ATTENUATED STRAIN OF SARS-CoV-2 CORONAVIRUS FOR DEVELOPMENT OF SPECIFIC PREVENTATIVE MEASURE AGAINST COVID-19

Abstract

FIELD: molecular biology; medical biotechnology.SUBSTANCE: intended to create a means that can be used for the specific prevention of COVID-19. The invention is an attenuated strain of SARS-CoV-2 F-F3 for the specific prevention of COVID-19, deposited under No. 39069 in the collection of the Unique Scientific Facility “Collection of Microorganisms of Pathogenicity Groups III and IV named after I.I. Mechnikov”.EFFECT: claimed strain is characterized by: evolutionary proximity to the current, circulating genovariants of the virus, since the attenuated mutant is obtained on the basis of an omicron-like strain of SARS-CoV-2; the formation of a protective immune response against SARS-CoV-2 even with a single intranasal immunization; the ability to provide protection against the disease when infected not only with a homologous, but also with a heterologous strain of SARS-CoV-2; safety, which is determined by the inability of the attenuated mutant to infect vital internal organs (lungs and brain) and the stability of the attenuated phenotype during long-term passaging; high reproductive activity of the attenuated mutant in a Vero CCL-81 cell culture at a temperature of 24° C, which allows obtaining viral material with high specific activity.1 cl, 2 dwg, 4 tbl, 5 ex

Description

[0001] Technical field

[0002] The invention relates to the field of medical biotechnology and is intended to create a means that can be used to create a means for the specific prevention of COVID-19.

[0003] Technology Level

[0004] The novel coronavirus infection caused by SARS-CoV-2 (species Betacoronavirus pandemicum, family Coronaviridae) has become the first recorded and comprehensively studied coronavirus pandemic in human history. Over the course of the pandemic, more than 7 million deaths from COVID-19 and its complications have been recorded worldwide, while the actual mortality rate is estimated to be even higher. This demonstrates the insufficient preparedness of national healthcare systems to counter such biological threats. In the spring of 2023, the World Health Organization declared the end of the global health emergency related to the COVID-19 pandemic. However, despite the end of the pandemic and the established high level of herd immunity against the novel coronavirus infection, the risk of the emergence of new epidemically significant SARS-CoV-2 variants, which could lead to a surge in COVID-19 cases, remains.In this regard, the problem of developing specific means of preventing COVID-19 remains relevant.

[0005] For the specific prevention of COVID-19, vaccines based on different technological platforms are used: inactivated vaccines (based on "killed" whole-virion preparations of SARS-CoV-2); vector vaccines (based on recombinant viruses containing genes for protective SARS-CoV-2 proteins); subunit vaccines (based on the use of individual purified viral protective antigens); DNA vaccines and mRNA vaccines (based on DNA or RNA molecules that encode information about protective viral antigens). Widespread clinical use of licensed vaccines has demonstrated their effectiveness against natural infection with homologous variants of the virus (i.e. infection with genovariants of the virus on which the vaccine is based) (Feikin et al., 2022).

[0006] The emergence and widespread dissemination of the Omicron variant and its descendants has led to a sharp decrease in the effectiveness of COVID-19 vaccination (Bowen et al., 2022; Dejnirattisai et al., 2022). A possible solution to this problem could be the development of a live attenuated vaccine (LAV) against COVID-19, capable of inducing a robust immune response to both structural and non-structural viral proteins and activating not only humoral but also cellular immunity (Golawski et al., 2022; Minor, 2015; Pollard and Bijker, 2021). Intranasal administration of LAV ensures the formation of local, mucosal immunity directly at the “entry gate” of infection – the respiratory tract mucosa. The use of live viral vaccines is justified not only by their high immunological efficacy but also by their economic feasibility, as they are characterized by low production costs. In recent years, considerable effort has been devoted to developing LAVs against COVID-19.Below are the patents and scientific publications that describe the closest analogues.

[0007] A solution is known for patent WO 2022011428 A1 (priority date - 16.07.2020), which proposes the use of an attenuated strain of SARS-CoV-2 as a vaccine, in which attenuation is achieved by deoptimizing the codons of the genomic region corresponding to ORF1a.

