Vaccine with reduced anti-vector antigenicity

Abstract

The present invention relates to a recombinant replication-deficient or replication-competent negative-strand RNA virus that can (i) present a heterologous antigen on its surface and induce mucosal immunity in a host, (ii) produce a heterologous antigen after invading host cells, thereby improving the host's immune response to the antigen, and (iii) have reduced anti-vector antigenicity compared to a wild-type vector, thereby being able to reduce the host's immune response to the vector.

Description

Technical Field

[0001] The inventors have constructed a recombinant Replicable type or replication-deficient negative-strand RNA virus that can (i) present a heterologous antigen on its surface, (ii) induce mucosal immunity in a host, (iii) produce a heterologous antigen after invading a host cell, thereby improving the host's immune response to the antigen, and (iv) have reduced anti-vector antigenicity compared to a wild-type vector, thereby reducing the host's immune response to the vector, i.e., inducing less anti-vector immunity.

[0002] The present invention relates to a recombinant replication-deficient negative-strand RNA virus comprising, in its genome, (a) a nucleotide sequence encoding a P polypeptide, the nucleotide sequence being modified compared to the wild type, the modification resulting in replication deficiency of the negative-strand RNA virus, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, the at least one heterologous polypeptide being presented on the surface of the viral particle and being capable of being expressed in an infected cell.

[0003] Another aspect of the present invention relates to a recombinant Have type negative-strand RNA virus comprising, in its genome, (a) a nucleotide sequence encoding a P polypeptide, where the P protein performs its original function Replicable in the viral replication cycle, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, the at least one heterologous polypeptide being presented on the surface of the viral particle and being capable of being expressed and replicated in an infected cell.

[0004] Another aspect of the present invention relates to a pharmaceutical composition, a vaccine, and an immunogenic composition comprising the recombinant Replicable type or replication-deficient negative-strand RNA virus of the present invention. A further aspect of the present invention relates to an RNA molecule, and the recombinant ReplicableThe present invention relates to DNA molecules encoding type or replication-deficient negative-strand RNA viruses. Another aspect relates to nucleocapsids containing the RNA molecules of the present invention. [Background technology]

[0005] background History teaches us that it is often second-generation vaccines that bring about groundbreaking progress in the complete control of persistently spreading viral infections. Even the polio era 70 years ago, sustained success was only achieved through second-generation vaccines that mimicked the natural enteral-mucosal transmission route. The first-generation vaccines that had been developed up to that point could only be injected into the muscles of the upper arm, but they mimicked the intestinal mucosa of the poliovirus. Infection The first-generation vaccine failed to prevent infection by poliovirus replication in the gastrointestinal tract, thus failing to provide "sterilizing immunity." As a result, vaccinated individuals could remain infected with poliovirus, and the progeny viruses produced in the intestines could transmit the disease to others through enteral excrement (feces). Consequently, the first-generation vaccine failed to establish sufficient herd immunity.

[0006] An overview of COVID-19 vaccines and candidate vaccines can be found on the WHO website ("COVID-19 Vaccine Tracking and Landscape"). https: / / www.who.int / publications / m / item / draft-landscape-of-covid-19-candidate-vaccines This can be found in [link / reference]. According to the WHO definition, all anti-SARS-CoV-2 vaccines approved to date belong to the first generation. These drugs are administered intramuscularly, usually by injection into the muscle of the upper arm. They do not enter the body through the respiratory tract, as SARS-CoV-2 infection does. First-generation SARS-CoV-2 vaccines produce an immune response sufficient to prevent most severe cases of COVID-19, but they do not provide sufficient immunity against respiratory tract infections.

[0007] This means that even those who have been vaccinated can still be infected (so-called breakthrough infections are frequently observed). Infection can occur through droplet transmission to unvaccinated individuals. This means that the first-generation SARS-CoV-2 vaccines approved to date cannot effectively break the chain of infection. Therefore, there is a strong need for a SARS-CoV-2 vaccine that induces respiratory "bactericidal immunity" against COVID-19.

[0008] Breakthrough infections are occurring frequently, potentially burdening hospitals. Herd immunity, which is urgently needed, is still a long way off. We will not succeed in returning to the “normal” life we ​​had before the pandemic, and this will affect every aspect of our lives.

[0009] Known vector-based (adenovirus-based) vaccines cannot incorporate the SARS-CoV-2 spike (S) protein into the envelope of the vector vaccine particle. In contrast to coronaviruses, adenovirus-based vaccines do not immediately provide recombinant S protein as an antigen. Rather, the S protein must be produced in muscle cells infected with the vector vaccine after intramuscular injection, resulting in a delayed immune response and reduced efficiency, particularly in the development of tracheal mucosal immunity. The same applies to mRNA-based vaccines administered intramuscularly.

[0010] If immunological protection from the anti-SARS-CoV-2 vaccine is not sufficiently sustained, repeated administration is essential. Currently, it is thought that three or more doses of vaccination are necessary, for example, a booster dose every six months. Furthermore, newly emerging virus subtypes require the administration of an adapted vaccine, thus necessitating repeated administration. In the case of vector vaccines, the adenovirus capsid is highly immunogenic and results in significant anti-vector immunity (anti-adenovirus immunity). Re-administration of the adenovirus vector can neutralize the vector due to this anti-vector immunity, reducing its effectiveness and potentially limiting the option of administering first-generation vector vaccines on a schedule requiring two or more doses. If anti-vector immunity is significant, the effect of booster immunity is neutralized, leading to loss of effectiveness or the need for high doses, increasing the labor and cost of production, as well as the risk of side effects.

[0011] Therefore, a SARS-CoV-2 vaccine is needed that at least partially overcomes the shortcomings of the first-generation vaccines mentioned above.

[0012] Paramyxoviruses, particularly Sendai viruses, are enveloped viruses possessing a helical nucleocapsid. The envelope contains a lipid membrane derived from the cell membrane of the host cell from which the virus was released. Transmembrane glycoproteins, namely fusion proteins (F) and hemagglutinin-neuraminidase (HN), are fixed to the viral envelope. Matrix proteins (M) are located on the inner side of the membrane. The nucleocapsid, or viral replication complex, consists of single-stranded RNA complexed with nucleoproteins (N). In each case, six nucleotides of the RNA are linked by one N protein, RNA-dependent RNA polymerase (L), and the coenzyme phosphoprotein (P), forming an RNA polymerase complex. During primary transcription, the genome is transcribed by viral RNA-dependent RNA polymerase (vRdRp), initiating de novo protein synthesis. It is thought that as soon as a sufficient amount of N protein is produced, vRdRp switches to replication mode and synthesizes the antigenome. From that template, a new genome encapsulated with the N protein is replicated.

[0013] The genomic RNA of the viral family, specifically the negative-strand RNA virus family, infects cells and, after this RNA is released into the cell, is not immediately translated, but usually forms a complex with a protein called a nucleoprotein (N protein or NP). Only this viral RNA-N protein complex is recognized by viral polymerase as a template for viral genome replication or viral transcription. During viral genome replication, newly synthesized genome or AntigenomeIt immediately complexes with the N protein. During viral transcription, mRNA that is not complexed with the N protein is synthesized. This interferes with translation at the ribosome. In other words, only the viral genome / antigenome complexes with the N protein. In the process of complexing viral RNA and the N protein, another viral protein, the P protein, is involved. The P protein itself forms a complex with the N protein. Only this Np complex enables the complexation of viral RNA and the N protein. If this complex between N and P is not formed due to mutations in the interaction site on the N or P protein, a complex of the newly synthesized viral genome / antigenome will not be formed. A new functional viral genome cannot be generated, and therefore, new functional viral particles cannot be generated. The virus replicates Defective This can occur. This replication defect can be achieved by mutating the binding site between the P protein and the N protein, specifically by deleting the nucleotides encoding amino acids 2-77 of the P protein (see also Wiegand et al., 2007).

[0014] The Sendai virus negative-strand RNA genome contains six structural protein genes in the following order: 3'-NPMF-HN-L-5'. The P gene encodes a total of eight proteins: phosphorylated structural proteins and all non-structural proteins known to date.

[0015] P, N, and L proteins are important for functional transcription and replication (Lamb et al., Paramyxoviridae: The Virus and their Replication. Fields Virology, 4th edition (2001), Lippincott, Williams & Wilkins, Philadelphia, 1305-1340).

[0016] WO 2006 / 084746 discloses replication-deficient and transcriptionally competent Sendai virus (SeV), which was used for transgene expression.

[0017] Wiegand (Journal of Virology 2017:91(10), e02298-16) showed that the ectodomain of SeV surface protein F can be substituted with the corresponding ectodomain of RSV virus F. The substitution between SeV F protein and RSV F protein forms a "family" of functionally and structurally equivalent sequences. Inside "It is a substitution."

[0018] To date, there have been no studies demonstrating such interfamily substitution of functionally essential proteins between paramyxoviruses and coronaviruses, two completely different viral families, without loss of function.

[0019] Attenuation of viral replication means that a particular vector construct is not under optimal conditions compared to the natural / wild type. While the latter has evolved to adapt to preferred host organisms or cell types, achieving high viral replication rates, attenuated viral vectors produce suboptimal titers during replication. There are various reasons why attenuation and suboptimal replication can occur. Attenuation can be caused by the viral vector itself, the host system, the replication conditions, or a combination of these factors. Of course, changes in the viral genome can significantly impact viral replication behavior because introduced mutations are unlikely to be the optimizations that the virus would naturally select for. The effects of single or multiple point mutations are usually already clearly recognizable by the affected gene or gene product. Mutations affecting the function of viral enzymes involved in viral replication can significantly impact viral replication. For example, an N-terminal deletion of the Sendai virus P gene prevents the L-protein from interacting with its cofactor, the P protein, in the way necessary for L-protein to interact with the viral genome and mediate the synthesis of copies of the viral RNA genome, thus hindering replication. DeficiencyThis can cause... Another type of mutation can affect the structural proteins of the virus, such as surface proteins or envelope proteins. Envelope proteins are necessary for the formation of new viral particles during viral replication and for the release of these particles from the host cell. Furthermore, these proteins are essential for the virus to interact with new host cells, bind to receptors and be taken into the cell, and mediate uptake mechanisms such as membrane fusion or uptake into the cell by vesicles. Different viruses have different uptake strategies. For example, the Sendai virus HN and F proteins in the membranous envelope of the viral particle enable binding to receptors, then fusion of the viral membrane with the host cell membrane, thereby releasing the viral genome into the host cell. SARS-CoV-2, on the other hand, enters host cells by uptake into vesicles via its surface protein S. Large mutations in these surface / envelope proteins, such as point mutations in essential amino acids or exchange of entire envelope proteins between genomes of different viral families, can have a significant impact on the normal viral life cycle and its replication efficiency. [Overview of the project]

[0020] Detailed explanation The inventors have developed a second-generation vaccine (particularly a vaccine against SARS-CoV-2) that meets the following requirements: • Because the vaccine vector exhibits maximum attenuation and is safe, it can be used not only in healthy subjects but also in partially immunosuppressed subjects in the pre-disease stage. This vector should efficiently penetrate the mucous layer of the mucosa and is therefore suitable as a vaccine vector for inducing respiratory mucosal immunity. • Vaccine vectors are administered via natural infection routes (particularly the respiratory mucosal route in the case of anti-SARS-CoV-2 vaccines). • Vaccine vectors induce immediate mucosal immunity by presenting antigens on the surface of the virus (especially spike (S) proteins in anti-SARS-CoV-2 vaccines). · If the vaccine vector induces little or at most reduced anti-vector immunity, repeated administration prevents loss of efficacy. · When the introduced gene is expressed in infected cells, the vector elicits a systemic immune response.

[0021] In accordance with the above requirements, the inventors have developed a vector-based vaccine in which the SARS-CoV-2 spike protein is carried on the surface of vaccine vector particles. With this design, during the process of intranasal / mucosal application, the protein is directly presented to the immune system, just like natural infection of the respiratory tract by SARS-CoV-2. Furthermore, when the virus invades host mucosal cells, it can produce the SARS-CoV-2 spike protein and present it on the cell surface. In this way, broad protection against the highly threatening COVID-19 disease, especially protection including "sterilizing immunity", is achieved and ensured.

[0022] The inventors have found that a suitable vector is the attenuated (replication-deficient) Sendai virus (SeV). The Sendai virus (Paramyxoviridae) usually infects rodents and causes highly infectious respiratory infections.

[0023] The Sendai virus (SeV, Paramyxoviridae) carries two proteins on its surface: (i) the hemagglutinin-neuraminidase (HN) protein, and (ii) the fusion (F) protein. The HN protein mediates the binding (adsorption) of SeV virus particles to cell receptors. The F protein mediates the fusion of the SeV viral membrane and the cytoplasmic membrane. After membrane fusion (penetration), the SeV replication complex (nucleocapsid) is released into the cell.

[0024] The biologically active forms of HN and F present on the SeV surface are both oligomers. When the virus matures, these oligomers concentrate in specific regions of the cytoplasmic membrane through interaction with the SeV matrix protein (M), which is a major prerequisite for the release ("budding") of newly formed virus particles from infected cells.

[0025] At the start of our testing, it was unclear whether recombinant proteins obtained from other virus families (for example, the S protein of SARS-CoV-2, a member of the coronavirus family) could be incorporated into the envelope of the SeV vaccine vector (Paramyxoviridae) without significantly adversely affecting the HN and F protein functions (adsorption and penetration) of SeV in infection of target cells.

[0026] Furthermore, it was completely unclear what modifications of recombinant proteins (e.g., chimeric SARS-CoV-2 S protein) are necessary for successful integration into the SeV envelope and presentation on its surface, what modifications are permissible, and whether they affect the biological function of recombinant proteins (e.g., the antigenicity of SARS-CoV-2 and its functionality in the infectious process).

[0027] The only polypeptide present on the surface of SARS-CoV-2 is the spike protein S. The S protein exists in oligomeric form. The S protein is the largest viral protein with fusion function (more than 1200 amino acid residues in length). Approximately 30-60 S protein molecules are fixed to the envelope of a single SARS-CoV-2 particle at an average distance of 15 nm. At the start of our experiments, it was unclear whether the distance of the S protein on the surface was essential for its function, whether the S protein would function in combination with SeV F and HN if recombinantly incorporated into the envelope, or whether complex presentation on the SeV surface would result in some or all loss of function.

[0028] Furthermore, it was unclear whether recombinant SARS-CoV-2 S protein could compensate for the function of the F and / or HN polypeptides if some or all of the sequences encoding them were deleted from the SeV genome.

[0029] From these points, it is clear that designing and constructing an SeV vector-based vaccine against SARS-CoV-2 is by no means trivial or simple, but requires considerable ingenuity.

[0030] The inventors constructed recombinant Sendai viruses displaying a recombinant antigen (e.g., SARS-CoV-2 spike protein) obtained from SARS-CoV-2 on their surface. Examples 1 and 2 describe cDNA constructs of recombinant Sendai virus (SeV) capable of expressing the coronavirus SARS-CoV-2 spike (S) protein. The S protein can be expressed as a chimeric protein containing the ectodomain of the S protein, the cytoplasmic domain of the SeV polypeptide, or the transmembrane domain of the SeV polypeptide or the SARS-CoV-2 spike. The inventors have shown that a vaccine candidate provided as cDNA can be obtained from GMP-certified Vero cells and proliferated in Vero cell-derived V3-10 helper cells. The inventors have shown that the SARS-CoV-2 S protein is incorporated into the candidate viral particles.

[0031] The inventors discovered that recombinant SARS-CoV-2 S protein is functionally presented on the surface of SeV. The inventors provided SeV vaccine vectors that (i) had reduced amounts of surface F and / or HN proteins, or (ii) did not contain F and HN proteins. The inventors recognized that coronavirus (positive-strand) S proteins can complement the adsorption and entry capabilities of negative-strand RNA viruses when the native (endogenous) genes mediating these capabilities are deleted in these viruses. In particular, the adsorption and penetration capabilities of negative-strand paramyxovirus F and HN proteins can be complemented by the adsorption and penetration capabilities of positive-strand coronavirus family virus S proteins. This is the first example showing that the two endogenous F and HN proteins of paramyxovirus (SeV) have been functionally replaced by a single coronavirus protein in an "interfamily" substitution.

[0032] The inventors of this invention have found that attenuation of the Sendai virus can be achieved by modifying the P protein, and as a result, a replication-deficient virus can be obtained.

[0033] The inventors also reproduce Possible type In SeV, the surface proteins F and / or HN are at least partially replaced by the SARS-CoV-2 surface protein S, or a fragment thereof, and the S protein of the fragment performs the function of the F and / or HN proteins. May have We found that attenuation can be achieved in this way. The inventors also found that such modified reproduction Possible type We found that SeV exhibits increased replication efficiency compared to the replication-deficient SeV of the present invention (Example 4).

[0034] The inventors also found that a specific mucosal immune response to SARS-CoV-2 can be stimulated by nasal immunization. Immunization with the virus of the present invention induced specific IgA antibodies in a dose-dependent manner (Example 5).

[0035] If a person who has received a vaccine has already acquired immunity to SARS-CoV-2 in their respiratory system due to, for example, a SARS-CoV-2 infection, then the subject's immune system is recombinant. ReplicableSeV vaccine vectors, particularly SeV vaccine vectors against SARS-CoV-2, can be recognized, and the vaccine can act as a booster immunizer. If a vaccinated subject has not yet acquired immunity to SARS-CoV-2 in the respiratory tract, the vaccine vector can induce immunity to SARS-CoV-2 through different immune stimulation pathways and interactions with the immune system. Firstly, vaccine vector particles exhibit immunogenicity against SARS-CoV-2 themselves because they display spike proteins on their surface. In this way, vaccine vectors function, for example, as nanoparticle vaccines or virus-like particle vaccines (VLPs). These particles are often recognized by antigen-presenting cells of the immune system, such as dendritic cells, or taken up by macrophages. Macrophages present a portion of the spike protein to T lymphocytes, which can then differentiate into helper and effector cells. Secondly, vaccine vectors can infect cells, such as those in the respiratory tract, and express the encoded spike protein within the cells. As a result, spike proteins may be expressed on the surface of these cells, leading to recognition of infection by the immune system. Furthermore, after standard proteolysis occurs within these cells, the spike protein peptide may be presented on the MHC-I molecule.

[0036] In light of experimental evidence obtained using paramyxoviruses (e.g., SeV) that recombinantly present an antigen (e.g., SARS-CoV-2 S protein) on their surface, the inventors have concluded that it is possible to construct recombinant negative-strand RNA viral vectors that carry recombinant antigens and exhibit little to no anti-vector immunity.

