyzer (Waltham, MA, USA) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Vilnius, Lithuania). The resulting sequences were analyzed using Lasergene® SeqMan Pro software version 7.0.0 (DNASTAR Inc., Madison, WI, USA).
2.9 Phylogenetic analysis
RNA sequences of viruses from this study and some other strains of WNV and TBEV were used in the phylogenetic analysis. The nucleotide sequences of the complete fragment of protein E were aligned using ClustalW. Phylogenetic analysis was conducted using the maximum likelihood method based on the General Time Reversible model in MEGA X with 1000 bootstrap replications (74, 75).
2.10 Statistical analysis
Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego CA, USA). For the NAb and virus titers comparisons, the Mann–Whitney U-test was used. Survival curves were analyzed by Kaplan–Meier curves using the log rank test with a Bonferroni correction for multiple comparisons. Differences in morbidity and mortality were assessed using Fisher’s exact test. Median survival time (MST) and incubation period (IP) are presented as median and range.
3 Results
3.1 In vivo immunogenicity and protectivity of mono-WNV vaccine against WNV strains with different virulences
It is known that WNV strains differ in terms of their propensity to induce lethal infections in both avian and mammalian hosts (76–81). Some previously characterized mutations in the genome are known to be responsible for virulence and interferon signaling, in particular in the glycosylation sites of surface protein E and in some positions in non-structural proteins (81–83). In the present study, we utilized five WNV strains belonging to lineages 1 and 2, which are the most pathogenic for humans and have been recently circulating in Europe and Russia (Table 2, Figure 1). The strains varied in virulence, with the most virulent strains in BALB/c mice being SHUA-1 and Strix nebulosa-12 (Table 2). Therefore, the immunogenicity of the WNV monovaccine was tested against all studied strains, and its protectiveness against the most pathogenic strains.
Table 2. Virulence of studied WNV strains in BALB/c mice.
Figure 1. A phylogenetic tree of WNV and TBEV isolates, constructed using the maximum likelihood method using the genome region encoding protein E. The strains used in this work are highlighted in green.
Immunogenicity was tested in a separate experiment. Mouse sera were collected two weeks after double immunization with inactivated WNV monovaccine, based on the SHUA-3 strain (67), and pooled. NAbs titers against all tested strains were greater than 1:10, including the HP-90 strain from a different lineage, when compared to the vaccine strain (Figure 2). The vaccine demonstrated 100% protection against WNV at a dose of 100 LD50 for each virus strain SHUA-1, Strix nebulosa 12 and Asio otus/RUS/14/2021, which were the most pathogenic in BALB/c mice.
Figure 2. Immunogenicity and protectivity of mono-WNV vaccine. (A) Neutralizing antibody titers in pooled serum (N = 3) of BALB/c mice twice immunized with mono-WNV vaccine against five WNV strains (SHUA-1, Strix Nebulosa 12, B-958, Asio_otus/RUS/14/2021, and HP-90). (B) Survival probability of BALB/c mice twice immunized with mono-WNV vaccine (straight line) and non-vaccinated (dashed line) infected with 100 LD50 SHUA-1 (green circles), 100 LD50 Strix nebulosa-12 (violet squares) and 100 LD50 Asio_otus/RUS/14/2021 (orange circles) WNV strains (N = 15 for each group of mice).
3.2 Cross-protectivity of TBE and WNV vaccines cultivated in Vero cell culture
A study of mouse sera obtained after double vaccination with TBE (Tick-E-Vac, manufactured by the Chumakov Federal Scientific Center, Russia) or WNV monovalent vaccines cultured on Vero cells without adjuvant, in the PRNT50 against TBEV and WNV strains used in the vaccine (67) showed an absence of cross-reactive NAbs. In experiments on BALB/c mice testing for vaccines cross-protectivity upon infection with WNV strain SHUA-1 and TBEV strain Sofjin at a dose of 100 LD50, a lack of any protection was also evident (Figure 3).
Figure 3. Survival probability of mice previously twice immunized with mono-TBE and -WNV vaccines. (A) Survival curves of BALB/c mice vaccinated with TBE vaccine (straight line) and non-vaccinated (dashed line) counterparts infected with 100 LD50 of WNV strain SHUA-1 (circles). (B) Survival curves of BALB/c mice vaccinated with WNV vaccine (straight line) and non-vaccinated (dashed line) counterparts infected with 100 LD50 TBEV strain Sofjin (squares) (N = 10 for each group of mice).
