RU2802058C1 - Recombinant influenza virus intended for the prevention of covid-19 and influenza, method of its production and use - Google Patents

Abstract

FIELD: medicine; biotechnology and virology.

SUBSTANCE: method of obtaining a recombinant influenza virus intended for the prevention of COVID-19 and influenza is described. The method is as follows: using a heterologous fragment containing, but not limited to, immunodominant and signal sequences of SEQ ID NO 1 and SEQ ID NO 2 borrowed from the N protein of the SARS-CoV-2 virus as part of the reading frame of the NS1 truncated protein of the influenza virus. The following is presented: a recombinant influenza virus obtained by this method, which is an influenza A virus with a NS1 protein truncated to 124 amino acids, containing the sequences of SEQ ID NO 1 and SEQ ID NO 2; recombinant influenza virus A/Guangdong/NS124_N (H1N1pdm09), family Orthomyxoviridae, genus Influenza Virus A, obtained by this method, deposited in the State collection of viruses under No. 2981; recombinant virus A/Cambodia/NS124_N (H3N2), family Orthomyxoviridae, genus Influenza Virus A, obtained by the above method, deposited in the State Collection of Viruses under No. 2982. A method of the prevention of COVID-19 and influenza is described, characterized by immunization of subjects with any of the presented recombinant influenza viruses by intranasal administration. The specified technical result is achieved due to the fact that the method for obtaining a recombinant influenza virus consists in using an influenza virus encoding a heterologous fragment containing, but not limited to, the sequences SEQ ID NO 1 and SEQ ID NO 2 borrowed from the N protein of the SARS-CoV-2 virus, which provide a higher level of reproduction of the recombinant virus in the main production substrates, more active expression of the heterologous transgene, and higher immunogenicity compared to similar recombinant viruses that do not contain these sequences.

EFFECT: obtaining highly productive genetically stable recombinant viral influenzas based on an influenza vector expressing a protein fragment of the N virus SARS-CoV-2, which in the mode of intranasal administration induce a pronounced post-vaccination N-specific humoral and T-cell immune response and provide cross-specific protection against infections with the SARS-CoV-2 virus and influenza.

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Description

The invention relates to the field of medicine, biotechnology and virology, and in particular to methods for obtaining recombinant influenza viruses encoding a protein fragment N of the SARS-CoV-2 virus, intended for specific prevention of COVID-19 and influenza using a mucosal vaccine based on them [A61P31/12 , A61P31/16, A61P31/00, A61K 39/00, A61K 39/12].

The development of specific prevention of COVID-19, which provides protection for various population groups, including against newly emerging mutant variants of the SARS-CoV-2 virus, is one of the priorities of public health.

Most of the currently used vaccines are intended for parenteral administration and are aimed at the formation of systemic neutralizing antibodies to the full-length coronavirus Spike protein or its S1 subunit containing the receptor-binding domain - RBD [J. S. Tregoning, K. E. Flight, S. L. Higham, Z. Wang, and B. F. Pierce, “Progress of the COVID-19 vaccine effect: viruses, vaccines and variants versus efficacy, effectiveness and escape.,” Nat. Rev. Immunol., vol. 21, no. 10, pp. 626–636, Oct. 2021, doi: 10.1038/s41577-021-00592-1.]. At the same time, the S/RBD protein, which plays an important role in the attachment, fusion, and penetration of the virus into the cell, is subject to the most pronounced antigenic changes as a result of increasing pressure from population immunity [K. Guruprasad, “Mutations in human SARS-CoV-2 spike proteins, potential drug binding and epitope sites for COVID-19 therapeutics development,” Curr. Res. Struct. Biol., vol. 4, pp. 41–50, 2022, doi: https://doi.org/10.1016/j.crstbi.2022.01.002.]. In this regard, the direction of research on the study of the vaccine potential of conservative proteins of the SARS-CoV-2 virus seems relevant, among which the most promising target is the nucleocapsid protein N, which is abundantly synthesized during infection, which regulates several important stages of the coronavirus life cycle [W. E. Matchett et al., “Cutting Edge: Nucleocapsid Vaccine Elicits Spike-Independent SARS-CoV-2 Protective Immunity.,” J. Immunol., vol. 207, no. 2, pp. 376-379, Jul. 2021, doi: 10.4049/jimmunol.2100421.; S. C. Oliveira, M. T. Q. de Magalhães, and E. J. Homan, “Immunoinformatic Analysis of SARS-CoV-2 Nucleocapsid Protein and Identification of COVID-19 Vaccine Targets,” Front. Immunol., vol. 11, 2020, doi: 10.3389/fimmu.2020.587615.].

Despite fundamental research data on the prevailing role of mucosal immunity in providing protection against antigenic variants of respiratory viruses, the vast majority of vaccines against COVID-19 are intended for parenteral use, and their action is aimed at generating a systemic immune response (mainly neutralizing antibodies in the blood serum). At the same time, mucosal-associated high molecular weight immunoglobulins of class A (IgA) with cross-protective activity, innate immunity factors, populations of long-lived respiratory CD4+ and CD8+ memory T-lymphocytes, as well as first-line cells play an important role in protection against infection. immune defense - resident Trm cells of the respiratory tract [V. Gauttieretal., “Tissue-residentmemory CD8 T-cell responses elicited by a single injection of a multi-target COVID-19 vaccine,” bioRxiv, p. Jan. 2020.08.14.240093 2020, doi: 10.1101/2020.08.14.240093.]. Respiratory memory T cells have been linked to a long-term cross-protection effect against various coronaviruses, including SARS-CoV-2 [J. Zhao et al., “Airway Memory CD4 + T Cells Mediate Protective Immunity against Emerging Respiratory Coronaviruses,” Immunity, vol. 44, no. 6 Jun. 2016, doi: 10.1016/j.immuni.2016.05.006.].

Vaccination against seasonal influenza is considered one of the most effective means of reducing the burden of a disease that in the pre-COVID-19 era caused an average of 250,000–500,000 deaths worldwide annually [Paget J., Spreeuwenberg P., Charu V., Taylor R.J., Iuliano A.D., Bresee J., Simonsen L., Viboud C. Global Seasonal Influenza-associated Mortality Collaborator Networkand GLaMOR Collaborating Teams. Global mortality associated with seasonal influenza epidemics: New burden estimates and predictors from the GLaMOR Project. J Glob. health. 2019;9:020421. doi: 10.7189/jogh.09.020421]. Although influenza virus circulation has declined sharply since 2020, the 2021/22 winter season in the northern hemisphere was characterized by the parallel circulation of both SARS-CoV-2 and influenza[World Health Organization (WHO) FluNet. Available online: https://www.who.int/tools/flunet]. The use of combined influenza and COVID-19 vaccines, along with the simultaneous administration of two vaccines, will increase vaccination coverage [World Health Organization (WHO) Coadministration of Seasonal Inactivated Influenza and COVID-19 Vaccines. Interim Guidance. [(accessed on February 7, 2022)]. Available online: https://www.who.int/publications/i/item/WHO-2019-nCoV-vaccines-SAGE_recommendation-coadministration-influenza-vaccines].