[0008] A solution is known for patent WO 2022037248 A1 (priority date - 19.08.2020), which proposes an attenuated SARS-CoV-2 strain Ca-DelMut-LASV (GenBank ID - MT862537) as a vaccine, characterized by a deletion of the amino acid sequence NSPRRARSVA (amino acids 679-688, NCBI Reference Sequence: YP 009724390.1) and amino acid substitutions P25L and V367F in the S protein relative to wild-type SARS-CoV-2, as well as amino acid substitutions A578V in the NSP3 protein, F20S in the E protein and V62L in ORF8. The Wuhan-Hu-1 strain (GenBank ID - NC 045512.2), isolated in December 2019, was used as a basis.

[0009] A patent solution known from WO 2022103870 A1 (priority date: November 10, 2020) describes the production of an attenuated SARS-CoV-2 strain with one or more attenuating mutations in the coding regions of the viral genome, which are deletions of at least 10 nucleotides and / or nucleotide substitutions. One aspect of the invention involves cloning the genome of the attenuated SARS-CoV-2 strain containing the attenuating mutations in the pY2B vector with regulatory sequences necessary for autonomous replication in yeast or bacterial cells.

[0010] A patent solution is known for WO 2023122731 A2 (priority date - December 22, 2021), in which the vaccine strain for LAV is obtained based on a recombinant attenuated strain of the epidemic mumps virus (strains JL1 or JL2). The patent considers variants of the recombinant epidemic mumps virus (rMuV), the genome of which can encode the SARS-CoV-2 S protein, fragments of the S protein, as well as other structural (M, N, and E) and non-structural proteins (nspl-nspl6) of SARS-CoV-2. The amino acid sequences of these viral proteins can correspond to different SARS-CoV-2 variants, including Alpha, Beta, Delta, Gamma, Lambda, and Omicron.

[0011] A known solution is patent WO 2023138770 A1 (priority date - 20.01.2022), in which the production of an attenuated strain of SARS-CoV-2 is based on the deoptimization of the S protein codons, and / or at least one of the following non-structural proteins of the virus - nsp7, nsp8, nsp9, nsp10, nsp11, ns1l2, nsp7, endoribonuclease, 2'-O-methyltransferase. The virulence of the virus with deoptimized codons is reduced due to a decrease in translation efficiency. Ultimately, codon deoptimization leads to a decrease in the production of viral proteins, viral reproduction, virulence and the production of an attenuated virus. Embodiments of the patent include the use of one of the SARS-CoV-2 variants - Alpha, Beta, Gamma, Delta or Omicron.

[0012] A known solution is patent WO 2023235863 A1 (priority date - 03.06.2022), according to which attenuation of SARS-CoV-2 to obtain a vaccine strain occurs by integrating into the viral genome such modifications as deletion of the DPRRA sequence in the S protein, substitution of K164A and HI 65A in non-structural protein 1 (Nspl); a mutation that prevents the expression of open reading frames ORF 6, 7a, 7b and 8. SARS-CoV-2 modified in this way is attenuated, but retains the ability to infect and replicate in mammalian cells. The patent also describes immunogenic compositions comprising attenuated SARS-CoV-2 and discusses a collection of plasmids encoding the full-length genome of recombinant attenuated SARS-CoV-2, and methods for producing attenuated SARS-CoV-2 using these plasmids and reverse genetics methods.

[0013] The scientific literature also describes studies that have produced attenuated SARS-CoV-2 strains for subsequent use in constructing LAVs. Current approaches to creating LAVs are described in a review article (Korchevaya E.R. et al., 2023). A number of authors are attenuating the virus using traditional approaches aimed at producing cold-adapted mutants through prolonged passaging in cell culture at low temperatures. Other authors are attenuating SARS-CoV-2 using genetic engineering and reverse genetics techniques, which allow for targeted modification of the viral genome to produce avirulent variants.

