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 competent (replicative) or replication-defective minus-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 host cells, 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-defective minus-strand RNA virus comprising, in its genome, (a) a nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, the modification resulting in replication deficiency of the minus-strand RNA virus, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle and can be expressed in infected cells.

[0003] Another aspect of the present invention relates to a recombinant competent minus-strand RNA virus comprising, in its genome, (a) a nucleotide sequence encoding a P polypeptide, wherein the P protein performs its original function in the viral replication cycle, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle and can be expressed and replicated in infected cells.

[0004] Other aspects of the invention relate to pharmaceutical compositions, vaccines and immunogenic compositions comprising a recombinant competent or replication-deficient negative-strand RNA virus of the invention. Further aspects of the invention relate to RNA molecules, and DNA molecules encoding a recombinant competent or replication-deficient negative-strand RNA virus of the invention. Another aspect relates to a nucleocapsid comprising an RNA molecule of the invention. BACKGROUND OF THE INVENTION

[0005] Background History has taught that it is often a second-generation vaccine that brings epoch-making progress in the complete control of a persistently spreading viral infection. Even in the case of the polio (poliomyelitis) countermeasure 70 years ago, sustained success was achieved only by a second-generation vaccine that mimicked the natural enteric-mucosal infection route. The first-generation vaccines developed until then could only be injected into the muscle of the upper arm at that time, but in the case of the enteric mucosal injection route of the poliovirus, it was not possible to prevent the infection of the poliovirus replicated in the digestive tract, so "sterilizing immunity" could not be obtained. Therefore, vaccinated people continued to be infected with the poliovirus, and the progeny virus produced in the intestine could transmit the disease to other people via enteric excreta (feces). For this reason, the first-generation vaccines could not establish sufficient herd immunity.

[0006] An overview of COVID-19 vaccines and candidate vaccines can be found on the WHO homepage (“COVID-19 Vaccine Tracker and Landscape”:[[]] https: / / www.who.int / publications / m / item / draft-landscape-of-covid-19-candidate-vaccines https: / / www.who.int / publications / m / item / draft-landscape-of-covid-19-candidate-vaccines ). According to the WHO definition, all anti-SARS-CoV-2 vaccines approved so far belong to the first generation. These drugs are administered intramuscularly, usually by injection into the muscle of the upper arm. In the case of an infection such as SARS-CoV-2, it does not enter the body from the respiratory organ. The first-generation SARS-CoV-2 vaccines elicit an immune response sufficient to mostly prevent the severe progression of COVID-19, but sufficient immunity against the respiratory tract is not obtained.

[0007] This means that vaccinated individuals are still at risk of infection (so-called breakthrough infections are frequently observed). There is a possibility of infecting unvaccinated individuals through droplet infection. This means that the first-generation SARS-CoV-2 vaccines approved so far cannot effectively interrupt the chain of infection. Therefore, there is a strong need for a SARS-CoV-2 vaccine that induces "sterilizing immunity" against the respiratory system for COVID-19.

[0008] Breakthrough infections occur frequently and can burden hospitals. The urgently needed herd immunity is still a long way off. Returning to "normal" life before the pandemic has not been successful, and it affects all aspects 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 particles. In contrast to coronaviruses, adenovirus-based vaccines do not immediately provide recombinant S protein as an antigen. Rather, after intramuscular injection, S protein must be produced in muscle cells infected with the vector vaccine, resulting in a delayed immune response and a decrease in efficiency, especially in the development of tracheal mucosal immunity. The same is true for intramuscularly administered mRNA-based vaccines.

[0010] If the immunological defense of the anti-SARS-CoV-2 vaccine does not last sufficiently, repeated administration is essential. Currently, more than three vaccinations are considered necessary, for example, receiving a booster vaccination once every six months. Furthermore, newly emerging virus subtypes require the administration of adapted vaccines, so repeated administration is necessary. In the case of vector vaccines, the adenovirus capsid has strong immunogenicity and brings about significant anti-vector immunity (anti-adenovirus immunity). When readministering an adenovirus vector, such anti-vector immunity neutralizes the vector, reducing its effectiveness, and the option of administering first-generation vector vaccines according to a schedule that requires more than two vaccinations may be limited. If anti-vector immunity is significant, the effect of booster immunity is neutralized, resulting in loss of effectiveness or the need for high-dose administration, increasing the labor and cost involved in production, as well as the risk of side effects.

[0011] Therefore, there is a need for a SARS-CoV-2 vaccine that at least partially overcomes the drawbacks of the above first-generation vaccines.

[0012] Paramyxoviruses, particularly Sendai virus, are enveloped viruses with a helical nucleocapsid. The envelope contains a lipid membrane and is derived from the cell membrane of the host cell from which the virus is released. Transmembrane glycoproteins, namely the fusion protein (F) and hemagglutinin-neuraminidase (HN), are anchored to the viral envelope. The matrix protein (M) is located inside the membrane. The nucleocapsid, i.e., the viral replication complex, consists of single-stranded RNA complexed with nucleoprotein (N), and in each case, 6 nucleotides of the RNA are bound by 1 N protein, the RNA-dependent RNA polymerase (L), and the phosphoprotein (p) of the coenzyme, forming an RNA polymerase complex. During primary transcription, the genome is transcribed by the viral RNA-dependent RNA polymerase (vRdRp), and de novo protein synthesis begins. As soon as a sufficient amount of N protein is produced, vRdRp is thought to switch to the replication mode and synthesize the antigenome. From that template, new genomes encapsulated with N protein are replicated.

[0013] The genomic RNA of a virus family, namely the negative-strand RNA virus family, infects cells and, although not immediately translated after the RNA is released into the cell, usually forms a complex with a protein called the nucleoprotein (N protein or NP). Only the complex of this viral RNA and the N protein is recognized by the viral polymerase as a template for viral genome replication or viral transcription. During replication of the viral genome, newly synthesized genomes or antigens immediately complex with the N protein. During viral transcription, mRNA that is not complexed with the N protein is synthesized. This inhibits translation by ribosomes. That is, only the viral genome / antigenome forms a complex with the N protein. Another viral protein, the P protein, is involved in the process of complexing the viral RNA and the N protein. The P protein itself forms a complex with the N protein. Only this N-P complex enables the complexing of the viral RNA and the N protein. If this complex between N and P is not formed due to mutations in the interaction sites on the N or P protein, etc., a complex of newly synthesized viral genomes / antigenomes is not formed. A functional new viral genome cannot be generated, and thus new functional viral particles cannot be generated either. The virus can become replication-deficient. 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 negative-strand RNA genome of Sendai virus contains the genes for six structural proteins in the following order: 3’-N-P-M-F-HN-L-5’. The P gene encodes a total of eight proteins, the phosphorylated protein which is a structural protein and all non-structural proteins known to date.

[0015] The 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 transcription-competent Sendai viruses (SeV), which have been used for the expression of transgenes.

[0017] Wiegand (Journal of Virology 2017:91(10), e02298-16) showed that the ectodomain of the surface protein F of SeV can be replaced with the corresponding ectodomain of the RSV virus F. The replacement of the SeV F protein with the RSV F protein is an "interfamiliar" replacement of sequences that are functionally and structurally equivalent.

[0018] To date, there have been no tests demonstrating such "interfamiliar" replacements of functionally essential proteins between completely different virus families such as paramyxoviruses and coronaviruses without loss of function.

[0019] Attenuation of virus replication means that a particular vector construct is not under optimal conditions as compared to the natural / wild type. While the latter has adapted to the preferred host organism or cell type during the process of evolution and achieved a high virus replication rate, the attenuated virus vector only generates non-optimal titers during replication. There are various reasons why attenuation and non-optimal replication can occur. Attenuation can occur in the virus vector itself, the host system, the conditions during replication, or a combination of these factors. Of course, changes in the virus genome can potentially have a significant impact on the replication behavior of the virus. This is because the introduced mutations are likely not optimized in the way the virus is naturally selected. The effects of single or multiple point mutations are usually already clearly recognizable depending on the gene or gene product affected. Mutations that affect the function of virus enzymes involved in virus replication can have a major impact on virus replication. For example, an N-terminal deletion in the Sendai virus P gene can cause replication deficiency because the L protein cannot interact with its cofactor, the P protein, in the form necessary for the L protein to interact with the virus genome and mediate the synthesis of copies of the virus RNA genome. 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 virus particles during virus propagation 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, be taken up into the cell, and mediate uptake mechanisms such as membrane fusion or uptake into vesicles by the cell. The uptake strategies vary depending on the virus. For example, the Sendai virus HN and F proteins in the membranous envelope of the virus particle enable binding to receptors and then enable fusion of the virus membrane with the host cell membrane, thereby releasing the virus genome into the host cell. SARS-CoV-2, rather, enters the host cell 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 the exchange of entire envelope proteins between genomes of different virus families, can have a major impact on the normal viral life cycle and its growth efficiency. Summary of the Invention

[0020] Detailed Description The inventors have developed a second-generation vaccine (particularly a vaccine against SARS-CoV-2) that meets the following requirements: · The vaccine vector shows maximum attenuation and is safe, so it can be used not only in healthy subjects but also in pre-disease, partially immunosuppressed subjects. · This vector should be able to efficiently penetrate the mucosal mucus layer and is thus suitable as a vaccine vector for inducing respiratory mucosal immunity. · The vaccine vector is administered via the natural infection route (particularly the respiratory mucosal route in the case of anti-SARS-CoV-2 vaccines). · The vaccine vector induces immediate mucosal immunity by presenting antigens on the virus surface (particularly spike (S) protein in the case of 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 causes a systemic immune response.

[0021] The inventors have developed a vector-based vaccine carrying the SARS-CoV-2 spike protein on the surface of vector vaccine particles according to the above requirements. With this design, the protein is directly presented to the immune system during the intranasal / mucosal application process, similar to the natural infection of the respiratory tract by SARS-CoV-2. Furthermore, when the virus invades the 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, particularly protection including "sterilizing immunity", is achieved and ensured.

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

[0023] Sendai virus (SeV, Paramyxoviridae) carries two proteins on its surface: (i) hemagglutinin-neuraminidase (HN) protein, and (ii) 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 virus membrane and the cytoplasmic membrane. After membrane fusion (penetration), the SeV replication complex (nucleocapsid) is released into the cell.

[0024] Both the biologically active forms of HN and F present on the surface of SeV are 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 beginning of the inventors' test, it was unclear whether recombinant proteins obtained from other virus families (such as the S protein of SARS-CoV-2, which is a member of the coronavirus family) could be incorporated into the envelope of the SeV vaccine vector (Paramyxoviridae) without significantly affecting the HN and F protein functions (adsorption and penetration) of SeV in the infection of target cells.

[0026] Moreover, it was completely unclear what modifications of the recombinant protein (e.g., chimeric SARS-CoV-2 S protein) were necessary to be successfully incorporated into the SeV envelope and presented on the surface, what modifications were acceptable, and whether they would have no impact on the biological functions of the recombinant protein (e.g., antigenicity of SARS-CoV-2 and functionality in the infection process).

[0027] The only polypeptide present on the surface of SARS-CoV-2 is the spike protein S. The S protein exists in an 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 immobilized on the envelope of one SARS-CoV-2 particle at an average distance of 15 nm. At the beginning of the inventors' tests, it was unclear whether the distance on the surface of the S protein was essential for function, whether the S protein would function in combination with SeV F and HN when recombinantly incorporated into the envelope, or whether the complex presentation on the SeV surface would result in partial or complete loss of function.

[0028] Furthermore, it was unclear whether the recombinant SARS-CoV-2 S protein could complement the functions of the F and / or HN polypeptides when part or all of the sequences encoding the F and / or HN polypeptides were deleted from the SeV genome.

[0029] From these points, it is clear that the design and construction of SeV vector-based vaccines against SARS-CoV-2 are by no means trivial and simple, and require considerable ingenuity.

[0030] The inventors constructed a recombinant Sendai virus presenting a recombinant antigen (e.g., SARS-CoV-2 spike protein) obtained from SARS-CoV-2 on its 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, and the transmembrane domain of the SeV polypeptide or SARS-CoV-2 spike. The inventors showed that the vaccine candidate provided as cDNA was obtained from GMP-compliant Vero cells and could be propagated in Vero cell-derived V3-10 helper cells. The inventors showed that the S protein of SARS-CoV-2 was incorporated into the candidate virus particles.

[0031] The inventors discovered that the recombinant SARS-CoV-2 S protein was presented in a functional form on the surface of SeV. The inventors provided a SeV vaccine vector in which (i) the amount of surface F and / or HN protein was reduced, or (ii) the F and HN proteins were absent. The inventors recognized that the S protein of coronaviruses (positive-strand) can complement the adsorption and entry capabilities of negative-strand RNA viruses when the natural (endogenous) genes mediating these capabilities are deleted in these viruses. In particular, the adsorption and penetration capabilities of the negative-strand paramyxovirus F and HN proteins can be complemented by the S protein of positive-strand coronaviruses having adsorption and penetration capabilities. This is the first example showing that two endogenous F and HN proteins of a paramyxovirus (SeV) have been functionally "interspecies" replaced by a single coronavirus protein.

[0032] The inventors found that attenuation of Sendai virus can be achieved by modifying the P protein, resulting in the obtainment of replication-deficient viruses.

[0033] The present inventors have also found that in SeV having replication ability, 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 attenuation can be achieved such that the S protein of the fragment can perform the functions of the F and / or HN proteins. The present inventors have also found that such a modified SeV having replication ability has an increased replication efficiency compared to the replication-deficient SeV of the present invention (Example 4).

