WO2024017682A1 - Rsv immunogens - Google Patents

Rsv immunogens Download PDF

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Publication number
WO2024017682A1
WO2024017682A1 PCT/EP2023/069010 EP2023069010W WO2024017682A1 WO 2024017682 A1 WO2024017682 A1 WO 2024017682A1 EP 2023069010 W EP2023069010 W EP 2023069010W WO 2024017682 A1 WO2024017682 A1 WO 2024017682A1
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rsv
protein
immunogen
seq
subject
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PCT/EP2023/069010
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French (fr)
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Johannes Petrus Maria Langedijk
Mark Johannes Gerardus BAKKERS
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Janssen Vaccines & Prevention B.V.
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Publication of WO2024017682A1 publication Critical patent/WO2024017682A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to immunogens, proteins, nucleic acids and vectors, as well as to the use thereof, e.g. as pharmaceutical agents, and to vaccines containing any of those.
  • RSV Human respiratory syncytial virus
  • subtype A and subtype B.
  • RSV replicates in the upper respiratory track and then spreads to the lower airways leading to bronchiolitis or pneumonia.
  • the virus causes inflammation, edema of the airways, increased mucus production, and breakdown of respiratory epithelium.
  • RSV-induced disease An estimated 64 million cases of respiratory illness and 160,000 deaths worldwide are attributable to RSV-induced disease. Severe RSV infection occurs most often in children and infants, especially in premature infants. Underlying health problems such as chronic lung disease or congenital heart disease can significantly increase the risk of serious illness. RSV infections also can cause serious illness in the elderly, individuals with chronic pulmonary disease and in immunocompromised adults, such as bone marrow transplant recipients.
  • Intravenous immunoglobulin (RSV-IGIV; RespiGam®) isolated from donors, and the monoclonal antibody palivizumab (SYNAGIS®) have been approved for RSV prophylaxis in high-risk premature infants.
  • a vaccine or commercially available treatment for RSV is not yet available.
  • Only ribavirin is approved for treatment of RSV infection.
  • high doses, repeated administrations and/or large volumes of antibody products, such as palivizumab are required due to low effectivity.
  • RSV has two major surface glycoproteins, F and G.
  • the F protein mediates fusion, allowing entry of the virus into the cell cytoplasm and facilitating the formation of syncytia in vitro.
  • the F protein sequence is well ( ⁇ 90%) conserved among RSV strains (Johnson and Collins, J Gen Virol. (1988) 69: 2623-2628).
  • the sole marketed monoclonal antibody palivizumab is directed against the F protein of RSV.
  • the G protein of RSV is a surface protein that is heavily glycosylated and functions as the attachment protein.
  • the G protein is quite variable across strains except for a central conserved domain (CCD), comprising amino acid residues 153-184 of the G protein of RSV A2 strain or corresponding amino acid residues in other strains. Both the central conserved domain and adjacent regions (residues 145-193) are flanked by rigid and heavy Ci- glycosylated mucin-like regions.
  • the N-terminal half of the central conserved domain contains a small region that is conserved among more than 700 strains.
  • the C-terminal half contains 4 conserved cysteines that are connected in a 1-4, 2-3 topology and folds into a cystine noose.
  • the present invention provides immunogens comprising at least one recombinant RSV G protein ectodomain, or fragment thereof, linked to a protein nanoparticle subunit, as well as nucleic acid molecules encoding the immunogens of the invention.
  • the invention provides methods for generating an immune response to
  • RSV G in a subject comprising administering to the subject an effective amount of the immunogenic composition according to the invention.
  • the invention provides methods for treating or preventing a RSV infection in a subject, comprising administering to the subject a therapeutically effective amount of the immunogenic composition according to the invention, thereby treating or preventing RSV infection in the subject.
  • the invention provides methods for detecting or isolating an RSV G binding antibody in a subject, comprising providing an effective amount of the immunogen according to the invention; contacting a biological sample from the subject with the immunogen under conditions sufficient to form an immune complex between the immunogen and the RSV G binding antibody; and detecting the immune complex, thereby detecting or isolating the RSV G binding antibody in the subject.
  • FIG 1 Schematic representation of the AaLS expression construct.
  • the N-terminal signal peptide (‘SP’) is cleaved off during protein maturation.
  • EPEA C-tag
  • the RSV G CCD mini-protein or other (mini-)proteins can potentially be incorporated into the monomer at three different sites: i) the ‘N-terminal fusion site’ between the SP and the AaLS monomer, ii) the ‘Internal fusion site’ at position 71 within the AaLS monomer, and iii) the ‘C-terminal fusion site’ at the C-terminus of the AaLS monomer (in the example shown the mini-protein is followed by the C-tag).
  • FIG 2 Design and expression in supernatant of AaLS fused to RSV G CCD peptides.
  • A Schematic representation of two AaLS backbones.
  • RSV190362 is a wildtype AaLS monomer, in RSV191645 the original initiating methionine at amino acid position 25, directly after the signal peptide, was removed.
  • B Schematic representation of AaLS monomers fused to the human RSV (HRSV) type A G CCD, either internally (construct RSV191646), N-terminally (construct RSV191647) or C-terminally (construct RS VI 91648) as based on the RSV191645 backbone.
  • FIG. 1 Schematic representation of AaLS monomers fused to the bovine RSV (BRSV) G CCD, either internally (construct RSV191649), N-terminally (construct RSV191650) or C-terminally (construct RSV191651) as based on the RSV191645 backbone.
  • D Western blot on reduced supernatants of expiHEK293 cells transfected with the different AaLS constructs depicted in Fig 2A-C. C-tag-based detection was used to confirm expression of the AaLS proteins.
  • FIG. 3 Addition of N-linked glycans boosts AaLS particle expression.
  • A Schematic representation of three AaLS constructs with 0, 1 or 2 N-linked glycosylation motifs (‘NST’) added within an internal loop. Constructs are based on backbone RSV191645.
  • B 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between 4.5 and 5.5 minutes corresponds to the AaLS particle.
  • FIG. 4 Addition of N-linked glycans allows N-terminal display of HRSV type A G CCD mini-protein.
  • A Schematic representation of the AaLS backbone with HRSV type A G CCD mini-protein fused to the N-terminus and with 0, 1, 2 or 3 N-linked glycosylation motifs (‘NST’) added within an internal loop.
  • B 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between 4.5 and 5.5 minutes corresponds to the AaLS particle.
  • C Amino acid alignment of the glycan loop insertion and the area surrounding it for two designs that both have three N-linked glycans, but differ in the design of the linker between the particle and glycans.
  • FIG. 5 Addition of N-linked glycans allows C-terminal display of HRSV type A G CCD mini-protein.
  • A Schematic representation of the AaLS backbone with HRSV type A G CCD mini-protein fused to the C-terminus and 0, 2 or 3 N-linked glycosylation motifs (‘NST’) added within an internal loop. Constructs RSV201064 and RSV201118 differ in the amino acid linker regions surrounding the N-linked glycosylation motifs.
  • B 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between 4.5 and 5.5 minutes corresponds to the AaLS particle.
  • FIG. 6 Addition of N-linked glycans allows internal display of HRSV type A G CCD miniprotein.
  • A Schematic representation of the AaLS backbone with HRSV type A G CCD peptide fused internally and flanked on either side of the G mini-protein with 0, 1 or 2 N-linked glycosylation motifs (‘NST’).
  • B 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between
  • FIG. 7 Addition of N-linked glycans allows dual display of HRSV G CCD mini-protein.
  • A Schematic representation of the AaLS backbone with HRSV type A G CCD peptide fused to the N-terminus and HRSV type B G CCD peptide fused to the C-terminus, with 1, 2, 3 or 4 N-linked glycosylation motifs (‘NST’) added within an internal loop.
  • B 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between 4.5 and 5.5 minutes corresponds to the AaLS particle.
  • FIG. 8 Addition of N-linked glycans allows C-terminal display of HRSV type B G CCD mini-protein.
  • A Schematic representation of the AaLS backbone with RSV-B G CCD peptide fused to the C-terminus and 1, 2, 3 or 4 N-linked glycans.
  • B 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between 4.5 and 5.5 minutes corresponds to the AaLS particle.
  • FIG. 9 Purification, characterization, and structure determination of an AaLS particle fused to HRSV type A G CCD mini-protein.
  • A Schematic representation of an AaLS design with HRSV type A G CCD mini-protein fused to the N-terminus of AaLS and with two N-linked glycosylation motifs (‘NST’) added within an internal loop.
  • B 450-A Analytical SEC profile of purified RSV200023 AaLS particle. The indicated peak (black arrow) between 4 and 6 minutes corresponds to the AaLS particle.
  • C Total yield of the purified particle and the weight of this particle calculated by Astra software compared to the calculated weight.
  • D The mass (in kDa) of the particle over time throughout the SEC -pattern of (B).
  • E Class averages of cryo-electron microscopy images of the purified particle of (B).
  • FIG. 10 Purification and characterization of a panel of AaLS particles fused to HRSV G CCD mini-protein.
  • A Schematic representation of a set of 5 AaLS designs with different HRSV G CCD mini-protein fusions that were selected for purification.
  • B-C 450-A Analytical SEC profiles of the purified AaLS particles displayed in (A). The indicated peak (black arrow) between 4 and 5 minutes corresponds to the AaLS particle.
  • D Total yield of the purified particles derived from constructs displayed in (A) and the weight of these particles calculated by Astra software compared to the theoretical weight.
  • E-F The mass (kDa) of the particles over time throughout the SEC-pattern of (B-C).
  • FIG. 11 Thermal stability of a panel of purified AaLS particles fused to HRSV G CCD miniprotein. Purified AaLS nanoparticles from Fig 10 were incubated for 30 minutes at different temperatures and then analyzed by 300- A analytical SEC to determine loss of particle content: 4°C (black line), 60°C (gray line), 70°C (light gray line), 80°C (dashed line) or 90°C (dotted line).
  • FIG. 12 Expression of AaLS particles with and without a C-tag.
  • A Schematic representation of a set of AaLS designs without a C-tag fused to the C-terminus.
  • B 500-A Analytical SEC profiles of AaLS particles derived from the constructs displayed in (A), as well as their counterparts with a C-tag, in crude cell supernatant. Designs with a C-tag (black, solid lines) were compared to their counterpart designs that lack a C-tag (dashed lines). The indicated peak (black arrow) between 4.5 and 5.5 minutes corresponds to the AaLS particle.
  • FIG. 13 Lectin-based purification and characterization of a panel of AaLS particles fused to HRSV G CCD mini-protein.
  • A Schematic representation of three tagless AaLS designs selected for purification with Galanthus nivalis lectin.
  • B 500-A Analytical SEC profiles of AaLS particles derived from monomers displayed in (A) in crude cell supernatant. The indicated peak (black arrow) between 4.5 and 5.5 minutes corresponds to the AaLS particle.
  • C 500-A Analytical SEC profiles of purified AaLS particles derived from the constructs displayed in (A).
  • D The mass (kDa) of the particles over time throughout the SEC-pattern of (C).
  • E Total yield of the purified particles derived from constructs displayed in (A) and the weight of these particles calculated by Astra software compared to the theoretical weight.
  • FIG. 14 Thermal stability of a panel of tag-less AaLS particles fused to HRSV G CCD miniprotein. 500-A SEC profiles after heat stress. Indicated purified proteins were incubated for 30 minutes at 4°C (black line), 60°C (gray line), 70°C (light gray line), 80°C (dashed line) or 90°C (dotted line). Samples were analyzed by 500-A analytical SEC to determine loss of particle content.
  • FIG. 15 A. RSV A G protein-binding antibody titers after prime induced by purified AaLS particles in mice.
  • RSV A G protein (Ga) binding antibody titers of serum samples isolated 20 days post prime were determined by ELISA using a peptide covering the CCD as target. Binding antibody titers we calculated relative to a standard amount of anti-G protein mAb taken along on every plate and displayed on a log 10 scale. Bars represent medium response per group.
  • Binding antibody titers we calculated relative to a standard amount of anti-G protein mAb taken along on every plate and displayed on a log 10 scale. Bars represent medium response per group.
