Vaccine composition against two respiratory viruses

Abstract

The present invention relates to a viral strain derived from a human metapneumovirus (hMPV) strain having a genomic sequence represented by sequence SEQ ID NO: 1 wherein the genomic sequence comprises the following genetic modifications: (i) inactivating an endogenous gene encoding an SH protein and / or a G protein, and (ii) the presence of an exogenous sequence encoding at least one extracellular domain of an F protein of a human respiratory syncytial virus (hRSV), the domain is of a wild type or mutant.

Description

Technical Field

[0001] The present invention relates to genetically modified virus strains comprising 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 viral infections, in particular respiratory viral infections. Background Art

[0003] Acute infections of the lower respiratory tract are one of the leading causes of morbidity and mortality worldwide. According to the World Health Organization, they are the second leading cause of death, especially among children under 5 years of age.

[0004] Most acute infections of the lower respiratory tract are associated with a variety of pathologies (from simple colds to severe pneumonia), especially those caused by viruses, such as those belonging to the following families:

[0005] (i) viruses of the family Orthomyxoviridae (including, in particular, influenza viruses);

[0006] (ii) viruses of the family Paramyxoviridae;

[0007] (iii) viruses of the family Adenoviridae;

[0008] (iv) viruses of the family Picornaviridae;

[0009] (v) Viruses of the family Coronaviridae and

[0010] (vi) Viruses of the family Pneumoviridae, including in particular human respiratory syncytial virus and human metapneumovirus.

[0011] Both human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) cause acute infections of the respiratory tract (such as bronchiolitis, bronchitis or pneumonia). They mainly affect risk groups, i.e. children under 5 years of age, the elderly and people with weakened immune systems. Both viruses are present on all continents. Their annual distribution is mainly in winter and spring. Virus transmission is mainly through the respiratory route, especially through the spread of saliva droplets in the air.

[0012] hRSV was first identified in a group of chimpanzees in 1956 and was subsequently confirmed to be a major human pathogen. hRSV was isolated in 1957. It is a virus with a single-stranded RNA genome of negative polarity and is of the "enveloped" type. Its RNA genome is linear, non-segmented, and is protected in a helical nucleocapsid. It contains 10 genes that encode 11 viral proteins. It belongs to the genus Orthopneumovirus of the family Pneumoviridae. Two subgroups of hRSV, A and B, have been identified based on differences in the F and G genes.

[0013] hRSV is the leading cause of bronchiolitis and pneumonia in infants younger than 1 year of age, with an increased incidence in infants younger than 6 months of age. hRSV causes 6.7% of deaths in children younger than 1 year of age and causes excess mortality levels comparable to influenza viruses in the elderly.

[0014] 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 belonging to the genus Metapneumovirus of the family Pneumoviridae.

[0015] hMPV is prevalent in infants with bronchiolitis and pneumonia, particularly affecting children aged 6 months to 3 years. The average age of children hospitalized for complications of hMPV infection is 6 to 12 months, i.e., later than the average age of children hospitalized for hRSV, which mainly occurs between 3 and 6 months.

[0016] The genome organization of hMPV and hRSV is close. Their genome is subdivided into several ORFs (open reading frames) encoding N, P, M, F, M2 (M2-1 and M2-2), SH, G and L proteins. In contrast to hMPV, hRSV also includes 2 additional non-structural genes NS1 and NS2. The general genome structure of virus strain rC-8543 and its attenuated version δSH is shown in Figure 1.

[0017] Three types of glycoproteins are expressed on the surface of viral particles: F, G and SH proteins.

[0018] The F fusion glycoprotein is highly conserved between different subgroups and is involved in the penetration of the virus into target cells and the formation of syncytia (large multinucleated cells derived from the fusion of individual cells after viral infection). The F glycoprotein is also referred to as F protein in this application and is considered to be the most antigenic protein in hRSV and hMPV viruses.

[0019] Currently, there are few preventive or therapeutic options for hRSV and / or hMPV infection. There are currently no licensed vaccines for hRSV or hMPV.

[0020] For treatment, ribavirin can be used occasionally to treat severe cases of hMPV or hRSV infection, but this therapy produces several side effects. The commonly used and highly regarded clinical approach is to provide symptomatic care for the infection: provide the patient with respiratory assistance (oxygen administration or mechanical ventilation), administer bronchodilators, corticosteroids, and / or antibiotics to prevent or treat bacterial superinfection, and prevent hypoxemia and electrolyte imbalances.

[0021] As a licensed preventive treatment, Palivizumab (a recombinant humanized hRSV anti-F protein monoclonal antibody) can be used to treat hRSV infection, especially for preventing severe disease in high-risk neonates. This preventive treatment is very expensive.

[0022] Currently, the scientific and clinical communities are deeply engaged in changing this situation of limited or no preventive or therapeutic options. New initiatives are underway, such as the establishment of RESCEU, a European consortium that aims to reintegrate all clinical data on hRSV infection and provide an infrastructure to test future RSV vaccine candidates and therapeutics.

[0023] Therefore, the development of a bivalent vaccine against hRSV and hMPV not only addresses a major public health challenge, but also a real socioeconomic issue, with the goal of reducing the high costs of treatment and hospitalization associated with these infections. In addition, the use of such a vaccine would help reduce the use of antibiotics in cases of bacterial superinfection, thereby limiting the emergence of antibiotic resistance in the population.

[0024] Prior art

[0025] Vaccines based on the use of live attenuated virus strains are called live attenuated vaccines (LAV) and have many advantages.

[0026] First, live attenuated viruses are able to 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 with administration of isolated recombinant antigens. In addition, for respiratory viruses, these LAVs can be administered via the intranasal route, thereby mimicking natural entry of the pathogen.

[0027] Live attenuated vaccines broadly stimulate innate, humoral, and cellular immunity both systemically and locally in the respiratory tract (mucosal immunity).

[0028] Due to this strong induction of the immune response, the use of additional adjuvants is usually not necessary.

[0029] Finally, this vaccination strategy would not produce any uncontrolled inflammatory responses like inactivated vaccines.

[0030] Therefore, it is considered the best vaccine strategy for preventing respiratory viral infections in infants and young children.

[0031] International application WO 2020 / 021180 discloses an attenuated hMPV strain obtained from clinical specimen C-85473, characterized by a significant fusion capacity, and genetically modified to exhibit reduced virulence, in particular by inhibiting the expression of small hydrophobic protein (ΔSH) and / or G protein (G).

[0032] In addition, international application WO 2020 / 120910 discloses a method for producing such an attenuated hMPV strain by infecting and culturing a specific avian immortalized cell line.

[0033] 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), which codon-deoptimized the nonstructural protein NS1 and NS2 genes, deleted the gene encoding the SH protein, and replaced the wild-type fusion (F) protein gene with the low-fusion RSV B subgroup F consensus sequence. In the same year, (Stobart et al., 2016) designed and described an attenuated virus strain named OE4 (RSV-A2-dNS1-dNS2-DSH-dGm-Gsnull-line19F). The 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 elimination of the secreted G. The safety and immunogenicity of the attenuated virus strain RSV / NS2 / 1313 / I1314L, which has a deletion of the interferon antagonist NS2 gene, a deletion of codon 1313 of the RSV polymerase gene, and a stabilizing missense mutation I1314L, have been shown in RSV seronegative children (Karron et al., 2020).

[0034] Among the most interesting viral antigens, the F protein is involved in viral entry into target cells and syncytia formation and has been extensively studied. In particular, antibodies against the F protein have been generated.

[0035] Currently, severe cases of the disease are prevented by administering a monoclonal antibody directed against the F protein of hRSV. Administering this humanized monoclonal antibody, palivizumab, to children significantly reduced the risk of hospitalization for RSV infection in high-risk children.

[0036] The F protein exploits the difference in folding energy between the two conformations to enable fusion of the viral and host cell membranes:

[0037] - the metastable state (pre-fusion state) adopted by the virus before interaction with the cell and

[0038] -The stable state that occurs after the virus and cell fuse (post-fusion state).

[0039] It has been noted that the major epitopes of the F protein of hRSV are found in the prefusion state of the F protein. For example, targeting antigenic sites have been developed These antibodies are 10 to 100 times more potent than palivizumab, which recognizes antigenic site II in both the prefusion and postfusion states.

[0040] Stable exposed antigenic sites have been designed Soluble variants of the F protein of the mitochondria (McLellan et al., 2013). These variants consist in the extracellular domain of the F protein and are stabilized in the prefusion state.

[0041] Another area of ​​research in the development of live attenuated vaccines is the design of bivalent vaccines, which contain antigens from two different pathogens.

[0042] For example, bivalent vaccines based on a combination of hRSV and hMPV are described in European patent application EP 3 868 874. In these recombinant viruses based on a reassortant strain of hRSV, the gene encoding the F protein of another virus (particularly a virus 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.

[0043] Nevertheless, efforts are still being made to provide a safe and effective bivalent vaccine for both hMPV and hRSV. The present invention relates to a novel virus strain with multiple genetic modifications, which is attenuated but still immunogenic, with significant fusion capacity. Advantageously, the attenuated phenotype is stable, i.e., irreversible during the replication cycle. The attenuated virus strain is capable of in vitro propagation in cells and can be used as a live attenuated vaccine in vivo to allow the generation of an immune response to both hMPV and hRSV in individuals to whom the vaccine is administered. Summary of the invention

[0044] The present invention relates to a bivalent vaccine that is capable of generating multiple immune responses against two viruses in an individual after administration. This vaccine induces a combined immune response against the two viruses, thereby reducing the number of injections required for an individual to obtain preventive immunity against different pathogens.

[0045] More specifically, the present invention relates to a virus strain derived from human metapneumovirus (hMPV) C-85473 strain having a genome sequence represented by SEQ ID NO: 1, wherein the genome sequence comprises the following genetic modifications:

[0046] (i) inactivating the endogenous gene encoding SH protein and / or G protein to obtain a strain with attenuated virulence, and

[0047] (ii) there is an exogenous sequence encoding at least one extracellular domain of the F protein of human respiratory syncytial virus (hRSV), said domain being wild type or mutated.

[0048] In particular, the present invention relates to the virus strain comprising an exogenous sequence, wherein the exogenous sequence comprises:

[0049] a. a sequence derived from hRSV, encoding at least one extracellular domain of the F protein, said domain being wild type or mutated; and

[0050] b. a sequence derived from hMPV, encoding at least one domain of the F protein, the domain being selected from a cytoplasmic domain and a transmembrane domain, the domain being wild type or mutated,

[0051] The exogenous sequence encodes a chimeric hRSV / hMPV F protein.