[0034] The present inventors have also found that a specific mucosal immune response against 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 vaccinated subject has already acquired immunity against SARS-CoV-2 in the respiratory tract, for example, due to SARS-CoV-2 infection, the subject's immune system can recognize a recombinant competent or replication-deficient negative-strand RNA virus vaccine vector, particularly a SeV vaccine vector against SARS-CoV-2, and the vaccine can act as a booster immunity. If a vaccinated subject has not yet acquired immunity against SARS-CoV-2 in the respiratory tract, the vaccine vector can induce immunity against SARS-CoV-2 through different immune stimulation pathways and interactions with the immune system. First, since the vaccine vector particles present spike proteins on their surface, they are themselves immunogenic against SARS-CoV-2. In this way, the vaccine vector functions as, for example, a nanoparticle vaccine or a virus-like particle vaccine (VLP). 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 part of the spike protein to T lymphocytes, which can then differentiate into helper cells and effector cells. Second, the vaccine vector can infect cells such as those in the respiratory tract and express the encoded spike protein intracellularly. As a result, spike proteins are expressed on the surface of these cells, recognized by the immune system as being infected, and after standard proteolysis within these cells, peptides of the spike protein can be presented on MHC-I molecules.

[0036] In view of the experimental evidence obtained using paramyxoviruses (e.g., SeV) that recombinantly present an antigen (e.g., the SARS-CoV-2 S protein) on the surface, the inventors concluded that a recombinant negative-strand RNA virus vector carrying a recombinant antigen and having little or no expression of anti-vector immunity can be constructed.

[0037] In a first aspect, the present invention has, in its genome, 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 replication deficiency of the negative-strand RNA virus, and (b) At least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle, a nucleotide sequence (relates to a recombinant replication-deficient negative-strand RNA virus comprising the same).

[0038] In the present invention, the term "virus" means an infectious substance that replicates only within living cells. Viruses have no independent metabolic mechanism. As used herein, the term "virus" includes all forms within host cells and virus particles (also called virions) outside host cells, i.e., those released from host cells and capable of infecting new host cells. RNA viruses may be single-stranded RNA viruses or double-stranded RNA viruses.

[0039] In the present invention, the terms "genome" or "viral genome" are used interchangeably and mean the entire nucleic acid present in the nucleocapsid. The genome can be non-segmented (a single nucleic acid molecule) or segmented (two or more nucleic acid molecules). Single-stranded viruses may be plus-strand viruses or minus-strand viruses. In minus-strand viruses, the nucleic acid complementary to the genome is called the antigenome. In minus-strand viruses, genes are transcribed into mRNA, which is then translated into polypeptides. In plus-strand viruses, the nucleic acid complementary to the genome is called the genome. In plus-strand viruses, the genome sequence is directly translated into polypeptides. In the present invention, the genome may be a recombinant genome. The recombinant replication-deficient negative-strand RNA virus described herein may contain a recombinant genome. The recombinant genome may contain a recombinant RNA molecule.

[0040] In the present invention, the terms "inter-family" and "intrafamiliar" refer to the relationship between viruses of different viral families or the same viral family. For example, an inter-family substitution of a nucleotide sequence is an exchange between viruses of different families, and an intrafamiliar substitution of a nucleotide sequence is an exchange between viruses of the same family.

[0041] As used herein, the term "heterologous" or "foreign" means a polypeptide and / or nucleic acid that is foreign to a particular virus, such as a negative-strand RNA virus. Heterologous (foreign) polypeptides and / or nucleic acids do not naturally occur in a particular virus and are introduced into the viral genome by artificial or recombinant means. For example, a heterologous (foreign) nucleotide sequence is a foreign sequence introduced into the viral genome. A heterologous (foreign) nucleotide sequence may be operably linked to a natural or endogenous viral sequence, such as an expression control sequence. A heterologous (foreign) nucleotide sequence can also be inserted in-frame into a natural or endogenous viral coding sequence so as to express a fusion protein or chimeric protein containing a heterologous (foreign) 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 original function particularly in the viral replication cycle, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the viral particle, comprising wherein the heterologous polypeptide comprises a viral antigen, and the viral antigen and the negative-strand RNA virus are selected from viruses of different families, relates to a recombinant replication-competent negative-strand RNA virus.

[0043] In particular, the recombinant competent negative-strand RNA virus is attenuated. In the recombinant competent negative-strand RNA virus, the P protein can 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 thereto. The wild-type P protein can have an amino acid sequence comprising 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 thereto.

[0044] In the present invention, the term "attenuated virus" relates to a virus with reduced or no pathogenicity, particularly a virus in which most of its replication is reduced or absent. An attenuated virus can induce an immune response without causing the specific disease caused by the non-attenuated virus.

[0045] In particular, the present invention also relates to replication-deficient negative-strand RNA viruses. Loss of replication ability means that in target cells that do not trans-produce the function deleted in the virus, no detectable viral genome replication is observed, in contrast to reduced replication ability or conditional replication deficiency, where there are no permissive conditions under which replication occurs. As used herein, the negative-strand RNA virus of the present invention is still infectious, i.e., it can adsorb to and enter the host cell. The infectious negative-strand RNA virus as used herein can also induce the expression of at least a heterologous gene in the host cell.

[0046] As used herein, the “recombinant-competent or replication-deficient minus-strand RNA virus according to the present invention” refers to two different viruses of the present invention: (i) a recombinant-competent minus-strand RNA virus, and (ii) a recombinant replication-deficient minus-strand RNA virus. The term “recombinant-competent or replication-deficient minus-strand RNA virus according to the present invention” or similar expressions (e.g., with respect to a specific virus species) are used herein, in particular, to describe features that can exist independently in (i) a recombinant-competent minus-strand RNA virus and (ii) a recombinant replication-deficient minus-strand RNA virus. Embodiments of the present invention regarding recombinant replication-deficient minus-strand RNA viruses are described in particular in paragraphs 1 to 69. Embodiments of the present invention regarding recombinant-competent minus-strand RNA viruses are described in particular in paragraphs 70 to 133.

[0047] The recombinant virus according to the present invention may be transcription-competent. That is, the virus can induce viral and heterologous mRNA synthesis and can be translated into viral polypeptides and heterologous polypeptides. In particular, the gene products encoded by the virus are transcribed after infection of the target cells, and the expression of viral proteins containing one or more heterologous gene products is carried out in the target cells. The heterologous polypeptide is presented on the surface of the target cells, i.e., the mucosal cells of the vaccinated subject, thereby inducing and / or improving the immune response against the heterologous polypeptide.

[0048] Transcription can include primary transcription and secondary transcription. As used herein, primary transcription begins with the minimum replication unit, i.e., the nucleocapsid that has penetrated the cell. The nucleocapsid contains a single-stranded RNA molecule complexed with nucleoprotein (N), RNA-dependent RNA polymerase (L), and phosphoprotein (P), and forms an RNA polymerase complex. Primary transcription can also start from a DNA molecule encoding the virus, particularly a cDNA molecule. During primary transcription, the genome is transcribed by RNA polymerase, and de novo protein synthesis begins. At the same time as the new genome is replicated, these templates are also transcribed. This stage of transcription is called secondary transcription and is much more efficient than primary transcription.

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

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

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

[0052] In the present invention, the term “P polypeptide” means the phosphoprotein present in negative-strand RNA viruses. The P polypeptide is a cofactor for the RNA-dependent RNA polymerase of these viruses and is involved in viral transcription and replication.

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

[0054] The recombinant competent or replication-deficient negative-strand RNA virus according to the present invention can be based on a natural negative-strand RNA virus. The recombinant competent or replication-deficient negative-strand RNA virus according to the present invention can be selected from the order Mononegavirales. Preferred families to which the recombinant competent or replication-deficient negative-strand RNA virus of the present invention can be based are the families Artoviridae, Bornaviridae, Filoviridae, Respirovirus, Mymonaviridae, Nyamiviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Sunviridae and Simoviridae. Preferred families to which the recombinant competent or replication-deficient negative-strand RNA virus of the present invention can be based are the families Paramyxoviridae (paramyxovirus), Rhabdoviridae (rhabdovirus), Filoviridae (Filoviridae), Bornaviridae (Bornaviridae), and their recombinant variants.

[0055] In the present invention, examples of single-stranded non-segmented negative-strand RNA viruses include the families Paramyxoviridae (paramyxovirus), Rhabdoviridae (rhabdovirus), Filoviridae (filovirus) and Bornaviridae (bornavirus).

[0056] Examples of paramyxoviruses include, but are not limited to, Sendai virus, human and bovine parainfluenza viruses such as human parainfluenza virus (hPIV) type 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, vesicular stomatitis virus (VSV).

[0057] Preferably, the recombinant competent or replication-deficient negative-strand RNA virus is a paramyxovirus. Preferably, the recombinant competent or replication-deficient negative-strand RNA virus of the present invention may be Sendai virus, such as the 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 encompassed by the present invention.

[0058] In the present invention, single-stranded positive-strand viruses include the family Coronaviridae. Examples of coronaviruses include MERS-CoV (Middle East respiratory syndrome coronavirus), SARS-CoV (severe acute respiratory syndrome-related coronavirus, also called SARS-CoV-1), SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2).

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

[0060] In the present invention, the term "viral envelope", if present, relates to the outer layer of a virus. The envelope can be derived from the host cell membrane and contains viral peptides, such as peptides that can adsorb to receptors of the host cell and mediate entry into the cell. These peptides can be anchored to the envelope by transmembrane domains and intracellular domains. In the present invention, the envelope of a recombinant competent or replication-deficient minus-strand RNA can 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 the present invention, the terms "peptide" and "polypeptide" can be used interchangeably.

[0062] In the present invention, the terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" can be used interchangeably.

[0063] In the present invention, the term "viral peptide" relates to peptides required for nucleic acid synthesis (e.g., transcription or replication, such as polymerase or polymerase complex), envelope structure (if an envelope is present), nucleocapsid structure, and surface structure (required for adsorption to and penetration into the host cell). There is no unified term for viral peptides and the genes encoding such peptides. In the present invention, references to specific genes and / or peptides in a particular virus or viral family are intended to include references to homologous genes and / or peptides in other virus species and / or families, particularly genes and / or peptides having the same function (also referred to herein as "functional homologs"). For example, a reference to the P peptide described herein particularly for Sendai virus includes a reference to any of the peptides described for single-stranded minus-strand RNA viruses that have essentially the same function as the P peptide of Sendai virus.

[0064] In the present invention, the nucleotide sequence (a) encoding the P polypeptide (also referred to as the P protein or phosphoprotein) may be modified compared to the wild type (i.e., the wild-type P sequence), and said modification results in replication deficiency of the negative-strand RNA virus. As used herein, the term "P polypeptide" refers to a polypeptide of a negative-strand single-stranded RNA virus that has essentially the same function as the P polypeptide of paramyxoviruses, particularly Sendai virus. As exemplary non-limiting examples of the P polypeptide (SEQ ID NO: 2) and the sequence encoding the P polypeptide (SEQ ID NO: 1), reference is made herein to the Sendai virus sequence encoding the P polypeptide.

[0065] As used herein, "modification" includes deletions, insertions, and / or substitutions in a nucleotide sequence or polynucleotide. When a nucleotide sequence includes a coding sequence, such modifications can result in a modified polypeptide.

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

[0067] In particular, the modification of the nucleotide sequence (a) is a deletion of the nucleotide sequence encoding the N-terminus of the P polypeptide. More specifically, the modification in the nucleotide sequence (a) is as follows: (i) Deletion of the nucleotide sequence encoding amino acids 2 - 77 of the P polypeptide ("deltaP 2 - 77", SEQ ID NO: 4) compared to the wild type, or (ii) The partial sequence of (i) that causes replication deficiency of RNA virus.

[0068] SEQ ID NO: 3 describes the nucleotide sequence encoding the SeV P protein lacking amino acids 2-77 of SEQ ID NO: 4.

[0069] In particular, there is no modification that impairs the transcriptional function in the C-terminal region (starting from amino acid 320) of the P polypeptide.

[0070] In the replication-competent or replication-deficient negative-strand RNA virus having replication ability described herein, the genome may further include the following: (c) A nucleotide sequence encoding a negative-strand RNA virus N polypeptide, (d) A nucleotide sequence encoding a negative-strand RNA virus M polypeptide, and (e) A nucleotide sequence encoding a negative-strand RNA virus L polypeptide.

[0071] As used herein, the terms "N polypeptide", "N protein" or "nucleocapsid protein" refer to a polypeptide of a negative-strand single-stranded RNA virus that has essentially the same function as the N polypeptide of paramyxovirus, particularly Sendai virus. Those skilled in the art know the appropriate N polypeptide of paramyxovirus and its function. In the nucleocapsid of a single-stranded RNA virus, the RNA molecule forms a complex with the N polypeptide. In this specification, the reference to the N polypeptide is not limited to paramyxovirus, but also includes functional and structural homologs of other virus families.

[0072] As used herein, the terms "M polypeptide", "M protein" or "matrix protein" refer to a polypeptide of a negative-sense single-stranded RNA virus that has essentially the same function as the M polypeptide of a paramyxovirus, particularly Sendai virus. Those skilled in the art know the appropriate M polypeptide of a paramyxovirus and its function. The M polypeptide is present on the inner surface of the viral envelope. As used herein, reference to an M polypeptide is not limited to paramyxoviruses, but also includes functional and structural homologs of other virus families.

