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WO2019092002A1 - Pharmaceutical compositions for treatment or prevention of viral infections - Google Patents

Pharmaceutical compositions for treatment or prevention of viral infections Download PDF

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Publication number
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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Prior art keywords
seq
protein
nucleic acid
hmpv
pharmaceutical composition
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PCT/EP2018/080424
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French (fr)
Inventor
Bruno Pitard
Melissa HANSON
Mehdi Lahmar
Fabien PERUGI
Klaus Schwamborn
Fabienne Guehenneux
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Valneva SE
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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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Abstract

Described herein are constructs and pharmaceutical compositions for the treatment or prevention of viral infections.

Description

PHARMACEUTICAL COMPOSITIONS FOR TREATMENT OR PREVENTION OF VIRAL
INFECTIONS
FIELD OF THE INVENTION The invention relates to constructs and compositions for use as prophylactic or therapeutic treatments against viral infections.
BACKGROUND OF THE INVENTION Human metapneumovirus (hMPV) 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. 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. (2006) Epidemiology of Human Metapneumovirus. Clinical Microbiol. Reviews 19(3):546-557). Hospitalization rates due to hMPV infection are comparable to those due to other common respiratory viruses such as RSV, parainfluenza virus (PIV) or influenza virus and viral co-infection is common (Tregoning and Schwarze (2010) Respiratory Viral Infections in Infants: Causes, Clinical Symptoms, Virology, and Immunology. Clin. Microbiol. Rev. 23(l):74-98).
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). 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). Similarly, the F-protein from the related Respiratory Syncytial virus (RSV) 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). For several decades, a vaccine has been sought for RSV. In the 1960s, a trial was conducted with alum- adjuvanted formalin-inactivated human RSV (hRSV). Not only did the vaccine not confer protection, it was associated with dramatically enhanced disease in infants upon natural infection with RSV and resulted in two deaths (Kim, et al. (1969) Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am. J. Epidemiol. 89:422-434). With regard to the more recently-discovered hMPV, similar deleterious effects of formalin-inactivated virus were observed in rhesus macaques and in the cotton rat model (Swart, et al , 2007, Immunization of macaques with formalin-inactivated human metapneumovirus induces hypersensitivity to hMPV infection. Vaccine 25:8518-8528 and Yim, et al , 2007, Human metapneumovirus: Enhanced pulmonary disease in cotton rats immunized with formalin -inactivated virus vaccine and challenged. Vaccine 25(27): 5034-5040, respectively). 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.
Although the deleterious effect of the inactivated RSV vaccine was initially hypothesized to be due to effects of inactivation on neutralizing epitopes, it has since been shown that inadequate affinity maturation of antibodies to critical epitopes was likely responsible. More specifically, the Th2-bias of the stimulated immune response may have contributed to the production of non-protective pathogenic antibodies in response to the vaccine (Delgado, et al. (2009) Lack of antibody affinity maturation due to poor Toll stimulation led to enhanced RSV disease. Nat. Med. 15(1):34-41). In this regard, Delgado et al. {supra) provided evidence that adjuvants which stimulate toll-like receptors may shift the immune response to inactivated RSV in a favorable direction; that is, by increasing the relative magnitude of the Thl response. This and other approaches to increasing the Thl/Th2 ratio in response to RSV antigens (and potentially hMPV antigens) may be key to providing a safe and effective vaccine. Several vaccination strategies for hMPV have been investigated in recent years, such as live -attenuated or live-vectored viruses and recombinant or DNA-encoding viral proteins. The successful development of a vaccine to hMPV presents major challenges, however. One is the need to induce strong and long-lasting immune responses which are greater than those induced by naturally-acquired immunity. Additionally, 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.
SUMMARY
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. The 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.
BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are not intended to be drawn to scale. The Figures are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
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 and CT: Cytoplasmic tail. The black points indicate cysteine residues taking part in intermolecular bonds (indicated by "S-S"), the triangles represent N-glycosylation sites; the arrow shows the proteolytic processing site. Also indicated is the Fl ectodomain, defined herein as the Fl domain without the transmembrane region and the cytoplasmic tail. Figure 2 schematically depicts the process of fusion of virus and host -cell membranes, which is mediated by paramyxovirus F-protein. (a) The pre -fusion F-protein trimer is depicted inserted into the viral membrane (via the transmembrane region) with a globular head connected to the transmembrane region through the HRB stalk, (b) Upon activation, the short a-helices of HRA refold into a long trimeric coiled- coil, inserting the fusion peptide of each subunit (encircled) into the host-cell membrane and forming the so-called pre -hairpin intermediate, (c) Collapse of this unstable intermediate draws the two membranes together, (d) Assembly of the six-helix-bundle (6-HB), formed by a core of three HRA a-helices surrounded by three antiparallel HRB α-helices completes the merging of the two membranes, resulting in the formation of the fusion pore and the transformation of the F-protein to the post-fusion conformation. The whole process probably requires the coordinated action of more than one F molecule; thus, two F-protein molecules are represented in the last two steps to emphasize the cooperation. (Figure is adapted from Melero and Mas, The Pneumovirinae fusion (F) protein: A common target for vaccines and antivirals (2015) Virus Research (209): 128-135.)
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.
The polynucleotide constructs for the DNA vaccine preparations as described herein do not encode a
Cleavage Site C or a His6 sequence for purification; these features are present, however, in the construct for in vitro production of the protein subunit. 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. FACS analysis of expression of sFhMPvAl-V and sPoFhMPvAl-MFur following transfection of CHO cells as shown by staining of permeabilized cells by MPE8 and DS7 monoclonal antibodies in flow cytometry. Figure 7 Neutralization capacity of pooled sera from mice vaccinated with DNA constructs encoding either post-fusion or full-length hMPV F-protein or with purified post-fusion hMPV F-protein. Neutralization of the Al hMPV strain is shown. IC50 values were calculated from the obtained curves and shown in the table. Figure 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.
Figure 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.
Figure 10 Comparison of immunogenicity of DNA vaccines and subunit vaccines in C57B1/6 mice by IC50 on day 56 following three immunizations (A) and IgGl titers on day 42 following two immunizations (B).
Figure 11 Comparison of immunogenicity of DNA vaccines and subunit vaccines in Balb/c mice by IC50 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).
Figure 12 Antibodies stimulated in Balb/c mice at day 42 following vaccination (2x at three week intervals) with post-fusion F-protein DNA (50 μg 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 μg sPoFhMPvAl-Mfur + alum) bound preferentially to the post- fusion trimer. 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 IC50 (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. In a preferred embodiment, 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. During viral particle fusion with the host cell, 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.
As used herein, in the 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. Further, the post-fusion I -protein combines to form a homotrimer comprising three F1/F2 heterodimers. 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). When the I -protei n is no longer inserted into the viral envelope via the transmembrane domain, 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. In one embodiment, one disulfide bond is formed between the I · I and I 2 regions of the hMPV post-fusion I -protein. In one embodiment, the one disulfide bond is formed between amino acid residues 60 and 1 82 of the hMPV post-fusion I -protei n. In one embodiment, 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.
In a preferred embodiment, 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):
-deletion of both the TM domain ( I'M ) and the cytoplasmic tail (CT),
-placement of a I F V protease cleavage site (SEQ II ) NO: 3) ( '-terminally to the heptad repeat B ( 1 1 KB ) ("cleavage site B"),
-placement of a trimerization domain ( l ibriti n T4 foldon; SEQ I I ) NO: 6) ( '-termi nal ly to the TEV protease cleavage site,
-deletion of the first nine amino acids of the fusion peptide (Δ103-111) to avoid aggregation of the heterodimers,
-insertion of an RSV furin cleavage site 11 (KKRKRR; SEQ II ) NO: 2) at the beginning of the fusion peptide (aa 102-107) for processing of F0 by co-expressed furin protease. Optionally, the F-protein i n post-fusion conformation as encoded by the nucleic acid of the invention further comprises:
-a serine endopeptidase factor Xa cleavage site (SEQ I I ) NO: 4) added ( '-terminal l to the trimerization domain ("cleavage site C"), -a ( "-termi nal I lis*, tag (SEQ II ) NO: 5) for purification purposes.
In one aspect, 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. As used herein, the terms "protein" and "polypeptide" are interchangeable. In a preferred embodiment, 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 amino acid sequence of SEQ II ) NO: 10 and b) the immunogenic I 2 domain consists of amino acid 20 to 101 of the hMPV I -protein from the A 1 genotype; i.e. the amino acid sequence of SEQ I I ) NO: 11. In a preferred embodiment, 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. amino acid sequence of SEQ I I ) NO: 13. In a further aspect, 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).
In one aspect, 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. In one aspect, 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. In one aspect, the alternative cleavage site is a furin cleavage site. In one aspect, the furin cleavage site is derived from the RSV I -protein. In one aspect, the RSV-de rived furin cleavage site is the RSV cleavage site II defined by SEQ NO: 2. In one aspect, 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. In one aspect, 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. In one aspect, the furin protease is stably expressed by the cell line used for expression of the post-fusion F-protein construct. In one aspect, the furin protease is a human furin protease. In one aspect, the human furin protease is defined by SEQ II ) NO: 26. In one aspect, the coding sequence of the human furin protease is defined by SEQ II ) NO: 31.
In one aspect, the polypeptide A encoded b the nucleic acid of the i nvention additionally comprises a trimerization domain ( '-terminal to the I · 1 ectodomain. In one aspect, 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). In a preferred aspect, the 4 foldon domain is defined by SEQ II ) NO: 6. In one aspect, 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. In a preferred aspect, the additional cleavage site B of polypeptide A is a cleavage site for TEV protease (Tobacco Etch Virus nuclear-inclusion-a endopeptidase). In one embodiment, the TEV protease cleavage site is of the general form EXXYXQ(G/S). In a preferred embodiment, the cleavage sequence for the TEV protease is ENLYFQG as defined by SEQ ID NO: 3.
In one aspect, the polypeptide A encoded by the nucleic acid of the invention additionally comprises a tag at the ( -terminal end of the trimerization domain. In a preferred aspect, the tag is a I lis. tag (SEQ II ) NO: 5). In one aspect, the polypeptide A encoded by the nucleic acid of the invention additionally comprises another cleavage site C between the trimerization domain and the tag. In a preferred aspect, the additional cleavage site C is a cleavage site of the serine endopeptidase factor Xa. In one aspect, the cleavage site of serine endopeptidase factor Xa is of the general form I(E/D)GR. In a preferred aspect, the serine endopeptidase factor Xa cleavage site is IEGR as defined by SEQ I I ) NO: 4.
In one aspect, 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. and a 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.
In one aspect, 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: 17, most preferably 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the polypeptide with SEQ ID NO: 17. In one aspect, 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. most preferably 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the polypeptide with SEQ ID NO: 15.
In one aspect, the current invention provides a vector which comprises the nucleic acid of the invention. In one aspect, 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. In one aspect, 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. In one aspect, 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. In one aspect, said vector optimized for use in DNA vaccines is a first generation DNA vaccine vector such as pVAXl or gWIS. In one aspect, said vector optimized for use i n DNA vaccines is a second-generation DNA vaccine vector. In a preferred aspect, the vector optimized for use in DNA vaccines is the pVAXl vector. In one aspect, the nucleic acid of the invention is comprised in a vector suitable for in vitro expression for subsequent (optional) purification of the encoded polypeptide. In one aspect, 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. In further embodiments, the vector is a viral vector, such as a recombinant viral vector. In one embodiment, the viral vector is a Newcastle Disease Virus ( NDV ). In one aspect, 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. In one aspect, the NDV is a moderate to high virulent strain of NDV, i.e., a mesogenic or velogenic strain, such as, e.g., AF2240. In one aspect, 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. In one aspect, the oncolytic strain is a LaSota strain of NDV. In a preferred aspect, the oncolytic strain is the highly virulent AF2240 strain of NDV. In some embodiments, the viral vector is a vaccinia virus vector. In one aspect the vaccinia virus vector is pRB21. In one aspect, the vector is a baculo virus vector. In a preferred embodiment, the vector suitable for in vitro expression is pVVS 137 1 (as defined by SEQ II ) NO: 29).
In some embodiments, 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. In a preferred embodiment, the host cells used for in vitro expression of the encoded polypeptide are Chinese Hamster Ovary (CHO) cells.
In one aspect, 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. In a further aspect, 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. In a further aspect, 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. In one aspect, 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. In a further aspect, 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. In a preferred aspect, the pharmaceutical composition for use is a vaccine. In a preferred embodiment, the vaccine of the invention is used for the prophylactic or therapeutic treatment of infection with one or more respiratory pathogens, such as viruses.
In one aspect, the disclosure provides a pharmaceutical composition comprising the nucleic acid, the polypeptide encoded by the nucleic acid or the vector of the invention. In one aspect, 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 μg, even more preferably between I μg and 200 μg, most preferably between 10 and 100 μg. 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. These administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory or genitourinary tracts. Although 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.
In one aspect, 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. In a preferred aspect, 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. In a preferred embodiment, the RSV I -proteins or variants thereof are selected from, the group consisting of post-fusion forms, monomelic native forms and profusion forms. In a preferred embodiment, 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 hksvS( '-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. In a preferred aspect, 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). In a preferred aspect, 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. In a preferred aspect, 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. In one aspect, 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. In a preferred embodiment, the pharmaceutically acceptable excipient comprises a nucleic acid delivery reagent. In a preferred embodiment, 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. There is further provided a pharmaceutical composition which is a vaccine, this vaccine may further comprise a pharmaceutically acceptable excipient. In a preferred embodiment, the excipient is a 704 or 704-M tetrafunctional non-ionic amphiphilic block copolymer.