[0052] Another object of the present invention is a gene cassette comprising one of the following nucleotide sequences:

[0053] - a sequence encoding a mutant extracellular domain corresponding to the stabilized prefusion state of the F protein of hRSV, and a sequence encoding a wild-type cytoplasmic domain and a wild-type transmembrane domain of the F protein of hMPV, in particular a sequence as shown in SEQ ID NO: 8 as a major part;

[0054] - A sequence encoding the wild-type extracellular domain of the hRSV protein, and a sequence encoding the wild-type cytoplasmic domain and wild-type transmembrane domain of the hMPV F protein, specifically a sequence as shown in SEQ ID NO: 9 as the main part.

[0055] The present invention also relates to a virus strain derived from a human metapneumovirus (hMPV) strain, comprising a gene cassette as defined above. Preferably, the virus strain has attenuated virulence.

[0056] The invention also relates to said virus strain for use as a medicament.

[0057] The present invention also relates to said virus strain for use in preventing and / or treating infection by at least one respiratory virus, in particular at least one, more particularly two, viruses of the Pneumoviridae family.

[0058] The present invention also relates to a vaccine composition comprising at least one viral strain as defined above in a pharmaceutically acceptable carrier and optionally an adjuvant. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1. Structure of hMPV genome and exogenous F protein, and cells infected with the virus strain

[0060] [1A] Structure of hMPV rC-85473 genome strain, attenuated The structure of the strain (ΔSH rC-85473 strain, genetically modified by deletion of the SH gene to attenuate the virulence of the strain), and the ΔSH rC-85473 strain into which foreign coding sequences encoding the v.1, v.2 and v.3 constructs were introduced. A cassette encoding green fluorescent protein (GFP) was inserted at the 3' position.

[0061] [1B] Schematic representation of the additional F RSV antigens expressed by the recombinant virus. Structure of the v.1, v.2, v.3 and v.4 proteins encoded by exogenous sequences introduced into the ΔSH rC-85473 genome. V.3 and v.4 are chimeric proteins that contain domains derived from the F proteins of hMPV and hRSV viruses.

[0062] CTD: cytoplasmic domain; TMD: transmembrane domain; ED: extracellular domain.

[0063] [1C] Infection Strains and bivalent - Fluorescence microscopy images of cells of RSV v.1, v.2 and v.3 constructs (magnification ×40 for the left image and ×100 for the right image).

[0064] Figure 2 .Recombinant bivalent -In vitro replication capacity of RSV virus.

[0065] LLC-MK2 cells were infected with the following recombinant viruses at a multiplicity of infection of 0.01:

[0066] -rC-85473 strain (solid black line);

[0067] -Two-price -RSV v.1 (dashed grey line);

[0068] -Two-price -RSV v.2 (dashed black line);

[0069] -Two-price -RSV v.3 (solid grey line).

[0070] Cell supernatants were collected daily for 7 consecutive days in triplicate and the viral load was assessed by TCID50 assay, which represents the final virus dilution at which 50% of the cell tissues showed visible cytopathic effect (50% tissue culture infectious dose).

[0071] Figure 3 : Recombinant bivalent - In vitro expression of exogenous F RSV protein on the surface of RSV virus infected cells.

[0072] Using wild-type recombinant rC-85473 HMPV strain, bivalent -RSV v.1, bivalent -RSVv.2 or bivalent - LLC-MK2 cells were infected with RSV v.3 (t=0) at an MOI of 0.01.

[0073] Hep-2 cells (second line) were infected with RSV A2 strain (t=0) at an MOI of 0.01.

[0074] Five days after infection, infected cell monolayers were fixed in formaldehyde solution and subjected to specific immunostaining using:

[0075] -HMPV24: monoclonal mouse antibody detecting F HMPV protein (MAb HMPV24 BioRad MCA 4674);

[0076] -Palivizumab: A monoclonal humanized antibody that detects the pre- and post-fusion forms of the F RSV protein (Palivizumab AstraZeneca);

[0077] - D25: a monoclonal human antibody that detects the prefusion form of the F RSV protein (D25 Mab, Creative Biolabs).

[0078] Figure 4: Exogenous F RSV protein in bivalent - In vitro expression on the surface of RSV particles.

[0079] [4A] Observation of three divalent -RSV v.1, bivalent -RSVv.2 and bivalent -RSV v.3 virus.

[0080] [4B] Observation of three divalent -RSV v.1, bivalent -RSVv.2 and bivalent -RSV v.3 viruses, which were combined with a monoclonal humanized antibody (palivizumab) that detects the F RSV protein ) and co-immunolabeled with polyclonal mouse sera (internal sera) detecting HMPV proteins.

[0081] [4C] The three bivalent -RSV v.1, bivalent -RSV v.2 and bivalent - Serum neutralization of RSV v.3 virus.

[0082] Figure 5: Three types of bivalent -Infection and replication capacity of RSV v.1, v.2 and v.3 viruses in ex vivo 3D reconstructed human respiratory epithelium.

[0083] [5A] Observation of each bivalent - 3D reconstructed human airway epithelium infected with RSV v.1, v.2 or v.3 and compared with monovalent All viruses contained a GFP encoding gene at the 3' position of their genome.

[0084] [5B] When using each bivalent - Transepithelial electrical resistance measured during infection of 3D reconstructed human airway epithelium with RSV v.1, v.2 or v.3.

[0085] [5C] Infection unit price (Black bar), Two-price -RSV v.1 (dark grey bar), bivalent -RSV v.2 (light grey bars) or bivalent - Quantification of viral genes (quantification of NHMPV gene and F RSV gene copies by RT-qPCR) at the apical surface of the epithelium after 1, 3, 5, 7 or 9 days (post-infection) of RSV v.3 (striped grey bars). The dashed line indicates the detection threshold.

[0086] [5D] Infection Bivalent -3D reconstruction of RSV v.1 virus Immunolabeling of surface expressed F RSV antigens of human respiratory epithelium. Immunostaining was performed using Palivizumab (monoclonal humanized antibody that detects pre- and post-fusion forms of F RSV protein). Dark arrows highlight F RSV protein expression at the apical surface of the epithelium.

[0087] Figure 6: Recombinant bivalent - In vivo characterization of RSV v.1, v.2 and v.3 viruses in a BALB / c mouse viral infection model.

[0088] [6A] Weight loss in BALB / c mice during in vivo experiments. Three constructs (bivalent -RSV v.1, v.2 and v.3).

[0089] [6B]Infected - Quantification of viral genes in lung tissues 5 days after RSV v.1, v.2 or v.3: Quantification of NHMPV genes (left panel) and F RSV genes (right panel) by RT-qPCR. The dotted line indicates the detection threshold.

[0090] Figure 7: In the BALB / c mouse model, after primary infection with HMPV or RSV, followed by bivalent -RSVv.1, v.2 and v.3 viruses boost infection and induce neutralizing antibody production in vivo.

[0091] [7A] Schematic diagram of the in vivo protocol, illustrating the first intranasal instillation of C-85473HMPV or RSV A wild-type virus strain (primary infection) and the second intranasal instillation of bivalent - RSV v.1, v.2 or v.3 (boost infection) to evaluate their efficacy in non-naive subjects Potential to induce HMPV- or RSV-specific antibody responses in BALB / c mice.

[0092] [7B] Compared with the group of mice boosted with OptiMEM (mock boost), BALB / c mice that were initially infected with HMPV showed a significant increase in the number of HMPV-positive mice after intranasal boosting with bivalent -Weight loss after RSV v.1, v.2 or v.3.

[0093] [7C] Compared with the group of mice boosted with OptiMEM (mock boost), BALB / c mice that were initially infected with RSV showed a significant increase in the number of mice that were boosted intranasally with bivalent -Weight loss after RSV v.1, v.2 or v.3.

[0094] [7D] BALB / c mice initially infected with HMPV were given a booster shot with a bivalent intranasal vaccination - Quantification of viral genes in lung tissues after 5 days of RSV v.1, v.2 or v.3 virus: Quantification of N HMPV gene (left panel) and F RSV gene (right panel) by RT-qPCR. The dotted line indicates the detection threshold.

[0095] [7E] RSV primary infection of BALB / c mice with intranasal booster vaccination - Quantification of viral genes in lung tissues after 5 days of RSV v.1, v.2 or v.3 virus: Quantification of N HMPV gene (left panel) and F RSV gene (right panel) by RT-qPCR. The dotted line indicates the detection threshold.

[0096] [7F] BALB / c mice infected with HMPV were given a bivalent intranasal booster vaccination. - Characterization of HMPV-specific neutralizing antibody responses in serum after RSV v.1, v.2 or v.3 virus or mock boost. Microneutralization assays were performed with serum collected 1 day before primary infection (-1), 20 days after primary infection (+20), and 42 days after primary infection / 21 days after intranasal boost infection (+42). Dashed lines indicate detection thresholds.

[0097] [7G] RSV primary infection of BALB / c mice with intranasal booster vaccination - Characterization of RSV-specific neutralizing antibody responses in serum after RSV v.1, v.2 or v.3 virus or mock boost. Microneutralization assays were performed with serum collected 1 day before primary infection (-1), 20 days after primary infection (+20), and 42 days after primary infection / 21 days after intranasal boost infection (+42) as described above. Dashed lines indicate detection thresholds.

[0098] Figure 8: Bivalent - In vivo protective properties of RSV v.3 virus against infectious challenge with a lethal dose of wild-type virus rC-85473HMPV strain

[0099] [8A] Schematic diagram below: Bivalent - Primary and boost immunizations with RSV v.3 virus in vivo followed by a lethal viral challenge with wild-type C-85473 HMPV virus (viral dose expected to induce >50% mortality). Each subsequent infection by intranasal instillation of virus was performed at intervals of three weeks.

[0100] [8B] Weight loss in BALB / c mice after infection with wild-type HMPV C-85473. or two-price The body weight of mice primed and boosted with RSV v.3 virus was compared with that of mice mock-immunized with OptiMEM medium.

[0101] [8C] Percentage of BALB / c mice that survived primary and booster immunizations after lethal challenge with HMPV C-85473.