[0073] As used herein, the terms "L polypeptide", "L protein" or "large protein" refer to a polypeptide of a negative-sense single-stranded RNA virus that has essentially the same function as the L polypeptide of a paramyxovirus, particularly Sendai virus. Those skilled in the art know the appropriate L polypeptide of a paramyxovirus and its function. The L polypeptide is the largest protein in the polymerase complex and has RNA-dependent polymerase activity. As used herein, reference to an L polypeptide is not limited to paramyxoviruses, but also includes functional and structural homologs of other virus families.

[0074] Furthermore, the genome of a recombinant-competent or replication-deficient negative-sense RNA virus may further comprise the following. (f) a nucleotide sequence encoding a negative-sense RNA virus F polypeptide, and (g) a nucleotide sequence encoding a negative-sense RNA virus HN polypeptide.

[0075] As used herein, the terms "F polypeptide", "F protein" or "fusion protein" refer to a polypeptide of a negative-sense single-stranded RNA virus that has essentially the same function as the F polypeptide of a paramyxovirus, particularly Sendai virus. Those skilled in the art know the appropriate F polypeptide of a paramyxovirus and its function. The F polypeptide mediates the fusion of viral particles with the host cell membrane. As used herein, the reference to an F polypeptide is not limited to paramyxoviruses, but also includes functional and structural homologs in other virus families.

[0076] As used herein, an F polypeptide or a sequence encoding an F polypeptide may include the sequences described herein.

[0077] SEQ ID NO: 6 describes the amino acid sequence of the Sendai virus (SeV) SeV F polypeptide. SEQ ID NO: 5 describes the nucleotide sequence encoding the Sendai virus (SeV) SeV F polypeptide of SEQ ID NO: 6.

[0078] As used herein, the terms "HN polypeptide", "HN protein" or "hemagglutinin-neuraminidase" refer to a polypeptide of a negative-sense single-stranded RNA virus that has essentially the same function as the HN polypeptide of a paramyxovirus, particularly Sendai virus. Those skilled in the art know the appropriate HN polypeptide of a paramyxovirus and its function. The HN polypeptide mediates the adsorption of viral particles to host cells. As used herein, the reference to an HN polypeptide is not limited to paramyxoviruses, but also includes functional and structural homologs in other virus families.

[0079] As used herein, an HN polypeptide or a sequence encoding an HN polypeptide may include the sequences described herein.

[0080] SEQ ID NO: 12 describes the amino acid sequence of the Sendai virus (SeV) SeV HN polypeptide. SEQ ID NO: 11 describes the nucleotide sequence encoding the Sendai virus (SeV) SeV HN polypeptide of SEQ ID NO: 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), more preferably one nucleotide sequence (b). “At least one nucleotide sequence (b)” and “nucleotide sequence (b)” may be used interchangeably herein.

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

[0083] At least one heterologous polypeptide encoded by the nucleotide sequence (b) may include a heterologous antigen, such as a heterologous antigen derived from a pathogen (such as a virus, bacterium, fungus or protozoan), a tumor antigen or a self-antigen. Those skilled in the art know antigens suitable for preparing immunogenic compositions and / or vaccines against pathogens such as viruses, bacteria, fungi, protozoa, etc. Examples of antigens suitable for preparing immunogenic compositions and / or vaccines against SARS-CoV-2 are the SARS-CoV-2 spike protein (S protein) or an immunogenic fragment thereof.

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

[0085] The heterologous polypeptide may contain an antigen derived from a virus that is not a negative-strand RNA virus.

[0086] The heterologous polypeptide may also contain an antigen derived from a positive-strand virus such as a coronavirus.

[0087] It is preferred that the single-stranded negative-strand RNA virus is a paramyxovirus such as Sendai virus, and the heterologous polypeptide contains an antigen derived from a positive-strand virus such as a coronavirus, particularly a coronavirus such as SARS CoV 2.

[0088] More preferably, the single-stranded negative-strand RNA virus is Sendai virus, and the heterologous polypeptide is an antigen derived from a coronavirus.

[0089] More preferably, the single-stranded negative-strand RNA virus is a paramyxovirus such as Sendai virus, and the heterologous polypeptide contains the spike protein (S protein) of a coronavirus, particularly SARS CoV 2, or an immunogenic fragment thereof.

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

[0091] At least one nucleotide sequence (b) may be located between two coding sequences in an RNA molecule, upstream of the 5'-terminal coding sequence and / or downstream of the 3'-terminal coding sequence. For example, in paramyxoviruses, 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. With this arrangement, at least one nucleotide sequence (b) present in the RNA molecule does not interfere with the endogenous viral sequence and its expression.

[0092] In the recombinant competent or replication-deficient RNA virus of the present invention, the genome (f) a nucleotide sequence encoding a minus-strand RNA virus F polypeptide, and (g) a nucleotide sequence encoding a minus-strand RNA virus HN polypeptide further comprises, and at least one nucleotide sequence (b) can independently be located in the RNA molecule at a position between two coding sequences, upstream of the 5'-terminal coding sequence and / or downstream of the 3'-terminal 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] In a type with replication ability or a replication-deficient negative-strand RNA virus, at least one nucleotide sequence (b) can be inserted into or at least partially replace at least one natural (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, at least one viral polypeptide is different from the P polypeptide. The viral polypeptide may be the N, M, F, HN and / or L of paramyxovirus and their homologs of other viral families. Preferably, at least one nucleotide sequence (b) can be inserted into or at least partially replace the F and / or HN of paramyxovirus and their homologs of other viral families such that the function or activity of the 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 a particular virus, particularly a negative-strand RNA virus.

[0095] One of ordinary skill in the art understands that constructs as described herein can be obtained in various ways from wild-type or modified negative-strand 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] For a replicable or replication-deficient negative-strand RNA virus, the heterologous sequences inserted without modifying the viral genes and their functions can be sequences that are not essential for propagation and / or infection. Therefore, there is a significant risk that such non-essential heterologous sequences may be lost during virus propagation, for example, due to incorrect transcription by RNA polymerase. For example, during nucleocapsid formation, 6 nucleotides of RNA can bind to one N protein in each case. Incorrect transcription cannot divide the length of the RNA molecule by 6, which may result in failure of nucleocapsid formation.

[0097] In the present invention, a replicable or replication-deficient negative-strand RNA virus may contain a genetically stable RNA molecule. As used herein, the term "genetically stable" relates to a nucleic acid molecule or genome that can be replicated and / or propagated without sequence changes during the process of replication and / or propagation. In particular, when essentially all coding sequences are essential for replication and / or propagation, i.e., when essentially all coding sequences are necessary for replication and / or propagation, the nucleic acid molecule is genetically stable. If there are changes in the base sequence, the essential base sequences may be missing, and thus the replication and / or propagation of the changed molecule may be inhibited.

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

[0099] In the recombinant competent or replication-deficient RNA virus of the present invention, the endogenous sequence encoding the F polypeptide and / or the endogenous sequence encoding the HN polypeptide can be modified such that their function or activity is at least partially or completely disrupted. The nucleotide sequence (b) can be inserted into the locus of the F and / or HN gene. Preferably, the nucleotide sequence (b) is inserted into the locus of the HN gene. More preferably, the nucleotide sequence (b) is inserted into the locus of the F gene.

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

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

[0102] For example, the sequence (b) encoding the ectodomain of the SARS-CoV-2 S protein can be inserted into the sequence encoding SeV F and / or HN, whereby the ectodomain of the SARS-CoV-2 S protein can be fused in-frame with the F transmembrane domain and / or cytoplasmic domain, and / or the HN transmembrane domain and / or cytoplasmic domain, where the function and / or activity of the native HN and / or F polypeptide is at least partially or completely disrupted.

[0103] As used herein, the transmembrane domain, cytoplasmic domain, or the sequence encoding such a domain of the F and HN polypeptides can each independently include the sequences described herein: SEQ ID NO: 8 describes the amino acid sequence of the transmembrane domain of the SeV F protein (subsequence of the SeV F protein of SEQ ID NO: 6). SEQ ID NO: 7 describes the nucleotide sequence encoding the SeV F protein transmembrane domain encoding SEQ ID NO: 8. SEQ ID NO: 10 describes the amino acid sequence of the cytoplasmic domain of the SeV F protein (subsequence of the SeV F protein of SEQ ID NO: 6). SEQ ID NO: 9 describes the nucleotide sequence encoding the SeV F protein cytoplasmic domain encoding SEQ ID NO: 10. SEQ ID NO: 16 describes the amino acid sequence of the transmembrane domain of the SeV HN protein (subsequence of the SeV HN protein of SEQ ID NO: 12). SEQ ID NO: 15 describes the nucleotide sequence encoding the SeV HN protein transmembrane domain encoding SEQ ID NO: 16. SEQ ID NO: 14 describes the amino acid sequence of the cytoplasmic domain of the SeV HN protein (a partial sequence of the SeV HN protein of SEQ ID NO: 12). SEQ ID NO: 13 describes the nucleotide sequence encoding the cytoplasmic domain of the SeV HN protein that encodes SEQ ID NO: 14. SEQ ID NO: 18 describes the amino acid sequence of the ectodomain of the SeV HN protein (a partial sequence of the SeV HN protein of SEQ ID NO: 12). SEQ ID NO: 17 describes the nucleotide sequence encoding the ectodomain of the SeV HN protein that encodes SEQ ID NO: 18.

[0104] The modified at least one coding sequence of a replication-competent or replication-defective negative-strand RNA virus can include two or more coding sequences, such as two, three, 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 includes the SeV F and HN coding sequences.

[0105] In the recombinant competent or replication-defective RNA virus of the present invention, the endogenous sequence encoding the HN polypeptide can be modified such that its function or activity is at least partially or completely disrupted. The nucleotide sequence (b) can be inserted at 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 at least partially or completely deleted. More specifically, in a recombinant competent or replication-defective RNA virus, the RNA molecule does not contain a sequence encoding the HN polypeptide. Examples of such constructs are described in the Examples ("Type B" constructs). In the recombinant competent or replication-defective RNA virus of the present invention, the endogenous sequence encoding the HN polypeptide can be at least partially or completely deleted, and the nucleotide sequence (b) can be inserted at the locus of the HN gene.

[0107] In the recombinant competent or replication-defective RNA virus of the present invention, the endogenous sequence encoding the F polypeptide can be modified such that its function or activity is at least partially or completely disrupted. The nucleotide sequence (b) can be inserted at 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 at least partially or completely deleted. More specifically, in the recombinant competent or replication-defective RNA virus, the RNA molecule does not contain the sequence encoding the F polypeptide. In the recombinant competent or replication-defective RNA virus of the present invention, the endogenous sequence encoding the F polypeptide can be at least partially or completely deleted, and the nucleotide sequence (b) may be inserted at the locus of the F gene.

[0109] In the recombinant competent or replication-defective RNA virus of the present invention, the endogenous sequence encoding the F polypeptide and the endogenous sequence encoding the HN polypeptide can be modified such that their functions or activities are at least partially or completely disrupted. The nucleotide sequence (b) can be inserted at the loci of the F and HN genes.

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

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

[0112] The recombinant competent or replication-defective negative-strand RNA virus of the present invention may further include a heterologous sequence encoding a reporter polypeptide (reporter gene), such as a fluorescent protein such as GFP, eGFP or a derivative thereof. The reporter gene may be present only for the in vitro evaluation of the RNA virus constructs described herein. A reporter gene such as eGFP may be located downstream of the 3' end of the L gene. The reporter gene has no functional meaning for the immune effect of the RNA virus construct. When the recombinant competent or replication-defective negative-strand RNA virus of the present invention is used for medical purposes, the reporter gene is not essential. Rather, the presence of the reporter gene may interfere with the medical use of the replication-capable or replication-defective negative-strand RNA virus of the present invention. The recombinant competent or replication-defective negative-strand RNA for medical use described herein preferably does not include a reporter gene. In particular, the recombinant competent or replication-defective negative-strand RNA for medical use described herein does not include a GFP gene or an eGFP gene, or a sequence derived therefrom.

[0113] In the present invention, the recombinant competent or replication-deficient negative-strand RNA virus of the present invention can adsorb to a host cell and penetrate into the host cell. The recombinant competent or replication-deficient negative-strand RNA virus of the present invention can also infect eukaryotic cells.

[0114] When a sequence encoding an HN protein is present in the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, adsorption to a host cell can be mediated by the HN protein. When a sequence encoding an F protein is present in the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, entry into the cell can be initiated by the F protein. When a sequence encoding a SARS-CoV-2 S protein is present in the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, adsorption and entry into a host cell can be mediated by the SARS-CoV-2 S protein

[0115] In the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, when a heterologous polypeptide is presented on the virus surface, it can mediate adsorption and entry into a host cell. In particular, the heterologous protein may be a SARS-CoV-2 S protein or may be a chimeric protein as described herein.

[0116] In particular, the host cell is a eukaryotic cell. The host cell may be any host cell that is susceptible to infection by a negative-strand RNA virus. Those skilled in the art know appropriate host cells. Appropriate 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 can be propagated in eukaryotic cells that are trans-complemented with the deletion sequence of the replication-deficient negative-strand RNA virus, particularly with the sequence encoding the P polypeptide. The cells may also be trans-complemented with the sequence encoding the N and / or L polypeptide. In particular, the recombinant replication-deficient negative-strand RNA virus of the present invention can be propagated in eukaryotic cells that are trans-complemented with (i) the P polypeptide and (ii) the sequences encoding the N and L polypeptides.