[0037] In a first embodiment, the present invention includes the following in its genome: (a) A nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in a replication defect of the negative-strand RNA virus, and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. This relates to recombinant replication-deficient negative-strand RNA viruses, including those mentioned above.

[0038] In this invention, the term "virus" means an infectious substance that replicates only within living cells. Viruses do not have their own metabolic mechanisms. As used herein, the term "virus" includes all forms within a host cell, as well as extracellular viral particles (also called virions), i.e., those released from a host cell that can infect new host cells. RNA viruses may be single-stranded RNA viruses or double-stranded RNA viruses.

[0039] In this invention, the terms “genome” and “viral genome” are used interchangeably and refer to the entire nucleic acid present in the nucleocapsid. The genome can be unsegmented (single nucleic acid molecule) or segmented (two or more nucleic acid molecules). A single-stranded virus may be a positive-sense virus or a negative-sense virus. In a negative-sense virus, the nucleic acid complementary to the genome is called the antigen. In a negative-sense virus, the gene is transcribed into mRNA, which is then translated into polypeptides. In a positive-sense virus, the nucleic acid complementary to the genome is called the genome. In a positive-sense virus, the genome sequence is directly translated into polypeptides. In this invention, the genome may be a recombinant genome. The recombinant replication-deficient negative-sense RNA virus described herein may contain a recombinant genome. The recombinant genome may contain recombinant RNA molecules.

[0040] In this invention, the terms “inter-family” and “intra-family” refer to relationships between viruses of different viral families or between viruses of the same viral family. For example, inter-family substitution of nucleotide sequences refers to an exchange between viruses of different families, while intra-family substitution of nucleotide sequences refers to an exchange between viruses of the same family.

[0041] As used herein, the terms “heterogeneous” or “exotic” mean polypeptides and / or nucleic acids that are foreign to a particular virus, such as a negative-strand RNA virus. Heterogeneous (exotic) polypeptides and / or nucleic acids do not exist naturally in a particular virus and are introduced into the viral genome by artificial or recombinant means. For example, a heterogeneous (exotic) nucleotide sequence is an exotic sequence introduced into the viral genome. Heterogeneous (exotic) nucleotide sequences may be operatively linked to natural or endogenous viral sequences, such as expression regulatory sequences. Heterogeneous (exotic) nucleotide sequences may also be inserted into a frame of natural or endogenous viral coding sequences to express a fusion protein or chimeric protein containing a heterogeneous (exotic) polypeptide sequence and a natural or endogenous amino acid sequence.

[0042] Yet another aspect of the present invention is that in its genome, (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein performs its intended function, particularly in the viral replication cycle. Have The nucleotide sequence, and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Includes, Here, the heterologous polypeptide contains a viral antigen, and the viral antigen and negative-strand RNA virus are selected from different families of viruses. Recombinant replication Possible type Regarding negative-strand RNA viruses.

[0043] In particular, recombinant Replicable Negative-strand RNA viruses have been attenuated. Recombinant ReplicableIn type 1 negative-strand RNA viruses, the P protein may be the wild-type P protein, for example, the P protein encoded by SEQ ID NO: 1, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to it. The wild-type P protein may have the amino acid sequence containing SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to it.

[0044] In this invention, the term "attenuated virus" refers to a virus whose pathogenicity is reduced or absent, and in particular a virus whose replication is reduced or almost completely lost. Attenuated viruses can induce an immune response without causing specific diseases caused by non-attenuated viruses.

[0045] In particular, the present invention also relates to replication-deficient negative-strand RNA viruses. Loss of replication means that in target cells that do not trans-produce the function deleted by the virus, no detectable replication of the viral genome is observed, and, in contrast to reduced replication capacity or conditional replication defects, there are no tolerant conditions under which replication occurs. The negative-strand RNA viruses of the present invention used herein are still infectious, that is, they can adsorb to and invade host cells. The infectious negative-strand RNA viruses used herein can also induce the expression of at least heterologous genes in host cells.

[0046] The term "recombination according to the present invention" as used herein Replicable "Type or replication-deficient negative-strand RNA virus" refers to two different viruses of the present invention: (i) recombinant Replicable (ii) Recombinant replication-deficient negative-strand RNA viruses are described. The term “recombinant according to the present invention” is used. Replicable The terms “type or replication-deficient minus-strand RNA virus” or similar expressions (e.g., relating to a specific virus species) are, in particular in this specification, (i) recombinantReplicable This is used to describe features that may exist independently of (ii) type negative-strand RNA viruses and (ii) recombinant replication-deficient negative-strand RNA viruses. Embodiments of the present invention relating to recombinant replication-deficient negative-strand RNA viruses are described in particular in sections 1 to 69. Recombinant Replicable Embodiments of the present invention relating to type negative-strand RNA viruses are described in particular in sections 70 to 133.

[0047] The recombinant virus according to the present invention may be transcriptionally competent. That is, the virus can induce viral and heterologous mRNA synthesis and translate into viral polypeptides and heterologous polypeptides. In particular, the gene product encoded by the virus is transcribed after infection in target cells, and the expression of a viral protein containing one or more heterologous gene products occurs in the target cells. The heterologous polypeptide is presented on the surface of the target cells, i.e., the mucosal cells of the vaccinated target, thereby inducing and / or improving the immune response to the heterologous polypeptide.

[0048] Transcription can include primary and secondary transcription. As used herein, primary transcription begins with the smallest replicating unit, i.e., a nucleocapsid that has permeated the cell, and the nucleocapsid comprises a single-stranded RNA molecule complexed with a nucleoprotein (N), RNA-dependent RNA polymerase (L), and phosphoprotein (P), forming an RNA polymerase complex. Primary transcription can also begin with a viral-coding DNA molecule, particularly a cDNA molecule. During primary transcription, the genome is transcribed by RNA polymerase, and de novo protein synthesis begins. As the new genome is replicated, these templates are also transcribed. This transcriptional stage is called secondary transcription and is far more efficient than primary transcription.

[0049] In this invention, the term "gene product" refers to a product obtained by gene expression. Gene products include nucleic acids (e.g., mRNA) transcribed from genes and polypeptides or proteins obtained by translation from mRNA.

[0050] The recombinant replication-deficient RNA virus according to the present invention can replicate in host cells trans-complemented with an exogenous sequence ("helper cell") encoding the deficient viral protein. As a result, replication-deficient viral particles are obtained. Eukaryotic host cells can be co-transfected with at least one eukaryotic expression vector encoding viral proteins necessary for the formation of the viral nucleocapsid, i.e., structural proteins and polymerases necessary for the replication of the cDNA sequence. For example, host cells can be transfected with a plasmid containing a replication-deficient viral genome, which is modified compared to the wild type, resulting in replication deficiency of the negative-strand RNA virus, and host cells can be co-transfected with three plasmids encoding the native viral proteins N (capsid), L, and P (which form RNA-dependent RNA polymerase complexes), respectively. The genome can be transcribed into antigenic RNA by an endogenous polymerase (e.g., RNA Pol II) in the host cell. The antigen can then be assembled into a nucleocapsid. Figure 1 illustrates an exemplary rescue strategy for replication-deficient Sendai virus.

[0051] For the initial production of replication-deficient negative-strand RNA viruses (referred to herein as “rescue” or “viral rescue”), eukaryotic host cells may be transfected with cDNA constituting the viral genome. In particular, host cells capable of viral rescue from the DNA molecule encoding the viral genome do not require transcomplement with an exogenous sequence encoding the deficient viral protein.

[0052] In this invention, the term "P polypeptide" refers to a phosphoprotein present in negative-strand RNA viruses. P polypeptides are cofactors of RNA-dependent RNA polymerases in these viruses and are involved in viral transcription and replication.

[0053] In this invention, the term "wild-type" refers to a recombinant minus-strand RNA virus that is essentially free from modifications such as recombinant sequences or deletions. The term "wild-type" in relation to a specific sequence refers to a sequence that is essentially free from modifications such as recombinant sequences or deletions. For example, the wild-type sequence of a P polypeptide is a P polypeptide sequence that is essentially free from modifications such as recombinant sequences or deletions. When "wild-type" refers to a specific sequence (such as the P sequence) of a recombinant minus-strand RNA virus, other sequences may be modified or may be unmodified wild-type sequences. Those skilled in the art know the wild-type P polypeptide sequence.

[0054] Recombination according to the present invention Replicable Negative-type or replication-deficient negative-strand RNA viruses can be based on naturally occurring negative-strand RNA viruses. Recombination according to the present invention Replicable The type or replication-deficient negative-strand RNA virus can be selected from the mononegaviral order. Recombination according to the present invention Replicable Preferred families (families) to which type or replication-deficient negative-strand RNA viruses can be based are Artoviridae, Bornaviridae, Filoviridae, Lyspiviridae, Maimonaviridae, Nyamiviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Sanviridae, and Simmoviridae. Replicable Preferred families of negative-strand RNA viruses that can be based on type or replication-deficient negative-strand RNA viruses are the paramyxovirus, rhabdovirus, filoviridae, bornaviridae, and their recombinant variants.

[0055] In the present invention, examples of single-stranded, unsegmented negative-sense RNA viruses include those of the Paramyxoviridae family (paramyxoviruses), Rhabdoviridae family (rhabdoviruses), Filoviridae family (filoviruses), and Bornaviridae family (bornaviruses).

[0056] Examples of paramyxoviruses include, but are not limited to, Sendai virus, human and bovine parainfluenza viruses, such as human parainfluenza virus (hPIV) types 1, 2, 3, 4a or 4b, Newcastle disease virus (NDV), mumps virus, measles virus, respiratory syncytial virus (RSV), and human respiratory syncytial virus (hRSV). Examples of rhabdoviruses include, but are not limited to, varicella-stomatitis virus (VSV).

[0057] Preferably, recombinant Replicable The negative-strand RNA virus of type or replication-deficient type is a paramyxovirus. Preferably, the recombinant RNA virus of the present invention. Replicable The negative-strand RNA virus (N-type or replication-deficient) may be Sendai virus, for example, Fushimi strain (ATCC VR105). Recombinant variants of the above viruses, such as those described in EP-A-702 085, EP-A-0 863 202, or WO 01 / 42445, are also included in the present invention.

[0058] In this invention, single-stranded positive-chain viruses include the Coronaviridae family. Examples of coronaviruses include MERS-CoV (Middle East Respiratory Syndrome Coronavirus), SARS-CoV (Severe Acute Respiratory Syndrome-related Coronavirus, also called SARS-CoV-1), and SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus type 2).

[0059] In this invention, the term "nucleocapsid" refers to a complex comprising viral nucleic acid and at least one viral protein ("capsid"). Nucleocapsids are present not only in viral particles (virions) but also in host cells.

[0060] In this invention, the term “viral envelope,” if present, refers to the outer layer of the virus. The envelope may originate from the host cell membrane and may contain viral peptides, such as peptides that can adsorb to receptors on the host cell and mediate entry into the cell. These peptides may be immobilized on the envelope by transmembrane and intracellular domains. In this invention, recombinant Replicable The envelope of a type or replication-deficient minus-strand RNA may contain at least one heterologous polypeptide, such as the SARS-CoV-2 spike protein or a fragment thereof, so as to be presented on the surface of the viral particle.

[0061] In this invention, the terms "peptide" and "polypeptide" may be used interchangeably.

[0062] In this invention, the terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” may be used interchangeably.

[0063] In this invention, the term “viral peptide” refers to peptides necessary for nucleic acid synthesis (e.g., transcription or replication, such as polymerase or polymerase complexes), envelope structure (if an envelope exists), nucleocapsid structure, and surface structure (necessary for adsorption to and penetration into host cells). There is no unified terminology for viral peptides and the genes encoding such peptides. In this invention, references to specific genes and / or peptides in a particular virus or viridae are intended to include references to homologous genes and / or peptides in other viral species and / or families, in particular genes and / or peptides having the same function (also referred to herein as “functional homologs”). For example, a reference herein to the P peptide specifically described for Sendai virus includes a reference to any of the peptides described for single-stranded negative-sense RNA viruses that have essentially the same function as the Sendai virus P peptide.

[0064] In the present invention, the nucleotide sequence (a) encoding the P polypeptide (also called the P protein or phosphoprotein) may be modified compared to the wild type (i.e., the wild-type P sequence), such modification resulting in a replication defect in the negative-strand RNA virus. As used herein, the term “P polypeptide” refers to a polypeptide of a paramyxovirus, particularly a negative-strand single-strand RNA virus having essentially the same function as the P polypeptide of the Sendai virus. As exemplary, but not limited, examples of the P polypeptide (SEQ ID NO: 2) and the sequence encoding the P polypeptide (SEQ ID NO: 1) are referenced herein to the Sendai virus sequence encoding the P polypeptide.

[0065] As used herein, “modification” includes deletions, insertions, and / or substitutions in nucleotide sequences or polynucleotides. If the nucleotide sequence includes a coding sequence, such modifications may result in a modified polypeptide.

[0066] In the nucleotide sequence (a) encoding the P polypeptide, modifications to nucleotide sequence (a) may be located in the nucleotide sequence encoding the N-terminus of the P polypeptide. At least the amino acid region 33-41 of the P polypeptide may be modified, which is important for replication ability. Modifications in the amino acid region 2-77 of the P polypeptide result in, for example, (i) deletion of amino acids 2-77 of the protein encoded by gene P, or (ii) deletion of the subsequence of (i) sufficient to cause loss of replication ability. Similar mutations can occur in the P protein of other negative-strand RNA viruses, such as other paramyxoviruses, such as hPIV3.

[0067] In particular, the modification of nucleotide sequence (a) is the deletion of the nucleotide sequence encoding the N-terminus of the P polypeptide. More specifically, the modifications in nucleotide sequence (a) are as follows: (i) Compared to the wild type, a deletion in the nucleotide sequence encoding amino acids 2-77 of the P polypeptide ("deltaP 2-77", SEQ ID NO: 4), or (ii) Replication of RNA viruses Deficiency The subarray of (i) that causes this.

[0068] Sequence ID 3 describes the nucleotide sequence encoding the SeV P protein, with amino acids 2-77 of Sequence ID 4 deleted.

[0069] In particular, the C-terminal region of the P polypeptide (starting from amino acid 320) does not contain any modifications that impair transcriptional function.

[0070] Reproductions described herein Possible type Alternatively, in replication-deficient negative-strand RNA viruses, the genome may further include: (c) Nucleotide sequence encoding a negative-strand RNA virus N polypeptide, (d) Nucleotide sequences encoding negative-strand RNA virus M polypeptides, and (e) Nucleotide sequence encoding a negative-strand RNA virus L polypeptide.

[0071] As used herein, the terms “N polypeptide,” “N protein,” or “nucleoprotein” refer to polypeptides of paramyxoviruses, particularly those of Sendai virus, that have essentially the same function as such polypeptides of minus-stranded single-stranded RNA viruses. Those skilled in the art are familiar with appropriate paramyxovirus N polypeptides and their functions. In the nucleocapsid of single-stranded RNA viruses, the RNA molecule forms a complex with the N polypeptide. In this specification, references to N polypeptides are not limited to paramyxoviruses but also include functional and structural homologs of other viral families.

[0072] As used herein, the terms “M polypeptide,” “M protein,” or “matrix protein” refer to polypeptides of paramyxoviruses, particularly those of Sendai virus, that have essentially the same function as such polypeptides of minus-stranded single-stranded RNA viruses. Those skilled in the art are familiar with appropriate M polypeptides of paramyxoviruses and their functions. M polypeptides are located on the inner surface of the viral envelope. In this specification, references to M polypeptides are not limited to paramyxoviruses but also include functional and structural homologs of other viral families.

[0073] As used herein, the terms “L polypeptide,” “L protein,” or “large protein” refer to polypeptides of paramyxoviruses, particularly those of Sendai virus, that have essentially the same function as such polypeptides of minus-stranded single-stranded RNA viruses. Those skilled in the art are familiar with appropriate L polypeptides of paramyxoviruses and their functions. L polypeptides are the largest proteins in polymerase complexes and possess RNA-dependent polymerase activity. In this specification, references to L polypeptides are not limited to paramyxoviruses but also include functional and structural homologs of other viral families.

[0074] Furthermore, recombinant Replicable The genome of a type or replication-deficient negative-strand RNA virus may further include the following: (f) Nucleotide sequences encoding negative-strand RNA virus F polypeptide, and (g) Nucleotide sequence encoding a negative-strand RNA virus HN polypeptide.

[0075] As used herein, the terms “F polypeptide,” “F protein,” or “fusion protein” refer to polypeptides of paramyxoviruses, particularly those of Sendai virus, that have essentially the same function as such polypeptides of minus-stranded single-stranded RNA viruses. Those skilled in the art are familiar with appropriate paramyxovirus F polypeptides and their functions. F polypeptides mediate the fusion of the viral particle to the host cell membrane. In this specification, references to F polypeptides are not limited to paramyxoviruses but also include functional and structural homologs in other virus families.

[0076] The F polypeptides or sequences encoding F polypeptides used herein may include the sequences described herein.

[0077] Sequence ID 6 describes the amino acid sequence of the Sendai virus (SeV)SeV F polypeptide. Sequence ID 5 describes the nucleotide sequence encoding the Sendai virus (SeV)SeV F polypeptide of Sequence ID 6.

[0078] As used herein, the terms “HN polypeptide,” “HN protein,” or “hemagglutinin-neuraminidase” refer to polypeptides of negative-stranded single-stranded RNA viruses that have essentially the same function as the HN polypeptides of paramyxoviruses, particularly Sendai virus. Those skilled in the art are familiar with appropriate paramyxovirus HN polypeptides and their functions. HN polypeptides mediate the adsorption of viral particles to host cells. In this specification, references to HN polypeptides are not limited to paramyxoviruses but also include functional and structural homologs in other virus families.

[0079] The HN polypeptides or sequences encoding HN polypeptides used herein may include the sequences described herein.

[0080] Sequence ID 12 describes the amino acid sequence of the Sendai virus (SeV)SeV HN polypeptide. Sequence ID 11 describes the nucleotide sequence encoding the Sendai virus (SeV)SeV HN polypeptide of Sequence ID 12.

[0081] As used herein, “at least one nucleotide sequence (b)” includes one, two, three, four, or more nucleotide sequences (b), each independently encoding a heterologous polypeptide. Preferably, “at least one nucleotide sequence (b)” includes one or two nucleotide sequences (b), and more preferably one nucleotide sequence (b). “At least one nucleotide sequence (b)” and “nucleotide sequence (b)” may be used interchangeably herein.

[0082] The at least one heterologous polypeptide encoded by the nucleotide sequence (b) may include any polypeptide suitable for therapy, such as an antigen that produces an immune response, or a therapeutic protein, such as a viral therapeutic protein.

