WO2019092002A1 - Compositions pharmaceutiques pour le traitement ou la prévention des infections virales - Google Patents

Compositions pharmaceutiques pour le traitement ou la prévention des infections virales Download PDF

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WO2019092002A1
WO2019092002A1 PCT/EP2018/080424 EP2018080424W WO2019092002A1 WO 2019092002 A1 WO2019092002 A1 WO 2019092002A1 EP 2018080424 W EP2018080424 W EP 2018080424W WO 2019092002 A1 WO2019092002 A1 WO 2019092002A1
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seq
protein
nucleic acid
hmpv
pharmaceutical composition
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PCT/EP2018/080424
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English (en)
Inventor
Bruno Pitard
Melissa HANSON
Mehdi Lahmar
Fabien PERUGI
Klaus Schwamborn
Fabienne Guehenneux
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Valneva Se
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18311—Metapneumovirus, e.g. avian pneumovirus
    • C12N2760/18322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18311—Metapneumovirus, e.g. avian pneumovirus
    • C12N2760/18334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to constructs and compositions for use as prophylactic or therapeutic treatments against viral infections.
  • Human metapneumovirus is a member of the Paramyxoviridae family, assigned to the genus Metapneumovirus in the subfamily of Pneumovirinae and is an enveloped, negative single-stranded RNA virus.
  • hMPV is closely related to respiratory syncytial virus (RSV), which is the most significant respiratory pathogen of infancy and early childhood.
  • RSV respiratory syncytial virus
  • the more recently discovered hMPV is also an important respiratory pathogen and is associated with significant morbidity in infants and other high-risk populations, such as immunocompromised patients and individuals with underlying conditions, including prematurity, asthma, and cardiopulmonary disease (Kahn, et al.
  • Isolates of hMPV are separated into two major lineages (A and B) and at least four subgroups (Al, A2, B l and B2) (van den Hoogen, et al. (2004) Antigenic and genetic variability of human metapneumoviruses. Emerg. Inf. Dis. 10:658-666).
  • the hMPV genome consists of a single negative strand of RNA of approximately 13 kb, containing eight genes presumed to encode nine different proteins. Of these, there are three hMPV surface glycoproteins: the attachment glycoprotein (G), which is involved in cell attachment, the fusion glycoprotein (F-glycoprotein or F-protein), which mediates fusion of the host cell and viral membranes and a small hydrophobic protein (SH).
  • G attachment glycoprotein
  • F-glycoprotein or F-protein fusion glycoprotein
  • Viral coat proteins are prime targets for neutralizing antibodies; however, studies regarding the induction of protective immunity to hMPV have demonstrated that only the highly-conserved F-protein elicited a high-titer neutralizing antibody response (Skiadopoulos, et al. (2006) Individual contributions of the human metapneumovirus F, G, and SH surface glycoproteins to the induction of neutralizing antibodies and protective immunity. Virology (345):492-501).
  • the F-protein from the related Respiratory Syncytial virus has also been shown to be a main target of neutralizing antibodies (Magro, et al , 2012, Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention PNAS 109(8):3089-3094).
  • RSV Respiratory Syncytial virus
  • hRSV lum- adjuvanted formalin-inactivated human RSV
  • Vaccine 25(27): 5034-5040 Natural infection with RSV does not result in enhanced disease upon reinfection; neither does it confer lasting protection (Kim, et al , supra). Similarly, natural infection with hMPV provides only transient protection and does not prevent further infections throughout the lifetime of the individual (Lenneke, et al. (2013) Human Metapneumovirus in Adults. Viruses 5:87-110). An effective vaccine to hMPV, therefore, must not only improve on natural immunity induced by infection, but must also simultaneously avoid harmful responses induced by the inactivated virus vaccine.
  • the vaccine must be sufficiently safe for use in high-risk populations such as immunocompromised patients and infants. Furthermore, induction of cross-reactive immunity against both clinically relevant isolates (A and B) would be highly desirable. In similar fashion, a combination vaccine simultaneously conferring protection against hMPV and RSV would be a valuable contribution to the field.
  • the current disclosure provides DNA molecules encoding hMPV F-proteins and variants thereof, particularly hMPV F-proteins in a trimeric post-fusion conformation, vectors comprising such DNA molecules, as well as purified hMPV F-protein polypeptides and variants thereof.
  • the disclosure further provides pharmaceutical compositions comprising the DNA molecules, vectors and/or polypeptides of the invention, particularly pharmaceutical compositions for stimulating an immune response in a subject, particularly an immune response which is protective against or neutralizes hMPV.
  • the disclosure further provides pharmaceutical compositions comprising the hMPV DNA molecules of the invention and DNA molecules encoding RSV antigens as combination vaccines.
  • DNA molecules, vectors, polypeptides and pharmaceutical compositions as disclosed herein are particularly suitable for use as a medicament, particularly for the prophylactic or therapeutic treatment of viral infections in a subject, especially metapneumo virus and/or respiratory syncytial virus infections.
  • Figure 1 shows the primary structure of the fusion glycoprotein (F-protein) of hMPV.
  • the full-length protein is 539 amino acids long and, during processing, becomes first the F0 form (aa 19-539) following removal of the signal peptide (aa 1-18), then a heterodimer of the F2 and Fl portions (aa 20-102 and 103- 539, respectively) linked by disulfide bonds, following proteolytic cleavage of F0.
  • SP Signal peptide
  • FP Fusion peptide
  • HRA Heptad repeat A
  • HRB Heptad repeat B
  • TM Transmembrane region
  • CT Cytoplasmic tail.
  • FIG. 1 schematically depicts the process of fusion of virus and host -cell membranes, which is mediated by paramyxovirus F-protein.
  • Figure 3 shows a schematic representation of a DNA sequence (A) of an expression construct for a post- fusion hMPV F-protein heterodimer of the invention and a processed protein (B) encoded by the expression construct.
  • the molecules depicted represent the subunit post-fusion hMPV F-protein of the invention, showing polypeptides A and B, containing the Fl ectodomain and F2 domain, respectively.
  • polynucleotide constructs for the DNA vaccine preparations as described herein do not encode a
  • Such a protein from an Al genotype of hMPV optionally contains a G294E substitution in the Fl portion.
  • the expressed heterodimeric protein forms homotrimers during processing, facilitated by the presence of the trimerization domain.
  • Figure 4 shows representative sequences for insertion into a protein expression plasmid comprising coding sequences of post-fusion hMPV F-protein heterodimers of the invention which are derived from (A) an Al strain of hMPV (isolate "NL/1/00") and (B) a Bl strain of hMPV (isolate "NL/1/99"). Major domains and restriction sites are indicated.
  • the preferred coding sequences for Al and B l subunit post- fusion F-proteins are provided as SEQ ID Nos: 18 and 19, respectively.
  • Figure 5 Confirmation of expression of post-fusion (sPoFhMPv), full-length (FIFhMPv) and soluble (sFhMPv) forms of hMPV F-protein following ICAFectin®441 transfection of Hela cells as shown by staining of permeabilized cells by the DS7 monoclonal antibody in flow cytometry.
  • Figure 6 A. Purified sPoFhMPvAl-MFur and sFhMPvAl-V hMPV F-proteins as visualized by coomassie staining and Western blot with anti-penta-His antibody (Qiagen). B.
  • FIG. 8 Comparison of the immunogenicity of FIFhMPv DNA and sPoFhMPv subunit as tested in flow cytometry with FlFhMPv/ICAFectin®441 transfected Hela cells. Each plot shows binding of serum antibodies from individual vaccinated mice, comparing day 0 (thicker trace) and day 56 responses.
  • FIG. 9 Comparison of the immunogenicity of sPoFhMPv DNA and sPoFhMPvAl-Mfur subunit as tested in flow cytometry with sPoFhMPv/ICAFectin®441 transfected Hela cells. Each plot shows binding of serum antibodies from individual vaccinated mice, comparing day 0 (thicker trace) and day 56 responses.