[0014] The production of an attenuated SARS-CoV-2 mutant by cold adaptation was first described by Seo S. and Jang Y. back in 2020 (Seo and Jang, 2020). The attenuated mutant CoV-2-CNUHV03-CA22DC, adapted to cultivation in Vero cell culture at 22°C, exhibited a temperature-sensitive (ts) phenotype and was characterized by 59 mutations in the viral genome, including 37 non-synonymous substitutions.

[0015] The attenuated cold-adapted SARS-CoV-2 mutant KaraVac, which was able to replicate in Vero cells at 25°C but not at 41°C, was characterized by two deletions in the S protein: one deletion in the S protein cleavage site at the S1 / S2 junction (PRRA motif), the second in the S1-NTD site (GTNGTKR motif) (Abdoli et al., 2022).

[0016] The attenuated SARS-CoV-2 mutant TS11 was adapted to growth in Vero E6 cell culture at 21°C, exhibited the ts phenotype, and differed from the parental strain by a 12-amino acid deletion at the furin cleavage site of the S protein, a 371-nt deletion encompassing the ORF7b-ORF8 genes, and a number of point mutations in the nsp16, S, E, orf7a, and N genes, and exhibited an attenuation phenotype in golden Syrian hamsters (hereinafter referred to as hamsters) (Xu et al., 2022).

[0017] The attenuated SARS-CoV-2 VAS5 mutant was obtained by serial passages in Vero cells at 37DC. It differed from the wild-type strain by a 7-amino acid deletion upstream of the S1 / S2 cleavage site of the S protein. It replicated significantly worse in human Caco-2 and Huh7 cell cultures and primary lung epithelial cells, and was non-pathogenic for K18-hACE2 transgenic mice (Li et al., 2022).

[0018] The only LAV against COVID-19 undergoing clinical trials is CoviLiv, formerly known as Covi-Vac (Wang et al., 2021), a joint development between Codagenix (USA) and the Serum Institute of India. CoviLiv is an attenuated virus with deoptimized codons in the S gene (Kaufmann et al., 2023).

[0019] The attenuated SARS-CoV-2 mutant d16 was obtained by introducing the D130A mutation into the NSP16 protein, which is a type I interferon antagonist and critical for methylation of the 5'-cap structure of viral mRNAs. Passaging of the d16 mutant in Vero E6 cell culture showed no signs of virulence reversal, indicating the stability of its attenuated (att) phenotype (Ye et al., 2022).

[0020] To generate the attenuated SARS-CoV-2 mutant Δ3678, the accessory protein genes ORF3, ORF6, ORF7, and ORF8 were deleted from the viral genome, and the transcriptional regulatory sequence TRS ACGAAC was replaced with CCGGAT (Y. Liu et al., 2022).

[0021] To obtain the attenuated mutant WAl-APRRA-AORF6-8-Nsp1 NI2S / K129E The sequence encoding the PRRA peptide upstream of the furin cleavage site was removed from the SARS-CoV-2 genome (isolate WA1 / 2020), the ORF6-ORF8 genes were removed, and K164A and H165A substitutions were introduced into the C-terminal domain of the NSP1 protein (S. Liu et al., 2022).

[0022] Another approach to attenuate SARS-CoV-2 involved generating a library of 659 mutant clones using reverse genetics techniques based on the clinical isolate of SARS-CoV-2 lineage B.1.1 (Pango) and selecting ts mutants from this library that did not induce CPE at 37°C (Yoshida et al., 2022).

[0023] To obtain attenuated mutants of sCDP9 and sCDP10, codon deoptimization was performed in different regions of the SARS-CoV-2 genome, after which attenuated mutants of the virus were selected that replicated more slowly than the parent strain in Vero E6 cell culture, while remaining genetically stable over 10 passages (Trimpert et al., 2021).

[0024] To obtain the attenuated ts mutant D-D2, the Wuhan-like SARS-CoV-2 Dubrovka strain was passaged for a long time in Vero CCL-81 monkey kidney cells at a temperature gradually lowered to 23°C. The D-D2 mutant reproduced efficiently at 23°C, but lost the ability to reproduce in cell culture at a temperature of 39°C and above, exhibiting the att phenotype for hamsters (Faizuloev et al., 2023; Faizuloev et al., 2024).