3.3 Seroconversion and immunogenicity of the combined WNV/TBEV vaccine
The combined vaccine was derived from purified inactivated whole virions of WNV and TBEV grown in Vero cells with an antigen (TBEV/WNV E protein) ratio of 180/180 ng per mouse with or without adjuvant Al(OH)3. The Al(OH)3 was chosen as an adjuvant because it is used in a licensed vaccine against TBEV, and has been shown to be both safe and effective (84). In the neutralization test, we first compared the seroconversion and immunogenicity of WNV and TBEV mono- and combined vaccines against the virus strain used in the vaccine. For the mono-WNV and combined WNV/TBEV vaccine and WNV/TBEV vaccine with Al(OH)3, seroconversion against the WNV strain SHUA-1 was 100, 90% and 100% respectively. For mono-TBEV and combined WNV/TBEV vaccines with or without Al(OH)3, seroconversion against the TBEV strain Sofjin was 100% for all (Figure 4). Also in the neutralization test, the immunogenicity of the monovalent and combined vaccines against WNV and TBEV did not differ and was greater than the expected protective titer (>1). No cross-protective antibodies were observed in the sera of mice after double immunization with the monovalent vaccines, either with the WNV vaccine against TBEV strain Sofjin or when immunized with TBE vaccine against WNV strain SHUA-1 (data not shown).
Figure 4. Seroconversion and immunogenicity of mono- and combined TBEV/WNV vaccines. (A) Seroconversion level of mono-WNV (green) or combined WNV/TBEV inactivated vaccines with (orange) and without Al(OH)3 (blue) against WNV strain SHUA-1 (N = 10). (B) NAb titers in the individual sera of BALB/c mice immunized with mono-WNV (green) or combined WNV/TBEV inactivated vaccines with (orange) and without Al(OH)3 (blue) against WNV strain SHUA-1 (N = 10). (C) Seroconversion level of mono-TBE (red) or combined WNV/TBEV inactivated vaccines with (orange) and without Al(OH)3 (blue) against TBEV strain Sofjin (N = 10). (D) NAb titers in the individual sera of BALB/c mice immunized with mono-TBE (red) or combined WNV/TBEV inactivated vaccines with (orange) and without Al(OH)3 (blue) against TBEV strain Sofjin (N = 10). The red dotted line indicates the protective titer of NAbs.
3.4 Protectivity of the combined WNV/TBEV vaccine against WNV and TBEV in vivo
The protective efficacy of the combined WNV/TBEV vaccine was evaluated in BALB/c mice against WNV strain SHUA-1 and TBEV strain Sofjin at a dose of 100 LD50. The immunogenicity data correlated with the protection data; the combined vaccine was 100% efficacious against the viruses tested and protecting against both morbidity and mortality (Figure 5). The absence of adjuvant (Al(OH)3) had no effect on vaccine efficacy.
Figure 5. Survival probability (A, C) and initial body weight percentage (B, D) of mice twice vaccinated with combined WNV/TBEV vaccine, with (orange squares) or without (blue circles) Al(OH)3, and infected with TBEV strain Sofjin (A, red triangles) or WNV strain SHUA-1 (C, D, green triangles) (N = 15 for each group of mice). Weight curves are presented only for surviving mice.
Unvaccinated control animals that had been infected with WNV strain SHUA-1 and survived (2/15) exhibited signs of disease; the presence of viral RNA in the brain of one of these survivors 36 days after infection. In the group of mice that were vaccinated with the WNV/TBEV combined vaccine, no mild signs of disease were observed (Figure 5D, Table 3). In groups of animals vaccinated with the combined WNV/TBEV vaccine, viral RNA was detected in only one mouse infected with TBEV 36 days after infection (Table 3).
Table 3. Effectiveness of WNV/TBEV combine vaccine candidate against WNV strain SHUA-1 and TBEV strain Sofjin following double immunization of mice.
3.5 Immunogenicity of the combined WNV/TBEV inactivated vaccine against a wide range of TBEV and WNV strains
In view of the fact that all TBEV subtypes and several WNV lineages are currently circulating in Russia, it is imperative that the vaccine is capable of protecting against all viral strains. Sera of mice twice immunized with mono-TBEV or WNV and combined WNV/TBEV vaccines were tested against TBEV strains belonging to five different subtypes and against WNV strains belonging to two different lineages. The monovalent vaccines demonstrated minimal cross-neutralizing antibodies against some strains of heterologous viruses and NAb titers were lower than 1log10. In contrast, combined vaccine generated board immunogenicity to all tested TBEV and WNV strains. (Figure 6). The NAb titers to different strains did not differ between mono- and combined TBEV or WNV and WNV/TBEV vaccines.
Figure 6. NAb titers in the pools of sera (N = 3) of BALB/c mice immunized with mono-TBEV (Mono-Vac(TBEV) group, red), mono-WNV (Mono-Vac(WNV) group, green) and combined WNV/TBEV vaccines (Vac (WNV/TBEV), blue) against different TBEV (A) and WNV (B) strains.