A CANDIDATE VACCINE DELTA-19 developed by Vivaldi Biosciences (USA, Austria) is known from the prior art [WO2022109068 - Influenza virus encoding a truncated NS1 protein and a SARS-CoV receptor binding domain, published: 05/27/2022]. The Delta-19 vaccine is made on the basis of an attenuated influenza vector with a shortened reading frame of the NS1 protein, in which the RBD fragment of the SARS-CoV-2 S virus S protein is encoded. The drug is intended for the prevention of COVID-19 and influenza. The vaccine is produced using Vero cell culture. The vaccine is currently undergoing preclinical studies [https://vivaldibiosciences.com/delta19]. A limitation of the present invention is the narrow spectrum of protection associated with the selection of the RBD domain of the SARS-CoV-2 virus as a target, which is subject to active mutational changes [K. Guruprasad, “Mutations in human SARS-CoV-2 spike proteins, potential drug binding and epitope sites for COVID-19 therapeutics development,” Curr. Res. Struct. Biol., vol. 4, pp. 41–50, 2022, doi: https://doi.org/10.1016/j.crstbi.2022.01.002]. The second limitation is the only possible substrate for vaccine production - interferon-defective Vero cells, since deltaNS1 vaccine strains are not able to replicate in interferon-competent systems, such as developing chick embryos (ECE). At the same time, in the Russian Federation, it is RCE that are used as the main substrate in the production of influenza vaccines from vaccine strains of the influenza virus, and the existing facilities can be used, among other things, for the production of vector vaccines based on the influenza virus.

The second close technical solution to the present invention is the DELNS1-2019-NCOV-RBD-OPT1 VACCINE CANDIDATE developed at the University of Hong Kong [WO2021160036- Compositions immunogenic against SARS coronavirus 2, methodsofmaking, andusingthereof; J. Chenetal., “A liveattenuatedvirus-based intranasal COVID-19 vaccine provides rapid, prolongation, and broad protection against SARS-CoV-2,” Sci. Bull., vol. 67, no. 13, pp. 1372-1387 Jul. 2022, doi: 10.1016/j.scib.2022.05.018, published: 19.08.2021]. The vaccine candidate is a temperature-sensitive (ts) vaccine strain based on the influenza A virus with a deleted NS1 protein, expressing the RBD domain, and is intended for intranasal use for the prevention of COVID-19. In the course of preclinical studies, the drug demonstrated high immunogenicity and good protective potential. At present, phases I and II clinical trials of the drug (ChiCTR2000037782; ChiCTR2000039715) have been completed, the start of phase III (ChiCTR2100051391), as well as phase II studies in the prime-boost scheme (NCT05200741) have been announced. The disadvantage of this vaccine candidate is also the choice of the RBD domain as a target. The range of possible substrates for the production of this vaccine candidate is expanded due to a mutation in the non-coding fragment of the M gene of the vaccine strain [WO2016074644 – Live attenuated vaccines for influenza viruses, published: 05/19/2016], which allows it to multiply not only in Vero cells, but also in MDCK cells ( the main substrate for this drug). The vector on the basis of which the vaccine candidate DelNS1-2019-nCoV-RBD-OPT1 was created was able to reproduce in RC, although worse than the wild-type virus; data on the reproduction of the vaccine candidate are not presented in the RC.

Also known are recombinant influenza viruses based on a domestic attenuation donor for a live influenza vaccine A/Leningrad/17/134/57 [Patent RU2782531; Isakova-Sivak I, Stepanova E, Matyushenko V, et al. Development of a T Cell-Based COVID-19 Vaccine Using a Live Attenuated Influenza Vaccine Viral Vector. Vaccines (Basel). 2022;10(7):1142. Published 2022 Jul 18. doi:10.3390/vaccines10071142, published: 10/28/2022]. These recombinant viruses have an attenuated phenotype and are able to reproduce in the chick embryo system. The limitation of the vaccine strain described in the patent is the narrow spectrum of protection associated with the choice of the RBD domain of the SARS-CoV-2 virus as a target, which is subject to active mutational changes, which necessitates frequent updating of the vaccine strain. A limitation of the vaccine candidates described in the open access publication is the use of a narrow set of epitopes in the heterogeneous insert, which is calculated for a specific set of major histocompatibility complex alleles. The advantage of the present invention is the use as a heterologous insert of a long region of the conserved N protein of the SARS-CoV-2 virus, containing multiple experimentally confirmed and theoretically predicted epitopes for a representative set of alleles.

The vaccine candidate "Coraflu", developed as part of an international collaboration between the University of Wisconsin-Madison and vaccine companies FluGen and Bharat Biotech, is known from the prior art. The vaccine candidate is based on a replication-defective M2 protein-deleted influenza vector (M2SR) incorporating a sequence encoding a SARS-CoV-2 fragment [UW–Madison, FluGen, Bharat Biotech to develop CoroFlu, a coronavirus vaccine. press release. https://news.wisc.edu/uw-madison-flugen-bharat-biotech-to-develop-coroflu-a-coronavirus-vaccine/; Nasal drop vaccine candidate for coronavirus from India, https://www.nature.com/articles/nindia.2020.59]. The limitation of this vaccine candidate is the only production substrate, the genetically modified M2CK cell line (MDCK line expressing the M2 viral protein) [US10119124B2, published: 04/06/2017].

An attenuated influenza A virus is known from the prior art, containing a chimeric NS fragment, including a shortened reading frame of the NS1 protein and a heterologous sequence of the NEP protein gene derived from a subtype of influenza A virus that differs from the subtype of the specified attenuated influenza A virus, where the specified shortened reading frame encodes a protein NS1 size 80-130 amino acid residues [RU 2628690, published: 08/21/2017, application EP3382010A4, application PCT/RU2016/050066]. The described strain is capable of reproducing in the chick embryo system. The description of the invention indicates that this virus can be used as a vector to create a recombinant strain in which the truncated reading frame of the NS1 protein is continued by inserting the sequence of at least one transgene encoding proteins or protein fragments of respiratory viruses. It is also indicated that the administration of such strains in an effective amount can be used to prevent infection caused by the respective respiratory viruses. However, since the patent application was filed before the start of the COVID-19 pandemic, the description of the invention could not and does not contain information about targets in the genome of the SARS-CoV-2 virus (which appeared only in 2019) that may have an advantage for use in as a transgene insert into an influenza vector, which is the essence of the present invention.

The prior art known attenuated recombinant influenza viruses DNA(RBD)-Flu, in which the NA protein sequence is deleted and which encode the RBD fragment of the SARS-CoV-2 virus in the modified NA gene segment [Loes A et al., Viruses. 2020 12:987]. The substrate for the production of recombinant viruses is the MDCK-SIAT1-TMPRSS2 cell culture. It has been shown that administration of recombinant viruses to mice leads to the formation of RBD-specific antibodies in animals that have neutralizing activity in the test with S-protein-encoding pseudolentiviruses. No information is available on the protective efficacy of these recombinant viruses. The disadvantages of these recombinant strains include the choice of a highly specific target, the RBD domain of the SARS-CoV-2 virus, as well as the need for adaptive mutations in the HA gene of the recombinant virus to maintain a high level of reproduction in cells. The literature shows that such mutations can reduce the engraftment and immunogenicity of constructs [Nakowitsch S. et al., Vaccine. 2011. 29:3517-3524].