  • C RSV B G protein (Gb) binding antibody titers of serum samples isolated 21 days post boost were determined by ELISA using a peptide covering the CCD as target. Binding antibody titers we calculated relative to a standard amount of anti-G protein mAb taken along on every plate and displayed on a log 10 scale. Bars represent medium response per group.
  • FIG. 16 Neutralization of HRS V A by mouse sera isolated 21 days after the boost in a virus neutralization assay on differentiated human airway epithelial cell cultures.
  • Primary human airway epithelial cells were cultured on an air-liquid interface and allowed to differentiate into a polarized tissue that mimics the human airway epithelium.
  • Infection with an HRSV type A virus encoding a GFP reporter in presence of a serial dilution of pooled mouse sera isolated 21 days after boost immunization leads to inhibition of HRSV infection and spread.
  • Controls taken along include uninfected cultures (‘Uninfected’), a virus-only control without serum (‘Virus only’), and a serum pool of phosphate buffered saline-vaccinated (‘Mock vaccinated’) mice at a 50-fold dilution.
  • a vaccine against RSV infection is currently not yet available.
  • One potential approach to producing a vaccine is providing a subunit vaccine based on RSV antigens.
  • Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines.
  • the development of such immunogens is often complicated by inefficiencies in their production.
  • the present invention provides RSV immunogens comprising at least one recombinant RSV G protein ectodomain, or fragment thereof, linked to a protein nanoparticle subunit.
  • the immunogens according to the invention are easy to produce since the designed fusion proteins self-assemble spontaneously into a particle that displays the immunogenic RSV G protein ectodomain or fragments thereof (e.g. the RSV G central conserved domain) on the surface.
  • Particle-based vaccines are known to be superior antigens and for the small RSV G domain, display on a larger particle is even more important for the induction of an immune response.
  • the protein nanoparticle subunit is a lumazine synthase.
  • Lumazine synthase has been used a carrier protein or nanoparticle by genetic linkage or fusion of proteins or peptides to the lumazine synthase.
  • the displayed proteins/peptides typically are covalently linked with the carrier protein.
  • 6,7-Dimethyl-8-ribityllumazine synthase (subsequently designated lumazine synthase) is an enzyme that catalyzes the penultimate step of vitamin B2 biosynthesis in microorganisms and plants.
  • Lumazine synthases from certain bacteria e.g. Escherichia coli, Bacillus subtilis, Aquifex aeolicus
  • Lumazine synthases from different microorganisms can be expressed efficiently in recombinant strains of Escherichia coli and Bacillus subtilis and it is assumed that the quaternary structures of the enzymes are highly similar.
  • the recombinant proteins can be isolated in high yield.
  • the N-terminus as well as the C-terminus are located at the surface of the icosahedral capsid molecule.
  • the lumazine synthase is an Aquifex aeolicus lumazine synthase (AaLS).
  • AaLS particles are known to be very stable.
  • the lumazine synthase comprises at least one introduced N-linked glycan.
  • the at least one glycan is introduced between the amino acid residues 70 and 71 of a lumazine synthase. It is to be understood that according to the present invention the numbering of the positions of the amino acid residues is according to the numbering of the amino acids in SEQ ID NO: 1.
  • the lumazine synthase comprises at least two introduced N- linked glycans.
  • the at least two glycans are introduced between the amino acid residues 70 and 71 of a lumazine synthase.
  • lumazine synthase has been used a carrier protein or nanoparticle by genetic linkage of proteins or peptides to the lumazine synthase.
  • the development of such fusion molecules is often complicated by inefficiencies in their production.
  • the glycans can be introduced by any suitable means known to those skilled in the art.
  • PCR common DNA methods
  • NXS/T N-linked glycosylation
  • the motif may or may not be flanked by short linkers (Gly/Ser).
  • the lumazine synthase comprises an amino acid sequence of SEQ ID NO: 1 or 2.
  • the lumazine synthase comprises a mammalian signal sequence for expression and secretion in a cell.
  • the immunogens comprise at least one recombinant RSV G protein ectodomain, or fragment thereof, linked to the protein nanoparticle subunit.
  • the RSV G protein ectodomain or fragment is from a human RSV G protein.
  • the RSV G ectodomain or fragment is derived from a G protein of a human RSV.
  • the RSV G protein ectodomain or fragment is from a bovine RSV G protein.
  • the RSV G ectodomain or fragment is derived from a G protein of a bovine RSV.
  • the RSV G protein ectodomain fragment is an RSV G central conserved domain (CCD) peptide.
  • CCD RSV G central conserved domain
  • the RSV G protein ectodomain can be an RSV Ga or RSV Gb protein ectodomain.
  • the RSV G protein ectodomain fragment comprises an amino acid sequence selected from the group consisting of DFHFEVFNFVP (SEQ ID NO: 104), DYHFEVFNFVP (SEQ ID NO: 105) , NDFHFEVFNFVPCSICSNNPTCWAICKRIPN (SEQ ID NO: 106) and DDYHFEVFNFVPCSICGNNQLCKSICKTIPS (SEQ ID NO: 107), or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 97%, most preferably at least 99% sequence identity to any of SEQ ID NO: 104-107.
  • the RSV G protein ectodomain fragment is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 3-6, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 97%, most preferably at least 99% sequence identity to any of SEQ ID NO: 3-6.
  • the at least one RSV G protein ectodomain or fragment thereof is linked to the N-terminal side of the lumazine synthase. In other embodiments, the at least one RSV G protein ectodomain or fragment thereof is linked to the C-terminal side of the lumazine synthase. According to the invention, it is also possible that the at least one RSV G protein ectodomain or fragment thereof is linked to the lumazine synthase at an internal fusion site.
  • the immunogen of the invention in certain embodiments comprise at least two RSV G proteins ectodomains or fragments thereof, i.e. one linked to N-terminal side and one linked to the C-terminal side, and/or one at an internal fusion site.
  • the at least two RSV G protein ectodomains or fragments thereof could be the same RSV G protein ectodomains or fragments or different.
  • the RSV G protein ectodomains or fragments are different RSV G proteins or fragments, i.e. one RSV G ectodomain or fragment from an RSV A strain and one from an RSV
  • the immunogens comprise a purification tag, including but not limited to a HIS-Tag, strep-tag or c-tag.
  • a His-Tag or polyhistidine-tag is an amino acid motif in proteins that consists of at least five histidine (H) residues;
  • a strep-tag is an amino acid sequence that consist of 8 residues (WSHPQFEK (SEQ ID NO: 108));
  • a c-tag is an amino acid motif that consists of 4 residues (EPEA; SEQ ID NO: 109).
  • the tags are often at the N- or C-terminus of a protein and are generally used for purification purposes.
  • the immunogens comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 8-14, 16-63, 67-69, and 72-103, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 97%, most preferably at least 99% sequence identity to any of these sequences.
  • nucleic acid molecule refers to a polymeric form of nucleotides (i.e. polynucleotides) and includes both DNA (e.g. cDNA, genomic DNA) and RNA, and synthetic forms and mixed polymers of the above. It is to be understood that numerous different nucleic acid molecules can encode the same protein as a result of the degeneracy of the genetic code. It is also understood that skilled persons can, using routine techniques, make nucleotide substitutions that do not affect the protein sequence encoded by the polynucleotides described there to reflect the codon usage of any particular host organism in which the proteins are to be expressed.
  • nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns. Sequences herein are provided from 5' to 3' direction, as custom in the art.
  • the nucleic acid molecules encoding the proteins according to the invention are codon-optimized for expression in cells, preferably human cells, bacterial and/or yeast cells. Methods of codon-optimization are known and have been described previously (e.g. WO 96/09378 for mammalian cells).
  • a sequence is considered codon-optimized if at least one non-preferred codon as compared to a wild type sequence is replaced by a codon that is more preferred.
  • a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon.
  • the frequency of codon usage for a specific organism can be found in codon frequency tables, such as in http://www.kazusa.or.jp/codon.
  • nucleic acid sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may or may not include introns.
  • Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScripts, Invitrogen, Eurofins).
  • the invention also provides vectors comprising a nucleic acid molecule as described above.
  • a nucleic acid molecule according to the invention thus is part of a vector.
  • Such vectors can easily be manipulated by methods well known to the person skilled in the art and can for instance be designed for being capable of replication in prokaryotic and/or eukaryotic cells.
  • the vector used can be any vector that is suitable for cloning DNA and that can be used for expression of a nucleic acid molecule of interest.
  • Suitable vectors according to the invention are viral vectors, e.g. adeno vectors, alphavirus, paramyxovirus, vaccinia virus, herpes virus, retroviral vectors etc.
  • Host cells comprising the nucleic acid molecules encoding the immunogens form also part of the invention.
  • the G particles may be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g. Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER. C6 cells, or yeast, or bacterial cells.
  • the cells are from a multicellular organism, in certain embodiments they are of vertebrate or invertebrate origin.
  • the cells are mammalian cells.
  • the cells are human cells.
  • the production of a recombinant proteins, such the immunogens of the invention, in a host cell comprises the introduction of a heterologous nucleic acid molecule encoding the protein in expressible format Y1 into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the immunogen in said cell.
  • the nucleic acid molecule encoding an immunogen in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like.
  • enhancer(s) promoter
  • polyadenylation signal and the like.
  • Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed.
  • the nucleic acid molecules according to the invention are operably linked to a promoter, including, but not limited to CMV, chicken beta actin.
  • the invention further provides immunogenic compositions comprising an immunogen and/or a nucleic acid molecule, and/or a vector, as described above, and one or more pharmaceutically acceptable excipients.
  • the invention may employ pharmaceutical compositions comprising an immunogen, a nucleic acid molecule and/or a vector as described herein, and a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered.
  • pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R.
  • the immunogens proteins, or nucleic acid molecules preferably are formulated and administered as a sterile solution although it may also be possible to utilize lyophilized preparations.
  • Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers.
  • the pH of the solution generally is in the range of pH 3.0 to 9.5, e.g.
  • the immunogens typically are in a solution having a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt.
  • stabilizing agent may be present, such as albumin.
  • detergent is added.
  • the immunogens proteins may be formulated into an injectable preparation.
  • a composition according to the invention further comprises one or more adjuvants.
  • Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant.
  • the terms “adjuvant” and “immune stimulant” are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system.
  • an adjuvant is used to enhance an immune response to the immunogens proteins of the invention.
  • suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in- water compositions), including squalene-water emulsions, such as MF59 (see e.g.
  • WO 90/14837 saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP- ribosylating bacterial toxins or mutants thereof, such as E.
  • MPL monophosphoryl lipid A
  • 3dMPL 3-O-deacylated MPL
  • CpG-motif containing oligonucleotides such as E.
  • compositions of the invention comprise aluminium as an adjuvant, e.g. in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or combinations thereof, in concentrations of 0.05 - 5 mg, e.g. from 0.075-1.0 mg, of aluminium content per dose.
  • the invention further provides methods for generating an immune response to RSV G in a subject, comprising administering to the subject an effective amount of the immunogenic composition as described herein to generate the immune response.
  • the invention also provides the use of an immunogen, a nucleic acid molecule, and/or a vector, according to the invention, for inducing an immune response against RSV G in a subject.
  • the invention also provides methods for treating or preventing a RSV infection in a subject, comprising administering to the subject a therapeutically effective amount of the immunogenic composition as described herein, thereby treating or preventing RSV infection in the subject.
  • the prevention and/or treatment may be targeted at patient groups that are susceptible RSV infection.
  • patient groups include, but are not limited to e.g., the elderly (e.g. > 50 years old, > 60 years old, and preferably > 65 years old), the young (e.g. ⁇ 5 years old, ⁇ 1 year old), pregnant women (for maternal immunization), hospitalized patients and patients who have been treated with an antiviral compound but have shown an inadequate antiviral response.
  • a therapeutically effective amount refers to an amount of an immunogen, nucleic acid molecule or vector, that is effective for preventing, ameliorating and/or treating a disease or condition resulting from infection by RSV.
  • Prevention encompasses inhibiting or reducing the spread of RSV, or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection by RSV.
  • Amelioration as used in herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of influenza infection.