3.6 Seroconversion and immunogenicity of WNV/TBEV combined vaccine against other ortoflaviviruses
The sera of mice twice vaccinated with WNV/TBEV combined inactivated vaccine mice were tested in PRNT50 against OHFV, POWV, JEV and YFV. We observed a low cross-immunogenity NAbs in mouse sera against OHFV and POWV, as well as an absence of cross-immunogenity NAbs against JEV and YFV (Figure 7).
Figure 7. Seroconversion and immunogenicity of mono- and combined TBEV/WNV vaccines against ortoflaviviruses circulating in Russia. (A) Seroconversion level of combined WNV/TBEV inactivated vaccines against WNV strain SHUA-1, TBEV strain Sofjin, Powassan virus strain Pow-24, Omsk hemorrhagic fever virus strain Nikitina and Japanese encephalitis viruses strain Jagar (N = 5). (B) NAb titers in the individual sera of BALB/c mice immunized with combined WNV/TBEV inactivated vaccines against WNV strain SHUA-1, TBEV strain Sofjin, Powassan virus strain Pow-24, Omsk hemorrhagic fever virus strain Nikitina and Japanese encephalitis viruses strain Jagar (N = 5).
4 Discussion
WNF incidence is sporadic and outbreaks are difficult to predict because of the multifactorial nature of the virus epidemiology (83). There are currently no licensed vaccines for human WNF infection; however, it is a rising concern because of the potential for long-term neurological impairment in patients and because of the expansion of the virus into new territories (84). A human WNV vaccine is currently being actively developed using a variety of platforms (62).
In our work, we have shown that the existing inactivated TBEV vaccine does not protect against experimental WNV infection, which prompted the development of a combination vaccine against two orthoflaviviruses at once.
For mono- WNV and combined WNV/TBEV inactivated vaccines was taken from the SHUA-3 strain and tested against strains belonging to lineages 1 and 2, as it is these that are responsible for the largest disease outbreaks. The relevance of the selected strain for the vaccine against WNV is highlighted by the fact that the SHUA-3 strain was isolated from a patient with fatal West Nile fever in 2021 (67). We investigated the pathogenicity of WNV strains in mice and identified the three most pathogenic strains (Asio_otus/RUS/14/2021, SHUA-1 and Strix nebulosa-12), which were further used to study the protectiveness of the mono-WNV vaccine. It was immunogenic against all five WNV strains tested, and showed 100% protection against lethal infection and morbidity after challenge with three pathogenic strains without Al(OH)3.
Due to the presence of several orthoflavivirus antigens at once, we expected that the combined WNV/TBEV vaccine would show higher immunogenicity against different strains of WNV and TBEV, as well as against other orthoflaviviruses. After the administration of two doses, the TBE and WNV monovaccines did not show a cross-reactive antibody response against different TBE and WNV strains, whereas the combined vaccine was immunogenic against a broad spectrum of both virus strains. The levels of NAbs did not differ from the monovaccine immunity. When sera were tested after double immunization with the combined WNV/TBEV vaccine, NAbs levels demonstrated low titers (~1 log10) against Powassan and OHF viruses were detected in 2/5 sera. No NAbs against JEV and YFV were observed.
The combined WNV/TBEV vaccine has been shown to be protective against both WNV strain SHUA-3 and TBEV strain Sofjin, also used for the vaccine preparation against TBEV, in in vivo experiments, indicating significant potential for its further development and use.
Because of the expanding range of WNV and the difficulty in predicting outbreak locations and timings, the development of a vaccine against WNV is an important public health challenge. The use of combined vaccines has many advantages, such as reducing the number of injections and providing protection against several pathogens at once. At the same time, the use of a combined WNV/TBEV vaccine would be relevant for use in areas where there are already viral co-habitats, or where there is a risk them in the future. Our study showed that both a mono-WNV and combined WNV/TBEV vaccines based on inactivated antigens had good immunogenicity and protected against both mortality and morbidity in mouse experiments.
Data availability statement
The data presented in the study are deposited in the GenBank repository, accession numbers SHUA-3 PX444460, Hp-90 PX444461, Asio_otus/RUS/14/2021 PX444462.
Ethics statement
The animals were kept and treated in accordance with the international recommendations for the treatment of laboratory animals (CIOMS recommendations, 1985, the Directive 2010/63/EU, and Appendix A to the European Convention ETS No. 123). The bioethics committee of Chumakov FSC R&D IBP RAS (Institute of Poliomyelitis) (protocol #13032023 from 15 March 2023) approved all experimental procedures performed on animals. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
KT: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. MV: Conceptualization, Data curation, Investigation, Project administration, Resources, Supervision, Writing – review & editing. YK: Formal analysis, Investigation, Writing – review & editing. GM: Investigation, Methodology, Writing – review & editing. IK: Formal analysis, Investigation, Visualization, Writing – original draft. AI: Investigation, Writing – review & editing. VK: Investigation, Writing – review & editing. AR: Resources, Writing – review & editing. GK: Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the Chumakov FSC R&D IBP RAS (Institute of Poliomyelitis) fundamental research assignment № FNZG-2024-0008.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declare that no Generative AI was used in the creation of this manuscript.
Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
References
1. Go YY, Balasuriya UBR, and Lee C. Zoonotic encephalitides caused by arboviruses: transmission and epidemiology of alphaviruses and flaviviruses. Clin Exp Vaccine Res. (2014) 3:58. doi: 10.7774/cevr.2014.3.1.58
2. Dobler G. Zoonotic tick-borne flaviviruses. Veterinary Microbiol. (2010) 140:221–8. doi: 10.1016/j.vetmic.2009.08.024
3. Zhang X, Jia R, Shen H, Wang M, Yin Z, and Cheng A. Structures and functions of the envelope glycoprotein in flavivirus infections. Viruses. (2017) 9:1–14. doi: 10.3390/v9110338
4. Rochlin I, Faraji A, Healy K, and Andreadis TG. West Nile virus mosquito vectors in North America. J Med Entomology. (2019) 56:1475–90. doi: 10.1093/jme/tjz146
5. Holicki CM, Ziegler U, Răileanu C, Kampen H, Werner D, Schulz J, et al. West Nile virus lineage 2 vector competence of indigenous Culex and Aedes mosquitoes from Germany at temperate climate conditions. Viruses. (2020) 12:561. doi: 10.3390/v12050561
6. May FJ, Davis CT, Tesh RB, and Barrett ADT. Phylogeography of West Nile virus: from the cradle of evolution in Africa to Eurasia, Australia, and the Americas. J Virol. (2011) 85:2964–74. doi: 10.1128/JVI.01963-10
7. D’amore C, Grimaldi P, Ascione T, Conti V, Sellitto C, Franci G, et al. West Nile Virus diffusion in temperate regions and climate change. A systematic review. Le infezioni Med. (2023) 31:20–30. doi: 10.53854/liim-3101-4
8. Mencattelli G, Ndione MHD, Rosà R, Marini G, Diagne CT, Diagne MM, et al. Epidemiology of West Nile virus in Africa: An underestimated threat. PLoS Negl Trop Dis. (2022) 16:e0010075. doi: 10.1371/journal.pntd.0010075
9. Lanciotti RS, Ebel GD, Deubel V, Kerst AJ, Murri S, Meyer R, et al. Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East. Virology. (1951) 298:96–105. doi: 10.1006/viro.2002.1449
10. Prow NA. The changing epidemiology of Kunjin virus in Australia. Int J Environ Res Public Health. (2013) 10:6255–72. doi: 10.3390/ijerph10126255
11. Rajaiah P, Mayilsamy M, and Kumar A. West Nile virus in India: An update on its genetic lineages. J. Vector Borne Dis. (2023) 60:225–37. doi: 10.4103/0972-9062.374039
12. Bakonyi T, Ivanics É, Erdélyi K, Ursu K, Ferenczi E, Weissenböck H, et al. Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe. Emerging Infect Dis. (2006) 12:618. doi: 10.3201/eid1204.051379
13. Bakonyi T, Hubálek Z, Rudolf I, and Nowotny N. Novel flavivirus or new lineage of West Nile virus, Central Europe. Emerging Infect Dis. (2005) 11:225. doi: 10.3201/eid1102.041028
14. Hubálek Z, Rudolf I, Bakonyi T, Kazdová K, Halouzka J, Šebesta O, et al. Mosquito (Diptera: Culicidae) surveillance for arboviruses in an area endemic for West Nile (Lineage Rabensburg) and Ťahyňa viruses in central Europe. J Med Entomology. (2010) 47:466–72. doi: 10.1603/me09219
15. Lvov DK, Butenko AM, Gromashevsky VL, Kovtunov AI, Prilipov AG, Kinney R, et al. West Nile virus and other zoonotic viruses in Russia: examples of emerging-reemerging situations. Arch virology. Supplementum. (2004) 18:85–96. doi: 10.1007/978-3-7091-0572-6_7
16. Fall G, Diallo M, Loucoubar C, Faye O, and Sall AA. Vector Competence of Culex neavei and Culex quinquefasciatus (Diptera: Culicidae) from Senegal for Lineages 1, 2, Koutango and a Putative New Lineage of West Nile virus. Am J Trop Med Hygiene. (2014) 90:747. doi: 10.4269/ajtmh.13-0405
17. Lobo JMd.A, Christofferson RC, and Mores CN. Investigations of Koutango virus infectivity and dissemination dynamics in Aedes aEgypti mosquitoes. Environ Health Insights. (2014) 8:9. doi: 10.4137/EHI.S16005
18. Pachler K, Lebl K, Berer D, Rudolf I, Hubalek Z, and Nowotny N. Putative new West Nile virus lineage in Uranotaenia unguiculata mosquitoes, Austria, 2013. Emerging Infect Dis. (2014) 20:2119–22. doi: 10.3201/eid2012.140921
19. Saiz JC, Martín-Acebes MA, Blázquez AB, Escribano-Romero E, Poderoso T, and Jiménez de Oya N. Pathogenicity and Virulence of West Nile virus revisited eight decades after its first isolation. Virulence. (2021) 12:1145–73. doi: 10.1080/21505594.2021.1908740
