Mrna-based immunobiological agent for preventing diseases caused by tick-borne encephalitis virus

Abstract

The group of inventions relates to the field of biotechnology, immunology and virology. What is provided is an immunobiological agent for preventing diseases caused by tick-borne encephalitis virus, said agent being based on tick-borne encephalitis virus mRNA containing a region that encodes antigenic pre-membrane (PrM) and envelope (E) proteins of the tick-borne encephalitis virus envelope.

Description

[0001] An immunobiological agent for the prevention of diseases caused by the tick-borne encephalitis virus based on mRNA.

[0002] Field of technology

[0003] This group of inventions relates to the fields of biotechnology, immunology, and virology. The proposed immunobiological agent can be used to prevent diseases caused by the tick-borne encephalitis virus.

[0004] State of the art

[0005] Tick-borne encephalitis (TBE) is a naturally occurring, transmissible viral infection with a pronounced seasonality. The incidence of the disease depends on the activity period of ixodid ticks, the primary vectors of the infection. The disease primarily affects the central nervous system (CNS) and is characterized by a polymorphic clinical course, with a high incidence of residual effects such as paralysis and mortality.

[0006] From a medical perspective, the tick-borne encephalitis virus (TBEV) is the most important representative of the Flavivirus genus, Flaviviridae family. Flaviviridae viruses are spherical, enveloped RNA viruses. The TBEV virion is spherical, 45-50 nm in diameter. The single-stranded, non-segmented (+)RNA genome of the TBEV is approximately 11,000 nucleotides long and encodes three structural genes (C, PrM, E) and seven non-structural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The E protein is the main structural protein of the virion, responsible for virion assembly, fusion with the eukaryotic cell membrane, and receptor binding. In immunized animals, it directly stimulates the synthesis of virus-neutralizing antibodies.Based on the nucleotide sequence of the gene encoding protein E, four TBEV genotypes are currently distinguished: Far Eastern (genotype 1, prototype strain Sof'in), European (Central European or Western, genotype 2, prototype strain Neudoerfl), Siberian (Ural-Siberian, genotype 3, prototype strains Vasilchenko and Zausaev), and the relatively recently described Baikal (prototype strain 886-84). Each TBEV genotype is characterized by an association with a specific tick vector species and a range of vertebrate hosts, different pathogenic potential for humans, and circulation within its own range, within which a certain genotype is noted to dominate.

[0007] TBE is endemic in central Europe and Asia, with circulation reaching France, Japan, and Albania. Currently, cases of TBE have been officially registered in 29 European countries and 6 Asian countries. Up to 12,000 cases of TBE are reported annually worldwide. In Eurasia, Russia is the primary natural focal point for TBE. Factors influencing the incidence of TBE include the length of time a person spends in the natural focal point (determined by professional, economic, and leisure activities), the duration of the tick bite, as well as environmental factors (climate change, resulting in increased tick populations and expansion of their ranges), and technological advances (improved diagnostics).

[0008] Currently, the most effective method of preventing tick-borne encephalitis is vaccination. However, despite the availability of vaccines, there is an increase in the incidence of tick-borne encephalitis due to insufficient vaccination coverage among at-risk groups. Several inactivated vaccines are approved for use in the Russian Federation to immunize the population against the tick-borne encephalitis virus: "Tick-borne encephalitis vaccine, purified, concentrated, inactivated, dry" and "Kleshch-E-Vak" produced by the Federal Scientific Center for Infectious Diseases and Prevention named after M. P. Chumakov Institute of Poliomyelitis of the Russian Academy of Sciences (Moscow, Russia), EnceVir® and EnceVir® Neo for Children manufactured by FSUE NPO Mikrogen of the Ministry of Health of the Russian Federation (Russia), FSME-Immun® and FSME-Immun® Junior manufactured by Pfizer Inc. (Austria), Encepur® and Encepur® for Children manufactured by GSK Vaccine GmbH (Germany).

[0009] Data on the annual incidence of TBE among vaccinated individuals indicate that current vaccines do not provide 100% protection against TBE. Vaccine ineffectiveness may be related to the properties of the vaccine itself, as well as the characteristics of the causative agent or susceptible host.