  • the invention provides methods for making a vaccine against respiratory syncytial virus (RSV), comprising providing an immunogen, nucleic acid or vector according to the invention and formulating it into a pharmaceutically acceptable composition.
  • RSV respiratory syncytial virus
  • the term "vaccine” refers to an agent or composition containing an active component effective to induce a certain degree of immunity in a subject against a certain pathogen or disease, which will result in at least a decrease (up to complete absence) of the severity, duration or other manifestation of symptoms associated with infection by the pathogen or the disease.
  • the vaccine comprises an effective amount of an immunogen and/or a nucleic acid molecule, and/or a vector comprising said nucleic acid molecule, which results in an immune response against the G protein of RSV.
  • the term “vaccine” implies that it is a pharmaceutical composition, and thus typically includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not comprise further active ingredients. In certain embodiments it may be a combination vaccine that further comprises other components that induce an immune response, e.g. against other proteins of RSV and/or against other infectious agents.
  • the administration of further active components may for instance be done by separate administration or by administering combination products of the vaccines of the invention and the further active components.
  • the invention further provides methods for detecting or isolating an RSV G binding antibody in a subject, comprising: providing an effective amount of the immunogen as described herein; contacting a biological sample from the subject with the immunogen under conditions sufficient to form an immune complex between the immunogen and the RSV G binding antibody; and detecting the immune complex, thereby detecting or isolating the RSV G binding antibody in the subject.
  • the immunogens of the invention may be used as diagnostic tool, for example to test the immune status of an individual by establishing whether there are antibodies in the serum of such individual capable of binding to the immunogen of the invention.
  • the invention thus also relates to an in vitro diagnostic method for detecting the presence of an RSV infection in a patient said method comprising the steps of a) contacting a biological sample obtained from said patient with a protein according to the invention; and b) detecting the presence of antibody-protein complexes.
  • the amino acid positions are given in reference to the sequence of lumazine synthase of SEQ ID NO: 1.
  • the wording “the amino acid residue at position “x” of the lumazine synthase thus means the amino acid corresponding to the amino acid at position “x” in the lumazine synthase of SEQ ID NO: 1.
  • Sequence alignments can be done using methods well known in the art, e.g. by CLUSTALW, Bioedit or CLC Workbench.
  • nucleotide sequences are provided from 5’ to 3’ direction, and amino acid sequences from N-terminus to C-terminus, as custom in the art.
  • An amino acid according to the invention can be any of the twenty naturally occurring (or ‘standard’ amino acids).
  • the standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties such as cysteine, that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that induces turns of the protein backbone, and glycine that is more flexible than other amino acids. Table 2 shows the abbreviations and properties of the standard amino acids.
  • EXAMPLE 1 BRSV G CCD, but not HRSV G CCD mini-protein can be fused to AaLS particles while retaining particle expression.
  • Plasmids corresponding to the wildtype Aquifex aeolicus Lumazine Synthase (AaLS) monomer that were equipped with a human CD 5 signal peptide to allow secretion into the cell supernatant and with a C-terminal C-tag were synthesized and codon-optimized at Genscript (FIG. 1). Indicated in figure 1 are also the three locations where peptides ( or ‘mini-proteins’) can be fused to the AaLS backbone. A series of AaLS particles fused to either the human RSV
  • the AaLS monomers were detected in crude supernatants using western blotting with a C-tag specific nanobody (CaptureSelectTM Biotin Anti-C-tag Conjugate, ThermoFisher) (Fig 2D). Both AaLS backbones (RSV190362 and RSV191645) showed a band at the expected height. In contrast, there was no AaLS expression detectable for the particles fused to HRSV G CCD miniprotein (RSV191646-48). For BRSV G CCD mini-protein-fused AaLS particles (RSV191649, RSV191650 and RSV191651) there wass a distinct band at the expected height.
  • the cleared crude cell culture supernatants were applied to a 500 A column, (Sepax Cat#235500-4615) with the corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min.
  • running buffer 150 mM sodium phosphate, 50 mM NaCl, pH 7.0
  • pMALS detectors were offline and analytical SEC data was analyzed using
  • EXAMPLE 2 Introduction of N-linked glycans boosts AaLS particle expression
  • Either 1 or 2 N-linked glycans were introduced at position 71 of the AaLS particle backbone (RSV191645) using standard methods widely known within the field involving site- directed mutagenesis and PCR and sequenced. This resulted in constructs RSV201131 (1 N- linked glycan) and RSV201132 (2 N-linked glycans). These two proteins, as well as the backbone without any glycans were expressed in the expi293F cell system.
  • Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 5 minutes at 300 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.
  • the cleared crude cell culture supernatants were applied to a 500 A column, (Sepax Cat#235500-4615) with the corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min.
  • running buffer 150 mM sodium phosphate, 50 mM NaCl, pH 7.0
  • pMALS detectors were offline and analytical SEC data was analyzed using Chromeleon 7.2.8.0 software package.
  • Fig 3A The empty backbone particle (Fig 3A) yielded a nanoparticle that could be detected in raw supernatant (Fig 3B).
  • Fig 3B The shift in retention times is caused by the increased diameter that is a consequence of the added amino acids and glycans.
  • EXAMPLE 3 Introduction of N-linked glycans allows expression of AaLS particles fused to HRSV G CCD mini-protein.
  • the series of AaLS particles fused to the HRSV G CCD mini-protein that did not express particles were equipped with varying amounts of N-linked glycans (Fig 4, N- terminal fusions; Fig 5, C-terminal fusions; Fig 6, internal fusions). Additionally, so-called ‘dual particles’ were designed that have HRSV type A G CCD mini-protein on the N-terminus, and HRSV type B G CCD mini-protein on the C-terminus (Fig 7). Additionally, particles were designed that have HRSV type B G CCD mini-protein on the C-terminus (Fig 8).
  • the cleared crude cell culture supernatants were applied to a 500 A column, (Sepax Cat#235500-4615) with the corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min.
  • running buffer 150 mM sodium phosphate, 50 mM NaCl, pH 7.0
  • pMALS detectors were offline and analytical SEC data was analyzed using Chromeleon 7.2.8.0 software package.
  • EXAMPLE 4 AaLS particles with N-linked glycans displaying HRS G CCD mini-proteins can be purified and have the desired structure. To confirm that the AaLS designs with glycans indeed formed the correct nanoparticles, one of the designs was purified to allow structure determination by EM. The design selected was RSV200023 (Fig 9A), which has two linked glycans and has the HRSV type A G CCD miniprotein fused to the N-terminus. This selected design was expressed in the expi293F cell system. Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 5 days at 37°C and 8% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 10 minutes at 600 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.
  • the supernatant was used in purification, where first C-tag affinity purification was performed with Capture Select C-tag XL (Thermo Scientific Cat#494307205). The eluted protein was thereafter further purified by size exclusion with Superdex200 (GE/Cytiva Cat# 28-9909- 44).
  • EXAMPLE 5 AaLS particles with N-linked glycans displaying HRS G CCD mini-proteins can be purified and are stable.
  • Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 5 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 10 minutes at 600 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.
  • the supernatant was used in purification, where first C-tag affinity purification was performed with Capture Select C-tag XL (Thermo Scientific Cat#494307205).
  • the eluted protein was thereafter further purified by size exclusion with Superdex200 (GE/Cytiva Cat# 28-9909- 44), followed by Superose 6 (GE/Cytiva Cat# 17-5172-01).
  • EXAMPLE 6 Tag-less AaLS particles with N-linked glycans displaying HRSV G CCD miniproteins have lower expression.
  • Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 5 minutes at 300 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.
  • the cleared crude cell culture supernatants were applied to a 500A column, (Sepax Cat#235500- 4615) with the corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min.
  • running buffer 150 mM sodium phosphate, 50 mM NaCl, pH 7.0
  • pMALS detectors were offline and analytical SEC data was analyzed using Chromeleon 7.2.8.0 software package.
  • EXAMPLE 7 Tag-less AaLS particles with N-linked glycans displaying HRSV G CCD miniproteins can be purified and are stable.
  • a set of designs that differ in the number of glycans and the length of the G miniprotein were selected for lectin-based purification.
  • the selected designs were expressed in the expi293F cell system.
  • Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 5 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 10 minutes at 600 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.
  • the supernatant was used in purification, where first Lectin-based affinity purification was performed with Galanthus nivalus lectin (Vector Labs Cat#AL-1243). The eluted protein was thereafter further purified by size exclusion with Superdex200 (GE/Cytiva Cat# 28-9909- 44), followed by Superose 6 (GE/Cytiva Cat# 29-0915-96). Analytical 500 A SEC-MALS was performed on the purified products and analyzed using Chromeleon 7.2.8.0 software package and Astra 7.3.2 to confirm particle formation (Fig 13C-E). Molecular weights were as expected. Yields are shown in Fig 13E.
  • EXAMPLE 8 AaLS particles with N-linked glycans displaying HRSV G CCD mini-proteins are immunogenic in mice, and sera are neutralizing in a virus neutralization assay on primary human airway epithelial cell cultures.
  • the immunogenicity of 5 AaLS particles fused to RSV Ga and/or RSV-Gb peptides were investigated.
  • the RSV G peptides were genetically fused N- or C- terminally to AalS and contained 2 or 3 added glycans required for expression (RSV201051, RSV201064 and RSV201214).
  • RSV201063 the internal fusion of G-peptide (RSV201063) is immunogenic. Since part of the G-peptide (CCD) is conserved between RSV-A and B, some level of crossreactivity was expected.
  • Serum samples were isolated 3 weeks after the prime immunization (day 20) and 3 weeks after the boost (day 42).
  • RSV-A (day 20 and 42 serum) and RSV-B (day 42 serum) G protein (Ga and Gb, respectively) binding antibody titers were measured with an ELISA. Biotinylated Ga and Gb peptides covering the CCD were captured by immobilized Streptavidin. After a washing step, two-fold serially diluted serum samples were added. After one-hour, unbound serum antibodies were washed away, and bound antibodies (IgG) were detected by anti-mouse- HRP followed by a fluorescence readout. Binding antibody titers were calculated relative to a standard concentration of anti-G mAb (MAB858-2, clone 131-2G, Sigma-Aldrich).
  • SEQ ID NO: 1 wildtype sequence
  • SEQ ID NO: 2 (2 point mutations for expression in mammalian cells) 154AA
  • SEQ ID NO: 7 RSV190362
  • SEQ ID NO: 8 RSV190363
  • SEQ ID NO: 10 RSV190365
  • SEQ ID NO: 12 RSV190367
  • SEQ ID NO: 13 RSV190368
  • SEQ ID NO: 14 RSV190369
  • SEQ ID NO: 16 RSV191646
  • SEQ ID NO: 20 RSV191650
  • SEQ ID NO: 21 RSV191651
  • SEQ ID NO: 22 RSV200020
  • SEQ ID NO: 24 RSV200022
  • SEQ ID NO: 27 RSV200025
  • SEQ ID NO: 30 RSV200028
  • SEQ ID NO: 36 RSV201051
  • SEQ ID NO: 42 RSV201057
  • SEQ ID NO: 45 RSV201060
  • SEQ ID NO: 46 RSV201061
  • SEQ ID NO: 48 RSV201063
  • SEQ ID NO: 50 RSV201214
  • SEQ ID NO: 58 RSV201125
  • SEQ ID NO: 62 RSV201129
  • SEQ ID NO: 68 RSV210158
  • SEQ ID NO: 72 RSV210162
  • SEQ ID NO: 77 RSV210167 MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
  • SEQ ID NO: 80 RSV210981
  • GGSGGSEPEA SEQ ID NO: 87: RSV210988
  • SEQ ID NO: 100 RSV211001
  • SEQ ID NO: 101 RSV211002

Abstract

The present invention relates to immunogens comprising at least one recombinant RSV G protein ectodomain or fragment thereof linked to a protein nanoparticle subunit, and to used thereof.

Description

RSV immunogens
The present invention relates to immunogens, proteins, nucleic acids and vectors, as well as to the use thereof, e.g. as pharmaceutical agents, and to vaccines containing any of those.