20. Beasley DWC, Li L, Suderman MT, and Barrett ADT. Mouse neuroinvasive phenotype of West Nile virus strains varies depending upon virus genotype. Virology. (2002) 296:17–23. doi: 10.1006/viro.2002.1372
21. Bin H, Grossman Z, Pokamunski S, Malkinson M, Weiss L, Duvdevani P, et al. West Nile fever in Israel 1999–2000 from Geese to humans. Ann N Y Acad Sci. (2001) 951:127–42. doi: 10.1111/j.1749-6632.2001.tb02691.x
22. Rappole JH and Hubálek Z. Migratory birds and West Nile virus. J Appl Microbiol Symposium Supplement. (2003) 94:47–58. doi: 10.1046/j.1365-2672.94.s1.6.x
23. Chancey C, Grinev A, Volkova E, and Rios M. The global ecology and epidemiology of West Nile virus. BioMed Res Int. (2015) 2015. doi: 10.1155/2015/376230
24. Hayes EB, Komar N, Nasci RS, Montgomery SP, O’Leary DR, and Campbell GL. Epidemiology and transmission dynamics of West Nile virus disease. Emerging Infect Dis. (2005) 11:1167. doi: 10.3201/eid1108.050289a
25. Platonov AE, Shipulin GA, Shipulina OY, Tyutyunnik EN, Frolochkina TI, Lanciotti RS, et al. Outbreak of West Nile virus infection, volgograd region, Russia, 1999. Emerging Infect Dis J. (2001) 7:128–32. doi: 10.3201/eid0701.010118
26. Putintseva EV, Udovichenko SK, Nikitin DN, Borodai NV, Shpak IM, Fomina VK, et al. West Nile fever: results of monitoring over the causative agent in the Russian federation in 2021, the incidence forecast for 2022. Problems Particularly Dangerous Infections. (2022) 28:43–53. doi: 10.21055/0370-1069-2022-1-43-53
27. Bouzalas IG, Diakakis N, Chaintoutis SC, Brellou GD, Papanastassopoulou M, Danis K, et al. Emergence of equine West Nile encephalitis in central Macedonia, Greece, 2010. Transboundary Emerging Dis. (2016) 63:e219–27. doi: 10.1111/tbed.12334
28. Camp JV and Nowotny N. The knowns and unknowns of West Nile virus in Europe: what did we learn from the 2018 outbreak? J. Travel Med. (2020) 18:145–54. doi: 10.1080/14787210.2020.1713751
29. Barzon L, Pacenti M, Montarsi F, Fornasiero D, Gobbo F, Quaranta E, et al. Rapid spread of a new West Nile virus lineage 1 associated with increased risk of neuroinvasive disease during a large outbreak in Italy in 2022. J Travel Med. (2022) 2022:1–11. doi: 10.1093/jtm/taac125
30. Aguilera-Sepúlveda P, Napp S, Llorente F, Solano Manrique C, Molina-López R, Obón E, et al. West Nile virus lineage 2 spreads westwards in Europe and overwinters in North-Eastern Spain (2017–2020). Viruses. (2022) 14:569. doi: 10.3390/v14030569
31. Sejvar JJ. Clinical manifestations and outcomes of West Nile virus infection. Viruses. (2014) 6:606–23. doi: 10.3390/v6020606
32. Byas AD and Ebel GD. Comparative pathology of West Nile virus in humans and non-human animals. Pathogens. (2020) 9:48. doi: 10.3390/pathogens9010048
33. Montgomery RR. Age-related alterations in immune responses to West Nile virus infection. Clin Exp Immunol. (2017) 187:26–34. doi: 10.1111/cei.12863
34. Montgomery RR and Murray KO. Risk factors for West Nile virus infection and disease in populations and individuals. Expert Rev Anti Infect Ther. (2015) 13:317–25. doi: 10.1586/14787210.2015.1007043
35. Hart J, Tillman G, Kraut MA, Chiang HS, Strain JF, Li Y, et al. West Nile virus neuroinvasive disease: Neurological manifestations and prospective longitudinal outcomes. BMC Infect Dis. (2014) 14:1–10. doi: 10.1186/1471-2334-14-248
36. Ronca SE, Ruff JC, and Murray KO. A 20-year historical review of West Nile virus since its initial emergence in North America: Has West Nile virus become a neglected tropical disease? PLoS Negl Trop Dis. (2021) 15:e0009190. doi: 10.1371/journal.pntd.0009190
37. Koraka P, Zeller H, Niedrig M, Osterhaus ADME, and Groen J. Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA. Microbes Infection. (2002) 4:1209–15. doi: 10.1016/S1286-4579(02)01647-7
38. Niedrig M, Sonnenberg K, Steinhagen K, and Paweska JT. Comparison of ELISA and immunoassays for measurement of IgG and IgM antibody to West Nile virus in human sera against virus neutralisation. J Virological Methods. (2007) 139:103–5. doi: 10.1016/j.jviromet.2006.09.009
