Vaccine composition against two respiratory viruses

Abstract

The present invention relates to a genome sequence represented by the sequence SEQ ID NO: 1, The following genetic modifications: (i) inactivation of endogenous genes encoding SH proteins and / or G proteins, and (ii) the presence of an exogenous nucleotide sequence encoding at least one extracellular domain of the F protein of human respiratory syncytial virus (hRSV), said domain being wild-type or mutated; The present invention relates to a virus strain derived from human metapneumovirus (hMPV) having the above genome sequence comprising:

Description

[Technical field]

[0001] The present invention relates to a genetically modified virus strain that contains antigenic portions from two different respiratory viruses.

[0002] The present invention relates to genetically modified viral strains and their use in the prophylactic or therapeutic treatment of infections with viruses, in particular respiratory viruses.

[0003] Introduction Acute infections of the lower respiratory tract are one of the leading causes of morbidity and mortality globally, especially in children under the age of 5, and according to the World Health Organization, they represent the second largest cause of mortality.

[0004] The majority of acute infections of the lower respiratory tract are associated with variable pathologies ranging from a simple cold to severe pneumonia, particularly in the following families: (i) viruses of the family Orthomyxoviridae (including, inter alia, influenza viruses); (ii) viruses of the family Paramyxoviridae; (iii) viruses of the Adenoviridae family; (iv) viruses of the Picornaviridae family; (v) viruses of the Coronaviridae family; and (vi) Viruses of the Pneumoviridae family, including, in particular, human respiratory syncytial virus and human metapneumovirus; It is caused by viruses such as those belonging to the genus .

[0005] Both human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are responsible for acute infections of the respiratory tract, such as bronchiolitis, bronchitis or pneumonia. These viruses mainly affect risk populations: young children under 5 years of age, elderly people and immunocompromised people. Both viruses are present on all continents. Their annual distribution is mainly in winter and spring. Viral infection occurs essentially by the respiratory route, in particular via dispersion of saliva droplets into the air.

[0006] hRSV was first identified in 1956 in chimpanzee communities and has since been demonstrated to be primarily a human pathogen. hRSV was isolated in 1957. hRSV is a virus with a single-stranded RNA genome of negative polarity and "enveloped". The RNA genome is linear, non-segmented, and protected within a helical nucleocapsid. The genome contains 10 genes that code for 11 viral proteins. hRSV belongs to the family Pneumoviridae and the genus Orthopneumovirus. Two hRSV subgroups, A and B, have been identified based on differences in the F and G genes.

[0007] hRSV is the major causative agent of bronchiolitis and pneumonia in infants under 1 year of age, with incidence increasing in those younger than 6 months of age. hRSV is responsible for 6.7% of deaths in children under 1 year of age and causes excess mortality in older adults at levels comparable to influenza virus.

[0008] hMPV was first isolated and described in the Netherlands in 2001 (van den Hoogen et al., 2001). The following year, several strains of hMPV were isolated from patients in North America (Peret et al., 2002). hMPV is a virus with negative single-stranded RNA that belongs to the family Pneumoviridae and the genus Metapneumovirus.

[0009] hMPV is a common cause of infantile bronchiolitis and pneumonia, particularly affecting children between 6 months and 3 years of age. The average age of children hospitalized due to complications of hMPV infections is 6-12 months, i.e. later than infections caused by hRSV, which occur mainly between 3 and 6 months of age.

[0010] The genome organization of hMPV and hRSV is similar. The genome is subdivided into several ORFs (open reading frames) encoding the N, P, M, F, M2 (M2-1 and M2-2), SH, G and L proteins. hRSV, unlike hMPV, also contains two additional nonstructural genes NS1 and NS2. In Figure 1, the general genome structure of the virus strain rC-8543 and its attenuated version delta SH is depicted.

[0011] Three types of glycoproteins, F, G and SH proteins, are expressed on the surface of the virus particle.

[0012] The F fusion glycoprotein, which is highly conserved among different subgroups, is involved in the entry of the virus into the target cell and in the formation of syncytia (large multinucleated cells resulting from the fusion of individual cells following their infection by the virus). The F glycoprotein, also referred to in this application as the F protein, is considered to be the most antigenic protein of the hRSV and hMPV viruses.

[0013] Today, there are very few prophylactic or therapeutic options available that are specific for hRSV and / or hMPV infection: there are no approved vaccines against hRSV or hMPV.

[0014] In treatment, ribavirin may occasionally be used in severely ill patients with infection by hMPV or hRSV, but this therapy exhibits several side effects. The usual and widely favored clinical approach is: symptomatic treatment of the infection by placing the patient on respiratory support (administration of oxygen or artificial ventilation), by administering bronchodilators, corticosteroids and / or antibiotics to prevent or treat bacterial superinfection, and by preventing hypoxemia and electrolyte abnormalities.

[0015] As an approved prophylactic treatment, palivizumab (recombinant humanized hRSV anti-F monoclonal antibody) is available for hRSV infection to prevent the severity of the disease, especially in high-risk newborns. This prophylactic treatment is very expensive.

[0016] Scientists and clinicians are now deeply committed to changing this situation of limited or non-existent preventive or therapeutic options. Efforts are underway, including the creation of RESCEU, a European consortium that aims to reorganize all clinical data on hRSV infection and provide the infrastructure to conduct clinical trials of future RSV candidate vaccines and therapeutics.

[0017] Therefore, the development of a bivalent vaccine against both hRSV and hMPV not only answers a major public health challenge, but also a real socio-economic problem in terms of reducing the high costs of treatment and hospitalization associated with these infections. Furthermore, the use of such a vaccine would show the advantage of reducing antibiotic use in the setting of bacterial superinfections, thus limiting the emergence of antibiotic resistance in the population. [Background technology]

[0018] prior art Vaccines based on the use of attenuated live virus strains, termed live attenuated vaccines (LAV), have a number of advantages.

[0019] First, live attenuated viruses can induce strong immune responses in vaccinated patients because they induce physiological responses similar to natural infection with wild-type viruses (albeit attenuated), which is not the case when isolated recombinant antigens are administered. Moreover, in the case of respiratory viruses, these LAVs can be administered by the intranasal route, thus mimicking the natural invasion of the pathogen.

[0020] Live attenuated vaccines broadly stimulate innate, humoral and cellular immunity both systemically and locally in the respiratory tract (mucosal immunity).

[0021] Due to this strong induced immune response, the use of additional adjuvants is usually not necessary.

[0022] Finally, this vaccination strategy does not generate any uncontrollable inflammatory responses that are possible with inactivated vaccines.

[0023] Therefore, this vaccination strategy is considered to be the optimal vaccine strategy for preventing respiratory viral infections in infants and young children.

[0024] International application WO2020 / 021180 discloses an attenuated hMPV strain obtained from clinical specimen C-85473, characterized by a pronounced membrane fusion ability and genetically modified to exhibit attenuated pathogenicity, in particular by suppression of the expression of the small hydrophobic protein (ΔSH) and / or the G protein (ΔG).

[0025] Furthermore, International Application WO2020 / 120910 discloses a method for producing this attenuated hMPV strain using infection and culture of a specific immortalized avian cell line.

[0026] Attenuated strains of hRSV have also been reported. For example, (Rostad et al., 2016) developed an attenuated virus strain: RSV-A2-dNS-ΔSH-BAF (DB1) using codon deoptimization of the genes for nonstructural proteins NS1 and NS2, deletion of the SH protein coding gene, and replacement of the wild-type fusion (F) protein gene with the low-fusion RSV subgroup BF consensus sequence. In the same year, an attenuated virus strain named OE4 (RSV-A2-dNS1-dNS2-DSH-dGm-Gsnull-line19F) was engineered and described in (Stobart et al., 2016). This OE4 strain expresses the line 19F protein and is attenuated by codon deoptimization of the nonstructural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon deoptimization of the attachment (G) gene, and excision of the secreted G. The safety and immunogenicity of the attenuated virus strain RSV / ΔNS2 / Δ1313 / I1314L, which harbors a deletion of the interferon antagonist NS2 gene, a deletion of codon 1313 in the RSV polymerase gene, and the stabilizing missense mutation I1314L, was demonstrated in RSV-seronegative children ( Karron et al., 2020 ).

[0027] Among the most interesting viral antigens, the F protein, which is involved in the entry of the virus into target cells and in the formation of syncytia, has been studied extensively and, in particular, antibodies against the F protein have been raised.

[0028] The administration of a monoclonal antibody directed against the F protein of hRSV is currently being used to prevent the severity of the disease. The administration of this humanized monoclonal antibody, palivizumab, to children has significantly reduced the risk of hospitalization related to RSV infection in high-risk children.

[0029] The F protein has two conformations: - A metastable state that occurs prior to the virus-cell interaction (prefusion state) - A stable state that occurs after the merger of a virus and a cell (post-fusion state) The virus merges with the host cell membrane by utilizing the difference in folding energy between the

[0030] It has been noted that the major epitopes of the hRSV F protein are found in the prefusion state of the F protein. For example, antibodies have been developed that target antigenic site O, a metastable site specific for the prefusion state of the F glycoprotein. These antibodies are 10-100 times more potent than palivizumab, which recognizes antigenic site II in the prefusion and postfusion states.

[0031] Soluble variants of the F protein have been engineered that stably expose the antigenic site O ( McLellan et al., 2013 ). These variants consist of the extracellular domain of the F protein, which is stabilized in the prefusion state.

[0032] In the development of live attenuated vaccines, another area of ​​research is the engineering of bivalent vaccines that contain antigens from two different pathogens.

[0033] Bivalent vaccines based on a combination of hRSV and hMPV are described, for example, in European Patent Application No. 3868874. In these recombinant viruses, which are based on rearranged virus strains of hRSV, the gene encoding the F protein of another virus, in particular belonging to the Pneumoviridae family, is (i) inserted between the genes encoding the G and F proteins of hRSV or (ii) replaces the gene encoding the F protein of hRSV.

[0034] Nevertheless, efforts are still ongoing to provide a safe and effective bivalent vaccine against both hMPV and hRSV.The present invention relates to a novel virus strain with multiple genetic modifications that is attenuated but still immunogenic and has significant membrane fusion ability.Advantageously, the attenuated phenotype is stable, i.e., irreversible during the replication cycle.This attenuated virus strain can replicate in vitro on cells and can be used in vivo as a live attenuated vaccine that generates an immune response against both hMPV and hRSV in the individual to whom it is administered. [Prior art documents] [Patent documents]

[0035] [Patent Document 1] International application WO2020 / 021180 [Patent Document 2] International application WO2020 / 120910 [Patent Document 3] European Patent Application No. 3868874 [Non-patent literature]

[0036] [Non-Patent Document 1] McLellan et al., 2013, doi:10.1126 / science.1243283 Summary of the Invention

[0037] The present invention relates to a bivalent vaccine capable of generating multiple immune responses in an individual against two viruses after administration. Such a vaccine induces a combined immune response against two viruses, thereby reducing the number of injections required in an individual to obtain protective immunity against different pathogens.