Background
Human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus of the family Pneumoviridae. There are two primary RSV subtypes: subtype A and subtype B. RSV replicates in the upper respiratory track and then spreads to the lower airways leading to bronchiolitis or pneumonia. The virus causes inflammation, edema of the airways, increased mucus production, and breakdown of respiratory epithelium.
An estimated 64 million cases of respiratory illness and 160,000 deaths worldwide are attributable to RSV-induced disease. Severe RSV infection occurs most often in children and infants, especially in premature infants. Underlying health problems such as chronic lung disease or congenital heart disease can significantly increase the risk of serious illness. RSV infections also can cause serious illness in the elderly, individuals with chronic pulmonary disease and in immunocompromised adults, such as bone marrow transplant recipients.
Several approaches to the prevention and treatment of RSV infection have been investigated. Intravenous immunoglobulin (RSV-IGIV; RespiGam®) isolated from donors, and the monoclonal antibody palivizumab (SYNAGIS®) have been approved for RSV prophylaxis in high-risk premature infants. A vaccine or commercially available treatment for RSV, however, is not yet available. Only ribavirin is approved for treatment of RSV infection. In order to be effective for treatment of RSV infection, high doses, repeated administrations and/or large volumes of antibody products, such as palivizumab, are required due to low effectivity.
RSV has two major surface glycoproteins, F and G. The F protein mediates fusion, allowing entry of the virus into the cell cytoplasm and facilitating the formation of syncytia in vitro. The F protein sequence is well (~ 90%) conserved among RSV strains (Johnson and Collins, J Gen Virol. (1988) 69: 2623-2628). The sole marketed monoclonal antibody palivizumab is directed against the F protein of RSV.
The G protein of RSV is a surface protein that is heavily glycosylated and functions as the attachment protein. In contrast to the F protein, the G protein is quite variable across strains except for a central conserved domain (CCD), comprising amino acid residues 153-184 of the G protein of RSV A2 strain or corresponding amino acid residues in other strains. Both the central conserved domain and adjacent regions (residues 145-193) are flanked by rigid and heavy Ci- glycosylated mucin-like regions. The N-terminal half of the central conserved domain contains a small region that is conserved among more than 700 strains. The C-terminal half contains 4 conserved cysteines that are connected in a 1-4, 2-3 topology and folds into a cystine noose.
In view of the severity of the respiratory illness caused by RSV, in particular in young children and in the elderly, there is an ongoing need for effective means to prevent and/or treat RSV infection.
Summary of the Invention
In a first aspect, the present invention provides immunogens comprising at least one recombinant RSV G protein ectodomain, or fragment thereof, linked to a protein nanoparticle subunit, as well as nucleic acid molecules encoding the immunogens of the invention. In another aspect, the invention provides methods for generating an immune response to
RSV G in a subject, comprising administering to the subject an effective amount of the immunogenic composition according to the invention.
In a further aspect, the invention provides methods for treating or preventing a RSV infection in a subject, comprising administering to the subject a therapeutically effective amount of the immunogenic composition according to the invention, thereby treating or preventing RSV infection in the subject.
In yet another aspect, the invention provides methods for detecting or isolating an RSV G binding antibody in a subject, comprising providing an effective amount of the immunogen according to the invention; contacting a biological sample from the subject with the immunogen under conditions sufficient to form an immune complex between the immunogen and the RSV G binding antibody; and detecting the immune complex, thereby detecting or isolating the RSV G binding antibody in the subject.
Brief description of the Figures
FIG 1: Schematic representation of the AaLS expression construct. The N-terminal signal peptide (‘SP’) is cleaved off during protein maturation. On the C-terminus a C-tag (‘EPEA’) is fused to the monomer which can be used for detection and purification purposes. The RSV G CCD mini-protein or other (mini-)proteins can potentially be incorporated into the monomer at three different sites: i) the ‘N-terminal fusion site’ between the SP and the AaLS monomer, ii) the ‘Internal fusion site’ at position 71 within the AaLS monomer, and iii) the ‘C-terminal fusion site’ at the C-terminus of the AaLS monomer (in the example shown the mini-protein is followed by the C-tag).
FIG 2: Design and expression in supernatant of AaLS fused to RSV G CCD peptides. (A) Schematic representation of two AaLS backbones. RSV190362 is a wildtype AaLS monomer, in RSV191645 the original initiating methionine at amino acid position 25, directly after the signal peptide, was removed. (B) Schematic representation of AaLS monomers fused to the human RSV (HRSV) type A G CCD, either internally (construct RSV191646), N-terminally (construct RSV191647) or C-terminally (construct RS VI 91648) as based on the RSV191645 backbone. (C) Schematic representation of AaLS monomers fused to the bovine RSV (BRSV) G CCD, either internally (construct RSV191649), N-terminally (construct RSV191650) or C-terminally (construct RSV191651) as based on the RSV191645 backbone. (D) Western blot on reduced supernatants of expiHEK293 cells transfected with the different AaLS constructs depicted in Fig 2A-C. C-tag-based detection was used to confirm expression of the AaLS proteins. (E-G) 500-A Analytical size-exclusion chromatography (SEC) profiles of supernatants of expiHEK293 cells transfected with the different AaLS constructs depicted in Fig 2A-C. The peak between 4.5 and 5.5 minutes corresponds to the AaLS particle (indicated with black arrow).
FIG. 3: Addition of N-linked glycans boosts AaLS particle expression. (A) Schematic representation of three AaLS constructs with 0, 1 or 2 N-linked glycosylation motifs (‘NST’) added within an internal loop. Constructs are based on backbone RSV191645. (B) 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between 4.5 and 5.5 minutes corresponds to the AaLS particle. FIG. 4: Addition of N-linked glycans allows N-terminal display of HRSV type A G CCD mini-protein. (A) Schematic representation of the AaLS backbone with HRSV type A G CCD mini-protein fused to the N-terminus and with 0, 1, 2 or 3 N-linked glycosylation motifs (‘NST’) added within an internal loop. (B) 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between 4.5 and 5.5 minutes corresponds to the AaLS particle. (C) Amino acid alignment of the glycan loop insertion and the area surrounding it for two designs that both have three N-linked glycans, but differ in the design of the linker between the particle and glycans.
FIG. 5: Addition of N-linked glycans allows C-terminal display of HRSV type A G CCD mini-protein. (A) Schematic representation of the AaLS backbone with HRSV type A G CCD mini-protein fused to the C-terminus and 0, 2 or 3 N-linked glycosylation motifs (‘NST’) added within an internal loop. Constructs RSV201064 and RSV201118 differ in the amino acid linker regions surrounding the N-linked glycosylation motifs. (B) 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between 4.5 and 5.5 minutes corresponds to the AaLS particle.
FIG. 6: Addition of N-linked glycans allows internal display of HRSV type A G CCD miniprotein. (A) Schematic representation of the AaLS backbone with HRSV type A G CCD peptide fused internally and flanked on either side of the G mini-protein with 0, 1 or 2 N-linked glycosylation motifs (‘NST’). (B) 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between
4.5 and 5.5 minutes corresponds to the AaLS particle.
FIG. 7: Addition of N-linked glycans allows dual display of HRSV G CCD mini-protein. (A) Schematic representation of the AaLS backbone with HRSV type A G CCD peptide fused to the N-terminus and HRSV type B G CCD peptide fused to the C-terminus, with 1, 2, 3 or 4 N-linked glycosylation motifs (‘NST’) added within an internal loop. (B) 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between 4.5 and 5.5 minutes corresponds to the AaLS particle.
FIG. 8: Addition of N-linked glycans allows C-terminal display of HRSV type B G CCD mini-protein. (A) Schematic representation of the AaLS backbone with RSV-B G CCD peptide fused to the C-terminus and 1, 2, 3 or 4 N-linked glycans. (B) 500-A Analytical SEC profiles of AaLS constructs shown in (A) in crude cell supernatant 3 days after transfection. The peak indicated with a black arrow between 4.5 and 5.5 minutes corresponds to the AaLS particle.
FIG. 9: Purification, characterization, and structure determination of an AaLS particle fused to HRSV type A G CCD mini-protein. (A) Schematic representation of an AaLS design with HRSV type A G CCD mini-protein fused to the N-terminus of AaLS and with two N-linked glycosylation motifs (‘NST’) added within an internal loop. (B) 450-A Analytical SEC profile of purified RSV200023 AaLS particle. The indicated peak (black arrow) between 4 and 6 minutes corresponds to the AaLS particle. (C) Total yield of the purified particle and the weight of this particle calculated by Astra software compared to the calculated weight. (D) The mass (in kDa) of the particle over time throughout the SEC -pattern of (B). (E) Class averages of cryo-electron microscopy images of the purified particle of (B).
FIG. 10: Purification and characterization of a panel of AaLS particles fused to HRSV G CCD mini-protein. (A) Schematic representation of a set of 5 AaLS designs with different HRSV G CCD mini-protein fusions that were selected for purification. (B-C) 450-A Analytical SEC profiles of the purified AaLS particles displayed in (A). The indicated peak (black arrow) between 4 and 5 minutes corresponds to the AaLS particle. (D) Total yield of the purified particles derived from constructs displayed in (A) and the weight of these particles calculated by Astra software compared to the theoretical weight. (E-F) The mass (kDa) of the particles over time throughout the SEC-pattern of (B-C).
FIG. 11: Thermal stability of a panel of purified AaLS particles fused to HRSV G CCD miniprotein. Purified AaLS nanoparticles from Fig 10 were incubated for 30 minutes at different temperatures and then analyzed by 300- A analytical SEC to determine loss of particle content: 4°C (black line), 60°C (gray line), 70°C (light gray line), 80°C (dashed line) or 90°C (dotted line).
FIG. 12: Expression of AaLS particles with and without a C-tag. (A) Schematic representation of a set of AaLS designs without a C-tag fused to the C-terminus. (B) 500-A Analytical SEC profiles of AaLS particles derived from the constructs displayed in (A), as well as their counterparts with a C-tag, in crude cell supernatant. Designs with a C-tag (black, solid lines) were compared to their counterpart designs that lack a C-tag (dashed lines). The indicated peak (black arrow) between 4.5 and 5.5 minutes corresponds to the AaLS particle.
FIG. 13: Lectin-based purification and characterization of a panel of AaLS particles fused to HRSV G CCD mini-protein. (A) Schematic representation of three tagless AaLS designs selected for purification with Galanthus nivalis lectin. (B) 500-A Analytical SEC profiles of AaLS particles derived from monomers displayed in (A) in crude cell supernatant. The indicated peak (black arrow) between 4.5 and 5.5 minutes corresponds to the AaLS particle. (C) 500-A Analytical SEC profiles of purified AaLS particles derived from the constructs displayed in (A). (D) The mass (kDa) of the particles over time throughout the SEC-pattern of (C). (E) Total yield of the purified particles derived from constructs displayed in (A) and the weight of these particles calculated by Astra software compared to the theoretical weight.
FIG. 14: Thermal stability of a panel of tag-less AaLS particles fused to HRSV G CCD miniprotein. 500-A SEC profiles after heat stress. Indicated purified proteins were incubated for 30 minutes at 4°C (black line), 60°C (gray line), 70°C (light gray line), 80°C (dashed line) or 90°C (dotted line). Samples were analyzed by 500-A analytical SEC to determine loss of particle content.
FIG. 15: A. RSV A G protein-binding antibody titers after prime induced by purified AaLS particles in mice. RSV A G protein (Ga) binding antibody titers of serum samples isolated 20 days post prime were determined by ELISA using a peptide covering the CCD as target. Binding antibody titers we calculated relative to a standard amount of anti-G protein mAb taken along on every plate and displayed on a log 10 scale. Bars represent medium response per group. B. RSV A G protein (Ga) binding antibody titers of serum samples isolated 21 days post boost were determined by ELISA using a peptide covering the CCD as target. Binding antibody titers we calculated relative to a standard amount of anti-G protein mAb taken along on every plate and displayed on a log 10 scale. Bars represent medium response per group. C. RSV B G protein (Gb) binding antibody titers of serum samples isolated 21 days post boost were determined by ELISA using a peptide covering the CCD as target. Binding antibody titers we calculated relative to a standard amount of anti-G protein mAb taken along on every plate and displayed on a log 10 scale. Bars represent medium response per group.