[0102] [8D] Use bivalent Quantification of viral genes in lung tissues of BALB / c mice primed and boosted with RSV v.3 5 days after lethal viral challenge with wild-type virus C-85473 HMPV strain: Quantification of N HMPV gene (left) and F RSV gene (right) by RT-qPCR. The dotted line indicates the detection threshold.

[0103] [8E] OptiMEM medium (mock), monovalent or two-price -Detection of HMPV-specific neutralizing antibodies in sera of BALB / c mice primed and boosted with RSV v.3 and challenged with wild-type C-85473 HMPV strain. Microneutralization assays were performed with sera collected 1 day before primary infection (-1), 20 days after primary infection (+20), 41 days after primary infection / 20 days after boost infection (+41), and 62 days after primary infection / 21 days after lethal virus challenge (+62), as described above. The dotted line indicates the detection threshold.

[0104] [8F] OptiMEM medium (mock), monovalent or two-price -RSV v.3 primary and booster immunizations, and detection of RSV-specific neutralizing antibodies in the serum of BALB / c mice attacked by wild-type C-85473HMPV strains. Microneutralization assays were performed with serum collected 1 day before primary infection (-1), 20 days after primary infection (+20), 41 days after primary infection / 20 days after booster infection (+41), and 62 days after primary infection / 21 days after lethal virus attack (+62), as described above. The dotted line represents the detection threshold.

[0105] Figure 9: Recombinant bivalent - In vivo characterization of RSV v.1 and v.3 viruses in a BALB / c mouse viral infection model.

[0106] [9A] Infection with HMPV WT virus (rC-85437) and monovalent live attenuated vaccine (LAV) candidate Compared with the infection bivalent - Quantification of viral genes in bronchoalveolar lavage fluid 2 days after RSV v.1 or v.3: Quantification of N HMPV gene (left panel) and F RSV gene (right panel) by RT-qPCR. The dotted line indicates the detection threshold.

[0107] [9B] Cumulative histopathological scores according to infection with HMPV WT virus (rC-85437), monovalent live attenuated vaccine (LAV) candidate Two-price -RSV v.1, bivalent - Inflammation assessment of lung tissues (interstitial, intraalveolar, peribronchial, intrabronchial, perivascular and pleural regions) of RSV v.3 or "mock" infected mice was measured. Five days after infection, lungs of infected mice were collected and fixed in formaldehyde.

[0108] Figure 10: Two-price - In vivo protective properties of RSV v.1 and v.3 viruses against infectious challenge with a lethal dose of wild-type virus rC-85473HMPV strain

[0109] At 21-day intervals, 5 × 10 5 TCID50 or two-price - BALB / c mice were immunized twice with RSV vaccine candidates v.1 or v.3 or with AddaVax via the IM route TM Three weeks after the last immunization, animals were inoculated with 2×10 6 TCID of r C-85473 virus.

[0110] [10A] Weight loss in BALB / c mice after infection with wild-type HMPV C-85473. Two-price - Body weights of mice primed and boosted with RSV v.1 or v.3 were compared with those of mice mock-immunized with OptiMEM medium or with adjuvanted lysate of HMPV WT virus (HMPV lysate, an alternative to vaccination with HMPV protein vaccine) by the intramuscular route.

[0111] [10B] Survival percentage of BALB / c mice after primary and booster immunizations and lethal challenge with HMPV C-85473.

[0112] [10C] Cumulative histopathological scores 5 days after challenge were calculated by -RSV v.1, bivalent -RSV v.3 or monovalent Inflammation assessment of lung tissues (interstitial, intraalveolar, peribronchial, intrabronchial, perivascular, and pleural regions) of mice primed and boosted with the virus was measured and compared with mice mock-immunized or immunized with adjuvanted HMPV lysate. Five days after lethal challenge, lungs of infected mice were collected and fixed in formaldehyde.

[0113] [10D] Using bivalent Quantification of viral genes in lung tissues of BALB / c mice primed and boosted with RSV v.1 and v.3 5 days after lethal viral challenge with wild-type virus C-85473 HMPV strain: Quantification of N HMPV gene (left) and F RSV gene (right) by RT-qPCR. The dotted line indicates the detection threshold.

[0114] [10E] OptiMEM medium (mock), monovalent Two-price -RSV v.1 or v.3 or HMPV lysate primary immunization and booster immunization, and detection of HMPV specific neutralizing antibodies in the serum of BALB / c mice attacked by wild-type C-85473 HMPV strain. Microneutralization assay was performed with serum collected 1 day before primary infection (-1), 20 days after primary infection (+20), 41 days after primary infection / 20 days after booster infection (+41), and 62 days after primary infection / 21 days after lethal virus attack (+62). Neutralizing antibody titers for homologous HMPV strains (A / rC-85473, left figure) and heterologous HMPV strains (B / CAN98-75, right figure) were measured by specific microneutralization assay.

[0115] [10F] OptiMEM medium (mock), monovalent Two-price -RSV v.1 or v.3, or HMPV lysate primary and boost immunization, and wild-type C-85473HMPV strain challenged BALB / c mice, using ELISA assay for HMPV-specific IgG antibody detection, serum collected from 1 day before primary infection (-1), 20 days after primary infection (+20) and 41 days after primary infection / 20 days after boost infection (+41) and 62 days after primary infection / 21 days after lethal virus challenge (+62). IgG titers are expressed in arbitrary units per mL, based on endpoint absorbance.

[0116] Figure 11: Two-price - In vivo protective properties of RSV v.1 and v.3 viruses against infectious challenge with recombinant RSV-LucWT virus expressing luciferase protein.

[0117] At 21-day intervals, 5 × 10 5 TCID50 BALB / c mice were immunized twice with either the RSV vaccine candidate or recombinant RSV (RSV WT) virus or mock immunized. Three weeks after the last immunization, the animals were inoculated with 1×10 5PFU of rRSV-Luc virus, which is a recombinant RSV A WT virus that expresses luminescent luciferase protein in vivo.

[0118] [11A] Bioluminescence from the ventral side of infected mice was imaged using an in vivo imaging system (IVIS) at 3 and 5 days post-challenge. Bioluminescence was measured after intranasal injection of 50 μl of D-luciferin. The scale on the right represents the average radiance (the sum of photons emitted per pixel in the region of interest per second, ps -1 cm -2 sr -1 ), expressed in grayscale.

[0119] [11B] Luciferase activity was quantified from bioluminescent images using the "Living Image" software and expressed as mean ± SEM photons per second (p / s). After intranasal injection of 50 μl of D-luciferin, mice were imaged ventrally using an in vivo imaging system (IVIS) at 3 and 5 days post-challenge to measure luciferase activity. One-way ANOVA and Dunnett's post hoc test were used to compare the activity of the luciferase in the infected mice. -***p<0.001 when RSV v.1, v.3, or RSV WT vaccinated groups were compared to mock-vaccinated conditions.

[0120] [11C] using divalent - Quantification of viral genes in lung tissue of BALB / c mice 4 days after challenge with RSV WT virus after primary and booster immunization with RSV v.1 and v.3: quantification of FRSV genes (left) and NHMPV genes (right) by RT-qPCR. The dotted line indicates the detection threshold.

[0121] [11D] OptiMEM medium (mock), bivalent -RSV v.1 or v.3 or recombinant RSV WT virus carries out primary immunity and booster immunization, and is subjected to the detection of RSV specific neutralizing antibody in the BALB / c mouse serum of rRSV-Luc virus attack.Micro-neutralization is carried out with serum collected 21 days (+62) after 1 day (-1) before initial infection, 20 days (+20) after initial infection and 41 days / boosted infection after initial infection and 62 days / lethal virus attack after initial infection before infection. Neutralizing antibody titer for homologous RSV A strain (RSV A Long, left figure) and heterologous RSV B strain (WV / 14617 / 85, right figure) is measured by specific micro-neutralization. DETAILED DESCRIPTION

[0122] Human metapneumovirus (hMPV) strains

[0123] The present invention is based on a strain of human metapneumovirus isolated from a Canadian patient sample, designated C-85473, and described in the literature (Hamelin et al., 2010).

[0124] The recombinant strain rC-85473 is derived from the virus strain C-85473 and obtained by reverse genetic engineering, characterized by a considerable fusion capacity. In addition, rC-85473 can penetrate into target cells at high frequency, that is, the infection level is high. The inventors do not wish to be bound by any theory and attribute these characteristics to a specific sequence of the F protein of the rC-85473 strain, which contains a unique five-amino acid peptide sequence not found in other F proteins of other hMPV strains (Dubois et al., 2017).

[0125] The rC-85473hMPV strain comprises the genome sequence shown in SEQ ID NO:1.

[0126] The first genetic modification (i) introduced into the rC-85473 strain is intended to attenuate the virulence of the strain. "Attenuated virulence" corresponds to the absence of pathogenicity and reduced inflammatory response in vivo after administration of an effective dose of this attenuated virus strain. (Dubois et al., 2019 and Le et al., 2019). Advantageously, this attenuation of virulence does not affect the ability of the virus strain to replicate in vitro, nor does it affect its ability to infect target cells.

[0127] This genetic modification consists essentially of the inactivation of at least one endogenous gene:

[0128] - the gene encoding the SH protein, or

[0129] -Gene encoding G protein.

[0130] The genetic modification may also be the inactivation of two genes.

[0131] In the sense of the present invention, "gene inactivation" refers to a genetic modification that induces loss of gene expression or induces expression of an inactive form of a coded protein. Gene inactivation can be achieved by all techniques known to those skilled in the art. Specifically, gene inactivation can be achieved by introducing point mutations into a gene, by partially or completely deleting the coding sequence of a gene, or by modifying the gene promoter. These different genetic modifications will be performed according to any molecular biological technique known to those skilled in the art.

[0132] In a specific embodiment of the present invention, in the virus strain of the present invention, the endogenous gene encoding the SH protein is deleted.

[0133] In the sense of the present invention, "gene deletion" means that a substantial part of the coding sequence of the gene has been removed, in particular:

[0134] - Partial deletion of a gene means that at least 50%, 60%, 70%, 80%, 90% or 95% of the coding sequence has been removed;

[0135] -Complete deletion of a gene means that 100% of the coding sequence has been removed.

[0136] According to a preferred embodiment, in the virus strain according to the invention, the gene encoding the SH protein is completely deleted, that is to say all (100%) coding sequences of the SH protein are removed from the original genomic sequence. In this case, the virus strain according to the invention comprises the nucleotide sequence shown in SEQ ID NO: 2. This particular attenuated virus strain is referred to as Also called "unit price ”.