[0083] The nucleotide sequence (b) encodes at least one heterologous polypeptide which may include heterologous antigens derived from a pathogen (such as a virus, bacterium, fungus, or protozoan), a tumor antigen, or an autoantigen. Those skilled in the art know antigens suitable for preparing immunogenic compositions and / or vaccines against pathogens such as viruses, bacteria, fungi, and protozoans. An example of an antigen suitable for preparing immunogenic compositions and / or vaccines against SARS-CoV-2 is the SARS-CoV-2 spike protein (S protein) or its immunogenic fragment.

[0084] The heterologous polypeptide may include a viral polypeptide, particularly a viral antigen. The viral polypeptide or viral antigen and the negative-strand RNA virus can be selected from different families of viruses. For example, the negative-strand RNA virus may be a paramyxovirus, and the heterologous antigen can be selected from viral antigens of families other than paramyxoviruses, such as viral polypeptides and antigens of the Rhabdoviridae, Filoviridae, Bornaviridae (negative-strand viruses), and Coronaviridae (positive-strand viruses).

[0085] Heterogeneous polypeptides may contain antigens derived from viruses other than negative-strand RNA viruses.

[0086] Heterogeneous polypeptides may also contain antigens derived from positive-strand viruses such as coronaviruses.

[0087] Preferably, the single-stranded negative-sense RNA virus is a paramyxovirus such as Sendai virus, and the heterologous polypeptide contains antigens derived from coronaviruses, particularly positive-sense viruses such as SARS-CoV-2.

[0088] It is more preferable that the single-stranded negative-sense RNA virus is the Sendai virus, and the heterologous polypeptide is an antigen derived from a coronavirus.

[0089] It is more preferable that the single-stranded negative-sense RNA virus is a paramyxovirus such as Sendai virus, and that the heterologous polypeptide contains a coronavirus, particularly a spike protein (S protein) or an immunogenic fragment derived from SARS-CoV-2.

[0090] The antigen may be a viral surface polypeptide or an immunogenic fragment thereof. Those skilled in the art know of suitable antigens, such as polypeptides present on viral particles. Examples of viral surface polypeptides include the SARS-CoV-2 spike protein (S protein) or its immunogenic fragment.

[0091] At least one nucleotide sequence (b) may be located between two coding sequences in the RNA molecule, upstream of the 5' coding sequence and / or downstream of the 3' coding sequence. For example, in paramyxovirus, at least one nucleotide sequence (b) can independently be located upstream of the 5' end of the N gene, between the N and P, P and M, M and F, F and HN, and HN and L genes, and / or downstream of the 3' end of the L gene. This arrangement ensures that at least one nucleotide sequence (b) present in the RNA molecule does not interfere with the endogenous viral sequence and its expression.

[0092] Recombination of the present invention Replicable In type or replication-deficient RNA viruses, the genome is, (f) Nucleotide sequences encoding negative-strand RNA virus F polypeptide, and (g) Nucleotide sequence encoding negative-strand RNA virus HN polypeptide It further includes, Then, at least one nucleotide sequence (b) can be independently located in the RNA molecule between two coding sequences, upstream of the 5' end coding sequence and / or downstream of the 3' end coding sequence, for example, upstream of the 5' end of the N gene, between the N gene and the P gene, the P gene and the M gene, the M gene and the F gene, the F gene and the HN gene, and the HN gene and the L gene, and / or downstream of the 3' end of the L gene. Examples of such constructs are described in the Examples ("Type A" constructs). Preferably, at least one nucleotide sequence (b) is located between the P gene and the M gene.

[0093] Reproduction Possible typeAlternatively, in replication-deficient negative-strand RNA viruses, at least one nucleotide sequence (b) may be inserted into or at least partially substituted into at least one native (endogenous) sequence encoding at least one viral polypeptide such that the function or activity of at least one endogenous viral polypeptide is at least partially or completely disrupted. In particular, the at least one viral polypeptide is different from the P polypeptide. The viral polypeptide may be N, M, F, HN and / or L of paramyxoviruses, and their homologs of other viral families. Preferably, at least one nucleotide sequence (b) may be inserted into or at least partially substituted into F and / or HN of paramyxoviruses and their homologs of other viral families such that the function or activity of F and / or HN is at least partially or completely disrupted.

[0094] As used herein, the terms “natural” or “endogenous” mean polypeptides and / or nucleic acids that are naturally present in certain viruses, particularly negative-strand RNA viruses.

[0095] Those skilled in the art will understand that constructs such as those described herein can be obtained in various ways from wild-type or modified minus-stranded single-stranded RNA viruses. For example, substitution modifications can be obtained by a single recombination event, or by deletion followed by insertion, or by insertion followed by deletion. In the present invention, these modifications may be equivalent.

[0096] Reproduction Possible typeAlternatively, heterologous sequences inserted into replication-deficient negative-strand RNA viruses without altering the viral gene or its function may be sequences that are not essential for replication and / or infection. Therefore, there is a considerable risk that such non-essential heterologous sequences may be lost during viral replication, for example, due to incorrect transcription by RNA polymerase. For instance, during nucleocapsid formation, six nucleotides of RNA may bind to one N protein in each case. Inaccurate transcription may fail to divide the length of the RNA molecule by six, potentially resulting in failure of nucleocapsid formation.

[0097] In the present invention, reproduction Possible type Alternatively, replication-deficient negative-strand RNA viruses may contain genetically stable RNA molecules. As used herein, “genetically stable” refers to nucleic acid molecules or genomes that can replicate and / or reproduce without altering their sequence during replication and / or proliferation. In particular, a nucleic acid molecule is genetically stable if essentially all coding sequences are essential for replication and / or proliferation, i.e., if essentially all coding sequences are required for replication and / or proliferation. Alterations in the base sequence can inhibit the replication and / or proliferation of the altered molecule because essential base sequences may be missing.

[0098] In the present invention, a nucleotide sequence (b) encoding at least one heterologous polypeptide may be essential for viral replication and / or proliferation. The at least one nucleotide sequence (b) encoding at least one heterologous polypeptide can provide the function or activity of at least one native viral polypeptide. In particular, nucleotide sequence (b) exhibits essentially the same function as at least one endogenous viral sequence. In the present invention, the function or activity of at least one endogenous viral sequence can be disrupted at least partially or completely to functionally express the heterologous polypeptide encoded by sequence (b). For example, the sequence encoding such a native viral polypeptide can be deleted at least partially or completely. The heterologous nucleotide sequence can substitute for the function of at least one native sequence, be essential for viral replication and proliferation, and contribute to the genetic stability of the construct. In particular, the heterologous polypeptide encoded by sequence (b) is presented on the surface in a biologically active form. In the present invention, for example, the SARS-CoV-2 S protein exhibits essentially the same function as the sequences encoding SeV F and HN.

[0099] Recombination of the present invention Replicable In type or replication-deficient RNA viruses, the endogenous sequences encoding the F polypeptide and / or the HN polypeptide may be modified such that their function or activity is at least partially or completely disrupted. Nucleotide sequence (b) can be inserted into the locus of the F and / or HN genes. Preferably, nucleotide sequence (b) is inserted into the locus of the HN gene. Even more preferably, nucleotide sequence (b) is inserted into the locus of the F gene.

[0100] In natural SeV viruses, F and HN are directly adjacent. In particular, the sequences encoding the F polypeptide and / or the HN polypeptide may be deleted at least partially or completely. Examples of such constructs are described in the examples ("Type B" construct and "Type C" construct).

[0101] For example, the nucleotide sequence (b) can replace at least one native (endogenous) sequence of the viral genome at least partially or completely. For example, the sequence (b) encoding the SARS-CoV-2S protein can replace at least a portion of the endogenous sequence encoding SeV F and / or HN.

[0102] For example, the sequence encoding the ectodomain of the SARS-CoV-2S protein (b) can be inserted into the sequence encoding SeV F and / or HN, thereby allowing the ectodomain of the SARS-CoV-2S protein to fuse in frame with the F transmembrane domain and / or cytoplasmic domain, as well as the HN transmembrane domain and / or cytoplasmic domain, where the function and / or activity of the native HN and / or F polypeptides are at least partially or completely disrupted.

[0103] The transmembrane domains, cytoplasmic domains, or sequences encoding such domains of F and HN polypeptides used herein may independently include the sequences described herein: Sequence ID 8 describes the amino acid sequence of the transmembrane domain of the SeV F protein (a partial sequence of the SeV F protein in Sequence ID 6). Sequence ID 7 describes the nucleotide sequence that encodes the transmembrane domain of the SeV F protein encoding Sequence ID 8. Sequence ID 10 describes the amino acid sequence of the cytoplasmic domain of the SeV F protein (a partial sequence of the SeV F protein in Sequence ID 6). Sequence ID 9 describes the nucleotide sequence that encodes the cytoplasmic domain of the SeV F protein encoding Sequence ID 10. Sequence ID 16 describes the amino acid sequence of the transmembrane domain of the SeV HN protein (a partial sequence of the SeV HN protein in Sequence ID 12). Sequence ID 15 describes the nucleotide sequence that encodes the transmembrane domain of the SeV HN protein encoding Sequence ID 16. Sequence ID 14 describes the amino acid sequence of the cytoplasmic domain of the SeV HN protein (a partial sequence of the SeV HN protein in Sequence ID 12). Sequence ID 13 describes the nucleotide sequence that encodes the cytoplasmic domain of the SeV HN protein encoding Sequence ID 14. Sequence ID 18 describes the amino acid sequence of the SeV HN protein ectodomain (a partial sequence of the SeV HN protein in Sequence ID 12). Sequence ID 17 describes the nucleotide sequence that encodes the SeV HN protein ectodomain that encodes Sequence ID 18.

[0104] Reproduction Possible type Alternatively, the modified at least one coding sequence of a replication-deficient negative-strand RNA virus may include two or more coding sequences, e.g., two, three, or four coding sequences, each encoding a distinct polypeptide. Preferably, the modified at least one coding sequence includes two coding sequences. For example, the modified at least one coding sequence may include SeV F and HN coding sequences.

[0105] Recombination of the present invention Replicable In type or replication-deficient RNA viruses, the endogenous sequence encoding the HN polypeptide may be modified such that its function or activity is at least partially or completely disrupted. The nucleotide sequence (b) can be inserted into the locus of the HN gene. The endogenous sequence encoding the F polypeptide may not be modified.

[0106] In particular, the sequence encoding the HN polypeptide can be deleted at least partially or completely. More specifically, recombinant Replicable In type or replication-deficient RNA viruses, the RNA molecule does not contain a sequence encoding an HN polypeptide. Examples of such constructs are described in the examples ("Type B" constructs). Recombination of the present invention ReplicableIn type or replication-deficient RNA viruses, the endogenous sequence encoding the HN polypeptide can be deleted at least partially or completely, and the nucleotide sequence (b) can be inserted into the locus of the HN gene.

[0107] Recombination of the present invention Replicable In type or replication-deficient RNA viruses, the endogenous sequence encoding the F polypeptide may be modified such that its function or activity is at least partially or completely disrupted. The nucleotide sequence (b) can be inserted into the locus of the F gene. The endogenous sequence encoding the HN polypeptide may not be modified.

[0108] In particular, the sequence encoding the F polypeptide may be deleted at least partially or completely. More specifically, recombinant Replicable In type or replication-deficient RNA viruses, the RNA molecule does not contain a sequence encoding the F polypeptide. Recombination according to the present invention. Replicable In type or replication-deficient RNA viruses, the endogenous sequence encoding the F polypeptide can be deleted at least partially or completely, and the nucleotide sequence (b) may be inserted into the locus of the F gene.

[0109] Recombination of the present invention Replicable In type or replication-deficient RNA viruses, the endogenous sequences encoding the F polypeptide and the HN polypeptide can be modified such that their function or activity is at least partially or completely disrupted. Nucleotide sequence (b) can be inserted into the loci of the F and HN genes.

[0110] In particular, the sequences encoding F and HN polypeptides can be deleted at least partially or completely. More specifically, recombinant Replicable In type or replication-deficient RNA viruses, the RNA molecule does not contain a sequence encoding an F polypeptide, and the RNA molecule does not contain a sequence encoding an HN polypeptide. Examples of such constructs are described in the Examples ("Type C" constructs). Recombination of the present inventionReplicable In type or replication-deficient RNA viruses, the endogenous sequences encoding the F polypeptide and the HN polypeptide can be deleted at least partially or completely, and the nucleotide sequence (b) can be inserted into the locus of the F gene and / or the HN gene.

[0111] In the present invention, nucleotide sequence (b) may be operatively linked to at least one endogenous expression regulatory sequence. Nucleotide sequence (b) may also include at least one heterologous expression regulatory sequence operatively linked to a heterologous coding sequence. Nucleotide sequence (b) may also be operatively linked to a combination of at least one endogenous viral expression regulatory sequence and at least one heterologous expression regulatory sequence. For example, an endogenous viral promoter sequence can be used. For example, at least one nucleotide sequence (b) may be operatively linked to the endogenous promoter of the F gene when inserted into the F locus. In another example, at least one nucleotide sequence (b) may be operatively linked to the endogenous promoter of the HN gene when inserted into the HN locus.

[0112] Recombinant mode of the present invention Possible The L-type or replication-deficient minus-strand RNA virus may further comprise a heterologous sequence encoding a reporter polypeptide (reporter gene), such as GFP, eGFP, or a derivative thereof, which is a fluorescent protein. The reporter gene may be present solely for the in vitro evaluation of the RNA virus construct described herein. Reporter genes, such as eGFP, may be located downstream of the 3' end of the L gene. The reporter gene has no functional significance to the immunological effect of the RNA virus construct. Recombination of the present invention Replicable When using type or replication-deficient negative-strand RNA viruses for medical purposes, a reporter gene is not essential. Rather, the presence of a reporter gene is necessary for the replication of the present invention. Possible type Alternatively, it may interfere with the medical use of replication-deficient negative-strand RNA viruses. The medical recombinants used herein as described herein ReplicableThe type or replication-deficient negative-strand RNA preferably does not contain a reporter gene. In particular, the medical recombinant RNA used herein is as described herein. ​ Type or replication-deficient negative-strand RNA does not contain the GFP gene or eGFP gene, or sequences derived therefrom.

[0113] In the present invention, the recombinant ​ Negative-type or replication-deficient negative-strand RNA viruses can adsorb to and enter host cells. Recombination of the present invention ​ Negative-type or replication-deficient negative-strand RNA viruses can also infect eukaryotic cells.

[0114] The sequence encoding the HN protein is recombinant according to the present invention. ​ When present in type or replication-deficient negative-strand RNA viruses, adsorption to host cells can be mediated by the HN protein. Recombination of the present invention ​ If a negative-strand RNA virus of type or replication-deficient type contains a sequence encoding the F protein, entry into cells can be initiated by the F protein. Recombination of the present invention ​ If a sequence encoding the SARS-CoV-2S protein is present in a type or replication-deficient negative-strand RNA virus, adsorption to and entry into host cells can be mediated by the SARS-CoV-2S protein.

[0115] Recombination of the present invention ​ In type 1 or replication-deficient negative-strand RNA viruses, heterologous polypeptides, when presented on the viral surface, can mediate adsorption to and entry into host cells. In particular, the heterologous protein may be the SARS-CoV-2S protein, or it may be a chimeric protein, as described herein.

[0116] In particular, the host cell is a eukaryotic cell. The host cell can be any host cell capable of infecting with negative-strand RNA viruses. Those skilled in the art know suitable host cells. Suitable host cells are described herein.

[0117] The recombinant replication-deficient negative-strand RNA virus of the present invention can be rescued from eukaryotic cells.

[0118] The recombinant replication-deficient negative-strand RNA virus of the present invention may be able to proliferate in eukaryotic cells that are trans-complemented with the deletion sequence of the replication-deficient negative-strand RNA virus, particularly with a sequence encoding a P polypeptide. The cells may also be trans-complemented with sequences encoding N and / or L polypeptides. In particular, the recombinant replication-deficient negative-strand RNA virus of the present invention may be able to proliferate in eukaryotic cells that are trans-complemented with (i) a P polypeptide and (ii) sequences encoding N and L polypeptides.

[0119] Recombination of the present invention ​ In type 1 or replication-deficient minus-strand RNA viruses, the heterologous polypeptide encoded by at least one nucleotide sequence (b) may consist of a spike polypeptide, particularly a fragment of the SARS-CoV-2 spike polypeptide. The SARS-CoV-2 S protein may also be a chimeric protein. In particular, the heterologous polypeptide encoded by at least one nucleotide sequence (b) may include the ectodomain of the SARS-CoV-2 spike polypeptide.

[0120] The heterologous polypeptide may be a chimeric SARS-CoV-2 spike polypeptide. Recombination of the present invention ​ In type 1 or replication-deficient negative-strand RNA viruses, the nucleotide sequence (b) is: (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) A transmembrane domain of SeV F or HN polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of SeV F or HN polypeptide, or a fragment thereof. It can be coded.

[0121] The nucleotide sequence (b) of this construct may include a partial sequence of the transmembrane domain of the SARS-CoV-2 spike polypeptide.

[0122] In this construct, nucleotide sequence (b) can operatively bind to endogenous SeV expression regulatory sequences, particularly SeV F or HN expression regulatory sequences such as SeV F or HN promoters.

[0123] Such structures are referred to as “Variant 1 structures” in this specification. Examples of Variant 1 structures are provided in the Examples.

[0124] In particular, in the variant 1 construct, the nucleotide sequence (b) is: (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) A transmembrane domain of a SeV F polypeptide, or a fragment thereof, and (iii) Cytoplasmic domain of SeV F polypeptide, or fragment thereof It can be coded.

[0125] The nucleotide sequence (b) of this construct may include a partial sequence of the transmembrane domain of the SARS-CoV-2 spike polypeptide.

[0126] In particular, nucleotide sequence (b) may include SEQ ID NO: 27 or 30. The heterologous polypeptide encoded by nucleotide sequence (b) may include SEQ ID NO: 31. The ectodomain of the SARS-CoV-2 spike polypeptide may be encoded by SEQ ID NO: 21 or 28. The transmembrane and cytoplasmic domains of SeV F may be encoded by SEQ ID NO: 29. The amino acid sequence of the ectodomain of the SARS-CoV-2 spike polypeptide may include SEQ ID NO: 22 or 32. The transmembrane and cytoplasmic domains of SeV F may include SEQ ID NO: 33.

[0127] In this construct, the nucleotide sequence (b) can be operably linked to an endogenous SeV expression regulatory sequence, particularly a SeV F expression regulatory sequence such as the SeV F promoter.