  • FIG 11 Comparison of immunogenicity of DNA vaccines and subunit vaccines in Balb/c mice by IC5 0 on day 56 following three immunizations (A) and IgG titers including IgG2a, IgGl and the IgG2a/IgGl ratio (Thl/Th2) on day 42 following two immunizations (B).
  • FIG. 12 Antibodies stimulated in Balb/c mice at day 42 following vaccination (2x at three week intervals) with post-fusion F-protein DNA (50 ⁇ g sPoFhMPv + 0.15% Nanotaxi® 1) bound to both post- fusion trimer (sPoFhMPvAl-Mfur) and pre -fusion trimer (sFhMPvAl-K) as well as to native monomer (sFhMPvAl-V) in an ELISA assay; whereas antibodies elicited by vaccination (2x at three week intervals) with post -fusion F-protein subunit (10 ⁇ g sPoFhMPvAl-Mfur + alum) bound preferentially to the post- fusion trimer.
  • post-fusion F-protein DNA 50 ⁇ g sPoFhMPv + 0.15% Nanotaxi® 1
  • nanotaxi® 1 Nanotaxi® 1
  • the monoclonal antibody DS7 which binds to both pre- and post-fusion forms, served as a control.
  • Figure 13 RSV antigen encoding vectors added in combination with hMPV post-fusion F-protein DNA vaccine do not influence the production of neutralizing hMPV antibodies. Characterization of anti-hMPV immunogenicity of DNA vaccines encoding hMPV antigen alone, RSV antigen alone, or both hMPV and RSV antigens in C57B1/6 mice is shown by hMPV neutralization curves (A) and IC5 0 (B) on day 56. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention relates to a nucleic acid encoding a heterodimeric protein consisting of a) a polypeptide A comprising an immunogenic I I ectodomain of the hM V F-protein; and b) a polypeptide B comprising an immunogenic I 2 domain of the hMPV F-protein, wherein the I ⁇ 1 and F2 domains are covalenily linked by at least one disulfide bond.
  • the encoded heterodimer of the invention combines to form a homotrimeric form.
  • the encoded F-protein heterodimers of the invention differ from wild type F-protein heterodimers at least in that they do not possess a transmembrane domain or a cytoplasmic tail.
  • the wild-type hMPV F-protein is a glycoprotein consisting of a signal peptide, an f 2 domain and an Fl domain (see Figure 1). After processing, the signal peptide is cleaved off and the F2 and I ⁇ 1 domains are proteolytically cleaved, but are joined covalently by disulfide bonds, forming a heterodimer.
  • the F- protein exists in trimeric form, each trimer consisting of three F-protein heterodimers, and is inserted into the viral envelope via the transmembrane domain with an outward orientation.
  • the hMPV F-protein trimer is processed first as a metastable pre -fusion form, which is capable of initiatin fusion of the viral membrane with host-cell membranes.
  • the pre -fusion form engages with the host cell membrane and the subsequent transformation of the F-protein to the post- fusion form facilitates the fusion of the two membranes (see Figure 2). Additionally, over time, even in the absence of fusion, the pre -fusion form of the F-protein spontaneously undergoes a conformational change to the more stable post-fusion form.
  • hMPV F-protein in trimeric post-fusion conformation is defined as a lully- processed hMPV F-protein, consisting of I ⁇ I and F2 regions, wherein the two regions have been proteolytically separated, but are covalently linked by at least one, preferably two, disulfide bonds, and wherein the transmembrane and cytoplasmic domains of the Fl region have been removed, resulting in a soluble protein in a post-fusion configuration.
  • the post-fusion I -protein combines to form a homotrimer comprising three F1/F2 heterodimers.
  • Fl eetodomain An Fl domain which lacks a transmembrane domain and a cytoplasmic tail is referred to herein as the "Fl eetodomain". It has been previously demonstrated that truncation of the RSV I -protein to remove the transmembrane domain and cytoplasmic tail (leaving only the eetodomain of the Fl region) results in a soluble form, of the I -protein which spontaneously folds into a post-fusion configuration (Magro, et al. (2012), supra).
  • heptad repeats A and B can come together to form a coiled-coil structure known as the six -helix bundle ( 6-1 I B; see Fig. 2), which is characteristic of the post-fusion form of the I -protein.
  • 6-1 I B six -helix bundle
  • one disulfide bond is formed between the I ⁇ I and I 2 regions of the hMPV post-fusion I -protein.
  • the one disulfide bond is formed between amino acid residues 60 and 1 82 of the hMPV post-fusion I -protei n.
  • the one disulfide bond is formed between amino acid residues 28 and 407 of the hMPV post-fusion I -protei n. In one embodiment, two disulfide bonds are formed between the I ⁇ 1 and I 2 regions of the hMPV post-fusion F-protein. In one embodiment, the two disulfide bonds are formed between amino acid residues 60 and 1 82 and amino acid residues 28 and 407 of the hMPV post-fusion F-protein.
  • the hMPV F-protein i n post-fusion conformation as encoded by the nucleic acid of the invention includes the following features (see Figure 3A):
  • the F-protein i n post-fusion conformation as encoded by the nucleic acid of the invention further comprises:
  • the hMPV I -protein I ⁇ I ectodomain and I 2 domain encoded by the nucleic acid of the invention are both selected from Al or B 1 strains of h PV.
  • the terms "protein” and “polypeptide” are interchangeable.
  • the encoded heterodimeric protein comprises an immunogenic Fl ectodomain consisting of: a) amino acids 112 to 489 of the hMPV I - protein from the A I genotype, especially wherein the sequence contains a G294E mutation; i.e.
  • the encoded heterodimeric protein comprises an immunogenic I ⁇ I ectodomain consisting of a) amino acids I 1 2 to 489 of the hMPV I -protein from the B I genotype; i.e. the amino acid sequence of SEQ II ) NO: 12 and b) the immunogenic I 2 domain consists of amino acid 20 to 101 of the hMPV I -protei n from the B I genotype; i.e.
  • the Fl ectodomain from the Al genotype is the wild-type sequence; i.e., does not contain a G294E mutat ion (SEQ II ) NO: 9).
  • the heterodimeric protein encoded by the nucleic acid of the invention contains one or more engineered changes for cleavage of the protein during processing or for enhancing or improving the purification or function of the encoded protein.
  • the native cleavage site for proteolytic processing of F0 (RQSR; SEQ I I ) NO: 1) which is sensitive to trypsin, is replaced by or overlapped with an alternative cleavage site.
  • the alternative cleavage site is a furin cleavage site.
  • the furin cleavage site is derived from the RSV I -protein.
  • the RSV-de rived furin cleavage site is the RSV cleavage site II defined by SEQ NO: 2.
  • the furin protease for proteolytic processing of F0 to F2 and Fl is provided by cloning a nucleic acid sequence encoding a furin protease int the same plasmid as the nucleic acid encoding the post-fusion form of the hMPV I -protein.
  • the nucleic acid sequence encoding the furin protease is provided separately in a different plasmid to be co-transfected with the post-fusion F-protein construct.
  • the furin protease is stably expressed by the cell line used for expression of the post-fusion F-protein construct.
  • the furin protease is a human furin protease.
  • the human furin protease is defined by SEQ II ) NO: 26.
  • the coding sequence of the human furin protease is defined by SEQ II ) NO: 31.
  • the polypeptide A encoded b the nucleic acid of the i nvention additionally comprises a trimerization domain ( ' -terminal to the I ⁇ 1 ectodomain.
  • the trimerization domain of the polypeptide A comprises or consists of the fibritin 1 4 foldon domain ( Bhardwaj. et al. (2008) Foldon- guided self-assembly of ultra-stable protein fibers Protein Science (2008), 17: 1475-1485).
  • the 4 foldon domain is defined by SEQ II ) NO: 6.
  • the polypeptide A encoded by the nucleic acid of the invention additionally comprises another cleavage site B between the 1 1 ectodomain and the trimerization domain.
  • the additional cleavage site B of polypeptide A is a cleavage site for TEV protease (Tobacco Etch Virus nuclear-inclusion-a endopeptidase).
  • TEV protease tobacco Etch Virus nuclear-inclusion-a endopeptidase
  • the TEV protease cleavage site is of the general form EXXYXQ(G/S).