[0119] In the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, 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 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. In the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, 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 SeV F or HN polypeptide, or a fragment thereof, and (iii) the cytoplasmic domain of the SeV F or HN polypeptide, or a fragment thereof may encode.

[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, the nucleotide sequence (b) can be operably linked to an endogenous SeV expression control sequence, particularly an SeV F or HN expression control sequence such as the SeV F or HN promoter.

[0123] Such a construct is referred to herein as a "variant 1 construct". Examples of the variant 1 construct are described in the Examples.

[0124] In particular, in the variant 1 construct, the nucleotide sequence (b) (i) the ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SeV F polypeptide, or a fragment thereof, and (iii) the cytoplasmic domain of the SeV F polypeptide, or a fragment thereof may encode.

[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, the nucleotide sequence (b) may include SEQ ID NO: 27 or 30. The heterologous polypeptide encoded by the 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 domain and cytoplasmic domain 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 domain and cytoplasmic domain 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 control sequence, particularly an SeV F expression control sequence such as the SeV F promoter.

[0128] In particular, in the variant 1 construct, the nucleotide sequence (b) may also (i) the ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) the transmembrane domain of the SeV HN polypeptide, or a fragment thereof, and (iii) the cytoplasmic domain of the SeV HN polypeptide, or a fragment thereof be capable of encoding.

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

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

[0131] As used herein, the term "fragment" includes a subsequence 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, "fragment of the S protein" or "fragment of the SARS-CoV-2 S protein" includes a polypeptide containing a subsequence of the full-length S protein. This fragment may have essentially the same activity and / or function as the full-length S protein. In particular, the 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 S protein. Examples of fragments of the S protein include the following: (i) subsequences of the ectodomain and transmembrane domain of the SARS-CoV-2 S protein, (ii) The ectodomain and transmembrane domain of the SARS-CoV-2 S protein, (iii) A partial sequence of the ectodomain, transmembrane domain, and cytoplasmic domain of the SARS-CoV-2 S protein, or / and (iv) The ectodomain, transmembrane domain, and cytoplasmic domain of the SARS-CoV-2 S protein, wherein 19 C-terminal amino acid residues of the cytoplasmic domain are deleted, the ectodomain, transmembrane domain, and cytoplasmic domain.

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

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

[0135] The ectodomain of the SARS-CoV-2 spike polypeptide contains amino acid residues (aa) 1 to 1,213 of SEQ ID NO: 20, or may contain SEQ ID NO: 22. The transmembrane domain of the SARS-CoV-2 spike polypeptide contains aa 1,214 to 1,234 of SEQ ID NO: 20, or may contain SEQ ID NO: 24, or SEQ ID NO: 41. The cytoplasmic domain of the SARS-CoV-2 spike polypeptide contains aa 1,235 to 1,273 of SEQ ID NO: 20, or may contain SEQ ID NO: 26, or SEQ ID NO: 43.

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

[0137] In the recombinant competent or replication-defective negative-strand RNA virus of the present invention, 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 SeV F or HN polypeptide, or a fragment thereof may encode.

[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, the nucleotide sequence (b) may be operably linked to an endogenous SeV expression control sequence, particularly a SeV F or HN expression control sequence, such as a SeV F or HN promoter.

[0140] Such a construct is referred to herein as a "variant 2 construct". Examples of the variant 2 construct are described in the examples.

[0141] In particular, in the variant 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) the cytoplasmic domain of the SeV F polypeptide, or a fragment thereof may encode.

[0142] The nucleotide sequence (b) of this construct may contain a partial sequence of the cytoplasmic domain of the SARS-CoV-2 spike polypeptide. In particular, the nucleotide sequence (b) may contain SEQ ID NO: 36, 44 or 46. The heterologous polypeptide encoded by the nucleotide sequence (b) may comprise SEQ ID NO: 37, 45 or 47, which are encoded by SEQ ID NO: 36, 44 or 46 respectively. The ectodomain of SARS-CoV-2 is encoded by SEQ ID NO: 21, 28 or 38. The ectodomain of SARS-CoV-2 may contain SEQ ID NO: 22, 32 or 39.

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

[0144] In particular, in the variant 2 construct, the nucleotide sequence (b) may 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) the cytoplasmic domain of the SeV HN polypeptide, or a fragment thereof may be encoded.

[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, the nucleotide sequence (b) may be operably contacted with an endogenous SeV expression control sequence, particularly a SeV HN expression control sequence such as the SeV HN promoter.

[0147] In the recombinant competent or replication-deficient minus-strand RNA virus of the present invention, 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 may encode.

[0148] Such constructs are referred to herein as "variant 3 constructs". Examples of variant 3 constructs are described in the Examples.

[0149] In this construct, the nucleotide sequence (b) may be operably linked to an endogenous SeV expression control sequence, particularly an SeV F or HN expression control sequence, such as the SeV F or HN promoter.

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

[0151] In the present invention, a recombinant competent or replication-deficient negative-strand RNA virus may have reduced anti-vector antigenicity compared to the wild type. In the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, the reduction in anti-vector antigenicity can be achieved by at least partial or complete deletion of the nucleotide sequence encoding the viral surface polypeptide, particularly F and / or HN, as described herein. For example, the recombinant competent or replication-deficient negative-strand RNA virus particles of the present invention may essentially not contain the endogenous (native) surface polypeptide on their surface. The recombinant competent or replication-deficient negative-strand RNA virus particles of the present invention 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 polypeptide can be supplemented by the S protein, particularly the SARS-CoV-2 S protein.

[0152] In the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, the ectodomain of the SARS-CoV-2 spike polypeptide may contain the S1 subunit, and the S1 subunit contains a receptor-binding domain (RBD) that can bind to the ACE-2 receptor of the host cell.

[0153] In the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, the S1 subunit can be cleaved by the ACE-2 protease of the host cell.

[0154] In the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, the ectodomain of the SARS-CoV-2 spike polypeptide may contain the S2 subunit that can fuse the SeV envelope with the host cell membrane or the endosome membrane.

[0155] In the recombinant competent or replication-deficient negative-strand RNA virus of the present invention, the sequence of the ectodomain of the S protein may include at least one sequence modification capable of stabilizing the ectodomain in the prefusion form. For example, the S protein or its ectodomain may be provided in a stabilized prefusion form that includes 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 / endosome membrane and the viral envelope after receptor binding. To mediate the fusion of the viral envelope and the cell / endosome membrane, a sequence encoding the F protein may be present.

[0156] The recombinant competent or replication-deficient negative-strand RNA virus of the present invention can induce a mucosal immune response against at least one heterologous polypeptide encoded by at least one nucleotide sequence (b) when administered, for example, by the intranasal and / or mucosal administration route. The recombinant competent or replication-deficient negative-strand RNA virus of the present invention, particularly the SeV vector of the present invention described herein, can easily penetrate the mucus layer of the respiratory tract and thus provides ideal conditions for delivering at least one heterologous polypeptide via the intranasal and / or mucosal administration route, for example, in the form of an intranasal / mucosal spray vaccine that is easy to handle and use.

[0157] Since the recombinant competent or replication-deficient negative-strand RNA virus of the present invention can display at least one heterologous polypeptide on its surface, the mucosal immune response may be an immediate response.

[0158] The recombinant competent or replication-deficient negative-strand RNA virus of the present invention can induce a protective immune response against a pathogen having a polypeptide antigen encoded by at least one nucleotide sequence (b), particularly against SARS-CoV-2. The protective immunity can be sterilizing immunity. As used herein, the term "sterilizing immunity" means an immune response against a pathogen, particularly the SARS-CoV-2 virus, that prevents the infection of the pathogen to another subject that has not yet been infected.

[0159] The recombinant competent or replication-deficient negative-strand RNA virus of the present invention can be directly presented to the mucosal immune system, particularly during the process of intranasal / mucosal application, in the same manner as the natural respiratory tract infection by SARS-CoV-2. Furthermore, when the virus invades the host mucosal cells, it can produce the SARS-CoV-2 spike protein and present it on the cell surface. In this way, a broad range of protection, particularly including sterilizing immunity, is achieved and ensured.

[0160] As used herein, the P protein can be encoded by the nucleotide sequence of 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 thereto.

[0161] As used herein, the P protein can include the amino acid sequence of SEQ 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 P 2-77 protein can be encoded by the nucleotide sequence of SEQ ID NO: 3, or 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 in this specification may include 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 in this specification may be encoded by the nucleotide sequence of SEQ ID NO: 5, or 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 in this specification 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 transmembrane domain of the SeV F protein used in this specification may be encoded by the nucleotide sequence of SEQ ID NO: 7, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0167] The transmembrane domain of the SeV F protein used in this specification may include the amino acid sequence of SEQ 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 cytoplasmic domain of the SeV F protein used in this specification may be encoded by the nucleotide sequence of SEQ ID NO: 9, or 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 domain used herein may include the amino acid sequence of SEQ 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 protein used herein may be encoded by the nucleotide sequence of SEQ ID NO: 11, or 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 protein used herein may include the amino acid sequence of SEQ 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 protein cytoplasmic domain used herein may be encoded by the nucleotide sequence of SEQ ID NO: 13, or 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 protein cytoplasmic domain used herein may be composed of the amino acid sequence of SEQ 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 protein transmembrane domain used herein may be encoded by the nucleotide sequence of SEQ ID NO: 15, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0175] As used herein, the SeV HN transmembrane domain may be composed 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] As used herein, the SeV HN ectodomain may be encoded by the nucleotide sequence of SEQ ID NO: 17, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0177] As used herein, the SeV HN ectodomain may include the amino acid sequence of SEQ 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] As used herein, the SARS-CoV-2 spike (S) protein may be encoded by the nucleotide sequence of SEQ ID NO: 19, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0179] As used herein, the SARS-CoV-2 spike (S) protein may include the amino acid sequence of SEQ 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] As used herein, the SARS-CoV-2 spike (S) ectodomain may be encoded by the nucleotide sequence of SEQ ID NO: 21, 28, or 38, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0181] As used herein, the SARS-CoV-2 spike (S) ectodomain comprises 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] As used herein, the SARS-CoV-2 spike (S) transmembrane domain can be encoded by the nucleotide sequence of SEQ ID NO: 23 or 40, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0183] As used herein, the SARS-CoV-2 spike (S) transmembrane domain can include the amino acid sequence of SEQ 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] As used herein, the SARS-CoV-2 spike (S) cytoplasmic domain can be encoded by the nucleotide sequence of SEQ ID NO: 25 or 42, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0185] As used herein, the SARS-CoV-2 spike (S) cytoplasmic domain comprises the amino acid sequence of SEQ 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 (variant 1 construct) consisting of the SARS-CoV-2 spike ectodomain, the SeV F transmembrane domain, and the SeV cytoplasmic domain used herein may be encoded by the nucleotide sequence of SEQ ID NO: 27, 30, or 34, or 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 (variant 1 construct) consisting of the SARS-CoV-2 spike ectodomain, the SeV F transmembrane domain, and the SeV cytoplasmic domain used herein may include 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 the SeV cytoplasmic domain used herein may be encoded by the nucleotide sequence of SEQ ID NO: 29, or 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 the SeV cytoplasmic domain used herein may be composed of the amino acid sequence of SEQ 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 (variant 2 construct) consisting of the SARS-CoV-2 spike ectodomain, transmembrane domain, and cytoplasmic domain (with 19 C-terminal amino acid residues of the cytoplasmic domain deleted), and the SeV F cytoplasmic domain used herein may be encoded by the nucleotide sequence of SEQ ID NO: 36, 44, or 46, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.

[0191] The chimeric protein used in this specification, which consists of the SARS-CoV-2 spike ectodomain, transmembrane domain and cytoplasmic domain (with 19 C-terminal amino acid residues of the cytoplasmic domain deleted), and the SeV F cytoplasmic domain (variant 2 construct), may include the amino acid sequence of SEQ ID NO: 37, 45 or 47, or a sequence 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 includes a nucleocapsid and an envelope, wherein the nucleocapsid has 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 replication deficiency of the negative-strand RNA virus, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle. It relates to a recombinant replication-deficient single-stranded negative-strand RNA virus particle comprising.

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

[0194] Another aspect of the present invention includes a nucleocapsid and an envelope, wherein the nucleocapsid has in its genome, (a) a nucleotide sequence encoding a SeV P polypeptide, wherein the P protein performs its original function particularly in the viral replication cycle. (b) At least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the viral particle, the nucleotide sequence It relates to a recombinant replication-competent single-stranded negative-strand RNA viral particle comprising

[0195] In particular, the recombinant replication-competent single-stranded negative-strand RNA viral particle is attenuated.

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

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

[0198] Another aspect of the present invention relates to a recombinant replication-deficient Sendai virus comprising 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 replication deficiency of the negative-strand RNA virus, the nucleotide sequence, and (b) At least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the Sendai virus particle, the nucleotide sequence.

[0199] The nucleotide sequence (a) encoding the P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the at least one nucleotide sequence (b) encoding at least one heterologous polypeptide 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] Yet another aspect of the invention relates to a recombinant competent Sendai virus comprising in its genome: (a) a nucleotide sequence encoding a P polypeptide, wherein the P protein performs its original function particularly in the viral replication cycle; (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle.

[0202] In particular, the replication-competent Sendai virus particles are attenuated.

[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 invention relates to recombinant replication-deficient Sendai virus particles comprising a nucleocapsid and an envelope, wherein the nucleocapsid comprises 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 replication deficiency of the Sendai virus; (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle and relates to recombinant replication-deficient Sendai virus particles.