In a preferred aspect, 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. Especially preferred are tetrafunctional non-ionic amphiphilic block copolymers comprising at least one hydrophilic block and at least one hydrophobic block. In a tetrafunctional non-ionic amphiphilic block co-polymer useful for the invention 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. Preferably, 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. Preferably, 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. Preferably, 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):
Figure imgf000016_0001
wherein RA, RB, RC, RD represent independently of one another
R1 R2 R3 R4 R5 R6
[CHCHO] -[CHCHO] i-[CHCHO]j-H
in which - 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,
- for R1 and R2, either (a) both are hydrogen or (b) one is hydrogen and the other is methyl,
- for R3 and R4 either (a) both are hydrogen or (b) one is hydrogen and the other is methyl, and
- if both of R3 and R4 are hydrogen, then one R5 and R6 is hydrogen and the other is methyl, or if one of R3 and R4 is methyl, then both of R5 and R6 are hydrogen.
More preferably, a non-ionic amphiphilic tetrafunctional block copolymer useful for the invention may be of formula (II):
Figure imgf000017_0001
in which
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
for each R1, R2 pair, R1 shall be hydrogen and R2 shall be a methyl group, and
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%.
In a preferred embodiment, 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. Preferably, 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, and 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%.
Further details of suitable 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.
In one embodiment, a non-ionic amphiphilic block copolymer suitable for the invention is selected from the group consisting of tetrafunctional non-ionic amphiphilic block copolymer 304 (defined as i = 4; j = 4), tetrafunctional non -ionic amphiphilic block copolymer 704 (defined as i = 13; j = 14), tetrafunctional non-ionic amphiphilic block copolymer 904 (defined as i = 15; j = 17), and tetrafunctional non-ionic amphiphilic block copolymer 1107 (defined as i = 59; j = 19) or mixtures thereof. In a more preferred embodiment, 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. Especially preferred are tetrafunctional non-ionic amphiphilic block copolymers. In one aspect, 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. In a preferred embodiment, the at least one glycosyl moiety comprises mannose or galactose, preferably mannose. Especially preferred are a 704 tetrafunctional non-ionic amphiphilic block copolymer (also referred to herein as Nanotaxi®l or 704), or a mannose-substituted 704 tetrafunctional non-ionic amphiphilic block copolymer (also referred to herein as Nanotaxi®2 or 704-M), both as described in WO2013128423, which is incorporated herein by reference in its entirety.
In a preferred embodiment, 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. (2015) Adjuvant effect of the human metapneumovirus (HMPV) matrix protein in HMPV subunit vaccines. J. of Gen. Virol. 96:767-774), or any combination of two or more of the above mentioned adjuvants. In a preferred aspect, the pharmaceutical composition comprises one or more of the 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 22), the 26-mer 5'-(dIdC)i3-3' (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. In one aspect, 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. In a preferred aspect, the viral infection is a metapneumovirus infection. In a preferred aspect, the viral infection is a human metapneumovirus infection. In a further aspect, 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. In a preferred aspect, the 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. In a preferred aspect, the pharmaceutical composition of the disclosure is useful in the prevention or treatment of RSV.
In one aspect, 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. In a preferred embodiment, 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. In a preferred embodiment, 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. In a preferred aspect, 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.
SEQUENCES
SEQID NO: 1
Native cleavage site for proteolytic processing of hMPV F-protein (trypsin cleavage site; amino acids 99-102) RQSR
SEQID NO: 2
Cleavage site II from RSV F-protein (furin cleavage site; amino acids 131-136)
KKRKRR SEQID NO: 3
Cleavage sequence forTEV protease
ENLYFQG
SEQID NO: 4
Cleavage sequence for serine endopeptidase factor Xa
IEGR
SEQID NO: 5 HHHHHH
SEQID NO: 6
Foldon domain of theT4 bacteriophage fibritin (Accession number: 1RF0_A)
GYIPEAPRDGQAYVRKDGEWVLLSTFL
SEQID NO: 7
F-protein of the Al hMPV isolate "NL/1/00" (Accession No.: AAK62968)
MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVG DVENLTCADGPSLIKTELDLTKSALRELRTVSADQ LAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTR AINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGF LIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIN VAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSK VEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSTMILVSVFIIIKKTKK PTGAPPELSGVTNNGFIPHN
SEQID NO: 8
F-protein of the Bl hMPV isolate "NL/1/99" (Accession No.: AAQ90145)
MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQ LAREEQIENPRQSRFVLGAIALGVATAAAVTAGIAIAKTIRLESEVNAIKGALKQTNEAVSTLGNGVRVLATAVRELKEFVSKNLTS AINRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGIL IGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTAAGINV AEQSRECNIN ISTTN YPCKVSTG RH PISMVALSPLGALVACYKGVSCSIGSNWVG 11 KQLPKGCSYITNQDADTVTI DNTVYQLSK VEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAEKGNTGFIIVVILVAVLGLTMISVSIIIIIKKTRK PTGAPPELNGVTNGGFIPHS
SEQID NO: 9
Fl ectodomain of the Al post-fusion hMPV F-protein heterodimer (aa 112-489)
VATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYMVQLPIFGVIDTP CWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVST GRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKF PEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNT
SEQID NO: 10
Fl ectodomain of the Al strain post-fusion hMPV F-protein heterodimer with a G294E mutation (aa 112-489) VATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYMVQLPIFGVIDTP CWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVST GRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKF PEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNT
SEQID NO: 11
F2 domain of the Al strain post-fusion heterodimer (aa 20-101)
KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQS SEQID NO: 12
Fl ectodomain of the Bl post-fusion hMPV F-protein heterodimer (aa 112-489)
VATAAAVTAGIAIAKTIRLESEVNAIKGALKQTNEAVSTLGNGVRVLATAVRELKEFVSKNLTSAINRNKCDIADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTP CWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTG RHPISMVALSPLGALVACYKGVSCSIGSNWVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPE DQFNVALDQVFESIENSQALVDQSNKILNSAEKGNT
SEQID NO: 13
F2 domain of the Bl post-fusion hMPV F-protein heterodimer (aa 20-101)
KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQS
SEQID NO: 14
Polypeptide A (Al strain) (with G294E mutation)
VATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYMVQLPIFGVIDTP CWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVST GRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKF PEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTSGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEG RHHHHHH
SEQID NO: 15
Polypeptide B (Al strain)
KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQSKKRKRR SEQID NO: 16
Polypeptide A (Bl strain)
VATAAAVTAGIAIAKTIRLESEVNAIKGALKQTNEAVSTLGNGVRVLATAVRELKEFVSKNLTSAINRNKCDIADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTP CWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTG RHPISMVALSPLGALVACYKGVSCSIGSNWVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPE DQFNVALDQVFESIENSQALVDQSNKILNSAEKGNTSGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR HHHHHH
SEQID NO: 17
Polypeptide B (Bl strain)
KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSKKRKRR SEQ ID NO: 18
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
GGTACCGTCGACGCTAGCGAATTCGCCGCCACCATGTCCTGGAAGGTCGTGATCATCTTCTCCCTGCTGATCACCCCCCAG CACGGCCTGAAAGAGTCCTACCTGGAAGAGAGCTGCTCCACCATCACCGAGGGCTACCTGTCTGTGCTGCGGACCGGCTG GTACACCAACGTGTTCACCCTGGAAGTGGGCGACGTGGAAAACCTGACCTGCGCCGATGGCCCCAGCCTGATCAAGACCG AGCTGGACCTGACCAAGTCCGCCCTGCGGGAACTGAGAACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGA GAACCCCCGGCAGTCCAAGAAACGGAAGCGGAGAGTGGCCACCGCCGCTGCTGTGACAGCTGGCGTGGCCATTGCCAAG ACCATCCGGCTGGAATCCGAAGTGACCGCCATCAAGAACGCCCTGAAAAAGACCAACGAGGCCGTGTCTACCCTGGGCAA TGGCGTGCGAGTGCTGGCTACAGCTGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACCCGGGCCATCAACAAG AACAAGTGTGATATCGCCGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTCCTGAACGTCGTGCGGCA GTTCTCTGACAACGCCGGCATCACCCCTGCCATCTCCCTGGATCTGATGACCGACGCCGAGCTGGCTAGAGCCGTGTCCAA CATGCCTACCTCTGCCGGCCAGATCAAGCTGATGCTGGAAAACCGGGCCATGGTGCGACGGAAGGGCTTCGGCTTTCTGA TCGGCGTGTACGGCTCCTCCGTGATCTACATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACCCCCTGCTGGATCGTGA AGGCCGCTCCTAGCTGCTCCGAGAAGAAGGGCAACTACGCCTGCCTGCTGAGAGAGGACCAGGGCTGGTACTGTCAGAA CGCCGGCTCCACCGTGTACTACCCCAACGAGAAGGACTGCGAGACACGGGGCGACCACGTGTTCTGTGATACCGCTGCTG GCATCAACGTGGCCGAGCAGTCCAAAGAGTGCAACATCAACATCTCCACCACCAACTACCCCTGCAAGGTGTCCACCGGCA GGCACCCCATCTCTATGGTGGCCCTGTCTCCTCTGGGCGCCCTGGTGGCTTGTTACAAGGGCGTGTCCTGCTCCATCGGCT CCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGACACCGTGACCATC GACAATACCGTGTATCAGCTGTCCAAGGTGGAAGGCGAGCAGCACGTGATCAAGGGCAGACCCGTGTCCTCCAGCTTCGA CCCCGTGAAGTTCCCCGAGGATCAGTTCAATGTGGCCCTGGACCAGGTGTTCGAGTCCATCGAGAACTCCCAGGCTCTGGT GGACCAGTCCAACCGGATCCTGTCCTCTGCCGAGAAGGGAAACACCTCCGGCAGAGAGAACCTGTATTTTCAAGGCGGCG GAGGCTCCGGCTACATCCCTGAGGCTCCTAGAGATGGCCAGGCCTACGTGCGGAAGGATGGCGAATGGGTGCTGCTGTC CACCTTCCTGGGCGGCATCGAGGGCAGACACCACCATCATCACCACTGATGATGACCATGGTTAATTAAGTTTAAAC SEQ I D NO: 19
Coding sequence for the Bl strain hM PV post-fusion F-protein heterodimer derived from isolate "N L/1/99" and codon optimized (with His-tag and other modifications as described herein, also shown in Fig. 4B)