39. Beck C, Jimenez-Clavero MA, Leblond A, Durand B, Nowotny N, Leparc-Goffart I, et al. Flaviviruses in Europe: complex circulation patterns and their consequences for the diagnosis and control of West Nile disease. Int J Environ Res Public Health. (2013) 10:6049–83. doi: 10.3390/ijerph10116049
40. Čabanová V, Kerlik J, Kirschner P, Rosochová J, Klempa B, Sláviková M, et al. o-circulation of West Nile, Usutu, and tick-borne encephalitis viruses in the same area: A great challenge for diagnostic and blood and organ safety. Viruses. (2023) 15:1–11. doi: 10.3390/v15020366
41. Shin A, Tukhanova N, Ndenkeh J, Shapiyeva Z, Yegemberdiyeva R, Yeraliyeva L, et al. Tick-borne encephalitis virus and West Nile fever virus as causes of serous meningitis of unknown origin in Kazakhstan. Zoonoses Public Health. (2022) 69:514–25. doi: 10.1111/zph.12941
42. Christova I, Panayotova E, Tchakarova S, Taseva E, Trifonova I, and Gladnishka T. A nationwide seroprevalence screening for West Nile virus and Tick-borne encephalitis virus in the population of Bulgaria. J Med Virol. (2017) 89:1875–8. doi: 10.1002/jmv.24855
43. García-Bocanegra I, Jurado-Tarifa E, Cano-Terriza D, Martínez R, Pérez-Marín JE, and Lecollinet S. Exposure to West Nile virus and tick-borne encephalitis virus in dogs in Spain. Transboundary Emerging Dis. (2018) 65:765–72. doi: 10.1111/tbed.12801
44. Gothe LMR, Ganzenberg S, Ziegler U, Obiegala A, Lohmann KL, Sieg M, et al. Horses as sentinels for the circulation of flaviviruses in eastern–central Germany. Viruses. (2023) 15:1108. doi: 10.3390/v15051108
45. Bournez L, Umhang G, Faure E, Boucher J-M, Boué F, Jourdain E, et al. Exposure of wild ungulates to the Usutu and tick-borne encephalitis viruses in France in 2009-2014: evidence of undetected flavivirus circulation a decade ago. Viruses. (2019) 12:10. doi: 10.3390/v12010010
46. Bażanów B, Jansen van Vuren P, Szymański P, Stygar D, Frącka A, Twardoń J, et al. A survey on West Nile and Usutu viruses in horses and birds in Poland. Viruses. (2018) 10:87. doi: 10.3390/v10020087
47. Egyed L, Nagy A, Lakos A, Zöldi V, and Lang Z. Tick-borne encephalitis epidemic in Hungary 1951–2021: The story and lessons learned. Zoonoses Public Health. (2023) 70:81–92. doi: 10.1111/zph.13003
48. Nagy A, Horvath A, Mezei E, Henczko J, Magyar N, Nagy O, et al. West Nile virus infections in Hungary: Epidemiological update and phylogenetic analysis of the Hungarian virus strains between 2015 and 2022. Acta Microbiologica Immunologica Hungarica. (2023) 70:111–8. doi: 10.1556/030.2023.02040
49. Kvapil P, Račnik J, Kastelic M, Pittermannová P, Avšič-Zupanc T, Bártová E, et al. Detection of antibodies against tick-borne encephalitis virus and other flaviviruses in a zoological collection in Slovenia. Front Veterinary Sci. (2021) 8:688904. doi: 10.3389/fvets.2021.688904
50. Udovichenko SK, Putintseva EV, Gusev EA, and Toporkov AV. Actual problems of epidemiological surveillance of West Nile fever in Russia and ways of its improvement. Epidemiologiya i Vaktsinoprofilaktika. (2024) 23:13–23. doi: 10.31631/2073-3046-2024-23-5-13-23
51. Erber W, Khan F, Zavadska D, Freimane Z, Dobler G, Böhmer MM, et al. Effectiveness of TBE vaccination in southern Germany and Latvia. Vaccine. (2022) 40:819–25. doi: 10.1016/j.vaccine.2021.12.028
52. Santonja I, Stiasny K, Essl A, Heinz FX, Kundi M, and Holzmann H. Tick-borne encephalitis in vaccinated patients: A retrospective case-control study and analysis of vaccination field effectiveness in Austria from 2000 to 2018. J Infect Dis. (2023) 227:512–21. doi: 10.1093/infdis/jiac075
53. Zens KD, Haile SR, Schmidt AJ, Altpeter ES, Fehr JS, and Lang P. Retrospective, matched case–control analysis of tickborne encephalitis vaccine effectiveness by booster interval, Switzerland 2006–2020. BMJ Open. (2022) 12:e061228. doi: 10.1136/bmjopen-2022-061228
54. Romanenko VV, Yesyunina M, and KilyaChina AS. [Experience in implementing the mass immunization program against tick-borne encephalitis in the Sverdlovsk region. Vopr. Virusol. (2007) 52:22–5.