[0025] In all of the above studies, attenuated SARS-CoV-2 mutants were obtained that protected susceptible laboratory animals from infection with a virulent strain and the development of viral pneumonia when immunized intranasally.

[0026] The authors of the claimed invention selected the attenuated SARS-CoV-2 mutant D-D2 as the prototype, as it was the closest in technical solution (Faizuloev et al., 2023; Faizuloev et al., 2024). After a single intranasal immunization of hamsters, the D-D2 mutant caused seroconversion in all immunized animals and provided highly effective protection against the development of productive infection and pneumonia when infected with both the parental Wuhan-like Dubrovka strain and heterologous strains related to the Delta (AY. 122) and Omicron (sublines B A. 1.1 and BA.5.2) variants. A significant drawback of the ts mutant D-D2 was its residual virulence for hamsters, manifested in limited virus reproduction in the lungs and mild focal interstitial pneumonia, which does not allow it to be characterized as completely safe.Furthermore, omicron-like SARS-CoV-2 genovariants are currently circulating worldwide. These genovariants possess altered antigenic properties and are capable of evading the immune response generated by a previous infection. Therefore, to create effective protection against new viral genovariants, it seems appropriate to use the omicron-like ts mutant of SARS-CoV-2, which is evolutionarily and antigenically similar to the currently circulating viral genovariants, for immunization.

[0027] It is important to note that cold adaptation of the virus to produce ts mutants is one of the traditional approaches to creating live vaccines that has proven its effectiveness (Maassab and DeBorde, 1985). For example, seasonal live influenza vaccines based on ts mutants, the principles of which were formulated more than 50 years ago, combine cross-protective activity, ease of intranasal administration, and the formation of mucosal immunity at the entry point of infection (Ghendon et al., 1984). Attenuation of the cold-adapted virus is based on the fact that the virus loses the ability to reproduce at a physiological temperature of 37°C or higher, acquiring the ts phenotype. Thus, the ts mutant is unable to affect the lungs and other vital internal organs and cause pathological changes in them, since their temperature exceeds 37°C, and in the brain it averages 38.5°C.

[0028] The problem solved by the proposed invention is to obtain an attenuated ts mutant of SARS-CoV-2, on the basis of which it is possible to create a means for inducing specific antiviral immunity.

[0029] To solve this problem, an attenuated SARS-CoV-2 F-F3 strain is proposed for the creation of a specific COVID-19 prophylaxis agent, deposited under No. 39069 in the collection of the Unique Scientific Facility "Collection of Microorganisms of Pathogenicity Groups III and IV at the I.I. Mechnikov Research Institute of Vaccines and Serums."

[0030] The technical result of the claimed invention is that the claimed strain is characterized by:

[0031] - evolutionary proximity to the current, currently circulating genovariants of the virus, since the attenuated mutant was obtained on the basis of an omicron-like strain of SARS-CoV-2;

[0032] - formation of a protective immune response against SARS-CoV-2 even with a single intranasal immunization;

[0033] - the ability to provide protection against the disease when infected with not only a homologous but also a heterologous strain of SARS-CoV-2;

[0034] - safety, which is determined by the inability of the attenuated mutant to infect vital internal organs (lungs and brain) and the stability of the attenuation phenotype during long-term passaging;

[0035] - high reproductive activity of the attenuated mutant in Vero CCL-81 cell culture at 24°C, which makes it possible to obtain viral material with high specific activity.

[0036] The proposed strain is a ts mutant of the omicron-like FEB2 strain (Omicron BA.5.2, GenBank ID OP920753.1), isolated in October 2022 from a patient with confirmed COVID-19. Strain F-F3 was obtained by passaging the FEB2 strain in Vero CCL-81 cells at a temperature gradually reduced to 24°C. As a result, it lost the ability to replicate in cell culture at 37°C, thereby acquiring the ts phenotype. Strain F-F3 lost the ability to infect hamster lungs and cause pneumonia. However, strain F-F3 retained the ability to replicate to a limited extent in upper respiratory tract epithelial cells, which determines its immunogenicity in hamsters. When selecting the SARS-CoV-2 strain for producing the ts mutant, we were guided by the fact that it should have similar antigenic properties to current wild strains, while at the same time being initially characterized by reduced virulence.The omicron-like strain FEB2, which is capable of causing pneumonia in hamsters without affecting the brain, met these requirements to the greatest extent (Gracheva et al., 2024).