GGTACCGTCGACGCTAGCGAATTCGCCGCCACCATGTCCTGGAAAGTGATGATCATTATCTCCCTGCTGATCACCCCCCAG CACGGCCTGAAAGAGTCCTACCTGGAAGAGAGCTGCTCCACCATCACCGAGGGCTACCTGTCTGTGCTGCGGACCGGCTG GTACACCAACGTGTTCACCCTGGAAGTGGGCGACGTGGAAAACCTGACCTGCACCGATGGCCCCAGCCTGATCAAGACCG AGCTGGACCTGACCAAGTCCGCCCTGCGCGAGCTGAAAACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGA GAACCCCCGGCAGTCCAAGAAACGGAAGCGGAGAGTGGCCACCGCCGCTGCTGTGACAGCTGGAATCGCTATCGCCAAG ACCATCCGGCTGGAATCCGAAGTGAACGCCATCAAGGGCGCTCTGAAGCAGACCAACGAGGCCGTGTCTACCCTGGGCAA TGGCGTGCGAGTGCTGGCTACAGCTGTGCGGGAACTGAAAGAATTCGTGTCCAAGAACCTGACCAGCGCCATCAACCGG AACAAGTGTGATATCGCCGACCTGAAGATGGCCGTGTCCTTCAGCCAGTTCAACCGGCGGTTCCTGAACGTCGTGCGGCA GTTCTCTGACAACGCCGGCATCACCCCTGCCATCTCCCTGGATCTGATGACCGACGCCGAGCTGGCTAGAGCCGTGTCTTA CATGCCTACCTCTGCCGGCCAGATCAAGCTGATGCTGGAAAACCGGGCCATGGTGCGACGGAAGGGCTTCGGCATCCTGA TCGGCGTGTACGGCTCCTCCGTGATCTACATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACCCCCTGCTGGATTATCAA GGCCGCTCCCAGCTGCTCCGAGAAGAACGGCAACTACGCCTGCCTGCTGAGAGAGGACCAGGGCTGGTACTGCAAGAAC GCCGGCTCCACCGTGTACTACCCCAACGAGAAGGACTGCGAGACACGGGGCGACCACGTGTTCTGTGATACCGCTGCTGG CATCAACGTGGCCGAGCAGTCCAGAGAGTGCAACATCAACATCTCCACCACCAACTACCCCTGCAAGGTGTCCACCGGCA GGCACCCCATCTCTATGGTGGCCCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTACAAGGGCGTGTCCTGCTCCATCGGCT CCAACTGGGTGGGAATCATCAAGCAGCTGCCCAAGGGCTGCAGCTACATCACCAACCAGGACGCCGACACCGTGACCATC GACAATACCGTGTATCAGCTGTCCAAGGTGGAAGGCGAGCAGCACGTGATCAAGGGCAGACCCGTGTCCAGCTCCTTCGA CCCCATCAAGTTCCCCGAGGATCAGTTCAATGTGGCCCTGGACCAGGTGTTCGAGTCCATCGAGAACTCCCAGGCTCTGGT GGACCAGTCCAACAAGATCCTGAACTCCGCCGAGAAGGGCAACACCTCCGGCAGAGAGAACCTGTATTTTCAAGGCGGCG GAGGCTCCGGCTACATCCCTGAGGCTCCTAGAGATGGCCAGGCCTACGTGCGGAAGGATGGCGAATGGGTGCTGCTGTC CACCTTCCTGGGCGGCATCGAGGGCAGACACCACCATCATCACCACTGATGATGACCATGGTTAATTAAGTTTAAAC SEQ I D NO: 20
DS7 VH - heavy chain variable region of DS7 monoclonal antibody specific to hM PV F-protein EVQLLESGGGLVQPGGSRRLSCAASGFTVSSSYMSWVRQTPGKGLEWISVFYSGGTTYYADAVKGRFSISMDTSKNTLHLQMN SLRVEDTAIYYCARVLSRASGMPDAFDIWGPGTMVTVSS
SEQID NO: 21
DS7 VL- light chain variable region of DS7 monoclonal antibody specific to hMPV F-protein
ELALIQPASVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQDSERPSGIPERFSGSNSGNTATLTISGTQAMDEADY YCQAWDSSTAVFGGGTTLTVLGQ
SEQID NO: 22
KLK peptide
KLKLLLLLKLK
SEQID NO: 23
5'-(dldC -3'
dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC
SEQID NO: 24
Matrix protein from the Al hMPV isolate "NL/1/00" (Accession No.: AAK62969)
MESYLVDTYQGIPYTAAVQVDLIEKDLLPASLTIWFPLFQANTPPAVLLDQLKTLTITTLYAASQNGPILKVNASAQGAAMSVLPK KFEVNATVALDEYSKLEFDKLTVCEVKTVYLTTMKPYGMVSKFVSSAKSVGKKTHDLIALCDFMDLEKNTPVTIPAFIKSVSIKESE SATVEAAISSEADQALTQAKIAPYAGLIMIMTMNNPKGIFKKLGAGTQVIVELGAYVQAESISKICKTWSHQGTRYVLKSR
SEQID NO: 25
Matrix protein from the Bl hMPV isolate "NL/1/99" (Accession No.: AAS92881)
MESYLVDTYQGIPYTAAVQVDLVEKDLLPASLTIWFPLFQANTPPAVLLDQLKTLTITTLYAASQNGPILKVNASAQGAAMSVLP KKFEVNATVALDEYSKLDFDKLTVCDVKTVYLTTMKPYGMVSKFVSSAKSVGKKTHDLIALCDFMDLEKNIPVTIPAFIKSVSIKES ESATVEAAISSEADQALTQAKIAPYAGLIMIMTMNNPKGIFKKLGAGTQVIVELGAYVQAESISRICKSWSHQGTRYVLKSR
SEQID NO: 26
Human furin (Accession No.: NP_001276753)
MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGFLNLGQIFGDYYHFWHRGVTKRSLSPHR PRHSRLQREPQVQWLEQQVAKRRTKRDVYQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPD LAGNYDPGASFDVNDQDPDPQPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGVRMLDGEVTDAVEARSLG LNPNHIHIYSASWGPEDDGKTVDGPARLAEEAFFRGVSQGRGGLGSIFVWASGNGGREHDSCNCDGYTNSIYTLSISSATQFG NVPWYSEACSSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIALTLEANKNLTWRDMQHLVVQTSKPAHLNA NDWATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTVAPQRKCIIDILTEPKDIGKRLEVRKTVTACLGEPNHITRLEHAQARL TLSYNRRGDLAIHLVSPMGTRSTLLAARPHDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVLYGTA PEGLPVPPESSGCKTLTSSQACVVCEEGFSLHQKSCVQHCPPGFAPQVLDTHYSTENDVETIRASVCAPCHASCATCQGPALTDC LSCPSHASLDPVEQTCSRQSQSSRESPPQQQPPRLPPEVEAGQRLRAGLLPSHLPEVVAGLSCAFIVLVFVTVFLVLQLRSGFSFR GVKVYTMDRGUSYKGLPPEAWQEECPSDSEEDEGRGERTAFIKDQSAL
SEQID NO: 27
Coding sequence for the Al strain hMPV post-fusion F-protein heterodimer (not containing a G294E mutation)
CGCGAATTCGGGATGTCTTGGAAAGTGGTGATCATTTTTTCATTGTTAATAACACCTCAACACGGTCTTAAAGAGAGCTACT
TAGAAGAGTCATGTAGCACTATAACTGAAGGATATCTCAGTGTTCTGAGGACAGGTTGGTACACCAATGTTTTTACACTGG AGGTAGGCGATGTAGAGAACCTTACATGTGCCGATGGACCCAGCTTAATAAAAACAGAATTAGACCTGACCAAAAGTGCA CTAAGAGAGCTCAGAACAGTTTCTGCTGATCAACTGGCAAGAGAGGAGCAAATTGAAAATCCCAGACAATCTAAGAAGAG GAAGAGAAGAGTTGCAACTGCAGCTGCAGTTACAGCAGGTGTTGCAATTGCCAAAACCATCCGGCTTGAAAGTGAAGTAA CAGCAATTAAGAATGCCCTCAAAAAGACCAATGAAGCAGTATCTACATTGGGGAATGGAGTTCGTGTGTTGGCAACTGCA GTGAGAGAGCTGAAAGATTTTGTGAGCAAGAATCTAACACGTGCAATCAACAAAAACAAGTGCGACATTGCTGACCTGAA AATGGCCGTTAGCTTCAGTCAATTCAACAGAAGGTTCCTAAATGTTGTGCGGCAATTTTCAGACAACGCTGGAATAACACC AGCAATATCTTTGGACTTAATGACAGATGCTGAACTAGCCAGAGCTGTTTCCAACATGCCAACATCTGCAGGACAAATAAA ACTGATGTTGGAGAACCGTGCAATGGTAAGAAGAAAAGGGTTCGGATTCCTGATAGGAGTTTACGGAAGCTCCGTAATTT ACATGGTGCAACTGCCAATCTTTGGGGTTATAGACACGCCTTGCTGGATAGTAAAAGCAGCCCCTTCTTGTTCAGGAAAAA AGGGAAACTATGCTTGCCTCTTAAGAGAAGACCAAGGATGGTATTGTCAAAATGCAGGGTCAACTGTTTACTACCCAAATG AAAAAGACTGTGAAACAAGAGGAGACCATGTCTTTTGCGACACAGCAGCAGGAATCAATGTTGCTGAGCAGTCAAAGGA GTGCAACATAAACATATCTACTACTAATTACCCATGCAAAGTTAGCACAGGAAGACATCCTATCAGTATGGTTGCACTATCT CCTCTTGGGGCTTTGGTTGCTTGCTACAAGGGAGTGAGCTGTTCCATTGGCAGCAACAGAGTAGGGATCATCAAGCAACT GAACAAAGGCTGCTCTTATATAACCAACCAAGACGCAGACACAGTGACAATAGACAACACTGTATACCAGCTAAGCAAAG TTGAAGGCGAACAGCATGTTATAAAAGGAAGGCCAGTGTCAAGCAGCTTTGACCCAGTCAAGTTTCCTGAAGATCAATTC AATGTTGCACTTGACCAAGTTTTCGAGAGCATTGAGAACAGTCAGGCCTTGGTGGATCAATCAAACAGAATCCTAAGCAGT GCAGAGAAAGGAAACACTTCTGGTCGTGAAAACTTATATTTCCAAGGTGGTGGTGGCAGCGGCTATATTCCGGAAGCGCC GCGCGATGGCCAGGCGTATGTGCGCAAAGATGGCGAATGGGTGCTGCTGAGCACCTTTCTGGGAGGCATAGAAGGAAG ACACCACCATCATCATCATTGATTCATCATTGTAATAATTCTAATTGCTGTCCTTGGCTCTACCATGATCCTAGTGAGTGTTTT TATCATAATAAAGAAAACAAAGAAACCCACAGGAGCACCTCCAGAGCTGAGTGGTGTCACAAACAATGGCTTCATACCAC ATAATTAG CCATG GCGT
SEQ ID NO: 28
pVAXl™ plasmid for DNA vaccine development (Invitrogen)
GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCC CCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTA TTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCT ATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCAC CCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTG ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTA CTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTC GGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAA ACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGA AGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGG GGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTAT GGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGC CCTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGA TGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTA TGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGT CAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTT CCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATC TCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGG CTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAG GATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCG AGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACT GTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGA ATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAG TTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACA GGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGA GACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCT TTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGA TCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCC GGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTA GCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGA AAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTG ATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTT TG CTCACATGTTCTT
SEQ ID NO: 29
Expression plasmid pVVS1371 (Valneva)
GAGCTCACGGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCC GCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGA AGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAA AGCTTTAGGCTGAAAGAGAGATTTAGAATGACAGAATCATAGAACGGCCTGGGTTGCAAAGGAGCACAGTGCTCATCCA GATCCAACCCCCTGCTATGTGCAGGGTCATCAACCAGCAGCCCAGGCTGCCCAGAGCCACATCCAGCCTGGCCTTGAATGC CTGCAGGGATGGGGCATCCACAGCCTCCTTGGGCAACCTGTTCAGTGCGTCACCACCCTCTGGGGGAAAAACTGCCTCCTC ATATCCAACCCAAACCTCCCCTGTCTCAGTGTAAAGCCATTCCCCCTTGTCCTATCAAGGGGGAGTTTGCTGTGACATTGTT GGTCTGGGGTGACACATGTTTGCCAATTCAGTGCATCACGGAGAGGCAGATCTTGGGGATAAGGAAGTGCAGGACAGCA TGGACGTGGGACATGCAGGTGTTGAGGGCTCTGGGACACTCTCCAAGTCACAGCGTTCAGAACAGCCTTAAGGATAAGA AGATAGGATAGAAGGACAAAGAGCAAGTTAAAACCCAGCATGGAGAGGAGCACAAAAAGGCCACAGACACTGCTGGTC CCTGTGTCTGAGCCTGCATGTTTGATGGTGTCTGGATGCAAGCAGAAGGGGTGGAAGAGCTTGCCTGGAGAGATACAGCT GGGTCAGTAGGACTGGGACAGGCAGCTGGAGAATTGCCATGTAGATGTTCATACAATCGTCAAATCATGAAGGCTGGAA AAGCCCTCCAAGATCCCCAAGACCAACCCCAACCCACCCACCGTGCCCACTGGCCATGTCCCTCAGTGCCACATCCCCACAG TTCTTCATCACCTCCAGGGACGGTGACCCCCCCACCTCCGTGGGCAGCTGTGCCACTGCAGCACCGCTCTTTGGAGAAGGT AAATCTTGCTAAATCCAGCCCGACCCTCCCCTGGCACAACGTAAGGCCATTATCTCTCATCCAACTCCAGGACGGAGTCAGT GAGAATATTGGATCCGGCGCGCCAGATCTGTCTAGACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGG TGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATG ACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGT CATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAG TCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCC CCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCCTCTCTTAAGGTAGCGGCCGCT GATCACAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGGCCAATAGAAACTGGGCTTGTCGAGACAGAGAAGATT CTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGGAAACTTGTCGACGCCGCCA CCATGGTATCCACCTCTCAGCTGCTGGGCCTGCTGCTGTTCTGGACCTCTGCCTCCAGATGCGACATCGTGATGACCCAGA GCCCCGCCACCCTGTCTGTGACCCCTGGCGATAGAGTGTCCCTGTCCTGCAGAGCCTCCCAGACCATCTCCGACTACCTGCA CTGGTACCAACAGAAGTCCCACGAGAGCCCTCGGCTGCTGATCAAGTTCGCCTCCCAGTCTATCAGCGGCATCCCCTCCAG ATTCTCCGGCAGCGGCTCTGGCTCTGACTTCACCCTGTCCATCAACTCCGTGGAACCCGAGGACGTGGGCGTGTACTACTG CCAGAACGGCCACGGCTTCCCCAGAACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCTCCG TGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACAGCCTCCGTCGTGTGCCTGCTGAACAACTTTTACCCCCG CGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAAAGCGTCACCGAGCAGGACTCC AAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAA GTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGATGATTAATTAAGATAAACCC GCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGG TGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGA TTTATTGGATCCGGCGCGCCAGATCTGTCTAGACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGG GTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGA CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA GTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGG TAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATC GCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCAT TGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCCTCTCTTAAGGTAGCGGCCGCTGATC ACAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGGCCAATAGAAACTGGGCTTGTCGAGACAGAGAAGATTCTTG CGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGGAAACTTGGTACCGCCGCCACCAT GAACTTCGGCCTGTCCCTGATCTTCCTGGCCCTGATCCTGAAGGGCGTGCAGTGCGAAGTGCAGCTGGTGGAATCTGGCG GCGACCTCGTGAAGCCTGGCGGCTCTCTGAAGCTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCGGCTACGGAATGTCCT GGGTGCGACAGACCCCTGACAAGCGGCTGGAATGGGTGGCCACAATCACCTCCGGCGGCACCTACACCTACTACCCCGAC TCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATCGACTCCCTGAAGTCCGA GGACACCGCCATCTACTTTTGCGCCAGATCCCTGGCCGGCAACGCCATGGATTATTGGGGCCAGGGCACCTCCGTGACCGT GTCCTCTGCTAGCACCAAGGGACCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCCACCTCTGGCGGCACAGCCGCCCT GGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGAGTGCATA CCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCTCCAGCCTGGGCACCCA GACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTCGAGCCCAAGTCCTGCGACAAGA CCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACAC CCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCATAATGCCAAGACCAAGCCCAGAGAGGAACAGTACAACTCCACCTACCGGGTGGT GTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCA GCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCCCAGGTGTACACACTGCCCCCTAGCCGGGA GGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAAGGCTTCTACCCCTCCGACATTGCCGTGGAATGGGAGT CCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGC TGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTAC ACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGC CAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGAT TGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGATTTAAATTCGGTGACATGTGAGCAAAAGGCCA GCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAA ATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTG CGCTCTCCTGTTCCGACCCTGCCGCTTACGGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCT CACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAG AACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAAC CACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT CTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCCGTCCTCGAGGTGGCACTT TTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACC CTGGTAAATGCTTCAATAATATTGAAAAAGGAAGAGTCCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGG GTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGC CCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATG CAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAA GATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGG TGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAG GGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTG GCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGC GAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGTCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCG GCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGG AAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAA GGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCC GCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTG CTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCT TCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATC ACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCT CCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCTAGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCG GAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGTGTTGGGCTCGAGGCATTTATCAGGGTTC GTCTCGTCCCGGTCTCCTCCCATGCATGGGATCCGGCGCGCCAGATCT SEQ ID NO: 30
Polypeptide A (Al strain) without G294E mutation VATAAAVTAGVAIAKTI RLESEVTAIKNALKKTN EAVSTLGNGVRVLATAVRELKDFVSKN LTRAI N KN KCDIADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN M PTSAGQI KLMLEN RAMVRRKGFGFLIGVYGSSVIYMVQLPIFGVI DTP CWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPN EKDCETRGDHVFCDTAAGI NVAEQSKECN I N ISTTNYPCKVST GRH PISMVALSPLGALVACYKGVSCSIGSN RVGI IKQLN KGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKF PEDQFNVALDQVFESI ENSQALVDQSN RILSSAEKGNTSGREN LYFQGGGGSGYI PEAPRDGQAYVRKDGEWVLLSTFLGGI EG RHH H HH H
SEQ ID NO: 31
CDS 381-2765 of the Homo sapiens Furin, Transcript variant 3, mRNA (N M_001289824.1)
ATGGAGCTGAGGCCCTGGTTGCTATGGGTGGTAGCAGCAACAGGAACCTTGGTCCTGCTAGCAGCTGATGCTCAGGGCCA GAAGGTCTTCACCAACACGTGGGCTGTGCGCATCCCTGGAGGCCCAGCGGTGGCCAACAGTGTGGCACGGAAGCATGGG TTCCTCAACCTGGGCCAGATCTTCGGGGACTATTACCACTTCTGGCATCGAGGAGTGACGAAGCGGTCCCTGTCGCCTCAC CGCCCGCGGCACAGCCGGCTGCAGAGGGAGCCTCAAGTACAGTGGCTGGAACAGCAGGTGGCAAAGCGACGGACTAAA CGGGACGTGTACCAGGAGCCCACAGACCCCAAGTTTCCTCAGCAGTGGTACCTGTCTGGTGTCACTCAGCGGGACCTGAA TGTGAAGGCGGCCTGGGCGCAGGGCTACACAGGGCACGGCATTGTGGTCTCCATTCTGGACGATGGCATCGAGAAGAAC CACCCGGACTTGGCAGGCAATTATGATCCTGGGGCCAGTTTTGATGTCAATGACCAGGACCCTGACCCCCAGCCTCGGTAC ACACAGATGAATGACAACAGGCACGGCACACGGTGTGCGGGGGAAGTGGCTGCGGTGGCCAACAACGGTGTCTGTGGT GTAGGTGTGGCCTACAACGCCCGCATTGGAGGGGTGCGCATGCTGGATGGCGAGGTGACAGATGCAGTGGAGGCACGC TCGCTGGGCCTGAACCCCAACCACATCCACATCTACAGTGCCAGCTGGGGCCCCGAGGATGACGGCAAGACAGTGGATGG GCCAGCCCGCCTCGCCGAGGAGGCCTTCTTCCGTGGGGTTAGCCAGGGCCGAGGGGGGCTGGGCTCCATCTTTGTCTGG GCCTCGGGGAACGGGGGCCGGGAACATGACAGCTGCAACTGCGACGGCTACACCAACAGTATCTACACGCTGTCCATCA GCAGCGCCACGCAGTTTGGCAACGTGCCGTGGTACAGCGAGGCCTGCTCGTCCACACTGGCCACGACCTACAGCAGTGGC AACCAGAATGAGAAGCAGATCGTGACGACTGACTTGCGGCAGAAGTGCACGGAGTCTCACACGGGCACCTCAGCCTCTGC CCCCTTAGCAGCCGGCATCATTGCTCTCACCCTGGAGGCCAATAAGAACCTCACATGGCGGGACATGCAACACCTGGTGGT ACAGACCTCGAAGCCAGCCCACCTCAATGCCAACGACTGGGCCACCAATGGTGTGGGCCGGAAAGTGAGCCACTCATATG GCTACGGGCTTTTGGACGCAGGCGCCATGGTGGCCCTGGCCCAGAATTGGACCACAGTGGCCCCCCAGCGGAAGTGCATC ATCGACATCCTCACCGAGCCCAAAGACATCGGGAAACGGCTCGAGGTGCGGAAGACCGTGACCGCGTGCCTGGGCGAGC CCAACCACATCACTCGGCTGGAGCACGCTCAGGCGCGGCTCACCCTGTCCTATAATCGCCGTGGCGACCTGGCCATCCACC TGGTCAGCCCCATGGGCACCCGCTCCACCCTGCTGGCAGCCAGGCCACATGACTACTCCGCAGATGGGTTTAATGACTGG GCCTTCATGACAACTCATTCCTGGGATGAGGATCCCTCTGGCGAGTGGGTCCTAGAGATTGAAAACACCAGCGAAGCCAA CAACTATGGGACGCTGACCAAGTTCACCCTCGTACTCTATGGCACCGCCCCTGAGGGGCTGCCCGTACCTCCAGAAAGCAG TGGCTGCAAGACCCTCACGTCCAGTCAGGCCTGTGTGGTGTGCGAGGAAGGCTTCTCCCTGCACCAGAAGAGCTGTGTCC AGCACTGCCCTCCAGGGTTCGCCCCCCAAGTCCTCGATACGCACTATAGCACCGAGAATGACGTGGAGACCATCCGGGCC AGCGTCTGCGCCCCCTGCCACGCCTCATGTGCCACATGCCAGGGGCCGGCCCTGACAGACTGCCTCAGCTGCCCCAGCCAC GCCTCCTTGGACCCTGTGGAGCAGACTTGCTCCCGGCAAAGCCAGAGCAGCCGAGAGTCCCCGCCACAGCAGCAGCCACC TCGGCTGCCCCCGGAGGTGGAGGCGGGGCAACGGCTGCGGGCAGGGCTGCTGCCCTCACACCTGCCTGAGGTGGTGGCC G G CCTCAG CTG CG CCTTCATCGTG CTG GTCTTCGTCACTGTCTTCCTG GTCCTG CAG CTG CG CTCTG G CTTTAGTTTTCG G G GGGTGAAGGTGTACACCATGGACCGTGGCCTCATCTCCTACAAGGGGCTGCCCCCTGAAGCCTGGCAGGAGGAGTGCCC GTCTGACTCAGAAGAGGACGAGGGCCGGGGCGAGAGGACCGCCTTTATCAAAGACCAGAGCGCCCTCTGA SEQ ID NO: 32
PoFhRsv: hRSV post-fusion F-Protein for DNA vaccine
With three naturally occurring substitutions in the ectodomain (P102A, 1379V, and M447V) and deletion of amino acids 137-146 of the fusion peptide
M ELLILKANAITTI LTAVTFCFASGQN ITEEFYQSTCSAVSKGYLSALRTGWYTSVITI ELSN I KEN KCNGTDAKVKLI KQELDKYKNAVTE LQLLMQSTPATN N RARRELPRFM NYTLN NAKKTNVTLSKKRKRRSAIASGVAVSKVLH LEGEVN KI KSALLSTN KAVVSLSNGVSVLT SKVLDLKNYIDKQLLPIVN KQSCSISN IETVI EFQQKN N RLLEITREFSVNAGVTTPVSTYM LTNSELLSLI N DM PITN DQKKLMSN NVQ IVRQQSYSI MSI IKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSN RVFC DTMNSLTLPSEVN LCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASN KN RGII KTFSNGCDYVSN KGVDTVSVGNTL YYVN KQEGKSLYVKGEPII N FYDPLVFPSDEFDASISQVN EKI NQSLAFIRKSDELLH NVNAGKSTTN
SEQ ID NO: 33 PoFhRsv: hRSV post-fusion F-Protein coding sequence human codon optimized for DNA
vaccineATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACAATCCTGACCGCCGTGACCTTTTGCTTCGCCAGCGGCCAG AACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGATATCTGTCTGCCCTGAGAACCGGCTGGTACAC CAGCGTGATCACCATCGAGCTGAGCAACATCAAAGAAAACAAGTGCAACGGCACCGACGCCAAAGTGAAGCTGATCAAGCAA GAGCTGGACAAGTACAAGAATGCCGTGACCGAACTGCAGCTGCTGATGCAGTCTACCCCTGCCACCAACAACCGGGCCAGAA GAGAACTGCCCAGATTCATGAACTACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGAGAAG ATCTGCCATTGCCAGCGGCGTGGCCGTGTCTAAAGTTCTGCATCTGGAAGGCGAAGTGAACAAGATCAAGAGCGCCCTGCTG AGCACCAACAAGGCCGTGGTTTCTCTGAGCAATGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGACCTGAAGAACTACATCG ACAAACAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCAGCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAA CAACCGGCTGCTGGAAATCACCCGCGAGTTTTCTGTGAATGCCGGCGTGACCACACCTGTGTCCACCTACATGCTGACCAACA GCGAGCTGCTGTCCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCG GCAGCAGAGCTACTCCATCATGAGCATTATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCTCTGTATGGCGTGATCG ATACCCCTTGCTGGAAGCTGCACACAAGCCCTCTGTGCACCACCAACACCAAAGAGGGCTCCAACATCTGCCTGACCAGAACC GATAGAGGCTGGTACTGCGATAATGCCGGCAGCGTCAGCTTCTTCCCACAAGCCGAGACATGCAAGGTGCAGAGCAACAGAG TGTTCTGCGACACCATGAACAGCCTGACACTGCCCTCCGAAGTGAATCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACT GCAAGATCATGACCTCCAAGACCGACGTGTCAAGCTCCGTGATCACATCTCTGGGCGCCATCGTGTCCTGCTACGGCAAGACA AAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAAGGCGTGG ACACCGTGTCTGTGGGCAACACCCTGTACTACGTGAACAAACAAGAGGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCAT CAACTTCTACGACCCTCTGGTGTTCCCCAGCGACGAGTTTGATGCCAGCATCTCCCAAGTGAACGAGAAGATCAACCAGAGCCT GGCCTTCATCAGAAAGTCCGATGAGCTGCTGCACAATGTGAACGCCGGCAAGAGCACCACAAAT
SEQ ID NO: 34
sPrFhRsvSC-DM : hRSV single-chain pre-fusion F-protein for DNA vaccine