[0128] In particular, in the variant 1 construct, the nucleotide sequence (b) is also, (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) Transmembrane domain of SeV HN polypeptide, or a fragment thereof, and (iii) Cytoplasmic domain of SeV HN polypeptide, or fragment thereof It can be coded.

[0129] The nucleotide sequence (b) of this construct may include a partial sequence of the transmembrane domain of the SARS-CoV-2 spike polypeptide.

[0130] In this construct, nucleotide sequence (b) can be operably linked to an endogenous SeV expression regulatory sequence, particularly a SeV HN expression regulatory sequence, such as a SeV HN promoter.

[0131] As used herein, the term “fragment” includes a partial sequence of a polynucleotide or polypeptide. A fragment may have essentially the same activity and / or function as the full-length polynucleotide or polypeptide. A fragment has at least 80%, preferably at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the full-length polynucleotide or polypeptide.

[0132] In particular, “S protein fragments” or “SARS-CoV-2 S protein fragments” include polypeptides containing a partial sequence of the full-length S protein. These fragments may have essentially the same activity and / or function as the full-length S protein. Specifically, the fragments have at least 80%, preferably at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the full-length S protein. Examples of S protein fragments include: (i) Partial sequences of the ectodomain and transmembrane domain of the SARS-CoV-2S protein, (ii) The ectodomain and transmembrane domain of the SARS-CoV-2 S protein, (iii) partial sequences of the ectodomain, transmembrane domain, and cytoplasmic domain of the SARS-CoV-2S protein, or / and (iv) The ectodomain, transmembrane domain, and cytoplasmic domain of the SARS-CoV-2S protein, wherein 19 C-terminal amino acid residues of the cytoplasmic domain are deleted.

[0133] Preferably, in these fragments of the SARS-CoV-2S polypeptide, the domains are directly adjacent.

[0134] SARS-CoV-2 spike polypeptide ​ This is encoded by nucleotides (nt) 1-3,639 of SEQ ID NO: 19, or by SEQ ID NO: 21 or 28. The SARS-CoV-2 spike polypeptide ​ This can be encoded by nt 3,640-3,702 of SEQ ID NO: 19, or by SEQ ID NO: 23, or SEQ ID NO: 40. The cytoplasmic domain of the SARS-CoV-2 spike polypeptide can be encoded by nt 3,703-3,819 of SEQ ID NO: 19, or by SEQ ID NO: 25, or by SEQ ID NO: 42. Positions 2820-2822 of SEQ ID NO: 19 describe a stop codon.

[0135] SARS-CoV-2 spike polypeptide ​ This may contain amino acid residues (aa)1-1,213 of SEQ ID NO: 20, or SEQ ID NO: 22. SARS-CoV-2 spike polypeptide ​ This may include aa1,214~1,234 of SEQ ID NO: 20, or SEQ ID NO: 24, or SEQ ID NO: 41. SARS-CoV-2 spike polypeptide ​ This may include aa1,235~1,273 of sequence number 20, or sequence number 26, or sequence number 43.

[0136] This fragment is, in particular, an immunogenic fragment, i.e., a polypeptide capable of inducing an immune response in a target. Certain fragments of the S protein, or fragments of the SARS-CoV-2 S protein, may be immunogenic fragments.

[0137] Recombination of the present invention ​ In type or replication-deficient negative-strand RNA viruses, the nucleotide sequence (b) is (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of SeV F or HN polypeptide, or a fragment thereof. It can be coded.

[0138] The nucleotide sequence (b) of this construct may contain a partial sequence of the cytoplasmic domain of the SARS-CoV-2 spike polypeptide.

[0139] In this construct, nucleotide sequence (b) may be operetically linked to an endogenous SeV expression regulatory sequence, particularly a SeV F or HN expression regulatory sequence, such as a SeV F or HN promoter.

[0140] Such structures are referred to herein as “Variant 2 structures.” Examples of Variant 2 structures are described in the Examples.

[0141] In particular, in mutant 2 construct, the nucleotide sequence (b) is, (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) Cytoplasmic domain of SeV F polypeptide, or fragment thereof It can be coded.

[0142] The nucleotide sequence (b) of this construct may include a subsequence of the cytoplasmic domain of the SARS-CoV-2 spike polypeptide. In particular, the nucleotide sequence (b) may include SEQ ID NOs. 36, 44, or 46. The heterologous polypeptide encoded by the nucleotide sequence (b) may include SEQ ID NOs. 37, 45, or 47, which are encoded by SEQ ID NOs. 36, 44, or 46, respectively. The ectodomain of SARS-CoV-2 is encoded by SEQ ID NOs. 21, 28, or 38. The ectodomain of SARS-CoV-2 may include SEQ ID NOs. 22, 32, or 39.

[0143] In this construct, the nucleotide sequence (b) may be operatively linked to an endogenous SeV expression regulatory sequence, particularly a SeV F expression regulatory sequence such as the SeV F promoter.

[0144] In particular, in the variant 2 construct, the nucleotide sequence (b) is also, (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) Cytoplasmic domain of SeV HN polypeptide, or fragment thereof It can be coded.

[0145] The nucleotide sequence (b) of this construct may contain a partial sequence of the cytoplasmic domain of the SARS-CoV-2 spike polypeptide.

[0146] In this construct, nucleotide sequence (b) can operatively contact endogenous SeV expression regulatory sequences, particularly SeV HN expression regulatory sequences such as the SeV HN promoter.

[0147] Recombination of the present invention ​ In type 1 or replication-deficient negative-strand RNA viruses, the nucleotide sequence (b) is: (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof. It can be coded.

[0148] Such structures are referred to herein as “Variant 3 structures.” Examples of Variant 3 structures are provided in the examples.

[0149] In this construct, nucleotide sequence (b) can be operetically linked to an endogenous SeV expression regulatory sequence, particularly a SeV F or HN expression regulatory sequence, such as a SeV F or HN promoter.

[0150] In this invention, the term “anti-vector antigenicity” as used herein refers to the ability of a vector to induce an immune response to the endogenous components of the vector when exposed to the host’s (i.e., the subject to which the vector is administered) immune system. Anti-vector antigenicity induces the host’s immune response to the vector, i.e., anti-vector immunity. In negative-strand RNA viruses, surface proteins such as F and / or HN polypeptides are particularly exposed to the host’s immune system. At least partial or complete deletion of the sequence encoding the surface polypeptide can reduce anti-vector antigenicity.

[0151] In the present invention, recombinant ​ Negative-type or replication-deficient negative-strand RNA viruses may have reduced anti-vector antigenicity compared to the wild type. Recombination of the present invention ​ In type or replication-deficient negative-strand RNA viruses, a reduction in antivector antigenicity can be achieved by at least partial or complete deletion of the viral surface polypeptide, particularly the nucleotide sequences encoding F and / or HN, as described herein. For example, the recombinant RNA of the present invention ​ Negative-type or replication-deficient negative-strand RNA virus particles do not necessarily have to contain endogenous (natural) surface polypeptides on their surface. Recombination of the present invention ​ Type or replication-deficient negative-strand RNA virus particles can present at least one heterologous polypeptide, such as the S protein described herein, on their surface. In these constructs, the function and / or activity of the F and / or HN polypeptides can be complemented by the S protein, in particular the SARS-CoV-2 S protein.

[0152] Recombination of the present invention ​ In type 1 or replication-deficient negative-strand RNA viruses, the ectodomain of the SARS-CoV-2 spike polypeptide may include an S1 subunit, the S1 subunit including a receptor-binding domain (RBD) capable of binding to the ACE-2 receptor of a host cell.

[0153] Recombination of the present invention ​In type 1 or replication-deficient negative-strand RNA viruses, the S1 subunit can be cleaved by the host cell's ACE-2 protease.

[0154] Recombination of the present invention ​ In type 1 or replication-deficient negative-strand RNA viruses, the ectodomain of the SARS-CoV-2 spike polypeptide may contain an S2 subunit that can fuse the SeV envelope to the host cell membrane or endosomal membrane.

[0155] Recombination of the present invention ​ In type 1 or replication-deficient minus-strand RNA viruses, the sequence of the ectodomain of the S protein may contain at least one sequence modification that can stabilize the ectodomain in a prefusion form. For example, the S protein or its ectodomain may be provided in a stabilized prefusion form containing two consecutive proline substitutions (K986P and V987P) at residues K986 and V987. In the prefusion form, the S protein cannot mediate the fusion of the cell membrane / endosomal membrane and the viral envelope after receptor binding. A sequence encoding the F protein may be present to mediate the fusion of the viral envelope and the cell / endosomal membrane.

[0156] Recombination of the present invention ​ Negative-type or replication-deficient negative-strand RNA viruses, when administered, for example, via intranasal and / or mucosal administration routes, can induce a mucosal immune response to at least one heterologous polypeptide encoded by at least one nucleotide sequence (b). Recombination of the present invention ​ Negative-type or replication-deficient negative-strand RNA viruses, particularly the SeV vectors of the present invention as described herein, are capable of readily penetrating the mucin layer of the respiratory tract and thus provide ideal conditions for delivering at least one heterologous polypeptide via intranasal and / or mucosal administration routes, for example, in the form of an easily handled and user-friendly intranasal / mucosal spray vaccine.

[0157] Recombination of the present invention​ Since type or replication-deficient negative-strand RNA viruses can present at least one heterologous polypeptide on their surface, the mucosal immune response may be an immediate response.

[0158] Recombination of the present invention ​ Negative-type or replication-deficient negative-strand RNA viruses can induce a protective immune response against pathogens having polypeptide antigens encoded by at least one nucleotide sequence (b), particularly SARS-CoV-2. Protective immunity is ​ This could be sterilizing immunity. The term used herein is “ ​ "Immunity" refers to the immune response to a pathogen, particularly the SARS-CoV-2 virus, which prevents the pathogen from infecting other uninfected targets.

[0159] Recombination of the present invention ​ Negative-type or replication-deficient negative-strand RNA viruses can be directly presented to the mucosal immune system, particularly during intranasal / mucosal application, as in natural respiratory infection with SARS-CoV-2. Furthermore, upon entering host mucosal cells, the virus can produce and present the SARS-CoV-2 spike protein on the cell surface. In this way, broad protection, particularly bactericidal immunity, is achieved and ensured.

[0160] The P protein used herein may be encoded by the nucleotide sequence of Sequence ID No. 1, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0161] The P protein used herein may include the amino acid sequence of Sequence ID No. 2, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0162] As used herein, the delta-P2-77 protein may be encoded by the nucleotide sequence of SEQ ID NO: 3, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0163] The delta-p 2-77 protein used herein may contain the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0164] The SeV F protein used herein may be encoded by the nucleotide sequence of Sequence ID No. 5, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0165] The SeV F protein used herein may include the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0166] The SeV F protein transmembrane domains used herein can be encoded by the nucleotide sequence of Sequence ID No. 7, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0167] The SeV F transmembrane domains used herein may include the amino acid sequence of Sequence ID No. 8, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0168] The SeV F protein cytoplasmic domain used herein may be encoded by the nucleotide sequence of Sequence ID No. 9, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0169] The SeV F protein cytoplasmic domains used herein may include the amino acid sequence of Sequence ID No. 10, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0170] The SeV HN proteins used herein may be encoded by the nucleotide sequence of Sequence ID No. 11, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0171] The SeV HN proteins used herein may include the amino acid sequence of Sequence ID No. 12, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0172] The SeV HN cytoplasmic domains used herein may be encoded by the nucleotide sequence of Sequence ID No. 13, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0173] The SeV HN cytoplasmic domains used herein may consist of the amino acid sequence of Sequence ID No. 14, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0174] The SeV HN transmembrane domains used herein may be encoded by the nucleotide sequence of Sequence ID No. 15, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0175] The SeV HN transmembrane domains used herein may consist of the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0176] The SeV HN ectodomains used herein may be encoded by the nucleotide sequence of Sequence ID No. 17, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0177] The SeV HN ectodomains used herein may include the amino acid sequence of Sequence ID No. 18, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0178] The SARS-CoV-2 spike (S) protein used herein may be encoded by the nucleotide sequence of Sequence ID No. 19, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0179] The SARS-CoV-2 spike (S) protein used herein may include the amino acid sequence of Sequence ID No. 20, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0180] The SARS-CoV-2 spike (S) ectodomain used herein may be encoded by the nucleotide sequence of Sequence ID No. 21, 28, or 38, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0181] The SARS-CoV-2 spike (S) ectodomains used herein comprise the amino acid sequence of SEQ ID NO: 22, 32, or 39, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0182] The SARS-CoV-2 spike (S) transmembrane domain used herein may be encoded by the nucleotide sequence of Sequence ID No. 23 or 40, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0183] The SARS-CoV-2 spike (S) transmembrane domains used herein may include the amino acid sequence of Sequence ID No. 24 or 41, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0184] The SARS-CoV-2 spike (S) cytoplasmic domain used herein may be encoded by the nucleotide sequence of Sequence ID No. 25 or 42, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0185] The SARS-CoV-2 spike (S) cytoplasmic domains used herein comprise the amino acid sequence of Sequence ID No. 26 or 43, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0186] The chimeric protein (mutant 1 construct) used herein, comprising a SARS-CoV-2 spike ectodomain, a SeV F transmembrane domain, and a SeV cytoplasmic domain, may be encoded by the nucleotide sequence of SEQ ID NO: 27, 30, or 34, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0187] The chimeric protein (mutant 1 construct) used herein, comprising a SARS-CoV-2 spike ectodomain, an SeV F transmembrane domain, and an SeV cytoplasmic domain, may contain the amino acid sequence of SEQ ID NO: 31 or 35, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0188] The SeV F transmembrane domain and SeV cytoplasmic domain used herein may be encoded by the nucleotide sequence of Sequence ID No. 29, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0189] The SeV F transmembrane domain and SeV cytoplasmic domain used herein may consist of the amino acid sequence of Sequence ID No. 33, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0190] The chimeric protein (mutant 2 construct) used herein, comprising the SARS-CoV-2 spike ecto, transmembrane domain and cytoplasmic domain (with 19 C-terminal amino acid residues deleted from the cytoplasmic domain), and the SeV F cytoplasmic domain, may be encoded by the nucleotide sequence of SEQ ID NOs. 36, 44, or 46, or by a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0191] The chimeric proteins used herein, comprising the SARS-CoV-2 spike ecto, transmembrane domain and cytoplasmic domain (with 19 C-terminal amino acid residues deleted from the cytoplasmic domain), and the SeV F cytoplasmic domain (mutant 2 construct), may contain the amino acid sequence of SEQ ID NOs. 37, 45, or 47, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0192] Another aspect of the present invention comprises a nucleocapsid and an envelope, wherein the nucleocapsid is present in its genome. (a) A nucleotide sequence encoding a SeV P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in a replication defect of the negative-strand RNA virus, and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. This relates to recombinant replication-deficient single-stranded negative-sense RNA virus particles, including those containing such particles.

[0193] A nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type (a), and at least one nucleotide sequence encoding at least one heterologous polypeptide (b) are described herein in the context of replication-deficient negative-strand RNA viruses.

[0194] Another aspect of the present invention comprises a nucleocapsid and an envelope, wherein the nucleocapsid is present in its genome. (a) A nucleotide sequence encoding the SeV P polypeptide, wherein the P protein performs its intended function particularly in the viral replication cycle. ​ nucleotide sequence (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Recombinant replication, including ​ Regarding single-stranded negative-sense RNA virus particles.

[0195] In particular, recombinant replication ​ Single-stranded negative-sense RNA virus particles are weakened.

[0196] In particular, this virus is the Sendai virus.

[0197] In particular, at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope presents the SARS-CoV-2 spike polypeptide or a fragment thereof on the surface of the virus particle.

[0198] A further aspect of the present invention relates to a recombinant replication-deficient Sendai virus whose genome contains the following: (a) A nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in a replication defect of the negative-strand RNA virus, and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein at least one heterologous polypeptide is presented on the surface of a Sendai virus particle.

[0199] A nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type (a), and at least one nucleotide sequence encoding at least one heterologous polypeptide (b) are described herein in the context of replication-deficient negative-strand RNA viruses.

[0200] In particular, in this embodiment, at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope presents the SARS-CoV-2 spike polypeptide or a fragment thereof on the surface of the Sendai virus particle.

[0201] Another aspect of the present invention is a recombinant genome containing the following: ​ Regarding type Sendai virus: (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein performs its intended function, particularly in the viral replication cycle. ​ nucleotide sequence (b) A nucleotide sequence comprising at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the viral particle.

[0202] In particular, duplication ​ The Sendai virus particles have been weakened.

[0203] In particular, in this embodiment, at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope presents the SARS-CoV-2 spike polypeptide or a fragment thereof on the surface of the Sendai virus particle.

[0204] Yet another aspect of the present invention comprises a nucleocapsid and an envelope, wherein the nucleocapsid is present in its genome. (a) A nucleotide sequence encoding a SeV P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in a Sendai virus replication defect. (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. This relates to recombinant replication-deficient Sendai virus particles, including those containing such particles.

[0205] A nucleotide sequence (a) encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and at least one nucleotide sequence (b) encoding at least one heterologous polypeptide are described herein in the context of replication-deficient minus-strand RNA viruses.

[0206] In particular, in this embodiment, at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope is formed by the SARS-CoV-2 spike polypeptide or a fragment thereof being presented on the surface of a Sendai virus particle.

[0207] Yet another aspect of the present invention comprises a nucleocapsid and an envelope, wherein the nucleocapsid is present in its genome. (a) A nucleotide sequence encoding a SeV P polypeptide, wherein the P protein performs its intended function particularly in the viral replication cycle. ​ The nucleotide sequence and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Recombinant ​ Regarding type Sendai virus particles.

[0208] In particular, duplication ​The Sendai virus has been weakened.

[0209] In particular, in this embodiment, at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope presents the SARS-CoV-2 spike polypeptide or a fragment thereof on the surface of the Sendai virus particle.

[0210] A further aspect of the present invention relates to a recombinant replication-deficient Sendai virus (SeV) particle comprising a nucleocapsid and an envelope, wherein the nucleocapsid contains in its genome, (a) A nucleotide sequence encoding a SeV P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in a Sendai virus replication defect. (b) At least one nucleotide sequence encoding the SARS-CoV-2 spike polypeptide or a fragment thereof This includes, where the SARS-CoV-2 spike polypeptide or a fragment thereof is presented on the surface of the virus particle. Regarding recombinant replication-deficient Sendai virus (SeV) particles.