[0010] Among the areas of development of vaccines for the prevention of TBEV infection, one can highlight the production of attenuated or inactivated vaccines (for example, RU2070929C1; RU2288266C2; RU2541784C2), DNA vaccines (for example, RU2150294C1; RU2112038C1; RU2202612C2) or the construction of recombinant viruses containing cloned genes of the most common flaviviruses (for example, RU2136312C1; RU2208635C2; RU2209082C2; RU2465326C2; EP0877086;

[0011] US6497884; US8828687B2; RU2527891C2). mRNA vaccines are a promising alternative to traditional vaccines due to their high efficacy, safety, rapid development, and relatively low cost of production. Recent technological advances have enabled the development of numerous mRNA vaccine platforms against infectious diseases and certain types of cancer. The resulting products have shown encouraging results in both animal models and humans (e.g., RU2746406C2; RU2022106357; RU2768829C2; US10933127B2). mRNA vaccines are mRNA preparations with modified nucleotides containing an antigen (Ag) sequence. In vitro transcription is typically used for mRNA synthesis. In this case, RNA polymerase of bacteriophage T3, T7 or SP6 and a linearized DNA molecule containing the sequence of the target antigen are used.In vitro transcription does not involve eukaryotic cells, so obtaining mRNA this way is simpler and faster than large-scale protein production and purification [Jain S, Venkataraman A, Wechsler ME, Peppas NA. Messenger RNA-based vaccines: Past, present, and future directions in the context of the CO VID-19 pandemic. Adv Drug Deliv Rev. 2021 Dec; 179: 114000.].

[0012] Upon reaching target cells, mRNA molecules can activate humoral and cellular immune responses through the expression of the target gene into protein products. Cellular immune responses, in which cytotoxic T cells play a primary protective role, have the ability to target infected cells, while direct neutralization or destruction of the pathogen (virus or bacteria) is achieved through humoral immunity. To induce cellular immune responses, the antigen must be delivered to the cytosol, where proteosomal processing occurs. Subsequently, the resulting peptides are delivered to the endoplasmic reticulum, where the antigenic peptides bind to major histocompatibility complex class I (MHC-I) molecules. The MHC-I / peptide complex is then transported to the cell surface for recognition by CD8+ T lymphocytes. Thus, delivery of immunogens to the antigen-processing pathway is a primary goal in modern vaccine development.Nucleic acid-based vaccines are particularly suitable for generating potent cellular immune responses because they ensure expression of the encoded protein in the cytosol of the antigen-presenting target cell [Pollard C, De Koker S, Saelens X, Vanham G, Grooten J. Challenges and advances towards the rational design of mRNA vaccines. Trends Mol Med. 2013 Dec;19(12):705–13. Jain S, Venkataraman A, Wechsler ME, Peppas NA. Messenger RNA-based vaccines: Past, present, and future directions in the context of the COVID-19 pandemic. Adv Drug Deliv Rev. 2021 Dec;179:114000].

[0013] The closest analog is document EP 3723796, which discloses an artificial nucleic acid and polypeptide suitable for use in the prevention of infection caused by flaviviruses, particularly infection caused by yellow fever virus or dengue virus, or a disorder associated with such an infectious disease. The nucleotide sequence of the PrM-E protein of yellow fever virus or dengue virus is used as the antigen for producing the immunogenic composition. This composition can be used to prevent infection by yellow fever virus or dengue virus, but not for the prevention of tick-borne encephalitis.

[0014] Document US20200368343A1, which discloses an artificial nucleic acid and polypeptide suitable for use in the prevention of infection caused by flaviviruses, in particular infection caused by the Zika virus, Chikungunya virus, or dengue virus, or a disorder associated with such an infectious disease, may also be considered as an analog. The nucleotide sequence of the PrM-E proteins of the Zika, Chikungunya, or dengue viruses is used as the antigen for producing the immunogenic composition. This composition can be used to prevent infection by the Zika virus, Chikungunya virus, or dengue virus, but not for the prevention of tick-borne encephalitis.

[0015] Thus, there is a need to create a new immunobiological agent based on TBEV mRNA, which can be used as a means of preventing diseases caused by TBEV.

[0016] Disclosure of the essence of the invention

[0017] The technical objective of the claimed group of inventions is to expand the arsenal of immunobiological agents based on TBEV mRNA for the prevention of diseases caused by TBEV.

[0018] The technical result consists in the creation of an immunobiological agent based on mRNA encoding PrM-E proteins of TBEV, which can be used to induce specific immunity against diseases caused by TBEV.

[0019] This technical result is achieved by creating an immunobiological agent for the prevention of diseases caused by TBEV. This immunobiological agent utilizes the genes encoding the premembrane (PgM) and envelope (E) proteins of the Sof'in TBEV strain for in vitro mRNA transcription. The synthesized mRNA encoding the PrM-E proteins of TBEV is packaged within lipid nanoparticles of TBEV mRNA. In this particular case, the immunobiological agent is a replicated mRNA of the tick-borne encephalitis virus.

