Immunobiological preparation for prevention of tick-borne encephalitis based on mRNA encoding prm-e proteins of krasny yar-8 strain

Abstract

FIELD: biotechnology; immunology; virology.SUBSTANCE: immunobiological preparation for the prevention of tick-borne encephalitis based on mRNA packaged in lipid nanoparticles encoding the prM and E proteins of the Krasny Yar-8 strain of tick-borne encephalitis virus (TBEV) is described. The use of an immunobiological preparation for the production of vaccines for the prevention of tick-borne encephalitis is presented.EFFECT: invention expands the range of means for the prevention of tick-borne encephalitis.3 cl, 6 dwg, 3 tbl, 4 ex

Description

[0001] Technical field

[0002] This group of inventions relates to the fields of immunology, biotechnology, and virology. It proposes the use of an immunological preparation based on mRNA encoding the prM-E proteins of the Krasny Yar strain for the prevention of tick-borne encephalitis.

[0003] Technology Level

[0004] Transmissible viral and bacterial natural focal infections, transmitted by ticks of the Ixodidae family, occupy a leading position in the structure of infectious diseases. Among tick-borne infections, tick-borne encephalitis virus (TBEV) ranks first in prevalence and significance in infectious pathology, due to the severity of the disease and the virus's extensive nosological distribution. Globally, approximately 700 million people are at risk of contracting TBEV annually, and approximately 10,000 become infected. In the Russian Federation, 46 regions are considered endemic for TBEV, with approximately 1,000-2,000 cases reported annually.

[0005] Several domestic and foreign vaccines are approved for use in Russia: "Tick-borne encephalitis vaccine, culture-based, purified, concentrated, inactivated, dry" and "Kleshch-E-Vak" produced by the Chumakov Federal Scientific Center for R&D in Poliomyelitis of the Russian Academy of Sciences (Moscow, Russia), "EnceVir®" and "EnceVir® Neo for children" produced by the Mikrogen Scientific and Production Association of the Ministry of Health of the Russian Federation (Russia), "FSME-Immun®" and "FSME-Immun® Junior" produced by Pfizer Inc. (Austria), "Encepur®" and "Encepur® for children" produced by GSK Vaccines GmbH (Germany). A vaccine for the prevention of tick-borne encephalitis (TBE) has been developed in China, "SenTaiBao" (Changchun, China), but it is not registered in Russia. All vaccines are inactivated. Despite their immunogenicity and safety, modern vaccines do not provide 100% protection. Cases of tick-borne encephalitis (TBE) are reported annually among vaccinated individuals.Even with a high level of vaccine immunity, vaccinated individuals may develop severe forms of TBE with a chronic course and even a fatal outcome.

[0006] Vaccines are biological compositions that prepare the immune system to fight infection. The main components of vaccines are antigens, which are obtained either directly from the pathogen of interest or through biotechnological means. Approaches to vaccine creation have evolved over many years, and today, several technologies and platforms for their production exist, including inactivated vaccines, toxoid vaccines, live attenuated vaccines, vaccines based on virus-like particles, synthetic peptides, polysaccharides and polysaccharide conjugates, viral vector vaccines, nucleic acid (DNA and mRNA)-based vaccines, and bacterial vector / synthetic antigen-presenting cells.

[0007] Recent advances in genetic engineering have enabled the development of next-generation nucleic acid-based vaccines, in which DNA or RNA encoding viral antigens can be incorporated into a vector and administered to the host. Improved stability of in vitro-synthesized mRNA and delivery methods have revolutionized mRNA vaccine development. When introduced into the host, in vitro-synthesized mRNA encoding viral antigens triggers transient expression of the viral protein antigen, thereby stimulating an immune response and creating protective immunological memory.

[0008] Vaccines for the prevention of tick-borne encephalitis can be attenuated or inactivated (e.g. RU 2070929 C1; RU 2288266 C2; RU 2541784 C2), DNA vaccines (e.g. RU 2150294 C1; RU 2112038 C1; RU 2202612 C2) or produced using recombinant viruses containing cloned genes of the most common flaviviruses (e.g. RU 2136312 C1; RU 2208635 C2; RU 2209082 C2; RU 2465326 C2; EP 0877086; US 6497884; US 8828687 B2; RU 2527891 C2).

[0009] mRNA vaccines are considered a promising vaccine option with high efficacy, safety, and rapid development. These vaccines have shown promising results in both animal models and humans (e.g., RU 2746406 C2; RU 2022106357; RU 2768829 C2; US 10933127 B2).

[0010] An immunobiological preparation for the prevention of tick-borne encephalitis based on mRNA encoding prM-E proteins of the Krasny Yar strain is an mRNA preparation with modified nucleotides, packaged in lipid nanoparticles.