The objective of the invention is to create a new recombinant strain based on the influenza vector, designed to obtain a vector vaccine against COVID-19 and influenza, which has a broader protective potential and is devoid of the shortcomings of the prototypes. The problem was solved using genetic engineering design of recombinant vaccine strains expressing a fragment of the N protein of the SARS-CoV-2 virus. A feature of the genetic design of vaccine strains is the shortening of the translated region of the NS1 protein to 124 amino acid residues and the replacement of its carboxyl part with a sequence encoding a fragment of the N protein of the SARS-CoV-2 virus, including dominant T-cell immunogenic epitopes and potential NES and NLS signal sequences. These features provide recombinant vaccine strains with a higher level of reproduction in the main production substrates, including RCE, more active transgene expression and higher immunogenicity compared to similar constructs that do not contain these features. The use of the N protein of the SARS-CoV-2 virus as a target provides recombinant vaccine strains with potentially broader cross-specific protection against infection caused by the SARS-CoV-2 virus compared to counterparts encoding the RBD fragment. The use of current subtypes of human influenza virus surface antigens as a source determines the possibility of using recombinant vaccine strains for the prevention of seasonal influenza.

Attenuation of recombinant vaccine strains is provided by the modification of the NS genomic fragment, which consists in shortening the NS1 protein to 124 amino acid residues. Shortening of the NS1 protein impairs the ability of the virus to inhibit the innate immune system, in particular, the production of type 1 interferons. This leads to the suppression of the ability of the virus to reproduce in vivo and the prevention of transmission of the vaccine strain. At the same time, intranasal immunization with the vaccine strain ensures the expression of the target antigen (SARS-CoV-2 virus N protein) along with influenza vector antigens in the epithelial and antigen-presenting cells of the respiratory tract. At the same time, increased production of interferons and other cytokines with immunoadjuvant properties occurs in the nasal cavity and associated mucous membranes of the respiratory tract, which causes a pronounced immune response to the target antigen and influenza vector with polarization towards the Th1 link and the formation of protection against COVID-19 and influenza. .

Despite the modification of the NS genomic fragment, a vaccine based on engineered recombinant vaccine strains can be produced in the RCE using standard technological schemes used for the production of classical influenza vaccines.

The technical result achieved in the implementation of the present invention is the production of highly productive genetically stable recombinant viral influenza based on an influenza vector expressing a protein fragment of the N virus SARS-CoV-2, which in the mode of intranasal administration induce a pronounced post-vaccination N-specific humoral and T-cell immune response and provide cross-specific protection against infection with the SARS-CoV-2 virus and influenza.

Recombinant influenza viruses A/Guangdong/NS124_N (H1N1pdm09) and A/Cambodia/NS124_N (H3N2) were deposited in the State Collection of Viruses of the National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamalei of the Ministry of Health of the Russian Federation under No. 2981 and No. 2982.

A method for obtaining a recombinant influenza virus, involving the use of an influenza virus encoding, in the reading frame of a NS1 protein truncated to 124 amino acid residues, a heterologous fragment containing, but not limited to, the immunodominant and signal sequences SEQIDNO 1 and SEQIDNO 2 borrowed from the N protein of the SARS-CoV-2 virus , which provide a higher level of reproduction of the recombinant virus in the main production substrates (MDCK, Vero, RCE), more active expression of the heterologous transgene and higher immunogenicity compared to similar recombinant viruses that do not contain these sequences.

At the same time, the influenza virus encoding, as part of the reading frame of the truncated NS1 protein, a heterologous fragment containing, but not limited to, the immunodominant and signal sequences SEQIDNO: 1 and SEQIDNO: 2, borrowed from the N protein of the SARS-CoV-2 virus, can be an influenza A virus of any subtype, including type A/H1N1, such as A/PR/8/34, type A/H1N1pdm09, such as A/Guangdong-Maonan/SWL1536/2019, type A/H3N2, such as A/Cambodia/e0826360/ 2020.

A possible implementation example is the use of the sequences SEQIDNO: 1 and SEQIDNO: 2 to enhance the reproductive, immunogenic and protective properties of a recombinant influenza virus when they are encoded in the reading frame of the truncated NS1 protein sequentially with antigens of other viruses/mycobacteria/bacteria in order to create vaccine strains against infections, caused by the respective microorganisms. Examples of suitable microorganisms can be influenza viruses, coronaviruses, respiratory syncytial virus, mycobacterium tuberculosis.

A possible implementation is also the use of the sequences SEQIDNO 1 and SEQIDNO 2 in a recombinant influenza virus when they are encoded in the reading frame of a truncated NS1 protein sequentially with reporter fluorescent or luminescent proteins in order to increase the expression of the reporter protein or the growth properties of the reporter virus.

The resulting recombinant influenza virus, based on an influenza vector expressing a fragment of the N protein of the SARS-CoV-2 virus, provides the possibility of preventing COVID-19 and influenza infection by intranasal administration of a pharmaceutical composition containing it.

Prophylactic use of a pharmaceutical composition containing the obtained recombinant influenza viruses is characterized by the administration of the composition once or twice with an interval of 14 to 28 days.

Prophylactic use of a pharmaceutical composition containing obtained recombinant influenza viruses is characterized by the administration of the composition in a prime-boost immunization schedule at intervals of 14 to 28 days sequentially with another type of COVID-19 vaccine preparations (inactivated or based on recombinant proteins) and the route of administration (intramuscular ).

The essence of the invention is explained with the help of graphic images, which are given for purposes of illustration only and are not intended to limit the scope of the invention.

On FIG. 1 shows the results of mapping signal sequences and T-cell epitopes in the N protein of the SARS-CoV-2 virus (GenBankid YP009724397). The fragment used as part of the heterologous insert in recombinant influenza viruses A/PR8/NS124_N211 (H1N1), A/Guangdong/NS124_N (H1N1pdm09), and A/Cambodia/NS124_N (H3N2) is framed. The marked fragment includes the nuclear/nucleolus localization signal (included in SEQ ID NO: 1, start is marked with a dotted arrow), as well as the immunodominant epitope (IEDB id 34851) and the NES nuclear export signal (included in SEQ ID NO: 2, start marked with a dotted arrow).

On FIG. Figure 2 shows the structure of the NS gene of recombinant influenza viruses encoding the N protein fragment of the SARS-CoV-2 virus as part of the NS1 truncated protein reading frame, using a fragment containing the immunodominant and signal sequences SEQ ID NO: 1 and SEQ ID NO: 2 as part of a heterologous insert. .

Figure 2 (A) shows a diagram of the NS gene segment of the wild-type influenza virus A/PR8 encoding the full-length NS1 protein.

Figure 2 (B) shows a diagram of the NS gene segment of the recombinant influenza virus A/PR8/NS124 encoding a truncated NS1 protein without inserting a heterologous antigen.

Figure 2 (B) shows a diagram of the NS gene segment of the recombinant influenza virus A/PR8/NS124_N231 encoding a truncated NS1 protein with an insert of a SARS-CoV-2 N protein fragment comprising the sequence of SEQ ID NO: 1.

Figure 2 (D) shows a diagram of the NS gene segment of the recombinant influenza virus A/PR8/NS124_N211 encoding a truncated NS1 protein with an insert of a protein fragment N of the SARS-CoV-2 virus, including the sequences SEQ ID NO: 1 and SEQ ID NO: 2 .