[0137] Virus strains comprising this first genetic modification (i) are referred to hereinafter as attenuated virus strains.

[0138] The second genetic modification (ii) introduced into the attenuated rC-85473 strain was aimed at obtaining the expression of antigens derived from the hRSV strain.

[0139] The genetic modification (ii) mainly involves introducing an exogenous coding sequence into the genome of the attenuated virus strain, for example, comprising a genome sequence as shown in SEQ ID NO: 2.

[0140] This introduction of exogenous coding sequences is not a replacement, but a true supplement to the genome of the attenuated rC-85473 strain. Therefore, even after the introduction of exogenous sequences, the F protein of the rC-85473 strain still exists in the strain genome and is expressed.

[0141] Therefore, the virus strain of the present 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.

[0142] In the sense of the present invention, "exogenous sequence encoding ..." or "exogenous coding sequence" or "exogenous nucleotide sequence" refers to a nucleic acid sequence that has been introduced into the viral genome, is under the control of a suitable promoter and encodes a protein or protein domain. In the present case, since the genome of hMPV consists of RNA, the introduced exogenous sequence will also consist of RNA. However, it should be understood that the preliminary step of introducing this exogenous sequence can use the corresponding DNA sequence (reverse genetics plasmid).

[0143] In the sense of the present invention, the term "F protein from hRSV" refers to a glycoprotein of hRSV from subgroup A or B of hRSV, preferably from subgroup A.

[0144] All known sequences of wild-type F proteins from hRSV can be found in public databases such as UniProt, for example under accession reference numbers P11209 or P03420 (precursor form).

[0145] Those skilled in the art are familiar with protein biology and can identify the three domains that make up a transmembrane protein: a cytoplasmic domain within the cell; a transmembrane domain inserted into the cell membrane; and an extracellular domain present on the surface of the cell membrane.

[0146] In the sense of the present invention, the phrase "encoding at least" means that the foreign sequence encodes at least one peptide domain, but in most cases also other domains.

[0147] In the sense of the present invention, the phrase "an extracellular domain of the F protein of hRSV" refers to the extracellular domain of any F protein from any hRSV subgroup (expressed on the surface of the virus particle), including wild-type domains and mutant domains.

[0148] As is known to the person skilled in the art, the extracellular domain is preferably combined with another peptide sequence to allow its anchoring into the viral particle, in particular at the surface of said viral particle, and / or to allow its anchoring into the membrane of the infected cell.

[0149] Therefore, in a preferred embodiment of the present invention, "an exogenous sequence encoding at least one extracellular domain of the F protein of human respiratory syncytial virus (hRSV), wherein the domain is wild type or mutated" refers to an exogenous sequence encoding a mutant or wild-type extracellular domain of the F protein of hRSV, which is associated with a sequence encoding at least one anchoring domain (in particular, encoding a cytoplasmic and / or transmembrane domain).

[0150] In a first embodiment of the invention, the domain of the F protein is a wild-type domain, ie, presents a peptide sequence of the domain from the native F protein of hRSV.

[0151] The term "wild-type" refers to the naturally occurring typical form of a protein (ie, its typical peptide sequence). In contrast, the term "mutated" refers to an atypical, non-standard form of the same protein.

[0152] In a specific embodiment, the exogenous nucleotide sequence comprises a sequence encoding the wild-type extracellular domain of the hRSV F protein.

[0153] In another specific embodiment, the exogenous nucleotide sequence consists mainly of a sequence encoding the wild-type extracellular domain of the hRSV F protein.

[0154] Specifically, the exogenous nucleotide sequence mainly comprises a sequence encoding the wild-type extracellular domain of the hRSV F protein from a subgroup A virus, and more specifically, encodes the wild-type extracellular domain of the hRSV F protein having a peptide sequence as shown in SEQ ID NO:3.

[0155] In a specific embodiment, the exogenous nucleotide sequence further comprises a sequence encoding the wild-type transmembrane domain of the hRSV F protein.

[0156] In a specific embodiment, the exogenous nucleotide sequence further comprises a sequence encoding the wild-type cytoplasmic domain of the hRSV F protein.

[0157] In another specific embodiment, the exogenous nucleotide sequence mainly comprises a sequence encoding three wild-type domains of the hRSV F protein (ie, the extracellular domain, the cytoplasmic domain, and the transmembrane domain constituting the entire F protein).

[0158] Specifically, the foreign nucleotide sequence mainly contains a sequence encoding a wild-type hRSV F protein from a subgroup A virus, and more specifically, encodes a wild-type hRSV F protein having a peptide sequence as shown in SEQ ID NO:4.

[0159] In a specific embodiment of the present invention, the exogenous nucleotide sequence is integrated into a specific site of the genome of the attenuated hMPV strain, for example:

[0160] - between the gene encoding the N protein and the gene encoding the P protein;

[0161] - between the gene encoding the P protein and the gene encoding the M protein;

[0162] - between the gene encoding the F protein and the gene encoding the M2 protein;

[0163] - between the gene encoding the SH protein and the gene encoding the G protein;

[0164] - between the gene encoding the M2 protein and the gene encoding the G protein (in the case of the ΔSH strain);

[0165] - between the gene encoding the G protein and the gene encoding the L protein.

[0166] According to one of these specific embodiments, advantageously, the exogenous nucleotide sequence is mainly composed of the sequence of the three wild-type domains of the hRSV F protein (ie, the extracellular, cytoplasmic and transmembrane domains constituting the entire F protein from hRSV).

[0167] A specific chimeric construct comprising the genome of an attenuated hMPV strain (represented by the sequence SEQ ID NO: 2) and an exogenous nucleotide sequence encoding a wild-type hRSV F protein (SEQ ID NO: 4) inserted between a gene encoding the F protein and a gene encoding the M2 protein is referred to in the Examples section as " -RSVv.1”.

[0168] The complete genome sequence of the virus strain combined with the GFP encoding gene is shown in SEQ ID NO:10.

[0169] In a specific embodiment, the present invention relates to a virus strain derived from a human metapneumovirus (hMPV) strain having a genome sequence represented by SEQ ID NO: 1, wherein the genome sequence comprises the following genetic modifications:

[0170] (i) inactivating endogenous genes encoding SH protein and / or G protein, and

[0171] (ii) the presence of an exogenous nucleotide sequence encoding a wild-type hRSV F protein of hRSV,

[0172] The foreign nucleotide sequence is inserted between the gene encoding the F protein and the gene encoding the M2 protein of the hMPV strain.

[0173] In a second embodiment of the present invention, the domain of the F protein is a mutant domain, that is, a peptide sequence presenting a domain of the wild-type F protein derived from hRSV, which peptide sequence contains at least one point mutation, that is, at least one residue in the wild-type peptide sequence is replaced by another residue.

[0174] In the context of the present invention, the term "mutant domain" or "mutant sequence" or "mutant protein" refers to a peptide sequence that exhibits at least 80% sequence identity with its corresponding standard wild-type peptide sequence after optimal alignment of the two sequences, and therefore exhibits at most 20% difference with its corresponding wild-type peptide sequence. In a preferred embodiment, in the mutant domain, the major antigenic epitope is conserved, i.e., presents its wild-type sequence.

[0175] In a preferred embodiment of the invention, the mutant 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. Preferably, in the mutant domain, the major antigenic epitope is conserved.

[0176] In a more specific embodiment of the invention, the mutant domain corresponds to a mutant domain that exhibits at least 97% sequence identity with the corresponding wild-type domain. The percentage identity referred to in the context of the present invention is determined by the optimal alignment of the sequences to be compared, which contain one or more differences, such as insertions, deletions, truncations and / or substitutions of amino acids.

[0177] The percent identity can be calculated by any sequence analysis method known to those skilled in the art.

[0178] The percent identity may be determined after global alignment of the sequences to be compared over their entire length.In addition to manual comparisons, global alignments may also be determined using the algorithm of Needleman and Wunsch (1970).

[0179] For peptide sequences, any software known to those skilled in the art can be used for sequence comparison, such as Needle software. The parameters used may be, in particular, the following parameters: "gap opening" equals 10.0, "gap extension" equals 0.5 and the BLOSUM62 matrix. Preferably, the percent identity is determined by a global alignment of the sequences over their full length.

[0180] In a specific embodiment, the exogenous nucleotide sequence comprises a sequence encoding a mutated extracellular domain of the hRSV F protein.

[0181] In another specific embodiment, the exogenous nucleotide sequence consists mainly of a sequence encoding a mutant extracellular domain of the hRSV F protein.

[0182] More preferably, the mutant domain corresponds to a stabilized prefusion state of the F protein, that is, its protein structure is constrained in the prefusion state.

[0183] The mutant extracellular domain comprises at least one of the following 14 mutations: S46G, K66E, E92D, Q101P, A149C, S155C, S190F, V203L, V207L, S215P, S290C, L373R, Y458C, K465Q, as described in (McLellan et al., 2013).

[0184] Preferably, the mutant extracellular domain comprises all 14 mutations described above. These mutations are incorporated by any technique known to those skilled in the art, in particular by site-directed mutagenesis.

[0185] In a specific embodiment, the mutant extracellular domain presents a sequence as shown in SEQ ID NO: 5. The mutant domain presents 14 point mutations over a total length of 513 residues, and thus has 499 common residues with the corresponding wild-type extracellular domain (residues 1-513 of SEQ ID NO: 4), which corresponds to a percent identity of 97.27% between the wild-type and mutant extracellular domains of the hRSV F protein.

[0186] In another embodiment, the exogenous nucleotide sequence consists mainly of sequences encoding a wild-type cytoplasmic domain, a wild-type transmembrane domain, and a mutant extracellular domain corresponding to the stabilized pre-fusion state of the F protein of hRSV.

[0187] A specific chimeric construct comprising the genome of an attenuated hMPV strain (represented by the sequence SEQ ID NO: 2) and an exogenous nucleotide sequence encoding a mutant hRSV F protein comprising a mutant extracellular domain corresponding to a stabilized prefusion state (SEQ ID NO: 5) is referred to in the Examples section as " -RSVv.2”.

[0188] Specifically, the mutant F protein presents the sequence shown in SEQ ID NO: 6. The sequence identity between the mutant protein and the wild-type hRSV F protein is 97.5% (560 common residues over a total length of 574 residues).

[0189] The complete genome sequence of the virus strain combined with the GFP encoding gene is shown in SEQ ID NO:11.