[0038] More particularly, the present invention relates to a method for the preparation of a genomic DNA comprising the steps of: (i) inactivation of the endogenous genes encoding the SH protein and / or the G protein to obtain a strain with attenuated virulence; and (ii) the presence of an exogenous sequence encoding at least the extracellular domain of the F protein of human respiratory syncytial virus (hRSV), said domain being wild-type or mutated; The present invention relates to a virus strain derived from human metapneumovirus (hMPV) C-85473 strain, including:

[0039] In particular, the present invention provides a. a sequence derived from hRSV encoding at least the extracellular domain of the F protein, said domain being wild-type or mutated; and b. encoding at least one domain of an F protein, said domain being selected from among a cytoplasmic domain and a transmembrane domain, said domain being wild-type or mutated; hMPV-derived sequence, wherein the exogenous sequence encodes a chimeric hRSV / hMPV F protein.

[0040] Another object of the present invention is to provide a method for the preparation of a nucleic acid sequence comprising the following nucleotide sequence: - a sequence encoding a mutated extracellular domain corresponding to a stabilized prefusion state of the F protein of hRSV, in particular consisting of the sequence shown in SEQ ID NO: 8, and a sequence encoding the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV; - a sequence encoding the wild-type extracellular domain of a protein of hRSV, in particular consisting of the sequence shown in SEQ ID NO: 9, and a sequence encoding the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV, It is a gene cassette containing one of the following:

[0041] The present invention also relates to a virus strain derived from a human metapneumovirus (hMPV) strain that contains a gene cassette as defined above. Preferentially, this virus strain exhibits an attenuated pathogenicity.

[0042] The present invention also relates to said virus strain for use as a medicament.

[0043] The present invention also relates to said virus strains for use in preventing and / or treating infections with at least one respiratory virus, in particular at least, and more particularly, two viruses of the Pneumoviridae family.

[0044] The present invention also relates to a vaccine composition comprising, in a pharma- ceutically acceptable medium, at least one virus strain as described above and, optionally, an adjuvant. [Brief description of the drawings]

[0045] [Figure 1] Structure of the hMPV genome and of the exogenous F protein, and of infected cells with said virus strains. [1A] Structure of the hMPV rC-85473 genome strain, an attenuated Metavac® strain (ΔSH rC-85473 strain, with genetic modification aimed at attenuating virulence by deletion of the SH gene), and the ΔSH rC-85473 strain with the introduction of exogenous coding sequences encoding the strains v.1, v.2 and v.3 constructs. A cassette encoding green fluorescent protein (GFP) is inserted at the 3' position. [1B] Schematic representation of the additional F RSV antigens expressed by the Metavac® recombinant virus. Structure of the v.1, v.2, v.3 and v.4 proteins encoded by exogenous sequences introduced into the genome of ΔSH rC-85473. v.3 and v.4 are chimeric proteins containing domains derived from the F proteins from hMPV and hRSV viruses. CTD: cytoplasmic domain; TMD: transmembrane domain; ED: extracellular domain [1C] Fluorescence micrographs of cells infected with Metavac® strains and bivalent Metavac®-RSV v.1, v.2 and v.3 constructs (×40 magnification in left panels, ×100 magnification in right panels). [Diagram 2]In vitro replication capacity of recombinant bivalent Metavac®-RSV viruses LLC-MK2 cells were infected separately with the following recombinant viruses:- rC-85473 strain (black solid line);- bivalent Metavac®-RSV v.1 (grey dotted line);- bivalent Metavac®-RSV v.2 (black dotted line);- bivalent Metavac®-RSV v.3 (grey solid line) at a multiplicity of infection of 0.01. Cell supernatants were collected in triplicate every day for 7 days and viral load was assessed by TCID50 assay, which represents the final viral dilution at which 50% of the tissues show visible cytopathic effect (50% tissue culture infectious dose). [Diagram 3] In vitro expression of exogenous F RSV protein on the surface of cells infected with recombinant bivalent Metavac®-RSV virus LLC-MK2 cells were infected with wild-type recombinant rC-85473 HMPV strain, bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2, or bivalent Metavac®-RSV v.3 at an MOI of 0.01 (t=0). Hep-2 cells (second line) were infected with RSV A2 strain at an MOI of 0.01 (t=0). Five days after infection, infected cell monolayers were fixed in formaldehyde solution and specific immunostaining was performed with: - HMPV24: a monoclonal mouse antibody detecting the F HMPV protein (MAb HMPV24 BioRad MCA 4674); - Palivizumab: a monoclonal humanized antibody detecting both the pre- and post-fusion forms of the F RSV protein (Palivizumab Synagis® AstraZeneca); - D25: a monoclonal human antibody detecting the pre-fusion form of the F RSV protein (D25 Mab, Creative Biolabs). [Figure 4]In vitro expression of exogenous F RSV protein on the surface of bivalent Metavac®-RSV particles [4A] Observation of three bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses by transmission electron microscopy [4B] Observation of three bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses co-immunolabeled with a monoclonal humanized antibody (Palivizumab Synagis®) detecting F RSV protein and a polyclonal mouse serum (homemade serum) detecting HMPV protein by transmission electron microscopy [4C] Observation of three bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses co-immunolabeled with a monoclonal humanized antibody (Palivizumab Synagis®) detecting F RSV protein and a polyclonal mouse serum (homemade serum) detecting HMPV protein by transmission electron microscopy [4D] Observation of three bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses co-immunolabeled with a monoclonal humanized antibody (Palivizumab Synagis®) detecting F RSV protein and a polyclonal mouse serum (homemade serum) detecting HMPV protein by transmission electron microscopy [4E] Observation of three bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses co-immunolabeled with a monoclonal humanized antibody (Palivizumab Synagis®) detecting F RSV protein and a polyclonal mouse serum (homemade serum) detecting HMPV protein by transmission electron microscopy [4F] Observation of three bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RS Serum neutralization of v.2 and bivalent Metavac®-RSV v.3 viruses [Diagram 5]Infection and replication capacity of three bivalent Metavac®-RSV v.1, v.2 and v.3 viruses in ex vivo 3D reconstituted human respiratory epithelium [5A] Fluorescence microscopy of 3D reconstituted human respiratory epithelium infected with each bivalent Metavac®-RSV v.1, v.2 or v.3 virus compared to monovalent Metavac® virus. All viruses contain a GFP-encoding gene at the 3' position of their genome. [5B] Transepithelial electrical resistance measured during infection of 3D reconstituted human respiratory epithelium with each bivalent Metavac®-RSV v.1, v.2 or v.3 virus. [5C] Viral gene quantification (quantification of N HMPV gene and F RSV gene copies performed by RT-qPCR) at the epithelial apical membrane side 1, 3, 5, 7 or 9 days (post-infection) after infection with monovalent Metavac® (black bars), bivalent Metavac®-RSV v.1 (dark grey bars), bivalent Metavac®-RSV v.2 (light grey bars) or bivalent Metavac®-RSV v.3 (striped grey bars). The dotted line represents the threshold of detection. [5D] Immunolabeling of F RSV antigen expressed at the surface of 3D reconstituted human respiratory epithelium infected with bivalent Metavac®-RSV v.1 virus. Immunostaining was performed using palivizumab, a monoclonal humanized antibody that detects both pre- and post-fusion forms of the F RSV protein. F RSV protein expression at the apical epithelial membrane is highlighted by the black arrowheads. [Figure 6] In vivo characterization of recombinant bivalent Metavac®-RSV v.1, v.2 and v.3 viruses in a BALB / c mouse virus infection model [6A] Weight loss of BALB / c mice during the time course of the in vivo experiment. Three constructs (bivalent Metavac®-RSV v.1, v.2 and v.3) are tested. [6B] Viral gene quantification in lung tissue 5 days after infection with Metavac®-RSV v.1, v.2 or v.3: Quantification of N HMPV genes (left graph) and F RSV genes (right graph) is performed by RT-qPCR. The dotted line represents the threshold of detection. [Figure 7]In vivo induction of neutralizing antibody production following HMPV- or RSV-prime infection in a BALB / c mouse model followed by boost infection with bivalent Metavac®-RSV v.1, v.2, and v.3 viruses. [7A] Schematic representation of the in vivo protocol illustrating the first intranasal instillation of C-85473 HMPV or RSV A wild-type virus strain (prime infection) and the second intranasal instillation of bivalent Metavac®-RSV v.1, v.2, or v.3 viruses after a 3-week interval to evaluate their potential to induce HMPV- or RSV-specific antibody responses in non-naive BALB / c mice. [7B] Bivalent Metavac®-RSV compared to a group of mice boosted with OptiMEM (mock boost). Weight loss in HMPV prime-infected BALB / c mice after intranasal boost with v.1, v.2 or v.3 viruses [7C] Weight loss in RSV prime-infected BALB / c mice after intranasal boost with bivalent Metavac®-RSV v.1, v.2 or v.3 viruses compared to groups of mice boosted with OptiMEM (mock boost) [7D] Viral gene quantification in lung tissue of HMPV prime-infected BALB / c mice 5 days after intranasal boost with bivalent Metavac®-RSV v.1, v.2 or v.3 viruses: Quantification of N HMPV genes (left graph) and F RSV genes (right graph) is performed by RT-qPCR. The dotted line represents the threshold of detection. [7E] Viral gene quantification in lung tissue of RSV primed infected BALB / c mice 5 days after intranasal boost with bivalent Metavac®-RSV v.1, v.2 or v.3 virus: Quantification of N HMPV genes (left graph) and F RSV genes (right graph) is performed by RT-qPCR. Dotted lines represent the threshold of detection. [7F] Characterization of HMPV-specific neutralizing antibody responses from serum of HMPV primed infected BALB / c after intranasal boost with bivalent Metavac®-RSV v.1, v.2 or v.3 virus, or mock boost.Microneutralization assays were performed with sera collected 1 day before prime infection (-1), 20 days after prime infection (+20), and 42 days after prime infection / 21 days after intranasal boost infection (+42). The dotted lines represent the threshold of detection. [7G] Characterization of RSV-specific neutralizing antibody responses from sera of RSV prime-infected BALB / c mice after intranasal boost with bivalent Metavac®-RSV v.1, v.2, or v.3 viruses, or mock boost. As previously described, microneutralization assays were performed with sera collected 1 day before prime infection (-1), 20 days after prime infection (+20), and 42 days after prime infection / 21 days after intranasal boost infection (+42). The dotted lines represent the threshold of detection. [Figure 8]In vivo protective properties of bivalent Metavac®-RSV v.3 virus against infectious challenge with a lethal dose of wild-type rC-85473 HMPV strain. [8A] Schematic of in vivo prime and boost immunization with bivalent Metavac®-RSV v.3 virus followed by lethal viral challenge (viral dose expected to induce >50% mortality) with wild-type C-85473 HMPV virus. Each successive infection by intranasal viral instillation is performed after an interval of 3 weeks. [8B] Weight loss of BALB / c mice after infectious challenge with wild-type HMPV C-85473. Body weight of mice primed and boosted with monovalent Metavac® or bivalent Metavac®-RSV v.3 virus is compared to a group of mice mock-immunized with OptiMEM culture medium. [8C] Survival percentage of prime- and boost-immunized BALB / c mice after lethal challenge with HMPV C-85473. [8D] Viral gene quantification in lung tissue of BALB / c mice prime- and boost-immunized with bivalent Metavac®-RSV v.3 5 days after lethal virus challenge with wild-type virus C-85473 HMPV strain: Quantification of N HMPV genes (left graph) and F RSV genes (right graph) is performed by RT-qPCR. The dotted line represents the threshold of detection. [8E] HMPV-specific neutralizing antibody detection in serum from BALB / c mice prime- and boost-immunized with OptiMEM culture medium (mock), monovalent Metavac® or bivalent Metavac®-RSV v.3 and challenged with wild-type C-85473 HMPV strain. As previously described, microneutralization assays were performed with sera collected 1 day before prime infection (-1), 20 days after prime infection (+20), 41 days after prime infection / 20 days after boost infection (+41), and 62 days after prime infection / 21 days after lethal virus challenge (+62). The dotted lines represent the threshold of detection.[8F] Detection of RSV-specific neutralizing antibodies in sera from BALB / c mice prime- and boost-immunized with OptiMEM culture medium (mock), monovalent Metavac®, or bivalent Metavac®-RSV v.3 and challenged with wild-type C-85473 HMPV strain. As previously described, microneutralization assays were performed with sera collected 1 day before prime infection (-1), 20 days after prime infection (+20), 41 days after prime infection / 20 days after boost infection (+41), and 62 days after prime infection / 21 days after lethal virus challenge (+62). The dotted lines represent the threshold of detection. [Figure 9] In vivo characterization of recombinant bivalent Metavac®-RSV v.1 and v.3 viruses in a BALB / c mouse viral infection model [9A] Viral gene quantification in bronchoalveolar lavage 2 days after infection with bivalent Metavac®-RSV v.1 or v.3 compared to infection with HMPV WT virus (rC-85437) and the monovalent live attenuated vaccine (LAV) candidate Metavac®: Quantification of N HMPV genes (left graph) and F RSV genes (right graph) is performed by RT-qPCR. The dotted line represents the threshold of detection. [9B] Cumulative histopathological score measured from inflammation assessment in lung tissue (interstitial, intraalveolar, peribronchial, endobronchial, perivascular and pleural compartments) of mice infected with HMPV WT virus (rC-85437), monovalent live attenuated vaccine (LAV) candidate Metavac®, bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.3 or «mock» infection. Lungs of infected mice were harvested 5 days post-infection and fixed in formaldehyde. [Figure 10]In vivo protective properties of bivalent Metavac®-RSV v.1 and v.3 viruses against infectious challenge with lethal doses of wild-type virus rC-85473 HMPV strain BALB / c mice were immunized twice, 21 days apart, with 5×105 TCID50 of Metavac® or bivalent Metavac®-RSV vaccine candidates v.1 or v.3 via the IN route or with HMPV C-85473 virus inactivated split adjuvanted with AddaVax™ via the IM route. Three weeks after the last immunization, animals were inoculated with 2×106 TCID of rC-85473 virus. [10A] Weight loss in BALB / c mice after infectious challenge with wild-type HMPV C-85473. The body weights of mice primed and boosted with monovalent Metavac®, bivalent Metavac®-RSV v.1 or v.3 viruses are compared to groups of mice mock-immunized with OptiMEM culture medium or groups of mice immunized via the intramuscular route with an adjuvanted split of HMPV WT virus (HMPV split), an alternative to vaccination with HMPV protein vaccines. [10B] Survival percentage of prime- and boost-immunized BALB / c mice following lethal challenge with HMPV C-85437. [10C] Cumulative histopathological scores 5 days after challenge measured from inflammation assessment in lung tissues (interstitial, intraalveolar, peribronchial, endobronchial, perivascular and pleural compartments) of mice primed- and boosted with bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.3 or monovalent Metavac® virus compared to mice mock-immunized or immunized with adjuvanted HMPV split. Lungs of infected mice were harvested 5 days after lethal challenge and fixed in formaldehyde.[10D] Viral gene quantification in lung tissue of BALB / c mice primed- and boosted with bivalent Metavac®-RSV v.1 or v.3 5 days after lethal virus challenge with wild-type C-85437 HMPV strain: Quantification of N HMPV genes (left graph) and F RSV genes (right graph) is performed by RT-qPCR. The dotted line represents the threshold of detection. [10E] HMPV-specific neutralizing antibody detection in sera from BALB / c mice primed- and boosted with OptiMEM culture medium (mock), monovalent Metavac®, bivalent Metavac®-RSV v.1 or v.3, or HMPV split and challenged with wild-type C-85473 HMPV strain. Microneutralization assays were performed with sera collected 1 day before prime infection (-1), 20 days after prime infection (+20), 41 days after prime infection / 20 days after boost infection (+41), and 62 days after prime infection / 21 days after lethal virus challenge (+62). Neutralizing antibody titers against a homologous HMPV strain (A / rC-85473, left graph) and a heterologous HMPV strain (B / CAN98-75, right graph) were measured by specific microneutralization assays. [10F] HMPV-specific IgG antibody detection in sera from BALB / c mice prime- and boost-immunized with OptiMEM culture medium (mock), monovalent Metavac®, bivalent Metavac®-RSV v.1 or v.3, or HMPV split and challenged with wild-type C-85473 HMPV strain was performed using an ELISA assay from sera collected 1 day before prime infection (-1), 20 days after prime infection (+20), 41 days after prime infection / 20 days after boost infection (+41), and 62 days after prime infection / 21 days after lethal virus challenge (+62). IgG titers were expressed as arbitrary units per mL based on endpoint absorbance. [Figure 11]In vivo protective properties of bivalent Metavac®-RSV v.1 and v.3 viruses against infection challenge with recombinant RSV-Luc WT virus expressing luciferase protein BALB / c mice were immunized twice via the IN route with 5×105 TCID50 of Metavac®-RSV vaccine candidate or recombinant RSV (RSV WT) virus or mock immunized with an interval of 21 days. Three weeks after the last immunization, animals were inoculated with 1×105 PFU of rRSV-Luc virus, a recombinant RSV A WT virus expressing the in vivo luminescent luciferase protein. [11A] Bioluminescence on the abdominal surface of infected mice was imaged on days 3 and 5 after challenge using an in vivo imaging system (IVIS). Bioluminescence was measured after intranasal injection of 50 μl of D-luciferin. The scale on the right indicates the mean brightness (total number of photons per second from each pixel inside the region of interest, ps-1cm-2sr-1) and is represented in shades of grey. [11B] Luciferase activity was quantified from bioluminescence images using "Living Image" software and expressed as mean ± SEM photons per second (p / s). Luciferase activity was measured from the abdominal surface of infected mice imaged on days 3 and 5 post-challenge using an in vivo imaging system (IVIS) after intranasal injection of 50 μl D-luciferin. ***p<0.001 when comparing Metavac®-RSV v.1, v.3 or RSV WT vaccinated groups to mock vaccinated condition using one-way ANOVA with Dunnett's post-hoc test. [11C] Viral gene quantification in lung tissue of BALB / c mice primed and boosted with bivalent Metavac®-RSV v.1 or v.3 4 days after viral challenge with RSV WT virus: Quantification of F RSV genes (left graph) and N HMPV genes (right graph) is performed by RT-qPCR. The dotted line represents the threshold of detection.[11D] Detection of RSV-specific neutralizing antibodies in sera from BALB / c mice primed and boosted with OptiMEM culture medium (mock), bivalent Metavac®-RSV v.1 or v.3, or recombinant RSV WT virus and challenged with rRSV-Luc virus. Microneutralization assays were performed with sera collected 1 day before prime infection (-1), 20 days after prime infection (+20), 41 days after prime infection / 20 days after boost infection (+41), and 62 days after prime infection / 21 days after virus challenge (+62). Neutralizing antibody titers against homologous RSV A strain (RSV A long, left graph) and heterologous RSV B strain (WV / 14617 / 85, right graph) were measured by specific microneutralization assays. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Human metapneumovirus (hMPV) strains The present invention is based on a human metapneumovirus strain isolated from a patient sample in Canada and described in the paper (Hamelin et al., 2010), designated C-85473.