[0205] A nucleotide sequence (a) encoding a P polypeptide, wherein the nucleotide sequence is a modified nucleotide sequence 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 a replication-defective negative-strand RNA virus.

[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 such that the SARS-CoV-2 spike polypeptide or a fragment thereof is presented on the surface of the Sendai virus particles.

[0207] Yet another aspect of the invention includes a nucleocapsid and an envelope, wherein the nucleocapsid has in its genome, (a) a nucleotide sequence encoding a SeV P polypeptide, wherein the P protein particularly exerts its original function in the viral replication cycle, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the viral particle, and relates to a recombinant competent Sendai virus particle.

[0208] In particular, the replication-competent Sendai virus is attenuated.

[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 is such that the SARS-CoV-2 spike polypeptide or a fragment thereof is presented on the surface of the Sendai virus particles.

[0210] Yet another aspect of the present invention relates to recombinant replication-deficient Sendai virus (SeV) particles comprising a nucleocapsid and an envelope, wherein the nucleocapsid has 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 replication deficiency of Sendai virus, and the nucleotide sequence (b) at least one nucleotide sequence encoding a SARS-CoV-2 spike polypeptide or a fragment thereof wherein the SARS-CoV-2 spike polypeptide or a fragment thereof is presented on the surface of the virus particle, relates to recombinant replication-deficient Sendai virus (SeV) particles.

[0211] Yet another aspect of the present invention relates to recombinant competent Sendai virus (SeV) particles comprising a nucleocapsid and an envelope, wherein the nucleocapsid has in its genome, (a) a nucleotide sequence encoding a SeV P polypeptide, wherein the P protein exhibits its original function particularly in the viral replication cycle, and the nucleotide sequence (b) at least one nucleotide sequence encoding a SARS-CoV-2 spike polypeptide or a fragment thereof, wherein the SARS-CoV-2 spike polypeptide or a fragment thereof is presented on the surface of the virus particle, relates to recombinant replication-deficient Sendai virus (SeV) particles.

[0212] In particular, the replication-competent Sendai virus is attenuated.

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

[0214] One aspect of the present invention relates to a pharmaceutical composition comprising a recombinant replication-deficient or replication-competent minus-strand RNA virus as described herein, and / or a recombinant replication-deficient or replication-competent minus-strand RNA virus particle as described herein.

[0215] In particular, the pharmaceutical composition comprises a recombinant replication-deficient or replication-competent Sendai virus as described herein, and / or a recombinant replication-deficient or replication-competent Sendai virus particle as described herein.

[0216] Yet another aspect of the present invention relates to a vaccine comprising a recombinant replication-deficient or replication-competent minus-strand RNA virus as described herein, and / or a recombinant replication-deficient or replication-competent minus-strand RNA virus particle as described herein. The vaccine is an anti-SARS-CoV-2 vaccine or a prophylactic vaccine against COVID 19.

[0217] Yet another aspect of the present invention relates to an immunogenic composition comprising a recombinant replication-deficient or replication-competent minus-strand RNA virus as described herein, and / or a recombinant replication-deficient or replication-competent minus-strand RNA virus particle as described herein. The immunogenic composition can elicit an immune response against SARS-CoV-2.

[0218] Yet another aspect of the present invention relates to a recombinant replication-deficient or replication-competent negative-strand RNA virus described herein for medical use, a recombinant replication-deficient or replication-competent negative-strand RNA virus particle described herein, a pharmaceutical composition described herein, a vaccine described herein, or an immunogenic composition described herein.

[0219] In particular, the present invention relates to a recombinant replication-deficient or replication-competent negative-strand RNA virus described herein, a recombinant replication-deficient or replication-competent negative-strand RNA virus particle described herein, a pharmaceutical composition described herein, a vaccine described herein, or an immunogenic composition described herein for use in a method of preventing SARS-CoV-2 infection.

[0220] Yet another aspect of the present invention relates to a recombinant replication-deficient or replication-competent negative-strand RNA virus described herein, a recombinant replication-deficient or replication-competent negative-strand RNA virus particle described herein, a pharmaceutical composition described herein, a vaccine described herein, or an immunogenic composition described herein for use in a method of preventing COVID 19.

[0221] Yet another aspect of the present invention relates to a recombinant replication-deficient or replication-competent negative-strand RNA virus described herein, a recombinant replication-deficient or replication-competent negative-strand RNA virus particle described herein, a pharmaceutical composition described herein, a vaccine described herein, or an immunogenic composition described herein for use as a vaccine. The vaccine is an anti-SARS-CoV-2 vaccine or a vaccine for preventing COVID 19.

[0222] The recombinant replication-deficient or replication-competent recombinant replication-deficient negative-strand RNA virus, the recombinant replication-deficient or replication-competent recombinant replication-deficient negative-strand RNA virus particle, the pharmaceutical composition, the vaccine or the immunogenic composition can be administered to a subject in need thereof by the intranasal and / or mucosal route.

[0223] Another aspect of the present invention relates to an RNA molecule encoding a recombinant replication-defective negative-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 comprises.

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

[0225] Another aspect of the present invention relates to an RNA molecule encoding a recombinant competent negative-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 P protein exerts its original function particularly in the viral replication cycle, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, comprises.

[0226] In particular, the replication-competent negative-strand RNA virus is attenuated.

[0227] In particular, the RNA molecule is (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 includes.

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

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

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

[0231] Yet another aspect of the present invention is a DNA molecule encoding the RNA molecule of the present 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 present invention is a host cell comprising a replication-competent or replication-deficient negative-strand RNA virus described herein, a recombinant competent or replication-deficient negative-strand RNA virus particle described herein, an RNA molecule described herein, a nucleocapsid described herein, and / or a DNA molecule described herein. In particular, the host cell is a eukaryotic cell. The host cell can be any host cell that is susceptible to infection by a negative-strand RNA virus. Those skilled in the art know the appropriate host cells.

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

[0234] Host cells, particularly V3-10 helper cells, can replicate the recombinant replication-defective negative-strand RNA virus of the present invention. Replication also includes the propagation of the replication-defective negative-strand RNA virus of the present invention. Examples of such eukaryotic cells that can replicate the recombinant replication-defective negative-strand RNA are cells that are complemented in trans with the defective sequences of the replication-defective negative-strand RNA virus, particularly (i) cells complemented in trans with a nucleic acid molecule encoding the P polypeptide. The cells can further be transfected with (ii) at least one nucleic acid molecule encoding the N and / or L polypeptides. An example is the V3-10 helper cells described herein.

[0235] Host cells, particularly V3-10 helper cells, can also rescue the replication-defective negative-strand RNA virus of the present invention after transfection with a nucleic acid molecule encoding the recombinant replication-defective 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 foreign sequences encoding the defective viral proteins.

[0236] The recombinant competent negative-strand RNA virus described herein can replicate in host cells without the need for transcomplementation. An example of such a cell is Vero 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. An example thereof is Vero cells.

[0238] The host cell may also be a prokaryotic cell such as a bacterium suitable for the maintenance and propagation of the DNA molecules described herein that encode a replicable or replication-defective negative-strand RNA virus of the invention. Suitable prokaryotic cells are known to those skilled in the art.

[0239] Yet another aspect of a method for preventing infection by SARS-CoV-2 and / or COVID 19 involves administering to a subject in need thereof a recombinant competent or replication-defective negative-strand RNA virus described herein, a recombinant competent or replication-defective negative-strand RNA virus particle described herein, a pharmaceutical composition described herein, a vaccine described herein, and / or an immunogenic composition described herein.

[0240] A further aspect of the invention relates to a method for manufacturing a pharmaceutical composition described herein, a recombinant competent or replication-defective recombinant competent or replication-defective negative-strand RNA virus particle described herein, a vaccine described herein, or an immunogenic composition described herein, or an immunogenic composition described herein, which comprises formulating a recombinant competent or replication-defective negative-strand RNA virus described herein, an RNA molecule described herein, and / or a nucleocapsid with at least one pharmaceutically acceptable excipient.

[0241] Yet another aspect of the invention is a method for producing a recombinant replication-defective negative-strand RNA virus described herein.

[0242] A method for producing a recombinant replication-defective negative-strand RNA virus or virus particle described herein may comprise the following steps: (a) Transfecting a eukaryotic host cell with a DNA molecule encoding an RNA molecule of the invention, said host cell being capable of expressing 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 Step of separating the viral particles of (c)(b).

[0243] The method for producing a recombinant replication-deficient negative-strand RNA virus or viral particles described herein 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 the DNA molecule described herein that encodes the recombinant replication-deficient RNA virus of the present invention, (b) Culturing the host cell under conditions such that the DNA molecule is transcribed and viral particles are formed, and (c) Step of separating the viral particles of (b), wherein the wild-type negative-strand RNA virus and the recombinant replication-deficient RNA virus may be derived from the same species.

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

[0245] Yet another aspect of the present invention relates to the use of the host cells described herein for the production of recombinant competent or replication-deficient negative-strand RNA viruses and / or recombinant competent or replication-deficient negative-strand RNA viral particles.

[0246] A further aspect of the present invention relates to the use of the recombinant competent or replication-deficient negative-strand RNA viruses described herein for the manufacture of a medicament for preventing infection with SARS-CoV-2 and / or COVID 19.

[0247] TIFF2025518078000001.tif228160 TIFF2025518078000002.tif248160 TIFF2025518078000003.tif232160

[0248] The present invention will be described in detail and illustrated by figures and examples, which are used for illustrative purposes only and are not intended to be limiting. From the present specification and examples, further aspects included in the present invention are also available to those skilled in the art.

Brief Description of the Drawings

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Example

[0250] Example 1 The examples describe a second-generation SARS-CoV-2 vaccine. This vaccine contains a Sendai virus vector having a 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: ● An important safety feature is to partially cleave the proteins of the SeV viral RNA polymerase complex to achieve complete replication deficiency. Thus, uncontrolled vector transmission in vaccinated subjects can be completely eliminated. Infection of subjects treated with SeV-based vaccines is completely blocked. ● The SeV vector is characterized by genetic stability against recombination phenomena, and no undesirable properties (e.g., transgenic) are introduced into the vector. ● The true ability of the SeV vector to easily penetrate the mucin layer in the respiratory tract provides ideal conditions for delivering the transgene antigen via the nasal / mucosal administration route in the form of an easy-to-handle and easy-to-use nasal / mucosal spray vaccine.

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

[0253] WO 2006 / 084746 describes SeV rescue and SeV propagation in different cell lines. In this example, the manufacturing system for the SeV vaccine candidate was changed so that the same cells can be used for SeV rescue and subsequent propagation. This significantly simplifies the manufacturing process of 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) Incorporated the S protein of SARS-CoV-2 onto the surface of the SeV-vaccine vector. (ii) Reduced the amounts of the SeV proteins F and HN on the surface of the SeV vaccine vector. For SARS-CoV-2 S and SeV F and HN, vector constructs called type A, type B, and type C are described in Table 1 below.

[0255] [Table 1]

[0256] If the vaccinated subject has already acquired immunity against SARS-CoV-2 in the respiratory system, for example, due to SARS-CoV-2 infection, the subject's immune system recognizes the SeV vaccine vector against SARS-CoV-2, and the SeV vaccine can act as a booster immunity. If the vaccinated subject has not yet acquired immunity against SARS-CoV-2 in the respiratory tract, the vaccine vector induces immunity against SARS-CoV-2 through different immune stimulation pathways and interactions with the immune system. First, since the vaccine vector particles present spike proteins on their surface, they are themselves immunogenic against SARS-CoV-2. Thus, the vaccine vector functions as, for example, a nanoparticle vaccine or a virus-like particle vaccine (VLP). 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 part of the spike protein to T lymphocytes, which can then differentiate into helper cells and effector cells. Second, the vaccine vector can infect cells such as those in the respiratory tract and express the encoded spike protein intracellularly. As a result, spike proteins are expressed on the surface of these cells and are recognized by the immune system as being infected. Also, after standard proteolysis occurs inside these cells, peptides of the spike protein 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 genomes of all SeV vaccine prototypes. The eGFP gene is present only for in vitro evaluation and has no functional meaning for the immune effect of the vaccine candidate. After positive evaluation of the vector vaccine candidate, the eGFP gene is removed from the construct. An example of the RNA virus of the present invention that does not contain the sequence encoding GFP is SeV88-1V2 (Example 5).

[0258] Type A: Express SARS-CoV-2 S protein in addition to the expression of SeV vector proteins F and HN (SARS-CoV-2 S + SeV F and HN) In the A construct, the S gene of SARS-CoV-2 was introduced between the P gene (truncated P mut ) and the M gene of SeV.

[0259] The A construct is designed to display a recombinant viral protein (e.g., the SARS-CoV-2 S protein) obtained from another family (coronavirus) on the envelope of a paramyxovirus (specifically, the envelope of SeV), in combination with the proteins F and HN. Such recombinant genes and / or proteins can interfere with the assembly of the virus and, when the protein is presented on the surface, can interact with F and / or HN, thus potentially affecting the adsorption and penetration ability.

[0260] Adsorption and penetration are essential steps in the infection of host cells, in the production of vaccines with replication-deficient viral vectors, and in eliciting the immune response of interest.

[0261] The S gene of SARS-CoV-2 has a length of more than 3 kb. It was necessary to determine whether the recombinant S gene was genetically stable in the SeV vector and thus permanently present in the SeV vaccine vector genome. This aspect is by no means trivial with respect to the production of vector vaccines. In the A viral vector, the SARS-CoV-2 gene / protein is not essential for replication and / or infection. Therefore, if such a non-essential gene has disadvantages from the perspective of the wild type, there is a significant risk that it will be lost or inactivated during virus replication, resulting in a decrease in the replication ability.