[0190] In another embodiment of the present invention, the exogenous nucleotide sequence encodes a chimeric protein comprising a domain of the F protein from hRSV and a domain of the F protein from hMPV.

[0191] Specifically, the exogenous nucleotide sequence encoding the chimeric hRSV / hMPV F protein comprises:

[0192] a. a sequence derived from hRSV, encoding at least one extracellular domain of the F protein, said domain being wild type or mutated; and

[0193] b. A sequence derived from hMPV, which encodes at least one domain of the F protein, the domain being selected from a cytoplasmic domain and a transmembrane domain, and the domain being wild type or mutated.

[0194] As mentioned above, in the context of the present invention, the term "mutant domain" refers to a domain having a peptide sequence with at least 80%, preferably at least 90%, more preferably at least 95%, 96% or 97% sequence identity with the peptide sequence of the corresponding wild-type domain. Preferably, the major antigenic epitope of the domain is conserved.

[0195] In other words, the present invention relates to a virus strain as described above, wherein the exogenous nucleotide sequence encodes a chimeric hRSV / hMPV F protein comprising:

[0196] a. a sequence derived from hRSV encoding at least one extracellular domain of the F protein, said domain being (i) wild-type or (ii) mutated and having at least 80% sequence identity with the peptide sequence of the corresponding wild-type domain, and

[0197] b. A sequence derived from hMPV, which encodes at least one domain of the F protein, wherein the domain is selected from a cytoplasmic domain and a transmembrane domain, and wherein the domain is (i) wild type or (ii) mutated and has at least 80% sequence identity with the peptide sequence of the corresponding wild type domain.

[0198] The object of the present invention is any attenuated virus strain as described above, the genome of which comprises foreign sequences according to any possible combination of (a) and (b).

[0199] For example, the combination comprises the following sequence encoding a chimeric hRSV / hMPV F protein, wherein the chimeric hRSV / hMPV F protein is mainly composed of:

[0200] - 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;

[0201] - 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;

[0202] - 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;

[0203] - mutated extracellular and wild-type transmembrane domains of the F protein from hRSV, and the wild-type cytoplasmic domain of the F protein from hMPV;

[0204] - mutated extracellular and wild-type cytoplasmic domains of the F protein from hRSV, and the wild-type transmembrane domain of the F protein from hMPV;

[0205] - A mutated extracellular domain of the F protein from hRSV, and wild-type transmembrane and cytoplasmic domains of the F protein from hMPV.

[0206] Advantageously, as described above, the “mutant” extracellular domain corresponds to a stabilized prefusion state of the F protein from hRSV, in particular exhibiting at least 97% sequence identity with the corresponding wild-type domain.

[0207] In a specific embodiment, the attenuated virus strain from hMPV comprises an exogenous nucleotide sequence, wherein the exogenous nucleotide sequence mainly comprises:

[0208] a. a sequence derived from hRSV encoding a mutant extracellular domain corresponding to the stabilized pre-fusion state of the F protein of hRSV; and

[0209] b. A sequence derived from hMPV, encoding the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV.

[0210] In a specific embodiment, the F protein of hMPV is from subgroup strain A1.

[0211] In another specific embodiment, the F protein of hMPV is from the rC-85473 strain and has the sequence shown in SEQ ID NO:7.

[0212] A specific chimeric construct comprising the genome of an attenuated hMPV strain (represented by the sequence SEQ ID NO: 2) and an exogenous nucleotide sequence encoding a chimeric protein comprising a mutant extracellular domain corresponding to the stabilized prefusion state of the F protein from hRSV (SEQ ID NO: 5) and wild-type cytoplasmic and transmembrane domains of the F protein from hMPV, the specific chimeric construct being referred to in the Examples section as " -RSV v.3”.

[0213] Specifically, the encoded chimeric protein presents the sequence shown in SEQ ID NO:8.

[0214] The complete genome sequence of the virus strain combined with the GFP encoding gene is shown in SEQ ID NO:12.

[0215] In a specific embodiment, the attenuated virus strain from hMPV comprises an exogenous nucleotide sequence, wherein the exogenous nucleotide sequence mainly comprises:

[0216] a. a sequence derived from hRSV encoding the wild-type extracellular domain of the F protein of hRSV; and

[0217] b. A sequence derived from hMPV, encoding the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV.

[0218] A specific chimeric construct comprising the genome of an attenuated hMPV strain (represented by sequence 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 of the F protein from hMPV. This specific chimeric construct is referred to as "v.4" in the Examples section.

[0219] Specifically, the encoded chimeric protein presents the sequence shown in SEQ ID NO:9.

[0220] In a specific embodiment, the virus strain of the invention exhibits the following genetic modifications:

[0221] (i) inactivation of the endogenous gene encoding the SH protein, and

[0222] (ii) there is an exogenous nucleotide sequence encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9.

[0223] Chimeric proteins and corresponding coding sequences, and viral strains containing them

[0224] Another aspect of the invention relates to a gene cassette encoding a chimeric protein comprising at least one domain from the F protein of hRSV and at least one domain from the F protein of hMPV.

[0225] Specifically, the gene cassette comprises:

[0226] a. a nucleotide sequence derived from hRSV, encoding at least one extracellular domain of the F protein, wherein the domain is wild type or mutated; and

[0227] b. a nucleotide sequence derived from hMPV, encoding at least one domain of the F protein, the domain being selected from a cytoplasmic domain and a transmembrane domain, the domain being wild type or mutated,

[0228] The exogenous sequence encodes a chimeric hRSV / hMPV F protein.

[0229] These gene cassettes also contain promoter sequences and all regulatory elements allowing transcription and translation into proteins of the nucleotide sequences (a) and (b).

[0230] All possible combinations of (a) and (b) are objects of the present invention.

[0231] The meaning of "mutant domain" has been defined above. Advantageously, the "mutant" extracellular domain corresponds to the stabilized prefusion state of the F protein from hRSV. In a specific embodiment, the mutant extracellular domain presents the sequence shown in SEQ ID NO:5.

[0232] For example, the combination comprises the following nucleotide sequence encoding a chimeric hRSV / hMPV F protein, wherein the chimeric hRSV / hMPV F protein is mainly composed of:

[0233] - the wild-type cytoplasmic domain of the F protein from hMPV;

[0234] - 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;

[0235] - 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;

[0236] - mutated extracellular and wild-type transmembrane domains of the F protein from hRSV, and the wild-type cytoplasmic domain of the F protein from hMPV;

[0237] - mutated extracellular and wild-type cytoplasmic domains of the F protein from hRSV, and the wild-type transmembrane domain of the F protein from hMPV;

[0238] - A mutated ectodomain of the F protein from hRSV, and the wild-type transmembrane and cytoplasmic domains of the F protein from hMPV. Advantageously, the "mutated" ectodomain corresponds to a stabilized prefusion state of the F protein from hRSV.

[0239] In a specific embodiment, the gene cassette comprises the following nucleotide sequence:

[0240] a. a sequence derived from hRSV encoding a mutant extracellular domain corresponding to the stabilized pre-fusion state of the F protein of hRSV; and

[0241] b. A sequence derived from hMPV encoding the wild-type cytoplasmic and transmembrane domains of the F protein of hMPV.

[0242] A specific chimeric construct according to this embodiment is referred to as "v.3" in the Examples section.

[0243] Specifically, the encoded chimeric protein presents the sequence shown in SEQ ID NO:8.

[0244] In a specific embodiment, the gene cassette comprises the following nucleotide sequence:

[0245] a. a sequence derived from hRSV encoding the wild-type extracellular domain of the F protein of hRSV; and

[0246] b. A sequence derived from hMPV, encoding the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV.

[0247] A specific chimeric construct according to this embodiment is referred to as "v.4" in the Examples section.

[0248] Specifically, the encoded chimeric protein presents the sequence shown in SEQ ID NO:9.

[0249] The nucleotide sequence may be in the form of DNA or RNA.

[0250] The above gene cassettes can be expressed by any system known to those skilled in the art. For example, the gene cassette according to the present invention can be incorporated into a plasmid, bacmid, liposome or any expression vector. The encoded chimeric protein can also be used in this way.

[0251] In another aspect, the present invention relates to a virus strain derived from a human metapneumovirus (hMPV) strain, the genome of which comprises a gene cassette as described above.

[0252] Preferably, the strain derived from the hMPV strain is further attenuated, ie, its virulence is reduced compared to the starting strain. For example, such attenuation of virulence is obtained by introducing a genetic modification in the genome sequence of the strain, as is known to those skilled in the art.

[0253] Virus strains used as medicines

[0254] The invention also relates to any viral strain as defined above for use as a medicament.

[0255] Indeed, this attenuated virus strain can be used in particular for the treatment and / or prevention of infections by at least one respiratory virus, more particularly by at least one virus of the Pneumoviridae family.

[0256] In the sense of the present invention, the term "treatment" refers to the fact of combating a viral infection in a human or animal organism. In the case of a viral infection, "treatment" refers to reducing the level of viral infection in an organism (infectious load), and preferably completely eradicating the virus in an organism. The term "treatment" also refers to the fact of alleviating symptoms associated with viral infection (respiratory syndrome, renal failure, fever, etc.).

[0257] In the sense of the present invention, the term "prevention" refers to the fact of avoiding or at least reducing the risk of an infection occurring in an organism. In the case of a viral infection, "prevention" means that the cells of the organism are less permissive to infection and are therefore less susceptible to infection by the virus. This also means that the immune system of the organism is ready to react quickly and effectively to fight the infection in the presence of the virus.

[0258] More specifically, the present invention relates to a virus strain as defined above for use in preventing and / or treating an infection by at least one respiratory virus, wherein the at least one respiratory virus is from the family Pneumoviridae, in particular human metapneumovirus and / or human respiratory syncytial virus.

[0259] In particular, the present invention relates to a virus strain as described above for use in preventing infection by two respiratory viruses, namely human metapneumovirus (hMPV) and human respiratory syncytial virus (hRSV).

[0260] The viral strain will preferably be incorporated into a vaccine composition comprising a pharmaceutically acceptable carrier suitable for suspension of the viral strain and for administration thereof.

[0261] The vaccine composition comprises at least one viral strain according to the invention, making it possible to stimulate the immune system of an organism in a specific manner.

[0262] Thus, the vaccine composition comprises at least one live attenuated virus strain that acts antigenically, ie it is recognized and induces a specific immune response in the organism, the memory of which is retained.