  • the cleavage sequence for the TEV protease is ENLYFQG as defined by SEQ ID NO: 3.
  • the polypeptide A encoded by the nucleic acid of the invention additionally comprises a tag at the ( -terminal end of the trimerization domain.
  • the tag is a I lis. tag (SEQ II ) NO: 5).
  • the polypeptide A encoded by the nucleic acid of the invention additionally comprises another cleavage site C between the trimerization domain and the tag.
  • the additional cleavage site C is a cleavage site of the serine endopeptidase factor Xa.
  • the cleavage site of serine endopeptidase factor Xa is of the general form I(E/D)GR.
  • the serine endopeptidase factor Xa cleavage site is IEGR as defined by SEQ I I ) NO: 4.
  • the trimeric configuration of the heterodimeric protein encoded b the nucleic acid according to the current disclosure comprises F-protein heterodimers consisting of a polypeptide A with SEQ I I ) NO: 14 or a functional variant thereof that is more than 80% identical to the polypeptide with SEQ ID NO: 14, especially more than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% identical to the polypeptide with SEQ ID NO: 14, most preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the polypeptide with SEQ I I ) NO: 14.
  • polypeptide B with SEQ I I ) NO: 15 or a functional variant thereof that is more than 80% identical to the polypeptide with SEQ II ) NO: 15, especially more than 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90% identical to the polypeptide with SEQ II ) NO: 1 , most preferably more than 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the polypeptide with SEQ II ) NO: 15.
  • the trimeric configuration of the heterodimeric protein encoded by the nucleic acid according to the current disclosure comprises F-protein heterodimers consisting of a polypeptide A with SEQ ID NO: 16 or a functional variant thereof that is more than 80% identical to the polypeptide with SEQ II ) NO: 16, especially more than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% identical to the polypeptide with SEQ ID NO: 16, most preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the polypeptide with SEQ I I ) NO: 16 and a polypeptide B with SEQ I I ) NO: 17 or a functional variant thereof that is more than 80% identical to the polypeptide with SEQ I I ) NO: 1 7 especially more than 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% identical to the polypeptide with SEQ ID NO
  • the trimeric configuration of the heterodimeric protein encoded by the nucleic acid according to the current disclosure comprises I -protein heterodimers consisting of a polypeptide A with SEQ ID NO: 29 or a functional variant thereof that is more than 80% identical to the polypeptide with SEQ ID NO: 29, especially more than 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% identical to the polypeptide with SEQ ID NO: 29, most preferably 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the polypeptide with SEQ ID NO: 29 and a polypeptide B with SEQ ID NO: 15 or a functional variant thereof that is more than 80% identical to the polypeptide with SEQ I NO: 15 especially more than 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% identical to the polypeptide with SEQ ID NO: 15.
  • the current invention provides a vector which comprises the nucleic acid of the invention.
  • the nucleic acid of the invention is comprised i n a vector suitable for use i n DNA vaccines, providing a vector suitable for inoculation of a subject.
  • said vector suitable for use in DNA vaccines contains an element or elements allowing propagation and selection i n a host cell, e.g., E. coli.
  • said vector optimized for use in DNA vaccines contains an element or elements that direct expression of the transgene in the target organism, e.g., a mammal such as a human.
  • said vector optimized for use in DNA vaccines is a first generation DNA vaccine vector such as pVAXl or gWIS.
  • said vector optimized for use i n DNA vaccines is a second-generation DNA vaccine vector.
  • the vector optimized for use in DNA vaccines is the pVAXl vector.
  • the nucleic acid of the invention is comprised in a vector suitable for in vitro expression for subsequent (optional) purification of the encoded polypeptide.
  • the vector suitable for in vitro expression of the encoded polypeptide is suitable for use in bacteria or in eukaryotic cells such as mammalian cells, avian cells, insect cells or yeast cells.
  • the vector is a viral vector, such as a recombinant viral vector.
  • the viral vector is a Newcastle Disease Virus ( NDV ).
  • NDV Newcastle Disease Virus
  • the NDV is a lentigenic strain of NDV, especially a LaSota or Hitchner B l strain.
  • a lentigenic strain is defined as having relatively lower virulence in birds.
  • the NDV is a moderate to high virulent strain of NDV, i.e., a mesogenic or velogenic strain, such as, e.g., AF2240.
  • the NDV strain is an oncolytic strain; i.e., a strain with capacity to selectively induce apoptosis in tumors or cancer cells in vivo or in vitro.
  • the oncolytic strain is a LaSota strain of NDV.
  • the oncolytic strain is the highly virulent AF2240 strain of NDV.
  • the viral vector is a vaccinia virus vector.
  • the vaccinia virus vector is pRB21.
  • the vector is a baculo virus vector.
  • the vector suitable for in vitro expression is pVVS 137 1 (as defined by SEQ II ) NO: 29).
  • the host cell used for in vitro expression of the encoded polypeptide is an insect cell, such as SF9, SF21 or Tni (e.g. , High Fives or Tn 368 cells), or a duck cell line, especially a duck cell line derived from duck retina or embryonic fibroblasts, such as those described in WO2005/042728, especially EB66.
  • the host cells used for in vitro expression of the encoded polypeptide are Chinese Hamster Ovary (CHO) cells.
  • the nucleic acid of the invention, the vector of the invention or the protein encoded by the nucleic acid of the invention is for use as a medicament.
  • the nucleic acid of the invention, the vector of the invention or the protein encoded by the nucleic acid of the invention is for use as a prophylactic or therapeutic treatment against a vi ral infection.
  • the nucleic acid of the invention, the vector of the invention or the protein encoded by the nucleic acid of the invention is comprised in a pharmaceutical composition.
  • the pharmaceutical composition comprising the nucleic acid of the invention, the vector of the invention or the protein encoded by the nucleic acid of the invention is used as a medicament.
  • the pharmaceutical composition comprising the nucleic acid of the invention, the vector of the invention or the protein encoded by the nucleic acid of the invention is used as a prophylactic or therapeutic treatment against a viral infection.
  • the pharmaceutical composition for use is a vaccine.
  • the vaccine of the invention is used for the prophylactic or therapeutic treatment of infection with one or more respiratory pathogens, such as viruses.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid, the polypeptide encoded by the nucleic acid or the vector of the invention.
  • the pharmaceutical composition comprises between I ng and 1 mg of nucleic acid, polypeptide or vector of the invention, preferably between 10 ng and 500 , more preferably between 100 ng and 400 ⁇ g, even more preferably between I ⁇ g and 200 ⁇ g, most preferably between 10 and 100 ⁇ g.
  • Such dose is preferably administered 1 to 3 times at intervals of 2 to 24 weeks.
  • the pharmaceutical compositions of the present invention may be used to protect a subject susceptible to hMPV infection or treat a subject with an hMPV infection, by means of administering said vaccine via a systemic or mucosal route.
  • administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory or genitourinary tracts.
  • the vaccine of the invention may be administered as a single dose, components thereof may also be co-administered together at the same time or at different times.
  • the disclosure provides a pharmaceutical composition wherein said pharmaceutical composition further comprises antigens or vectors encoding said antigens from further respiratory pathogens, in particular, RSV and/or parainfluenza virus (PIV) antigens.
  • the further antigens from RSV are RSV I -proteins or variants thereof or, more preferably, nucleic acids encoding RSV I -proteins or variants thereof, most preferably vectors comprising such nucleic acids.
  • the RSV I -proteins or variants thereof are selected from, the group consisting of post-fusion forms, monomelic native forms and profusion forms.
  • the RSV I -proteins are selected from the group consisting of a post-fusion form of liKSV as defined by SEQ II ) NO: 32 (referred to herein as sPoFhRsv; encoded by SEQ II ) NO: 33); a profusion liKSV I -protein as defined by SEQ II ) NO: 34 (referred to herein as sPrl hk svS( ' -l )M; encoded by SEQ II ) NO: 35) or a prolusion liKSV I - protein as defined by SEQ ID NO: 36 (referred to herein as sPrFhRsvDS-Cavl ; encoded by SEQ ID NO: 37).