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

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

[0264] To date, there has been no study demonstrating such "inter-family" substitution of functionally essential proteins without loss of function between two completely different virus families, paramyxoviruses and coronaviruses. 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 (fusion) into cells 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. The inventors have demonstrated that a SeV virus in which some or all of the F and HN proteins are replaced with the S protein of SARS-CoV-2 forms recombinant SeV virus particles capable of adsorbing to target cells and entering cells, and is suitable as an anti-SARS-CoV-2 vaccine.

[0265] Furthermore, in the type B candidate vaccine (SARS-CoV-2 S + SeV-F), since SeV-HN is absent from the SeV vaccine vector genome, the amount of SeV surface protein decreases, leading to a decrease in anti-SeV vector immunity.

[0266] The entry mechanisms are different for paramyxoviruses (fusion with the host cell membrane) and coronaviruses (mostly endocytosis). Although entry by membrane fusion of coronaviruses has been reported, it seems to be less efficient than endocytosis (Jackson, C.B., 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 regard, “within-family” substitutions such as the above can have an adverse effect on the penetration mechanism and thus on the replication effect.

[0267] The adsorption of SARS-CoV-2 begins with the virus docking in a “pre-bound form” to the ACE-2 receptor of the host (target) cell. In the pre-bound form, the receptor-binding domain (RBD) is located inside the S protein due to its natural folding properties. However, through movements such as those of a hinge, the RBD domain repeatedly moves to the surface of the S protein, thereby enabling it to bind to the ACE-2 receptor of the target cell. When binding to the ACE-2 receptor, the conformation of the S protein changes from an unstable pre-binding conformation to a highly stable post-binding conformation, which promotes / stabilizes 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 domain and the S2 domain of the S protein of SARS-CoV-2. S1 is thereby cleaved, and S2 fuses with the host cell membrane. The S1 subunit contains the RBD and binds to the receptor of the target cell (host cell). The S2 subunit mediates the fusion of the viral envelope with the cell membrane / endosome membrane after receptor binding. Random structural changes in the RBD are affected by the ACE-2 receptor. The ACE-2 receptor binds to the RBD and stabilizes the post-fusion form more than the pre-fusion form.

[0268] For many viral fusion glycoproteins, pre-binding stabilization by sequence modification can be induced by specific exchange of amino acids at the cleavage site, as described herein, and generally can protect a more conformationally stable post-binding structure. Thus, pre-binding stabilization typically significantly increases the yield in the recombinant expression of many viral fusion glycoproteins. Furthermore, virus glycoproteins stabilized in the pre-binding state are generally better / stronger immunogens than their wild-type counterparts that are not stabilized because antibody neutralization corresponds to the pre-binding conformation rather than the post-binding conformation.

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

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

[0271] In the 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 S gene of SARS-CoV-2 is an essential gene of the SeV vaccine vector, and its deletion or damage completely abolishes the replication ability in trans-complementing cells of the recombinant SeV vaccine virus.

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

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

[0274] Type C: Expression of only the SARS-CoV-2 S protein (SARS-CoV-2 S “only”); No expression of the SeV vector proteins F and HN In the C-type construct, the SeV HN gene and F gene were deleted. The S gene of SARS-CoV-2 was introduced between the M gene and L gene of SeV, that is, instead of the F gene and HN gene.

[0275] This approach (SARS-CoV-2 S “only”) was considered the most interesting among approaches A, B, and C because all surface proteins of the SeV vector were removed and did not function as antigens for anti-vector immunity.

[0276] Due to incomplete replication, the vaccine vector does not grow within the subject. Importantly, since the type C vaccine vector does not present vector proteins on its surface, the immunity acquired by the subject against the vaccine vector (e.g., in the case of an adenovirus vector) does not interfere with the acquisition of immunity against SARS-CoV-2.

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

[0278] Due to the deletion of the SeV genes F and HN, the type C chimeric SeV vector has a genome length almost the same as that of the SeV wild-type virus. The adsorption ability and entry ability of the SARS-CoV-2 protein are equivalent to those of the SeV F and HN proteins, and it replicates with the same efficiency as the SeV wild-type.

[0279] Preferably, the type C construct does not have a pre-binding stabilization sequence. In the type C construct, the SARS CoV-2 S protein can be provided in a form that can undergo conformational changes between the pre-binding form and the post-binding form and can be cleaved by the ACE-2 receptor protease of the host cell.

[0280] Example 2 Variant of the S protein of SARS-CoV-2: A chimeric construct combining the ectodomain of the S protein and the cytoplasmic domain and / or transmembrane domain 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 S protein of SARS-CoV-2 was incorporated into the envelope of the SeV vector and in what arrangement. In addition to the matrix protein (M) of SeV, if the surface proteins of SeV are involved in the concentration of the host cell plasma membrane, the incorporation of the S protein of SARS-CoV-2 into the envelope of the vaccine vector may have an adverse effect on the budding process.

[0281] It is necessary to test whether a part of the SeV vector surface proteins F and HN is required for interaction with the SeV vector protein M (matrix protein), for example, in the assembly of the virus. Three different design types (A - C) of vaccine candidates each contain (i) an ectodomain derived from the S protein of SARS-CoV-2 ecto , (ii) a transmembrane domain derived from SARS-CoV-2 or SeV tm , and / or (iii) a cytoplasmic domain derived from SARS-CoV-2 or SeV ct and are designed using three different S proteins (Table 2). As a result, the following three variations are obtained for each of the A - type to C - type constructs:

[0282]

Table 2

[0283] As an example of the construct SeV85E 1V2, SEQ ID NO: 36 is cited. A chimeric polypeptide containing the SARS-CoV-2 spike ecto, transmembrane domain, and cytoplasmic domain (19 C-terminal amino acid residues are deleted), as well as the 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 number of possible combinations, a meaningful and promising selection (ranking) of SeV vaccine constructs had to be made. SeV85E-2V1 (type A), SeV85E-1V1 (type C), and SeV85E-1V2 (type C) were evaluated in the 6 steps outlined in Table 3:

[0286] [Table 3]

[0287] Step 1 Expression of the chimeric SARS-CoV-2 S protein of the ranked SeV vaccine candidates using a plasmid in cell culture (control experiment for verifying the structurally correct expression of the chimeric SARS-CoV-2 S protein) Expression in cell culture using plasmids of the chimeric SARS-CoV-2 S proteins of SeV85E-2V1, SeV85E-1V1, and SeV85E-1V2, which are SeV vaccine candidates, confirmed the expression of the correct structure of the chimeric SARS-CoV-2 S protein.

[0288] Vero cells were transfected with eukaryotic expression plasmids pcDNA3.1 CoV-2 S2V1 (type A), pcDNA3.1 CoV-2 S1V1 (type C), and pcDNA3.1 CoV-2 S1V2 (type C), which have the coding sequences of SARS-CoV-2 S2V1, SARS-CoV-2 S1V1, and SARS-CoV-2 S1V2, respectively, or were not transfected (MOCK = negative control). 48 hours after transfection, the cells were stained with a primary antibody against the SARS-CoV-1 / 2 S protein. Goat anti-mouse Alexa Fluor 546 was used as the secondary antibody. The structural integrity of any 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] Project 2 Rescue, i.e., initial production of SeV vaccine candidates in GMP-compliant Vero cells and V3-10 helper cells Rescue, i.e., initial production of recombinant virus particles, was successful for both SeV vaccine candidate SeV85E-1V2 (type C) and SeV vaccine candidate SeV85E-2V1 (type A) using GMP-compliant Vero cells (Figs. 4 and 5).

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

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

[0292] Project 3 To supply a sufficient amount of virus for subsequent in vivo tests (animal models, clinical trials), the SeV vaccine candidates are grown in helper cells transfected with a nucleic acid molecule encoding at least the SeV P protein in trans. Growth in V3-10 helper cells was successful for the SeV vaccine candidate SeV85E-1V2 (type C). Using this type C prototype, the titers required for animal experiments have already been achieved (Figs. 8 and 9).

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

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

[0295] Step 6 Characterization of the immunological response after infecting mouse experimental animals with SeV vaccine candidates The humoral and cellular immune responses of mice vaccinated with the SeV vector construct were measured.

[0296] Conclusion This example shows that the recombinant SeV candidate vaccine presents the chimeric SARS-CoV-2 S protein on the surface and at the same time the expression of the SeV surface antigen is significantly reduced. The SeV vaccine candidate could be rescued from GMP-compliant Vero cells. The SeV candidate vaccine can be propagated in helper cells (e.g., cell line V3-10) derived from Vero cells transfected with a nucleic acid molecule encoding at least the SeV P protein in trans. Cell culture in Vero cell-derived helper cells can supply a sufficient amount of virus for in vivo tests (animal models and clinical trials).

[0297] Example 4 Attenuation of replication-competent SeV Here, the inventors confirmed the difference in attenuation between two viral vector constructs of the SARS-CoV-2 vaccine. Both of these two viral vector constructs contain an attenuation mutation that completely replaces both Sendai surface proteins F and HN with the SARS-CoV-2 surface protein S. One vaccine candidate (1) has replication ability and still contains the unmodified Sendai P gene, while the other vaccine candidate (2) contains an additional attenuating deletion of amino acids 2-77 in the P protein and is replication-deficient. Compared with the unmodified recombinant Sendai viral vector, a significant decrease in replication efficacy is observed due to the addition of attenuation mutations. The following virus titers were obtained from virus replication studies under cell culture conditions using Vero cells (SeV with replication ability) and V3-10 helper cells (replication-deficient SeV).

[0298] TIFF2025518078000007.tif54160

[0299] In conclusion, SeV(1) with replication ability in which surface proteins F and HN are replaced with the SARS-CoV-2 surface protein S is attenuated compared to unmodified SeV with replication ability, and such recombinant viruses are suitable as immunogenic agents or vaccines against SARS-CoV-2.

[0300] Furthermore, such modified SeV(1) with replication ability has improved production efficiency compared to replication-deficient SeV(2).

[0301] Example 5 Immunogenic effect of SeV88-1V2 BAL specimens are lower respiratory tract mucosal tissue specimens, and NW specimens are upper respiratory tract mucosal tissue specimens. Both samples contain components of the immune system, such as macrophages, lymphocytes, neutrophils, or antibodies released from cells and immune cells surrounding the tissue, and thus are useful for explaining the immunological status of these tissues. Specific antibodies against pathogens contribute to the defensive 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 5 and immunized intranasally (IN) 2 - 3 times every 3 weeks while increasing the vaccine dosage. To collect bronchoalveolar lavage fluid (BAL) and nasal wash fluid (NW), the mice were sacrificed by cervical dislocation under anesthesia 2 weeks after the last immunization.

[0303] IgA antibodies against the SARS-CoV-2 spike protein were measured by enzyme-linked immunosorbent assay (ELISA) in microtiter format. Serial two-fold dilutions were prepared and incubated in antigen pre-coated microtiter plates. Subsequently, horseradish peroxidase-labeled anti-mouse IgA was added. After 1 hour, the substrate 3,3’,5,5’-tetramethylbenzidine (TMB) was added, and the reaction was stopped after 30 minutes with NaH 2 SO 4 and the colorimetric conversion was measured at 450 nm with a spectrophotometer. The titer is represented as the reciprocal of the average value.

[0304] TIFF2025518078000008.tif50160 TIFF2025518078000009.tif50160

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

[0306] The present invention also includes the following items: 1. A recombinant replication-defective negative-strand RNA virus, wherein in its genome, (a) A nucleotide sequence encoding a P polypeptide, wherein said nucleotide sequence is modified compared to the wild type, and said modification results in replication deficiency of a negative-strand RNA virus, and (b) At least one nucleotide sequence encoding at least one heterologous polypeptide, wherein said at least one heterologous polypeptide is presented on the surface of said virus particle, A recombinant replication-deficient negative-strand RNA virus comprising 2. The recombinant replication-deficient negative-strand RNA virus according to item 1, which is a paramyxovirus, an Artovirus, a bornavirus, a filovirus, a respivirus, a mimivirus, a nyamivirus, a pneumovirus, a rhabdovirus, a sunvirus, or a simovirus. 3. The recombinant replication-deficient negative-strand RNA virus according to item 1 or 2, which is Sendai virus. 4. The recombinant replication-deficient negative-strand RNA virus according to any one of the preceding items, wherein the modification of the nucleotide sequence (a) is located in the nucleotide sequence encoding the N-terminus of the P polypeptide. 5. The recombinant replication-deficient negative-strand RNA virus according to any one of the preceding items, 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) A deletion of the nucleotide sequence encoding amino acids 2-77 of the P polypeptide compared to the wild type, or (ii) A partial sequence of (i) that causes replication deficiency of an RNA virus The recombinant replication-deficient negative-strand RNA virus according to any one of the preceding items. 7. The recombinant replication-deficient negative-strand RNA virus according to any one of the preceding items, which has transcription ability. 8. The genome is (c) A nucleotide sequence encoding a negative-strand RNA virus N polypeptide, (d) A nucleotide sequence encoding a minus-strand RNA virus M polypeptide, and (e) A nucleotide sequence encoding a minus-strand RNA virus L polypeptide The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, further comprising 9. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide comprises a heterologous antigen. 10. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide comprises a viral antigen. 11. The recombinant replication-defective minus-strand RNA virus according to item 10, wherein the viral antigen and the minus-strand RNA virus are selected from viruses of different families. 12. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide comprises an antigen derived from a virus other than a minus-strand RNA virus. 13. The recombinant replication-defective negative-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide comprises an antigen derived from a plus-strand virus such as a coronavirus. 14. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the minus-strand RNA virus is a paramyxovirus such as Sendai virus, and the heterologous polypeptide comprises an antigen derived from a coronavirus. 15. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide comprises the spike protein (S protein) of a coronavirus or an immunogenic fragment thereof. 16. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the coronavirus is SARS CoV 2. 17. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein at least one nucleotide sequence (b) is located upstream of the 5'-terminal coding sequence and / or downstream of the 3'-terminal coding sequence in an RNA molecule between two coding sequences. 18. The genome is (f) a nucleotide sequence encoding a minus-strand RNA virus F polypeptide, and (g) a nucleotide sequence encoding a minus-strand RNA virus HN polypeptide The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, further comprising 19. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein at least one nucleotide sequence (b) is inserted into or at least partially replaces at least one natural (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. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the nucleotide sequence (b) encoding at least one heterologous polypeptide is essential for virus replication and / or propagation.