On FIG. Figure 3 shows the results of electrophoretic analysis of the results of RT-PCR with the determination of the length of the heterologous insert in the chimeric NS gene in recombinant influenza viruses of various subtypes encoding the N protein fragment of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein, obtained as a result of passages in developing chicken embryos. . The target RT-PCR fragment is marked with an arrow; the lengths of the marker fragments (MM) are presented in the number of nucleotide pairs.

On FIG. 4 shows the expression of the NS1 protein in MDCK cells infected with recombinant influenza A/PR8/NS124_N231 and A/PR8/NS124_N211 viruses encoding the N protein fragment of the SARS-CoV-2 virus in the reading frame of the truncated NS1 protein, the control virus without the A/PR8/ insert. NS124, as well as the wild type A/PR8 influenza virus containing the full-length NS1 protein.

On FIG. Figure 5 shows N protein expression in Vero cells infected with recombinant influenza viruses A/PR8/NS124_N231 and A/PR8/NS124_N211 encoding the SARS-CoV-2 N protein fragment in the NS1 truncated protein reading frame and the control virus without the A/PR8 insert. /NS124.

On FIG. Figure 6 shows the dynamics of changes in body weight of mice after immunization with attenuated recombinant influenza virus A/PR8/NS124_N211 (encoding the N protein fragment of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein), compared with the "empty" vector A/PR8/NS124 and the wild-type A/PR8 influenza virus pathogenic for mice.

On FIG. 7 shows stimulation of production of antigen-specific CD8+ effector memory T-lymphocytes in the lungs of mice 10 days after intranasal immunization with recombinant influenza viruses A/PR8/NS124_N231 [N231] and A/PR8/NS124_N211 [N211] encoding the SARS-CoV N protein fragment. -2 in the reading frame of the truncated NS1 protein compared to intact animals [Intact]. The relative content (%) of memory effector T-cells producing IFNγ/TNFα/IL-2 after stimulation with SARS-CoV-2 virus N protein peptide cocktail is shown.

On FIG. 8 shows suppression of SARS-CoV-2 virus activity in the lungs and nasal passages of infected hamsters following a single intranasal immunization with recombinant A/PR8/NS124_N211 virus.

On FIG. 9 shows the protective efficacy of recombinant viruses against SARS-CoV-2 variant beta coronavirus infection. To assess the protective efficacy, mice were immunized intranasally with recombinant A/Guangdong/NS124_N (H1N1pdm09) and A/Cambodia/NS124_N (H3N2) viruses in a double dose regimen [NS124_N twice] or in a prime-boost regimen with formalin-inactivated SARS-CoV-2 virus. [Prime Boost]. In the comparison groups, mice were immunized twice with vectors without inserting the corresponding subtypes [NS124], or they were injected once with formalin-inactivated SARS-CoV-2 virus with aluminum hydroxide [FI coronavirus]. Mice from the challenge control group were given a placebo. Mice were challenged with live SARS-CoV-2 beta strain 3 weeks after the second vaccination. The figures show the dynamics of the body weight of immunized and control mice after infection with SARS-CoV-2 (A) and the viral load in the lungs of infected animals on the 5th day of infection (B).

On FIG. 10 shows the protective efficacy of recombinant viruses against infection caused by influenza viruses of various subtypes. To evaluate the efficacy, mice were intranasally immunized with recombinant A/Guangdong/NS124_N (H1N1pdm09) and A/Cambodia/NS124_N (H3N2) viruses in a double dose [NS124_N twice] regimen. Mice from the challenge control group were given a placebo. 3 weeks after the second vaccination, mice were challenged with influenza viruses at a lethal dose. The graphs show the dynamics of body weight and survival of animals after infection with strains A/Aichi/2/68 (H3N2) (A, B) and A/California/07/2009 (H1N1pdm09) (C, D). The graphs show p-values for group comparisons in the ANOVA test (Sidak's test) for % animal weight and the Mantel-Cox test for survival.

The present invention is illustrated by the following examples , which are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 . The construction of plasmids that provide the expression of fragments of the SARS-CoV-2 virus N protein in the reading frame of the truncated influenza virus NS1 protein.

In order to obtain recombinant influenza viruses encoding the N protein fragment of the SARS-CoV-2 virus as part of the NS1 truncated protein reading frame by reverse genetics, plasmids encoding the gene segments of recombinant viruses were constructed. Plasmids encoding the gene segments HA, NA, PB2, PB1, PA, NP and M of the virus A/PR/8/34 (H1N1) [A/PR8] were obtained as described previously [RU 2759054, published: 09.11.2021, RU 2726106, published: 07/09/2020]. Plasmids encoding the HA and NA genes of epidemic influenza viruses A/Guangdong/NS124_N (H1N1pdm09) and A/Cambodia/NS124_N (H3N2) were obtained by cloning full-length RT-PCR-amplified gene fragments into a synthetic vector based on pHW2000 as described previously. [EA200201164 (A1), published: 10/30/2003].

The pHW-PR8-NS124_NanoLuc plasmid encoding the chimeric NS gene of the recombinant influenza virus A/PR/8/34, which ensures the expression of NanoLuc as part of the NS1 truncated protein reading frame [RU 2726106], was used as a basis for creating plasmids encoding chimeric NS genes. Using PCR (Table 1) and site-directed mutagenesis, the sequence encoding the NanoLuc protein was removed from the pHW-PR8-NS124_NanoLuc plasmid and the XhoI restriction site was added. Thus, the working vector pHW-PR8-NS124_XhoI_428 was obtained.

Table 1 - Primers for vector amplification with the introduction of a restriction site

Name sequence 5' ->3' n Tm XhoI to pNS F2 TTCTCGAGaacttcgatcttctgaaactg 29 67 XhoI to pNSR2 TTTCCGAGtcctcccatgatcgcct 25 69

Further, using RT-PCR with specific primers (Table 2), fragments of the N gene of the SARS-CoV-2 virus (strain hCoV-19/Russia/SPE-RII-3524V/2020, number in the GISAID database EPI_ISL_415710) were amplified. At the same time, the first amplified fragment [N231] contained an extended fragment of the N protein of the SARS-CoV-2 virus containing multiple B- and T-cell epitopes (shown in Fig. 1), including the potential signal sequence KKPRQKRTA (SEQIDNO: 1), and the second amplified fragment [N211] in addition to the above also included the immunodominant sequence LALLLLDRLNQL (SEQIDNO: 1). The amplified fragments at the ends contained the XhoI and NotI restriction site sequences for cloning into the working vector pHW-PR8-NS124_XhoI428.

Table 2 - Primers for cloning fragments of the N protein of the SARS-CoV-2 virus

fragment name primer name sequence 5' ->3' n Tm N211 N cloning XhoI F ttctcgagGCTGGCAATGGCGGTG 25 71 N cloning NotI R ttgcggccgcttaTTTAGGCTCTGTTGGT 29 72 N231 deltaNS-Nepitopes 2 F tttctcgagGAGAGCAAAATGTCTGGTAAAGG N cloning NotI R ttgcggccgcttaTTTAGGCTCTGTTGGT

Further, the amplified fragments and the working vector were treated with XhoI and NotI restriction enzymes (Thermo), purified by preparative agarose gel electrophoresis, and ligated for 10 minutes at room temperature using T4 ligase (Thermo). E. coli DH5α cells were electroporated with the ligation mixture. Colonies carrying plasmid DNA with the required insert were identified by PCR using cloning primers (Table 2), and plasmid sequences in selected clones were verified by Sanger sequencing.