[0037] Thus, the F-F3 strain we obtained exhibited a pronounced ts phenotype. When hamsters were infected intranasally, it lacked the lung-infecting ability of the parent strain but retained viability in the nasal passages. Although the F-F3 strain's nasal replication rate was lower than that of the parent strain, all animals developed seroconversion with the production of both total and virus-neutralizing antibodies three weeks post-immunization (p.i.). Strain F-F3 exhibited significantly lower immunogenicity than the parent strain FEB2, which is typical of vaccine strains and can be considered an additional marker of viral attenuation.

[0038] A single intranasal immunization with the F-F3 mutant protected hamsters from weight loss, productive lung infection, and pneumonia development when challenged with both the homologous omicron-like FEB2 strain and the heterologous Wuhan-like Dubrovka strain.

[0039] An important characteristic of the F-F3 strain is the stability of its ts phenotype during long-term cultivation at 33°C, which roughly corresponds to the conditions of the upper respiratory tract. Since the ts phenotype determines viral attenuation, the obtained results also indicate the stability of the attenuation phenotype of the F-F3 mutant, which minimizes the likelihood of virulence reversal and suggests the safety of clinical use of LAV.

[0040] Strain F-F3 is grown in Vero CCL-81 cell culture at 24°C, reaching a titer of 7.0-8.0 lg TCID 50 / ml, while for effective immunization of hamsters a dose of 5.0 lg TCD is sufficient 50 / head. The ability to produce a highly active F-F3 strain preparation in cell culture indirectly indicates the low cost of a single vaccine dose, as the virus-containing culture fluid must be standardized to a working titer by multiple dilutions to obtain the finished vaccine. In other words, one volume of virus-containing culture fluid will correspond to 10-100 volumes of the finished standardized vaccine preparation.

[0041] The invention is illustrated by the following graphic materials.

[0042] Fig. 1. Weight distribution of hamsters immunized with the F-F3 strain and unimmunized ones from day 1 to day 4 after infection with (A) the Dubrovka strain and (B) the FEB2 strain. Bars in the histograms represent SEM.

[0043] Fig. 2 Virus titer in the lungs, brain and nasal passages of hamsters immunized (1) with strain F-F3 and (2) non-immunized, on day 4 p.i.

[0044] Authentication: FEB2 - infection with FEB2 strain, Dubrovka - infection with Dubrovka strain. Bars in the histograms represent SEM.

[0045] Examples of the invention

[0046] Example 1. Production and characterization of the ts mutant F-F3 of the SARS-CoV-2 strain FEB2

[0047] To generate ts mutants, we selected the previously obtained omicron-like SARS-CoV-2 strain FEB2 (Omicron BA.5.2, GenBank ID OP920753.1), which exhibits high reproductive activity in Vero CCL-81 cells. For cold adaptation, the FEB2 strain was cultured for 24 passages in Vero CCL-81 cells at a temperature gradually reduced from 37°C to 24°C. This resulted in a cold-adapted virus preparation (FEB2-ca) capable of efficient replication at 24°C. Based on the FEB2-ca virus preparation, six virus clones (F-F1, F-F3, F-D3, F-D12, F-C5, and F-C9) capable of reproducing at 24°C were obtained by threefold cloning using the limiting dilution method in 96-well plates. Four of the six mutant clones (F-F1, F-F3, F-D3, and F-D12) exhibited a pronounced ts phenotype, i.e., they lost the ability to reproduce at 37°C and 39°C (Table 1).From the resulting ts mutants, clone F-F3 (hereinafter referred to as strain F-F3), sensitive to temperatures of 37°C and above, was selected for further study. Viral material from strain F-F3 was accumulated in Vero CCL-81 cell culture at 24°C, packaged in cryovials, characterized for infectious activity, and stored at -80°C.