[0307] 21. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide encoded by sequence (b) is functionally expressed (expressed in a biologically active form). 22. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the endogenous sequence encoding the F polypeptide and / or 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 F locus and / or the HN locus. 23. The recombinant replication-defective minus-strand RNA virus according to item 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. The recombinant replication-defective negative-strand RNA virus according to item 22, wherein the endogenous sequence encoding the F polypeptide is modified such that its function or activity is at least partially or completely destroyed, and the nucleotide sequence (b) is inserted into the locus of the F gene. The recombinant replication-defective negative-strand RNA virus according to item 22, wherein the endogenous sequence encoding the F polypeptide and the endogenous sequence encoding the HN polypeptide are modified such that their functions or activities are at least partially or completely destroyed, and the nucleotide sequence (b) is inserted into the F gene locus and the HN gene locus. The recombinant replication-defective negative-strand RNA virus according to any one of the preceding items, which, when presented on the surface of the virus, can adsorb to a host cell and enter the host cell. The recombinant replication-defective negative-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide, when presented on the surface of the virus, mediates adsorption to a host cell and entry into the host cell. The recombinant replication-defective negative-strand RNA virus according to item 27, wherein the host cell is a eukaryotic cell. The recombinant replication-defective negative-strand RNA virus according to any one of the preceding items, which is capable of infecting a eukaryotic cell. The recombinant replication-defective negative-strand RNA virus according to any one of the preceding items, which can be rescued from a eukaryotic cell. The recombinant replication-defective negative-strand RNA virus according to any one of the preceding items, which is capable of growing in a eukaryotic cell, wherein the cell is trans-complemented with the sequence that is defective in the replication-defective negative-strand RNA virus, particularly the sequence encoding the P polypeptide. 32. (i) The P polypeptide, and / or (ii) The N and L polypeptides The recombinant replication-defective negative-strand RNA virus according to any one of the preceding items, which is capable of growing in a eukaryotic cell trans-complemented with the sequence encoding the same. 33. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the heterologous polypeptide encoded by at least one nucleotide sequence (b) comprises a fragment of the spike polypeptide. 34. 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 SeV F or HN polypeptide, or a fragment thereof, and (iii) the cytoplasmic domain of the SeV F or HN polypeptide, or a fragment thereof and encodes the recombinant replication-defective minus-strand RNA virus according to any one of the preceding items. 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 the SeV F or HN polypeptide, or a fragment thereof and encodes the recombinant replication-defective minus-strand RNA virus according to any one of the preceding items. 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 and encodes the recombinant replication-defective minus-strand RNA virus according to any one of the preceding items. 37. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the anti-vector antigenicity is reduced. 38. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, 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 the host cell. 39. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the S1 subunit can be cleaved by the ACE-2 protease of the host cell. 40. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the ectodomain of the SARS-CoV-2 spike polypeptide comprises an S2 subunit capable of fusing the SeV envelope with the host cell membrane or the endosomal membrane.

[0308] 41. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, wherein the sequence of the ectodomain comprises at least one sequence modification capable of stabilizing the ectodomain in the pre-bound form. 42. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, which can induce a mucosal immune response against at least one heterologous polypeptide encoded by at least one nucleotide sequence (b). 43. The recombinant replication-defective minus-strand RNA virus according to any one of the preceding items, which can induce a protective immune response against a pathogen having a polypeptide antigen encoded by at least one nucleotide sequence (b). 44. A recombinant replication-defective minus-strand RNA virus particle, comprising a nucleocapsid and an envelope, wherein the nucleocapsid comprises the following in its genome: (a) A nucleotide sequence encoding the SeV P polypeptide, wherein the nucleotide sequence is modified compared to the wild type, and the modification results in replication deficiency of the minus-strand RNA virus, a nucleotide sequence, and (b) At least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle, the nucleotide sequence. 45. The recombinant replication-defective negative-strand RNA virus particle according to item 44, 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 the Sendai virus particle. 46. A recombinant replication-defective Sendai virus, wherein in its genome, (a) A nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified as compared to the wild type, and the modification results in replication deficiency of the negative-strand RNA virus, the nucleotide sequence, and (b) At least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle, the nucleotide sequence A recombinant replication-defective Sendai virus comprising. 47. The recombinant replication-defective Sendai virus (SeV) according to item 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 the Sendai virus particle, the recombinant replication-defective Sendai virus (SeV). 48. A recombinant replication-defective Sendai virus particle comprising a nucleocapsid and an envelope, wherein the nucleocapsid has in its genome, (a) A nucleotide sequence encoding a SeV P polypeptide, wherein the nucleotide sequence is modified as compared to the wild type, and the modification results in replication deficiency of the negative-strand RNA virus, the nucleotide sequence, and (b) At least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of Sendai virus particles, nucleotide sequence A recombinant replication-deficient Sendai virus particle comprising 49. The recombinant replication-deficient Sendai virus (SeV) particle according to item 48, wherein at least one nucleotide sequence (b) encodes a SARS-CoV-2 spike polypeptide or a fragment thereof, and the envelope contains the SARS-CoV-2 spike polypeptide or a fragment thereof presented on the surface of the Sendai virus particle, recombinant replication-deficient Sendai virus (SeV) particle. 50. A pharmaceutical composition comprising the recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43 and / or the recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49. 51. A vaccine comprising the recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43 and / or the recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49. 52. An immunogenic composition comprising the recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43 and / or the recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49. 53. For pharmaceutical use, the recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43, the recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49, the pharmaceutical composition according to item 50, the vaccine according to item 51, or the immunogenic composition according to item 52. 54. For use in a method of preventing SARS-CoV-2 infection, the recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43, the recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49, the pharmaceutical composition according to item 50, the vaccine according to item 51, or the immunogenic composition according to item 52. 55. For use in a method of preventing COVID-19, a recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43, a recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49, a pharmaceutical composition according to item 50, a vaccine according to item 51, or an immunogenic composition according to item 52. 56. For use as a vaccine, a recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43, a recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49, a pharmaceutical composition according to item 50, a vaccine according to item 51, or an immunogenic composition according to item 52. 57. For use according to any one of items 53 to 56, a recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43, a recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49, a pharmaceutical composition according to item 50, a vaccine according to item 51, or an immunogenic composition according to item 52, which is administered via the intranasal route and / or the mucosal route. 58. An RNA molecule encoding a recombinant replication-deficient negative-strand RNA virus, wherein the RNA molecule (a) a nucleotide sequence encoding a P polypeptide, wherein the nucleotide sequence is modified as compared to the wild type, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide is included. 59. (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 is further included in the RNA molecule according to item 58. 60. A nucleocapsid comprising the RNA molecule according to item 58 or 59. 61. A DNA molecule encoding the RNA molecule according to item 58 or 59.

[0309] A host cell comprising the replication-deficient negative-strand RNA virus according to any one of items 1 to 43, the recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49, the RNA molecule according to item 58 or 59, the nucleocapsid according to item 60, and / or the DNA molecule according to item 61. A method for preventing infection with SARS-CoV-2 and / or COVID 19, the method comprising administering to a subject in need thereof the recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43, the recombinant replication-deficient negative-strand RNA virus particle according to any one of items 44 to 49, the pharmaceutical composition according to item 50, the vaccine according to item 51, or the immunogenic composition according to item 52. A method for producing the pharmaceutical composition according to item 50, the vaccine according to item 51, or the immunogenic composition according to item 52, the method comprising formulating the recombinant replication-deficient negative-strand RNA virus according to any one of items 1 to 43, or the 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 virus particle according to any one of items 1 to 45, the method comprising the following steps: (a) Transfecting a eukaryotic host cell with the DNA molecule according to item 61, wherein the host cell is capable of expressing 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). The method comprising the above steps. A method for producing a recombinant negative-strand RNA virus or virus particle according to any one of items 1 to 45, the method comprising the following steps: (a) Infecting a eukaryotic host cell with a wild-type negative-strand RNA virus and transfecting the eukaryotic host cell with the DNA molecule according to item 61 encoding a recombinant replication-deficient RNA virus; (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). A method comprising: 67. The method according to claim 66, wherein the wild-type negative-strand RNA virus and the recombinant replication-deficient RNA virus are derived from the same species. 68. Use of the 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 a recombinant replication-deficient negative-strand RNA virus particle according to any one of claims 44 to 49. 69. Use of a recombinant replication-deficient negative-strand RNA virus according to any one of claims 1 to 43 for the manufacture of a medicament for preventing infection with SARS-CoV-2 and / or COVID 19. 70. A negative-strand RNA virus having recombinant replication ability, wherein in its genome, (a) a nucleotide sequence encoding a P polypeptide, wherein the P protein exerts its original function particularly in the viral replication cycle; (b) at least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle; wherein the heterologous polypeptide comprises a viral antigen, and the viral antigen and the negative-strand RNA virus are selected from viruses of different families; A recombinant replication-competent negative-strand RNA virus. 71. The recombinant competent negative-strand RNA virus according to claim 68, which is attenuated. 72. The recombinant replication-deficient negative-strand RNA virus according to any one of claims 70 to 71, which is a paramyxovirus, an Artovirus, a bornavirus, a filovirus, a respivirus, a mimivirus, a nairovirus, a pneumovirus, a rhabdovirus, a thogotovirus, or a simovirus. 73. A recombinant competent negative-strand RNA virus according to any one of items 70 to 72, which is Sendai virus. 74. A negative-strand RNA virus having recombinant replication ability according to any one of items 70 to 73, wherein the genome further includes the following: (c) A nucleotide sequence encoding a negative-strand RNA virus N polypeptide, (d) A nucleotide sequence encoding a negative-strand RNA virus M polypeptide, and (e) A nucleotide sequence encoding a negative-strand RNA virus L polypeptide. 75. A recombinant competent negative-strand RNA virus according to any one of items 70 to 74, wherein the heterologous polypeptide contains a heterologous antigen. 76. A recombinant competent negative-strand RNA virus according to any one of items 70 to 75, wherein the heterologous polypeptide contains a viral antigen. 77. The recombinant competent negative-strand RNA virus according to item 76, wherein the viral antigen and the negative-strand RNA virus are selected from viruses of different families. 78. A recombinant competent negative-strand RNA virus according to any one of items 70 to 77, wherein the heterologous polypeptide contains an antigen derived from a virus that is not a negative-strand RNA virus. 79. A negative-strand RNA virus having recombinant replication ability according to any one of items 70 to 78, wherein the heterologous polypeptide contains an antigen derived from a positive-strand virus such as a coronavirus. 80. A negative-strand RNA virus having recombinant replication ability according to any one of items 70 to 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.