[0047] The recombinant strain rC-85473, derived from this virus strain C-85473 and obtained by reverse genetic engineering, is characterized by a considerable membrane fusion ability. Moreover, rC-85473 is able to enter target cells at a high frequency, i.e., at a high infectivity. Without wishing to be bound by any theory, the inventors attribute these properties to a specific sequence of the F protein of the rC-85473 strain, which contains a unique peptide sequence of five amino acids that is not found among other F proteins of other hMPV strains (Dubois et al., 2017).

[0048] The rC-85473 hMPV strain contains the genomic sequence shown in SEQ ID NO:1.

[0049] The first genetic modification (i) introduced into the rC-85473 strain aims to attenuate the virulence of said strain. "Attenuated virulence" corresponds to the absence of pathogenicity and a reduced inflammatory response in vivo after administration of an effective amount of such an attenuated virus strain (Dubois et al., 2019 and Le et al., 2019).

[0050] Advantageously, this attenuation of virulence does not affect the ability of the viral strain to replicate in vitro or its ability to infect target cells. The genetic modification involves the alteration of at least one endogenous gene: - a gene encoding an SH protein, or - Genes that code for G proteins It consists of inactivation of

[0051] This genetic modification may be the inactivation of both genes. In the context of the invention, "gene inactivation" refers to a genetic modification that leads to the disappearance of the expression of a gene or to the expression of an inactive form of the encoded protein. This inactivation of a gene may be carried out by all techniques known to those skilled in the art. In particular, the inactivation of a gene may be obtained by the introduction of a point mutation in the gene, by partial or total deletion of the coding sequence of the gene, or by modification of the gene promoter. These different genetic modifications are carried out according to any one of the molecular biology techniques known to those skilled in the art.

[0052] In a particular embodiment of the invention, the endogenous gene encoding the SH protein is deleted in the virus strain of the invention.

[0053] In the context of the invention, a "deleted gene" means that a substantial part of the coding sequence of this gene has been removed, in particular: - a partial deletion of a gene means that at least 50%, 60%, 70%, 80%, 90% or 95% of the coding sequence has been removed; - A complete gene deletion means that 100% of the coding sequence has been removed.

[0054] According to a preferred embodiment, in the virus strain according to the invention, the gene encoding the SH protein is completely deleted, i.e. all (100%) of the coding sequence for the SH protein is removed from the original genome sequence. In this case, the virus strain according to the invention comprises the nucleotide sequence as depicted in SEQ ID NO: 2. This particular attenuated virus strain is designated in the Examples section by the name "Metavac®" and is also designated "monovalent Metavac®".

[0055] A viral strain that contains this first genetic modification (i) is referred to below as an attenuated viral strain.

[0056] The second genetic modification (ii) introduced into the attenuated rC-85473 strain aims to obtain the expression of an antigen derived from the hRSV strain.

[0057] This genetic modification (ii) consists of the introduction of an exogenous coding sequence into the genome of said attenuated virus strain, comprising, for example, the genomic sequence as represented in SEQ ID NO:2.

[0058] The introduction of the exogenous coding sequence is an actual addition to the genome of the attenuated rC-85473 strain, rather than a replacement, so that the F protein of the rC-85473 strain is still present in the genome of the strain and is expressed even after the introduction of the exogenous sequence.

[0059] Thus, the virus strain of the invention comprises in its genome a sequence encoding at least one extracellular domain of the F protein of human respiratory syncytial virus (hRSV), said domain being wild-type or mutated.