[0211] Yet another aspect of the present invention relates to recombinants comprising a nucleocapsid and an envelope. ​ With respect to type Sendai virus (SeV) particles, the nucleocapsid is contained in its genome. (a) A nucleotide sequence encoding the SeV P polypeptide, wherein the P protein performs its intended function particularly in the viral replication cycle. ​ The nucleotide sequence and (b) At least one nucleotide sequence encoding the SARS-CoV-2 spike polypeptide or a fragment thereof, This includes, where the SARS-CoV-2 spike polypeptide or a fragment thereof is presented on the surface of the virus particle. Regarding recombinant replication-deficient Sendai virus (SeV) particles.

[0212] In particular, duplication ​ The Sendai virus has been weakened.

[0213] A further aspect of the present invention relates to the medical use of the recombinant replication-deficient single-stranded negative-sense RNA virus of the present invention. As used herein, the term “virus” includes RNA molecules, nucleocapsids, and viral particles capable of infecting new host cells. In these embodiments, the recombinant replication-deficient negative-sense RNA virus of the present invention is preferably provided in the form of viral particles.

[0214] One aspect of the present invention relates to recombinant replication defects or replication defects as described herein. ​ Type negative-strand RNA viruses, and / or recombinant replication defects or replications as described herein. ​ This relates to a pharmaceutical composition containing negative-strand RNA virus particles.

[0215] In particular, pharmaceutical compositions are recombinant replication defects or replication defects as described herein. ​ Type Sendai virus, and / or recombinant replication defects or replications as described herein. ​ Contains Sendai virus particles.

[0216] Further aspects of the present invention include recombinant replication defects or replication defects as described herein. ​ Type negative-strand RNA viruses, and / or recombinant replication defects or replications as described herein. ​ This concerns vaccines containing negative-strand RNA virus particles. The vaccine is an anti-SARS-CoV-2 vaccine, or a vaccine for the prevention of COVID-19.

[0217] Further aspects of the present invention include recombinant replication defects or replication defects as described herein. ​ Type negative-strand RNA viruses, and / or recombinant replication defects or replications as described herein. ​This invention relates to an immunogenic composition containing negative-strand RNA virus particles. The immunogenic composition may induce an immune response to SARS-CoV-2.

[0218] A further aspect of the present invention relates to a recombinant replication-deficient or replication-deficient type described herein for medical use. ​ Type negative-strand RNA virus, recombinant replication-deficient type or replication-deficient type as described herein. ​ This relates to negative-strand RNA virus particles, the pharmaceutical composition described herein, the vaccine described herein, or the immunogenic composition described herein.

[0219] In particular, the present invention relates to recombinant replication-deficient or replication-deficient variants described herein for use in methods for preventing SARS-CoV-2 infection. ​ Type negative-strand RNA virus, recombinant replication-deficient type or replication-deficient type as described herein. ​ This relates to negative-strand RNA virus particles, the pharmaceutical composition described herein, the vaccine described herein, or the immunogenic composition described herein.

[0220] A further aspect of the present invention relates to recombinant replication-deficient or replication-deficient types described herein for use in methods for preventing COVID-19. ​ Type negative-strand RNA virus, recombinant replication-deficient type or replication-deficient type as described herein. ​ This relates to negative-strand RNA virus particles, the pharmaceutical composition described herein, the vaccine described herein, or the immunogenic composition described herein.

[0221] Yet another aspect of the present invention is the recombinant replication-deficient or replication-deficient type described herein, for use as a vaccine. ​ Type negative-strand RNA virus, recombinant replication-deficient type or replication-deficient type as described herein. ​ This specification relates to negative-strand RNA virus particles, the pharmaceutical compositions described herein, the vaccines described herein, or the immunogenic compositions described herein. The vaccines are anti-SARS-CoV-2 vaccines or vaccines for the prevention of COVID-19.

[0222] Recombinant replication-deficient type or replication ​ Recombinant replication-deficient negative-strand RNA virus, recombinant replication-deficient or replication-deficient ​ Recombinant replication-deficient negative-strand RNA virus particles, pharmaceutical compositions, vaccines, or immunogenic compositions may be administered to subjects requiring them via the nasal cavity and / or mucosal route.

[0223] A further aspect of the present invention relates to an RNA molecule encoding a recombinant replication-deficient minus-strand RNA virus as described herein. The RNA molecule may be an isolated RNA molecule. In particular, the RNA molecule is (a) A nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide Includes.

[0224] A nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type (a), and at least one nucleotide sequence encoding at least one heterologous polypeptide (b) are described herein in the context of replication-deficient negative-strand RNA viruses.

[0225] Further aspects of the present invention are described herein, recombinant ​ This relates to RNA molecules encoding negative-strand RNA viruses. The RNA molecules may be isolated RNA molecules. In particular, the RNA molecules are... (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein performs its intended function particularly in the viral replication cycle. ​ The nucleotide sequence, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, Includes.

[0226] In particular, duplication​ The negative-strand RNA virus is attenuated.

[0227] In particular, the RNA molecule (c) a nucleotide sequence encoding an N polypeptide, (d) a nucleotide sequence encoding an M polypeptide, and (e) a nucleotide sequence encoding an L polypeptide further comprises.

[0228] The nucleotide sequences (c), (d) and (e) are described herein in the context of a replication ​ competent or replication-deficient negative-strand RNA virus.

[0229] Yet another aspect of the invention is a nucleocapsid comprising the RNA molecule of the invention.

[0230] The nucleocapsid may consist of a complex of single-stranded RNA and nucleoprotein (N). In particular in each case, 6 nucleotides of the RNA may bind to one N protein. The nucleocapsid may also contain an RNA-dependent RNA polymerase (L) and a cofactor phosphorylated protein (P) that form an RNA polymerase complex.

[0231] Yet another aspect of the invention is a DNA molecule encoding the RNA molecule of the invention. The DNA molecule may be an isolated DNA molecule. In particular, the DNA molecule may be a cDNA molecule. The cDNA can constitute the genome of a replication ​ competent or replication-deficient negative-strand RNA virus described herein. This cDNA can form virus particles in a suitable helper cell. The cDNA can be provided in the form of a plasmid such as an expression plasmid that is transcribable into an RNA molecule encoding a replication ​ competent or replication-deficient negative-strand RNA virus described herein.

[0232] Yet another aspect of the invention is a replication ​Type or replication-deficient negative-strand RNA virus, recombinant as described herein ​ A host cell comprising a negative-strand RNA virus particle of type or replication-deficient type, an RNA molecule as described herein, a nucleocapsid as described herein, and / or a DNA molecule as described herein. In particular, the host cell is a eukaryotic cell. The host cell may be any host cell capable of infecting with a negative-strand RNA virus. Those skilled in the art know suitable host cells.

[0233] For example, (i) Vero cells or cells derived therefrom that have been transfected with a nucleic acid molecule transcoding at least the SeV P protein, such as V3-10 helper cells. In particular, the cells may be further transfected with (ii) at least one nucleic acid molecule transcoding the native viral proteins N and / or L.

[0234] Host cells, particularly V3-10 helper cells, can replicate the recombinant replication-deficient negative-strand RNA virus of the present invention. Replication includes the proliferation of the replication-deficient negative-strand RNA virus of the present invention. Examples of such eukaryotic cells capable of replicating recombinant replication-deficient negative-strand RNA are cells trans-complemented with the deletion sequence of the replication-deficient negative-strand RNA virus, particularly cells trans-complemented with a nucleic acid molecule encoding a (i)P polypeptide. Cells can further be transfected with at least one nucleic acid molecule encoding an N and / or L polypeptide. An example is the V3-10 helper cells described herein.

[0235] Host cells, particularly V3-10 helper cells, can also rescue the replication-deficient negative-strand RNA virus of the present invention after being transfected with a nucleic acid molecule encoding the recombinant replication-deficient negative-strand RNA virus of the present invention, such as the DNA molecule described herein. In particular, host cells capable of virus rescue are not transcomplemented with an exogenous sequence encoding the deficient viral protein.

[0236] Recombinant organisms as described herein ​ Negative-type RNA viruses can replicate within host cells without requiring transcomplementation. Vero cells are an example of such cells.

[0237] The host cell may be a GMP-compliant eukaryotic cell. Suitable GMP-compliant eukaryotic cells are known to those skilled in the art. One example is the Vero cell.

[0238] The host cell also replicates the present invention. ​ These may be prokaryotic cells, such as bacteria, that are suitable for maintaining and growing the DNA molecules described herein, encoding type or replication-deficient negative-strand RNA viruses. Suitable prokaryotic cells are known to those skilled in the art.

[0239] Further embodiments of methods for preventing infection with SARS-CoV-2 and / or COVID-19 are recombinant methods described herein. ​ Type or replication-deficient negative-strand RNA virus, recombinant as described herein ​ This includes administering a negative-strand RNA virus particle of type or replication-deficient type, a pharmaceutical composition as described herein, a vaccine as described herein, and / or an immunogenic composition as described herein to a subject requiring such treatment.

[0240] Further aspects of the present invention include pharmaceutical compositions as described herein, and recombinants as described herein. ​ Type or missing copy ​ This invention relates to negative-strand RNA virus particles, the vaccine described herein, or a method for producing the immunogenic composition described herein, or an immunogenic composition described herein, wherein the recombinant RNA virus particle described herein is used. ​ The process involves formulating a type or replication-deficient negative-strand RNA virus, an RNA molecule described herein, or / or a nucleocapsid together with at least one pharmaceutically acceptable excipient.

[0241] Yet another aspect of the present invention is a method for producing recombinant replication-deficient negative-strand RNA viruses as described herein.

[0242] The method for producing a recombinant replication-defective negative-strand RNA virus or virus particles described in this specification may include the following steps: (a) Transfecting a eukaryotic host cell with a DNA molecule encoding the RNA molecule of the present invention, wherein the host cell can express SeV polypeptides N, P, and L, (b) Culturing the host cell under conditions such that the DNA molecule is transcribed and virus particles are formed, and (c) Separating the virus particles of (b).

[0243] The method for producing a recombinant replication-defective negative-strand RNA virus or virus particles described in this specification may also include the following steps: (a) Infecting a eukaryotic host cell with a wild-type negative-strand RNA virus and transfecting the eukaryotic host cell with a DNA molecule described in this specification encoding the recombinant replication-defective RNA virus of the present invention, (b) Culturing the host cell under conditions such that the DNA molecule is transcribed and virus particles are formed, and (c) Separating the virus particles of (b), wherein the wild-type negative-strand RNA virus and the recombinant replication-defective RNA virus may be derived from the same species.

[0244] Yet another aspect of the present invention is the recombinant ​ method for producing a negative-strand RNA virus or virus particles as described in this specification, the method comprising: (a) Transfecting a eukaryotic host cell with a DNA molecule described in this specification, (b) Culturing the host cell under conditions such that the DNA molecule is transcribed and virus particles are formed, and (c) Separating the virus particles of (b).

[0245] Yet another aspect of the present invention is a recombinant ​ type or replication-defective negative-strand RNA virus and / or recombinant ​This relates to the use of the host cells described herein for the production of type or replication-deficient negative-strand RNA virus particles.

[0246] Further aspects of the present invention relate to recombinants described herein for the manufacture of pharmaceuticals for preventing infection with SARS-CoV-2 and / or COVID-19. ​ Regarding the use of type or replication-deficient negative-strand RNA viruses.

[0247] TIFF2023227758000001.tif223157 TIFF2023227758000002.tif234157 TIFF2023227758000003.tif234154

[0248] The present invention will be described in detail and illustrated by figures and examples, which are for illustrative purposes only and are not intended to limit it. Further embodiments also included in the present invention are available to those skilled in the art through this specification and examples. [Brief explanation of the drawing]

[0249] ​ Figure 1: Rescue of recombinant SeV in the RNA Pol II helper system. ​ Figure 2: SeV-based candidate cDNA constructs for SARS-CoV-2. ​ Figure 3: Immunofluorescence staining of Vero cells transfected with eukaryotic expression plasmids encoding the nucleotide sequences of SARS-CoV-2 S1V1 or SARS-CoV-2 S2V1 revealed that both mutants were successfully expressed. ​ Figure 4: The SeV85E-1V2 vaccine vector genome (encoding the SARS-CoV-2 S protein as a surface protein instead of SeV proteins F and HN) was successfully rescued in Vero cells. ​Figure 5: The SeV85E-2V1 vaccine vector genome (encoding SARS-CoV-2S protein instead of SeV protein F and HN as surface proteins) was successfully rescued in Vero cells. ​ Figure 6: The SeV85E-1V2 vaccine vector genome (encoding SARS-CoV-2S protein instead of SeV protein F and HN as surface proteins) successfully rescued V3-10 helper cells. ​ Figure 7: The SeV85E-2V1 vaccine vector genome (encoding SARS-CoV-2S protein instead of SeV protein F and HN as surface proteins) successfully rescued V3-10 helper cells. ​ Figure 8: The SeV85E-1V2 vaccine (encoding the SARS-CoV-2S protein instead of SeV protein F and HN as surface proteins) successfully proliferated in V3-10 helper cells, as reflected by the time-dependent increase in fluorescence indicating viral spread during the observation period. ​ Figure 9: The SeV85E-1V2 vaccine (encoding SARS-CoV-2S protein instead of SeV protein F and HN as surface proteins) successfully proliferated in V3-10 helper cells for passages 2-4 at the specified MOI. ​Figure 10: Immunoblots of SeV85E-1V2 virus particles (purified by ultracentrifugation (UC)) showed expression of the SARS-CoV-2S protein, indicating that the SARS-CoV-2S protein was directly incorporated into the SeV85E-1V2 virus particles. Purified virus particles of SeV85E-1V2 p1 and SeV85E p1 (a control virus that does not encode the chimeric SARS-CoV-2 protein) were separated by SDS-PAGE under reducing conditions (+β-ME) or non-reducing conditions (-β-ME). The expression of the virus-encoded proteins SARS-CoV-2S and EGFP was analyzed by immunoblotting. Two antibodies (Sigma, GeneTex) were used to detect the SARS-CoV-2S protein. ​ Figure 11: Dose-dependent IgA levels in nasal lavage fluid (A) and bronchoalveolar lavage fluid (B) from mice immunized with SeV88-1V2. [Examples]

[0250] Example 1 The examples describe a second-generation SARS-CoV-2 vaccine. This vaccine contains a Sendai virus vector with the SARS-CoV-2 antigen on its surface.

[0251] Quality and safety The candidate vaccine includes the quality and safety characteristics of the Sendai virus (SeV) vector described in WO 2006 / 084746: A key safety feature is that it partially cleaves the SeV viral RNA polymerase complex protein, achieving complete replication loss. Therefore, uncontrolled vector transmission in vaccinated subjects can be completely eliminated. Infection in subjects treated with SeV-based vaccines is completely blocked. ●SeV vectors are characterized by their genetic stability against recombination, and undesirable properties (e.g., transgenicity) are not introduced into the vector. ● The SeV vector's genuine ability to easily penetrate the mucin layer in the respiratory tract provides ideal conditions for delivering the gene-transfer antigen via the nasal / mucosal administration route in the form of an easy-to-handle and user-friendly nasal / mucosal spray vaccine.

[0252] Virus rescue and propagation For the primary production of SeV vectors (the so-called SeV rescue process), a system supported by intracellular RNA polymerase II has been established to provide flexibility in the selection of rescue cells and allow the use of cells already approved for vaccine production. This reduces cumbersome testing procedures and alleviates the burden on regulatory authorities.

[0253] WO 2006 / 084746 describes SeV rescue and SeV transmission in different cell lines. In this example, the production system for the SeV vaccine candidate was modified so that the same cells could be used for both SeV rescue and subsequent proliferation. This significantly simplifies the production process for the SeV vaccine candidate.

[0254] SARS-CoV-2 vaccine candidate To achieve an efficient second-generation anti-SARS-CoV-2 vaccine, the inventors did the following: (i) The SARS-CoV-2 S protein was incorporated into the surface of the SeV vaccine vector. (ii) The amount of SeV proteins F and HN on the surface of the SeV vaccine vector was reduced. With respect to SARS-CoV-2S and SeV F and HN, vector constructs referred to as type A, type B, and type C in this specification are listed in Table 1 below.

[0255] [Table 1]

[0256] If a vaccinated individual has already acquired immunity to SARS-CoV-2 in their respiratory system, for example through SARS-CoV-2 infection, their immune system can recognize the SeV vaccine vector against SARS-CoV-2, and the SeV vaccine can act as a booster immunity. If a vaccinated individual has not yet acquired immunity to SARS-CoV-2 in their respiratory system, the vaccine vector induces immunity against SARS-CoV-2 through different immune stimulation pathways and interactions with the immune system. Firstly, because vaccine vector particles display spike proteins on their surface, they themselves exhibit immunogenicity against SARS-CoV-2. Thus, vaccine vectors function, for example, as nanoparticle vaccines or virus-like particle vaccines (VLPs). These particles are often recognized by antigen-presenting cells of the immune system, such as dendritic cells, or taken up by macrophages. Macrophages present a portion of the spike protein to T lymphocytes, which can then differentiate into helper and effector cells. Secondly, vaccine vectors can infect cells such as those in the respiratory tract, causing them to express the encoded spike protein within the cells. As a result, the spike protein is expressed on the surface of these cells, which is recognized as an infection by the immune system. Furthermore, after standard proteolytic degradation occurs within these cells, the spike protein peptide can be presented on MHC-I molecules.

[0257] To facilitate the detection of newly generated SeV vaccine particles, the gene for enhanced green fluorescent protein (eGFP) was inserted into the genome of all SeV vaccine prototypes. The eGFP gene is present solely for in vitro evaluation and has no functional significance to the immune efficacy of the vaccine candidate. After positive evaluation of the vector vaccine candidate, the eGFP gene is removed from the construct. An example of an RNA virus of the present invention that does not contain a GFP-encoding sequence is SeV88-1V2 (Example 5).

[0258] Type A: In addition to expressing SeV vector proteins F and HN, it also expresses the SARS-CoV-2S protein (SARS-CoV-2S + SeV F and HN). In the A-type construct, the P gene of SeV (cleaved P) mut The SARS-CoV-2 S gene was introduced between the M gene and the ) gene.

[0259] The A construct involves combining recombinant viral proteins (e.g., SARS-CoV-2S protein) obtained from other families (coronaviruses) with proteins F and HN on the envelope of a paramyxovirus (particularly the SeV envelope). ​ Such recombinant genes and / or proteins may interfere with viral assembly and, if the proteins are presented on the surface, may interact with F and / or HN, thus negatively affecting their adsorption and penetration capabilities.

[0260] Adsorption and penetration are essential processes in the infection of host cells, in the production of vaccines using replication-deficient viral vectors, and in eliciting an immune response from the target.