[0020] The said technical result is also achieved by the fact that the immunobiological agent can be used to produce vaccines for the prevention of tick-borne encephalitis.

[0021] Brief description of the drawings

[0022] Fig. 1 shows a scheme for obtaining a linear plasmid based on the linear bacterial vector pJAZZ-OK. a) Vector pJAZZ-OK (telN - proteomerase gene, gerA

[0023] - origin of replication, cB - replication repressor, KanR - kanamycin resistance gene, black hemispheres - terminal protelomeric hairpins); b) ColEl origin of replication followed by a restriction site for SmaI restriction enzyme (ori - origin); c) Structural components for in vitro transcription (T7P - promoter for bacteriophage RNA polymerase, 5' and 3' UTRs - 5' and 3' non-coding regions of mRNA, SmaI

[0024] - restriction site, Stop - translation terminator, pow(A) - segmented poly-A tail; d) Schematic representation of the resulting linear plasmid with the open reading frame of the target gene.

[0025] Figure 2 shows the pJAZZ-OK plasmid, which contains the origin of replication, structural components, and genes encoding the PrM-E proteins of TBEV for in vitro transcription.

[0026] Fig. 3 shows the structure of the mRNA component molecule of the immunobiological agent: cap is a modification of the 5'-end of mRNA carried out by the enzyme guanylyltransferase, 5' and 3' UTRs are the 5'- and 3'-non-coding regions of mRNA, respectively; PrM-E TBEV is the sequence encoding PrM-E proteins of TBEV, poly(A) is the segmented poly-A tail.

[0027] Figure 4 shows the results of assessing the level of virus-neutralizing antibodies in model animals immunized with different doses of the mRNA preparation encoding the PrM-E proteins of TBEV. ** - the level of virus-neutralizing antibodies is statistically significantly different from the control group.

[0028] Fig. 5 shows the survival dynamics of animals immunized with the mRNA preparation after experimental infection with TBEV. Implementation of the invention

[0029] The first step in developing an immunobiological agent against the TBEV coronavirus was the selection of a vaccine antigen. A literature review revealed that the PrM and E proteins of the viral envelope were the most promising antigens for creating a candidate mRNA vaccine. These are transmembrane type I glycoproteins responsible for the binding, fusion, and penetration of viral particles into cells. It has been shown to induce neutralizing antibodies (Zimna M et al., Functional characterization and immunogenicity of a novel vaccine candidate against tick-borne encephalitis virus based on Leishmania-derived virus-like particles. Antiviral Res. 2023 Jan:209:105511. doi: 10.1016 / j.antiviral.2022.105511. Epub 2022 Dec 27.).

[0030] TBEV RNA contains one open reading frame (ORF), which is translated into a large polyprotein, which is then co- and post-translationally cleaved by cellular and viral proteases to produce three structural (E, C, and M) and seven non-structural proteins involved in the viral replication cycle inside the cell (Barrows NJ et al. Biochemistry and molecular Biology of flaviviruses Chem. Rev., 118 (8) (2018), pp. 4448-4482). Two viral proteins (glycoprotein E and small membrane protein M) play an important role in viral penetration into target cells. Envelope glycoprotein E, as the most exposed structural element of virions, is involved in the assembly of infectious particles and plays a role in viral penetration, since it ensures interaction with specific cell surface receptors and induces fusion of the viral envelope and the host cell membrane.It consists of three structural domains and a transmembrane domain, which is necessary for anchoring the protein in the lipid membrane. Domain I contains the N-glycosylation site, and the fusion loop peptide is located in domain II (Lattova E et al. Comprehensive N-glycosylation mapping of envelope glycoprotein from tick-borne encephalitis virus grown in human and tick cells. Sci. Rep. [Internet], 10 (1) (2020), pp. 1-10, 10.1038 / s41598-020-70082-2). Domains I and II together are responsible for the dimerization of protein E. Immunoglobulin-like domain III is the most likely candidate for interaction with cellular receptors. It has also been shown that during infection, the majority of neutralizing antibodies are directed against domain III of glycoprotein E (Zhang X et al. Structures and functions of the envelope glycoprotein in flavivirus infections. Viruses, 9 (11) (2017), pp. 1-14).The PrM / M glycoprotein is a small membrane protein that is cleaved to the Pr peptide and M protein present in mature virions during maturation of viral particles. The Pr fragment contains one N-glycosite. The exact role of the PrM protein in flaviviruses has not been fully determined, but it is believed to be a chaperone-like protein that promotes the correct folding of the E glycoprotein. This protein is also required for pH-dependent rearrangements during virion maturation and protection against premature fusion with cellular membranes (Roby JA et al. Post-translational regulation and modifications of flavivirus structural proteins. J. Gen. Virol., 96 (7) (2015), pp. 1551–1569, 2015).