M ELLILKANAITTI LTAVTFCFASGQN ITEEFYQSTCSAVSKGYLSALRTGWYTSVITI ELSN I KKI KCNGTDAKI KLI KQELDKYKNAVTEL QLLMQSTPATN NQARGSGSGRSLGFLLGVGSAIASGVAVSKVLH LEGEVN KI KSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQL LPIVN KQSCSIPN IETVI EFQQKN N RLLEITREFSVNAGVTTPVSTYMLTNSELLSLI N DMPITN DQKKLMSN NVQIVRQQSYSI MSI IKE EVLAYVVQLPLYGVI DTPCWKLHTSPLCTTNTKEGSN ICLTRTDRGWYCDNAGSVSFFPQAETCKVQSN RVFCDTMNSLTLPSEVN L CNVDI FN PKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKN RGI IKTFSNGCDYVSN KGVDTVSVGNTLYYVN KQEGKSLYVK GEPII N FYDPLVFPSDEFDASISQVN EKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO: 35
sPrFhRsvSC-DM : hRSV single-chain pre-fusion F-protein coding sequence human codon optimized for DNA vaccine ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACAATCCTGACCGCCGTGACCTTTTGCTTCGCCAGCGGCCAGAACATC ACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGATATCTGTCTGCCCTGAGAACCGGCTGGTACACCAGCGT GATCACCATCGAGCTGAGCAACATCAAGAAAATCAAGTGCAACGGCACCGACGCCAAGATCAAGCTGATCAAGCAAGAGCTG GACAAGTACAAGAATGCCGTGACCGAACTGCAGCTGCTGATGCAGTCTACCCCTGCCACCAACAATCAGGCCAGAGGCTCTGG ATCTGGCAGAAGCCTGGGATTTCTGCTCGGCGTGGGATCTGCTATTGCTTCTGGCGTGGCCGTGTCTAAGGTGCTGCATCTGG AAGGCGAAGTGAACAAGATTAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTTTCTCTGAGCAATGGCGTGTCCGTGCT GACCAGCAAGGTGCTGGACCTGAAGAACTACATCGACAAACAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCC AACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTTTCTGTGAATGCCGGCG TGACCACACCTGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGTCCCTGATCAACGACATGCCCATCACCAACGACCAGA AAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGAGCATCATCAAAGAAGAGGTGCTGGC CTACGTGGTGCAGCTGCCTCTGTATGGCGTGATCGATACCCCTTGCTGGAAGCTGCACACAAGCCCTCTGTGCACCACCAACAC CAAAGAGGGCAGCAACATCTGCCTGACCAGAACCGATAGAGGCTGGTACTGCGATAATGCCGGCAGCGTCAGCTTCTTCCCAC AAGCCGAAACATGCAAGGTGCAGAGCAACAGAGTGTTCTGCGACACCATGAACAGCCTGACACTGCCCTCCGAAGTGAATCT GTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCAAGCTCCGTGATCACATC TCTGGGCGCCATCGTGTCCTGCTACGGCAAGACAAAGTGCACCGCCAGCAACAAGAACCGGGGAATCATCAAGACCTTCAGC AACGGCTGCGACTACGTGTCCAACAAAGGCGTGGACACCGTGTCTGTGGGCAACACCCTGTACTACGTGAACAAACAAGAGG GCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCTCTGGTGTTCCCCAGCGACGAGTTTGATGCCAGC ATCAGCCAAGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGTCCGATGAGCTGCTGAGCGCCATCGGCGGCT ATATCCCTGAAGCTCCTAGAGATGGACAGGCCTATGTGCGGAAGGATGGCGAATGGGTGCTGCTGTCTACATTTCTG
SEQ ID NO: 36 sPrFhRsvDS-Cavl: hRSV pre-fusion F-protein for DNA vaccine
M ELLILKANAITTI LTAVTFCFASGQN ITEEFYQSTCSAVSKGYLSALRTGWYTSVITI ELSN I KEN KCNGTDAKVKLI KQELDKYKNAVTE LQLLMQSTPATN N RARRFLGFLLGVGSAIASGVAVCKVLH LEGEVN KI KSALLSTN KAVVSLSNGVSVLTFKVLDLKNYI DKQLLPI LN K QSCSISN IETVI EFQQKN N RLLEITREFSVNAGVTTPVSTYM LTNSELLSLIN DMPITN DQKKLMSN NVQIVRQQSYSIMCI IKEEVLAY VVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSN RVFCDTMNSLTLPSEVN LCNVDI FN PKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASN KN RGI I KTFSNGCDYVSNKGVDTVSVGNTLYYVN KQEGKSLYVKGEPI IN FYDPLVFPSDEFDASISQVN EKINQSLAFI RKSDELLSAIGGYI PEAPRDGQAYVRKDGEWVLLSTFL
SEQ ID NO: 37
sPrFhRsvDS-Cavl: hRSV pre-fusion F-protein coding sequence human codon optimized for DNA vaccine
ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACAATCCTGACCGCCGTGACCTTTTGCTTCGCCAGCGGCCAGAACATC ACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGATATCTGTCTGCCCTGAGAACCGGCTGGTACACCAGCGT GATCACCATCGAGCTGAGCAACATCAAAGAAAACAAGTGCAACGGCACCGACGCCAAAGTGAAGCTGATCAAGCAAGAGCTG GACAAGTACAAGAATGCCGTGACCGAACTGCAGCTGCTGATGCAGTCTACCCCTGCCACCAACAACCGGGCCAGAAGATTTCT GGGCTTTCTGCTCGGCGTGGGCTCTGCTATTGCTAGCGGAGTGGCTGTGTGCAAGGTGCTGCATCTGGAAGGCGAAGTGAAC AAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTTTCTCTGAGCAATGGCGTGTCCGTGCTGACCTTTAAGGTGCT GGACCTGAAGAACTACATCGACAAACAGCTGCTGCCCATCCTGAACAAGCAGTCCTGCAGCATCAGCAACATCGAGACAGTGA TCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTTTCTGTGAATGCCGGCGTGACCACACCTGTGTCC ACCTACATGCTGACCAACAGCGAGCTGCTGTCCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAA CAACGTGCAGATCGTGCGGCAGCAGAGCTACAGCATCATGTG CATTATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTG CCTCTGTATGGCGTGATCGATACCCCTTGCTGGAAGCTGCACACAAGCCCTCTGTGCACCACCAACACCAAAGAGGGCTCCAA CATCTGCCTGACCAGAACCGATAGAGGCTGGTACTGCGATAATGCCGGCAGCGTCAGCTTCTTCCCACAAGCCGAAACATGCA AAGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACACTGCCCTCCGAAGTGAATCTGTGCAACGTGGACATC TTCAACCCTAAGTACGACTGCAAGATCATGACCAGCAAGACCGACGTGTCAAGCTCCGTGATCACATCTCTGGGCGCCATCGT GTCCTGCTACGGCAAGACAAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTAC GTGTCCAACAAAGGCGTGGACACCGTGTCTGTGGGCAACACCCTGTACTACGTGAACAAACAAGAGGGCAAGAGCCTGTACG TGAAGGGCGAGCCCATCATCAACTTCTACGACCCTCTGGTGTTCCCCAGCGACGAGTTTGATGCCAGCATCTCCCAAGTGAACG AGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGTCCGATGAGCTGCTGAGCGCCATCGGCGGCTATATCCCTGAAGCTCCT AGAGATGGACAGGCCTATGTGCGGAAGGATGGCGAATGGGTGCTGCTGTCTACATTTCTG SEQ ID NO: 38
sFhMPv: Soluble native F-protein for DNA vaccine
MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVG DVEN LTCADGPSLI KTELDLTKSALRELRTVSADQLAR EEQIEN PRQSKKRKRRVATAAAVTAGVAIAKTI RLESEVTAI KNALKKTN EAVSTLGNGVRVLATAVRELKDFVSKN LTRAI N KN KCDI ADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNM PTSAGQI KLM LEN RAMVRRKGFGFLIGVYGSSVIYM VQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPN EKDCETRGDHVFCDTAAGI NVAEQSKECN IN ISTT NYPCKVSTGRH PISMVALSPLGALVACYKGVSCSIGSN RVGI IKQLN KGCSYITNQDADTVTI DNTVYQLSKVEGEQHVI KGRPVSSSF DPVKFPEDQFNVALDQVFESIENSQALVDQSN RILSSAEKGNTG
SEQ ID NO: 39
sFhMPv: Soluble native F-protein coding sequence human codon optimized for DNA vaccine
ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCACCCCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTG CAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTG GAAAACCTGACCTGTGCCGATGGACCCAGCCTGATCAAGACCGAGCTGGATCTGACAAAGAGCGCCCTGAGAGAACTGAGGA CCGTGTCTGCAGATCAGCTGGCCAGAGAGGAACAGATCGAGAACCCCAGACAGAGCAAGAAACGGAAGCGGAGAGTGGCCA CAGCTGCTGCTGTTACAGCTGGCGTGGCCATTGCCAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAAGAACGCCCT GAAAAAGACCAACGAGGCCGTGTCTACACTCGGCAATGGCGTTAGAGTGCTGGCTACAGCCGTGCGCGAGCTGAAGGACTTC GTGTCCAAAAATCTGACCCGGGCCATCAACAAGAACAAGTGCGACATTGCCGACCTGAAGATGGCCGTGTCCTTTAGCCAGTT CAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTTCTGACAACGCCGGCATCACACCAGCCATCAGCCTGGACCTGATGACAG ATGCTGAGCTGGCAAGAGCCGTGTCCAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTGGAAAACAGAGCCATGGT CCGACGGAAAGGCTTCGGCTTTCTGATCGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGA TCGACACCCCTTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGA CCAAGGCTGGTATTGTCAGAATGCCGGCAGCACCGTGTACTACCCCAACGAGAAGGATTGCGAGACACGGGGCGATCACGTG TTCTGTGATACAGCCGCCGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACGAACTACCCCTG CAAGGTGTCCACAGGCAGACACCCTATCTCTATGGTGGCCCTGTCTCCTCTGGGAGCCCTGGTTGCTTGTTACAAGGGCGTGTC CTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGAT ACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCA GCAGCTTCGACCCTGTGAAGTTCCCCGAGGATCAGTTCAACGTGGCCCTGGATCAGGTGTTCGAGAGCATCGAGAATAGCCAG GCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAATACCGGA
SEQ ID NO: 40
FI FhMPV: Full length F-Protein of the Al hMPV isolate "N L/1/00" (Accession No.: AAK62968)
MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVG DVEN LTCADGPSLI KTELDLTKSALRELRTVSADQLAR EEQIEN PRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKN LTRAIN KN KCDIADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN MPTSAGQI KLM LEN RAMVRRKG FGFLIGVYGSS VIYMVQLPI FGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPN EKDCETRGDHVFCDTAAGI NVAEQSKECN I N ISTTNYPCKVSTG RH PISMVALSPLGALVACYKGVSCSIGSN RVGI IKQLN KGCSYITNQDADTVTIDNTVYQLSKVEG EQHVIKGRP VSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RI LSSAEKGNTGFI IVI ILIAVLGSTM ILVSVFI II KKTKKPTGAPPELSGVTN N GFI PH N
SEQ ID NO: 41
FI FhMPv: Full length F-Protein coding sequence of the Al hM PV isolate "N L/1/00" human codon optimized for DNA vaccine
ATGAGCTGGAAGGTGGTGATCATCTTCAGCCTGCTGATCACCCCCCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGAGCT GCAGCACCATCACCGAGGGCTACCTGAGCGTGCTGCGGACCGGCTGGTACACCAACGTGTTCACCCTGGAAGTGGGCGACGT GGAAAACCTGACCTGCGCCGACGGCCCCAGCCTGATCAAGACCGAGCTGGACCTGACCAAGAGCGCCCTGCGCGAGCTGAGA ACCGTGTCCGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAACCCCCGGCAGAGCAGATTCGTGCTGGGCGCCATTGCCC TGGGCGTGGCCACAGCTGCTGCTGTGACAGCCGGCGTGGCCATTGCCAAGACCATCCGGCTGGAAAGCGAAGTGACCGCCAT CAAGAACGCCCTGAAAAAGACCAACGAGGCCGTGTCCACCCTGGGCAACGGCGTGCGGGTGCTGGCTACAGCCGTGCGGGA ACTGAAGGACTTCGTGTCCAAGAACCTGACCCGGGCCATCAACAAGAACAAGTGCGATATCGCCGACCTGAAGATGGCCGTG TCCTTCAGCCAGTTCAACCGGCGGTTCCTGAACGTGGTGCGCCAGTTCAGCGACAACGCCGGCATCACCCCCGCCATCAGCCT GGACCTGATGACCGATGCCGAGCTGGCCAGGGCCGTGTCTAACATGCCTACCTCTGCCGGCCAGATCAAGCTGATGCTGGAA AACCGGGCCATGGTCCGCCGGAAGGGCTTCGGCTTCCTGATCGGCGTGTACGGCAGCAGCGTGATCTACATGGTGCAGCTGC CCATCTTCGGCGTGATCGACACCCCCTGCTGGATCGTGAAGGCCGCTCCCAGCTGCAGCGGCAAGAAGGGCAACTACGCCTGC CTGCTGAGAGAGGACCAGGGCTGGTACTGCCAGAACGCCGGCTCCACCGTGTACTACCCCAACGAGAAGGACTGCGAGACAC GGGGCGACCACGTGTTCTGCGACACCGCCGCTGGCATCAACGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCAC CACCAACTACCCCTGCAAGGTGTCCACCGGCAGACACCCCATCAGCATGGTGGCCCTGAGCCCTCTGGGAGCCCTGGTGGCCT GTTACAAGGGCGTGTCCTGCAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCAC CAACCAGGACGCCGACACCGTGACCATCGACAACACCGTGTACCAGCTGAGCAAGGTGGAAGGCGAGCAGCACGTGATCAA GGGCAGACCCGTGTCCAGCAGCTTCGACCCCGTGAAGTTCCCCGAGGACCAGTTCAATGTGGCCCTGGACCAGGTGTTCGAG AGCATCGAGAACAGCCAGGCCCTGGTGGACCAGAGCAACCGGATCCTGAGCAGCGCCGAGAAGGGAAACACCGGCTTCATC ATCGTGATCATCCTGATCGCCGTGCTGGGCAGCACCATGATCCTGGTGTCCGTGTTCATCATCATCAAGAAAACAAAGAAGCCC ACAGGCGCCCCTCCCGAGCTGAGCGGCGTGACCAACAATGGCTTCATCCCCCACAAC
SEQ ID NO: 42
sPoFhMPV: Post-Fusion F-protein (Al) for DNA vaccine
MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVG DVEN LTCADGPSLI KTELDLTKSALRELRTVSADQLAR EEQIEN PRQSKKRKRRVATAAAVTAGVAIAKTI RLESEVTAI KNALKKTN EAVSTLGNGVRVLATAVRELKDFVSKNLTRAI N KN KCDI ADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNM PTSAGQI KLM LEN RAMVRRKGFGFLIGVYGSSVIYM VQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPN EKDCETRGDHVFCDTAAGI NVAEQSKECN IN ISTT NYPCKVSTGRH PISMVALSPLGALVACYKGVSCSIGSN RVGI IKQLN KGCSYITNQDADTVTI DNTVYQLSKVEGEQHVI KGRPVSSSF DPVKFPEDQFNVALDQVFESI ENSQALVDQSN RI LSSAEKGNTSGREN LYFQGGGGSGYI PEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO: 43