[0261] The SARS-CoV-2 S gene is over 3kb long. It was necessary to determine whether the recombinant S gene is genetically stable in the SeV vector and therefore permanently present in the SeV vaccine vector genome. This aspect is by no means trivial when it comes to the production of vector vaccines. In type A viral vectors, the SARS-CoV-2 gene / protein is not essential for replication and / or infection. Therefore, if such non-essential genes are disadvantageous compared to the wild type, there is a considerable risk that they may be lost or inactivated during viral replication, leading to reduced replication capacity.

[0262] In the type A construct, the SARS-CoV-2S protein may be provided in a stabilized pre-binding form, for example, by two consecutive proline substitutions at residues K986 and V987 (K986P and V987P).

[0263] Type B: Expression of SARS-CoV-2S protein in addition to SeV-vector protein F (SARS-CoV-2S + SeV-F) In the type B construct, the SeV HN gene is deleted. The SARS-CoV-2 S gene was introduced between the SeV F and L genes, that is, in place of the HN gene.

[0264] To date, no studies have demonstrated the "interfamily" substitution of functionally essential proteins between paramyxoviruses and coronaviruses, two entirely different viral families, without loss of function. Importantly, the functions of the surface proteins of paramyxoviruses and coronaviruses are not equivalent. Adsorption to host cells is mediated by the HN protein, and entry into the cell (fusion) is initiated by the F protein. In contrast, in coronaviruses, particularly SARS-CoV-2, adsorption and entry are mediated by a single surface protein, the S protein. We have demonstrated that SeV viruses in which some or all of the F and HN proteins are replaced with the S protein of SARS-CoV-2 form recombinant SeV virus particles capable of adsorption to target cells and entry into cells, making them suitable as an anti-SARS-CoV-2 vaccine.

[0265] Furthermore, in the candidate vaccine for type B (SARS-CoV-2S + SeV-F), the absence of SeV-HN in the SeV vaccine vector genome led to a decrease in the amount of SeV surface protein, resulting in a decline in anti-SeV vector immunity.

[0266] The entry mechanisms differ between paramyxoviruses (fusion with the host cell membrane) and coronaviruses (mostly endocytosis). While entry by membrane fusion has been reported for coronaviruses, it appears to be less efficient than endocytosis (Jackson, CB, Farzan, M., Chen, B. et al. Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol 23, 3-20 (2022). https: / / doi.org / 10.1038 / s41580-021-00418-x). In this respect, intrafamily substitution as described above can negatively affect the osmotic mechanism and, consequently, the replication effect.

[0267] SARS-CoV-2 adsorption begins with the virus docking to the ACE-2 receptor on the host (target) cell in its “pre-binding form.” In the pre-binding form, the receptor-binding domain (RBD) is located inside the S protein due to its innate folding properties. However, through hinge-like movement, the RBD domain repeatedly moves to the surface of the S protein, thereby enabling it to bind to the ACE-2 receptor on the target cell. Upon binding to the ACE-2 receptor, the S protein's conformation changes from the unstable pre-binding conformation to the more stable post-binding conformation, promoting / stabilizing subsequent membrane fusion. However, the ACE-2 protein acts not only as a receptor for the S protein but also as an extracellular peptidase that cleaves between the S1 and S2 domains of the SARS-CoV-2 S protein. S1 is cleaved, and S2 fuses with the host cell membrane. The S1 subunit contains the RBD, which binds to the receptor on the target cell (host cell). After receptor binding, the S2 subunit mediates the fusion of the viral envelope with the cell membrane / endosomal membrane. Random structural changes in the RBD are influenced by the ACE-2 receptor. The ACE-2 receptor binds to the RBD, stabilizing the fused morphology compared to the pre-fusion morphology.

[0268] In many viral fusion glycoproteins, pre-binding stabilization by sequence modification can be induced by specific amino acid exchange at the cleavage site, as described herein, and generally protects a more conformationally stable post-binding structure. Therefore, pre-binding stabilization usually significantly increases the yield in recombinant expression of many viral fusion glycoproteins. Furthermore, pre-binding-stabilized viral glycoproteins are usually better / stronger immunogens than their unstabilized wild-type counterparts because antibody neutralization corresponds to the pre-binding conformation rather than the post-binding conformation.

[0269] In the type B construct, the SARS-CoV-2S protein may be provided in a stabilized pre-binding form, for example, by two consecutive proline substitutions at residues K986 and V987 (K986P and V987P).

[0270] The type B candidate vaccine (expressing the pre-binding stabilized SARS-CoV-2S protein together with SeV vector protein F, while not expressing SeV HN (=SARS-CoV-2S+SeV F)) is designed to achieve more efficient vector production and improved immunogenicity against the SARS-CoV-2S protein by stabilizing it with the pre-binding conformation. At the same time, by removing SeV vector protein HN, the proportion of SeV vector protein in the SeV vaccine particles is reduced, significantly decreasing the anti-vector vaccine immunogenicity and enabling more efficient revaccination (e.g., booster vaccination).

[0271] In the type B SeV construct, the SARS-CoV-2 S protein performs receptor binding instead of the SeV HN protein. The S protein is an essential component of the SeV vaccine vector. The SARS-CoV-2 S gene is an essential gene for the SeV vaccine vector, and its deletion or damage completely disables the replication ability of recombinant SeV vaccine virus in transcomplement cells.

[0272] In the type B SeV construct, the SARS-CoV-2 S protein performs receptor binding instead of the SeV HN protein. The S protein is an essential component of the SeV vaccine vector. The SARS-CoV-2 S gene is an essential gene for the SeV vaccine vector, and its deletion or damage can completely disable the replication ability of the recombinant SeV vaccine virus in transcomplicating cells.

[0273] In the type B construct, the HN coding sequence may not be present. In the type B construct, the adsorption of SeV vector particles to the ACE-2 receptor of target cells may be mediated by the pre-fusion stabilized SARS-CoV-2S protein. In the type B construct, the SeV F protein may mediate the fusion of the viral envelope to the cell membrane or endosomal membrane. However, it was unclear to the inventors whether the pre-fusion stabilized S protein and the SeV F protein functionally interact to enable the type B construct containing the pre-fusion stabilized SARS-CoV-2S protein to adsorb to and fuse with host cells.

[0274] Type C: Expression of SARS-CoV-2S protein only (SARS-CoV-2S "only"); no expression of SeV vector proteins F and HN. In the C construct, the SeV HN and F genes were deleted. The SARS-CoV-2 S gene was introduced between the SeV M and L genes, i.e., in place of the F and HN genes.

[0275] This approach (SARS-CoV-2S "only") was considered the most interesting of approaches A, B, and C because all surface proteins of the SeV vector are removed, rendering it ineffective as an antigen for anti-vector immunity.

[0276] Reproduction ​Therefore, the vaccine vector does not proliferate within the target. Importantly, since type C vaccine vectors do not display vector proteins on their surface, the immunity acquired by the target against the vaccine vector (for example, in the case of an adenovirus vector) does not interfere with the acquisition of immunity against SARS-CoV-2.

[0277] In type C vaccine vectors, the SARS-CoV-2 S protein is responsible for adsorption to and entry into target cells. The function of the S protein is necessary for the replication / proliferation of the vaccine vector. In other words, the vaccine vector cannot replicate unless the S gene is intact, and intact S genes are selected during proliferation.

[0278] Due to deletions in the SeV gene F and HN, the type C chimeric SeV vector has a genome length nearly identical to that of the wild-type SeV virus. The adsorption and entry capabilities of the SARS-CoV-2 protein are comparable to those of the SeV F and HN proteins, and it replicates with similar efficiency to wild-type SeV.

[0279] Preferably, the type C construct does not contain a pre-binding stabilization sequence. In the type C construct, the SARS-CoV-2S protein may be provided in a form that is conformationally changeable between pre-binding and post-binding forms and can be cleaved by the host cell's ACE-2 receptor protease.

[0280] Example 2 SARS-CoV-2 S protein variants: Chimeric constructs combining the ectodomain of the S protein with the cytoplasmic and / or transmembrane domains of SeV F and / or HN. In parallel with considerations regarding the combination of surface proteins (i.e., the arrangement, distance, and number of surface protein molecules on the viral particle), it was necessary to investigate whether the SARS-CoV-2 S protein is incorporated into the envelope of the SeV vector, and if so, in what sequence. If SeV surface proteins, in addition to the SeV matrix protein (M), are involved in enrichment into the cytoplasmic membrane of host cells, then the incorporation of the SARS-CoV-2 S protein into the envelope of the vaccine vector could adversely affect the budding process.

[0281] It is necessary to test whether parts of the SeV vector surface proteins F and HN are required for interaction with the SeV vector protein M (matrix protein), for example, in viral assembly. The three different design types (AC) of the vaccine candidate each (i) are derived from the ectodomain of the SARS-CoV-2 S protein. ​ (ii) Transmembrane domains derived from SARS-CoV-2 or SeV ​ , and / or (iii) cytoplasmic domains derived from SARS-CoV-2 or SeV ​ It was designed using three different S proteins, including (Table 2). As a result, the following three variations are obtained for each A-C construct:

[0282] [Table 2]

[0283] An example of construct SeV85E 1V2 is SEQ ID NO: 36. A chimeric polypeptide containing the SARS-CoV-2 spike ecto, transmembrane domain and cytoplasmic domain (with 19 C-terminal amino acid residues deleted), and SeV F cytoplasmic domain is encoded by SEQ ID NO: 36 and has the amino acid sequence of SEQ ID NO: 37.

[0284] A total of nine SeV vaccine constructs were designed (Figure 2).

[0285] Example 3 From a multitude of possible combinations, a meaningful and promising selection (prioritization) of SeV vaccine constructs had to be made. SeV85E-2V1 (type A), SeV85E-1V1 (type C), and SeV85E-1V2 (type C) were evaluated using the six steps outlined in Table 3:

[0286] [Table 3]

[0287] Process 1 Expression of prioritized SeV vaccine candidates and their chimeric SARS-CoV-2S proteins in cell culture using plasmids (control experiment to verify the structurally correct expression of chimeric SARS-CoV-2S proteins) Expression of chimeric SARS-CoV-2S proteins of the SeV vaccine candidates SeV85E-2V1, SeV85E-1V1, and SeV85E-1V2 in cell culture confirmed the correct structural expression of the chimeric SARS-CoV-2S proteins.

[0288] Vero cells were either transfected with eukaryotic expression plasmids pcDNA3.1 CoV-2 S2V1 (type A), pcDNA3.1 CoV-2 S1V1 (type C), or pcDNA3.1 CoV-2 S1V2 (type C), each containing the coding sequences for SARS-CoV-2 S2V1, SARS-CoV-2 S1V1, or SARS-CoV-2 S1V2, respectively, or not transfected (MOCK = negative control). 48 hours after transfection, cells were stained with a primary antibody against SARS-CoV-1 / 2S protein. Goat anti-mouse Alexa Fluor 546 was used as the secondary antibody. Structural integrity of the coding chimeric sequences of SARS-CoV-2 S2V1, SARS-CoV-2 S1V1, or SARS-CoV-2 S1V2 is indicated by a red signal (Figure 3, middle and right panels).

[0289] Process 2 Rescue, i.e., initial production of SeV vaccine candidates in GMP-qualified Vero cells and V3-10 helper cells. Rescue, i.e., initial production of recombinant virus particles, was successful with both SeV vaccine candidates SeV85E-1V2 (type C) and SeV vaccine candidate SeV85E-2V1 (type A) using GMP-compliant Vero cells (Figures 4 and 5).

[0290] Furthermore, rescue was achieved with the SeV vaccine candidates SeV85E-1V2 (type C) and SeV85E-2V1 (type A) in the helper cell line V3-10 (Figures 6 and 7).

[0291] V3-10 cells are Vero-based helper cells stably transfected with plasmids encoding at least SeV proteins N and P. This helper cell line is transfected with replication-deficient SeV vectors (here, P). mut It can be used for the rescue and propagation of ) P mut This is the N-terminal cleavage form of the P protein.

[0292] Process 3 To supply a sufficient amount of virus for subsequent in vivo trials (animal models, clinical trials), the SeV vaccine candidate is grown in helper cells transfected with at least one nucleic acid molecule that trans-encodes the SeV P protein. Proliferation in V3-10 helper cells was successful with the SeV vaccine candidate SeV85E-1V2 (type C). Using this type C prototype, the titer required for animal experiments can already be achieved (Figures 8 and 9).

[0293] Process 4 Evidence of SARS-CoV-2S protein in SeV vaccine candidate virus particles Immunoblots of SeV85E-1V2(type C) virus particles (purified by ultracentrifugation) showed expression of the SARS-CoV-2S protein, indicating that the SARS-CoV-2S protein is directly incorporated into the SeV85E-1V2(type C) virus particles (Figure 10).

[0294] Process 5 Stability of SARS-CoV-2S protein expression by SeV vaccine candidates in cell culture. The stability of SARS-CoV-2 S protein expression was demonstrated by immunoblotting analysis of supernatants and whole cell lysates collected from various viral passages.

[0295] Process 6 Characterization of the immunological response after infecting mouse experimental animals with a SeV vaccine candidate. We measured the humoral and cellular immune responses of mice vaccinated with SeV vector constructs.

[0296] conclusion This example demonstrates that the recombinant SeV candidate vaccine displays a chimeric SARS-CoV-2S protein on its surface while simultaneously exhibiting significantly reduced expression of the SeV surface antigen. The SeV vaccine candidate could be rescued from GMP-compliant Vero cells. The SeV candidate vaccine can be grown in Vero cell-derived helper cells (e.g., cell line V3-10) transfected with a nucleic acid molecule that trans-encodes at least the SeV P protein. Cell culture in Vero cell-derived helper cells can supply sufficient amounts of virus for in vivo testing (animal models and clinical trials).

[0297] Example 4 Reproduction ​ SeV attenuation Here, the inventors confirmed the difference in attenuation between two viral vector constructs of the SARS-CoV-2 vaccine. Both of these viral vector constructs contain attenuating mutations that completely replace both the Sendai surface proteins F and HN with the SARS-CoV-2 surface protein S. One vaccine candidate (1) replicates ​ Yes, it still contains the unmodified Sendai P gene, but the other vaccine candidate (2) contains additional attenuation deletions of amino acids 2-77 in the P protein, and replication ​ The results are as follows. Compared to an unmodified recombinant Sendai virus vector, a significant decrease in replication efficacy is observed due to the addition of attenuating mutations. The following viral titers are from Vero cells (replication). ​ This was obtained from viral replication studies under cell culture conditions using SeV and V3-10 helper cells (replication-deficient SeV).

[0298] TIFF2023227758000007.tif52153

[0299] In conclusion, the replication was in which surface proteins F and HN were replaced with SARS-CoV-2 surface protein S. ​ SeV(1) is an unqualified copy. ​Since it is attenuated from the perspective of SeV, such recombinant viruses are suitable as immunogenic agents or vaccines against SARS-CoV-2.

[0300] Furthermore, such modified copies ​ SeV(1) shows improved production efficiency compared to replication-deficient SeV(2).

[0301] Example 5 Immunogenic effects of SeV88-1V2 BAL samples are mucosal tissue samples from the lower respiratory tract, while NW samples are mucosal tissue samples from the upper respiratory tract. Both samples contain components of the immune system, such as macrophages, lymphocytes, neutrophils, or antibodies released from surrounding cells and immune cells, and are therefore useful in describing the immunological state of these tissues. Specific antibodies against pathogens contribute to the protective immune response by binding to and neutralizing invading pathogens such as viruses.

[0302] method Female BALB / c mice (4 weeks old) were grouped into sets of five and immunized intranasally (IN) 2-3 times every three weeks, increasing the vaccine dose each time. To collect bronchoalveolar lavage (BAL) and nasal lavage (NW), the mice were sacrificed by cervical dislocation under anesthesia two weeks after the last immunization.

[0303] IgA antibodies against the SARS-CoV-2 spike protein were measured using microtiter-based enzyme-linked immunosorbent assay (ELISA). Serial 2-fold dilutions were prepared and incubated on antigen-precoated microtiter plates. Horseradish peroxidase-labeled anti-mouse IgA was then added. After 1 hour, the substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added, and the reaction was stopped with NaH2SO4 after 30 minutes. The colorimetric conversion was then measured at 450 nm using a spectrophotometer. Titer is expressed as the reciprocal of the mean value.

[0304] TIFF2023227758000008.tif47148 TIFF2023227758000009.tif47149

[0305] result Searching for specific IgA in inactivated immunized mice demonstrated that this immunization can stimulate a specific mucosal immune response against SARS-CoV-2. SeV88-1V2 induced specific IgA antibodies in a dose-dependent manner. Based on specific IgA concentration, IgA concentrations were higher in the bronchoalveolar (BAL) group than in the neuron (NW) group. In particular, groups G4 and G5 showed the highest titers compared to groups immunized with low or small doses of SeV88-1V2 (Figure 11).