[0060] In the context of the invention, "exogenous sequence encoding" or "exogenous coding sequence" or "exogenous nucleotide sequence" refers to a nucleic acid sequence that is introduced into the viral genome, which is under the control of a suitable promoter and which codes for a protein or a protein domain. In this case, since the genome of hMPV is made of RNA, the introduced exogenous sequence is also composed of RNA. However, it is understood that a preliminary step for introducing this exogenous sequence may be to use the corresponding DNA sequence (reverse gene plasmid).

[0061] In the sense of the invention, the term "F protein from hRSV" refers to the glycoprotein from hRSV subgroup A or B, preferentially from hRSV of subgroup A.

[0062] The sequences of all known wild-type F proteins from hRSV can be found in public databases such as UniProt, for example under accession references P11209 or P03420 (precursor type).

[0063] Those skilled in the art are familiar with protein biology and can identify three domains that make up a transmembrane protein: a cytoplasmic domain inside the cell; a transmembrane domain that is inserted into the cell membrane; and an extracellular domain that is present on the surface of the cell membrane.

[0064] In the context of the invention, the phrase "encodes at least" means that the exogenous sequence encodes at least one peptidic domain, but in most cases also other domains.

[0065] In the context of the invention, the phrase "an extracellular domain of the F protein of hRSV" refers to any extracellular domain of the F protein (expressed on the surface of the viral particle) from any hRSV subgroup, including wild-type and mutated domains.

[0066] As is well known to those skilled in the art, it is preferred to combine the extracellular domain with another peptide sequence that allows its anchoring in the viral particle, in particular at the surface of said viral particle, and / or that allows its anchoring in the membrane of an infected cell.

[0067] Thus, in a preferred embodiment of the invention, "an exogenous sequence encoding at least an extracellular domain of the F protein of human respiratory syncytial virus (hRSV), said domain being wild-type or mutated" refers to an exogenous sequence encoding a mutated or wild-type extracellular domain of the F protein of hRSV, in relation to a sequence encoding at least one anchoring domain, in particular the cytoplasmic and / or transmembrane domain.

[0068] In a first embodiment of the invention, the domain of the F protein is a wild-type domain, i.e. it exhibits the peptide sequence of the domain of the naturally occurring F protein from hRSV.

[0069] The term "wild type" refers to the typical form of a protein as it occurs in nature (i.e., its typical peptide sequence). In contrast, the term "mutated" refers to an atypical, non-standard form of the same protein.

[0070] In certain embodiments, the exogenous nucleotide sequence comprises a sequence encoding the wild-type extracellular domain of the hRSV F protein.

[0071] In another specific embodiment, the exogenous nucleotide sequence consists of a sequence encoding the wild-type extracellular domain of the hRSV F protein.

[0072] In particular, the exogenous nucleotide sequence comprises a sequence encoding the wild-type extracellular domain of a hRSV F protein from a subgroup A virus, more particularly, a sequence encoding the wild-type extracellular domain of a hRSV F protein having the peptide sequence set forth in SEQ ID NO:3.

[0073] In certain embodiments, the exogenous nucleotide sequence further comprises a sequence encoding a wild-type transmembrane domain of the hRSV F protein.

[0074] In certain embodiments, the exogenous nucleotide sequence further comprises a sequence encoding the wild-type cytoplasmic domain of the hRSV F protein.

[0075] In another specific embodiment, the exogenous nucleotide sequence is composed of a sequence encoding the three wild-type domains of the hRSV F protein, i.e., the extracellular, cytoplasmic and transmembrane domains that make up the entire F protein.

[0076] In particular, the exogenous nucleotide sequence comprises a sequence encoding a wild-type hRSV F protein from a subgroup A virus, more particularly, a sequence encoding a wild-type hRSV F protein having the peptide sequence set forth in SEQ ID NO:4.

[0077] In certain embodiments of the invention, the exogenous nucleotide sequence is located at a specific site in the genome of the attenuated hMPV virus strain, such as: - between the gene coding for the N protein and the gene coding for the P protein; - between the gene coding for the P protein and the gene coding for the M protein; - between the gene encoding the F protein and the gene encoding the M2 protein; - between the gene encoding the SH protein and the gene encoding the G protein; - between the gene encoding the M2 protein and the gene encoding the G protein (in the case of ΔSH strains); - between the gene encoding the G protein and the gene encoding the L protein; is incorporated into.

[0078] According to one of these specific embodiments, the exogenous nucleotide sequence advantageously comprises the three wild-type domains of the hRSV F protein, i.e., the extracellular, cytoplasmic and transmembrane domains that constitute the entire F protein from hRSV.

[0079] A particular chimeric construct comprising the genome of an attenuated hMPV strain (sequence represented by SEQ ID NO:2) and an exogenous nucleotide sequence encoding a wild-type hRSV F protein (SEQ ID NO:4) inserted between the gene encoding the F protein and the gene encoding the M2 protein is designated "Metavac®-RSV v.1" in the Examples section.

[0080] The complete genome sequence of this virus strain, combined with the GFP-encoding gene, is shown in SEQ ID NO:10.

[0081] In a particular embodiment, the present invention provides a genomic sequence represented by SEQ ID NO:1, The following genetic modifications: (i) inactivation of endogenous genes encoding SH proteins and / or G proteins, and (ii) the presence of an exogenous nucleotide sequence encoding a wild-type hRSV F protein of hRSV, the exogenous nucleotide sequence being inserted between a gene encoding the F protein and a gene encoding the M2 protein of the hRSV strain; The present invention relates to a viral strain derived from a human metapneumovirus (hMPV) strain having the above genome sequence comprising:

[0082] In a second embodiment of the invention, the domain of the F protein is a mutated domain, i.e., it exhibits a peptide sequence derived from the domain of the wild-type F protein from hRSV, which comprises at least one point mutation, i.e., a substitution of at least one residue in the wild-type peptide sequence with another residue.

[0083] In the context of the present invention, the terms "mutated domain" or "mutated sequence" or "mutated protein" all refer to a peptide sequence that exhibits at least 80% sequence identity with its corresponding standard wild-type peptide sequence after optimal alignment of both sequences, and therefore exhibits a maximum of 20% difference from its corresponding standard wild-type peptide sequence. In a preferred embodiment, in said mutant domain, the main antigenic epitopes are conserved, i.e., they exhibit their wild-type sequence.

[0084] In a preferred implementation of the invention, the mutated domain exhibits at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97% sequence identity with its corresponding wild-type domain. Preferentially, in said mutated domain, major antigenic epitopes are preserved.

[0085] In a more specific embodiment of the invention, the mutated domain corresponds to a mutated domain exhibiting at least 97% sequence identity with the corresponding wild-type domain. The percent identity referred to in the context of the present invention is determined upon optimal alignment of the sequences to be compared, which sequences include one or more differences such as insertions, deletions, truncations and / or substitutions of amino acids.

[0086] This percent identity may be calculated by any sequence analysis method known to those skilled in the art.

[0087] The percent identity may be determined after global alignment of the sequences to be compared within the sequences taken in their entirety over their entire length. In addition to manual comparison, it is possible to determine global alignment using the algorithm of Needleman and Wunsch (1970).

[0088] For peptide sequences, sequence comparisons may be performed using any software well known to those skilled in the art, such as the Needle software. The parameters used may be, inter alia, the following: "Gap open" equal to 10.0, "Gap extend" equal to 0.5, and the BLOSUM62 matrix. Preferably, the percent identity is determined via a global alignment of the sequences being compared over their entire length.

[0089] In a specific embodiment, the exogenous nucleotide sequence comprises a sequence encoding a mutant extracellular domain of the hRSV F protein.

[0090] In another specific embodiment, the exogenous nucleotide sequence comprises a sequence encoding a mutated extracellular domain of the hRSV F protein.

[0091] More preferentially, this mutated domain corresponds to a stabilized pre-fusion state of the F protein, ie has a protein conformational constraint under the pre-fusion state.

[0092] The mutant extracellular domain contains at least one of the following 14 mutations as described in (McLellan et al., 2013): S46G, K66E, E92D, Q101P, A149C, S155C, S190F, V203L, V207L, S215P, S290C, L373R, Y458C, K465Q.

[0093] Preferentially, the mutated extracellular domain comprises all 14 mutations listed above. These mutations are incorporated by any technique known to those skilled in the art, in particular by directed mutagenesis.

[0094] In certain embodiments, the mutant extracellular domain has the sequence set forth in SEQ ID NO: 5. This mutant domain exhibits 14 point mutations across its full length of 513 residues and thus shares 499 residues in common with the corresponding wild-type extracellular domain (residues 1-513 of SEQ ID NO: 4), which corresponds to a percentage identity between the wild-type and mutant extracellular domains of the hRSV F protein of 97.27%.

[0095] In another embodiment, the exogenous nucleotide sequence is composed of a sequence encoding a wild-type cytoplasmic domain, a wild-type transmembrane domain, and a mutant extracellular domain corresponding to a stabilized prefusion state of the hRSV F protein.

[0096] A particular chimeric construct comprising the genome of an attenuated hMPV strain (sequence represented by SEQ ID NO:2) and an exogenous nucleotide sequence encoding a mutant hRSV F protein containing a mutant extracellular domain corresponding to a stabilized prefusion state (SEQ ID NO:5) is designated in the Examples section as "Metavac®-RSV v.2."

[0097] In particular, the mutant F protein has the sequence set forth in SEQ ID NO: 6. This mutant protein exhibits 97.5% sequence identity with the wild-type hRSV F protein (560 common residues over a full length of 574 residues).

[0098] The complete genome sequence of this virus strain, combined with the GFP-encoding gene, is shown in SEQ ID NO:11.

[0099] In another embodiment of the invention, the exogenous nucleotide sequence encodes a chimeric protein comprising a domain derived from the F protein of hRSV and a domain derived from the F protein of hMPV.

[0100] In particular, the exogenous nucleotide sequence encoding the chimeric hRSV / hMPV F protein comprises: a. a sequence from hRSV encoding at least one extracellular domain of the F protein, said domain being wild-type or mutated; and b. a sequence from hMPV encoding at least one domain of the F protein, selected between the cytoplasmic domain and the transmembrane domain, said domain being wild type or mutated; As mentioned above, in the context of the present invention, the term "mutated domain" refers to a domain having a peptide sequence that has at least 80%, preferentially at least 90%, and even more preferentially at least 95%, 96% or 97% sequence identity to the peptide sequence of the corresponding wild-type domain. Preferentially, the main antigenic epitopes of said domain are conserved.

[0101] In other words, the present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: a. a sequence from hRSV encoding at least one extracellular domain of an F protein, said domain having at least 80% sequence identity to the peptide sequence of the corresponding wild-type domain and being either (i) wild-type or (ii) mutated; and b. a sequence from hMPV encoding at least one domain of the F protein, selected between the cytoplasmic domain and the transmembrane domain, said domain having at least 80% sequence identity to the peptide sequence of the corresponding wild-type domain and being either (i) wild-type or (ii) mutated; Any of the above attenuated virus strains, comprising in their genome an exogenous sequence according to any possible combination of (a) and (b), is the subject of the present invention.