As a result, plasmids pHW-PR8-NS124_N211 and pHW-PR8-NS124_N231 encoding chimeric NS gene segments were obtained. The chimeric gene segment PR8-NS124_N211 included the coding region, as well as the 5'-UTR and 3'-UTR non-coding regions of the nucleotide sequence of the NS gene segment of the influenza virus A/PR/8/34 (H1N1); while the open reading frame of the NS1 protein was shortened to 124 amino acids, followed by the GGLE linker, then a heterologous sequence encoding fragment 211-369 of the N protein of the SARS-CoV-2 virus, terminated by a stop codon; while 30 nucleotides following the end of the reading frame of the NS124_N chimeric protein were removed and replaced with a unique NotI restriction site, the remaining 100 nucleotides before the start of the NS2(NEP) frame were saved for the splicing process. The total length of the PR8-NS124_N211 chimeric gene segment was 1360 nucleotides. The total length of the plasmid was 4327 nucleotides. A map of the PR8-NS124_N211 chimeric gene segment is shown in FIG. 2, the amino acid sequence of the NS124_N211 chimeric protein is shown in the Sequence Listing SEQ ID NO: 3.

The chimeric PR8-NS124_N211 gene segment differed from the segment described above by the insertion of a shorter SARS-CoV-2 N protein fragment that included amino acids 231-369. The total length of the PR8-NS124_N231 chimeric gene segment was 1300 nucleotides. A map of the PR8-NS124_N231 chimeric gene segment is shown in FIG. 2, the amino acid sequence of the NS124_N231 chimeric protein is shown in the Sequence Listing SEQ ID NO: 4.

The resulting plasmids were accumulated in E. coli DH5alpha cells and purified using the EndofreeMaxiKit kit (Qiagen) to isolate endotoxin-free plasmids. The nucleotide sequences of the plasmids were verified by Sanger sequencing.

Example 2 . A method for obtaining genetically stable recombinant influenza viruses of various subtypes encoding a fragment of the N protein of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein.

A set of 8 plasmids (Table 3) obtained as described in Example 1 were transfected into Vero cells (ATCC CCL-81) adapted to growth in serum-free OptiPro SFM medium (Gibco). For one transfection, 3*10 ⁶ cells were used, resuspended in 100 μl of buffer mixture from Nucleofector Kit V (Lonza). Electroporation was performed using Amaxa Nucleofector II (Lonza) program U-020. Transfected cells were incubated at 37°C, 5% CO2 in 6-well plates. After 2 days, the beginning of the development of the cytopathic action (CPE) of the virus was observed in the formed monolayer of cells. After 3 days, the destruction of the monolayer was 100%. The culture fluid contained recombinant influenza viruses (passage V0). To obtain the main and working bank of viruses, as well as to assess the genetic stability, the resulting recombinant viruses were passaged in the EC system using the limiting dilution method. Chicken embryos were infected into the allantoic cavity with 0.2 ml of virus-containing material and incubated at 34°C for 48 h, then cooled. The collected allantoic fluid was clarified, aliquoted and stored at -70°C.

Table 3 - Sets of plasmids (1 µg each) used to obtain recombinant influenza viruses of various subtypes encoding the N protein fragment of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein

Virus/
gene segment Prototype strains Vaccine strains A/PR8/NS124_N231 (H1N1) A/PR8/NS124_N211 (H1N1) A/Guangdong/NS124_N(H1N1pdm09) A/Cambodia/NS124_N (H3N2) HA pHW-PR8-HA pHW-PR8-HA pHW-Guangdong-HA pHW-Cambodia-HA NA pHW-PR8-NA pHW-PR8-NA pHW-Guangdong-NA pHW-Cambodia-NA PB2 pHW-PR8-PB2 pHW-PR8-PB2 pHW-PR8-PB2 pHW-PR8-PB2 PB1 pHW-PR8-PB1 pHW-PR8-PB1 pHW-PR8-PB1 pHW-PR8-PB1 PA pHW-PR8-PA pHW-PR8-PA pHW-PR8-PA pHW-PR8-PA NP pHW-PR8-NP pHW-PR8-NP pHW-PR8-NP pHW-PR8-NP M pHW-PR8-M pHW-PR8-M pHW-PR8-M pHW-PR8-M NS pHW-PR8-NS124_N231 pHW-PR8-NS124_N211 pHW-PR8-NS124_N211 pHW-PR8-NS124_N211

Those skilled in the art will understand that the HA and NA genes of influenza viruses and other subtypes (H2N2, H5N1, H7N9, and others) can be used to construct such recombinant strains, as well as the sequences of these N protein fragments of evolutionarily altered human coronaviruses, including SARS variants. -CoV-2 beta, omicron, cerberus, etc., as well as recombinant variants of SARS-CoV-2.

The control of genetic stability was carried out by RT-PCR by determining the size of the heterologous insert in the NS gene segment after several successive passages in the RCE system.

Viral RNA was isolated from 150 µl of virus-containing fluid using the QIAmpViral RNA kit (Qiagen). RT-PCR was performed by a "one-step" method using the AgPath-ID One-Step RT-PCR Reagents kit (Ambion, USA). Primer for reverse transcription was preliminarily annealed: 2 μl of RNA and 2 μl of Uni12 primer (100 pmol/μl, Beagle, Russia) were mixed [Zhou B, Donnelly ME, Scholes DT et al. J Virol. (2009) 83: 10309], then incubated at 70°C for 4 min, after which they were cooled in ice.

The composition of the reaction mixture for RT-PCR included the following components:

2x buffer 12.5 µl Direct primer 0.25 µl reverse primer 0.25 µl Emix Enzyme Blend 1 µl MgCl2 1 µl NWF water 6 µl RNA+Uni12 4 µl Total reaction volume 25 µl

The primer sequences used are shown below:

Name Subsequence Uni12 AGCRAAAGCAGG PR8-NS1-332F TGGCAGGCCCTCTTTGTA PR8-NS1-523R TGACATCCTCAGCAGTATGTCC

The thermal profile of the reaction included the following steps:

1: 42°С - pause

2: 42 °C - 60 min

3: 95°C - 15 min

4: 95°C - 15 s

5: 58°C - 30 s

6: 72°C - 1 min

7: Jump to 4, repeat 39 times

8: 72°C - 5 min

9: 8°C - storage

To analyze the results of RT-PCR, horizontal electrophoresis of samples was performed in 2% agarose gel (Thermo Scientific, USA) in 1X TBE buffer (Thermo Scientific, EU) containing 5 μg/ml ethidium bromide (Ethidium Bromide, Amresco, USA). M28 (SibEnzyme, Russia) was used as a molecular weight marker. Electrophoresis was carried out in an SE-2 chamber (Helicon, Russia) at 150 V for 1.5 hours. To detect the results of EF, a ChemiDoc gel-documentation system (Bio-Rad, USA) was used.

The electrophoresis results are shown in Fig. 3. The results obtained demonstrate that the length of the NS gene in all clones of recombinant viruses corresponds to the length of the corresponding segment in the control plasmid and thus confirms the genetic stability of the viruses. Genetic stability for vaccine strains A/Cambodia/NS124_N (H3N2) and A/Guangdong/NS124_N (H1N1pdm09) was demonstrated during 6 passages in the RC, which complies with the requirements of regulatory documentation (at least 5 passages).