  • the pharmaceutical composition as provided herein is also suitable for use as a medicament, particularly as a vaccine for preventing or treating an infection caused by human metapneumovirus (hMPV), particularly an hMPV from A and/or B genospecies.
  • the pharmaceutical composition as provided herein is particularly suitable for use in a method of treating or preventing an hMPV infection, particularly an hMPV infection caused by genotype A and/or B hMPV, such as genotype Al, A2, Bl and/or B2 hMPV.
  • the pharmaceutical composition of the invention is additionally for use as a vaccine for preventing or treating an infection cause by human Respiratory Syncytial Virus (RSV).
  • RSV Respiratory Syncytial Virus
  • the pharmaceutical composition according to the current invention is for use in a method of treating or preventing a human Respiratory Syncytial Virus (RSV) infection.
  • the pharmaceutical composition according to the current disclosure may contain one or more suitable auxiliary substances, such as buffer substances, pharmaceutical excipients, stabilizers or further active ingredients, especially ingredients known in connection with a pharmaceutical composition and/or vaccine production.
  • the pharmaceutical composition of the disclosure further comprises an adjuvant and/or other pharmaceutically acceptable carriers or excipients, such as buffer substances, stabilizers or further active ingredients, especially ingredients known in connection with pharmaceutical compositions and/or vaccine production.
  • the pharmaceutically acceptable excipient comprises a nucleic acid delivery reagent.
  • the nucleic acid delivery reagent comprises tetrafunctional non-ionic amphiphilic block copolymers comprising at least one hydrophilic block and at least one hydrophobic block.
  • a preferable carrier or excipient for the nucleic acid molecules according to the present invention in their diverse embodiments is an immunostimulatory compound such as an adjuvant for further stimulating the immune response to the polypeptide encoded by the nucleic acid molecule(s) herein disclosed.
  • a pharmaceutical composition which is a vaccine this vaccine may further comprise a pharmaceutically acceptable excipient.
  • the excipient is a 704 or 704-M tetrafunctional non-ionic amphiphilic block copolymer.
  • one or more synthetic delivery systems will be used in the formulation.
  • a preferred compound is one of the class known as Nanotaxi®, which allow for very high in vivo antigen delivery and stimulation of the innate immune system, eliciting a powerful immune response.
  • tetrafunctional non-ionic amphiphilic block copolymers comprising at least one hydrophilic block and at least one hydrophobic block.
  • the hydrophilic block may be selected in the group consisting of polyoxyalkylenes, polyvinyl alcohols, polyvinyl-pyrrolidones, poly(2-methyl-2-oxazoline), or saccharides, and the hydrophobic block that may be selected in the group consisting of polyoxyalkylenes, aliphatic chains, alkylidene polyesters, polyethylene glycol with a benzyl polyether head, and cholesterol.
  • the hydrophilic blocks of a block copolymer are comprised of, and preferably consist in, polyethylene oxide units.
  • the hydrophobic blocks of a block copolymer are comprised of, and preferably consist, in polypropylene oxide units.
  • Especially preferred block copolymer comprises hydrophilic blocks comprising, and preferably consisting in, polyethylene oxide units, and hydrophobic blocks comprising, and preferably consisting in, polypropylene oxide units.
  • a preferred compound is a tetrafunctional non-ionic amphiphilic block copolymer comprising at least one terminal hydrophilic block.
  • a "terminal hydrophilic block” is a block located at one end of a copolymer, and in particular at a distal end of a branch of a tetraiunctional polymer.
  • a tetraiunctional non- ionic amphiphilic block copolymer comprises at least two, preferably three, and more preferably four terminal hydrophilic blocks.
  • a preferred compound is a block copolymer comprising at least one, preferably two, even preferably three, and more preferably four terminal oxyethylene unit(s), each at one end of each branch of the polymer.
  • a tetraiunctional non-ionic amphiphilic block copolymer comprises hydrophilic and hydrophobic blocks in a ratio hydrophilic block/hydrophobic block ranging from 0.7 to 1.5, preferably from 0.8 to 1.3, and more preferably from 0.8 to 1.2.
  • a tetraiunctional non-ionic amphiphilic tetraiunctional block copolymer useful for the invention may be a (A-B)n-C branched block copolymers, with A representing an hydrophilic block, B representing an hydrophobic block, C representing a linking moiety, and n being 4 and figuring the number of (A-B) group linked to C.
  • the hydrophilic block A is a polyoxyethylene block
  • the hydrophobic block B is a polyoxypropylene block.
  • the linking moiety C may be an alkylene diamine moiety, and preferably is an ethylene diamine moiety.
  • a tetraiunctional non -ionic amphiphilic block copolymer useful for the invention may be of formula (I):
  • RA, RB, RC, RD represent independently of one another
  • - i has values from about 5 to about 125, in particular from about 10 to about 100, and more particularly from about 10 to about 60, and
  • - j has values from 5 to about 85, in particular from about 10 to about 50, in particular from about 10 to about 20, and more particularly equal to or greater than 13,
  • R* is an alkylene of 2 to 6 carbons, a cycloalkylene of 5 to 8 carbons or a phenylene, and preferably is an ethylene
  • R 1 and R 2 either (a) both are hydrogen or (b) one is hydrogen and the other is methyl,
  • R 3 and R 4 either (a) both are hydrogen or (b) one is hydrogen and the other is methyl, and
  • R 3 and R 4 are hydrogen, then one R 5 and R 6 is hydrogen and the other is methyl, or if one of R 3 and R 4 is methyl, then both of R 5 and R 6 are hydrogen.
  • a non-ionic amphiphilic tetrafunctional block copolymer useful for the invention may be of formula (II):
  • i has values from about 5 to about 125, in particular from about 10 to about 100, and more particularly from about 10 to about 60, and
  • j has values from about 5 to about 85, in particular from about 10 to about 50, and more particular from about 10 to about 20, and
  • R 1 shall be hydrogen and R 2 shall be a methyl group
  • the molecular weight ranges from about 4000 to about 35000, in particular from about 4500 to about 30000, more particularly from about 5000 to about 25000, and
  • the ethylene-oxide unit content is about 30% to about 80%, in particular about 35% to 50%, more preferably about 40%.
  • said tetrafunctional block co-polymer has an i-value of 13, a j -value of 14, a molecular weight of 5500 and an ethylene-oxide unit content of 40%; i.e., is a 704 block copolymer.
  • i may range from about 5 to about 125, in particular from about 10 to about 100, and more particularly from about 10 to about 60
  • j may range from about 5 to about 50, in particular from about 10 to about 25, in particular from about 10 to about 20, and more particularly equal to or greater than 13.
  • a block copolymer useful for the invention may have a molecular weight ranging from 4000 to 35000 and in particular ranging from 4500 to 30000 and more particularly ranging from 5000 to 25000.
  • a block copolymer useful for the invention may comprise, and preferably consist in, an ethylene -oxide units content from about 40%, in particular from about 45%, in particular ranging from about 45 to about 80%, in particular ranging from about 45 to 70%, and more particularly from about 45 to about 60%, and more preferably of about 50%.
  • non-ionic amphiphilic block copolymers for the invention can be found in Surfactant Systems, Eds. Attwood and Florence, Chapman and Hall, London 1983, p 356-361 ; in The Condensed Encyclopaedia of Surfactants, Ed. Ash and Ash, Edward Arnold, London, 1989, in Non-ionic Surfactants, pp. 300-371, Ed. Nace, Dekker, New York, 1996, in Santon, Am. Perfumer Cosmet. 72(4):54-58 (1958); (Dekker, N.Y., 1967), or in US 6,353,055.
  • the non-ionic amphiphilic block copolymer suitable for the invention is selected from the group consisting of 704 and 904 or a mixture thereof. In a preferred embodiment, the non-ionic amphiphilic block copolymer suitable for the invention is a 704 copolymer.
  • Another preferred compound is at least one block of a block copolymer useful for the invention, and preferably a hydrophilic block, is conjugated with a glycosyl moiety.