FIG. 16: Neutralization of HRS V A by mouse sera isolated 21 days after the boost in a virus neutralization assay on differentiated human airway epithelial cell cultures. Primary human airway epithelial cells were cultured on an air-liquid interface and allowed to differentiate into a polarized tissue that mimics the human airway epithelium. Infection with an HRSV type A virus encoding a GFP reporter in presence of a serial dilution of pooled mouse sera isolated 21 days after boost immunization leads to inhibition of HRSV infection and spread. Controls taken along include uninfected cultures (‘Uninfected’), a virus-only control without serum (‘Virus only’), and a serum pool of phosphate buffered saline-vaccinated (‘Mock vaccinated’) mice at a 50-fold dilution. Detailed description of the Invention
As described above, a vaccine against RSV infection is currently not yet available. One potential approach to producing a vaccine is providing a subunit vaccine based on RSV antigens. Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production.
The present invention provides RSV immunogens comprising at least one recombinant RSV G protein ectodomain, or fragment thereof, linked to a protein nanoparticle subunit. The immunogens according to the invention are easy to produce since the designed fusion proteins self-assemble spontaneously into a particle that displays the immunogenic RSV G protein ectodomain or fragments thereof (e.g. the RSV G central conserved domain) on the surface. Particle-based vaccines are known to be superior antigens and for the small RSV G domain, display on a larger particle is even more important for the induction of an immune response.
In certain embodiments, the protein nanoparticle subunit is a lumazine synthase.
Lumazine synthase has been used a carrier protein or nanoparticle by genetic linkage or fusion of proteins or peptides to the lumazine synthase. The displayed proteins/peptides typically are covalently linked with the carrier protein.
6,7-Dimethyl-8-ribityllumazine synthase (subsequently designated lumazine synthase) is an enzyme that catalyzes the penultimate step of vitamin B2 biosynthesis in microorganisms and plants. Lumazine synthases from certain bacteria (e.g. Escherichia coli, Bacillus subtilis, Aquifex aeolicus) represent highly symmetrical, icosahedral complexes of 60 subunits with a molecular weight of approximately 1 M Dalton. Lumazine synthases from different microorganisms can be expressed efficiently in recombinant strains of Escherichia coli and Bacillus subtilis and it is assumed that the quaternary structures of the enzymes are highly similar. The recombinant proteins can be isolated in high yield. The N-terminus as well as the C-terminus are located at the surface of the icosahedral capsid molecule.
In certain preferred embodiments, the lumazine synthase is an Aquifex aeolicus lumazine synthase (AaLS). AaLS particles are known to be very stable.
In certain preferred embodiments, the lumazine synthase comprises at least one introduced N-linked glycan. Preferably, the at least one glycan is introduced between the amino acid residues 70 and 71 of a lumazine synthase. It is to be understood that according to the present invention the numbering of the positions of the amino acid residues is according to the numbering of the amino acids in SEQ ID NO: 1.
In further embodiments, the lumazine synthase comprises at least two introduced N- linked glycans. Preferably, the at least two glycans are introduced between the amino acid residues 70 and 71 of a lumazine synthase. As described above, lumazine synthase has been used a carrier protein or nanoparticle by genetic linkage of proteins or peptides to the lumazine synthase. However, the development of such fusion molecules is often complicated by inefficiencies in their production. According to the present invention it has been found that by introducing one or more N-linked glycans, the glycans increase expression and assembly of AaLS fused to RSV G when produced in mammalian cells.
The glycans can be introduced by any suitable means known to those skilled in the art.
For example, use can be made of common DNA methods (PCR) to introduce ‘NXS/T’ motifs that allow N-linked glycosylation in mammalian cells. The motif may or may not be flanked by short linkers (Gly/Ser).
In certain preferred embodiments, the lumazine synthase comprises an amino acid sequence of SEQ ID NO: 1 or 2.
In certain embodiments, the lumazine synthase comprises a mammalian signal sequence for expression and secretion in a cell.
According to the invention, the immunogens comprise at least one recombinant RSV G protein ectodomain, or fragment thereof, linked to the protein nanoparticle subunit.
In certain embodiments, the RSV G protein ectodomain or fragment is from a human RSV G protein. Thus, the RSV G ectodomain or fragment is derived from a G protein of a human RSV.
In certain other embodiments, the RSV G protein ectodomain or fragment is from a bovine RSV G protein. Thus, the RSV G ectodomain or fragment is derived from a G protein of a bovine RSV.
In certain embodiments, the RSV G protein ectodomain fragment is an RSV G central conserved domain (CCD) peptide. The CCD is very small and linking this to a particle may increase its immunogenicity.
According to the invention the RSV G protein ectodomain can be an RSV Ga or RSV Gb protein ectodomain.
In certain preferred embodiments, the RSV G protein ectodomain fragment comprises an amino acid sequence selected from the group consisting of DFHFEVFNFVP (SEQ ID NO: 104), DYHFEVFNFVP (SEQ ID NO: 105) , NDFHFEVFNFVPCSICSNNPTCWAICKRIPN (SEQ ID NO: 106) and DDYHFEVFNFVPCSICGNNQLCKSICKTIPS (SEQ ID NO: 107), or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 97%, most preferably at least 99% sequence identity to any of SEQ ID NO: 104-107.
In certain embodiments, the RSV G protein ectodomain fragment is derived from an amino acid sequence selected from the group consisting of SEQ ID NO: 3-6, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 97%, most preferably at least 99% sequence identity to any of SEQ ID NO: 3-6.
In certain embodiments, the at least one RSV G protein ectodomain or fragment thereof is linked to the N-terminal side of the lumazine synthase. In other embodiments, the at least one RSV G protein ectodomain or fragment thereof is linked to the C-terminal side of the lumazine synthase. According to the invention, it is also possible that the at least one RSV G protein ectodomain or fragment thereof is linked to the lumazine synthase at an internal fusion site.
The immunogen of the invention, in certain embodiments comprise at least two RSV G proteins ectodomains or fragments thereof, i.e. one linked to N-terminal side and one linked to the C-terminal side, and/or one at an internal fusion site.
According to the invention, the at least two RSV G protein ectodomains or fragments thereof could be the same RSV G protein ectodomains or fragments or different. In certain embodiments, the RSV G protein ectodomains or fragments are different RSV G proteins or fragments, i.e. one RSV G ectodomain or fragment from an RSV A strain and one from an RSV
B strain. Two identical RSV G protein domain could possibly reduce the dose of vaccine needed to induce a protective immune response. Advantage of both A and B domain on one particle is simplicity of the vaccine: only one particle would be needed to protect against A and B virus instead of 2 particles.
In certain embodiments, the immunogens comprise a purification tag, including but not limited to a HIS-Tag, strep-tag or c-tag. A His-Tag or polyhistidine-tag is an amino acid motif in proteins that consists of at least five histidine (H) residues; a strep-tag is an amino acid sequence that consist of 8 residues (WSHPQFEK (SEQ ID NO: 108)); a c-tag is an amino acid motif that consists of 4 residues (EPEA; SEQ ID NO: 109). The tags are often at the N- or C-terminus of a protein and are generally used for purification purposes.
In certain embodiments, the immunogens comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 8-14, 16-63, 67-69, and 72-103, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 97%, most preferably at least 99% sequence identity to any of these sequences.
The present invention further provides nucleic acid molecules encoding the immunogens according to the invention. The term “nucleic acid molecule” as used in the present invention refers to a polymeric form of nucleotides (i.e. polynucleotides) and includes both DNA (e.g. cDNA, genomic DNA) and RNA, and synthetic forms and mixed polymers of the above. It is to be understood that numerous different nucleic acid molecules can encode the same protein as a result of the degeneracy of the genetic code. It is also understood that skilled persons can, using routine techniques, make nucleotide substitutions that do not affect the protein sequence encoded by the polynucleotides described there to reflect the codon usage of any particular host organism in which the proteins are to be expressed. Therefore, unless otherwise specified, a “nucleic acid molecule encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns. Sequences herein are provided from 5' to 3' direction, as custom in the art.
In preferred embodiments, the nucleic acid molecules encoding the proteins according to the invention are codon-optimized for expression in cells, preferably human cells, bacterial and/or yeast cells. Methods of codon-optimization are known and have been described previously (e.g. WO 96/09378 for mammalian cells). A sequence is considered codon-optimized if at least one non-preferred codon as compared to a wild type sequence is replaced by a codon that is more preferred. Herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in codon frequency tables, such as in http://www.kazusa.or.jp/codon. Preferably more than one non-preferred codon, preferably most or all non-preferred codons, are replaced by codons that are more preferred. Preferably the most frequently used codons in an organism are used in a codon-optimized sequence. Replacement by preferred codons generally leads to higher expression.
It will be understood by a skilled person that numerous different polynucleotides and nucleic acid molecules can encode the same protein as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the protein sequence encoded by the nucleic acid molecules to reflect the codon usage of any particular host organism in which the proteins are to be expressed. Therefore, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may or may not include introns.
Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScripts, Invitrogen, Eurofins).
The invention also provides vectors comprising a nucleic acid molecule as described above. In certain embodiments, a nucleic acid molecule according to the invention thus is part of a vector. Such vectors can easily be manipulated by methods well known to the person skilled in the art and can for instance be designed for being capable of replication in prokaryotic and/or eukaryotic cells. The vector used can be any vector that is suitable for cloning DNA and that can be used for expression of a nucleic acid molecule of interest. Suitable vectors according to the invention are viral vectors, e.g. adeno vectors, alphavirus, paramyxovirus, vaccinia virus, herpes virus, retroviral vectors etc.
Host cells comprising the nucleic acid molecules encoding the immunogens form also part of the invention. The G particles may be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g. Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER. C6 cells, or yeast, or bacterial cells. In certain embodiments, the cells are from a multicellular organism, in certain embodiments they are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. In general, the production of a recombinant proteins, such the immunogens of the invention, in a host cell comprises the introduction of a heterologous nucleic acid molecule encoding the protein in expressible format Y1 into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the immunogen in said cell. The nucleic acid molecule encoding an immunogen in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like. The person skilled in the art is aware that various promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed. In certain embodiments, the nucleic acid molecules according to the invention are operably linked to a promoter, including, but not limited to CMV, chicken beta actin.
The invention further provides immunogenic compositions comprising an immunogen and/or a nucleic acid molecule, and/or a vector, as described above, and one or more pharmaceutically acceptable excipients. For administering to subjects, such as humans, the invention may employ pharmaceutical compositions comprising an immunogen, a nucleic acid molecule and/or a vector as described herein, and a pharmaceutically acceptable carrier or excipient. In the present context, the term "pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The immunogens proteins, or nucleic acid molecules, preferably are formulated and administered as a sterile solution although it may also be possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g. pH 5.0 to 7.5. The immunogens typically are in a solution having a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt. Optionally stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, the immunogens proteins may be formulated into an injectable preparation.
In certain embodiments, a composition according to the invention further comprises one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant. The terms “adjuvant” and "immune stimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the immunogens proteins of the invention. Examples of suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in- water compositions), including squalene-water emulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP- ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (e.g. antibodies or fragments thereof (e.g. directed against the antigen itself or CDla, CD3, CD7, CD80) and ligands to receptors (e.g. CD40L, GMCSF, GCSF, etc), which stimulate immune response upon interaction with recipient cells. In certain embodiments the compositions of the invention comprise aluminium as an adjuvant, e.g. in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or combinations thereof, in concentrations of 0.05 - 5 mg, e.g. from 0.075-1.0 mg, of aluminium content per dose.
The invention further provides methods for generating an immune response to RSV G in a subject, comprising administering to the subject an effective amount of the immunogenic composition as described herein to generate the immune response.
The invention also provides the use of an immunogen, a nucleic acid molecule, and/or a vector, according to the invention, for inducing an immune response against RSV G in a subject.
The invention also provides methods for treating or preventing a RSV infection in a subject, comprising administering to the subject a therapeutically effective amount of the immunogenic composition as described herein, thereby treating or preventing RSV infection in the subject.