[0263] The present invention also relates to a vaccine composition comprising at least one virus strain according to the present invention in a pharmaceutically acceptable carrier and optionally an adjuvant.

[0264] In the sense of the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or excipient, i.e., a compound that does not have any specific effect on the infection under consideration. These carriers or excipients are pharmaceutically acceptable, meaning that they can be administered to an individual without causing significant harmful effects or unacceptable adverse reactions.

[0265] The vaccine composition according to the invention comprises at least one virus strain in an effective amount. In the sense of the present invention, an "effective amount" refers to an amount of a virus strain that is sufficient to trigger an immune response in an organism to which it is administered.

[0266] The vaccine composition of the present invention is suitable for oral, sublingual, inhalation, subcutaneous, intramuscular or intravenous administration.

[0267] According to a particular embodiment of the invention, the vaccine composition is in galenic form intended for administration by inhalation.

[0268] Inhalation refers to absorption through the respiratory tract. Specifically, it is a method used to absorb compounds of therapeutic interest involving certain substances in the form of gases, droplets in suspension, or powders.

[0269] Administration of pharmaceutical or veterinary compositions by inhalation (ie, through the nasal and / or buccal passages) is well known to those skilled in the art.

[0270] Two types of administration by inhalation are distinguished:

[0271] - when the composition is in powder form, by insufflation, and

[0272] - Administration by nebulization when the composition is in the form of an aerosol (suspension) or in the form of a solution (eg a pressurized aqueous solution). It would then be recommended to use a nebulizer or spray to administer the pharmaceutical or veterinary composition.

[0273] Thus, the pharmaceutical forms contemplated here are advantageously chosen from: powders, aqueous suspensions of droplets or pressurized solutions.

[0274] The target population for respiratory viruses is the pediatric population, consisting of individuals under the age of 18, more specifically young children (under 5 years old) and infants. Administration by inhalation is advantageous because it is non-invasive.

[0275] The present invention also relates to a vaccine composition as described above for use in preventing and / or treating infection by 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.

[0276] Such vaccine compositions can be used as prophylactic vaccines, that is, vaccines intended to stimulate a specific immune response before the virus infects an organism.

[0277] Such a vaccine composition may also be used as a therapeutic vaccine, that is to say, is intended to stimulate a specific immune response while the virus infects an organism.

[0278] The present invention also relates to a method for preventing infection with at least one virus of the Pneumoviridae family, in particular hMPV and / or hRSV, comprising administering the above-described vaccine composition to an individual susceptible to infection with such viruses.

[0279] The present invention also relates to a method for treating infection with a Pneumoviridae virus, in particular hMPV and / or hRSV, comprising administering the above-described vaccine composition to an individual infected with at least one of these viruses.

[0280] In particular, such individuals are children and infants.

[0281] As shown in the Examples section below, vaccine compositions comprising the virus strains of the present invention induce neutralizing antibodies against multiple hMPV strains (HMPV A and HMPV B, see Example 10) and multiple hRSV strains (RSV A and RSV B, see Example 11).

[0282] Table 1. Summary of sequences shown in the sequence listing

[0283]

[0284] Example

[0285] Example 1. Constructs

[0286] Four F RSV protein constructs were designed as follows Figure 1A Shown are native F RSV forms (v.1), stabilized prefusion F RSV forms (v.2), chimeric HMPV / RSV stabilized F prefusion forms (v.3), or chimeric HMPV / RSV native F RSV forms (v.4).

[0287] The F RSV v.1 sequence corresponds to the native F RSV gene of the RSV A2 strain available to those skilled in the art.

[0288] 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) are described as stabilizing the glycoprotein F of the metastable form before fusion (McLellan et al., 2013doi:10.1126 / science.1243283).

[0289] 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).

[0290] 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).

[0291] The corresponding coding sequence was inserted into a plasmid encoding the full-length genome of rC-85473HMPV virus (SEQ ID NO: 1).

[0292] Insertion of the F RSV coding sequence was made at several genomic locations, as described in Table 2 .

[0293] In these cases, insertion between the HMPV genes N and P, or between P and M, or between F and M2, allows the rescue of the virus after reverse genetics, following protocols known to those skilled in the art. The virulence of HMPV is attenuated by the deletion of the gene encoding the SH protein (ΔSH), and the construct consisting of the insertion of the F RSV coding sequence between the F and M2 HMPV genes is the only construct compatible with significant rescue of the recombinant virus, efficient virus propagation and amplification.

[0294] Table 2 - Description and rescue efficacy of bivalent HMPV / RSV constructs

[0295] F RSV gene location Flanking HMPV genes Virus rescue efficacy 1A 3'-GFP none 1B GFP-N none 2 NP yes 3 PM yes 5 F-M2 yes 7 SH-G yes

[0296] Prepared Figure 1B The genetic construct shown in FIG. 1 was constructed so that the recombinant virus could be detected by the expression of GFP (green fluorescent protein), and the gene encoding the SH protein was deleted (ΔSH, named SEQ ID NO: 13, containing GFP gene) and has attenuated virulence, and encodes an exogenous viral gene encoding the F fusion protein of respiratory syncytial virus (RSV), the F RSV coding sequence is inserted between the hMPV F and M2 genes, especially the three different gene versions.

[0297] The complete sequences of these genetic constructs are shown in SEQ ID NO: 13 (GFPΔSH-rC-85473), SEQ ID NO: 10 (bivalent RSV v.1), SEQ ID NO: 11 (bivalent RSV v.2) and SEQ ID NO: 12 (bivalent RSV v.3).

[0298] exist Figure 1C In, use The virus or the resulting bivalent recombinant virus was used to infect LLC-MK2 cells at a multiplicity of infection (MOI) of 0.01 (×40 magnification). The bivalent recombinant virus was: -RSV v.1, bivalent -RSV v.2 and bivalent -RSV v.3.

[0299] Cells were observed by fluorescence microscopy ten days after infection.

[0300] GFP expression indicates that the recombinant virus is fully functional and replicable, as well as having the expected fusion phenotype (inducing cellular syncytia by efficient expression of the F fusion protein), which is attenuated. Intrinsic characteristics of recombinant strains.

[0301] These results show The recombinant virus has the ability to accept foreign F RSV genes, thereby expressing a fully functional F fusion protein and generating a recombinant virus with proliferation and replication (by reverse genetics rescue), especially when the corresponding coding sequence is inserted between the F and M2 HMPV genes.

[0302] Example 2: Three recombinant bivalent -In vitro replication capacity of RSV virus.

[0303] LLC-MK2 cells were infected with the following recombinant viruses at a multiplicity of infection of 0.01:

[0304] -rC-85473 strain;

[0305] -Two-price -RSV v.1;

[0306] -Two-price -RSV v.2;

[0307] -Two-price -RSV v.3.

[0308] Cell supernatants were collected daily for 7 consecutive days in triplicate and the viral load was evaluated by TCID50 assay (a virological technique well known to those skilled in the art), which represents the final virus dilution at which 50% of the cell tissues show a visible cytopathic effect (50% tissue culture infectious dose).

[0309] Figure 2 A shows the insertion of attenuated Different kinetics, replication and production capacity are generated by the different nature of the foreign F RSV coding sequences in the genetic backbone. The positive control is the non-attenuated rC-85473 strain, which shows the same strains with similar abilities.

[0310] Recombinant virus bivalent -RSV v.1 appears to be more bivalent than the virus -RSV v.2 and bivalent -RSV v.3 has better replication ability.

[0311] The following table (Table 3) shows the bivalent -RSV v.1, bivalent -RSV v.2 and bivalent - Average loads of virus stocks generated and concentrated from RSV v.3, indicating that all three recombinant bivalent candidates have similar production yields despite different replication kinetics.

[0312]

[0313] These results show that bivalent -RSV v.1, bivalent -RSV v.2 and bivalent -Replication capacity of RSV v.3 virus.

[0314] Example 3: Recombinant bivalent - In vitro expression of exogenous F RSV protein on the surface of RSV virus infected cells.

[0315] Immunostaining assay

[0316] Use two price -RSV v.1, bivalent -RSV v.2 and bivalent LLC-MK2 cells were infected with RSV v.3 virus or wild-type recombinant rC-85473 HMPV strain (t=0) at an MOI of 0.01. Hep-2 cells were infected with RSV A2 strain (t=0) at an MOI of 0.01. Five days after infection, the infected cell monolayers were fixed in formaldehyde solution and specific immunostaining was performed using the following:

[0317] - monoclonal mouse antibody detecting F HMPV protein (MAb HMPV24 BioRad MCA 4674);

[0318] - Monoclonal humanized antibodies (palivizumab) that detect pre- and post-fusion forms of the F RSV protein AstraZeneca);

[0319] - Monoclonal human antibody (D25 Mab, Creative Biolabs) detecting the prefusion form of the F RSV protein.

[0320] The results are shown in Figure 3 middle.

[0321] Specific labeling was observed after peroxidase development. Representative images show that both HMPV F (after HMPV24 immunostaining) and RSV F (after palivizumab immunostaining) fusion proteins were present in the infection bivalent -RSV v.1, or bivalent -RSV v.2 and bivalent -RSV v.3 virus was expressed and detected on cells.

[0322] As expected for the controls, no immunostaining for MAb HMPV24 was observed on cells infected with RSV A2 strain (which does not express the HMPV F fusion protein), and no immunostaining for palivizumab was observed on cells infected with rC-85473 HMPV (which does not express the RSV F fusion protein). or immunostaining with D25 Mab.

[0323] Immunostaining with monoclonal D25 in infected bivalent -RSV v.2 and bivalent -RSV v.3 virus was detected on cells with a higher intensity than the infection bivalent F RSV protein form -RSV v.1 cells, which supports a stronger expression and exposure of the stabilized prefusion F RSV protein on the cell surface, as expected.

[0324] Flow cytometry assay

[0325] Use two price -RSV v.1, bivalent -RSV v.2 and bivalent LLC-MK2 cells were infected with RSV v.3 virus or rC-85473HMPV (t=0) at an MOI of 0.1.

[0326] 48 hours after infection, infected cell monolayers were trypsinized, resuspended, and the expression of FHMPV or F RSV proteins was quantified by flow cytometry.