Determination of the reproductive properties of the obtained recombinant viruses was carried out in the RCE system, the main substrate for the production of the vaccine. Embryos were infected with a series of falling dilutions of viral material in phosphate-buffered saline DPBS supplemented with 100 U/ml penicillin/100 µg/ml streptomycin and 5 µg/ml amphotericin B (Biolot, Russia). RCE was infected into the allantoic cavity with 0.2 ml of the material and incubated at 34°C for 48 hours. Then the RCE was cooled and opened. The presence of the virus in the allantoic fluid was determined by a positive hemagglutination test in a volume of 50 μl with an equivalent amount of 0.5% suspension of chicken erythrocytes. The infectious activity of the virus was calculated according to the method of Reed-Mench (Reed, Muench, 1938) and expressed in decimal logarithms of 50% embryonic infectious dose (lgEID50). All strains obtained in the framework of the present invention had a high reproductive activity in the RCE system (table 4), which meets the requirements of regulatory documentation for industrial vaccine strains.

Table 4 - Infectious activity in RCE of recombinant influenza viruses of various subtypes encoding a fragment of the N protein of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein

Strain Infectious activity, lgEID50/ml
mean ± SD A/PR8/NS124_N231 (H1N1) 8.7 ± 0.4 A/PR8/NS124_N211 (H1N1) 8.2 ± 0.5 A/Guangdong/NS124_N(H1N1pdm09) 8.3±0.4 A/Cambodia/NS124_N (H3N2) 7.8± 0.3

Example 3 . Influence of the insert-fragment construct on the expression of viral proteins upon infection of cells with recombinant influenza viruses encoding the SARS-CoV-2 virus N protein fragment as part of the NS1 truncated protein reading frame

Determination of protein expression upon infection of sensitive cells with recombinant and control viruses was carried out by immunofluorescence analysis by staining with antigen-specific antibodies.

Vero cells in 96-well plates were infected with recombinant viruses at a dose of 1-10 EID50/cell diluted in OptiPro medium (Gibco) supplemented with 2% GlutaMax (Gibco) and 1% antibiotic/antimycotic (Gibco). The plate with infected cells was incubated for 24 hours at +37°C and 5% CO2. The next day, the medium was removed from the plate with a dispenser and 100 μl of 80% acetone was added to the well to fix and permeabilize the cells. The tablet was incubated for 15-20 minutes at +2-8°C. The plate was then washed using an ELX50 automatic washer (BioTek Instruments) twice with a PBS-T solution (PBS (Biolot) supplemented with 0.1% Tween-20 (Serva)) followed by the addition of a blocking solution (5% skimmed milk powder (LLC "TF-Ditol") in PBS-T) 200 µl per well and incubated for 2 hours at room temperature. Then the plate was washed again twice and 100 μl of dilutions of FITC-labeled monoclonal antibodies to the NP protein of the influenza A 6D11 virus (1:40) or polyclonal mouse antibodies to the N protein of the SARS-CoV-2 virus (1:1000) were added to certain wells. or to polyclonal mouse antibodies to the NS1 protein of the influenza virus (1:1000) (the latter were obtained by immunizing mice with the appropriate recombinant proteins). The plate was incubated for 1.5 h at room temperature. The plate was then washed four times. Wells with antibodies to NP were flooded with PBS-T. The remaining wells were filled with a fluorescent conjugate of anti-mouse IgG antibodies labeled with AlexaFluor 488 (ab150113, Abcam) at a dilution of 1:300. The plate was incubated for 1 hour. Washing was repeated six times and the whole plate was filled with PBS-T. Cell photographs were obtained using an Axio Vert.A1 fluorescent microscope (Zeiss) and an Axio ICc5 built-in camera (Zeiss) at 40x magnification.

The results shown in FIG. 4 demonstrate differences in NS1 protein localization in infected cells. When cells are infected with the wild type A/PR8-NSwt virus, the NS1 protein is actively expressed and localized throughout the cell, including the cytoplasm, nucleus, and nucleoli. Shortening of the NS1 protein to 124 amino acids (virus A/PR8-NS124) leads to a change in its localization - the protein loses its ability to localize in the nucleoli of infected cells and is localized in the cytoplasm in the form of granules. Previously, similar localization was described for other strains with a shortened NS1 [Pulkina A.A. etal. Evidence for the extracellular delivery of influenza NS1 protein // MIR Journal. 2021;8(1):27-37. doi: 10.18527/2500-2236-2021-8-1-27-37]. This is most likely due to the loss of the NLS/NoLS signal located in the C-terminal domain of NS1 [Hale BG, Randall RE, Ortín J, Jackson D. The multifunctional NS1 protein of influenza A viruses. J Gen Virol. 2008; 89(Pt 10):2359-2376. doi:10.1099/vir.0.2008/004606-0]. At the same time, it is also noticeable from published works that the addition of various proteins (for example, the Luc genes, ESAT-6) to the shortened NS1 reading frame did not restore the localization of the NS1 protein in the nucleoli [Pulkina A.A. et al. Evidence for the extracellular delivery of influenza NS1 protein // MIR Journal. 2021; 8(1):27-37. doi: 10.18527/2500-2236-2021-8-1-27-37; Kuznetsova I, Shurygina AP, Wolf B, et al. Adaptive mutation in nuclear export protein allows stable transgene expression in a chimaeric influenza A virus vector. J Gen Virol. 2014; 95(Pt 2): 337-349. doi:10.1099/vir.0.056036-0]. The absence in the nucleoli of infected cells of the NS1 protein involved in the splicing of other viral proteins may be one of the reasons for a significant decrease in the expression of these proteins, which was previously demonstrated in relation to the M1 and M2 proteins in viruses with truncated NS1 [Egorov A, Brandt S, Sereinig S et al. Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells. J Virol. 1998; 72(8):6437-6441. doi:10.1128/JVI.72.8.6437-6441.1998]. A decrease in the expression of M1 and M2 proteins can significantly affect the reproductive properties of recombinant viruses with a truncated NS1 protein. The prior art describes an approach to increase the reproductive properties of a virus with a deleted NS1 by restoring the expression of the M2 protein, due to a mutation in the non-coding region that affects the splicing of the corresponding mRNA [WO2016074644].

For the recombinant viruses described in the present invention A/PR8-NS124_N231 and A/PR8-NS124_N211, the localization of the truncated NS1 protein in the nucleoli was restored (Fig. 4), which is associated with the use of the SEQIDNO: 1 fragment containing the signal in the NS1 open reading frame NoLS derived from the N protein of the SARS-CoV-2 virus. Thus, the use of the SEQIDNO: 1 sequence in the construction of influenza viruses with a truncated NS1 affects the localization of the NS1 protein in the cell and thus can enhance the reproductive properties of viruses by restoring the splicing of other viral proteins.

The results shown in FIG. 5 demonstrate that the recombinant viruses A/PR8-NS124_N231 and A/PR8-NS124_N211 described in the present invention provide expression of the N antigen of the SARS-CoV-2 virus upon infection of cells and thus can present this antigen for the development of a specific immune response.

Example 4. Influence of the insert fragment design on the formation of a SARS-CoV-2 N-specific T-cell immune response during intranasal immunization of laboratory mice with recombinant influenza viruses encoding the N protein fragment of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein.