sPoFhMPV: Post-Fusion F-protein (Al) human codon optimized DNA Sequence for DNA vaccine ATGAGCTGGAAGGTGGTGATCATCTTCAGCCTGCTGATCACCCCCCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGAGCT GCAGCACCATCACCGAGGGCTACCTGAGCGTGCTGCGGACCGGCTGGTACACCAACGTGTTCACCCTGGAAGTGGGCGACGT GGAAAACCTGACCTGCGCCGACGGCCCCAGCCTGATCAAGACCGAGCTGGACCTGACCAAGAGCGCCCTGCGCGAGCTGAGA ACCGTGTCCGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAACCCCCGGCAGAGCAAGAAACGGAAGCGGAGAGTGGCC ACCGCCGCTGCCGTGACAGCTGGCGTGGCCATTGCCAAGACCATCCGGCTGGAAAGCGAAGTGACCGCCATCAAGAACGCCC TGAAAAAGACCAACGAGGCCGTGTCCACCCTGGGCAACGGCGTGCGGGTGCTGGCTACAGCCGTGCGGGAACTGAAGGACT TCGTGTCCAAGAACCTGACCCGGGCCATCAACAAGAACAAGTGCGATATCGCCGACCTGAAGATGGCCGTGTCCTTCAGCCAG TTCAACCGGCGGTTCCTGAACGTGGTGCGCCAGTTCAGCGACAACGCCGGCATCACCCCCGCCATCAGCCTGGACCTGATGAC CGATGCCGAGCTGGCCAGGGCCGTGTCTAACATGCCTACCTCTGCCGGCCAGATCAAGCTGATGCTGGAAAACCGGGCCATG GTCCGCCGGAAGGGCTTCGGCTTCCTGATCGGCGTGTACGGCAGCAGCGTGATCTACATGGTGCAGCTGCCCATCTTCGGCGT GATCGACACCCCCTGCTGGATCGTGAAGGCCGCTCCCAGCTGCAGCGAGAAGAAGGGCAACTACGCCTGCCTGCTGAGAGAG GACCAGGGCTGGTACTGCCAGAACGCCGGCTCCACCGTGTACTACCCCAACGAGAAGGACTGCGAGACACGGGGCGACCAC GTGTTCTGCGACACCGCTGCCGGCATCAACGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTACCC CTGCAAGGTGTCCACCGGCAGACACCCCATCAGCATGGTGGCCCTGAGCCCTCTGGGAGCCCTGGTGGCCTGTTACAAGGGC GTGTCCTGCAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACG CCGACACCGTGACCATCGACAACACCGTGTACCAGCTGAGCAAGGTGGAAGGCGAGCAGCACGTGATCAAGGGCAGACCCGT GTCCAGCAGCTTCGACCCCGTGAAGTTCCCCGAGGACCAGTTCAATGTGGCCCTGGACCAGGTGTTCGAGAGCATCGAGAACA GCCAGGCCCTGGTGGACCAGAGCAACCGGATCCTGAGCAGCGCCGAGAAGGGCAATACCAGCGGCAGAGAGAACCTGTATT TTCAAGGCGGAGGCGGCAGCGGCTACATCCCCGAAGCCCCTAGAGATGGCCAGGCCTACGTGCGGAAGGACGGCGAGTGGG TG CTG CTG AG CACCTTCCTG
SEQID NO: 44
sPoFhMPvAl-Mfur: post-fusion configuration F-protein polypeptide with His-tag for purification (encoded by SEQ ID NO: 18)
KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQSKKRKRR VATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYMVQLPIFGVIDTP CWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVST GRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKF PEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTSGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEG RHHHHHH
SEQID NO: 45
sFhMPvAl-V: soluble configuration F-protein polypeptide in monomer form for purification
MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVG DVENLTCADGPSUKTELDLTKSALRELRTVSADQ LAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTR AINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGF LIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIN VAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSK VEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTGHHHHHH
SEQID NO: 46
sFhMPvAl-K soluble F-protein subunit polypeptide in prefusion trimer form for purification
MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVG DVENLTCADGPSLIKTELDLTKSALRELRTVSADQ LAREEQIENPRQSRFVLGAIALGVCTAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLAFAVRELKDFVSKNLTR ALNKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGF LIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTACGIN VAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSK VEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAESAIGGYIPEAPRDGQAYVRKDGEWVLLST FLGGLVPRGSHHHHHHSAWSHPQFEK
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)
MSWKVM IIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVEN LTCTDGPSLI KTELDLTKSALRELKTVSADQ LAREEQIEN PRQSKKRKRRVATAAAVTAGIAIAKTI RLESEVNAIKGALKQTN EAVSTLGNGVRVLATAVRELKEFVSKNLTSAIN R N KCDIADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLEN RAMVRRKGFGI LIGVY GSSVIYMVQLPIFGVI DTPCWI I KAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPN EKDCETRGDHVFCDTAAGI NVAEQS RECN IN ISTTNYPCKVSTGRH PISMVALSPLGALVACYKGVSCSIGSNWVGII KQLPKGCSYITNQDADTVTI DNTVYQLSKVEGE QHVIKGRPVSSSFDPI KFPEDQFNVALDQVFESIENSQALVDQSN KILNSAEKGNTSGREN LYFQGGGGSGYIPEAPRDGQAYVR KDGEWVLLSTFL SEQ ID NO: 48
sPoFhMPv: Post-Fusion hMPV F-protein (Bl) adapted from the sequence of the "Arg/2/02" isolate (Accession number DQ362937.1)
MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVG DVEN LTCADGPSLI KTELDLTKSALRELRTVSADQ LAREEQIEN PRQSKKRKRRVATAAAVTAGVAIAKTI RLESEVTAIKNALKKTN EAVSTLGNGVRVLATAVRELKDFVSKN LTRAI N K N KCDIADLKMAVSFSQFN RRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSN MPTSAGQI KLM LEN RAMVRRKGFGFLIGV YGSSVIYMVQLPI FGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPN EKDCETRGDHVFCDTAAGINVAE QSKECN IN ISTTNYPCKVSTGRH PISMVALSPLGALVACYKGVSCSIGSN RVGII KQLN KGCSYITNQDADTVTI DNTVYQLSKVEG EQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RILSSAEKGNTSGREN LYFQGGGGSGYI PEAPRDGQAY VRKDGEWVLLSTFL
SEQ ID NO: 49
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
ATGAGCTGGAAGGTGGTGATCATCTTCAGCCTGCTGATCACCCCCCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGAG CTGCAGCACCATCACCGAGGGCTACCTGAGCGTGCTGCGGACCGGCTGGTACACCAACGTGTTCACCCTGGAAGTGGGCG ACGTGGAAAACCTGACCTGCGCCGACGGCCCCAGCCTGATCAAGACCGAGCTGGACCTGACCAAGAGCGCCCTGCGCGA GCTGAGAACCGTGTCCGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAACCCCCGGCAGAGCAAGAAACGGAAGCG GAGAGTGGCCACCGCCGCTGCCGTGACAGCTGGCGTGGCCATTGCCAAGACCATCCGGCTGGAAAGCGAAGTGACCGCC ATCAAGAACGCCCTGAAAAAGACCAACGAGGCCGTGTCCACCCTGGGCAACGGCGTGCGGGTGCTGGCTACAGCCGTGC GGGAACTGAAGGACTTCGTGTCCAAGAACCTGACCCGGGCCATCAACAAGAACAAGTGCGATATCGCCGACCTGAAGAT GGCCGTGTCCTTCAGCCAGTTCAACCGGCGGTTCCTGAACGTGGTGCGCCAGTTCAGCGACAACGCCGGCATCACCCCCG CCATCAGCCTGGACCTGATGACCGATGCCGAGCTGGCCAGGGCCGTGTCTAACATGCCTACCTCTGCCGGCCAGATCAAG CTGATGCTGGAAAACCGGGCCATGGTCCGCCGGAAGGGCTTCGGCTTCCTGATCGGCGTGTACGGCAGCAGCGTGATCTA CATGGTGCAGCTGCCCATCTTCGGCGTGATCGACACCCCCTGCTGGATCGTGAAGGCCGCTCCCAGCTGCAGCGAGAAGA AGGGCAACTACGCCTGCCTGCTGAGAGAGGACCAGGGCTGGTACTGCCAGAACGCCGGCTCCACCGTGTACTACCCCAAC GAGAAGGACTGCGAGACACGGGGCGACCACGTGTTCTGCGACACCGCTGCCGGCATCAACGTGGCCGAGCAGAGCAAA GAGTGCAACATCAACATCAGCACCACCAACTACCCCTGCAAGGTGTCCACCGGCAGACACCCCATCAGCATGGTGGCCCTG AGCCCTCTGGGAGCCCTGGTGGCCTGTTACAAGGGCGTGTCCTGCAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCA GCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGACACCGTGACCATCGACAACACCGTGTACCAGCTGAGCA AGGTGGAAGGCGAGCAGCACGTGATCAAGGGCAGACCCGTGTCCAGCAGCTTCGACCCCGTGAAGTTCCCCGAGGACCA GTTCAATGTGGCCCTGGACCAGGTGTTCGAGAGCATCGAGAACAGCCAGGCCCTGGTGGACCAGAGCAACCGGATCCTG AGCAGCGCCGAGAAGGGCAATACCAGCGGCAGAGAGAACCTGTATTTTCAAGGCGGAGGCGGCAGCGGCTACATCCCCG AAGCCCCTAGAGATGGCCAGGCCTACGTGCGGAAGGACGGCGAGTGGGTGCTGCTGAGCACCTTCCTG EXAMPLES
Materials and Methods
DNA sequences encoding F-proteins for DNA vaccines
Three 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.
Three 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.
Synthesis and subcloning ofcodon optimized F-protein DNA sequences
Because examination of the native sequence of the hMPV F-protein revealed numerous rare mammalian codons, AT -rich sequences, mRNA instability motifs including poly(A) regions and cryptic splice sites, as well as several potential RNA secondary structural elements, 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. coli (DH5a™), 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
Figure imgf000035_0001
protein (SFIAIPV) the ectodomain in native form with only a G294E mutation. The C- terminus is truncated, removing the transmembrane domain and
cytoplasmic tail. Additionally added was a KKRKRR cleavage site
for enhanced processing. The sequence is modified from "fusion
protein [Human metapneumo irus]" with accession number
AAK62968.
Soluble single-chain Soluble form of hRSV F-protein in a pre- fusion configuration
prefusion hRSV F- (Krarup et al., 2015), including a modified ectodomain with three
Protein naturally occurring substitutions (found in other RSV isolates) to
(sPrFhRsvSC-DM) enhance expression (P102A, I379V, and M447V) as well as four
mutations to improve expression and to stabilize the prefusion state
35 34 (E66K, N67I, V76I and S215P). Further, the P27 interchain region
from amino acids 108-134 were replaced by a linker (GSGSG) and
two substitutions to impair furin cleavage (R106Q and F137S). This
is a single chain (SC) mutant, which is resistant to cleavage into Fl
and F2 fragments.
Soluble pre-fusion Soluble form of hRSV F-protein (McLellan et al., 2013), including
hRSV F-Protein an ectodomain to which several modifications have been made
(Disulfide-Cavity- including three substitutions to enhance expression (P102A, I379V,
filling) and M447V), the addition of a cysteine pair (S155C; S290C) and
37 36
(PrFhRsvDS-Cavl) cavity-filling hydrophobic substitutions (S190F; V207L) to stabilize
the prefusion state and, further, the P27 interchain region from
amino acids 108-134 were removed by keeping only the first furin
site intact.
Post-fusion hRSV F- Post-fusion form of hRSV F-protein, including a truncated
Protein ectodomain (TM and CT removed) in which three naturally
(POFIIRSV) occurring substitutions were introduced to enhance expression
(P102A, I379V, and M447V), additionally, a part of the fusion 33 32 peptide has been removed (aa 137 to 146). Notably, this RSV post- fusion ectodomain properly trimerizes without the addition of
foldon (McLellan et al., 2011; Swanson et al., 2011)
Plasmidfor F-protein DNA vaccines
The plasmid pVAXl (SEQ ID NO: 28) from Invitrogen was developed for use in DNA vaccines by modification of the pcDNA™3.1 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 pVAXl™ vector was used for all DNA constructs used in the DNA vaccination studies described herein and contains:
-a human cytomegalovirus immediate -early (CMV) promoter for high-level expression in a wide range of mammalian cells
-a bovine growth hormone (BGH) polyadenylation signal for efficient transcription termination and polyadenylation of mRNA
-a kanamycin resistance gene for selection in E. coli.
Constructs for hMPV F-protein subunit vaccines
In nature, 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).
Modifications to the subunit F -proteins
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). In addition, 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. Furthermore, in order to avoid aggregation of the mature post-fusion hMPV F-protein homotrimer, the first eight amino acids of the fusion peptide were deleted (Δ103-111). A His6-tag (SEQ ID NO: 5) was added downstream of the trimerization domain for purification purposes. Finally, also added were 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 (sFhMPvAl-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.)