[0102] For example, the combination is - the wild-type extracellular and transmembrane domains of the F protein from hRSV and the wild-type cytoplasmic domain of the F protein from hMPV; - the wild-type extracellular and cytoplasmic domains of the F protein from hRSV and the wild-type transmembrane domain of the F protein from hMPV; - the wild-type extracellular domain of the F protein from hRSV and the wild-type transmembrane and cytoplasmic domains of the F protein from hMPV; - a mutated extracellular and wild-type transmembrane domain of the F protein from hRSV and a wild-type cytoplasmic domain of the F protein from hMPV; - a mutated extracellular and wild-type cytoplasmic domain of the F protein from hRSV and the wild-type transmembrane domain of the F protein from hMPV; - a mutated extracellular domain of the F protein from hRSV and the wild-type transmembrane and cytoplasmic domains of the F protein from hMPV; The chimeric hRSV / hMPV F protein comprises the following sequence:

[0103] Advantageously, the "mutated" extracellular domain corresponds to a stabilized pre-fusion state of the F protein from hRSV as described above, in particular exhibiting at least 97% sequence identity to the corresponding wild-type domain.

[0104] In a particular embodiment, the attenuated virus strain derived from hMPV is a. a sequence derived from hRSV encoding a mutated extracellular domain corresponding to a stabilized prefusion state of the F protein of hRSV; and b. a sequence derived from hMPV encoding the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV; The exogenous nucleotide sequence comprises:

[0105] In a specific embodiment, the F protein of the hMPV is from subgroup strain A1.

[0106] In another specific embodiment, the F protein of hMPV is from the rC-85473 strain and exhibits the sequence shown in SEQ ID NO:7.

[0107] A particular chimeric construct comprising an exogenous nucleotide sequence encoding the genome of an attenuated hMPV strain (sequence represented by SEQ ID NO: 2) and a chimeric protein comprising a mutated extracellular domain corresponding to a stabilized prefusion state of the F protein from hRSV (SEQ ID NO: 5) and the wild-type cytoplasmic and transmembrane domains of the F protein from hMPV is designated in the Examples section as "Metavac®-RSV v.3."

[0108] In particular, the encoded chimeric protein exhibits the sequence shown in SEQ ID NO:8.

[0109] The complete genome sequence of this virus strain, combined with the GFP-encoding gene, is shown in SEQ ID NO:12.

[0110] In a particular embodiment, the attenuated virus strain derived from hMPV is a. a sequence from hRSV encoding the wild-type extracellular domain of the F protein of hRSV; and b. A sequence derived from hMPV that encodes the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV. The exogenous nucleotide sequence comprises:

[0111] A particular chimeric construct comprising the genome of an attenuated hMPV strain (sequence represented by SEQ ID NO:2) and an exogenous sequence encoding a chimeric protein comprising the wild-type extracellular domain of the F protein from hRSV (SEQ ID NO:3) and the wild-type cytoplasmic and transmembrane domains from the F protein of hMPV is designated "v.4" in the Examples section.

[0112] In particular, the encoded chimeric protein exhibits the sequence shown in SEQ ID NO:9.

[0113] In a particular embodiment, the viral strain of the invention comprises the following genetic modifications: (i) inactivation of the endogenous gene encoding the SH protein, and (ii) the presence of an exogenous nucleotide sequence encoding a polypeptide having a sequence selected among SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:9; Shows.

[0114] Chimeric proteins and corresponding coding sequences, and virus strains containing same Another aspect of the invention relates to a gene cassette encoding a chimeric protein comprising at least one domain derived from the F protein of hRSV and at least one domain derived from the F protein of hMPV.

[0115] In particular, the gene cassette comprises: a. a nucleotide sequence from hRSV encoding at least the extracellular domain of the F protein, said domain being wild-type or mutated; and b. a nucleotide sequence from hMPV encoding at least one domain of the F protein, selected between the cytoplasmic domain and the transmembrane domain, said domain being wild-type or mutated; Including, The exogenous sequence encodes a chimeric hRSV / hMPV F protein.

[0116] These gene cassettes also encapsulate the promoter sequence and all the regulatory elements allowing the transcription and translation of the nucleotide sequences (a) and (b) into proteins.

[0117] All possible combinations of (a) and (b) are within the scope of the present invention.

[0118] The meaning of "mutated domain" has been previously defined. Advantageously, the "mutated" extracellular domain corresponds to the stabilized pre-fusion state of the F protein from hRSV. In a specific embodiment, the mutant extracellular domain has the sequence shown in SEQ ID NO:5.

[0119] For example, the combination is - the wild-type cytoplasmic domain of the F protein from hMPV; - the wild-type extracellular and cytoplasmic domains of the F protein from hRSV and the wild-type transmembrane domain of the F protein from hMPV; - the wild-type extracellular domain of the F protein from hRSV and the wild-type transmembrane and cytoplasmic domains of the F protein from hMPV; - a mutated extracellular and wild-type transmembrane domain of the F protein from hRSV and a wild-type cytoplasmic domain of the F protein from hMPV; - a mutated extracellular and wild-type cytoplasmic domain of the F protein from hRSV and the wild-type transmembrane domain of the F protein from hMPV; - a mutated extracellular domain of the F protein from hRSV and the wild-type transmembrane and cytoplasmic domains of the F protein from hMPV; The chimeric hRSV / hMPV F protein comprises the following nucleotide sequence: ##STR00011## Advantageously, the "mutated" extracellular domain corresponds to the stabilized pre-fusion state of the F protein from hRSV.

[0120] In a particular embodiment, the gene cassette comprises the following nucleotide sequence: a. a sequence derived from hRSV encoding a mutated extracellular domain corresponding to a stabilized prefusion state of the F protein of hRSV; and b. a sequence derived from hMPV encoding the wild-type cytoplasmic and transmembrane domains of the F protein of hMPV; Includes.

[0121] A particular chimeric construct according to this embodiment is designated "v.3" in the Examples section.

[0122] In particular, the encoded chimeric protein exhibits the sequence shown in SEQ ID NO:8.

[0123] In a particular embodiment, the gene cassette comprises the following nucleotide sequence: a. a sequence from hRSV encoding the wild-type extracellular domain of the F protein of hRSV; and b. a sequence derived from hMPV encoding the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV; Includes.

[0124] A particular chimeric construct according to this embodiment is designated "v.4" in the Examples section.

[0125] In particular, the encoded chimeric protein exhibits the sequence shown in SEQ ID NO:9.

[0126] The nucleotide sequences described can be in the form of DNA or RNA.

[0127] The gene cassettes described above can be expressed by any system known to those skilled in the art. For example, the gene cassettes according to the present invention can be incorporated into plasmids, bacmids, liposomes, or any vector of expression. The encoded chimeric proteins can also be used as such.

[0128] In another aspect, the invention relates to a virus strain derived from a human metapneumovirus (hMPV) strain, which comprises in its genome a gene cassette as described above.

[0129] Preferentially, the virus strain derived from the hMPV strain is further attenuated, i.e. its pathogenicity is reduced compared to that of the original virus strain, this attenuation of the pathogenicity being obtained, for example, by introducing genetic modifications into the genomic sequence of this virus strain, as is well known to those skilled in the art.

[0130] Viral strains for their use as medicines The present invention also relates to any of the above mentioned virus strains for use as a medicament.

[0131] Indeed, this attenuated virus strain may be used, inter alia, to treat and / or prevent infection with at least one respiratory virus, and more particularly with at least one virus of the Pneumoviridae family.

[0132] In the context of the invention, the term "treat" refers to the fact of combating infection by a virus in a human or animal organism. In the case of a viral infection, "treat" refers to the reduction of the level of viral infection (infectious load) in the organism, and preferably the complete eradication of the virus from the organism. The term "treat" also refers to the fact of attenuating the symptoms associated with viral infection (respiratory syndrome, renal failure, fever, etc.).

[0133] In the context of the invention, the term "prevent" refers to the fact of avoiding an infection in an organism, or at least reducing the risk of the occurrence of an infection. In the case of a viral infection, "prevent" means that the organism's cells are not permissive to the infection and are therefore best placed not to be infected by said virus. "Prevent" also means that the organism's immune system is primed to react rapidly and efficiently to the presence of the virus in order to combat the infection.

[0134] More specifically, the present invention relates to a viral strain as described above for use in preventing and / or treating infection with at least one respiratory virus, wherein the at least one respiratory virus is from the Pneumoviridae family, in particular human metapneumovirus and / or human respiratory syncytial virus.

[0135] In particular, the invention relates to said virus strains for use in preventing infection with two respiratory viruses, human metapneumovirus (hMPV) and human respiratory syncytial virus (hRSV).

[0136] This virus strain is preferably incorporated into a vaccine composition comprising a pharma- ceutically acceptable vehicle suitable for suspending said virus strain and for its administration.

[0137] Said vaccine composition comprises at least one virus strain according to the invention, making it possible to stimulate the immune system of the body in a specific manner.

[0138] This vaccine composition therefore comprises at least one live attenuated virus strain that acts as an antigen, ie is recognised in the organism and induces a specific immune response, thereby preserving its memory.

[0139] The present invention also relates to a vaccine composition comprising at least one virus strain according to the invention and, optionally, an adjuvant, in a pharma- ceutically acceptable vehicle.

[0140] In the context of the invention, the term "pharmaceutical acceptable vehicle" refers to vehicles or excipients, i.e. compounds that do not have any specific action on the infections considered here. These vehicles or excipients are pharmaceutical acceptable, meaning that they can be administered to an individual without the risk of significant adverse or prohibitive undesirable effects.

[0141] The vaccine composition according to the invention comprises at least one effective amount of a virus strain. "Effective amount" is understood in the context of the invention to mean an amount of the virus strain sufficient to trigger an immune response in the organism to which it is administered.

[0142] The vaccine composition of the invention is suitable for oral, sublingual, inhalation, subcutaneous, intramuscular or intravenous administration.

[0143] According to a particular embodiment of the invention, the vaccine composition is in a galenic form intended for administration by inhalation.

[0144] Inhalation refers to absorption by the respiratory tract. Inhalation is a method for the absorption of certain substances, particularly compounds of therapeutic interest, in the form of gases, microdroplets or powders in suspension.

[0145] The administration of pharmaceutical or veterinary compositions by inhalation, ie, via the nasal and / or buccal passageways, is well known to those skilled in the art.

[0146] There are two types of administration by inhalation: - administration by insufflation if the composition is in powder form; and - Administration by nebulization if the composition is in the form of an aerosol (suspension) or in the form of a solution, e.g. a compressed aqueous solution Secondly, the use of a nebulizer or spray is recommended for the administration of pharmaceutical or veterinary compositions.

[0147] The pharmaceutical forms considered here are therefore advantageously chosen from powders, aqueous suspensions of droplets or compressed solutions.

[0148] The target population for respiratory viruses is primarily the pediatric population, consisting of individuals under the age of 18, and more specifically young children (under 5 years of age) and infants. Administration by inhalation is advantageous as it is non-invasive.

[0149] The present invention also relates to a vaccine composition as described above for use in preventing and / or treating infection with at least one respiratory virus, wherein the at least one respiratory virus is from the Pneumoviridae family, in particular human metapneumovirus and / or human syncytial respiratory virus.

[0150] Such vaccine compositions could be used as prophylactic vaccines, ie, to stimulate a specific immune response prior to infection of the organism by the virus.

[0151] Such vaccine compositions could also be used as therapeutic vaccines, ie to stimulate a specific immune response upon infection of the organism with the virus.