Testing of the recombinant influenza viruses A/PR8-NS124_N231 and A/PR8-NS124_N211, encoding the N protein fragment of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein, for the ability to stimulate an N-specific immune response was performed on C57/black strained black mice . The studies used healthy animals on which no previous tests had been performed. Immunization was carried out intranasally at 6.0 lgEID ₅₀ /mouse at 30 μl/mouse of the virus. Intact mice were used as controls. Ten days after immunization, mice were assessed for T-specific T-cell response in the lungs.

For this, the animals were sacrificed by cervical dislocation, the left ventricle was perfused with 10 ml of cold DPBS, after which the lungs were removed. The organs were homogenized using a Tissue Lyser II homogenizer (Qiagen) and treated with a mixture of collagenase (Sigma) and DNase I (Sigma) for 30 min at 37°C. Tissue homogenates were passed through a 70 μm cell filter. Cells were washed once (500g, 7 min) in DPBS containing 2.5% fetal calf serum (Gibco). Lysis of erythrocytes was performed using RBC Lysis Buffer (Biolegend). Cells were washed again in DPBS + 2.5% FBS. Counting was performed using a Cytoflex flow cytometer (Beckman Coulter). To assess the adaptive virus-specific immune response, cells were seeded into flat-bottomed plates at a density of 2 * 10^6 cells/100 µl and stimulated with a cocktail of SARS-CoV-2 virus N protein peptides (PepTivator® SARS-CoV-2 Prot_N, MiltenyiBiotec). Simultaneously, the cell transport inhibitor brefeldin A (BD Biosciences) was added to the medium. Stimulation was performed for 6 h in the presence of anti-CD28 costimulatory antibodies (Biolegend). All of the listed reagents were added to the control wells, except for the specific stimulant.

After stimulation, cells were stained with fluorochrome-conjugated antibodies CD8-PE/Cy7, CD44-BV510, CD62L-APC/Cy7, IFNγ-FITC, TNFα-BV421, IL2-PE. Zombie Red markers were used to identify cell viability. Blocking of non-specific binding of antibodies was carried out using the True Stain reagent containing antibodies to CD16/CD32 (Biolegend). Staining was performed using the Cytofix/Cytoperm intracellular staining kit (BD Biosciences) according to the manufacturer's instructions. Staining results were detected on a Cytoflex flow cytometer (Beckman Coulter). The results were analyzed using Kaluza Analisis 2.1 software (Beckman Coulter).

The results obtained in Fig. 7 demonstrate significant differences in the ability of recombinant strains A/PR8-NS124_N231 and A/PR8-NS124_N211 to stimulate a CD8 N-specific T cell response in laboratory animals. Thus, in mice immunized with A/PR8-NS124_N231, the average level of N-specific cytokine-producing CD8+ T-lymphocytes was at the level of intact mice within 1% of the total number of T-cells. In mice immunized with A/PR8-NS124_N211, mean CD8+ levels were significantly higher. High levels of CD8+ antigen-specific cells were noted in all mice immunized with the A/PR8-NS124_N211 strain and only in one mouse from the A/PR8-NS124_N231 group. This difference is due to the presence in the heterologous insert in the NS gene of the A/PR8-NS124_N211 virus of the sequence SEQIDNO: 2 containing an immunodominant epitope (IEDB epitope ID 34851), which is relevant both for C57/black mice (H2-Db allele) and for people with the most common HLA-A*02:01 allele. Thus, the use of SEQ ID NO: 2 in constructing NS1 truncated influenza viruses and inserting the SARS-CoV-2 N protein fragment increases the ability of these viruses to stimulate a T-cell immune response.

Example 5. Attenuation in laboratory mice of a recombinant influenza virus encoding a fragment of the N protein of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein.

Safety was assessed based on body weight dynamics and mortality in groups of C57/black laboratory mice immunized with the prototype strain A/PR8-NS124_N211 (as described in Example 4) compared with wild-type influenza virus A/PR8-NSwt and attenuated viral vector without insert A/PR8-NS124. Analysis of the body weight dynamics of vaccinated animals using the Dunnett multiple comparison test showed a significantly smaller decrease in body weight of animals that received A/PR8-NS124_N211 compared to the group of animals that received wild-type influenza virus, starting from the 2nd day after immunization (p <0.05). The decrease in body weight of animals in the A/PR8-NS124_N211 group was comparable to the attenuated vector group without insert (Fig. 6). The data obtained indicate an attenuated phenotype of the vaccine candidate compared to the wild-type influenza virus. It also shows no increase in the pathogenicity of the vaccine candidate compared to an attenuated viral vector without an insert.

The pharmacokinetics of the vaccine candidate was analyzed in terms of such characteristics as reproduction at the injection site with the formation of infectious progeny and dissemination of the viral vector from the site of application to distant organs (lungs and bronchi, nasal turbines, spleen, brain) using RT-PCR and / or isolation of infectious progeny in cell culture. Comparison of pharmacokinetic parameters was performed with a group of animals that received a well-characterized attenuated influenza vector without an A/PR8-NS124 insert. On the 4th day of the study, the average level of viral load in the tissues of the lungs in animals that received the A/PR8-NS124_N211 virus was 4.8 ± 0.4 lgTID50 and did not significantly differ from that in the A/PR8-NS124 empty vector group (4.6 ± 0.1). In both groups, the amount of viral RNA in the tissues of the lungs was higher than in the tissues of the upper respiratory tract (ΔCt ranged from 5.8 to 10.6). Influenza virus RNA was not detected in spleen and brain tissue homogenates in any of the experimental animals. Thus, according to the indicators of reproduction in the respiratory tract of vaccinated animals (lungs, nasal turbines), the prototype strain A/PR8-NS124_N211 showed an attenuated phenotype at the level of the previously characterized influenza vector without an insert, while no dissemination of the strain to distant organs was recorded during intranasal application.

Example 6 Protective Efficacy of the Prototype Recombinant Influenza Virus Encoding the SARS-CoV-2 Virus N Protein Fragment in the Reading Frame of the Shortened NS1 Protein in the Model of Experimental Infection of Syrian Hamsters with the SARS-CoV-2 Virus

The study used female Syrian hamsters at the age of 3-4 weeks at the time of the first vaccination. Animals were immunized intranasally under anesthesia, injecting virus preparations or placebo into both nasal passages, there were 6 animals in each group. The dose of the prototype strain and control vector without insert was 7.0 lgEID50/animal. Animals were infected 4 weeks after immunization. The SARS-CoV-2 virus (clinical isolate, collection number 11308A, clyde GR, lineage B.1.1) was injected intranasally into both nasal passages at a dose of 5 lgTCID50/animal in a volume of 100 µl/animal. After infection and before euthanasia, a daily clinical examination was performed with registration of the weight and body temperature of the animals. 3 and 6 days after infection, 3 animals from the group were euthanized with the collection of sera and organs to assess the immunological parameters and the severity of the infection.

Virus excretion from the lower and upper respiratory tract of animals was studied on the 3rd day after infection by the infectious activity of the virus in organ suspension, measured by titration in Vero cells. Control animals (placebo group) showed high titers of the SARS-CoV-2 virus in the lungs (up to 8.5 lgTCID50/ml), trachea (up to 6.7 lgTCID50/ml) and nasal passages (up to 8.0 lgTCID50/ml) . Statistically significant suppression of infectious activity of the virus in the lungs and nasal passages was observed in animals immunized with the prototype strain A/PR8-NS124_N211, which amounted to 0.9 lg in the lungs and 1.2 lg in the nasal passages (Fig. 8).