55. Shcherbinina MS, Barkhaleva OA, Dorokhova OS, and Movsesyants AA. Effectiveness of specific prevention of tick-Borne encephalitis. BIOpreparations. Prevention Diagnosis Treat. (2020) 20:174–86. doi: 10.30895/2221-996X-2020-20-3-174-186
56. Chernokhaeva LL, Rogova YV, Vorovitch MF, Romanova LI, Kozlovskaya LI, Maikova GB, et al. Protective immunity spectrum induced by immunization with a vaccine from the TBEV strain Sofjin. Vaccine. (2016) 34:2354–61. doi: 10.1016/j.vaccine.2016.03.041
57. Šmit R and Postma MJ. Review of tick-borne encephalitis and vaccines: clinical and economical aspects. Expert Rev Vaccines. (2015) 14:737–47. doi: 10.1586/14760584.2015.985661
58. Maikova GB, Chernokhaeva LL, Rogova YV, Kozlovskaya LI, Kholodilov IS, Romanenko VV, et al. Ability of inactivated vaccines based on far-eastern tick-borne encephalitis virus strains to induce humoral immune response in originally seropositive and seronegative recipients. J Med Virol. (2019) 91:190–200. doi: 10.1002/jmv.25316
59. Prochorova O, Romanenko V, and Zlobin V. Comparative characteristics of immunological activity of tick-borne encephalitis vaccines used during a mass immunization program in the Sverdlovsk region. Èpidemiol Vaktsinoprofilaktika. (2006) 29:33–6.
60. Erber W and Schmitt HJ. Self-reported tick-borne encephalitis (TBE) vaccination coverage in Europe: Results from a cross-sectional study. Ticks tick-borne Dis. (2018) 9:768–77. doi: 10.1016/j.ttbdis.2018.02.007
61. Vorovitch MF, Grishina KG, Volok VP, Chernokhaeva LL, Grishin KV, Karganova GG, et al. Evervac: phase I/II study of immunogenicity and safety of a new adjuvant-free TBE vaccine cultivated in Vero cell culture. Hum Vaccin Immunother. (2020) 16:2123–30. doi: 10.1080/21645515.2020.1757990
62. Diamond MS. Development of effective therapies against West Nile virus infection. Expert Rev Anti Infect Ther. (2005) 3:931–44. doi: 10.1586/14787210.3.6.931
63. Kaiser JA and Barrett ADT. Twenty years of progress toward West Nile virus vaccine development. Viruses. (2019) 11:823. doi: 10.3390/v11090823
64. Sambri V. West Nile virus in Europe: Emergence, epidemiology, diagnosis, treatment, and prevention. Clin Microbiol Infect. (2013) 19:699–704. doi: 10.1111/1469-0691.12211
65. Ng T, Hathaway D, Jennings N, Champ D, Chiang YW, and Chu HJ. Equine vaccine for West Nile virus. Developments Biologicals. (2003) 114:221–7.
66. Cortese V, Hankins K, Holland R, and Syvrud K. Serologic responses of West Nile virus seronegative mature horses to West Nile virus vaccines. J Equine Veterinary Sci. (2013) 33:1101–5. doi: 10.1016/j.jevs.2013.04.010
67. Vorovitch MF, Tuchynskaya KK, Kruglov YA, Peunkov NS, Mostipanova GF, Kholodilov IS, et al. An inactivated West Nile virus vaccine candidate based on the lineage 2 strain. Vaccines. (2024) 12:1398–2024. doi: 10.3390/vaccines12121398
68. Dutta SK and Langenburg T. A perspective on current flavivirus vaccine development: A brief review. Viruses. (2023) 15:860. doi: 10.3390/v15040860
69. Thomas S, Smatti MK, Ouhtit A, Cyprian FS, Almaslamani MA, Al Thani A, et al. Antibody-Dependent Enhancement (ADE) and the role of complement system in disease pathogenesis. Mol Immunol. (2022) 152:172–82. doi: 10.1016/j.molimm.2022.11.010
70. Romanova LI, Gmyl AP, Dzhivanian TI, Bakhmutov DV, Lukashev AN, Gmyl LV, et al. Microevolution of tick-borne encephalitis virus in course of host alternation. Virology. (2007) 362:75–84. doi: 10.1016/j.virol.2006.12.013
71. Tuchynskaya K, Volok V, Illarionova V, Okhezin E, Polienko A, Belova O, et al. Experimental assessment of possible factors associated with tick-borne encephalitis vaccine failure. Microorganisms. (2021) 9:1–21. doi: 10.3390/microorganisms9061172
72. Reed LJ and Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol. (1938) 27:493–7. doi: 10.1093/oxfordjournals.aje.a118408
73. Lorenz RJ and Bögel K. Laboratory techniques in rabies: methods of calculation. Monograph series. World Health Organ. (1973), 321–35.