  • a glycosylated tetrafunctional non- ionic amphiphilic block copolymer useful for the invention comprises at least one terminal block, and preferably one terminal hydrophilic block, conjugated with at least one glycosyl moiety. More preferably, at least 25%, in particular at least 50%, in particular at least 75% and more particularly at least 100% of terminal blocks of a block copolymer of the invention are conjugated with a glycosyl moiety.
  • a non-glycosylated or glycosylated tetrafunctional non-ionic amphiphilic block copolymer may be used in an amount ranging from 0.01 to 10% by weight of the total weight of a composition containing it, in particular in a range from about 0.02 to 5%, more particularly from about 0.05 to 2%, more preferably from about 0.07 to 1%, more preferably from about 0.075 to 0.3%, and most preferably at about 0.15%, by weight of the total weight of a composition containing it.
  • tetrafunctional non-ionic amphiphilic block copolymers are especially preferred.
  • the tetrafunctional block co-polymers comprise at least one glycosyl moiety, wherein said at least one glycosyl moiety is a single glycosyl unit or a linear or branched polymer of glycosyl units.
  • the at least one glycosyl moiety comprises mannose or galactose, preferably mannose.
  • a 704 tetrafunctional non-ionic amphiphilic block copolymer also referred to herein as Nanotaxi®l or 704
  • a mannose-substituted 704 tetrafunctional non-ionic amphiphilic block copolymer also referred to herein as Nanotaxi®2 or 704-M
  • the pharmaceutical composition further comprises an immunostimulatory substance such as an adjuvant.
  • the adjuvant can be selected based on the method of administration and may include polycationic substances, especially polycationic peptides, immunostimulatory nucleic acids molecules, preferably immunostimulatory oligo-deoxynucleotides (ODNs), especially the 26- mer oligo(dIdC) i3 (also known as "5'-(dIdC)i3-3"), peptides containing at least two KLK motifs separated by a linker of 3 to 7 hydrophobic amino acids, especially peptide KLKLLLLLKLK, a combination of KLK peptide and oligo(dIdC) i3 (also known as IC31 ® ), alum, full-length M protein from hMPV or fragments thereof (Aerts, et al.
  • ODNs immunostimulatory oligo-deoxynucleotides
  • the pharmaceutical composition comprises one or more of the 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 22), the 26-mer 5'-(dIdC)i 3 -3' (SEQ ID NO: 23), alum or an hMPV M protein or fragment thereof, especially an M protein derived from an Al or Bl strain of hMPV, especially an M protein as defined by SEQ ID NO: 24 or SEQ ID NO: 25.
  • the nucleic acid of the invention or a protein as encoded by the nucleic acid of the invention or the pharmaceutical composition of the disclosure is used to treat or prevent a viral infection.
  • the viral infection is a metapneumovirus infection.
  • the viral infection is a human metapneumovirus infection.
  • the viral infection is an hMPV infection caused by an A or B strain of hMPV, especially an hMPV infection caused by one or more of the group of strains selected from Al, A2, Bl and B2 strains of hMPV.
  • nucleic acid of the invention or a protein as encoded by the nucleic acid of the invention or the pharmaceutical composition according to the invention provides cross -protection against more than one hMPV strain.
  • the pharmaceutical composition of the disclosure is useful in the prevention or treatment of RSV.
  • the invention provides a process for the production of a pharmaceutical composition of the disclosure comprising the steps of: a) providing a nucleic acid sequence encoding a polypeptide A and polypeptide B as defined above in a suitable vector; b) combining said nucleic acid with an optional adjuvant, nucleic acid delivery reagent and/or pharmaceutically acceptable excipient in order to obtain said pharmaceutical composition.
  • the process is performed using a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 43 and 49.
  • the current disclosure further provides an hMPV F-protein complex in trimeric post-fusion conformation, wherein said complex consists of three hMPV F-protein heterodimers, and wherein said heterodimer comprises; a) a polypeptide A comprising an immunogenic Fl ectodomain of the hMPV F-protein; and b) a polypeptide B comprising an immunogenic F2 domain of the hMPV F-protein; wherein the Fl and F2 domains are covalently linked by at least one disulfide bond for use as a medicament.
  • the said hMPV F-protein heterodimer comprised in the trimeric post-fusion complex is defined as SEQ ID NO: 44 or 47, preferably SEQ ID NO: 44.
  • the hMPV F-protein complex in trimeric post-fusion conformation as disclosed herein is used as a subunit for vaccination against hMPV infection.
  • the current disclosure further provides a process for producing a pharmaceutical composition comprising an hMPV F-protein complex in trimeric post-fusion conformation as defined herein, comprising the steps of: a) providing a nucleic acid sequence encoding a polypeptide A and a polypeptide B of the invention in a suitable vector; b) expressing said vector in a suitable host cell to yield polypeptide A and polypeptide B; c) optionally, purifying said hMPV F-protein complex; and d) combining said hMPV F-protein complex with an optional adjuvant and/or other suitable excipient(s) in order to obtain said pharmaceutical composition.
  • Polypeptide A (Al strain) (with G294E mutation)
  • Coding sequence for the Al strain hMPV post-fusion F-protein heterodimer subunit (sPoFhMPvAl-Mfur; SEQ ID NO: 44; also shown in Fig. 4A) codon optimized for expression in CHO cells
  • Matrix protein from the Al hMPV isolate "NL/1/00" (Accession No.: AAK62969)
  • Matrix protein from the Bl hMPV isolate "NL/1/99" (Accession No.: AAS92881)
  • sPrFhRsvSC-DM hRSV single-chain pre-fusion F-protein coding sequence human codon optimized for DNA vaccine
  • sPrFhRsvDS-Cavl hRSV pre-fusion F-protein coding sequence human codon optimized for DNA vaccine
  • sFhMPv Soluble native F-protein coding sequence human codon optimized for DNA vaccine
  • FI FhMPV Full length F-Protein of the Al hMPV isolate "N L/1/00" (Accession No.: AAK62968)
  • FI FhMPv Full length F-Protein coding sequence of the Al hM PV isolate "N L/1/00" human codon optimized for DNA vaccine
  • sFhMPvAl-V soluble configuration F-protein polypeptide in monomer form for purification
  • SEQID NO: 47 sPoFhMPv Post-Fusion hM PV F-protein (Bl) adapted from the sequence of the "N L/1/99" isolate (Accession number AY304361.1)
  • sPoFhMPv Post-Fusion hMPV F-protein (Bl) adapted from the sequence of the "Arg/2/02" isolate (Accession number DQ362937.1)
  • sPoFhMPv Post-Fusion hM PV F-protein (Bl) adapted from the sequence of the "Arg/2/02" isolate (Accession number DQ362937.1) human codon optimized DNA Sequence for DNA vaccine
  • Nanotaxi®-DNA hMPV vaccine candidate constructs encoding hMPV F-proteins were designed to trigger expression of proteins by the host cell as outlined in Table 1A.
  • Nanotaxi®-DNA hRSV vaccine candidate constructs were designed for combination hMPV/RSV vaccine candidates as detailed in Table 1A.
  • the constructs trigger the expression of secreted (soluble) forms of the pre- or post-fusion conformations of the hRSV F-protein as a trimer complex by the host cells.
  • the amino acid sequences of the different hRSV F-proteins are derived from the sequence of the F- protein of the hRSV A2 strain (Pubmed accession No.: P03420) belonging to the A2 sublineage.
  • codon optimization of the DNA sequences for use in DNA vaccination was performed to increase expression in humans.
  • the codon- optimized sequences for the six F-protein constructs in Table 1A below were de novo synthesized and spliced into the immunization plasmid (pVaxl, Invitrogen; SEQ ID NO: 28) using Hindlll and Xhol restriction sites.
  • the resulting vector DNA was amplified in E.
  • DH5aTM purified with a HiSpeed Plasmid Giga EF Kit (Qiagen S.A., Courtaboeuf, France) according to the manufacturer's protocol and quantified by measuring the absorbance (260 nm) of diluted plasmid solution (1/250 & 1/500) in triplicate. The plasmids were additionally checked by enzymatic digestion, followed by agarose gel electrophoresis.