The obtained results demonstrate the protective efficacy against COVID-19 of the recombinant influenza virus encoding the N protein fragment of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein when prophylactically administered to naive animals.

Example 7 Protective Efficacy of Vaccine Strains Encoding the SARS-CoV-2 Virus N Protein Fragment as a Reading Frame of the Shortened NS1 Protein in the Model of Experimental Infection of Mice with the SARS-CoV-2 Variant Beta Virus

BALB/c mice (females, 6-8 weeks old, Pushchino Laboratory Animal Nursery, Russian Academy of Sciences) were used in the study. In each experimental group there were 15 animals, in the intact group there were 5 animals. The first group of mice was inoculated twice intranasally with vaccine strains at a dose of 6.0 lgEID50 (strains A/Cambodia/NS124_N (H3N2) and A/Guangdong/NS124_N (H1N1pdm09). As a control for nonspecific activation of immunity, a group of mice was inoculated with an attenuated influenza vector without an insert at a similar dose. For the prime-boost regimen, mice were first vaccinated intramuscularly with purified formalin-inactivated coronavirus adsorbed on aluminum hydroxide, then boosted 3 weeks later with the A/Guangdong/NS124_N (H1N1pdm09) vaccine strain. only "prime" - immunization with inactivated coronavirus, in a similar dose - 2.5 μg with 10 μg of aluminum hydroxide. The infection control group received a placebo (phosphate-buffered saline) intranasally twice. The study also contained a group of intact mice to control background immunological reactions.

3 weeks after the second immunization, all but intact mice were infected with the novel coronavirus SARS-CoV-2 of the beta genetic line (South African variant), which is capable of infecting mice without prior adaptation [R. Kant et al., “Common Laboratory Mice Are Susceptible to Infection with the SARS-CoV-2 Beta Variant,” Viruses, vol. 13, no. 11, 2021, doi: 10.3390/v13112263].

After infection in animals, body weight was determined daily (Fig. 9A). Placebo-treated control animals lost significant weight during infection, losing up to 85% of their original weight on day 3 post-infection. Mice primed intramuscularly with inactivated coronavirus showed a rapid weight loss on the first day after infection, which increased on the second day, after which the weight gradually recovered. Mice vaccinated with vaccine strains or a control vector without an insert lost less weight during infection than animals from the placebo group and completely regained weight by day 5. Mice vaccinated according to the "prime-boost" scheme practically did not lose weight against the background of infection. The results obtained may indicate a significant role of boosting intranasal immunization in reducing the severity of the infection.

On the 5th day after infection in control and immunized animals, the viral load in the lungs was determined. The amount of virus was assessed by titration of samples of broncho-alveolar lavage in Vero cell culture (Fig. 9B). Double immunization with vaccine strains significantly reduced the amount of infectious virus (by 1.4 lgTID50) in the lungs of mice. The greatest decrease in viral load was recorded in mice immunized with inactivated coronavirus or in the prime-boost scheme; on the 5th day after infection, no infectious virus was detected in the BAL.

The obtained results demonstrate the protective efficacy against COVID-19 of vaccine strains based on recombinant influenza virus encoding the N protein fragment of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein, with a double prophylactic administration to naive animals and a single administration to primed animals.

Example 8 . Protective efficacy of vaccine strains encoding the N protein fragment of the SARS-CoV-2 virus as part of the NS1 truncated protein reading frame in a model of lethal influenza infection in mice.

White outbred BALB/c mice (females, 6-8 weeks old, Rappolovo Laboratory Animal Nursery) were used in the cross-protection study. Each experimental group included 10 animals. Mice were infected 28 days after the second vaccination intranasally at a dose of 10 MLD50/animal in a volume of 30 µl. For infection, strains A/Aichi/2/68 (H3N2) and A/California/07/2009 (H1N1pdm09), pathogenic for mice, obtained from the collection of viruses of the Federal State Budgetary Institution “Influenza Research Institute named after N.N. A.A. Smorodintsev" of the Ministry of Health of Russia. All of the viruses used caused a lethal infection in mice in the placebo group, resulting in 60% to 70% deaths. At the same time, in the group of vaccinated animals, the death of mice was not observed (Fig. 10 B and D). In the placebo group, infected mice lost an average of 20 to 40% of their body weight. In groups of mice immunized with vaccine strains, a slight decrease in body weight of mice was observed, which did not exceed 5-7% of the initial values and was significantly lower than in the placebo group (Fig. 10.A and B).

The obtained results demonstrate the protective efficacy against influenza infection of vaccine strains based on the recombinant influenza virus encoding the N protein fragment of the SARS-CoV-2 virus as part of the reading frame of the truncated NS1 protein, with a double prophylactic administration to naive animals.

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</INSDSeq_feature-table>

<INSDSeq_sequence>MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGST

LGLDIETATRAGKQIVERILKEESDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPKQKVAGPLCIRM

DQAIMGGLEAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVT

QAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDD

KDPNFKDQVILLNKHIDAYKTFPPTEPK</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceDatasequenceIDNumber="4">

<INSDSeq>

<INSDSeq_length>267</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..267</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q7">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Chimearic protein constituent truncated

NS1 (1-124) from Influenza A virus and (231-369) fragment of

SARS-CoV-2 nucleoprotein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..267</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q8">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>RVADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETATRAGKQIVERIL

KEESDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPKQKVAGPLCIRMDQAIMGGLEAGNGGDAALAL

LLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQEL

IRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYK

TFPPTEPK</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (7)

1. A method for obtaining a recombinant influenza virus intended for the prevention of COVID-19 and influenza, characterized by the use of a heterologous fragment containing, but not limited to, immunodominant and signal sequences of SEQ ID NO 1 and SEQ ID NO 2 as part of the reading frame of the truncated NS1 protein of the influenza virus , borrowed from the N protein of the SARS-CoV-2 virus. 2. The recombinant influenza virus obtained by the method of claim 1 is an influenza A virus with a NS1 protein truncated to 124 amino acids containing the sequences SEQ ID NO 1 and SEQ ID NO 2. 3. Recombinant influenza virus A / Guangdong / NS124_N (H1N1pdm09), family Orthomyxoviridae, genus Influenza Virus A, obtained by the method according to claim 1, deposited in the State Collection of Viruses under No. 2981. 4. Recombinant virus A/Cambodia/NS124_N (H3N2), family Orthomyxoviridae, genus Influenza Virus A, obtained by the method according to claim 1, deposited in the State Collection of Viruses under No. 2982. 5. A method for preventing COVID-19 and influenza, characterized by immunizing subjects with the recombinant influenza virus according to claims 2-4 via intranasal administration. 6. The method of preventing COVID-19 and influenza according to claim 5, characterized by immunization with the recombinant influenza virus according to claims 2-4 in immunologically naive and immunologically primed subjects. 7. The method of preventing COVID-19 and influenza according to claim 5 or 6, characterized by immunizing subjects with the recombinant influenza virus according to claims 2-4, by single or double administration with an interval of 14 to 28 days.

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