[0152] The present invention also relates to a method for preventing infection with at least one virus from the Pneumoviridae family, in particular hMPV and / or hRSV, comprising administration of the above-mentioned vaccine composition to an individual susceptible to infection with such a virus.

[0153] The present invention also relates to a method for treating infections with viruses from the Pneumoviridae family, in particular hMPV and / or hRSV, comprising administration of the above-described vaccine composition to an individual infected with at least one of these viruses.

[0154] In particular, the individuals are children and infants.

[0155] As shown in the Examples section below, vaccine compositions comprising virus strains according to the invention induce the production of neutralizing antibodies against multiple strains of hMPV (HMPV A and B, see Example 10) and multiple strains of hRSV (RSV A and B, see Example 11).

[0156] [Table 1] EXAMPLES

[0157] Example 1 Constructs Four F RSV protein constructs representing the native F RSV type (v.1), the stabilized prefusion F RSV type (v.2), the chimeric HMPV / RSV stabilized F prefusion type (v.3) or the chimeric HMPV / RSV native F RSV type (v.4) were designed as depicted in FIG. 1A.

[0158] The F RSV v.1 sequence corresponds to the native F RSV gene from the RSV A2 strain available to those of skill in the art.

[0159] The F RSV v.2 sequence corresponds to the F RSV v.1 gene into which 14 mutations (*) have been incorporated by directed mutagenesis. These mutations (S46G, K66E, E92D, Q101P, A149C, S155C, S190F, V203L, V207L, S215P, S290C, L373R, Y458C, K465Q) have been described to stabilize glycoprotein F in its prefusion metastable form (McLellan et al., 2013 doi:10.1126 / science.1243283).

[0160] The F RSV v.3 sequence corresponds to the F RSV v.2 gene in which the region encoding the F RSV protein transmembrane and cytoplasmic domains (amino acid positions 514-574) has been replaced by the corresponding coding sequence of the F C-85473 HMPV gene (amino acid positions 482-539).

[0161] The F RSV v.4 sequence corresponds to the F RSV v.1 gene in which the region encoding the F RSV protein transmembrane and cytoplasmic domains (amino acid positions 514-574) has been replaced by the corresponding coding sequence of the F C-85473 HMPV gene (amino acid positions 482-539).

[0162] The corresponding coding sequence was inserted into a plasmid encoding the full-length genome of the rC-85473 HMPV virus (SEQ ID NO:1).

[0163] Insertions of F RSV coding sequences were performed at several genomic locations, as resumed in Table 2.

[0164] Under these conditions, insertion between HMPV genes N and P, or P and M, or F and M2 allowed virus rescue after reverse genetics, according to experimental protocols known to those skilled in the art. Since HMPV pathogenicity is attenuated by deletion of the gene encoding the SH protein (ΔSH), the construct constituted by insertion of the F RSV coding sequence between the F and M2 HMPV genes is the only construct compatible with significant rescue of recombinant virus, efficient virus growth and amplification.

[0165] [Table 2]

[0166] The genetic construct depicted in FIG. 1B , in which the recombinant virus is detectable by expression of GFP (green fluorescent protein) and has attenuated pathogenicity due to deletion of the gene encoding the SH protein (ΔSH, named Metavac®, SEQ ID NO: 13, which contains the GFP gene), encodes an exogenous viral gene encoding an F fusion protein derived from respiratory syncytial virus (RSV), in which the F RSV coding sequence was inserted between the HMPV F and M2 genes, and in particular, three different genetic versions were prepared as described.

[0167] The complete sequences of these genetic constructs are shown in SEQ ID NO: 13 (GFP ΔSH-rC-85473), SEQ ID NO: 10 (bivalent Metavac®-RSV v.1), SEQ ID NO: 11 (bivalent Metavac®-RSV v.2) and SEQ ID NO: 12 (bivalent Metavac®-RSV v.3).

[0168] In FIG. 1C, LLC-MK2 cells were infected with Metavac® virus or bivalently produced recombinant viruses: bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2, and bivalent Metavac®-RSV v.3 at a multiplicity of infection (MOI) of 0.01 (×40 magnification).

[0169] Cells are observed by fluorescence microscopy 10 days after infection.

[0170] GFP expression revealed fully functional and replicating recombinant virus and the expected membrane fusion phenotype (induction of cell syncytia via efficient expression of the F fusion protein), which are intrinsic characteristics of attenuated Metavac® recombinant strains.

[0171] These results demonstrate the ability of the Metavac® recombinant virus to accept an exogenous F RSV gene, resulting in the expression of a fully functional F fusion protein and the production of a productive replicative recombinant virus (rescued by reverse genetics), particularly when the corresponding coding sequence is inserted between the F and M2 HMPV genes.

[0172] Example 2 In vitro replication capacity of three recombinant bivalent Metavac®-RSV viruses LLC-MK2 cells were transfected with the following recombinant viruses: - rC-85473 strain; - bivalent Metavac®-RSV v.1; - bivalent Metavac®-RSV v.2; - Bivalent Metavac®-RSV v.3 The mice were separately infected with 100 μg / mL of 10 ...

[0173] Cell supernatants were collected in triplicate daily for 7 days and viral load was assessed by TCID50 assay, a virology technique well known to those skilled in the art, which represents the final viral dilution at which 50% of tissues show visible cytopathic effects (50% tissue culture infectious dose).

[0174] Figure 2A shows the differential kinetics, replication and production capacity as a function of the nature of the exogenous F RSV coding sequence inserted into the attenuated Metavac® gene backbone. The positive control is the non-attenuated rC-85473 strain, which shows similar performance to the attenuated Metavac® strain.

[0175] The recombinant virus bivalent Metavac®-RSV v.1 appears to have a better replication capacity than the viruses bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3.

[0176] In the table below (Table 3), the average amount of concentrated virus stock produced for each recombinant virus bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 is presented, showing that all three recombinant bivalent candidates result in similar production yields despite variable replication kinetics.

[0177] [Table 3]

[0178] These results demonstrate the replication competence of bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses.

[0179] Example 3 In vitro expression of exogenous F RSV protein on the surface of cells infected with recombinant bivalent Metavac®-RSV virus Immunostaining assay LLC-MK2 cells were infected with bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses or with wild-type recombinant rC-85473 HMPV strain at an MOI of 0.01 (t=0). Hep-2 cells were infected with RSV A2 strain at an MOI of 0.01 (t=0). Five days after infection, infected cell monolayers were fixed in formaldehyde solution and specific immunostaining was performed. - F monoclonal mouse antibody detecting HMPV protein (MAb HMPV24 BioRad MCA 4674); - a monoclonal humanized antibody that detects both pre- and post-fusion F RSV proteins (Palivizumab Synagis® AstraZeneca); - A monoclonal human antibody detecting the prefusion form of the F RSV protein (D25 Mab, Creative Biolabs) The results are shown in Figure 3.

[0180] Specific labeling was observed after peroxidase revelation, and representative images show that both HMPV F (after HMPV24 immunostaining) and RSV F (after palivizumab immunostaining) fusion proteins are expressed and detected on cells infected with bivalent Metavac®-RSV v.1, or bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses.

[0181] As expected for the controls, no immunostaining was observed with MAb HMPV24 on cells infected with the RSV A2 strain (which does not express the HMPV F fusion protein), and no immunostaining was observed with palivizumab Synagis® or D25 MAb on cells infected with rC-85473HMPV (which does not express the RSV F fusion protein).

[0182] Using monoclonal D25 immunostaining, the pre-fusion F RSV protein type was detected with higher intensity on cells infected with bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses than with bivalent Metavac®-RSV v.1, arguing, as expected, for stronger expression and exposure of the stabilized pre-fusion F RSV protein at the cell surface.

[0183] Flow cytometry assay LLC-MK2 cells were infected with bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses or with rC-85473HMPV at an MOI of 0.1 (t=0).

[0184] Forty-eight hours post-infection, infected cell monolayers were trypsinized and resuspended, and quantification of F HMPV or F RSV protein expression was performed by flow cytometry.

[0185] Infected cells were detected by GFP fluorescence, and detection of F HMPV and F RSV expression was performed by the following immunolabeling: - a monoclonal humanized antibody that detects both pre- and post-fusion F RSV proteins conjugated to the R-phycoerythrin (PE) fluorescent dye (Palivizumab Synagis® AstraZeneca); - a monoclonal mouse antibody detecting the F HMPV protein conjugated to the Alexa Fluor™ 647 (A647) fluorescent dye (MAb HMPV24 BioRad MCA 4674); The results are presented in Table 4 below.

[0186] [Table 4]

[0187] The results reported in Table 4 show that greater than 85% of infected cells exposed the F HMPV protein on their surface, and 64.5%, 54.6%, and 47.2% of cells exposed the F RSV protein when cells were infected with bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2, and bivalent Metavac®-RSV v.3 viruses, respectively.

[0188] These results demonstrate the ability of the three bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses to express and expose both F RSV and F HMPV proteins on the surface of infected cells, in particular, the stabilized pre-fusion F RSV form is expressed by the bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses.

[0189] Example 4 In vitro expression of exogenous F RSV protein on the surface of bivalent Metavac®-RSV particles Viral suspensions of each bivalent Metavac®-RSV virus were prepared, 0.45 μm filtered, concentrated by ultracentrifugation, and then resuspended in NaCl. Viral suspensions were adsorbed onto 200 Mesh coated nickel grids and either unlabeled (FIG. 4A) or with antibodies: Primary antibody: - a monoclonal humanized antibody that detects both pre- and post-fusion F RSV proteins (Palivizumab Synagis® AstraZeneca); - Polyclonal mouse serum (autologous serum) detecting HMPV proteins Secondary antibody: - 15 nm gold particle-conjugated goat anti-human IgG detecting anti-F RSV monoclonal humanized antibody; - 5 nm gold particle-conjugated goat anti-mouse IgG to detect antibodies in anti-HMPV mouse serum; The results were observed by transmission electron microscopy using co-immunolabeling with , and the results are presented in Figure 4.

[0190] In FIG. 4A, the presence of typical pleomorphic virus particles coated with transmembrane glycoproteins was observed for all three bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses.

[0191] In Figure 4B, the F RSV protein is detected on the surface of some viral particles for all three bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses as revealed by immunolabeling with 15 nm gold particles (highlighted by black arrows). These proteins are also immunolabeled by specific anti-HMPV sera (represented by 5 nm gold particles).

[0192] In FIG. 4C, virus stocks of each bivalent Metavac®-RSV virus were preincubated with specific anti-HMPV or specific anti-RSV neutralizing mouse serum before infection of LLC-MK2 cells. The % of specific serum neutralization was calculated by measuring the intensity of GFP fluorescent signal from infected cells. Neutralization of infection means that glycoproteins on the surface of virus particles, more specifically F protein, are efficiently recognized by neutralizing antibodies present in the serum, thereby inhibiting virus attachment to cell receptors and subsequent virus infection. Bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses are highly neutralized by anti-HMPV serum and significantly neutralized by anti-RSV serum compared to wild-type hMPV rC-85473 strain. These results confirm the exposure of both F HMPV and F RSV proteins on the surface of bivalent Metavac®-RSV v.1, v.2 and v.3 viral particles.