In both constructs above, a G294E mutation was introduced to potentially improve the cell surface exposure and fusion characteristics of the F-protein (Mas et al. (2011) Residues of the Human Metapneumo virus Fusion (F) Protein Critical for Its Strain -Related Fusion Phenotype: Implications for the Virus Replication Cycle. J. Virol. 85(23): 12650-12661). The prefusion trimeric hMPV F-protein construct (sFhMPvAl-K) assumes a prefusion trimeric configuration. Table IB. Sequences for hMPV F-protein subunit preparation; DNA sequences were codon optimized for expression in CHO cells and sub-cloned into pVVS 1371. For the post-fusion hMPV F-protein, the pVVS 1371 plasmid also contained the furin coding sequence of SEQ ID NO: 31 to cleave the introduced furin site. (The
Figure imgf000038_0001
Plasmid for expression of hMPV F -proteins
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:
-an HS4 insulator sequence from chicken β-globin locus,
-two cytomegalovirus (CMV) promoters,
-two chimeric introns, downstream from the CMV promoter, composed of the 5 '-donor site from the first intron of the human β-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,
-the bovine growth hormone polyadenylation signal sequence (bgh-polyA),
-the 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,
-a kanamycin resistance gene under the regulation of a bacterial promoter, and
-a pUC origin of replication. Constructs for DNA vaccines
All DNA sequences used for DNA vaccines (see Table 1A) were cloned into the pVAXl plasmid backbone containing a CMV promoter to control gene expression. Plasmids were purified from recombinant E. coli using the EndoFree plasmid purification kit (Qiagen, France). Construction of expression vectors for production of hMPV F-protein polypeptides
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 ( Ί ΙΟ 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. Briefly, 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 DH5a™ competent cells. Selected clones were checked for mutation by sequencing.
Because proteolytic cleavage by furin is key to the proteolytic processing of the sPoFhMPv Al-Mfur protein (due to the introduced furin cleavage site) and only very low levels of furin are expressed by CHO cells, 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. For tandem expression, 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.
Cell lines, bacteria and viruses
Vero and HeLa cell lines were acquired from ATCC, CHO cells from ECACC and LLC-MK2 cells from HP A Culture Collections. Max Efficiency™ DH5a™ 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.
Animals
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).
Monoclonal antibodies to F -proteins
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).
Nanotaxi® and preparation of DNA formulations
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.
Two 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/Th2 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. In the Examples, 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 μg to 50 μg were performed in both anterior tibial muscles of C57B1/6 mice.
Example 1 Expression of protein from DNA pVAXl constructs in HeLa cells
Preparation of ICAfectin®441 transfection solution
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% CO2 / 95% air containing atmosphere.
Thirty minutes before transfection, medium was removed and replaced by 400 of fresh complete medium and 100 μΕ of ICAfectin®441/DNA complexes were added to the HeLa cells. After 24h of incubation at 37°C, 5% CO2, samples of cells (105-106) were collected and re-suspended in phosphate- buffered saline (PBS). The resulting cell suspension was assayed for the expression of hMPV F-protein by flow cytometry analysis using a FACSverse flow cytometer (Becton Dickinson) after labelling by anti- F antibody DS7. All measurements were performed in triplicate. Results are shown in Figure 5. Example 2 Construction, expression, characterization and formulation of F-protein polypeptides for subunit vaccines and/or use in ELISA
Expression in Chinese Hamster Ovary ( CHO) cells
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% CO2. Following the recovery period, cells were resuspended at high density in EX -CELL ACF CHO medium (Sigma Aldrich). Post-electroporation, cells were incubated at 37°C with 8% CO2 and orbital shaking for 24 hours.
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 EfficientFeed™ (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 μιη sterile filtered and stored at -80°C before protein purification.
Concentration and purification
After a period of storage at -80°C, 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 Na2HP04 buffer at pH 8.0, 300 mM NaCl. Purification of the protein was performed using Immobilized metal ion affinity chromatography (IMAC) as described below, followed by diafiltration on a Slide-A-Lyzer™ Dialysis Cassette (ThermoFisher Scientific; lOkDa MWCO) against a storage buffer (50mM Na2HP04, 300mM NaCl, 5mM EDTA, 10% sucrose, 0.05% Tween-20, pH 8).
Immobilized metal ion affinity chromatography (IMAC) For metal ion affinity chromatography (IMAC), agarose resin containing Ni2+ (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. The column was washed extensively until the A280 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.
Coomassie blue staining and Western Blot
The sPoFhMPvAl-Mfur and sFhMPvAl -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.
When the gel was used to perform a Western blot, 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 (50μg/ml; Qiagen, ref. 34660). Then the membrane was washed three times with stirring for 5 minutes in 0.1% Tween-20/PBS. The antigen- antibody complexes were detected by incubation with a-Ig specific to the peroxidase-conjugated species, and after washing the membrane again in 0.1% Tween-20/PBS, it was developed with the luminescent substrate ECL (ECL Prime Western Blotting Detection Reagent, GE Healthcare) following the instructions of the manufacturer. The bands were visualized using an Imaging System camera (Imager 600RGB, GE HealthCare). Results are shown in Figure 6A. FACS analysis of expressed subunit proteins
The presence of 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. Cells were washed with BD Perm wash buffer and incubated with a Goat anti-mouse IgG and IgM FITC (Jackson ImmunoResearch, JIR 115-096-068) for one hour at 4°C. After washing, the cells were resuspended in 100-150 μΙ^ΛνεΙΙ of PBS-SVF 2% for FACS analysis. Example 3 Immunogenicity comparison of Post-fusion hMPV and full-length hMPV DNA vaccines versus post-fusion hMPV F-protein subunit vaccine in mice
To investigate the immunogenicity of recombinant Nanotaxi®-associated DNA, 8 groups of 5 mice (C57B1/6; Groups A, B and C: sPoFhMPv; Groups D, E and F: FIFhMPv, Group G: subunit sPoFhMPvAl- Mfur; Group H: empty vector at the highest DNA dose) were used to determine humoral responses. The immunization protocol was carried out as described in Table 2. Briefly, 6 groups of 5 mice (6-8 weeks of age) were injected with different doses (5, 10 and 50μg) of both DNA vector constructs (sPoFhMPv and FIFhMPv). All DNA vaccines were formulated with a final concentration of 0.15% 704 (Nanotaxi®l). For the DNA vector constructs and the negative control (empty vector), three immunizations by intramuscular route (anterior tibial muscle) were performed at DO, D21 and D42. In parallel, the empty vector and alum- adjuvanted sPoFhMPv subunit vaccine were injected as a negative control and a benchmark, respectively, in two additional groups of 5 mice. The sub-unit vaccine was delivered three times at two week intervals.
During the study, 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.
Additionally, individual terminal sera were assessed for binding to hMPV-FL ICAFectin®441 transfected
HeLa cells by flow cytometry. FIFhMPv DNA and sPoFhMPvAl-Mfur subunit vaccination results are shown in Figure 8; sPoFhMPv DNA and sPoFhMPvAl-Mfur subunit vaccination results are shown in Figure 9.
Each FACS plot as shown represents the binding results of sera from individual vaccinated mice at day 0 and day 56 after immunization. Table 2. Immunization protocol for Post-fusion and Full-length hMPV F-protein-encoding DNA constructs formulated with Nanotaxi® 1 (0.15%) and compared with alum-adjuvanted post-fusion subunit (polypeptide) vaccination.
Figure imgf000045_0001
*see Fig. 3; polypeptide formulated in Alhydrogel® adjuvant 2%, Brenntag Table 3. PRNT protocol
Figure imgf000045_0002
*with or without trypsin depending on the virus strain used
**serum or control (DS7) antibody diluted in EMEM-2mM L-glutamine + 1% NEAA
Example 4 Analysis of immunogenic response to hMPV F-protein-encoding DNA constructs with two Nanotaxi® formulations in two different strains of mice
To investigate the immunogenicity of recombinant Nanotaxi® -formulated hMPV F-protein DNA vectors, the immunization protocol as outlined in Table 4 was used. Briefly, 18 groups of 5 animals each (Groups 1 to 4: Benchmark 1 [subunit sPoFhMPvAl-MFur]; Groups 5 to 8: Post-fusion hMPV F-protein DNA [sPoFhMPv]; Groups 9 to 12: full length hMPV F-protein DNA [FIFhMPv] ; Groups 13 and 14: Benchmark 2 [subunit sFhMPvAl-V] ; Groups 15 to 18: soluble hMPV F-protein DNA [sFhMPv]) were used to compare three different hMPV F-protein-encoding DNA constructs combined with two different Nanotaxi® DNA delivery systems at one dose of DNA/injection (50 μg) in two mouse strains (C57BL/6 and BALB/c). Three immunizations by subcutaneous or intramuscular routes (anterior tibial muscle) as indicated were performed at DO, D14 and D28 for subunit vaccines and DO, D21, D42 for Nanotaxi®/DNA formulations (and one subunit group: Group 4). During the study, mice were bled 4 times: DO before immunization, D14, D28 and D42 (termination day) or DO, D21, D42 and D56 (termination day), depending on the treatment. At D56, splenocytes and bronchoalveolar lavage (BALs) cells were also collected. All samples from mice were stored at -80°C until analysis. Antibody titers as well as PRNT values were assessed.
Table 4. Experimental design for comparison of hMPV F-protein DNA constructs combined with 0.15% 704 (Nanotaxi®l) or 704-M (Nanotaxi®2) in Balb/c and C57B1/6 mice. Four experiments in total were done as indicated, using two different subunit (polypeptide) vaccines as Benchmarks (shaded grey).
Figure imgf000047_0001
*5 μΐ Al ydrogel (Brenntag)
"comprised in pVAXl vector
Comparison of antibody responses in Balb/c and C57BI/6 mice
For antibody titer determination, 96-well half-area plates were coated with 1 μg/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 IgG2A 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. The substrate reaction was stopped by addition of 85% orthophosphoric acid and signal was detected by optical density reading at 450 nm. For IgGi and IgG2A isotype ELISAs, 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 IgG2A-HRP (Abeam) at 1 : 10,000 dilution. For total IgG ELISA, 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.
As shown in Figure 10A, in comparison with the subunit vaccines, the hMPV neutralizing titers in C57B1/6 mice on day 56 were generally higher in response to the DNA vaccine preparations, with the exception of the post-fusion subunit delivered at three week intervals. These observations are in contrast to those in Balb/c mice on day 56, as shown in Figure 11A, which showed somewhat higher neutralizing antibody titers in response to subunit vaccines. The IgG2a/IgGl ratio (i.e. Thl/Th2 ratio) in Balb/c mice on d42, however, was more favorable with the DNA vaccine formulations (i.e. >1) (see Figure 11B), demonstrating the ability of DNA vaccination to generate strong Thl responses even in a Th2-biased mouse model. The IgGl titers in C57B1/6 mice on d42, as shown in Figure 10B, indicated that both subunit vaccine and DNA vaccine preparations can generate strong IgGl/Th2 immune responses despite the well-established Thl bias of C57B1/6 mice (Watanabe, et al., 2004, Innate Immune Response in Thl- and Th2-Dominant Mouse Strains, SHOCK 22(5): 460-466). In sum, the results demonstrated that DNA vaccines performed robustly in both Thl- and Th2-biased mouse models.
Finally, 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 (sFhMPvAl-K; SEQ ID NO: 46) and a native monomeric form of hMPV F-protein (sFhMPvAl-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.
As shown in Figure 12A, it was observed that the antibodies raised in sPoFhMPv-DNA-vaccinated mice bound approximately equally to all three forms of the protein in vitro. By contrast, however, the sera raised in sPoFhMPvAl-Mfur subunit-vaccinated mice bound preferentially to the post-fusion trimeric form (sPoFhMPvAl-Mfur) of the protein in vitro. This observation is shown quantitatively by endpoint titer in the Table in Figure 12B. This finding suggests that the DNA -delivered coding sequence of the post- fusion form of the hMPV F-protein is superior to the subunit vaccine in terms of diversity of epitope presentation in vivo, which would likely translate to a more robust protective response.
Example 5 Testing of hMPV/RSV combination DNA vaccine in mice
To investigate immunogenicity of recombinant hMPV and RSV F-protein Nanotaxi®-associated DNA vectors, 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 μg of the indicated DNA constructs (single or in combination), to a total of 50 μg of DNA in combination with 0.15% Nanotaxi® 704 (Nanotaxi®l). pVAXl vector comprising DNA encoding sPoFhMPv 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. For immunizations comprising a single construct, the 25 μg of recombinant DNA plasmid was supplemented with 25 μg empty pVAXl plasmid. Three immunizations by the intramuscular route (anterior tibial muscle) were performed at DO, D21 and D42. During the study, 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.