74. Nei M and Kumar S. Molecular Evolution and Phylogenetics Vol. 1–2c. . New York, NY, USA: Oxford University Press (2000).
75. Kumar S, Stecher G, Li M, Knyaz C, and Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. (2018) 35:1547–9. doi: 10.1093/molbev/msy096
76. Brault AC, Langevin SA, Bowen RA, Panella NA, Biggerstaff BJ, Miller BR, et al. Differential virulence of West Nile strains for American crows. Emerging Infect Dis. (2004) 10:2161. doi: 10.3201/eid1012.040486
77. Frost MJ, Zhang J, Edmonds JH, Prow NA, Gu X, Davis R, et al. Characterization of virulent West Nile virus Kunjin strain, Australia, 2011. Emerging Infect Dis. (2012) 18:792. doi: 10.3201/eid1805.111720
78. Perez-Ramírez E, Llorente F, Del Amo J, Fall G, Sall AA, Lubisi A, et al. Pathogenicity evaluation of twelve West Nile virus strains belonging to four lineages from five continents in a mouse model: discrimination between three pathogenicity categories. J Gen Virol. 98:662–70. doi: 10.1099/jgv.0.000743
79. Beasley DWC, Whiteman MC, Zhang S, Huang CY-H, Schneider BS, Smith DR, et al. Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains. J Virol. (2005) 79:8339–47. doi: 10.1128/JVI.79.13.8339-8347.2005
80. Donadieu E, Lowenski S, Servely JL, Laloy E, Lilin T, Nowotny N, et al. Comparison of the neuropathology induced by two West Nile virus strains. PLoS One. (2013) 8:e84473. doi: 10.1371/journal.pone.0084473
81. Rawle DJ, Setoh YX, Edmonds JH, and Khromykh AA. Comparison of attenuated and virulent West Nile virus strains in human monocyte-derived dendritic cells as a model of initial human infection. Virol J. (2015) 12:1–5. doi: 10.1186/s12985-015-0279-3
82. Setoh YX, Prow NA, Rawle DJ, Tan CSE, Edmonds JH, Hall RA, et al. Systematic analysis of viral genes responsible for differential virulence between American and Australian West Nile virus strains. J Gen Virol. (2015) 96:1297–308. doi: 10.1099/vir.0.000069
83. Fiacre L, Lowenski S, Bahuon C, Dumarest M, Lambrecht B, Dridi M, et al. Evaluation of NS4A, NS4B, NS5 and 3′UTR genetic determinants of WNV lineage 1 virulence in birds and mammals. Viruses. (2023) 15:1094–2023. doi: 10.3390/v15051094
84. Domnich A, Panatto D, Klementievna Arbuzova E, Signori A, Avio U, Gasparini R, et al. Immunogenicity against Far Eastern and Siberian subtypes of tick-borne encephalitis (TBE) virus elicited by the currently available vaccines based on the European subtype: Systematic review and meta-analysis. Hum Vaccines Immunotherapeutics. (2014) 10:2819–33. doi: 10.4161/hv.29984
Keywords: WNV, TBEV, inactivated vaccine, combined vaccine, orthoflaviviruses, flaviviruses
Citation: Tuchynskaya K, Vorovitch M, Kruglov Y, Mostipanova G, Kholodilov I, Ivanova A, Kuchina V, Rogova A and Karganova G (2025) Mono-WNV and combined WNV/TBEV inactivated vaccine efficacy against a wide range of WNV and TBEV strains. Front. Immunol. 16:1715371. doi: 10.3389/fimmu.2025.1715371
Received: 29 September 2025; Accepted: 24 November 2025; Revised: 14 November 2025;
Published: 12 December 2025.
Edited by:
Ang Lin, China Pharmaceutical University, ChinaReviewed by:
Kyra Zens, University of Zurich, Institute for Experimental Immunology, SwitzerlandAlbert To, University of Hawaii at Manoa, United States
Copyright © 2025 Tuchynskaya, Vorovitch, Kruglov, Mostipanova, Kholodilov, Ivanova, Kuchina, Rogova and Karganova. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Galina Karganova, a2FyZ2Fub3ZhQGJrLnJ1