[0306] The present invention also includes the following: 1. Recombinant replication-deficient negative-strand RNA virus, wherein its genome contains, (a) A nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in a replication defect of the negative-strand RNA virus, and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Recombinant replication-deficient negative-strand RNA viruses, including those containing this virus. 2. Recombinant replication-deficient minus-strand RNA viruses as described in item 1, which are paramyxovirus, artovirus, bornavirus, filovirus, rispivirus, mymonavirus, nyamivirus, pneumovirus, rhabdovirus, sunvirus, or simmovirus. 3. A Sendai virus, which is a recombinant replication-deficient negative-strand RNA virus as described in item 1 or 2. 4. A recombinant replication-deficient minus-strand RNA virus according to any one of the preceding items, wherein the modification of nucleotide sequence (a) is located in the nucleotide sequence encoding the N-terminus of the P polypeptide. 5. The recombinant replication-deficient minus-strand RNA virus according to any one of the preceding paragraphs, wherein the modification in the nucleotide sequence (a) is a deletion in the nucleotide sequence encoding the N-terminus of the P polypeptide. 6. The modification in the nucleotide sequence (a) is (i) Compared to the wild type, deletion of the nucleotide sequence encoding amino acids 2-77 of the P polypeptide, or (ii) Replication of RNA viruses ​ (i) subarray that causes A recombinant replication-deficient negative-strand RNA virus as described in any one of the preceding paragraphs. 7. A recombinant replication-deficient negative-strand RNA virus according to any one of the preceding items, which has transcriptional ability. 8. The genome is (c) Nucleotide sequence encoding a negative-strand RNA virus N polypeptide, (d) Nucleotide sequences encoding negative-strand RNA virus M polypeptides, and (e) Nucleotide sequences encoding negative-strand RNA virus L polypeptide A recombinant replication-deficient negative-strand RNA virus as described in any one of the preceding paragraphs, further comprising: 9. The recombinant replication-deficient negative-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide contains a heterologous antigen. 10. A recombinant replication-deficient negative-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide contains a viral antigen. 11. The recombinant replication-deficient negative-strand RNA virus according to item 10, wherein the viral antigen and the negative-strand RNA virus are selected from viruses of different families. 12. The recombinant replication-deficient negative-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide contains an antigen derived from a virus other than a negative-strand RNA virus. 13. The recombinant replication-deficient negative-chain RNA virus described in any one of the preceding paragraphs, wherein the heterologous polypeptide contains an antigen derived from a positive-chain virus such as a coronavirus. 14. A recombinant replication-deficient negative-strand RNA virus as described in any one of the preceding paragraphs, wherein the negative-strand RNA virus is a paramyxovirus such as Sendai virus, and the heterologous polypeptide contains an antigen derived from a coronavirus. 15. The recombinant replication-deficient minus-strand RNA virus according to any one of the preceding paragraphs, wherein the heterologous polypeptide comprises the spike protein (S protein) or an immunogenic fragment thereof of a coronavirus. 16. A recombinant replication-deficient minus-strand RNA virus as described in any one of the preceding paragraphs, wherein the coronavirus is SARS-CoV-2. 17. A recombinant replication-deficient minus-strand RNA virus according to any one of the preceding paragraphs, wherein at least one nucleotide sequence (b) is located upstream of the 5' end coding sequence and / or downstream of the 3' end coding sequence in an RNA molecule between two coding sequences. 18. The genome is (f) Nucleotide sequences encoding negative-strand RNA virus F polypeptide, and (g) Nucleotide sequence encoding negative-strand RNA virus HN polypeptide A recombinant replication-deficient negative-strand RNA virus as described in any one of the preceding paragraphs, further comprising: 19. A recombinant replication-deficient minus-strand RNA virus according to any one of the preceding paragraphs, wherein at least one nucleotide sequence (b) is inserted into or at least partially substituted into at least one native (endogenous) sequence encoding at least one viral polypeptide such that the function or activity of at least one endogenous viral polypeptide is at least partially or completely disrupted. 20. A recombinant replication-deficient minus-strand RNA virus according to any one of the preceding paragraphs, wherein a nucleotide sequence (b) encoding at least one heterologous polypeptide is essential for viral replication and / or proliferation.

[0307] 21. A recombinant replication-deficient minus-strand RNA virus as described in any one of the preceding paragraphs, wherein a heterologous polypeptide encoded by sequence (b) is functionally expressed (expressed in a biologically active form). 22. Recombinant replication-deficient minus-strand RNA virus as described in any one of the preceding paragraphs, wherein the endogenous sequence encoding the F polypeptide and / or the endogenous sequence encoding the HN polypeptide are modified such that their function or activity is at least partially or completely disrupted, and the nucleotide sequence (b) is inserted into the F locus and / or HN locus. 23. A recombinant replication-deficient minus-strand RNA virus as described in subsection 22, wherein the endogenous sequence encoding the HN polypeptide is modified such that its function or activity is at least partially or completely disrupted, and the nucleotide sequence (b) is inserted into the locus of the HN gene. 24. A recombinant replication-deficient minus-strand RNA virus as described in subsection 22, wherein the endogenous sequence encoding the F polypeptide is modified such that its function or activity is at least partially or completely disrupted, and the nucleotide sequence (b) is inserted into the locus of the F gene. 25. Recombinant replication-deficient minus-strand RNA virus as described in subsection 22, wherein the endogenous sequence encoding the F polypeptide and the endogenous sequence encoding the HN polypeptide are modified such that their function or activity is at least partially or completely disrupted, and the nucleotide sequence (b) is inserted into the F locus and the HN locus. 26. A recombinant replication-deficient negative-strand RNA virus as described in any one of the preceding paragraphs, which, when presented on the surface of a virus, can adsorb to a host cell and enter the host cell. 27. A recombinant replication-deficient negative-strand RNA virus according to any one of the preceding paragraphs, wherein the heterologous polypeptide, when presented on the surface of the virus, mediates the adsorption to and entry into a host cell. 28. Recombinant replication-deficient negative-strand RNA viruses as described in item 27, whose host cell is a eukaryotic cell. 29. Recombinant replication-deficient negative-strand RNA viruses described in any one of the preceding paragraphs, which are capable of infecting eukaryotic cells. 30. Recombinant replication-deficient negative-strand RNA viruses described in any one of the preceding paragraphs, which can be rescued from eukaryotic cells. 31. Recombinant replication-deficient negative-strand RNA virus according to any one of the preceding items, which is capable of proliferating in eukaryotic cells, wherein the cells are trans-complemented with a sequence missing in the replication-deficient negative-strand RNA virus, and in particular with a sequence encoding a P polypeptide. 32. (i) P polypeptides, and / or (ii) N and L polypeptides A recombinant replication-deficient negative-strand RNA virus according to any one of the preceding items, which is capable of growing in eukaryotic cells trans-complemented with a sequence encoding the above. 33. A recombinant replication-deficient minus-strand RNA virus according to any one of the preceding paragraphs, wherein a heterologous polypeptide encoded by at least one nucleotide sequence (b) comprises a fragment of a spike polypeptide. 34. The nucleotide sequence (b) is, (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) A transmembrane domain of SeV F or HN polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of SeV F or HN polypeptide, or a fragment thereof. A recombinant replication-deficient negative-strand RNA virus as described in any one of the preceding paragraphs, which encodes the aforementioned virus. 35. The nucleotide sequence (b) is, (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of SeV F or HN polypeptide, or a fragment thereof. A recombinant replication-deficient negative-strand RNA virus as described in any one of the preceding paragraphs, which encodes the aforementioned virus. 36. The nucleotide sequence (b) is, (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof. A recombinant replication-deficient negative-strand RNA virus as described in any one of the preceding paragraphs, which encodes the aforementioned virus. 37. Recombinant replication-deficient negative-strand RNA viruses as described in any one of the preceding paragraphs, wherein anti-vector antigenicity is reduced. 38. A recombinant replication-deficient minus-strand RNA virus according to any one of the preceding paragraphs, wherein the ectodomain of the SARS-CoV-2 spike polypeptide comprises an S1 subunit containing a receptor-binding domain (RBD) capable of binding to the ACE-2 receptor of a host cell. 39. A recombinant replication-deficient minus-strand RNA virus according to any one of the preceding paragraphs, wherein the S1 subunit can be cleaved by the ACE-2 protease of the host cell. 40. A recombinant replication-deficient minus-strand RNA virus according to any one of the preceding paragraphs, wherein the ectodomain of the SARS-CoV-2 spike polypeptide includes an S2 subunit that can fuse the SeV envelope with the host cell membrane or endosomal membrane.

[0308] 41. A recombinant replication-deficient minus-strand RNA virus according to any one of the preceding paragraphs, wherein the sequence of the ectodomain includes at least one sequence modification that can stabilize the ectodomain in the pre-binding form. 42. A recombinant replication-deficient minus-strand RNA virus according to any one of the preceding items, which can induce a mucosal immune response to at least one heterologous polypeptide encoded by at least one nucleotide sequence (b). 43. A recombinant replication-deficient negative-strand RNA virus according to any one of the preceding paragraphs, which can induce a protective immune response against a pathogen having a polypeptide antigen encoded by at least one nucleotide sequence (b). 44. Recombinant replication-deficient negative-strand RNA virus particles comprising a nucleocapsid and an envelope, wherein the nucleocapsid contains the following in its genome: (a) A nucleotide sequence encoding a SeV P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in a replication defect of the negative-strand RNA virus, and (b) A nucleotide sequence comprising at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the viral particle. 45. The recombinant replication-deficient minus-strand RNA virus particle according to claim 44, wherein the at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope comprises presenting the SARS-CoV-2 spike polypeptide or a fragment thereof on the surface of the Sendai virus particle. 46. ​​Recombinant replication-deficient Sendai virus, in which its genome contains, (a) A nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in a replication defect of the negative-strand RNA virus, and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Recombinant replication-deficient Sendai virus, including 47. Recombinant replication-deficient Sendai virus (SeV) as described in Section 46, wherein at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope comprises a SARS-CoV-2 spike polypeptide or a fragment thereof presented on the surface of a Sendai virus particle. 48. Recombinant replication-deficient Sendai virus particle comprising a nucleocapsid and an envelope, wherein the nucleocapsid contains within its genome, (a) A nucleotide sequence encoding a SeV P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in a replication defect of the negative-strand RNA virus, and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of a Sendai virus particle. Recombinant replication-deficient Sendai virus particles, including those containing this particle. 49. Recombinant replication-deficient Sendai virus (SeV) particle as described in Section 48, wherein at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope comprises a SARS-CoV-2 spike polypeptide or a fragment thereof presented on the surface of the Sendai virus particle. 50. A pharmaceutical composition comprising a recombinant replication-deficient negative-strand RNA virus as described in any one of items 1 to 43 and / or recombinant replication-deficient negative-strand RNA virus particles as described in any one of items 44 to 49. 51. A vaccine comprising a recombinant replication-deficient negative-strand RNA virus as described in any one of items 1 to 43 and / or a recombinant replication-deficient negative-strand RNA virus particle as described in any one of items 44 to 49. 52. An immunogenic composition comprising a recombinant replication-deficient negative-strand RNA virus as described in any one of items 1 to 43 and / or recombinant replication-deficient negative-strand RNA virus particles as described in any one of items 44 to 49. 53. Recombinant replication-deficient negative-strand RNA viruses as described in any one of claims 1 to 43, recombinant replication-deficient negative-strand RNA virus particles as described in any one of claims 44 to 49, pharmaceutical compositions as described in claim 50, vaccines as described in claim 51, or immunogenic compositions as described in claim 52, for pharmaceutical use. 54. Recombinant replication-deficient negative-strand RNA virus as described in any one of items 1 to 43, recombinant replication-deficient negative-strand RNA virus particles as described in any one of items 44 to 49, a pharmaceutical composition as described in item 50, a vaccine as described in item 51, or an immunogenic composition as described in item 52, for use in a method for preventing SARS-CoV-2 infection. 55. Recombinant replication-deficient negative-strand RNA virus as described in any one of items 1 to 43, recombinant replication-deficient negative-strand RNA virus particles as described in any one of items 44 to 49, a pharmaceutical composition as described in item 50, a vaccine as described in item 51, or an immunogenic composition as described in item 52, for use in methods of preventing COVID-19. 56. Recombinant replication-deficient negative-strand RNA virus as described in any one of items 1 to 43, recombinant replication-deficient negative-strand RNA virus particles as described in any one of items 44 to 49, a pharmaceutical composition as described in item 50, a vaccine as described in item 51, or an immunogenic composition as described in item 52, for use as a vaccine. 57. Recombinant replication-deficient negative-strand RNA viruses as described in any one of the items 1 to 43, recombinant replication-deficient negative-strand RNA virus particles as described in any one of the items 44 to 49, pharmaceutical compositions as described in item 50, vaccines as described in item 51, or immunogenic compositions as described in item 52, administered via an intranasal route and / or mucosal route for use as described in any one of the items 53 to 56. 58. An RNA molecule encoding a recombinant replication-deficient negative-strand RNA virus, wherein the RNA molecule is (a) A nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide RNA molecules, including those containing this molecule. 59. (c) Nucleotide sequence encoding an N polypeptide, (d) Nucleotide sequences encoding the M polypeptide, and (e) Nucleotide sequence encoding L polypeptide The RNA molecules described in item 58, further including the RNA molecules described in item 58. 60. A nucleocapsid containing the RNA molecule described in section 58 or 59. 61. A DNA molecule encoding an RNA molecule as described in section 58 or 59.

[0309] 62. A host cell comprising a replication-deficient negative-strand RNA virus as described in any one of items 1 to 43, a recombinant replication-deficient negative-strand RNA virus particle as described in any one of items 44 to 49, an RNA molecule as described in item 58 or 59, a nucleocapsid as described in item 60, and / or a DNA molecule as described in item 61. 63. A method for preventing infection with SARS-CoV-2 and / or COVID-19, comprising administering to a subject in need thereof a recombinant replication-deficient negative-strand RNA virus as described in any one of items 1 to 43, recombinant replication-deficient negative-strand RNA virus particles as described in any one of items 44 to 49, a pharmaceutical composition as described in item 50, a vaccine as described in item 51, or an immunogenic composition as described in item 52. 64. A method for producing a pharmaceutical composition according to item 50, a vaccine according to item 51, or an immunogenic composition according to item 52, comprising the step of formulating a recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43, or a recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49, together with at least one pharmaceutically acceptable excipient. A method for producing a recombinant negative-strand RNA virus or viral particle as described in any one of paragraphs 65-45, comprising the following steps: (a) A step of transfecting eukaryotic host cells with the DNA molecules described in item 61, wherein the host cells are capable of expressing SeV polypeptides N, P and L. (b) A step of culturing host cells under conditions such that the DNA molecule is transcribed and virus particles are formed, and (c) The process of separating the virus particles from (b) A method that includes A method for producing a recombinant negative-strand RNA virus or viral particle as described in any one of paragraphs 66.1 to 45, comprising the following steps: (a) A step of infecting a eukaryotic host cell with a wild-type negative-strand RNA virus and transfecting the eukaryotic host cell with a DNA molecule described in item 61 that encodes a recombinant replication-deficient RNA virus, (b) A step of culturing host cells under conditions such that the DNA molecule is transcribed and virus particles are formed, and (c) The process of separating the virus particles from (b) A method that includes 67. The method described in item 66, wherein the wild-type negative-strand RNA virus and the recombinant replication-deficient RNA virus originate from the same species. 68. Use of a host cell according to claim 62 for the production of a recombinant replication-deficient negative-strand RNA virus according to any one of claims 1 to 43, and / or recombinant replication-deficient negative-strand RNA virus particles according to any one of claims 44 to 49. 69. Use of recombinant replication-deficient minus-strand RNA viruses as described in any one of subsections 1 to 43 for the manufacture of a medicine for the prevention of infection with SARS-CoV-2 and / or COVID-19. 70. Recombinant Reproduction ​ It is a negative-strand RNA virus, and in its genome, (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein performs its intended function, particularly in the viral replication cycle. ​ nucleotide sequence (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. This includes, where the heterologous polypeptide comprises a viral antigen, and the viral antigen and the negative-strand RNA virus are selected from different families of viruses. Recombinant ​ Negative-strand RNA virus. 71. Recombinant, attenuated, as described in item 68. ​ Negative-type RNA virus. 72. Recombinant replication-deficient minus-strand RNA viruses as described in any one of items 70-71, which are paramyxovirus, artovirus, bornavirus, filovirus, rispivirus, mymonavirus, nyamivirus, pneumovirus, rhabdovirus, sunvirus, or simmovirus. 73. A recombinant Sendai virus as described in any one of items 70-72. ​ Negative-type RNA virus. 74. Recombinant replication as described in any one of sections 70-73, wherein the genome further comprises the following: ​ Negative-strand RNA virus: (c) Nucleotide sequence encoding a negative-strand RNA virus N polypeptide, (d) Nucleotide sequences encoding negative-strand RNA virus M polypeptides, and (e) Nucleotide sequence encoding a negative-strand RNA virus L polypeptide. 75. Recombination according to any one of items 70 to 74, wherein the heterologous polypeptide contains a heterologous antigen. ​ Negative-type RNA virus. 76. Recombinant polypeptide as described in any one of items 70 to 75, wherein the heterologous polypeptide contains a viral antigen. ​ Negative-type RNA virus. 77. The recombinant virus described in item 76, wherein the viral antigen and the negative-strand RNA virus are selected from different families of viruses. ​Negative-type RNA virus. 78. Recombination according to any one of items 70-77, wherein the heterologous polypeptide contains an antigen derived from a virus that is not a negative-strand RNA virus. ​ Negative-type RNA virus. 79. Recombinant replication of a heterologous polypeptide containing an antigen derived from a forward-chain virus such as a coronavirus, as described in any one of items 70 to 78. ​ A chain RNA virus. 80. Recombinant replication as described in any one of items 70-79, wherein the negative-strand RNA virus is a paramyxovirus such as Sendai virus, and the heterologous polypeptide contains an antigen derived from a coronavirus. ​ Negative-strand RNA virus.

[0310] 81. Recombination according to any one of items 70 to 80, wherein the heterologous polypeptide comprises the coronavirus spike protein (S protein) or an immunogenic fragment thereof. ​ Negative-type RNA virus. 82. Recombinant coronavirus as described in any one of paragraphs 70-81, wherein the coronavirus is SARS-CoV-2. ​ Negative-type RNA virus. 83. Recombinant replication as described in any one of the items 70-82, wherein at least one nucleotide sequence (b) is located in the RNA molecule between two coding sequences, upstream of the 5' end coding sequence and / or downstream of the 3' end coding sequence. ​ Negative-strand RNA virus 84. The genome, (f) Nucleotide sequences encoding negative-strand RNA virus F polypeptide, and (g) Nucleotide sequence encoding negative-strand RNA virus HN polypeptide Recombinant replication as described in any one of paragraphs 70 to 83, further including ​ Negative-strand RNA virus. 85. Recombination according to any one of the claims 70-84, wherein at least one nucleotide sequence (b) is inserted into or at least partially substituted into at least one native (endogenous) sequence encoding at least one viral polypeptide such that the function or activity of at least one endogenous viral polypeptide is at least partially or completely disrupted. ​ Negative-type RNA virus. 86. Recombinant replication as described in any one of subsections 70-85, wherein at least one nucleotide sequence (b) encoding a heterologous polypeptide is essential for viral replication and / or proliferation. ​ Negative-strand RNA virus. 87. Recombinant replication as described in any one of the following clauses, 70-86: The heterologous polypeptide encoded by sequence(b) is functionally expressed (expressed in a biologically active form). ​ Negative-strand RNA virus. 88. Recombinant replication as described in any one of sections 70-87, wherein an endogenous sequence encoding an F polypeptide and / or an endogenous sequence encoding an HN polypeptide is modified such that their function or activity is at least partially or completely disrupted, and nucleotide sequence (b) is inserted into the F locus and / or HN locus. ​ Negative-strand RNA virus. 89. Recombinant replication as described in Section 88, wherein the endogenous sequence encoding the HN polypeptide is modified such that its function or activity is at least partially or completely disrupted, and the nucleotide sequence (b) is inserted into the locus of the HN gene. ​ Negative-strand RNA virus. 90. Recombinant replication as described in item 88, wherein an endogenous sequence encoding the F polypeptide is modified such that its function or activity is at least partially or completely disrupted, and the nucleotide sequence is inserted into the locus of the F gene. ​ Negative-strand RNA virus. 91. Recombinant replication as described in item 88, wherein the endogenous sequence encoding the F polypeptide and the endogenous sequence encoding the HN polypeptide are modified such that their function or activity is at least partially or completely disrupted, and the nucleotide sequences are inserted into the F locus and the HN locus. ​ Negative-strand RNA virus. 92. Recombinant replication as described in any one of items 70-91, which, when presented on the surface of the virus, can adsorb to and invade host cells. ​ Negative-strand RNA virus. 93. Heterogeneous polypeptides on the surface of the virus ​ If this occurs, the recombinant organism described in any one of the items 70 to 92 mediates adsorption to and entry into host cells. ​ Negative-type RNA virus. 94. Recombinant cell type as described in item 93, where the host cell is a eukaryotic cell. ​ Negative-type RNA virus. 95. Recombinant cells capable of infecting eukaryotic cells, as described in any one of items 70-94. ​ Negative-type RNA virus. 96. Recombinant replication as described in any one of sections 70-95, which can be rescued from eukaryotic cells. ​ Negative-strand RNA virus. 97. Recombinant cells capable of proliferating in eukaryotic cells, as described in any one of items 70-96. ​ A negative-strand RNA virus, wherein the cell replicates ​ In negative-strand RNA viruses, the deficient sequence is complemented in trans, and in particular, the sequence encoding the P polypeptide is complemented in trans, recombinant ​ Negative-type RNA virus. 98. A heterologous polypeptide encoded by at least one nucleotide sequence (b) comprising a fragment of a spike polypeptide, as described in any one of the items 70 to 97. ​ Negative-type RNA virus. 99. The nucleotide sequence (b) is, (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) A transmembrane domain of SeV F or HN polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of SeV F or HN polypeptide, or a fragment thereof. Recombination described in any one of paragraphs 70-98, which codes for ​ Negative-type RNA virus. 100. The nucleotide sequence (b) is, (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of SeV F or HN polypeptide, or a fragment thereof. Recombination encoding as described in any one of paragraphs 70-99. ​ Negative-type RNA virus.