[0310] 81. A recombinant competent negative-strand RNA virus according to any one of items 70 to 80, wherein the heterologous polypeptide contains the spike protein (S protein) of a coronavirus or an immunogenic fragment thereof. 82. The recombinant competent negative-strand RNA virus according to any one of items 70 to 81, wherein the coronavirus is SARS CoV 2. 83. The recombinant replication-capable negative-strand RNA virus according to any one of items 70 to 82, wherein at least one nucleotide sequence (b) is located in an RNA molecule between two coding sequences, at a position upstream of the 5'-terminal coding sequence, and / or at a position downstream of the 3'-terminal coding sequence. 84. The genome is (f) a nucleotide sequence encoding a negative-strand RNA virus F polypeptide, and (g) a nucleotide sequence encoding a negative-strand RNA virus HN polypeptide The recombinant replication-capable negative-strand RNA virus according to any one of items 70 to 83, further comprising. 85. The recombinant competent negative-strand RNA virus according to any one of items 70 to 84, wherein at least one nucleotide sequence (b) is inserted into or at least partially replaces at least one natural (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. 86. The recombinant competent negative-strand RNA virus according to any one of items 70 to 85, wherein the nucleotide sequence (b) encoding at least one heterologous polypeptide is essential for virus replication and / or propagation. 87. The recombinant replication-capable negative-strand RNA virus according to any one of items 70 to 86, wherein the heterologous polypeptide encoded by sequence (b) is functionally expressed (expressed in a biologically active form). 88. The recombinant replication-capable negative-strand RNA virus according to any one of items 70 to 87, wherein the endogenous sequence encoding the F polypeptide and / or 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 F locus and / or the HN locus. 89. The minus-strand RNA virus having recombinant replication ability according to item 88, wherein the endogenous sequence encoding the HN polypeptide is modified so that its function or activity is at least partially or completely destroyed, and the nucleotide sequence (b) is inserted into the locus of the HN gene. 90. The minus-strand RNA virus having recombinant replication ability according to item 88, wherein the endogenous sequence encoding the F polypeptide is modified so that its function or activity is at least partially or completely destroyed, and the nucleotide sequence is inserted into the locus of the F gene. 91. The minus-strand RNA virus having recombinant replication ability according to item 88, wherein the endogenous sequence encoding the F polypeptide and the endogenous sequence encoding the HN polypeptide are modified so that their functions or activities are at least partially or completely destroyed, and the nucleotide sequence is inserted into the F locus and the HN locus. 92. The negative-strand RNA virus having recombinant replication ability according to any one of items 70 to 91, which can adsorb to a host cell and penetrate into the host cell when presented on the virus surface. 93. The recombinant competent minus-strand RNA virus according to any one of items 70 to 92, wherein the heterologous polypeptide mediates adsorption to a host cell and penetration into the host cell when displayed on the surface of the virus. 94. The recombinant competent minus-strand RNA virus according to item 93, wherein the host cell is a eukaryotic cell. 95. The recombinant competent minus-strand RNA virus according to any one of items 70 to 94, which is capable of infecting a eukaryotic cell. 96. The minus-strand RNA virus having recombinant replication ability according to any one of items 70 to 95, which can be rescued from a eukaryotic cell. 97. The recombinant competent minus-strand RNA virus according to any one of items 70 to 96, which is capable of growing in a eukaryotic cell, wherein the cell is trans-complemented with a sequence that is defective in the replication-competent type minus-strand RNA virus, in particular, trans-complemented with a sequence encoding the P polypeptide. 98. The recombinant competent minus-strand RNA virus according to any one of items 70 to 97, wherein the heterologous polypeptide encoded by at least one nucleotide sequence (b) contains a fragment of the spike polypeptide. 99. 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 SeV F or HN polypeptide, or a fragment thereof, and (iii) the cytoplasmic domain of the SeV F or HN polypeptide, or a fragment thereof The recombinant competent minus-strand RNA virus according to any one of items 70 to 98, which encodes 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 the SeV F or HN polypeptide, or a fragment thereof The recombinant competent minus-strand RNA virus according to any one of items 70 to 99, which encodes

[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 a fragment thereof The recombinant competent minus-strand RNA virus according to any one of items 70 to 100, which encodes 102. A recombinant competent minus-strand RNA virus according to any one of items 70 to 101, having reduced anti-vector antigenicity. 103. A recombinant competent minus-strand RNA virus according to any one of items 70 to 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. 104. A recombinant replication-competent negative-strand RNA virus according to any one of items 70 to 103, wherein the S1 subunit can be cleaved by the ACE-2 protease of a host cell. 105. A recombinant competent minus-strand RNA virus according to any one of items 70 to 104, wherein the ectodomain of the SARS-CoV-2 spike polypeptide comprises an S2 subunit capable of fusing the SeV envelope with the host cell membrane or endosomal membrane. 106. A recombinant replication-competent minus-strand RNA virus according to any one of items 70 to 105, wherein the sequence of the ectodomain comprises at least one sequence modification capable of stabilizing the ectodomain in the pre-bound form. 107. A recombinant competent minus-strand RNA virus according to any one of items 70 to 106, capable of inducing a mucosal immune response against at least one heterologous polypeptide encoded by at least one nucleotide sequence (b). 108. A recombinant competent minus-strand RNA virus according to any one of items 70 to 107, capable of inducing a protective immune response against a pathogen having a polypeptide antigen encoded by at least one nucleotide sequence (b). 109. A recombinant competent minus-strand RNA virus particle comprising a nucleocapsid and an envelope, wherein the nucleocapsid has in its genome (a) a nucleotide sequence encoding a P polypeptide, wherein the P protein exerts its original function particularly in the viral replication cycle, the nucleotide sequence (b) At least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle, the nucleotide sequence A recombinant competent negative-strand RNA virus particle comprising 110. The recombinant competent negative-strand RNA virus particle 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 contains the SARS-CoV-2 spike polypeptide or a fragment thereof presented on the surface of the Sendai virus particle. 111. A recombinant competent Sendai virus, in its genome (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein particularly exerts its original function in the viral replication cycle, the nucleotide sequence (b) At least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of the virus particle, the nucleotide sequence A recombinant competent Sendai virus comprising 112. The recombinant competent Sendai virus (SeV) 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 contains the SARS-CoV-2 spike polypeptide or a fragment thereof presented on the surface of the Sendai virus particle. 113. A recombinant competent Sendai virus particle comprising a nucleocapsid and an envelope, wherein the nucleocapsid has in its genome (a) A nucleotide sequence encoding a P polypeptide, wherein the P protein particularly exerts its original function in the viral replication cycle, the nucleotide sequence (b) At least one nucleotide sequence encoding at least one heterologous polypeptide, wherein the at least one heterologous polypeptide is presented on the surface of Sendai virus particles, the nucleotide sequence A recombinant competent Sendai virus particle comprising 114. The recombinant competent Sendai virus (SeV) particle 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 the SARS-CoV-2 spike polypeptide or a fragment thereof presented on the surface of the Sendai virus particle. 115. The recombinant competent Sendai virus or particle according to any one of items 111 to 114, which is attenuated. 116. A pharmaceutical composition comprising the recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, or the recombinant competent Sendai virus or virus particle according to any one of items 111 to 115. 117. A vaccine comprising the recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, or the recombinant competent Sendai virus or virus particle according to any one of items 111 to 115. 118. An immunogenic composition comprising the recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, or the recombinant competent Sendai virus or virus particle according to any one of items 111 to 115. 119. For pharmaceutical use, the recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, or the recombinant competent Sendai virus or virus particle according to any one of items 111 to 115, the pharmaceutical composition according to item 116, the vaccine according to item 117, or the immunogenic composition according to item 118. For use in a method of preventing SARS-CoV-2 infection, a recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, a recombinant competent Sendai virus or virus particle according to any one of items 111 to 115, a pharmaceutical composition according to item 116, a vaccine according to item 117, or an immunogenic composition according to item 118.

[0312] For use in a method of preventing COVID 19, a recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, a recombinant competent Sendai virus or virus particle according to any one of items 111 to 115, a pharmaceutical composition according to item 116, a vaccine according to item 117, or an immunogenic composition according to item 118. For use as a vaccine, a recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, a recombinant competent Sendai virus or virus particle according to any one of items 111 to 115, a pharmaceutical composition according to item 116, a vaccine according to item 117, or an immunogenic composition according to item 118. For use according to any one of items 119 to 122, a recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, a recombinant competent Sendai virus or virus particle according to any one of items 111 to 115, a pharmaceutical composition according to item 116, a vaccine according to item 117, or an immunogenic composition according to item 118, which is administered by the intranasal and / or mucosal route. An RNA molecule encoding a recombinant competent negative-strand RNA virus, wherein the RNA molecule (a) a nucleotide sequence encoding a P polypeptide, wherein the P protein exerts its original function particularly in the viral replication cycle, and (b) at least one nucleotide sequence encoding at least one heterologous polypeptide An RNA molecule comprising. 125. (a) A nucleotide sequence encoding an N polypeptide, (b) A nucleotide sequence encoding an M polypeptide, and (c) A nucleotide sequence encoding an L polypeptide The RNA molecule according to claim 124, further comprising. 126. A nucleocapsid comprising the RNA molecule according to any one of claims 124 to 125. 127. A DNA molecule encoding the RNA molecule according to any one of claims 124 to 125. 128. A host cell comprising a recombinant competent negative-strand RNA virus or virus particle according to any one of claims 70 to 110, a recombinant competent Sendai virus or virus particle according to any one of claims 111 to 115, an RNA molecule according to any one of claims 124 to 125, the nucleocapsid according to claim 126 and / or the DNA molecule according to claim 127. 129. A method for preventing infection with SARS-CoV-2 and / or COVID 19, comprising administering to a subject in need thereof a recombinant competent negative-strand RNA virus or virus particle according to any one of claims 70 to 110, a recombinant competent Sendai virus or virus particle according to any one of claims 111 to 115, the pharmaceutical composition according to claim 116, the vaccine according to claim 117, or the immunogenic composition according to claim 118. 130. A method for producing the pharmaceutical composition according to claim 116, the vaccine according to claim 117, or the immunogenic composition according to claim 118, comprising formulating a recombinant competent negative-strand RNA virus or virus particle according to any one of claims 70 to 110, or a recombinant competent Sendai virus or virus particle according to any one of claims 111 to 115, together with at least one pharmaceutically acceptable excipient. The method for producing a recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, or a recombinant competent Sendai virus or virus particle according to any one of items 111 to 115, comprising: (a) Transfecting a eukaryotic host cell with the DNA molecule according to item 127; (b) Culturing the host cell under conditions such that the DNA molecule is transcribed and virus particles are formed; and (c) Isolating the virus particles of (b). A method comprising the above steps. Use of the host cell according to item 128 for the production of a recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, or a recombinant competent Sendai virus or virus particle according to any one of items 111 to 115. Use of a recombinant competent negative-strand RNA virus or virus particle according to any one of items 70 to 110, or a recombinant competent Sendai virus or virus particle according to any one of items 111 to 115 for the manufacture of a medicament for preventing infection with SARS-CoV-2 and / or COVID 19.

Claims

**Claim 1** A recombinant replication-defective negative-strand RNA virus, wherein in its genome, (a) a nucleotide sequence encoding a P polypeptide, the nucleotide sequence being modified as 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 virus particle is included, wherein the heterologous polypeptide includes a viral antigen, and the viral antigen and the negative-strand RNA virus are selected from viruses of different families, a recombinant replication-defective negative-strand RNA virus. **Claim 2** The modification in the nucleotide sequence (a) is (i) a deletion of the nucleotide sequence encoding amino acids 2-77 of the P polypeptide as compared to the wild type, or (ii) a partial sequence of (i) that causes replication deficiency of the RNA virus The recombinant replication-defective negative-strand RNA virus according to claim 1. **Claim 3** A recombinant competent negative-strand RNA virus, wherein in its genome, (a) a nucleotide sequence encoding a P polypeptide, the P protein exerting a natural function in the viral replication cycle (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 virus particle is included, wherein the heterologous polypeptide includes a viral antigen, and the viral antigen and the negative-strand RNA virus are selected from viruses of different families, a negative-strand RNA virus having recombinant replication ability. **Claim 4** The recombinant competent negative-strand RNA virus according to claim 3, which is attenuated. **Claim 5** The recombinant replication-defective or replication-competent negative-strand RNA virus according to any one of claims 1 to 4, having reduced anti-vector antigenicity. **Claim 6** The recombinant replication-defective or replication-competent negative-strand RNA virus according to any one of claims 1 to 5, which is Sendai virus. **Claim 7** A recombinant replication-deficient or replication-competent negative-strand RNA virus according to any one of claims 1 to 6, which is transcription-competent.

8. A recombinant replication-deficient or replication-competent negative-strand RNA virus according to any one of claims 1 to 7, wherein the heterologous polypeptide comprises a spike protein (S protein) of a coronavirus or an immunogenic fragment thereof.

9. An endogenous sequence encoding an F polypeptide and / or an endogenous sequence encoding an 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 F gene and / or the HN gene, A recombinant replication-deficient or replication-competent negative-strand RNA virus according to any one of claims 1 to 8.

10. A recombinant replication-deficient or replication-competent negative-strand RNA virus according to any one of claims 1 to 9, which adsorbs to a host cell and can penetrate the host cell when presented on the virus surface.

11. The nucleotide sequence (b) is as follows: A. (i) The ectodomain of the SARS-CoV-2 spike polypeptide, or a fragment thereof, (ii) The transmembrane domain of the SeV F polypeptide or HN polypeptide, or a fragment thereof, and (iii) The cytoplasmic domain of the SeV polypeptide F or HN polypeptide, or a fragment thereof, or B. (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 SeV polypeptide F or HN polypeptide, or a fragment thereof, or C. (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 encoding a recombinant replication-deficient or replication-competent negative-strand RNA virus according to any one of claims 1 to 10.

12. A recombinant replication-deficient or replication-competent negative-strand RNA virus according to any one of claims 1 to 11, which can induce a mucosal immune response against at least one heterologous polypeptide encoded by at least one nucleotide sequence (b).

13. A pharmaceutical composition comprising a recombinant replication-deficient or replication-competent negative-strand RNA virus according to any one of claims 1 to 12.

14. A recombinant replication-deficient or replication-competent negative-strand RNA virus according to any one of claims 1 to 12, or a pharmaceutical composition according to claim 13, for use in a method of preventing SARS-CoV-2 infection.

15. A recombinant replication-deficient or replication-competent negative-strand RNA virus according to any one of claims 1 to 12, or a pharmaceutical composition according to claim 13 for use according to claim 14, which is administered intranasally and / or by the mucosal route.

16. An RNA molecule or a DNA molecule encoding a recombinant replication-deficient negative-strand RNA virus or a replication-competent negative-strand RNA virus according to any one of claims 1 to 12.

17. A nucleocapsid comprising the RNA molecule according to claim 16.

18. A host cell comprising a replication-deficient or replication-competent negative-strand RNA virus according to any one of claims 1 to 12, the RNA molecule according to claim 16, the nucleocapsid according to claim 17, and / or the DNA molecule according to claim 16.

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