In certain embodiments, the prevention and/or treatment may be targeted at patient groups that are susceptible RSV infection. Such patient groups include, but are not limited to e.g., the elderly (e.g. > 50 years old, > 60 years old, and preferably > 65 years old), the young (e.g. < 5 years old, < 1 year old), pregnant women (for maternal immunization), hospitalized patients and patients who have been treated with an antiviral compound but have shown an inadequate antiviral response.
A therapeutically effective amount refers to an amount of an immunogen, nucleic acid molecule or vector, that is effective for preventing, ameliorating and/or treating a disease or condition resulting from infection by RSV. Prevention encompasses inhibiting or reducing the spread of RSV, or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection by RSV. Amelioration as used in herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of influenza infection.
In certain embodiments, the invention provides methods for making a vaccine against respiratory syncytial virus (RSV), comprising providing an immunogen, nucleic acid or vector according to the invention and formulating it into a pharmaceutically acceptable composition. The term "vaccine" refers to an agent or composition containing an active component effective to induce a certain degree of immunity in a subject against a certain pathogen or disease, which will result in at least a decrease (up to complete absence) of the severity, duration or other manifestation of symptoms associated with infection by the pathogen or the disease. In the present invention, the vaccine comprises an effective amount of an immunogen and/or a nucleic acid molecule, and/or a vector comprising said nucleic acid molecule, which results in an immune response against the G protein of RSV. This provides a method of preventing serious lower respiratory tract disease leading to hospitalization and the decrease in frequency of complications such as pneumonia and bronchiolitis due to RSV infection and replication in a subject. The term “vaccine” according to the invention implies that it is a pharmaceutical composition, and thus typically includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not comprise further active ingredients. In certain embodiments it may be a combination vaccine that further comprises other components that induce an immune response, e.g. against other proteins of RSV and/or against other infectious agents. The administration of further active components may for instance be done by separate administration or by administering combination products of the vaccines of the invention and the further active components.
The invention further provides methods for detecting or isolating an RSV G binding antibody in a subject, comprising: providing an effective amount of the immunogen as described herein; contacting a biological sample from the subject with the immunogen under conditions sufficient to form an immune complex between the immunogen and the RSV G binding antibody; and detecting the immune complex, thereby detecting or isolating the RSV G binding antibody in the subject. Thus, the immunogens of the invention may be used as diagnostic tool, for example to test the immune status of an individual by establishing whether there are antibodies in the serum of such individual capable of binding to the immunogen of the invention. The invention thus also relates to an in vitro diagnostic method for detecting the presence of an RSV infection in a patient said method comprising the steps of a) contacting a biological sample obtained from said patient with a protein according to the invention; and b) detecting the presence of antibody-protein complexes.
As used throughout the present application, the amino acid positions are given in reference to the sequence of lumazine synthase of SEQ ID NO: 1. As used in the present invention, the wording “the amino acid residue at position “x” of the lumazine synthase thus means the amino acid corresponding to the amino acid at position “x” in the lumazine synthase of SEQ ID NO: 1. Sequence alignments can be done using methods well known in the art, e.g. by CLUSTALW, Bioedit or CLC Workbench. As used throughout the present application nucleotide sequences are provided from 5’ to 3’ direction, and amino acid sequences from N-terminus to C-terminus, as custom in the art.
An amino acid according to the invention can be any of the twenty naturally occurring (or ‘standard’ amino acids). The standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties such as cysteine, that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that induces turns of the protein backbone, and glycine that is more flexible than other amino acids. Table 2 shows the abbreviations and properties of the standard amino acids.
It will be appreciated by a skilled person that the mutations can be made to the proteins by routine molecular biology procedures.
Examples
EXAMPLE 1: BRSV G CCD, but not HRSV G CCD mini-protein can be fused to AaLS particles while retaining particle expression.
Plasmids corresponding to the wildtype Aquifex aeolicus Lumazine Synthase (AaLS) monomer that were equipped with a human CD 5 signal peptide to allow secretion into the cell supernatant and with a C-terminal C-tag were synthesized and codon-optimized at Genscript (FIG. 1). Indicated in figure 1 are also the three locations where peptides ( or ‘mini-proteins’) can be fused to the AaLS backbone. A series of AaLS particles fused to either the human RSV
(HRSV) or bovine RSV (BRSV) G central-conserved domain (CCD) mini-protein were designed (Fig 2A-C) The constructs were cloned into pCDNA2004 by standard methods widely known within the field involving site-directed mutagenesis and PCR and sequenced. Proteins were expressed in the expi293F cell system. Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 5 minutes at 300 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.
The AaLS monomers were detected in crude supernatants using western blotting with a C-tag specific nanobody (CaptureSelect™ Biotin Anti-C-tag Conjugate, ThermoFisher) (Fig 2D). Both AaLS backbones (RSV190362 and RSV191645) showed a band at the expected height. In contrast, there was no AaLS expression detectable for the particles fused to HRSV G CCD miniprotein (RSV191646-48). For BRSV G CCD mini-protein-fused AaLS particles (RSV191649, RSV191650 and RSV191651) there wass a distinct band at the expected height. Employing analytical size-exclusion chromatography (analytical SEC) on raw cell culture supernatants the assembled 60-meric AaLS particle could be detected. To this end an ultra high-performance liquid chromatography system (Vanquish, Thermo Scientific) and pDAWN TREOS instrument (Wyatt) coupled to an Optilab pT-rEX Refractive Index Detector (Wyatt), in combination with an in-line Nanostar DLS reader (Wyatt) were used for performing the analytical SEC experiment. The cleared crude cell culture supernatants were applied to a 500 A column, (Sepax Cat#235500-4615) with the corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min. When analyzing supernatant samples, pMALS detectors were offline and analytical SEC data was analyzed using
Chromeleon 7.2.8.0 software package. Both empty backbone particles (Fig 2E) as well as the BRSV (Fig 2F) fusions yielded nanoparticles that could be detected in raw supernatant. In line with the western blot results, there was no detection of the AaLS nanoparticles that are fused to HRSV G CCD mini-protein (Fig 2G).
EXAMPLE 2: Introduction of N-linked glycans boosts AaLS particle expression
Either 1 or 2 N-linked glycans were introduced at position 71 of the AaLS particle backbone (RSV191645) using standard methods widely known within the field involving site- directed mutagenesis and PCR and sequenced. This resulted in constructs RSV201131 (1 N- linked glycan) and RSV201132 (2 N-linked glycans). These two proteins, as well as the backbone without any glycans were expressed in the expi293F cell system. Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 5 minutes at 300 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.
Employing analytical size-exclusion chromatography (analytical SEC) on raw cell culture supernatants the assembled 60-meric AaLS particle can be detected. An ultra high-performance liquid chromatography system (Vanquish, Thermo Scientific) and pDAWN TREOS instrument (Wyatt) coupled to an Optilab pT-rEX Refractive Index Detector (Wyatt), in combination with an in-line Nanostar DLS reader (Wyatt) were used for performing the analytical SEC experiment. The cleared crude cell culture supernatants were applied to a 500 A column, (Sepax Cat#235500-4615) with the corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min. When analyzing supernatant samples, pMALS detectors were offline and analytical SEC data was analyzed using Chromeleon 7.2.8.0 software package.
The empty backbone particle (Fig 3A) yielded a nanoparticle that could be detected in raw supernatant (Fig 3B). When adding 1 or 2 glycans (Fig 3A) a significant increase in particle expression in the supernatant (Fig 3B) was seen. The shift in retention times is caused by the increased diameter that is a consequence of the added amino acids and glycans.
EXAMPLE 3: Introduction of N-linked glycans allows expression of AaLS particles fused to HRSV G CCD mini-protein.
The series of AaLS particles fused to the HRSV G CCD mini-protein that did not express particles (Fig 2B, G) were equipped with varying amounts of N-linked glycans (Fig 4, N- terminal fusions; Fig 5, C-terminal fusions; Fig 6, internal fusions). Additionally, so-called ‘dual particles’ were designed that have HRSV type A G CCD mini-protein on the N-terminus, and HRSV type B G CCD mini-protein on the C-terminus (Fig 7). Additionally, particles were designed that have HRSV type B G CCD mini-protein on the C-terminus (Fig 8). All constructs were cloned into pCDNA2004 by standard methods widely known within the field involving site-directed mutagenesis and PCR and sequenced. Proteins were expressed in the expi293F cell system. Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 5 minutes at 300 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use. Employing analytical size-exclusion chromatography (analytical SEC) on raw cell culture supernatants the assembled 60-meric AaLS particle can be detected. To this end, an ultra high- performance liquid chromatography system (Vanquish, Thermo Scientific) and pDAWN TREOS instrument (Wyatt) coupled to an Optilab pT-rEX Refractive Index Detector (Wyatt), in combination with an in-line Nanostar DLS reader (Wyatt) were used for performing the analytical SEC experiment. The cleared crude cell culture supernatants were applied to a 500 A column, (Sepax Cat#235500-4615) with the corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min. When analyzing supernatant samples, pMALS detectors were offline and analytical SEC data was analyzed using Chromeleon 7.2.8.0 software package.
When adding N-linked glycans particle expression in the supernatant was achieved. The number of glycans required to have successful particle production depended on the specific design. In all cases it was seen that more glycans gives more particle expression, until a plateau is reached (compare for example RSV210981 (with 3 glycans) and RSV210982 (with 4 glycans)) (Fig 8). The exact composition of the N-linked glycan motif insertion is not pivotal: compare for example RSV201048 (Tong’ linker)and RSV201051 (‘short’ linker) which both have 3 glycans but differ in the design of the linker regions between the AaLS particle and the glycans (Fig 4C). Both designs were successfully produced as particles, though there are differences in overall expression levels (Fig 4B).
EXAMPLE 4: AaLS particles with N-linked glycans displaying HRS G CCD mini-proteins can be purified and have the desired structure. To confirm that the AaLS designs with glycans indeed formed the correct nanoparticles, one of the designs was purified to allow structure determination by EM. The design selected was RSV200023 (Fig 9A), which has two linked glycans and has the HRSV type A G CCD miniprotein fused to the N-terminus. This selected design was expressed in the expi293F cell system. Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 5 days at 37°C and 8% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 10 minutes at 600 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.
The supernatant was used in purification, where first C-tag affinity purification was performed with Capture Select C-tag XL (Thermo Scientific Cat#494307205). The eluted protein was thereafter further purified by size exclusion with Superdex200 (GE/Cytiva Cat# 28-9909- 44).
An ultra high-performance liquid chromatography system (Vanquish, Thermo Scientific) and pDAWN TREOS instrument (Wyatt) coupled to an Optilab pT-rEX Refractive Index Detector (Wyatt), in combination with an in-line Nanostar DLS reader (Wyatt), was used for performing analytical SEC-MALS on the purified product (Fig 9B). The purified product was applied to a 450A column, (Waters Cat#l 86006851) with the corresponding guard column (Waters) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min. Analytical SEC-MALS data was analyzed using Chromeleon 7.2.8.0 software package and Astra 7.3.2. The yield of the purified particle was 4.92 mg/L (Fig. 9C). The molecular weight was 1477 kDa, close to the expected value of 1377 kDa (Fig. 9C and D). Structure was determined using negative stain electron microscopy (nsEM) (Fig 9E). Class averages of the particles at different angles are shown (Fig 9E) and confirm that the correct nanoparticles are formed.
EXAMPLE 5: AaLS particles with N-linked glycans displaying HRS G CCD mini-proteins can be purified and are stable.
Next, we selected a set of designs that differ in the number of glycans and the location and number of fusions of the G mini-protein to AaLS (Fig 10A). The selected designs were expressed in the expi293F cell system. Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 5 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 10 minutes at 600 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.