[0311] 101. The nucleotide sequence (b) is, (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of the SARS-CoV-2 spike polypeptide, or its fragment Recombination encoding as described in any one of paragraphs 70 to 100. ​ Negative-type RNA virus. 102. Recombinant having reduced antivector antigenicity as described in any one of items 70 to 101. ​ Negative-type RNA virus. 103. Recombination according to any one of items 70-102, wherein the ectodomain of the SARS-CoV-2 spike polypeptide comprises an S1 subunit containing a receptor-binding domain (RBD) capable of binding to the ACE-2 receptor of a host cell.​ Negative-type RNA virus. 104. Recombinant replication as described in any one of sections 70-103, in which the S1 subunit can be cleaved by the ACE-2 protease of the host cell. ​ A chain RNA virus. 105. Recombination according to any one of items 70-104, wherein the ectodomain of the SARS-CoV-2 spike polypeptide contains an S2 subunit capable of fusing the SeV envelope to the host cell membrane or endosomal membrane. ​ Negative-type RNA virus. 106. Recombinant replication according to any one of the items 70 to 105, wherein the sequence of the ectodomain includes at least one sequence modification that can stabilize the ectodomain in the pre-jointed form. ​ Negative-strand RNA virus. 107. Recombination according to any one of the following clauses, 70-106, which can induce a mucosal immune response to at least one heterologous polypeptide encoded by at least one nucleotide sequence (b). ​ Negative-type RNA virus. 108. Recombination according to any one of sub-sub ​ Negative-type RNA virus. 109. Recombinant including nucleocapsid and envelope ​ A negative-strand RNA virus particle, wherein the nucleocapsid is contained within its genome. (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein performs its intended function, particularly in the viral replication cycle. ​ nucleotide sequence, (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Recombinant ​ Negative-type RNA virus particles. 110. The recombinant according to item 109, wherein the at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope comprises a SARS-CoV-2 spike polypeptide or a fragment thereof presented on the surface of a Sendai virus particle. ​ Negative-type RNA virus particles. 111. Recombination ​ It is a type Sendai virus, and in its genome, (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein performs its intended function, particularly in the viral replication cycle. ​ nucleotide sequence (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Recombinant ​ Type Sendai virus. 112. The recombinant according to item 111, wherein the at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope comprises a SARS-CoV-2 spike polypeptide or a fragment thereof presented on the surface of a Sendai virus particle. ​ Sendai virus type (SeV). 113. Recombination ​ A type Sendai virus particle comprising a nucleocapsid and an envelope, wherein the nucleocapsid contains within its genome, (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein performs its intended function, particularly in the viral replication cycle. ​ nucleotide sequence (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of a Sendai virus particle. Recombinant ​ Type Sendai virus particles. 114. Recombination according to item 113, wherein at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope comprises a SARS-CoV-2 spike polypeptide or a fragment thereof presented on the surface of a Sendai virus particle. ​ Sendai virus (SeV) particle. 115. Recombinant organisms described in any one of items 111-114, which have been attenuated. ​ Type Sendai virus or particles. 116. Recombination described in any one of paragraphs 70-110 ​ Type negative-strand RNA virus or viral particle, or recombinant as described in any one of items 111-115. ​ A pharmaceutical composition containing type Sendai virus or virus particles. 117. Recombinant modifications as described in any one of paragraphs 70-110. ​ Type negative-strand RNA virus or viral particle, or recombinant as described in any one of items 111-115. ​ A vaccine containing type Sendai virus or viral particles. 118. Recombination described in any one of paragraphs 70-110 ​ Type negative-strand RNA virus or viral particle, or recombinant as described in any one of items 111-115. ​ An immunogenic composition containing type Sendai virus or viral particles. 119. Recombinant compounds used for pharmaceutical purposes as described in any one of items 70 to 110. ​ Type negative-strand RNA virus or viral particle, or recombinant as described in any one of items 111-115. ​ Sendai virus type or viral particles, the pharmaceutical composition described in item 116, the vaccine described in item 117, or the immunogenic composition described in item 118. 120. Recombinant preparations described in any one of sections 70 to 110 for use in methods for preventing SARS-CoV-2 infection. ​ Type negative-strand RNA virus or viral particle, recombinant as described in any one of items 111-115. ​Sendai virus type or viral particles, the pharmaceutical composition described in item 116, the vaccine described in item 117, or the immunogenic composition described in item 118.

[0312] 121. Recombinant for use in methods of preventing COVID-19, as described in any one of subsections 70 to 110. ​ Type negative-strand RNA virus or viral particle, recombinant as described in any one of items 111-115. ​ Sendai virus type or viral particles, the pharmaceutical composition described in item 116, the vaccine described in item 117, or the immunogenic composition described in item 118. 122. Recombinant vaccines as described in any one of paragraphs 70 to 110. ​ Type negative-strand RNA virus or viral particle, recombinant as described in any one of items 111-115. ​ Sendai virus type or viral particles, the pharmaceutical composition described in item 116, the vaccine described in item 117, or the immunogenic composition described in item 118. 123. For use as described in any one of paragraphs 119-122, the recombinants described in any one of paragraphs 70-110 are administered intranasally and / or via a mucosal route. ​ Type negative-strand RNA virus or viral particle, recombinant as described in any one of items 111-115. ​ Sendai virus type or viral particles, the pharmaceutical composition described in item 116, the vaccine described in item 117, or the immunogenic composition described in item 118. 124. Recombination ​ An RNA molecule encoding a type negative-strand RNA virus, wherein the RNA molecule is (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein performs its intended function particularly in the viral replication cycle. ​ The nucleotide sequence, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide RNA molecules, including those containing this molecule. 125. (a) Nucleotide sequence encoding an N polypeptide, (b) Nucleotide sequence encoding M polypeptide, and (c) Nucleotide sequence encoding L-polypeptide The RNA molecules described in item 124, further including the RNA molecules described in item 124. 126. A nucleocapsid containing an RNA molecule as described in any one of items 124-125. 127. A DNA molecule that encodes an RNA molecule as described in any one of items 124-125. 128. Recombinant functions as described in any one of paragraphs 70-110. ​ Type negative-strand RNA virus or viral particle, recombinant as described in any one of items 111-115. ​ A host cell comprising type Sendai virus or viral particles, an RNA molecule as described in any one of items 124-125, a nucleocapsid as described in item 126, and / or a DNA molecule as described in item 127. 129. Methods for preventing infection with SARS-CoV-2 and / or COVID-19, comprising the recombinant method described in any one of sub-sub ​ Type negative-strand RNA virus or viral particle, recombinant as described in any one of items 111-115. ​ A method comprising the step of administering to a subject requiring the use of type Sendai virus or viral particles, a pharmaceutical composition as described in item 116, a vaccine as described in item 117, or an immunogenic composition as described in item 118. 130. A method for producing a pharmaceutical composition as described in item 116, a vaccine as described in item 117, or an immunogenic composition as described in item 118, wherein the recombinant composition is described in any one of items 70 to 110. ​ Type negative-strand RNA virus or viral particle, or recombinant as described in any one of items 111-115. ​ A method comprising the step of formulating a type Sendai virus or viral particles together with at least one pharmaceutically acceptable excipient. 131. Recombination described in any one of paragraphs 70-110 ​ Type negative-strand RNA virus or viral particle, or recombinant as described in any one of items 111-115. ​A method for producing type Sendai virus or virus particles, (a) Transfect eukaryotic host cells with the DNA molecules described in item 127, (b) Culturing host cells under conditions such that the DNA molecule is transcribed and virus particles are formed, and (c) Isolating the virus particles in (b) Methods that include... 132. Recombinant organisms as described in any one of paragraphs 70 to 110. ​ Type negative-strand RNA virus or viral particle, or recombinant as described in any one of items 111-115. ​ Use of host cells as described in item 128 for the production of type Sendai virus or viral particles. 133. Recombinant compounds as described in any one of sub-sub ​ Type negative-strand RNA virus or viral particle, or recombinant as described in any one of items 111-115. ​ Use of type Sendai virus or virus particles. Further aspects of the present invention are described below: [Section 1] A recombinant replication-deficient negative-strand RNA virus, in which its genome contains, (a) a nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in a replication defect in the negative-strand RNA virus; and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Includes, Here, the heterologous polypeptide comprises a viral antigen, and the viral antigen and negative-strand RNA virus are selected from different families of viruses. Recombinant replication-deficient negative-strand RNA virus. [Section 2] Modifications in the nucleotide sequence (a) (i) Compared to the wild type, deletion of the nucleotide sequence encoding amino acids 2-77 of the P polypeptide, or (ii) A partial sequence of (i) that causes replication defects in RNA viruses The recombinant replication-deficient negative-strand RNA virus described in item 1. [Section 3] A recombinant, reproducible negative-strand RNA virus, whose genome contains, (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein has an innate function in the viral replication cycle. (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Includes, Here, the heterologous polypeptide comprises a viral antigen, and the viral antigen and negative-strand RNA virus are selected from different families of viruses. Recombinant, reproducible negative-strand RNA virus. [Section 4] A weakened recombinant, reproducible negative-strand RNA virus as described in item 3. [Section 5] A recombinant replication-deficient or replication-capable negative-strand RNA virus as described in any one of items 1 to 4, having reduced anti-vector antigenicity. [Section 6] A Sendai virus, which is a recombinant replication-deficient or replication-capable negative-strand RNA virus as described in any one of items 1 to 5. [Section 7] A transcriptionally transcribed recombinant replication-deficient or replication-capable negative-strand RNA virus as described in any one of items 1 to 6. [Section 8] A recombinant replication-deficient or replication-capable minus-strand RNA virus as described in any one of items 1 to 7, wherein the heterologous polypeptide comprises the coronavirus spike protein (S protein) or an immunogenic fragment thereof. [Section 9] A recombinant replication-deficient or replication-capable minus-strand RNA virus as described in any one of items 1 to 8, wherein the endogenous sequence encoding the F polypeptide and / or the endogenous sequence encoding the HN polypeptide is modified such that their function or activity is at least partially or completely disrupted, and the nucleotide sequence (b) is inserted into the locus of the F gene and / or the HN gene. [Section 10] A recombinant replication-deficient or replication-capable negative-strand RNA virus as described in any one of items 1 to 9, which can adsorb to a host cell and invade the host cell when presented on the viral surface. [Section 11] The nucleotide sequence (b) is as follows: A. (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) A transmembrane domain of a SeVF polypeptide or an HN polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of SeV polypeptide F or HN polypeptide, or a fragment thereof or B.(i) Ectodomain of SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of SeV polypeptide F or HN polypeptide, or a fragment thereof or C.(i) Ectodomain of SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of the SARS-CoV-2 spike polypeptide, or a fragment thereof. A recombinant replication-deficient or replication-capable minus-strand RNA virus, as described in any one of items 1 to 10, which codes for a specific RNA virus. [Section 12] A recombinant replication-deficient or replication-capable minus-strand RNA virus according to any one of claims 1 to 11, capable of inducing a mucosal immune response to at least one heterologous polypeptide encoded by at least one nucleotide sequence (b). [Section 13] A pharmaceutical composition comprising a recombinant replication-deficient or replication-capable negative-strand RNA virus as described in any one of items 1 to 12. [Section 14] A recombinant replication-deficient or replication-capable negative-strand RNA virus as described in any one of items 1 to 12, or a pharmaceutical composition as described in item 13, for use in a method of preventing SARS-CoV-2 infection. [Section 15] A recombinant replication-deficient or replication-capable negative-strand RNA virus as described in any one of items 1 to 12, or a pharmaceutical composition as described in item 13 for use as described in item 14, administered intranasally and / or via a mucosal route. [Section 16] An RNA molecule or DNA molecule encoding a recombinant replication-deficient minus-strand RNA virus or a replication-capable minus-strand RNA virus as described in any one of items 1 to 12. [Section 17] A nucleocapsid containing the RNA molecule described in item 16. [Section 18] A host cell comprising a replication-deficient or replication-capable negative-strand RNA virus as described in any one of items 1 to 12, an RNA molecule as described in item 16, a nucleocapsid as described in item 17, and / or a DNA molecule as described in item 16.

Claims

1. A recombinant replication-deficient negative-strand RNA virus, in which its genome contains, (a) a nucleotide sequence encoding a P polypeptide, wherein the P polypeptide is modified compared to a wild-type P polypeptide, the modification resulting in a replication defect in the negative-strand RNA virus; and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Includes, Here, the heterologous polypeptide comprises a viral antigen, and the viral antigen and negative-strand RNA virus are selected from different families of viruses. Recombinant replication-deficient negative-strand RNA virus.

2. Modifications in the nucleotide sequence (a) (i) Deletion of the nucleotide sequence encoding amino acids 2-77 of the P polypeptide compared to the wild-type P polypeptide, or (ii) Deletion, insertion, or substitution of the nucleotide sequence encoding amino acids 33-41 of the wild-type P polypeptide. The recombinant replication-deficient negative-strand RNA virus according to claim 1.

3. A recombinant, reproducible negative-strand RNA virus, whose genome contains, (a) A nucleotide sequence encoding a P polypeptide, wherein the P polypeptide has the same function as the wild-type P polypeptide in the viral replication cycle, or is a wild-type P polypeptide having the sequence of Sequence ID No. 2, and (b) A nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. Includes, Here, the heterologous polypeptide comprises a viral antigen, and the viral antigen and negative-strand RNA virus are selected from different families of viruses. Recombinant, reproducible negative-strand RNA virus.

4. A recombinant, reproducible negative-strand RNA virus according to claim 3, which has been weakened.

5. A recombinant replication-deficient or replication-capable minus-strand RNA virus according to any one of claims 1 to 4, wherein no fusion (F) polypeptide and / or hemagglutinin-neuraminidase (HN) polypeptide are presented on the surface of the viral particle.

6. A Sendai virus, which is a recombinant replication-deficient or replication-capable negative-strand RNA virus according to any one of claims 1 to 4.

7. A recombinant replication-deficient or replication-capable minus-strand RNA virus according to any one of claims 1 to 4, wherein the virus is transcriptional or the wild-type P polypeptide has the sequence of SEQ ID NO:

2.

8. A recombinant replication-deficient or replication-capable minus-strand RNA virus according to any one of claims 1 to 4, wherein the heterologous polypeptide comprises the spike protein (S protein) or an immunogenic fragment thereof of a coronavirus.

9. The recombinant replication-deficient or replication-capable minus-strand RNA virus according to any one of claims 1 to 4, wherein the endogenous sequence encoding the F polypeptide and / or the endogenous sequence encoding the HN polypeptide are modified such that their function or activity is at least partially or completely disrupted, and the nucleotide sequence (b) is inserted into the locus of the F gene and / or the HN gene.

10. A recombinant replication-deficient or replication-capable negative-strand RNA virus according to any one of claims 1 to 4, which can adsorb to a host cell and invade the host cell when presented on the surface of the virus.

11. The nucleotide sequence (b) is as follows: A. (i) Ectodomain of SARS-CoV-2 spike polypeptide, or a fragment thereof (ii) A transmembrane domain of a SeVF polypeptide or an HN polypeptide, or a fragment thereof, and (iii) Cytoplasmic domain of SeV polypeptide F or HN polypeptide, or a fragment thereof or B. (i) Ectodomain of SARS-CoV-2 spike polypeptide, or a fragment thereof (ii) Transmembrane domain of SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) Cytoplasmic domain of SeV polypeptide F or HN polypeptide, or a fragment thereof or C. (i) Ectodomain of SARS-CoV-2 spike polypeptide, or a fragment thereof (ii) Transmembrane domain of SARS-CoV-2 spike polypeptide, or a fragment thereof, and (iii) Cytoplasmic domain of SARS-CoV-2 spike polypeptide, or fragment thereof A recombinant replication-deficient or replication-capable negative-strand RNA virus according to claim 8, which encodes the virus.

12. A recombinant replication-deficient or replication-capable negative-strand RNA virus according to any one of claims 1 to 4, which can induce a mucosal immune response to at least one heterologous polypeptide encoded by at least one nucleotide sequence (b).

13. A pharmaceutical composition comprising a recombinant replication-deficient or replication-capable negative-strand RNA virus as described in any one of claims 1 to 4.

14. The pharmaceutical composition according to claim 13 for use in a method for preventing SARS-CoV-2 infection.

15. A recombinant replication-deficient or replication-capable negative-strand RNA virus according to any one of claims 1 to 4, formulated for administration via the nasal cavity and / or mucosal route.

16. An RNA molecule or DNA molecule encoding a recombinant replication-deficient negative-strand RNA virus or a replication-capable negative-strand RNA virus according to any one of claims 1 to 4.

17. A nucleocapsid comprising the RNA molecule described in claim 16.

18. A host cell comprising a replication-deficient or replication-capable negative-strand RNA virus according to any one of claims 1 to 4.

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