  • Table 1A Sequences for hMPV F-protein DNA vaccine preparation and hRSV F-protein DNA vaccine preparation. All DNA sequences were codon optimized for human expression and sub-cloned into pVAXl
  • Soluble single-chain Soluble form of hRSV F-protein in a pre- fusion configuration Soluble single-chain Soluble form of hRSV F-protein in a pre- fusion configuration
  • SC single chain
  • Soluble pre-fusion Soluble form of hRSV F-protein (McLellan et al., 2013), including
  • the plasmid pVAXl (SEQ ID NO: 28) from Invitrogen was developed for use in DNA vaccines by modification of the pcDNATM3.1 vector according to considerations put forth by the FDA Center for Biologies Evaluation and Research (CBER) in the document "Points to Consider on Plasmid DNA Vaccines for Preventive Infectious Diseases Indications" (2007; Docket no. 96N-0400).
  • the pVAXlTM vector was used for all DNA constructs used in the DNA vaccination studies described herein and contains:
  • CMV cytomegalovirus immediate -early
  • BGH bovine growth hormone
  • the mature F-protein is a homotrimer of F2/F1 heterodimers covalently linked by two disulfide bridges.
  • Each F2/F1 heterodimer is initially expressed as a single polypeptide precursor, designated FO ( Figure 1).
  • the FO precursor proteins form trimers in the endoplasmic reticulum and are proteolytically processed by extracellular proteases at a single site located immediately upstream of the hydrophobic fusion peptide, which lies at the N-terminus of the Fl domain.
  • the mature trimeric F-protein adopts a metastable pre-fusion conformation in the mature virus particle that is triggered to undergo a conformational change when the viral and target-cell membranes are brought into proximity.
  • Final refolding of the paramyxovirus F-protein into a stable post -fusion conformation leads to the merging of the viral and host cell membranes and the formation of the fusion pore ( Figure 2).
  • the post-fusion hMPV F-protein construct (sPoFhMPv) in trimeric form is stabilized by fusion of the ectodomain (F-protein excluding the C-terminal transmembrane and cytoplasmic tail domains) to a foldon trimerization domain derived from the T4 phagehead fibritin (SEQ ID NO: 6).
  • the polybasic cleavage site II of hRSV (KKRKRR; SEQ ID NO: 2) was added after the native proteolytic cleavage site present in the hMPV F-protein (RQSR; SEQ ID NO: 1) to facilitate proteolytic processing in the absence of added trypsin.
  • the first eight amino acids of the fusion peptide were deleted ( ⁇ 103-111).
  • a His6-tag (SEQ ID NO: 5) was added downstream of the trimerization domain for purification purposes.
  • a TEV protease cleavage site (SEQ ID NO: 3) for the removal of the foldon if desired and an Xa cleavage site (SEQ ID NO: 4) to remove the His6 tag after purification if desired, for example, for use in humans, and the sequence was codon optimized for expression in CHO cells.
  • the soluble monomeric subunit hMPV F-protein construct (sF hMP vAl-V) is both soluble and monomeric by virtue of truncation of the transmembrane and cytoplasmic tail domains.
  • the construct contains also a G294E substitution for enhanced production and a His6-tag (SEQ ID NO: 5) for purification purposes. (Herfst et al. (2007) Journal of General Virology, 88:2702-2709.)
  • the plasmid pVVS1371 (SEQ ID NO: 28) was designed at Valneva for transient or stable expression of one or, optionally, two proteins of interest in CHO cells.
  • the plasmid contains:
  • CMV cytomegalovirus
  • -two chimeric introns downstream from the CMV promoter, composed of the 5 '-donor site from the first intron of the human ⁇ -globin gene and the branch and 3 '-acceptor sites from the intron of an immunoglobulin gene heavy chain variable region.
  • the sequences of the donor and acceptor sites, along with the branchpoint site, were changed to match the consensus sequences for splicing.
  • the intron is located upstream of the cDNA insert in order to prevent utilization of possible cryptic 5'- donor splice sites within the cDNA sequence,
  • bovine growth hormone polyadenylation signal sequence (bgh-polyA)
  • neomycin phosphotransferase gene from Tn5 under the regulation of the SV40 enhancer and early promoter region.
  • An HSV TK polyadenylation signal based on the highly efficient polyadenylation signal of the thymidine kinase gene of Herpes Virus is located downstream of the neomycin phosphotransferase gene. Expression of the neomycin phosphotransferase gene in mammalian cells confers resistance to the antibiotic G-418,
  • the coding sequences for the post -fusion hMPV F-protein construct sPoFhMPvAl-MFur (polynucleotide sequence provided by SEQ ID NO: 18), the soluble monomeric hMPV F-protein construct sFhMPvAl-V (polypeptide of SEQ ID NO: 45), the prefusion trimeric hMPV F-protein construct sFhMPvAl-K (polypeptide of SEQ ID NO: 46) as well as the coding sequence for human furin (polynucleotide sequence provided by SEQ ID NO: 31) were cloned into pVVS1371 for transient or stable protein expression in ( ⁇ ⁇ cells.
  • the human furin protease for processing F0 into Fl and F2 was provided by cloning a furin coding sequence (SEQ ID NO: 31) into the same plasmid under the EF1 promoter (see below for details).
  • the hMPV F-protein coding sequences for Al-Mfur (SEQ ID NO: 18), Al-V and Al-K were-inserted between the chimeric intron and the bGH A polyadenylation site of pVVS1371 vector using the restriction sites Sail and Pad.
  • vector and synthetic coding sequences for recombinant F- proteins (synthesis done by GeneArt) were double-digested with Sail and Pad followed by purification following separation on an agarose gel.
  • the vector and coding sequence fragments were ligated with T4 DNA ligase and the ligation product was used to transform Max efficiency DH5aTM competent cells. Selected clones were checked for mutation by sequencing.
  • a codon optimized coding sequence for the human furin gene (SEQ ID NO: 31) was inserted into the same plasmid downstream from the recombinant hMPV post-fusion F-protein sequence for co-expression of the two proteins.
  • the expression of the furin gene (accession No.: NP_001276753; encoded by SEQ ID NO: 26) was under the regulation of an EF1 promoter and a bovine growth hormone polyadenylation signal (bgh-polyA).
  • the synthetic gene including a bgh- polyA sequence, the EF1 promoter sequence and the hFUR gene were cloned between the HS4 insulator and the bgh-polyA sequence by using Xball/Pmel restriction sites following the same steps as described previously.
  • the same synthetic gene was cloned between the hMPV F-protein coding sequence and the bgh-polyA sequence using Pacl/Pmel restriction sites following the same steps as described previously.
  • Vero and HeLa cell lines were acquired from ATCC, CHO cells from ECACC and LLC-MK2 cells from HP A Culture Collections. Max EfficiencyTM DH5aTM Competent Cells (ThermoFisher), a chemically-competent E. coli strain, were used for amplification of the plasmids used in this work. Strain Al hMPV was a kind gift from CHU Caen.
  • mice e.g. , C57B1/6 and BALB/c, Janvier were used for immunogenicity studies of the vaccine candidates.
  • Cotton rats for use in hMPV and/or RSV challenge studies were purchased from Sigmovir Biosy stems (Rockville, MD).
  • DS7 Antibody A neutralizing mouse monoclonal antibody that specifically binds to an epitope on hMPV F-protein that is present on both pre- and post-fusion conformations of the MPV F-protein.
  • the DS7 antibody and methods for its production have been described previously (e.g. , Wen, et al., 2012, Structure of the Human Metapneumovirus Fusion Protein with Neutralizing Antibody Identifies a Pneumovirus Antigenic Site. Nat. Struct. Mol. Biol. 19:461-463).
  • the amino acid sequences of the heavy and light variable regions of the DS7 antibody are provided as SEQ ID NOs: 20 and 21, respectively, and are deposited in PDB as Nos.
  • 4DAG_H (DS7 VH) and 4DAG_L (DS7 VL), each of which is incorporated by reference herein as present in the database.
  • the DS7 antibody was manufactured in-house.