[0048] Accumulation of the F-F3 strain in Vero CCL-81 cell culture at 24°C allowed us to obtain highly active viral material with a titer of 7.0 to 8.0 lg TCID 50 / ml. For subsequent immunization of hamsters, the resulting virus-containing culture liquid was diluted to a working titer of 6.0 lg TCID 50 / ml (intranasal immunization, 100 μl / head, dose 5.0 lg TCID 50 ).

[0049]

[0050] Culture fluid samples were collected 4 days after infection at 37°C and 39°C, and 9 days after infection at 24°C. Values ​​from two independent experiments are presented (exp. 1, exp. 2).

[0051] Comparative analysis of the genomes of the F-F3 mutant and its parent strain FEB2 showed that as a result of adaptation to growth in Vero CCL-81 cell culture at 24°C, it acquired 23 nucleotide substitutions (C3154T, C10175T, C10801T, C11422T, C13132T, T15353C, G17535A, G17915T, C19515T, C19863T, T22772G, G23754T, A24339T, C24348A, C24349G, G26316A, G26488A, C26501T, A26668G, G28507T, C28626A, C29070A, C29312T), of which 20 resulted in amino acid substitutions in proteins NSP3 (P971L), NSP5 (T3520I, A3727V), NSP6 (T4297I), NSP12 (S5038P), NSP13 (C5765Y, G5892C), NSP14 (S6425L, A6541V), S (N414K, D742Y, T937S, P940S), E (R38Q), M (D3N, T7I, T63A), N (R95L, P132T, Q280K).

[0052] Example 2. Evaluation of the virulence of strain F-F3

[0053] To study the virulence of SARS-CoV-2 strains F-F3 and FEB2, female hamsters weighing 40-50 g were used, which were infected intranasally with the virus at a dose of 5.0 lg TCID 50 / head. The hamsters were observed daily, weight monitoring was performed from 0 to 4 days post infection (p.i.). After 4 days p.i., the animals were humanely euthanized under chloroform anesthesia. The lungs, brain, and other organs were removed, homogenized in DMEM medium with gentamicin (40 μg / ml), and centrifuged at 10,000 rpm for 5 min at +4°C. The supernatant was collected and stored at -80°C for subsequent titration and determination of viral RNA. Virulence was assessed by comparing the dynamics of animal weight and the viral load in the organs of animals infected with the virulent strain FEB2 and its ts mutant F-F3.

[0054] Intranasal infection of hamsters with strain F-F3 at a dose of 5.0 lg TCID 50 / head did not cause pathological changes in the lungs, delayed weight gain, or behavioral changes. Reproduction of the F-F3 strain on the 4th day p.i. was detected only in the nasal passages (4.4±4.5 lg TCD 50 / ml), whereas no infectious activity of the virus was detected in lung and brain homogenates, which was in good agreement with the viral RNA content in these organs (Table 2). At the same time, the parental strain FEB2 replicated effectively both in the nasal passages (5.7±5.7 lg TCID 50 / ml), and in the lungs (5.3±5.1 lg TCD 50 / ml), but did not infect the brain. The viral load of the F-F3 strain in the nasal passages of hamsters on the 4th day p.i. was 1.3 lg TCD 50and 1.6 lg RNA copies lower than after infection with the FEB2 strain at a similar dose (p < 0.01). Thus, the F-F3 strain, after intranasal infection, limitedly replicated only in the nasal passages, while the virulent parental strain FEB2 effectively replicated in both the nasal passages and the lungs. These data indicate that the F-F3 strain has an attenuation phenotype.