[0193] These results demonstrate efficient expression of both the hMPV F protein and the hRSV F protein on the surface of the viral particles of the three bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses.

[0194] Example 5 Infection and replication capacity of three bivalent Metavac®-RSV v.1, v.2 and v.3 viruses in ex vivo 3D reconstituted human respiratory epithelium 3D reconstituted human respiratory epithelium (MucilAir® HAE, Epithelix) was cultured at the air-liquid interface according to the supplier's instructions.

[0195] The epithelia were then infected with monovalent Metavac® or bivalent Metavac®-RSV v.1, v.2 or v.3 viruses at an MOI of 0.5.

[0196] Representative photographs of viral growth were taken 3, 5 and 7 days after infection with monovalent Metavac®, bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses.

[0197] FIG. 5A shows expanded GFP expression in human respiratory epithelia, revealing that bivalent Metavac®-RSV v.1, v.2 and v.3 viruses are infectious and replicative, although their propagation into tissues appears to be slower than that of monovalent Metavac®, given the delay in GFP fluorescence detection.

[0198] In Figure 5B, transepithelial electrical resistance (TEER) measurements were performed during the time course of infection. TEER measurements represent a relevant marker of epithelial integrity. Similar to monovalent Metavac®, limited variation in TEER was observed following infection with bivalent Metavac®-RSV v.1, v.2 and v.3 viruses, correlating with an attenuated viral phenotype and preservation of epithelial integrity.

[0199] In FIG. 5C, viral genome quantification at the apical surface confirmed efficient production of viral progeny from infection with each of the bivalent Metavac®-RSV.

[0200] In FIG. 5D, immunolabeling with palivizumab Synagis® reveals expression of F RSV protein at the apical membrane side of 3D reconstituted human respiratory epithelium infected with bivalent Metavac®-RSV v.1 virus.

[0201] These results demonstrate the ability of three different bivalent Metavac®-RSV v.1, v.2 and v.3 viruses to infect and grow in ex vivo human airway epithelial tissue, and further demonstrate the ability of these bivalent Metavac®-RSV viruses to express exogenous F RSV antigens on the surface of 3D reconstituted human respiratory epithelium.

[0202] Example 6 In vivo characterization of recombinant bivalent Metavac®-RSV v.1, v.2, and v.3 viruses in a BALB / c mouse viral infection model BALB / c mice were immunized with 5×10 5 Mice were infected by intranasal instillation with TCID50 of recombinant bivalent Metavac®-RSV v.1, v.2 or v.3 viruses. Body weight and survival of infected mice were monitored daily for 10 days (weight mean ± SEM for 5 mice).

[0203] Additionally, 5 days after infection, 2 mice per group were euthanized for measurement of lung viral load by RT-qPCR.

[0204] In FIG. 6A, no significant weight loss was observed during the time course of infection with bivalent Metavac®-RSV v.1, v.2, or v.3 viruses, as would be expected for an attenuated virus strain.

[0205] Figure 6B shows the results of viral gene quantification in mouse lungs 5 ​​days after infection. High levels of gene expression were observed for both N HMPV and F RSV, confirming the replication properties of the three bivalent Metavac®-RSV v.1, v.2, or v.3 viruses and their ability to express F RSV genes in vivo.

[0206] These results demonstrate the ability of the three bivalent Metavac®-RSV viruses to infect and replicate in vivo and to express exogenous F RSV genes at levels similar to those of N HMPV genes into the lung tissue of infected mice.

[0207] Example 7 In vivo induction of neutralizing antibody production following HMPV- or RSV-prime infection followed by boost infection with bivalent Metavac®-RSV v.1, v.2, and v.3 viruses in a BALB / c mouse model BALB / c mice were infected with a non-lethal dose of wild-type HMPV rC-85473 strain (1 × 10 6 TCID50) or wild-type RSV A Long strain (1 × 10 6 The mice were infected with 1000 PFU by intranasal instillation.

[0208] Three weeks later, HMPV- or RSV-primed BALB / c mice were immunized with 5×10 5Mice were infected with TCID50 of recombinant bivalent Metavac®-RSV v.1, v.2 or v.3 viruses by intranasal (boost) instillation.

[0209] Mouse weight and survival were monitored daily for 10 days (weight mean ± SEM for 8 mice).

[0210] Additionally, 5 days after intranasal boost infection, 3 mice per group were euthanized for measurement of lung viral load by RT-qPCR.

[0211] In Figures 7D-E, quantification of N HMPV and F RSV genes by RT-qPCR shows that pulmonary replication of bivalent Metavac®-RSV v.1, v.2, or v.3 viruses was very weak in mice that were non-naive to HMPV virus and was high in mice that were non-naive to RSV.

[0212] In Figures 7F-G, characterization of HMPV- or RSV-specific neutralizing antibody responses by serum neutralization assays shows that the three bivalent Metavac®-RSV v.1, v.2, or v.3 viruses were able to induce strong HMPV- or RSV-directed antibody responses depending on the initial non-naive HMPV or RSV serological status.

[0213] These results demonstrate the ability of the three bivalent Metavac®-RSV v.1, v.2 or v.3 viruses to induce potent and specific neutralizing antibody responses against HMPV and RSV viruses in vivo.

[0214] Example 8 In vivo protective properties of bivalent Metavac®-RSV v.3 virus against infectious challenge with a lethal dose of wild-type virus rC-85473 HMPV strain BALB / c mice received 5×10 immunization doses based on the active immunization dose determined for monovalent Metavac® virus compared to a group of mice mock-immunized with culture medium (mock). 5 Mice were immunized twice with TCID50 of recombinant monovalent Metavac® or bivalent Metavac®-RSV v.3 virus by intranasal instillation, 21 days apart.

[0215] Twenty-one days after the boost infection, mice were vaccinated with 3 × 10 6 They survived a lethal hMPV virus challenge by intranasal instillation of TCID50 of wild-type HMPV rC-85473 (the infectious dose that produces >50% mortality in mice).

[0216] Mouse weight and survival were monitored daily for 14 days (weight mean ± SEM for 8 mice).

[0217] In FIG. 8B, the two immunized mice showed no significant weight loss after lethal virus challenge, indicating complete survival, whereas the mock-immunized mice showed highly significant weight loss, with all mice dying by day 8 after lethal challenge (FIG. 8C).

[0218] Five days after virus challenge, four mice per group were euthanized for measurement of lung viral load by RT-qPCR.

[0219] In FIG. 8D, gene copy quantification of both N HMPV and F RSV demonstrated that two-dose immunization with monovalent Metavac® or bivalent Metavac®-RSV v.3 virus was efficient in significantly suppressing HMPV viral replication in the lungs compared to mock-immunized mice.

[0220] Finally, in Figures 8E-8F, quantification of HMPV- or RSV-specific neutralizing antibody responses was performed from sera collected prior to the prime intranasal and 21 days after the boost instillation (except for the mock-immunized group).

[0221] As expected, high levels of HMPV neutralizing antibodies were measured in the sera of mice immunized with two doses of monovalent Metavac® virus.

[0222] In mice doubly immunized with bivalent Metavac®-RSV v.3 virus, high neutralizing antibody titers against both HMPV and RSV viruses were detected 21 days after the boost immunization and persisted up to 21 days after HMPV virus challenge.

[0223] These results demonstrate the ability of the bivalent Metavac®-RSV candidate (bivalent Metavac®-RSV v.3) to induce strong specific neutralizing antibody responses against both HMPV- and RSV viruses in vivo and fully protect mice challenged with a lethal dose of HMPV wild-type virus.

[0224] Example 9 In vivo characterization of recombinant bivalent Metavac®-RSV v.1 and v.3 viruses in a BALB / c mouse viral infection model BALB / c mice were immunized with 5×10 5 Mice were infected with TCID50 of recombinant bivalent Metavac®-RSV v.1 or v.3 virus by intranasal instillation.

[0225] Figure 9A shows the results of viral gene quantification in mouse bronchoalveolar lavage 5 days after infection. High levels of both N HMPV and F RSV gene expression were observed, confirming the presence and replication properties of bivalent Metavac®-RSV v.1 and v.3 virus in the lower respiratory tract of infected animals and its ability to express F RSV genes in vivo.

[0226] Furthermore, 5 days after infection, three mice per group were euthanized for lung tissue collection, and the intact lungs were fixed in formaldehyde solution for further histopathological analysis.

[0227] In Figure 9B, a low inflammatory profile was described after infection with bivalent Metavac®-RSV v.1 or v.3 viruses in contrast to the images of low inflammatory scores measured after infection with the monovalent LAV candidate Metavac® and the high pro-inflammatory scores measured after infection with HMPV WT.

[0228] Overall, as expected from a live attenuated vaccine candidate, these results highlight the ability of both bivalent Metavac®-RSV viruses to infect and replicate in vivo, express the exogenous F RSV gene, and induce a low inflammatory response in the lung tissue of infected mice.

[0229] Example 10 In vivo protective properties of bivalent Metavac®-RSV v.1 and v.3 viruses against infectious challenge with a lethal dose of the wild-type rC-85473 HMPV strain BALB / c mice received 5×10 immunization doses based on the active immunization dose determined for monovalent Metavac® virus compared to groups of mice mock-immunized with culture medium (mock) or groups of mice immunized via the intramuscular route with an adjuvanted split of HMPV WT virus (HMPV split) as an alternative to vaccination with HMPV protein vaccines. 5 Mice were immunized twice, 21 days apart, by intranasal instillation with TCID50 of recombinant monovalent Metavac® or bivalent Metavac®-RSV v.1 or v.3 viruses.

[0230] Twenty-one days after the boost infection, mice were vaccinated with 2 × 10 6 They survived a lethal HMPV virus challenge by intranasal instillation of TCID50 of wild-type HMPV rC-85473 (the infectious dose that produces >50% mortality in mice).

[0231] Mouse weight and survival were monitored daily for 14 days (weight mean ± SEM for 8 mice).

[0232] In Figures 10A and 10B, immunized mice (vaccinated with recombinant monovalent Metavac® or bivalent Metavac®-RSV v.1 or v.3 viruses or HMPV split) showed significantly reduced weight loss and complete survival after lethal virus challenge, while mock-immunized mice showed highly significant weight loss and all mice had died by day 6 after lethal challenge (Figure 10B).

[0233] Five days after virus challenge, three mice per group were euthanized for lung tissue collection for further histopathological analysis, and two or four mice per group were euthanized for measurement of lung viral load by RT-qPCR.

[0234] In Figure 10C, different inflammatory profiles are described depending on the vaccine candidate and route of administration. After lethal HMPV challenge, reduced interstitial, peribronchial, intraalveolar and pleural inflammation appears to be induced in mice vaccinated with bivalent Metavac®-RSV v.1 or v.3 viruses, with the difference being the high inflammation scores measured induced after immunization with HMPV split, corresponding to the enhanced disease syndrome described in the literature after immunization with inactivated vaccines.

[0235] In FIG. 10D, gene copy quantification of both N HMPV and F RSV shows that two-dose immunization with monovalent Metavac® or bivalent Metavac®-RSV v.1 and v.3 viruses was efficient in significantly suppressing HMPV viral replication in the lungs compared to mock-immunized and HMPV split-immunized mice.