  • MPE8 Antibody A neutralizing monoclonal antibody that binds selectively to the prefusion form of F- proteins of both hRSV and hMPV (Corti et al., 2013, Cross-neutralization of four paramyxoviruses by a human monoclonal antibody Nature 501, 439-443).
  • the 704 tetrafunctional non-ionic block copolymers (Nanotaxi®) in non-glycosylated (704) and mannosylated (704-M) form were provided by In-Cell-Art (Nantes, France).
  • Stock solutions of the 704 tetrafunctional non-ionic amphiphilic block copolymers were prepared at 2% in sterile deionized water and stored at 4°C.
  • Formulations of DNA with 704 or 704-M tetrafunctional block copolymers were prepared by mixing equal volumes of tetrafunctional block copolymer working solution at 0.3% in water with plasmid DNA solution at the desired concentration in buffered solutions.
  • Nanotaxi® delivery systems (Nanotaxi®l and Nanotaxi®2, i.e., copolymer 704 without or with mannose; i.e., 704 and 704-M, respectively) were used in the immunization studies based upon their ability to stimulate the innate immune response and to trigger a balanced Thi/Th 2 response.
  • the Nanotaxi® 1 (704) was used in the first immunogenicity study.
  • Each plasmid was separately formulated with each Nanotaxi® prior to injection into mice. Briefly, stock solutions of the 704 tetrafunctional non-ionic amphiphilic block copolymers were prepared at 2% in sterile deionized water and stored at 4°C.
  • Plasmid DNAs were amplified and purified using endotoxin free kit and controlled by enzymatic restriction analysis and concentration measured by optical density.
  • Formulations of DNA with non-glycosylated or glycosylated tetrafunctional block copolymers were prepared by mixing equal volumes of tetrafunctional block copolymer stock solution in water with plasmid DNA solution at the desired concentrations in buffered solutions.
  • the 704 and 704-M working solutions were at a concentration of 0.3% for a final concentration of 0.15%.
  • Intramuscular injections of glycosylated or not tetrafunctional non-ionic block copolymer formulating various amounts of plasmid DNA ranging from 5 ⁇ g to 50 ⁇ g were performed in both anterior tibial muscles of C57B1/6 mice.
  • Formulations of nucleic acids with ICAfectin®441 were prepared by mixing equal volume of DNA solution containing 0 ⁇ g per p24-well plate with ICAfectin®441 as recommended by the provider (In- Cell-Art, France). Twenty four hours prior to transfection HeLa cells were seeded in 24-well culture plates at a density of 55 000 cells per well in 1 mL of complete medium and incubated at 37°C in a humidified 5% CO 2 / 95% air containing atmosphere.
  • Subunit protein production was based on transient transfection of CHO cells using a MaxCyte ® STX Scalable Transfection System device and following experimental recommendations of the supplier. Briefly, prior to electroporation, CHO cells were pelleted, suspended in MaxCyte® electroporation buffer and mixed with corresponding expression plasmid DNA. The cell-DNA mixture was transferred to a cassette processing assembly and loaded onto the MaxCyte® STX Scalable Transfection System. Cells were electroporated using the preprogrammed "CHO" protocol and immediately transferred to culture flasks and incubated for 30 to 40 minutes at 37°C with 8% CO 2 .
  • the production kinetics consisted of decreasing the culture temperature to 32°C and feeding the transfected cells daily with a fed-batch medium developed for transient protein expression in CHO cells (CHO CD EfficientFeedTM (ThermoFisher Scientific), supplemented with yeastolate, glucose and glutaMax). After 7 or 14 days of culture, cell viability was checked and conditioned medium was harvested after cell clarification corresponding to two runs of centrifugation at maximum speed for 10 minutes. Clarified product was 0.2 ⁇ sterile filtered and stored at -80°C before protein purification.
  • the clarified supernatant was brought to room temperature and concentrated 50-60 times the initial volume with a tangential flow system of 50 kDa (Vivaflow 200, Sartorius). Subsequently, it was equilibrated with 50 mM Na 2 HP0 4 buffer at pH 8.0, 300 mM NaCl.
  • IMAC Immobilized metal ion affinity chromatography
  • Immobilized metal ion affinity chromatography For metal ion affinity chromatography (IMAC), agarose resin containing Ni 2+ (His-Select Nickel Affinity Gel, Sigma) was manually packed into chromatography columns. The resin was washed with two volumes of deionized water and equilibrated with three volumes of equilibration and wash buffer (50 mM sodium phosphate, pH 8.0, with 0.3 M sodium chloride and 10 niM imidazole) as indicated by the manufacturer. The sample was loaded onto the column via a peristaltic pump having a flow rate of about 0.8 mL/minute. After all extract was loaded, the column was washed with wash buffer at a flow rate of about 10-20 column volumes/hour.
  • IMAC Immobilized metal ion affinity chromatography
  • the column was washed extensively until the A 280 of the eluate was stable and near that of the wash buffer.
  • the His-tagged protein was eluted from the column using 3-10 column volumes of elution buffer as indicated by the manufacturer (50 mM sodium phosphate, pH 8.0, with 0.3 M sodium chloride and 250 mM imidazole).
  • the fractions (0.5 mL) with the highest absorbance were pooled and concentrated to a volume of 0.5 mL with Amicon Ultra-4 centrifugal filters (Millipore, Merck) having a pore size of 50 kDa.
  • the sPoF hMP vAl-Mfur and sF hMP vAl -V proteins were diluted in sample buffer (0.08 M Tris-HCl at pH 8.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue), boiled for five minutes and electrophoretically separated on polyacrylamide gels (Criterion XT precast gel Bis-Tris 12% 12+2 wells) in the presence of 100 mM dithiothreitol (DTT). The gels were stained for 1 hour in a solution of 0.05% Coomassie blue, 45% methanol and 7% acetic acid in water. The excess stain was removed with 25% methanol and 7% acetic acid in water. Results are shown in Figure 6A.
  • the proteins were electro-transferred to Immobilon- P paper (Millipore) in transfer buffer (25 mM Tris, 192 mM glycine, 0.1% SDS and 20% methanol) for 2 hours at 250 mA. Nonspecific binding sites were blocked for one hour at room temperature or overnight at 4°C with 2% Membrane blocking agent (GE Healthcare) in 0.01% Tween-20/PBS. The membrane was incubated with stirring for one hour at room temperature with antibodies diluted in blocking solution (monoclonal IgGl anti Penta-His (50 ⁇ g/ml; Qiagen, ref. 34660).
  • F-protein in the transfected CHO cells was confirmed by flow cytometry ( Figure 6) by staining with anti-F antibodies MPE8 and DS7. Analysis by flow cytometry was done as follows: Seven days after transfection, cells were washed once in PBS and fixed in 4% paraformaldehyde for 10 minutes at room temperature. After two washes in PBS, cells were permeabilized in BD Perm wash buffer for 15 minutes at room temperature. Then the cells were stained with anti-F primary antibodies in BD Perm wash buffer for one hour at 4°C.
  • mice were bled 5 times: at DO (before and after immunization), D21, D42 and D56 (termination day) or dO, dl4, d28, d42 for the subunit-vaccinated mice. Pooled sera from each group were collected and terminal pools (d56 for Nanotaxi groups; d42 for subunit group) were analyzed in virus neutralization (PRNT) assays as outlined in Table 3 below. The results are shown in Figure 7.
  • PRNT virus neutralization
  • Each FACS plot as shown represents the binding results of sera from individual vaccinated mice at day 0 and day 56 after immunization.
  • DO DO before immunization
  • D14, D28 and D42 terminal day
  • DO DO
  • D21, D42 and D56 terminal day
  • BALs bronchoalveolar lavage
  • 96-well half-area plates were coated with 1 ⁇ g/mL of capture antigen overnight at 4°C in pH 9.6 carbonate -bicarbonate buffer. After washing in PBS 0.05% Tween-20, 1 hour of blocking in PBS/ 0.05% Tween-20/ 5% bovine serum albumin (BSA) at 37°C and another wash step, serially diluted sera (or control DS7 IgG 2A antibody) were added to the plates and incubated for 1 hour at 37°C. Subsequently, plates were washed, incubated for 1 hour at 37°C with secondary antibodies, washed and finally incubated in TMB substrate solution (KPL) for 20 minutes at RT.