[0055]

[0056]

[0057] Example 3. Evaluation of the immunogenicity of strain F-F3

[0058] To assess the immunogenicity, female hamsters weighing 40-50 g were intranasally immunized with the F-F3 strain (n=12) and the FEB2 strain (n=6) at a dose of 5.0 lg TCID 50 / head. After 21 days p.i., all immunized animals showed seroconversion, while the IgG titer to SARS-CoV-2 in the group of animals immunized with the F-F3 strain was, on average, 9.5 times lower (1117±850) than after immunization with the parental strain FEB2 (10667±3350) (p<0.05, Mann-Whitney U-test) (Table 3). Virus-neutralizing antibodies (VNA) to the FEB2 strain were detected in the sera of all hamsters immunized with the F-F3 strain in titers ranging from 80 to 640 (on average, 333±207). Antibodies to the virus were not detected in the sera of non-immunized animals.

[0059]

[0060]

[0061]

[0062] Example 4. Evaluation of the protective activity of strain F-F3

[0063] 28 days after immunization with the F-F3 strain, 6 hamsters were infected with the Dubrovka strain (Wuhan-like) and 6 hamsters with the FEB2 strain (Omicron B A. 5.2) at a dose of 5.0 lg TCID 50per head. The hamsters were observed daily, weight monitoring was performed from 0 to 4 days post-infection (p.i.). After 4 days p.i., the animals were humanely euthanized under chloroform anesthesia. The lungs, brain, and other organs were removed, homogenized in DMEM medium with gentamicin (40 μg / ml), and centrifuged at 10,000 rpm for 5 min at 4°C. The supernatant was collected and stored at -80°C for subsequent titration and determination of viral RNA. The effectiveness of immunization was assessed by comparing the dynamics of weight and viral load in the organs of immunized and non-immunized animals.

[0064] When infected with the heterologous highly virulent Dubrovka strain, immunized animals were protected from weight loss - on day 4 p.i. they gained an average of 1.8% in weight, while non-immunized animals lost 7.6% in weight (the difference is significant, p<0.05, Mann-Whitney U-test) (Fig. 1). When infected with the homologous FEB2 strain, non-immunized animals showed a significant delay in weight gain by 1.8% compared to immunized animals on day 3 p.i. (p<0.05, Mann-Whitney U-test), while on day 4 these groups no longer had significant differences in weight.

[0065] Visual assessment of the lung condition of hamsters on day 4 p.i. showed that a single intranasal immunization with the F-F3 mutant protected hamsters from the development of pneumonia when infected with both the homologous FEB2 strain and the heterologous Dubrovka strain. When infected with the homologous FEB2 strain, immunized animals were protected from productive infection in the lungs and nasal passages. When heterologously infected with the Dubrovka strain, immunization protected animals from productive infection in the lungs and brain, while infectious virus was detected in the nasal passages of immunized animals, but at a titer of 1.8 lg TCID. 50 / ml lower (p<0.01, Mann-Whitney U-test) than in non-immunized animals (Fig. 2).

[0066] Example 5. Evaluation of the stability of the attenuation phenotype of strain F-F3

[0067] An important characteristic of a vaccine strain is the stability of its attenuation phenotype, as this minimizes the likelihood of virulence reversion and determines the safety of the vaccine's clinical use. In strain F-F3, the primary attenuation marker is the ts phenotype, so the stability of the attenuation phenotype was determined using this indicator. To assess the stability of the ts phenotype, nine passages of strain F-F3 were performed in Vero CCL-81 cells at 33°C and 37°C. At 37°C, F-F3 infection was abortive—the virus did not replicate or induce CPE, exhibiting the ts phenotype. At 33°C, strain F-F3 replicated at all passage levels, causing pronounced CPE, accompanied by the accumulation of viral RNA in the culture fluid at a concentration of at least 7.0 lg RNA copies / ml. It is important to note that 33°C roughly corresponds to the conditions found in the upper respiratory tract.The viral progeny obtained after nine passages at 33°C (F-F3-33) were tested for sensitivity to 37°C and 39°C. While the parental strain FEB2 and the F-F3-33 variant replicated equally efficiently in Vero CCL-81 cells at 33°C, only the FEB-2 strain was capable of replication at 37°C and 39°C, as evidenced by an increase in viral RNA concentration and the development of CPE (Table 4). The absence of signs of reproduction of the F-F3-33 variant at 37°C and 39°C indicates the stability of the ts phenotype of the F-F3 strain under long-term cultivation conditions at 33°C.

[0068]

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