[0236] In Figure 10E, quantification of homologous or heterologous HMPV-specific neutralizing antibody responses was performed from sera collected along the time course of the protocol. As expected, high levels of HMPV neutralizing antibodies were measured in sera from mice immunized with two doses of monovalent Metavac® virus. In mice dually immunized with bivalent Metavac®-RSV v.1 and v.3 viruses, similar high neutralizing antibody titers against both HMPV A and B viruses were detected 21 days after the boost immunization and persisted or increased until 21 days after HMPV virus challenge.

[0237] Finally, in Figure 10F, quantification of HMPV-specific IgG antibodies was performed from sera collected along the time course of the protocol. As expected, given the neutralizing antibody titers, high levels of IgG antibodies were measured in the sera of mice immunized with two doses of monovalent Metavac® or bivalent Metavac®-RSV v.1 and v.3 viruses. In contrast, immunization with adjuvanted HMPV split appears to induce higher levels of neutralizing and IgG antibodies as soon as 21 days after the second immunization, but the antibody response appears to decrease after HMPV challenge.

[0238] These results demonstrate the ability of the bivalent Metavac®-RSV candidates (both Metavac®-RSV v.1 and v.3) to induce strong and specific neutralizing antibody responses against both HMPV A and B viruses in vivo and fully protect mice challenged with a lethal dose of HMPV wild-type virus.

[0239] Example 11 In vivo protective properties of bivalent Metavac®-RSV v.1 and v.3 viruses against infection challenge with recombinant RSV-Luc WT virus expressing luciferase protein BALB / c mice were immunized with either a group of mock-immunized mice (mock) or 5 × 10 5Based on the active immunization dose determined for the monovalent Metavac® virus, compared to a group of mice immunized by intranasal instillation with PFU of recombinant RSV WT virus (RSV-mCh), 5×10 5 Mice were immunized twice, 21 days apart, by intranasal instillation with TCID50 of bivalent Metavac®-RSV v.1 or v.3 virus.

[0240] Twenty-one days after the boost infection, mice were administered 1 × 10 of rRSV-Luc virus, a recombinant RSV A WT virus that expresses the luminescent luciferase protein in vivo. 5 They survived RSV infection challenge by intranasal instillation of PFU ( Rameix-Welti et al., 2014 ).

[0241] The animals' weights were monitored daily for 14 days and there was no weight loss, as would be expected from RSV infection in a mouse model.

[0242] In Figures 11A and 11B, immunized mice (vaccinated with bivalent Metavac®-RSV v.1 or v.3 viruses or RSV WT) show significantly reduced luciferase activity and bioluminescence in the nose and lungs after virus challenge, while mock-immunized mice show high intensity luminescence, especially in the lungs, 5 days after challenge as a result of RSV virus replication in lung tissue.

[0243] Four days after virus challenge, four mice per group were euthanized for measurement of lung viral load by RT-qPCR.

[0244] In Figure 11C, consistent with the previous luminescence results, F RSV gene copy quantification shows that two-dose immunization with bivalent Metavac®-RSV v.1 and v.3 viruses tends to reduce and suppress RSV viral replication in the lungs compared to mock-immunized mice, and no residual N HMPV genetic material is detectable in the lungs of mice more than 3 weeks after boost immunization with bivalent Metavac®-RSV candidate.

[0245] In FIG. 11D, quantification of homologous and heterologous RSV-specific neutralizing antibody responses was performed from sera collected along the time course of the protocol.

[0246] As expected, induction of RSV neutralizing antibodies was measured in the sera of mice immunized with two doses of bivalent Metavac®-RSV v.1 and RSV WT viruses, and higher levels of neutralizing antibodies specific for RSV A and B strains appear to be induced by vaccination with the bivalent Metavac®-RSV v.3 candidate 21 days after RSV virus challenge.

[0247] These results demonstrate the ability of the bivalent Metavac®-RSV candidates (bivalent Metavac®-RSV v.1 and v.3) to induce strong specific neutralizing antibody responses against both RSV A and B viruses in vivo and suppress RSV replication in the upper and lower respiratory tract of mice challenged with RSV virus. (References) Patent Literature WO 2020 / 021180 WO 2020 / 120910 EP 3 868 874 References van den Hoogen BG, of Jong JC, Groen J, Kuiken T, of Groot R, Fouchier RA, et al. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med. 2001;7(6):719-24 Peret TC, Boivin G, Li Y, Couillard M, Humphrey C, Osterhaus AD, Erdman DD, Anderson LJ. Characterization of human metapneumoviruses isolated from patients in North America. J Infect Dis. 2002 Jun 1;185(11):1660-3. Rostad CA, Stobart CC, Gilbert BE, Pickles RJ, Hotard AL, Meng J, Blanco JCG, Moin SM, Graham BS, Piedra PA, Moore ML. A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats. J Virol. 2016 Jul 27;90(16):7508-7518. Stobart CC, Rostad CA, Ke Z, Dillard RS, Hampton CM, Strauss JD, Yi H, Hotard AL, Meng J, Pickles RJ, Sakamoto K, Lee S, Currier MG, Moin SM, Graham BS, Boukhvalova MS, Gilbert BE, Blanco JC, Piedra PA, Wright ER, Moore ML. A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation. Nat Commun. 2016 Dec 21;7:13916. Karron RA, Luongo C, Mateo JS, Wanionek K, Collins PL, Buchholz UJ. Safety and Immunogenicity of the Respiratory Syncytial Virus Vaccine RSV / ΔNS2 / Δ1313 / I1314L in RSV-Seronegative Children. J Infect Dis. 2020 Jun 16;222(1):82-91. McLellan JS, Chen M, Joyce MG, Sastry M, Stewart-Jones GB, Yang Y, Zhang B, Chen L, Srivatsan S, Zheng A, Zhou T, Graepel KW, Kumar A, Moin S, Boyington JC, Chuang GY, Soto C, Baxa U, Bakker AQ, Spits H, Beaumont T, Zheng Z, Xia N, Ko SY, Todd JP, Rao S, Graham BS, Kwong PD. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science. 2013 Nov 1;342(6158):592-8. Needleman SB, Wunsch CD. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970 Mar;48(3):443-53. doi: 10.1016 / 0022-2836(70)90057-4. PMID: 5420325. Hamelin ME, Gagnon C, Prince GA, Kiener P, Suzich J, Ulbrandt N, Boivin G. Prophylactic and therapeutic benefits of a monoclonal antibody against the fusion protein of human metapneumovirus in a mouse model. Antiviral Res. 2010 Oct;88(1):31-7. Dubois J, Cavanagh MH, Terrier O, Hamelin ME, Lina B, Shi R, et al. Mutations in the fusion protein heptad repeat domains of human metapneumovirus impact on the formation of syncytia. The Journal of general virology. 2017;98(6):1174-80. Le VB, Dubois J, Couture C, Cavanagh MH, Uyar O, et al. Human metapneumovirus activates NOD-like receptor protein 3 inflammasome via its small hydrophobic protein which plays a detrimental role during infection in mice. (2019) PLOS Pathogens 15(4): e1007689. https: / / doi.org / 10.1371 / journal.ppat.1007689 Dubois J, Pizzorno A, Cavanagh M-H, Padey B, Nicolas de Lamballerie C, Uyar O, Venable M-C, Carbonneau J, Traversier A, Julien T, Lavigne S, Couture C, Lina B, Hamelin M-E, Terrier O, Rosa-Calatrava M, Boivin G. Strain-Dependent Impact of G and SH Deletions Provide New Insights for Live-Attenuated HMPV Vaccine Development. Vaccines. 2019; 7(4):164. https: / / doi.org / 10.3390 / vaccines7040164 Rameix-Welti, MA., Le Goffic, R., Herve, PL. et al. Visualizing the replication of respiratory syncytial virus in cells and in living mice. Nat Commun 5, 5104 (2014). https: / / doi.org / 10.1038 / ncomms6104

Claims

1. A virus strain derived from a human metapneumovirus (hMPV) strain having a genome sequence depicted in SEQ ID NO:1, said genome sequence comprising the following genetic modifications: (i) inactivation of endogenous genes encoding SH proteins and / or G proteins, and (ii) the presence of an exogenous nucleotide sequence comprising a sequence encoding at least one extracellular domain of the F protein of human respiratory syncytial virus (hRSV), said domain being wild-type or mutated; , including viral strains.

2. The virus strain of claim 1, wherein the endogenous gene encoding the SH protein is deleted.

3. The virus strain of claim 1 or 2, wherein the exogenous nucleotide sequence comprises a sequence encoding a wild-type extracellular domain of the F protein of hRSV.

4. The virus strain of claim 3, wherein the exogenous nucleotide sequence is composed of a sequence encoding the three wild-type domains (cytoplasmic, transmembrane and extracellular) of the F protein of hRSV.

5. The virus strain of claim 1 or 2, wherein the exogenous nucleotide sequence comprises a sequence encoding a mutated domain of the F protein of hRSV, in particular a mutated extracellular domain corresponding to a stabilized prefusion state of the F protein.

6. The virus strain of claim 5, wherein the exogenous nucleotide sequence is composed of a sequence encoding a wild-type cytoplasmic domain, a wild-type transmembrane domain, and a mutated extracellular domain corresponding to a stabilized prefusion state of the hRSV F protein.

7. The exogenous nucleotide sequence is a. a sequence derived from hRSV encoding at least the extracellular domain of the F protein, said domain being wild-type or mutated; and b. a sequence derived from hMPV encoding at least one domain of the F protein, said domain being selected from among the cytoplasmic domain and the transmembrane domain, said domain being wild type or mutated; 3. The virus strain of claim 1 or 2, wherein the exogenous sequence encodes a chimeric hRSV / hMPV F protein.

8. The exogenous nucleotide sequence is a. a sequence derived from hRSV encoding a mutated extracellular domain corresponding to a stabilized prefusion state of the F protein of hRSV; and b. A sequence derived from hMPV that encodes the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV. The virus strain of claim 7, comprising:

9. The exogenous nucleotide sequence is a. a sequence derived from hRSV encoding the wild-type extracellular domain of the F protein of hRSV; and b. A sequence derived from hMPV that encodes the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV. The virus strain of claim 7, comprising:

10. The nucleotide sequence: - a sequence encoding a mutated extracellular domain corresponding to a stabilized prefusion state of the F protein of hRSV, and a sequence encoding the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV, in particular a sequence consisting of the sequence shown in SEQ ID NO: 8; - a sequence coding for the wild-type extracellular domain of a protein of hRSV and a sequence coding for the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV, in particular a sequence that is composed of the sequence shown in SEQ ID NO: 9 A gene cassette containing one of the following:

11. 11. A virus strain derived from a human metapneumovirus (hMPV) strain comprising in its genome the gene cassette of claim 10, said virus strain being preferentially further attenuated.

12. 12. A virus strain according to any one of claims 1 to 9 or 11 for use as a medicament.

13. 12. A virus strain according to any one of claims 1 to 9 or 11 for use in preventing and / or treating infection with at least one respiratory virus.

14. 14. The virus strain of claim 13, wherein the at least one respiratory virus is from the Pneumoviridae family, in particular human metapneumovirus (hMPV) and / or human respiratory syncytial virus (hRSV).

15. 12. A vaccine composition comprising at least one of the virus strains according to any one of claims 1 to 9 or 11 in a pharma- ceutically acceptable vehicle, and optionally an adjuvant.

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