[0031] To obtain mRNA containing the genes for the PrM-E proteins of TBEV, a linear plasmid created on the basis of the pJAZZ-OK vector, which is a linear plasmid system based on the N15 bacteriophage genome, was used as a template for subsequent in vitro transcription. To obtain a plasmid suitable for subsequent in vitro transcription, several sequential clonings were performed from assemblies of intermediate plasmids. The scheme of sequential cloning and assemblies is shown in Figs. 1a-g. The scheme of the final linear plasmid pJAZZ-OK- Ori 5'Sofyin_PrM-E, which includes the origin of replication, structural components, and genes encoding the PrM-E proteins of TBEV for in vitro transcription, is shown in Fig. 2.

[0032] The resulting plasmid DNA was used for subsequent in vitro transcription and cotranscriptional copying. The resulting mRNA was then encapsulated in lipid nanoparticles using a self-assembly process in which an aqueous solution of mRNA at pH 4.0 is rapidly mixed with a solution of lipids dissolved in ethanol.

[0033] The ability of TBEV mRNA packaged in lipid nanoparticles to stimulate the production of virus-neutralizing antibodies against tick-borne encephalitis was assessed using animal models. It was found that serum from immunized animals specifically neutralizes the Sof'in strain of TBEV at dilutions ranging from 1 / 20 to 1 / 1500, suggesting the development of virus-neutralizing antibodies in the animals. TBEV mRNA can be used to prevent TBEV-induced disease. The invention is supported by the following examples.

[0034] Example 1. In order to obtain a plasmid suitable for subsequent in vitro transcription, a SmaI restriction site was first added to the ColEl origin of replication at one end and inserted into pJAZZ-OK to increase the amount of plasmid. The resulting intermediate plasmid was then digested with SmaI, and the polymerase chain reaction products containing mRNA elements (T7 promoter, 5'-UTR, 3'-UTR and poly(A) tail) were assembled using the Gibson Assembly method. Then, before the next assembly, the SmaI site was inserted between the 5'- and 3'-UTRs; after digestion of the resulting plasmid, SmaI PrM-E was cloned directly between the 5'- and 3'-UTRs, also using the Gibson assembly, with the simultaneous removal of the SmaI site. The scheme of sequential cloning and assemblies is shown in Fig. 1a-d.

[0035] Example 2. The diagram of the final linear plasmid pJAZZ-OK-Ori_5'Sofyin_PrM-E, which includes the origin of replication, structural components and genes encoding the PrM-E proteins of TBEV for in vitro transcription is shown in Fig. 2.

[0036] Example 3. DNA of the pJAZZ-OK-Ori_5'Sofyin_PrM-E plasmid for in vitro transcription was isolated and purified from E. coli culture using the Plasmid Maxi Kit (QIAGEN). For in vitro transcription, a reaction mixture was prepared containing 3 μg of DNA template (pJAZZ-OK-Ori_5'Sofyin_PrM-E), 3 μl of T7 RNA polymerase (Biolabmix) and Uxbuffer (TriLink), 4 mM trinucleotide cap analog 1 ((3'-OMe-m7G)-5'-ppp-5 (2'-OMeA)pG)) (Biolabmix), 5 mM ml TP (Biolabmix), and 5 mM GTP, ATP, and CTP. After 2 h of incubation at 37°C, 6 μl of DNase I (Thermo Fisher Scientific) were added for another 15 min, followed by precipitation of mRNA with 2 M LiCl (incubation for 1 h on ice and centrifugation for 10 min at 14,000 g, 4°C). c) and thoroughly washed with 80% ethanol. RNA integrity was assessed by electrophoresis in 8% denaturing PAAT. In vitro transcription resulted in the production of mRNA, a schematic representation of which is shown in Fig. 3.