[0011] Document RU 2823754 C1 can be considered the closest analog. This document describes the development of an immunobiological agent for the prevention of diseases caused by the tick-borne encephalitis virus, based on tick-borne encephalitis virus mRNA containing a region encoding the antigenic premembrane (PrM) and envelope (E) proteins of the tick-borne encephalitis virus envelope. The immunobiological agent was created using mRNA encoding the prM-E proteins of the Sof'in strain.

[0012] 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, may also be considered a close analogue. 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.

[0013] 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 disorders associated with such infectious diseases, may also be adopted 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.

[0014] Disclosure of the essence of the invention

[0015] 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.

[0016] The technical result consists in the creation of an immunobiological drug for the prevention of tick-borne encephalitis based on mRNA encoding prM-E proteins of the Krasny Yar strain, which can be used to induce specific immunity against diseases caused by tick-borne encephalitis.

[0017] The specified technical result is achieved by creating an immunobiological preparation for the prevention of TBEV, in which mRNA of the PrM-E protein genes of the Krasny Yar strain of TBEV, synthesized during in vitro transcription, is packaged into lipid nanoparticles.

[0018] Also, the specified technical result is achieved by the fact that the immunobiological preparation can be used to obtain vaccines for the prevention of tick-borne encephalitis.

[0019] Brief description of figures

[0020] Figure 1 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 the PrM-E proteins of TBEV, poly(A) is the segmented poly-A tail.

[0021] Figure 2 shows a heat map with the results of a cross-assessment of the level of virus-neutralizing antibodies in model animals immunized with variants of mRNA preparations encoding PrM-E proteins of TBEV Krasny Yar and Sofjin.

[0022] Figure 3 shows the results of assessing the average level of virus-neutralizing antibodies in model animals immunized with different variants of mRNA preparations encoding PrM-E proteins of TBEV. * - the level of virus-neutralizing antibodies differs statistically significantly.

[0023] Figure 4 shows the signal-to-noise ratio (S / N) values ​​for assessing the level of binding antibodies in model animals immunized with variants of mRNA preparations encoding PrM-E proteins of TBEV Krasny Yar and Sofjin.

[0024] Figure 5 shows the geometric mean titer (GMT) values ​​of binding antibodies in model animals immunized with variants of mRNA preparations encoding PrM-E proteins of TBEV Krasny Yar and Sofjin.

[0025] Figure 6 shows the survival dynamics of animals immunized with the mRNA preparation after experimental infection with TBEV.

[0026] Implementation of the invention

[0027] During the first stage of developing an immunobiological drug against TBEV, a vaccine antigen was selected. An analysis of the literature revealed that the PrM and E proteins of the viral envelope were the most promising antigens for creating a candidate mRNA vaccine. Two viral proteins (glycoprotein E and small membrane protein M) play a key role in viral penetration into target cells.

[0028] Example 1. To obtain mRNA containing the genes for the PrM-E proteins of TBEV, a linear plasmid created based on the pJAZZ-OK vector was used. The plasmid was prepared as previously described in patent RU 2823754 C1. In vitro-transcribed mRNA encoding the prM-E proteins of the TBEV strains Sof'in and Krasny Yar-8 was 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. 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 3:1 volume ratio (aqueous: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 formulation was tested for particle size, zeta potential, and RNA encapsulation. The main physicochemical characteristics of the lipid nanoparticles are presented in Table 1.

[0029] Table 1. Main physicochemical characteristics of mRNA-LNP vaccine preparations

[0030] Name of the drug Particle size, d, (nm) (average of three dimensions) PDI (average of three dimensions) Zeta, mV Concentration of packaged mRNA, C (ng / µl) Encapsulation efficiency, E% Krasny Yar 79,5 0,115 -4,79 92 88 Sofyin 79 0,128 -4,4 90 80

[0031] Example 2. The ability of the obtained mRNA preparations to stimulate the formation of virus-neutralizing antibodies against tick-borne encephalitis was assessed using model animals.

[0032] BalbC females were pre-immunized intramuscularly with 5 μg of mRNA per animal. A second (booster) dose was administered 2 weeks later, at the same dose for each group. Serum samples for the neutralization assay were collected 2 weeks after the second immunization. In the control group, saline was used as a placebo for immunization. Table 2 shows the animal groups and the immunization schedule.