[0327] Infected cells were detected by GFP fluorescence, and F HMPV and F RSV expression were detected by the following immunolabeling:

[0328] - Monoclonal humanized antibodies (palivizumab) that detect the pre- and post-fusion forms of the F RSV protein AstraZeneca), conjugated with R-phycoerythrin (PE) fluorescent dye;

[0329] - Monoclonal mouse antibody detecting F HMPV protein (MAb HMPV24 BioRad MCA 4674), conjugated with AlexaFluor TM 647 (A647) fluorescent dye conjugated.

[0330] The results are shown in Table 4 below.

[0331]

[0332] NA: Not applicable

[0333] The results reported in Table 4 show that when infected with bivalent -RSV v.1, bivalent -RSVv.2 and bivalent When infected with WT-RSV v.3 virus, more than 85% of infected cells exposed F HMPV protein on their surface, while 64.5%, 54.6% and 47.2% of cells exposed F RSV protein.

[0334] These results showed that three bivalent -RSV v.1, bivalent -RSV v.2 and bivalent - The ability of RSV v.3 to express and expose F RSV and F HMPV proteins on the surface of infected cells, especially by the bivalent -RSV v.2 and bivalent - Stabilized prefusion F RSV form expressed by RSV v.3 virus.

[0335] Example 4: Exogenous F RSV protein in bivalent - In vitro expression on the surface of RSV particles.

[0336] Prepare each bivalent -Virus suspension of RSV virus was filtered at 0.45 μm, concentrated by ultracentrifugation, and then resuspended in NaCl. The virus suspension was adsorbed on a 200-mesh coated nickel grid and observed by transmission electron microscopy without labeling ( Figure 4A ) or co-immunolabeling using antibodies:

[0337] Primary Antibody:

[0338] - Monoclonal humanized antibodies (palivizumab) that detect pre- and post-fusion forms of the F RSV protein AstraZeneca);

[0339] - Polyclonal mouse sera (in-house sera) tested for HMPV proteins.

[0340] Secondary Antibody:

[0341] - Goat anti-human IgG conjugated to 15 nm gold particles to detect anti-F RSV monoclonal humanized antibodies;

[0342] - Goat anti-mouse IgG conjugated to 5 nm gold particles to detect antibodies in anti-HMPV mouse sera.

[0343] The results are shown in Figure 4.

[0344] exist Figure 4A For all three bivalent -RSV v.1, bivalent -RSV v.2 and bivalent -RSV v.3 virus, the presence of typical pleomorphic virus particles covered by transmembrane glycoproteins was observed.

[0345] exist Figure 4B In the , immunolabeling with 15 nm gold particles (highlighted by black arrows) showed that in all three bivalent -RSV v.1, bivalent -RSV v.2 and bivalent -F RSV protein was detected on the surface of some virus particles of RSV v.3 virus. They were also immunolabeled with specific anti-HMPV serum (represented by 5 nm gold particles).

[0346] exist Figure 4C Before infecting LLC-MK2 cells, each bivalent -RSV virus stock solution was pre-incubated with specific anti-HMPV or specific anti-RSV neutralizing mouse serum. The percentage of specific serum neutralization was calculated by measuring the intensity of the GFP fluorescence signal from infected cells. Infection neutralization means that the glycoproteins on the surface of the virus particles, more specifically the F protein, are effectively recognized by the neutralizing antibodies present in the serum, thereby inhibiting the virus from attaching to the cell receptors and subsequent viral infection. Compared with the wild-type hMPV rC-85473 strain, the bivalent -RSV v.1, bivalent -RSV v.2 and bivalent -RSV v.3 virus was highly neutralized by anti-HMPV serum and significantly neutralized by anti-RSV serum. These results confirm that both F HMPV and F RSV proteins are exposed to the bivalent - The surface of RSV v.1, v.2 and v.3 virus particles.

[0347] These results demonstrate that hMPV F protein and hRSV F protein are bivalent in three -RSV v.1, bivalent -RSV v.2 and bivalent -RSV v.3 virus is efficiently expressed on the surface of viral particles.

[0348] Example 5: Three bivalent -Infection and replication capacity of RSV v.1, v.2 and v.3 viruses in ex vivo 3D reconstructed human respiratory epithelium.

[0349] 3D reconstructed human airway epithelium was cultured at the air-liquid interface according to the supplier's instructions ( HAE, Epithelix).

[0350] Then use the unit price or two-price - Epithelial cells were infected with RSV v.1, v.2 or v.3 viruses at an MOI of 0.5.

[0351] In infection unit price Two-price -RSV v.1, bivalent -RSV v.2 and bivalent -RSV v.3 virus 3, 5 and 7 days later, representative pictures of virus proliferation were taken.

[0352] Figure 5A showed prolonged GFP expression in human airway epithelium, indicating bivalent - RSV v.1, v.2, and v.3 viruses are infectious and replicate, although their proliferation in tissues appears to be slower than monovalent Proliferation should be slow, which accounts for the delay in GFP fluorescence detection.

[0353] exist Figure 5B Transepithelial electrical resistance (TEER) measurements were performed during infection. TEER measurements correspond to markers related to epithelial integrity. Similarly, in the two-price -Limited changes in TEER were observed after infection with RSV v.1, v.2, and v.3 viruses, which correlated with the attenuated viral phenotype and maintenance of epithelial integrity.

[0354] exist Figure 5C Quantification of viral genomes on the apical surface confirmed that each bivalent -RSV infection can effectively produce viral progeny.

[0355] exist Figure 5D In patients with Immunolabeling showed that - RSV v.1 virus-infected 3D reconstructed human airway epithelial cells expressing F RSV protein on the apical surface.

[0356] These results showed that three different bivalent - The ability of RSV v.1, v.2, and v.3 viruses to infect and proliferate in isolated human respiratory epithelial tissue, and the ability of these bivalent -The ability of RSV virus to express exogenous F RSV antigen on the surface of 3D reconstructed human respiratory epithelium.

[0357] Example 6: Recombinant Bivalent - In vivo characterization of RSV v.1, v.2 and v.3 viruses in a BALB / c mouse viral infection model.

[0358] Based on unit price The active immunization dose of the virus was determined by intranasal instillation with 5 × 10 5 TCID50 of recombinant bivalent - BALB / c mice were infected with RSV v.1, v.2 or v.3. The body weight and survival rate of infected mice were monitored daily for 10 days (mean body weight ± SEM of 5 mice).

[0359] In addition, 2 mice per group were euthanized 5 days after infection, and lung viral load was measured by RT-qPCR.

[0360] exist Fig. 6A In the two-price - No significant weight loss was observed during RSV v.1, v.2 or v.3 virus infection, as expected for attenuated virus strains.

[0361] Figure 6B Results of viral gene quantification in mouse lungs 5 ​​days after infection are shown. High levels of NHMPV and F RSV gene expression were observed, confirming the presence of three bivalent - The replication characteristics of RSV v.1, v.2 or v.3 viruses and their ability to express the F RSV gene in vivo.

[0362] These results suggest that three bivalent -RSV virus has the ability to infect and replicate in vivo and has the ability to express foreign F RSV genes in the lung tissue of infected mice at levels similar to those of NHMPV genes.

[0363] Example 7: In the BALB / c mouse model, after primary infection with HMPV or RSV, bivalent -Boosted infection with RSV v.1, v.2 and v.3 viruses induces neutralizing antibody production in vivo.

[0364] A non-lethal dose of wild-type HMPV rC-85473 (1 × 10 6 TCID50) or wild-type RSV A Long strain (1×10 6 PFU) to infect BALB / c mice to induce primary seroconversion in infected mice.

[0365] Three weeks later, based on the unit price Active immunization doses determined by virus were administered by intranasal (boost) instillation with 5 × 10 5 TCID50 of recombinant bivalent - RSV v.1, v.2 or v.3 virus infection of BALB / c mice that were initially infected with HMPV or RSV.

[0366] The body weight and survival rate of mice were monitored daily for 10 days (mean body weight ± SEM of 8 mice).

[0367] In addition, 5 days after intranasal boost infection, 3 mice in each group were euthanized and lung viral load was measured by RT-qPCR.

[0368] exist Figure 7D-Figure 7E Quantification of the N HMPV and F RSV genes by RT-qPCR showed that bivalent - RSV v.1, v.2 or v.3 viruses replicated very poorly in the lungs, whereas they replicated very high in RSV non-naive mice.

[0369] exist Figure 7F-7G In the study, HMPV- or RSV-specific neutralizing antibody responses were characterized by serum neutralization assays, indicating that three bivalent - RSV v.1, v.2 or v.3 viruses were able to induce strong HMPV- or RSV-directed antibody responses, depending on the starting non-naive HMPV or RSV serological status.

[0370] These results show that three bivalent - RSV v.1, v.2 or v.3 viruses have the ability to induce strong and specific neutralizing antibody responses against HMPV and RSV viruses in vivo.

[0371] Example 8: Bivalent - In vivo protective properties of RSV v.3 virus against infectious challenge with a lethal dose of wild-type virus rC-85473HMPV strain

[0372] Based on the unit price at 21-day intervals Active immunization dose determined by virus, 5 × 105 TCID50 of recombinant unit price or two-price BALB / c mice were immunized twice with RSV v.3 virus and compared with a group of mice mock-immunized with medium (Mock).

[0373] 21 days after the booster infection, 3 × 10 6 Mice were challenged with lethal hMPV virus at TCID50 of wild-type HMPV rC-85473 (the infectious dose that causes more than 50% mortality in mice).

[0374] The body weight and survival rate of mice were monitored daily for 14 days (mean body weight ± SEM of 8 mice).

[0375] exist Figure 8B In the study, mice immunized twice showed no significant weight loss after lethal viral challenge and fully survived, whereas mice immunized with mock immunization showed very significant weight loss, resulting in the death of all mice within 8 days after lethal challenge ( Figure 8C ).

[0376] Five days after viral challenge, four mice in each group were euthanized to measure lung viral load by RT-qPCR.

[0377] exist Fig.8D Quantification of N HMPV and F RSV gene copies in mice immunized with monovalent or two-price -Two doses of immunization with RSV v.3 virus effectively and significantly inhibited HMPV viral replication in the lungs.

[0378] Finally, in Figure 8E-8F In the present study, HMPV- or RSV-specific neutralizing antibody responses were quantified in sera collected before the primary intranasal vaccination and 21 days after the booster instillation (excluding the mock-immunized group).

[0379] As expected, the 2-dose High levels of HMPV neutralizing antibodies were measured in the sera of mice immunized with the virus.