  • BSA bovine serum albumin
  • the substrate reaction was stopped by addition of 85% orthophosphoric acid and signal was detected by optical density reading at 450 nm.
  • the capture antigen was Post-Fusion subunit hMPV F- protein (sPoFhMPvAl-Mfur) and the secondary antibodies were anti-mouse IgGi-HRP (horseradish peroxidase; Bio-rad) and anti-mouse IgG 2A -HRP (Abeam) at 1 : 10,000 dilution.
  • the capture antigen was either post-fusion subunit hMPV F-protein (sPoFhMPvAl-Mfur), native monomer hMPV F-protein (sFhMPvAl-V), or prefusion trimer protein (sFhMPvAl-K).
  • the secondary antibody anti- mouse IgG-HRP (Covalab) was used at 1 :5000 dilution.
  • the hyperimmune sera from Balb/c groups immunized two times at three week intervals (d42 sera) with either subunit post-fusion hMPV F-protein or DNA encoding post-fusion hMPV F-protein were compared for their binding ability to different bound antigens in vitro by ELISA.
  • the coating antigens used were a post -fusion trimeric form (sPoFhMPvAl-Mfur; SEQ ID NO: 44), a pre -fusion trimeric form (sF hMP vAl-K; SEQ ID NO: 46) and a native monomeric form of hMPV F-protein (sF hMP vAl-V; SEQ ID NO: 45).
  • the mAb DS7 which binds both pre- and post-fusion forms of hMPV F- protein, was used as a control.
  • C57B1/6 mice were immunized as outlined in Table 5. Briefly, 7 groups of 5 mice (C57B1/6; 6-8 weeks of age) were immunized with 25 ⁇ g of the indicated DNA constructs (single or in combination), to a total of 50 ⁇ g of DNA in combination with 0.15% Nanotaxi ® 704 (Nanotaxi ® l).
  • pVAXl vector comprising DNA encoding sPoF hMP v as described above, as well as three pVAXl constructs containing the coding sequences for three different hRSV F-proteins; soluble pre -fusion hRSV F-protein "SC-DM” (sPrFhRsvSC-DM), soluble pre-fusion hRSV F-protein "DS-Cavl” (sPrFhRsvDS-Cavl) and post-fusion hMPV F-protein (PoFhRsv) were used.
  • mice were bled 4 times: at DO (before immunization), D21, D42 and D56 (termination day). At D56, splenocytes and bronchoalveolar lavage (BALs) cells were collected. All samples from mice were stored at -80°C until analysis. Humoral responses in pooled D56 serum samples were assessed by neutralization of hMPV as outlined in Table 3. Sera collected at DO from group 1 were used as a negative control.
  • Results are shown in Figure 13. The findings indicated that the addition of RSV F-protein encoding vectors did not reduce the immune response to post-fusion hMPV F-protein encoding vectors.
  • hMPV virus isolates are grown on LLC-MK2 cells, banked and used for animal challenge experiments.
  • Cotton rats previously vaccinated as indicated above are challenged intra-nasally on day 28 with 10 5 PFU of the hMPV viruses.
  • a few days later, the animals are sacrificed and individual serum samples are prepared and frozen.
  • Nasal and lung tissues are harvested separately, weighed and either snap frozen in liquid nitrogen and conserved for viral titer determination or fixed with 4% buffered formalin for histopathological examination after paraffin embedding and staining with hematoxylin and eosin.
  • Viral load in nasal and lung tissues is determined by virus foci immunostaining as described above.
  • PCR is used to determine viral load in the harvested tissues.
  • immunogenicity is determined as described for mouse studies (above).
  • hMPV F-protein complex in trimeric post-fusion conformation, wherein said complex consists of three hMPV F-protein heterodimers, and wherein said heterodimer comprises
  • a polypeptide A comprising an immunogenic Fl ectodomain of the hMPV F-protein
  • a polypeptide B comprising an immunogenic F2 domain of the hMPV F-protein
  • the complex according to aspect 1 for use as a prophylactic or therapeutic treatment against a viral infection.
  • a pharmaceutical composition comprising the complex according to aspect 1 for use as a medicament.
  • a pharmaceutical composition comprising the complex according to aspect 1 for use as a prophylactic or therapeutic treatment against a viral infection.
  • the amino acid sequence of SEQ ID NO: 12 and (ii) the immunogenic F2 domain consists of amino acid 20 to 101 of the hMPV F-protein (B l genotype), i.e. amino acid sequence of SEQ ID NO: 13.
  • the complex or composition for use according to aspect 10 wherein the cleavage site B is a TEV protease cleavage site.
  • the complex or composition for use according to aspect 16 wherein the cleavage site C is a factor Xa cleavage site.
  • the complex or composition for use according to aspect 17, wherein the factor Xa cleavage site is SEQ ID NO: 4.
  • heterodimer consists of a polypeptide A with SEQ ID NO: 14 or a functional variant thereof that is more than 80% identical to the polypeptide with SEQ ID NO: 14, and a polypeptide B with SEQ ID NO: 15 or a functional variant thereof that is more than 80% identical to the polypeptide with SEQ ID NO: 15.
  • composition for use according to aspect 22, wherein the hMPV M protein is defined by SEQ ID NO: 24 or SEQ ID NO: 25.
  • composition for use according to any of aspects 3 to 27, wherein the composition is a vaccine comprising the steps of: a. providing a nucleic acid sequence encoding a polypeptide A and polypeptide B as defined in aspects 1 to 20 in a suitable vector;
  • nucleic acid sequence is selected from the group consisting of

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L'invention concerne des constructions et des compositions pharmaceutiques pour le traitement ou la prévention des infections virales.
PCT/EP2018/080424 2017-11-07 2018-11-07 Compositions pharmaceutiques pour le traitement ou la prévention des infections virales WO2019092002A1 (fr)

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RU2811991C2 (ru) * 2019-05-20 2024-01-22 Вальнева Се Субъединичная вакцина для лечения или предотвращения инфекции дыхательных путей
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine
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CN114127101A (zh) * 2019-05-20 2022-03-01 瓦尔尼瓦公司 用于治疗或预防呼吸道感染的亚单位疫苗
GB2598494A (en) * 2019-05-20 2022-03-02 Valneva Se A subunit vaccine for treatment or prevention of a respiratory tract infection
GB2598494B (en) * 2019-05-20 2024-07-24 Valneva Se A subunit vaccine for treatment or prevention of a respiratory tract infection
WO2020234300A1 (fr) * 2019-05-20 2020-11-26 Valneva Se Vaccin sous-unitaire pour le traitement ou la prévention d'une infection des voies respiratoires
RU2811991C2 (ru) * 2019-05-20 2024-01-22 Вальнева Се Субъединичная вакцина для лечения или предотвращения инфекции дыхательных путей
US11964013B2 (en) 2020-02-13 2024-04-23 Institut Pasteur Nucleic acid vaccine against the SARS-CoV-2 coronavirus
WO2021160346A1 (fr) 2020-02-13 2021-08-19 Institut Pasteur Vaccin à base d'acide nucléique contre le coronavirus sars-cov-2
US11759516B2 (en) 2020-02-13 2023-09-19 Institut Pasteur Nucleic acid vaccine against the SARS-CoV-2 coronavirus
US11969467B2 (en) 2020-02-13 2024-04-30 Institut Pasteur Nucleic acid vaccine against the SARS-CoV-2 coronavirus
US11911462B2 (en) 2020-02-13 2024-02-27 Institut Pasteur Nucleic acid vaccine against the SARS-CoV-2 coronavirus
WO2021205017A1 (fr) * 2020-04-09 2021-10-14 Valneva Austria Gmbh Améliorations apportées à des formulations de vaccin à usage médical
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US11547673B1 (en) 2020-04-22 2023-01-10 BioNTech SE Coronavirus vaccine
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US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

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