[0033] Table 2. Animal groups and immunization schedule

[0034] group Dose 1 of immunization Animals in a group dose / interval mRNA-PrM / E-TBEV (Sofjin) 5 mcg / animal 6 2 in 2 weeks mRNA-PrM / E-TBEV (Krasny Yar-8) 5 mcg / animal 6 2 in 2 weeks

[0035] The virus neutralization assay was performed in 96-well plates (TPP, Switzerland, USA). For this, serial two-fold dilutions of animal sera were prepared and mixed with 100 TCID 50tick-borne encephalitis virus (strains Sof'in, Krasny Yar-8, 205, Zmeinogorsk-1, Lesopark-11, Aina-1448, Vologda-2, Buzuuchuk, 178-79, 288-83). The plates were incubated for 1 hour at 37°C. Then, a mixture of serum dilutions with the virus was transferred to a monolayer of SPEV cells. Inhibition of the virus-induced cytopathic effect by the serum dilutions was assessed after 4 days using the MTT test. 50% neutralizing titer of serum (NT 50 ) was calculated using regression analysis using GraphPad Prism 9 (GraphPad Software Inc., USA).

[0036] The neutralization test showed that serum from immunized animals specifically neutralizes both homologous genovariants and heterologous variants of the TE virus with HT values 50at dilutions ranging from 1 / 20 to 1 / 160, which suggests 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 Figs. 2 and 3.

[0037] Example 3. The ability of the obtained mRNA preparations to stimulate the formation of binding antibodies against tick-borne encephalitis was assessed using model animals (as described in Example 2).

[0038] The enzyme-linked immunosorbent assay was performed in 96-well plates (NEST, China). For this, the plates were coated with recombinant protein E at a concentration of 0.5 μg / ml. Antibodies specific to protein E were analyzed; four variants of the protein E of TBEV - 205, Lesopark-11, Vologda-2, 886 / 84 were used in the analysis. The studied animal sera were serially diluted from 1:100 to 1:100,000 in a 1% BSA solution in 0.01 M PBS and added to the plates; intact mouse serum (PBS group) was used as a negative control (K-). The plates were incubated for 1 hour at 37°C and then washed with 0.01 M phosphate-buffered saline containing 0.05% Tween-20 (PBST). Next, 0.1 ml of peroxidase-conjugated anti-mouse IgG solution was added to each well and the plates were incubated for 45 minutes at 25°C.After incubation, the plates were washed with PBST and 0.1 ml of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added to each plate. The plates were incubated for 10 minutes at room temperature. The reaction was stopped by adding 0.1 ml of 1 M sulfuric acid. Endpoint antibody titers (EPTs) were determined in serially diluted serum samples.

[0039] Analysis of the binding antibody level showed that the positive response was maintained at different dilutions of serum samples for all four antigens (protein E) (Fig. 4). The results were interpreted using the signal-to-noise ratio (S / N), where S is the optical density of the sample and N is the average optical density of the negative control. A cutoff value of S / N > 3 was chosen as the criterion for a positive reaction. To evaluate and compare the titer of binding antibodies, the geometric mean titer (GMT) of the serum at which S / N > 3 was observed was calculated (Fig. 5).

[0040] Example 4. The ability of the obtained mRNA preparations to protect animals from TBEV infection was assessed using BALB / c mice after double immunization.

[0041] Female BALB / c mice were pre-immunized with two doses of mRNA, 2 weeks apart. The immunization dose was 5 μg of mRNA per animal. In the control group, saline was used as a placebo. Table 3 provides a description of the animal groups and the schedule.

[0042] Table 3. Animal groups and immunization schedule

[0043] group Dose Animals in a group dose / interval mRNA-PrM / E-TBEV (Sofjin) 5 mcg / animal 6 2 in 2 weeks mRNA-PrM / E-TBEV (Krasny Yar-8) 5 mcg / animal 6 2 in 2 weeks placebo 50 µl of saline solution 6 2 in 2 weeks

[0044] Four weeks after the first immunization, the animals were intraperitoneally infected with 7 different strains of TBEV (Sofjin, Krasny Yar-8, 205, Zmeinogorsk-1, Lesopark-11, 178-79, 288-83) at a dose of 100 LD 50and observed the animals' condition for 4 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. 6).

[0045] When assessing the cross-virus neutralizing activity against TBEV of different genovariants, as well as the level of binding antibodies in the mRNA-PrM / E-TBEV ELISA (Krasny Yar-8), it showed the highest efficiency both in terms of variant coverage and in terms of the level of virus-neutralizing and binding antibodies.

[0046] Thus, it can be concluded that the developed immunobiological agent can be used to induce specific immunity against diseases caused by TBEV.

Claims

1. An immunobiological preparation for the prevention of tick-borne encephalitis based on mRNA packaged in lipid nanoparticles encoding the prM and E proteins of the Krasny Yar-8 strain of the tick-borne encephalitis virus (TBEV).

2. An immunobiological preparation according to claim 1, characterized in that it contains capped mRNA encoding the prM and E proteins of the Krasny Yar-8 strain of tick-borne encephalitis virus (TBEV).

3. Use of an immunobiological preparation according to any of paragraphs 1, 2 for obtaining vaccines for the prevention of tick-borne encephalitis.

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