[0380] For the two-price -In mice dually immunized with RSV v.3, high neutralizing antibody titers against HMPV and RSV were detected 21 days after booster immunization, and persisted until 21 days after HMPV virus challenge.

[0381] These results suggest that bivalent -RSV candidate (bivalent -RSV v.3) has the ability to induce strong specific neutralizing antibody responses against HMPV and RSV viruses in vivo and has the ability to completely protect mice challenged with a lethal dose of HMPV wild-type virus.

[0382] Example 9: Recombinant Bivalent - In vivo characterization of RSV v.1 and v.3 viruses in a BALB / c mouse viral infection model.

[0383] Based on unit price The active immunization dose of the virus was determined by intranasal instillation with 5 × 10 5 TCID50 of recombinant bivalent - BALB / c mice were infected with RSV v.1 or v.3.

[0384] Fig.9A Results of viral gene quantification in bronchoalveolar lavage fluid of mice 5 days after infection are shown. High levels of NHMPV and F RSV genes were observed, confirming the bivalent - The presence and replication properties of RSV v.1 and v.3 viruses in the lower respiratory tract of infected animals and their ability to express FRSV genes in vivo.

[0385] In addition, 3 mice in each group were euthanized 5 days after infection to collect lung tissues. The intact lungs were fixed with formaldehyde solution for further histopathological analysis.

[0386] exist Fig. 9B In the infection of monovalent LAV candidates The low inflammation score image measured later depicts the infection bivalent - Hypoinflammatory profile after RSV v.1 or v.3 infection, in contrast to the high pro-inflammatory scores measured after HMPV WT infection.

[0387] Overall, these results highlight the potential of two bivalent -RSV virus has the ability to infect and replicate in vivo, express foreign F RSV genes, and induce a low inflammatory response in the lung tissue of infected mice, as expected for a live attenuated vaccine candidate.

[0388] Example 10: Bivalent - In vivo protective properties of RSV v.1 and v.3 viruses against infectious challenge with a lethal dose of wild-type virus rC-85473HMPV strain

[0389] Based on the unit price at 21-day intervals Active immunization dose determined by virus, 5 × 10 5TCID50 of recombinant unit price or two-price -BALB / c mice were immunized twice with RSV v.1 or v.3 viruses and compared with a group of mice mock-immunized with medium (Mock) or with a group of mice immunized with adjuvanted HMPV WT virus lysate (HMPV lysate) by the intramuscular route as an alternative to vaccination with HMPV protein vaccines.

[0390] 21 days after the booster infection, 2 × 10 6 Mice were challenged with lethal HMPV virus at TCID50 of wild-type HMPV rC-85473 (the infectious dose that causes more than 50% mortality in mice).

[0391] The body weight and survival rate of mice were monitored daily for 14 days (mean body weight ± SEM of 8 mice).

[0392] exist Fig. 10A and Fig. 10B In the immunized mice (using recombinant monovalent or two-price -RSV v.1 or v.3 virus or HMPV lysate vaccination) showed significant weight loss and fully survived the lethal viral challenge, while mock-immunized mice showed very significant weight loss, resulting in the death of all mice within 6 days after the lethal challenge ( Fig. 10B ).

[0393] Five days after viral challenge, three mice in each group were euthanized to collect lung tissue for further histopathological analysis, and two or four mice in each group were euthanized to measure lung viral load by RT-qPCR.

[0394] exist Fig. 10C In the literature, different inflammatory profiles were described, depending on the vaccine candidate and route of administration. - Mice immunized with RSV v.1 or v.3 viruses appeared to induce reduced interstitial, peribronchial, intraalveolar, and pleural inflammation, which was in contrast to the high inflammation scores measured after immunization with HMPV lysate, which corresponded to the enhanced disease syndrome described in the literature after immunization with inactivated vaccines.

[0395] exist Fig. 10D Quantification of N HMPV and F RSV gene copies in mice immunized with monovalent or two-price - Two doses of immunization with RSV v.1 and v.3 viruses effectively and significantly inhibited HMPV viral replication in the lungs.

[0396] exist Fig.10E Homologous or heterologous HMPV-specific neutralizing antibody responses were quantified in sera collected during the protocol. High levels of HMPV neutralizing antibodies were measured in the sera of mice immunized with the virus. - In mice doubly immunized with RSV v.1 and v.3 viruses, similar high neutralizing antibody titers against HMPV A and HMPV B viruses were detected 21 days after booster immunization, which persisted or increased until 21 days after HMPV virus challenge.

[0397] Finally, in Fig.10F HMPV-specific IgG antibodies were quantified in sera collected during the protocol. As expected, given the neutralizing antibody titers, HMPV-specific IgG antibodies were quantified in sera collected during the protocol. or two-price - High levels of IgG antibodies were measured in the sera of mice immunized with RSV v.1 and v.3 viruses. In contrast, immunization with adjuvanted HMPV lysate appeared to induce higher levels of neutralizing and IgG antibodies 21 days after the second immunization, but the antibody response seemed to decline after HMPV challenge.

[0398] These results suggest that bivalent -RSV Candidates ( - RSV v.1 and v.3) have the ability to induce strong specific neutralizing antibody responses against HMPV A and HMPV B viruses in vivo and have the ability to completely protect mice challenged with a lethal dose of HMPV wild-type virus.

[0399] Example 11: Bivalent - In vivo protective properties of RSV v.1 and v.3 viruses against infectious challenge with recombinant RSV-Luc WT virus expressing luciferase protein.

[0400] Based on the interval of 21 days Active immunization dose determined by virus, 5 × 10 5 TCID50 divalent BALB / c mice were immunized twice with RSV v.1 or v.3 and compared with mice mock-immunized with medium (mock) or by intranasal instillation of 5 × 10 5 The mice group (RSV-mCh) immunized with PFU of recombinant RSV WT virus was compared.

[0401] 21 days after the booster infection, 1 × 10 5 Mice were challenged with RSV infection with PFU of rRSV-Luc virus (a recombinant RSV A WT virus that expresses luminescent luciferase protein in vivo, Rameix-Welti et al., 2014).

[0402] Animal body weight was monitored daily for 14 days and there was no weight loss, as expected for RSV infection in the mouse model.

[0403] exist Fig.11A and Fig. 11B In the experiment, mice immunized with bivalent -RSV v.1 or v.3 virus or RSV WT) showed significantly reduced luciferase activity and bioluminescence in the nose and lungs after virus challenge, while mock-immunized mice showed high intensity luminescence (a result of RSV virus replication in lung tissue), especially in the lungs 5 ​​days after challenge.

[0404] Four days after viral challenge, four mice in each group were euthanized, and lung viral load was measured by RT-qPCR.

[0405] exist Fig. 11C In accordance with previous luminescence results, quantification of F RSV gene copy number showed that compared with mock-immunized mice, mice immunized with bivalent - Two doses of RSV v.1 and v.3 tend to reduce and limit RSV viral replication in the lungs, and the use of bivalent Three weeks after boosting with the -RSV candidate, no residual NHMPV genetic material was detected in the lungs of mice.

[0406] exist Fig.11D In this study, homologous or heterologous RSV-specific neutralizing antibody responses were quantified in sera collected during the protocol.

[0407] As expected, the two-dose bivalent - Induction of RSV neutralizing antibodies was measured in the sera of mice immunized with RSV v.1 and RSV WT viruses. 21 days after RSV virus challenge, -Vaccination with the RSV v.3 candidate appeared to induce higher levels of neutralizing antibodies specific for RSV A and RSV B strains.

[0408] These results suggest that bivalent -RSV candidate (bivalent -RSV v.1 and v.3) have the ability to induce strong specific neutralizing antibody responses against RSV A and RSV B viruses in vivo and have the ability to limit RSV replication in the upper and lower respiratory tracts of mice challenged with RSV viruses.

[0409] References

[0410] patent

[0411] WO 2020 / 021180

[0412] WO 2020 / 120910

[0413] EP 3 868 874

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Claims

1. A virus strain derived from a human metapneumovirus (hMPV) strain having a genome sequence represented by SEQ ID NO: 1, wherein the genome sequence comprises the following genetic modifications: (i) inactivating endogenous genes encoding SH protein and / or G protein, and (ii) there is 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.

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

3. The virus strain according to any one of claims 1 to 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 according to claim 3, wherein the exogenous nucleotide sequence is mainly composed of sequences encoding three wild-type domains (cytoplasmic, transmembrane and extracellular) of the F protein of hRSV.

5. according to the virus strain described in any one in claim 1 to 2, wherein the exogenous nucleotide sequence comprises the sequence of the mutant domain of the F protein encoding hRSV, particularly corresponding to the mutant extracellular domain of the stabilized pre-fusion state of the F protein.

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

7. The virus strain according to any one of claims 1 to 2, wherein the exogenous nucleotide sequence comprises: a. a sequence derived from hRSV, encoding at least one 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, the domain being selected from a cytoplasmic domain and a transmembrane domain, the domain being wild type or mutated, The exogenous sequence encodes a chimeric hRSV / hMPV F protein.

8. The virus strain according to claim 7, wherein the exogenous nucleotide sequence is mainly composed of: a. a sequence derived from hRSV encoding a mutant extracellular domain corresponding to the stabilized pre-fusion 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.

9. The virus strain according to claim 7, wherein the exogenous nucleotide sequence is mainly composed of: 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, encoding the wild-type cytoplasmic domain and the wild-type transmembrane domain of the F protein of hMPV.

10. A gene cassette comprising one of the following nucleotide sequences: - a sequence encoding a mutant extracellular domain corresponding to the stabilized prefusion state of the F protein of hRSV, and a sequence encoding a wild-type cytoplasmic domain and a wild-type transmembrane domain of the F protein of hMPV, in particular a sequence as shown in SEQ ID NO: 8 as a major part; - A sequence encoding the wild-type extracellular domain of hRSV protein, and a sequence encoding the wild-type cytoplasmic domain and wild-type transmembrane domain of the F protein of hMPV, especially a sequence as shown in SEQ ID NO: 9 as a main part.

11. A virus strain derived from a human metapneumovirus (hMPV) strain, the genome of which comprises the gene cassette of claim 10, preferably, wherein the virus strain is further attenuated.

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

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

14. The virus strain for use according to 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. A vaccine composition comprising at least one viral strain according to any one of claims 1 to 9 or 11 in a pharmaceutically acceptable carrier, and optionally an adjuvant.

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