Table 5. Immunization protocol for combination hMPV/RSV DNA vaccines in C57B1/6 mice. Three different hRSV F-protein DNA coding sequences in the pVAXl vector were delivered alone or together with a pVAXl vector comprising a DNA sequence encoding a post-fusion form of hMPV F-protein. All treatments were combined with all with 0.15% Nanotaxi®! (704). Immunization Day of
Group Treatment (IM) Dose/site Volume/ site
Schedule terminal bleed sPoFhMPv encoding
1 25 μg* 30 μΐ / tibia
DNA/Nanotaxi®l
SPOFRSV encoding
2 25 μg* 30 μΐ / tibia
DNA/Nanotaxi®l
sPrFhRsvSC-DM encoding
3 25 μg* 30 μΐ / tibia
DNA/Nanotaxi®l
sPrFhRsvDS-Cavl encoding
4 25 μg* 30 μΐ / tibia D0+D21+D42 D56
DNA/Nanotaxi®l
sPoFhMPv + POFRSV encoding 25 μg +
5 30 μΐ / tibia
DNAs/Nanotaxi® 1 25 μg
sPoFhMPv + sPrFHRSvSC-DM 25 μg +
6 30 μΐ / tibia
encoding DNAs/Nanotaxi® 1 25 μg
sPoFhMPv + sPrFhRsvDS-Cavl 25 μg +
7 30 μΐ / tibia
encoding DNAs/Nanotaxi® 1 25 μ8
♦Includes additionally 25 μg empty pVAXl vector for a final dose of 50 μg total DNA
Example 6 Virus challenge studies following immunization
Al, B l and B2 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 105 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. Alternatively or additionally, PCR is used to determine viral load in the harvested tissues. Optionally, immunogenicity is determined as described for mouse studies (above).
Preferred aspects:
1. 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 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 complex for use according to aspect 1 or 2 or the composition for use according to aspect 3 or 4, wherein both domains, i.e. the Fl ectodomain and the F2 domain, are selected from an Al strain or a B 1 strain of hMPV. The complex for use according to aspect 1 or 2 or the composition for use according to aspect 3 or 4, wherein the immunogenic Fl ectodomain consists of (i) amino acids 112 to 489 of the hMPV F- protein (Al genotype), i.e. the amino acid sequence of SEQ ID NO: 10 and (ii) the immunogenic F2 domain consists of amino acid 20 to 101 of the hMPV F-protein (Al genotype), i.e. amino acid sequence of SEQ ID NO: 11. The complex for use according to aspect 1 or 2 or the composition for use according to aspect 3 or 4, wherein the immunogenic Fl ectodomain consists of (i) amino acids 112 to 489 of the hMPV F- protein (B l genotype), i.e. 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 for use according to aspects 1 or 2 or the composition for use according to any of aspects 3 to 7, wherein the polypeptide A additionally comprises a trimerization domain C-terminal to the Fl ectodomain. The complex or composition for use according to aspect 8, wherein the trimerization domain comprises or consists of the fibritin T4 foldon domain (SEQ ID NO: 6). The complex or composition for use according to aspect 8 or 9, wherein the polypeptide A additionally comprises another cleavage site B between the Fl ectodomain and the trimerization domain. 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 11, wherein the TEV protease cleavage site is defined by SEQ ID NO: 3. The complex or composition for use according to any of aspects 1 to 5 or 8 to 12, wherein the strain Al Fl ectodomain is the wild-type sequence; i.e., does not contain a G294E mutation (SEQ ID NO: 9). The complex or composition for use according to any of aspects 1 to 13, wherein the polypeptide A additionally comprises a tag at the C-terminal end of the trimerization domain. The complex or composition for use according to aspect 14, wherein the tag is a His6 tag (SEQ ID NO: 5). The complex or composition for use according to aspect 14 or 15, wherein the polypeptide A additionally comprises another cleavage site C between the trimerization domain and the tag. 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. The complex or composition for use according to any of aspects 1 to 6 or 8 to 18 wherein said 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. The complex or composition for use according to any of aspects 1 to 6 or 8 to 18 wherein said heterodimer consists 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 ID NO: 16, and a polypeptide B with SEQ ID NO: 17 or a functional variant thereof that is more than 80% identical to the polypeptide with SEQ ID NO: 17. The composition for use according to any of aspects 3 to 20, additionally comprising an adjuvant and/or other pharmaceutically acceptable excipient(s). The composition for use according to aspect 21, wherein the adjuvant is an hMPV M protein and/or alum and/or IC31. The composition for use according to aspect 22, wherein the hMPV M protein is defined by SEQ ID NO: 24 or SEQ ID NO: 25. The complex or composition for use according to any of aspects 2 or 4 to 23, wherein the viral infection is an hMPV infection. The composition for use according to aspect 24, wherein the viral infection is an hMPV infection caused by the A or B strain of hMPV. The composition for use according to aspect 25, wherein the viral infection is an hMPV infection caused by one or more of the group of strains selected from the Al, A2, Bl and B2 strains of hMPV. The composition for use according to any of aspects 3 to 26, wherein the composition further comprises RSV and/or PIV antigens. The composition for use according to any of aspects 3 to 27, wherein the composition is a vaccine. A process for producing the composition for use according to any of aspects 3 to 28 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;
b. expressing said vector in a suitable host cell to yield polypeptide A and polypeptide B;
c. optionally, purifying resulting complex; and
d. combining said complex with an optional adjuvant and/or other suitable excipient(s) in order to obtain said pharmaceutical composition. The process according to aspect 29, wherein the nucleic acid sequence is selected from the group consisting of
a. SEQ ID NO: 18,
b. SEQ ID NO: 27 and
c. SEQ ID NO: 19. The process according to aspect 29 or 30, wherein the vector is pVVS1371 (SEQ ID NO: 28). The process according to any of aspect 29 to 31 , wherein the host cells are Chinese Hamster Ovary (CHO) cells.

Claims

1 . A nucleic acid which encodes a single polypeptide capable of being processing into two fragments consisting of
a) a fragment A comprising an immunogenic I · I ectodomain of an hMPV F-protein, wherein fragment A does not comprise the transmembrane domain; and
b) a fragment B comprising an immunogenic I 2 domain of an hMPV F-protein,
2. The nucleic acid according to claim 1 , wherein said nucleic acid encodes four cysteine residues which can form disulfide bridges between said Fl ectodomain and F2 domain, resulting in the formation of a soluble heterodimeric protein,
3. The nucleic acid according to claim 1 or 2, wherein said encoded heterodimeric protein combines to form a trimeric post-fusion conformation,
4. The nucleic acid according to any of claims 1 to 3, wherein said encoded immunogenic Fl ectodomain and said encoded immunogenic F2 domain, are selected from a genotype A or a genotype B hMPV, such as a genotype Al , A2, B l or B2 hMPV.
5. The nucleic acid according to any of claims 1 to 4, wherein said encoded immunogenic Fl ectodomain consists of (i) amino acids 112 to 489 of an A 1 genotype hMPV F-protein with a G294E point mutation, i.e. the amino acid sequence of SEQ ID NO: 10 and (ii) said encoded immunogenic I 2 domain consists of amino acids 20 to 101 of an A I genotype hMPV F-protein, i.e. the amino acid sequence of SEQ ID NO: 1 1.
6. The nucleic acid according to any of claims I to 5, wherein said encoded immunogenic I I ectodomain consists of (i) ami no acids I 1 2 to 489 of a B l genotype hMPV I -protein, i.e. the ami no acid sequence of SEQ ID NO: 1 2 and (ii) said encoded immunogenic 1 2 domain consists of ami no acids 20 to 101 of a B I genotype hMPV F-protein, i.e. the amino acid sequence of SEQ II ) NO: 13.
7. The nucleic acid according to any of claims 1 to 6, wherein said encoded polypeptide A additionally comprises a trimerization domain ( -termi nal to the Fl ectodomain.
8. The nucleic acid according to claim 7, wherein said encoded trimerization domai n comprises or consists of the fibritin T4 foidon domain, i.e. the amino acid sequence of SEQ ID NO: 6.
9. The nucleic acid according to any of claims 1 to 8, wherein said encoded polypeptide B additionally comprises a cleavage site A at its C-terminal end.
10. The nucleic acid according to claim 9, wherein said encoded cleavage site A comprises or consists of the KSV cleavage site I I. i.e. the amino acid sequence of SEQ I I ) NO: 2 (KKRKRR).
11. The nucleic acid according to any of claims 7 to 10, wherein said encoded polypeptide A additionally comprises another cleavage site B between the I · I ecu domai n and the trimerization domain.
12. The nucleic acid according to claim 11 , wherein said encoded cleavage site B comprises or consists of a TEV protease cleavage site, preferably as defined by the amino acid sequence of SEQ ID NO: 3 (ENLYFQG).
1 . The nucleic acid according to any of claims 1 to 12, wherein said encoded I · I ectodomain from the A I genotype is the wild-type sequence not containing a G294E mutation, i.e., the amino acid sequence of SEQ ID NO: 9.
1 4. The nucleic acid according to any of claims 1 to 13 wherein said encoded heterodimeric protein consists of a polypeptide A with the amino acid sequence of SEQ II ) NO: 1 4 or a functional variant thereof that is more than 80% identical to the polypeptide defined by SEQ I I ) NO: 14, and a polypeptide B with the amino acid sequence of SEQ I I ) NO: 15 or a functional variant thereof that is more than 80% identical to the polypeptide defined by SEQ II ) NO: 15.
15. The nucleic acid according to any of claims 1 to 1 3 wherein said encoded heterodimeric protein consists of a polypeptide A with the amino acid sequence of SEQ I I ) NO: 16 or a functional variant thereof that is more than 80% identical to the polypeptide defined by SEQ ID NO: 16, and a polypeptide B with the amino acid sequence of SEQ ID NO: 17 or a functional variant thereof that is more than 80% identical to the polypeptide defined by SEQ II ) NO: 17.
16. A vector comprising the nucleic acid according to any of claims 1 to 15.
17. The vector according to claim 16, wherein said vector is preferably pVAXl vector as provided by SEQ ID NO: 28 or a vector for in vitro expression of the heterodimeric protein encoded by the nucleic acid of any of claims 1 to 14.
18. The vector according to claim 16 or 17, wherein said vector optionally comprises at least one additional coding sequence.
19. The vector according to claim 18, wherein said at least one additional coding sequence encodes an additional antigen or a furin coding sequence according to SEQ ID NO: 31.
20. The nucleic acid according to any of claims 1 to 15 or the vector according to any of claims 16 to 19 for use as a medicament.
21. The nucleic acid according to any of claims 1 to 15 or the vector according to any of claims 16 to 19 for use as a prophylactic or therapeutic treatment against a viral infection, preferably an hMPV infection.
22. A pharmaceutical composition comprising the nucleic acid according to any of claims 1 to 15 or the vector according to any of claims 16 to 19.
23. A pharmaceutical composition comprising a nucleic acid encoding an Al post-fusion protein and a B l post -fusion protein, preferably an Al post-fusion protein as defined by SEQ ID NO: 44 and a B l post-fusion protein as defined by SEQ ID NO: 47 or 48.
24. The pharmaceutical composition according to claim 22 or 23, additionally comprising an adjuvant, a nucleic acid delivery reagent and/or a pharmaceutically acceptable excipient.
25. The pharmaceutical composition according to claim 24, wherein said nucleic acid delivery reagent is a tetrafunctional block co-polymer.
26. The pharmaceutical composition according to claim 25, wherein said tetrafunctional block copolymer is a non-ionic amphiphilic tetrafunctional block copolymer of the formula:
Figure imgf000058_0001
in which
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
for each R1, R2 pair, R1 shall be hydrogen and R2 shall be a methyl group, and 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%.
27. The pharmaceutical composition according to claim 26, wherein said tetrafunctional block copolymer has an i-value of 13, a j-vaiue of 14, a molecular weight of 5500 and an ethylene -oxide unit content of 40%.
28. The pharmaceutical composition according to claim 26 or 27, wherein said tetrafunctional block co-polymer is a 704 block co-polymer.
29. The pharmaceutical composition according to any of claims 26 to 28 wherein said tetrafunctional block co-polymer optionally comprises 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.
30. The pharmaceutical composition according to claim 29 wherein said at least one glycosyl moiety comprises mannose or galactose, preferably mannose.
31. The pharmaceutical composition according to any of claims 26 to 30, wherein said tetrafunctional block co-polymer is present at about 0.01 to 5%, preferably at about 0.05 to 2%, preferably at about 0.1 to 1%, most preferably at about 0.15 to 1%.
32. The pharmaceutical composition according to any of clai ms 22 to 1 . wherein said composition further comprises a nucleic acid encoding an KSV antigen, preferably an KSV 1 -protein.
33. The pharmaceutical composition according to clai m 32, wherein said encoded KSV antigens are selected from the group consisti ng of SEQ II ) NO: 33, SEQ II ) NO: 35 and SEQ I I ) NO: 37.
34. A pharmaceutical composition comprisi ng SEQ I I ) NO: 43 and/or SEQ II ) NO: 49, optionall comprisi ng about 0.15 to 1% 704 or 704-M.
35. The pharmaceutical composition according to any of clai ms 22 to 34, wherein said composition is a vaccine.
36. The pharmaceutical composition accordi ng to any of claims 22 to 35. for use as a medicament.
37. The pharmaceutical composition according to any of clai ms 22 to 35. for use in a method of treatment or prevention against a viral infection.
38. The pharmaceutical composition according to clai m 37, wherein said viral infection is n hMPV infection and/or and KSV infection.
39. The pharmaceutical composition according to claims 37 or 38, wherein said viral infection is an hMPV infection.
40. The pharmaceutical composition according to claim 39, wherein said hMPV infection is caused by A and/or B genotypes of hMPV.
41 . A process for producing the pharmaceutical composition according to any of claims 22 to 40 comprising the steps of:
a) providing a nucleic acid sequence accordi ng to any of claims I to 15 ;
b) combi ni ng 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 according to claim 41. wherein the nucleic acid sequence is selected from the group consisting of
a) SEQ ID NO: 43, and
b) a nucleic acid sequence encoding the protein according to SEQ ID NO: 49.
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