[0037] Example 4. In vitro transcribed mRNA encoding TBEV PrM-E proteins was encapsulated into lipid nanoparticles using a self-assembly process in which an aqueous solution of mRNA at pH 4.0 is rapidly mixed with a solution of lipids dissolved in ethanol. For this purpose, lipids were dissolved in ethanol at a molar ratio of 46.3:9:42.7:1.6 (ionizable lipid:distearoyl-PC:cholesterol:PEG lipid). The lipid mixture was combined with an acidifying buffer of 10 mM sodium citrate (pH 4.0) containing mRNA (0.2 mg / mL) in a volume ratio of 3:1 (aqueous solution:ethanol) using a Nanoassemblr Spark (Precision NanoSystems). The ratio of ionizable nitrogen atoms in the ionizable lipid to the number of phosphate groups in the mRNA (N:P ratio) was set to 6 for each formulation. The formulations were dialyzed against PBS (pH 7.2) in Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific) for at least 24 hours.The formulations were concentrated using Amicon ultracentrifugal filters (EMD Millipore), then passed through a 0.22 µm filter and stored at 4°C (PBS) until use. The formulations were tested for particle size, zeta potential, and RNA encapsulation. The main physicochemical characteristics of the lipid nanoparticles are presented in Table 1. Table 1. Main physicochemical characteristics of the particles.

[0038] ± SD (standard deviation)

[0039] Example 4. The ability of the obtained mRNA preparation to stimulate the formation of virus-neutralizing antibodies against the tick-borne encephalitis virus was assessed using inbred BALB / c mice and a two-dose immunization regimen.

[0040] Previously, BALB / c females were immunized intramuscularly with the mRNA preparation at a dose of 5 μg or 10 μg. A second (booster) equivalent dose was administered two weeks after the first immunization. Serum was collected two weeks after the second immunization to determine virus-neutralizing activity. The control group of animals received saline solution as a placebo. Table 2 shows the animal groups and the immunization schedule.

[0041] Table 2. Animal groups and immunization schedule

[0042] The virus neutralization assay was performed in 96-well plates (SPL, USA). Serial two-fold dilutions of animal sera were prepared and mixed with 100 TCID50 of tick-borne encephalitis virus (Sofjin strain). The plates were incubated for 1 hour at 37°C. The mixture of serum dilutions with the virus was then transferred to a monolayer of Spev cells. Inhibition of the virus-induced cytopathic effect by the serum dilutions was assessed 4 days later using the MTT assay. The 50% neutralizing titer of serum (HT50) was calculated by regression analysis using GraphPad Prism 9 (GraphPad Software Inc., USA).

[0043] Neutralization assays revealed that serum from immunized animals specifically neutralized the Sof'in strain of TBE virus with HT50 values ​​ranging from 1 / 40 to 1 / 1600, suggesting the development of virus-neutralizing antibodies in the animals. The results of a comparison of virus-neutralizing antibody levels in the serum of immunized animals are presented in Figure 4.

[0044] Example 5. The ability of the obtained mRNA preparation to protect animals from TBEV infection was assessed using inbred BALB / c mice and a two-dose immunization regimen.

[0045] BALB / c female mice were pre-immunized intramuscularly with 5 μg of the mRNA preparation per animal. A second (booster) dose was administered two weeks later, at the same dose. In the control group, saline solution was used as a placebo. Table 3 shows the animal groups and the immunization schedule.

[0046] Table 3. Animal groups and immunization schedule

[0047] Four weeks after the initial immunization, animals were infected intraperitoneally with the Sofjin TBEV strain at a dose of 100 LD50 and monitored for four weeks after infection. During the observation period, only animal deaths were observed in the placebo group. The survival rate of animals in the mRNA group was 100% (Fig. 5).

[0048] Thus, it can be concluded that the developed immunobiological agent can be used to prevent infection with TBEV.

Claims

Invention formula 1. An immunobiological agent for the prevention of diseases caused by the tick-borne encephalitis virus based on the mRNA of the tick-borne encephalitis virus, containing a region encoding the antigenic premembrane (PgM) and envelope (E) proteins of the tick-borne encephalitis virus envelope.

2. An immunobiological agent according to paragraph 1, in which the genes of the premembrane (PgM) and envelope (E) proteins of the Sofjin strain of tick-borne encephalitis virus were used for the synthesis of mRNA.

3. An immunobiological agent according to paragraphs 1-2, which is a copied mRNA of the tick-borne encephalitis virus.

4. An immunobiological agent according to paragraphs 1-3, which is mRNA of the tick-borne encephalitis virus packaged in lipid nanoparticles.

5. Use of an immunobiological agent according to paragraphs 1-4 for obtaining vaccines for the prevention of tick-borne encephalitis.

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