The supernatant was used in purification, where first C-tag affinity purification was performed with Capture Select C-tag XL (Thermo Scientific Cat#494307205). The eluted protein was thereafter further purified by size exclusion with Superdex200 (GE/Cytiva Cat# 28-9909- 44), followed by Superose 6 (GE/Cytiva Cat# 17-5172-01). An ultra high-performance liquid chromatography system (Vanquish, Thermo Scientific) and pDAWN TREOS instrument (Wyatt) coupled to an Optilab pT-rEX Refractive Index Detector (Wyatt), in combination with an in-line Nanostar DLS reader (Wyatt), was used for performing analytical SEC-MALS on the purified products to confirm particle formation (Fig 10B-F). The purified product was applied to a 450A column, (Waters Cat#l 86006851) with the corresponding guard column (Waters) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min. Analytical SEC-MALS data was analyzed using Chromeleon 7.2.8.0 software package and Astra 7.3.2. Molecular weights are as expected. Yields are shown in Fig 10D. Next, we determined stability of the protein nanoparticles by incubating them at different temperatures for 30 min (Fig 11), followed by analytical 300A SEC-MALS (Sepax Cat# 231300-4615, with the corresponding guard column). This data showed that even after exposure to 90°C for 30 min, in all cases (a portion of) particles remain.
EXAMPLE 6: Tag-less AaLS particles with N-linked glycans displaying HRSV G CCD miniproteins have lower expression.
To determine the effect of removing the C-tag, a number of particle designs was selected and the region encoding the C-tag was removed from the plasmids by standard methods widely known within the field involving site-directed mutagenesis and PCR. Proteins were expressed in the expi293F cell system. Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 5 minutes at 300 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use.
Employing analytical size-exclusion chromatography (analytical SEC) on raw cell culture supernatants the assembled 60-meric AaLS particle can be detected. An ultra high-performance liquid chromatography system (Vanquish, Thermo Scientific) and pDAWN TREOS instrument (Wyatt) coupled to an Optilab pT-rEX Refractive Index Detector (Wyatt), in combination with an in-line Nanostar DLS reader (Wyatt) was used for performing the analytical SEC experiment. The cleared crude cell culture supernatants were applied to a 500A column, (Sepax Cat#235500- 4615) with the corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min. When analyzing supernatant samples, pMALS detectors were offline and analytical SEC data was analyzed using Chromeleon 7.2.8.0 software package.
The particles with a C-tag (Fig 12B, solid lines) all had higher expression in supernatant compared to particles that lack a C-tag (Fig 12B, dashed lines), however, in most cases particles were still being formed. The shift in retention times was caused by the decreased diameter that is the consequence of removing the C-tag.
EXAMPLE 7: Tag-less AaLS particles with N-linked glycans displaying HRSV G CCD miniproteins can be purified and are stable.
Next, a set of designs that differ in the number of glycans and the length of the G miniprotein (Fig 13 A) were selected for lectin-based purification. The selected designs were expressed in the expi293F cell system. Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 5 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 10 minutes at 600 g. The clarified supernatant was subsequently sterile filtered using a 0.22 um vacuum filter and stored at 4°C until use. An ultra high-performance liquid chromatography system (Vanquish, Thermo Scientific) and pDAWN TREOS instrument (Wyatt) coupled to an Optilab pT-rEX Refractive Index Detector (Wyatt), in combination with an in-line Nanostar DLS reader (Wyatt), was used for performing analytical SEC on the filtered clarified supernatant to check on particle expression presence (Fig 13B). The cleared supernatants were applied to a 500 A column, (Sepax Cat#235500-4615) with the corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL/min. Analytical SEC data was analyzed using Chromel eon 7.2.8.0 software.
The supernatant was used in purification, where first Lectin-based affinity purification was performed with Galanthus nivalus lectin (Vector Labs Cat#AL-1243). The eluted protein was thereafter further purified by size exclusion with Superdex200 (GE/Cytiva Cat# 28-9909- 44), followed by Superose 6 (GE/Cytiva Cat# 29-0915-96). Analytical 500 A SEC-MALS was performed on the purified products and analyzed using Chromeleon 7.2.8.0 software package and Astra 7.3.2 to confirm particle formation (Fig 13C-E). Molecular weights were as expected. Yields are shown in Fig 13E. Next, we determined stability of the protein nanoparticles by incubating them at different temperatures for 30 min (Fig 14), followed by analytical 500A SEC- MALS. This data show that also the tag-less particles are exceptionally stable, and that only RSV210984 shows some reduction in particle content after exposure to 90°C.
EXAMPLE 8: AaLS particles with N-linked glycans displaying HRSV G CCD mini-proteins are immunogenic in mice, and sera are neutralizing in a virus neutralization assay on primary human airway epithelial cell cultures.
The immunogenicity of 5 AaLS particles fused to RSV Ga and/or RSV-Gb peptides (as shown in Fig. 10A) were investigated. The RSV G peptides were genetically fused N- or C- terminally to AalS and contained 2 or 3 added glycans required for expression (RSV201051, RSV201064 and RSV201214). In addition, it was investigated if the internal fusion of G-peptide (RSV201063) is immunogenic. Since part of the G-peptide (CCD) is conserved between RSV-A and B, some level of crossreactivity was expected. However, the immunogenicity of AaLS particles containing both RSV-A and RSV-B derived G-peptides (RSV201056) was explored as well for optimal coverage of both subtypes. To determine the immunogenicity of the different RSV G-AaLS particles, female BALB/c mice (6-8 weeks) received 1 or 10 pg AaLS particles containing RSV-A G-peptide or a combination of RSV-A and B G-peptide. The particles were adjuvanted with 2% Adjuplex and administrated via the i.m. route at day 0 and 21 according to the experimental design shown in Table 1.
Table 1. Experimental design of the study
Figure imgf000033_0001
Serum samples were isolated 3 weeks after the prime immunization (day 20) and 3 weeks after the boost (day 42). RSV-A (day 20 and 42 serum) and RSV-B (day 42 serum) G protein (Ga and Gb, respectively) binding antibody titers were measured with an ELISA. Biotinylated Ga and Gb peptides covering the CCD were captured by immobilized Streptavidin. After a washing step, two-fold serially diluted serum samples were added. After one-hour, unbound serum antibodies were washed away, and bound antibodies (IgG) were detected by anti-mouse- HRP followed by a fluorescence readout. Binding antibody titers were calculated relative to a standard concentration of anti-G mAb (MAB858-2, clone 131-2G, Sigma-Aldrich).
Neutralizing antibody responses against RSV-A were measured in pooled serum samples (day 42) in a primary human airway epithelial cell-based VNA (hAEC) assay [Harrison G. Jones, et al, PLoS Pathog. 2018 Mar],
Robust RSV binding antibody responses were induced after prime and prime-boost immunization against RSV Ga peptide. RSV201051 induced significantly higher RSV Ga peptide binding antibody titers after the prime (day 20) compared to RSV201064 and RSV201214 (pO.OOOl, Tobit model) (Figure 15A) and only significantly higher Ga peptide binding antibody titers after the boost (day 42) (Fig. 15B) compared to RSV201214 (p=0.0322 Tobit model).
Three weeks after boost, levels of cross-reactive RSV Gb antibodies were detected, and those were not different between RSV201051, RSV201064 and RSV201214 (Figure 15C).
RS V201056, containing both RSV Ga and Gb peptides induced similar levels of Ga (Figure 15A and 15B), but significantly higher levels of RSV Gb directed antibody titers (pO.OOOl, Tobit model compared to RSV201051; which was the most potent RSV Ga particle) (Figure 15C).
In the hAEC VNA assay, the pooled sera from all groups showed potent neutralization towards RSV A2 with a GFP reporter (Figure 16).
Table 2. Standard amino acids, abbreviations and properties
Figure imgf000034_0001
Figure imgf000035_0001
SEQUENCES
SEQ ID NO: 1 (wildtype sequence) 154AA
MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKE
DIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAAL
SAIEMANLFKSLR
SEQ ID NO: 2 (2 point mutations for expression in mammalian cells) 154AA
MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKE
DIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAAL
SAIEMANLFKSLR
SEQ ID NO: 3
KQRQNKPPNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKK
SEQ ID NO: 4
KQRQNKPQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKK
SEQ ID NO: 5
KPRPKSPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNK
SEQ ID NO: 6:
KPRPKNPPKKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTTKPTNK
SEQ ID NO: 7: RSV190362
MPMGSLQPLATLYLLGMLVASVLAMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAI
DAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEV
SKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGG
SGGSGGSEPEA
SEQ ID NO: 8: RSV190363
MPMGSLOPLATLYLLGMLVASVLAMOIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAI
DAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSNKPNNDFHFEVFNFVPCSICSN
NPTCWAICKRIPNKKPGKGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPI
TFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEPEA SEQ ID NO: 9: RSV190364
MPMGSLQPLATLYLLGMLVASVLAMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAI
DAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSKQRQNKPQNKPNNDFHFEVFN
FVPC SICSNNPTCWAIC I<RIPNI<I<PGI<I<TTTI<GGSDIDAVIAIGVLIRGATPHFDYIASEVS
KGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGS GGSGGSEPEA
SEQ ID NO: 10: RSV190365
MPMGSLOPLATLYLLGMLVASVLAMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAI
DAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSTTTTQILPSKPTTKQRQNKPQN
KPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKPTLKTTKKDPKP
QTTGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIE
RAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 1E RSV190366
MPMGSLQPLATLYLLGMLVASVLANKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPN
I<I<PGI<GGSMQIYEGI<LTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLV
RVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITF
GVITADTLEQAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 12: RSV190367
MPMGSLQPLATLYLLGMLVASVLANKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPN KKPGKKTTTKPTKKPTLKTTKKDPKPQTTGGSMQIYEGKLTAEGLRFGIVASRFNHALV
DRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPH
FDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMAN
LFKSLRGGSGGSGGSEPEA
SEQ ID NO: 13: RSV190368
MPMGSLQPLATLYLLGMLVASVLAMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAI
DAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEV
SKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGG
SNI<PNNDFHFEVFNFVPC SICSNNPTCWAIC I<RIPNI<I<PGI<I<TGGSGGSGGSEPEA
SEQ ID NO: 14: RSV190369
MPMGSLQPLATLYLLGMLVASVLAMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAI
DAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEV
SKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGG
STTTTQILPSKPTTKQRQNKPQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPG
KKTGGSGGSGGSEPEA
SEQ ID NO: 15: RSV191645
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVS KGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGS
GGSGGSEPEA
SEQ ID NO: 16: RSV191646
MPMGSLQPLATL YLLGMLV AS VLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSNDFHFEVFNFVPCSICSNNPTCW
AICKRIPNGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLE
QAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 17: RSV191647
MPMGSLQPLATL YLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAI
ERAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 18: RSV191648
MPMGSLQPLATL YLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVS
KGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGS
NDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSGGSGGSEPEA SEQ ID NO: 19: RSV191649
MPMGSLQPLATL YLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSENHQDHNNSQTLPHVPCSTCEG
NPACLSLCQIGPESASSRAGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKP
ITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEPEA
SEQ ID NO: 20: RSV191650
MPMGSLQPLATL YLLGMLVASVLAENHQDHNNSQTLPHVPCSTCEGNPACLSLCQIGPE
SASSRAGGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVR
VPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFG
VITADTLEQAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 21: RSV191651
MPMGSLQPLATL YLLGMLV AS VLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVS
KGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGS
ENHQDHNNSQTLPHVPCSTCEGNPACLSLCQIGPESASSRAGGSGGSGGSEPEA
SEQ ID NO: 22: RSV200020
MPMGSLQPLATL YLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGDGGDGGDGNDFHFEVFNFVPCSIC SNNPTCWAICKRIPNGDGGDGGDGDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLEL
RKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEPEA
SEQ ID NO: 23: RSV200021
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGDGG
DGGDGQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGS
WEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITA
DTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEPEA
SEQ ID NO: 24: RSV200022
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVS
KGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGD
GGDGGDGNDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSGGSGGSEPEA
SEQ ID NO: 25: RSV200023
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQ
LSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGG
SEPEA SEQ ID NO: 26: RSV200024
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGDGG
DGGDGQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGS
WEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASE
VSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRG GSGGSGGSEPEA
SEQ ID NO: 27: RSV200025
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSENHQDHNNSQTLPHVPCSTCEGNPACLSLCQIGPESASSRAGGSDIDAVI
AIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGW
EAALSAIEMANLFKSLRGGSGGSGGSEPEA
SEQ ID NO: 28:
RSV200026MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICK
RIPNGGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPG
SWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVIT
ADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSENHQDHNNSQTLPHVPCS
TCEGNPACLSLCQIGPESASSRAGGSGGSGGSEPEA SEQ ID NO: 29: RSV200027
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGDGG
DGGDGQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGS
WEIPVAAGELARKEGGSENHQDHNNSQTLPHVPCSTCEGNPACLSLCQIGPESASSRAG
GSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGT
I<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 30: RSV200028
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGDGG
DGGDGQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAID AIVRHGGREEDITLVRVPGS
WEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITA
DTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSENHQDHNNSQTLPHVPCST
CEGNPACLSLCQIGPESASSRAGGSGGSGGSEPEA
SEQ ID NO: 31: RSV201046
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSNSTGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVI
TADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEPEA SEQ ID NO: 32: RSV201047
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSNSTGGSNSTGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRK
PITFGVITADTLEQAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 33: RSV201048
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSNSTGGSNSTGGSNSTGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQL
SLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGS EPEA
SEQ ID NO: 34: RSV201049
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKENSTDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLE
QAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA SEQ ID NO: 35: RSV201050
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKENSTSNSTDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITA DTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEPEA
SEQ ID NO: 36: RSV201051
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA GELARKENSTSNSTSNSTDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFG
VITADTLEQAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 37: RSV201052
MPMGSLQPLATLYLLGMLVASVLANKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPN GGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWE
IPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSK
GLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSG
GSGGSEPEA SEQ ID NO: 38: RSV201053
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAIC I<RIPNI<I<PG
I<I<GGSQIYEGI<LTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGS
WEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASE
VSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRG
GSGGSGGSEPEA
SEQ ID NO: 39: RSV201054
MPMGSLQPLATLYLLGMLVASVLANKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPN
I<I<PGI<I<GGSQIYEGI<LTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVR
VPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDY
IASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFK
SLRGGSGGSGGSEPEA
SEQ ID NO: 40: RSV201055
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQ
LSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSDDYHF
EVFNFVPCSICGNNQLCKSICKTIPSGGSGGSGGSEPEA SEQ ID NO: 41: RSV201056
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKENSTSNSTSNSTDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFG
VITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRDDYHFEVFNFVPCSICGNN QLCKSICKTIPSGGSGGSGGSEPEA
SEQ ID NO: 42: RSV201057
MPMGSLQPLATLYLLGMLVASVLADDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQ
LSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGG SEPEA
SEQ ID NO: 43: RSV201058
MPMGSLQPLATLYLLGMLVASVLADDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSNSTGGSNSTGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRK
PITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEPEA SEQ ID NO: 44: RSV201059
MPMGSLQPLATLYLLGMLVASVLADDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKENSTSNSTSNSTDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFG
VITADTLEQAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 45: RSV201060
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSNSTGGSNDFHFEVFNFVPCSICS
NNPTCWAICKRIPNGGSNSTGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELR
KPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEPEA
SEQ ID NO: 46: RSV201061
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGNSTGNDFHFEVFNFVPCSICSNNPT
CWAICKRIPNGNSTGDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVIT
ADTLEQAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 47: RSV201062
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSNSTGGSNSTGGSNDFHFEVFNFV
PCSICSNNPTCWAICKRIPNGGSNSTGGSNSTGGSDIDAVIAIGVLIRGATPHFDYIASEVS KGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGS GGSGGSEPEA
SEQ ID NO: 48: RSV201063
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGNSTSNSTGNDFHFEVFNFVPCSICS
NNPTCWAICKRIPNGNSTSNSTGDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELR
I<PITFGVITADTLEQAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 49: RSV201064
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSNDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSGGSG GSEPEA
SEQ ID NO: 50: RSV201214
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKENSTSNSTSNSTDIDAVIAIGVLIRGAT
PHFDYIASEVSI<GLAQLSLELRI<PITFGVITADTLEQAIERAGTI<HGNI<GWEAALSAIEM
ANLFKSLRGGSNDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSGGSGGSEPEA SEQ ID NO: 51: RSV201118
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFI<SLRGGSNI<PNNDFHFEVFNFVPCSICSNNPTCWAICT<RIPNGGS GGSGGSEPEA
SEQ ID NO: 52: RSV201119
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSNSTGGSNSTGGSNKPNNDFHFE
VFNFVPCSICSNNPTCWAICKRIPNGGSNSTGGSNSTGGSDIDAVIAIGVLIRGATPHFDYI
ASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKS LRGGSGGSGGSEPEA
SEQ ID NO: 53: RSV201120
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGNSTSNSTGNKPNNDFHFEVFNFVPC
SICSNNPTCWAICKRIPNGNSTSNSTGDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSL
ELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEP
EA SEQ ID NO: 54: RSV201121
MPMGSLQPLATLYLLGMLVASVLANKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPN
GGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWE
IPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSK
GLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSD
DYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSGGSGGSEPEA
SEQ ID NO: 55: RSV201122
MPMGSLQPLATLYLLGMLVASVLANKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPN
GGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWE
IPVAAGELARKENSTSNSTSNSTDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRK
PITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVP
CSICGNNQLCKSICKTIPSGGSGGSGGSEPEA
SEQ ID NO: 56: RSV201123
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQ
LSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSKKPKD
DYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSGGSGGSEPEA SEQ ID NO: 57: RSV201124
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKENSTSNSTSNSTDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFG
VITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSKKPKDDYHFEVFNFVP
CSICGNNQLCKSICKTIPSGGSGGSGGSEPEA
SEQ ID NO: 58: RSV201125
MPMGSLQPLATLYLLGMLVASVLANKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPN
GGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWE
IPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSK
GLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSK
KPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSGGSGGSEPEA
SEQ ID NO: 59: RSV201126
MPMGSLQPLATLYLLGMLVASVLANKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPN
GGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWE
IPVAAGELARKENSTSNSTSNSTDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRK
PITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSKKPKDDYHFEV
FNFVPCSICGNNQLCKSICKTIPSGGSGGSGGSEPEA SEQ ID NO: 60: RSV201127
MPMGSLQPLATLYLLGMLVASVLADDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSNSTGGSNSTGGSNDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSNSTG
GSNSTGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQA
IERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEPEA
SEQ ID NO: 61 : RSV201128
MPMGSLQPLATLYLLGMLVASVLADDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGNSTSNSTGNDFHFEVFNFVPCSICSNNPTCWAICKRIPNGNSTSNSTGDIDAV
IAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKG
WEAALSAIEMANLFKSLRGGSGGSGGSEPEA
SEQ ID NO: 62: RSV201129
MPMGSLQPLATLYLLGMLVASVLAKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPS
GGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWE
IPVAAGELARKEGGSNSTGGSNSTGGSNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIP
NGGSNSTGGSNSTGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVI
TADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSEPEA SEQ ID NO: 63: RSV201130
MPMGSLQPLATLYLLGMLVASVLAKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPS
GGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWE
IPVAAGELARKEGNSTSNSTGNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNGNSTS
NSTGDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERA
GTI<HGNI<GWEAALSAIEMANLFI<SLRGGSGGSGGSEPEA
SEQ ID NO: 64: RSV201131
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSNSTGGSDIDAVIAIGVLIRGATPH
FDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMAN
LFKSLRGGSGGSGGSEPEA
SEQ ID NO: 65: RSV201132
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSGGSGGSEPEA
SEQ ID NO: 66: RSV210156
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVS
KGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR
SEQ ID NO: 67: RSV210157
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSENHQDHNNSQTLPHVPCSTCEG
NPACLSLCQIGPESASSRAGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKP
ITFGVITADTLEQAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLR
SEQ ID NO: 68: RSV210158
MPMGSLQPLATLYLLGMLVASVLAENHQDHNNSQTLPHVPCSTCEGNPACLSLCQIGPE
SASSRAGGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVR
VPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFG
VITADTLEQAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLR
SEQ ID NO: 69: RSV210159
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVS
KGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGS
ENHQDHNNSQTLPHVPCSTCEGNPACLSLCQIGPESASSRA SEQ ID NO: 70: RSV210160
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSNSTGGSDIDAVIAIGVLIRGATPH
FDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMAN LFKSLR
SEQ ID NO: 71: RSV210161
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK GWEAALSAIEMANLFKSLR
SEQ ID NO: 72: RSV210162
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKENSTSNSTSNSTDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFG
VITADTLEQAIERAGTI<HGNI<GWEAALSAIEMANLFI<SLR
SEQ ID NO: 73: RSV210163
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDA VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSNDFHFEVFNFVPCSICSNNPTCWAICKRIPN
SEQ ID NO: 74: RSV210164
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKENSTSNSTSNSTDIDAVIAIGVLIRGAT
PHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEM
ANLFKSLRGGSNDFHFEVFNFVPCSICSNNPTCWAICKRIPN
SEQ ID NO: 75: RSV210165
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSNSTGGSNSTGGSNDFHFEVFNFV
PCSICSNNPTCWAICKRIPNGGSNSTGGSNSTGGSDIDAVIAIGVLIRGATPHFDYIASEVS
KGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR
SEQ ID NO: 76: RSV210166
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGNSTSNSTGNDFHFEVFNFVPCSICS
NNPTCWAICKRIPNGNSTSNSTGDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELR
KPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR
SEQ ID NO: 77: RSV210167 MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKENSTSNSTSNSTDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFG
VITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRDDYHFEVFNFVPCSICGNN QLCKSICKTIPS
SEQ ID NO: 78: RSV210979
MPMGSLQPL ATE YLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGSGGSDIDAVIAIGVLI
RGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALS
AIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSGGSGGSEPEA
SEQ ID NO: 79: RSV210980
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSGGSG GSEPEA
SEQ ID NO: 80: RSV210981
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGGNSTGGSG
GSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGT
KHGNKGWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPSG
GSGGSGGSEPEA
SEQ ID NO: 81: RSV210982
MPMGSLQPL ATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNH AL VDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGGNSTGGGN
STGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQA
IERAGTKHGNKGWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSIC
KTIPSGGSGGSGGSEPEA
SEQ ID NO: 82: RSV210983
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHAL VDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGSGGSDIDAVIAIGVLI
RGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALS
AIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPS
SEQ ID NO: 83: RSV210984
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHAL VDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDA VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPS
SEQ ID NO: 84: RSV210985
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGGNSTGGSG
GSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGT
KHGNKGWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPS
SEQ ID NO: 85: RSV210986
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGGNSTGGGN
STGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQA lERAGTKHGNKGWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSIC KTIPS
SEQ ID NO: 86: RSV210987
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGS
GGSGGSEPEA SEQ ID NO: 87: RSV210988
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPS
SEQ ID NO: 88: RSV210989
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGGNSTGGSG
GSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGT
KHGNKGWEAALSAIEMANLFKSLRGGSKKPKDDYHFEVFNFVPCSICGNNQLCKSICKT
IPSGGSGGSGGSEPEA
SEQ ID NO: 89: RSV210990
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGGNSTGGSG
GSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGT
KHGNKGWEAALSAIEMANLFKSLRGGSKKPKDDYHFEVFNFVPCSICGNNQLCKSICKT
IPS SEQ ID NO: 90: RSV210991
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRK
PITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVP
CSICGNNQLCKSICKTIPSGGSGGSGGSEPEA
SEQ ID NO: 91: RSV210992
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGGNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEV
SKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGG
SDDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSGGSGGSEPEA
SEQ ID NO: 92: RSV210993
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGGNSTGGGNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHF DYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANL
FKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSGGSGGSEPEA
SEQ ID NO: 93: RSV210994
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQ
LSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSDDYHF
EVFNFVPCSICGNNQLCKSICKTIPS
SEQ ID NO: 94: RSV210995
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRK
PITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVP
CSICGNNQLCKSICKTIPS
SEQ ID NO: 95: RSV210996
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGGNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHFDYIASEV SKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGG
SDDYHFEVFNFVPCSICGNNQLCKSICKTIPS
SEQ ID NO: 96: RSV210997
MPMGSLQPLATLYLLGMLVASVLANDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSQI
YEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA
GELARKEGGSGGSNSTGGGNSTGGGNSTGGGNSTGGSGGSDIDAVIAIGVLIRGATPHF
DYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANL
FKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPS
SEQ ID NO: 97: RSV210998
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGDGGSNSTGGGNSTGGDGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPS
SEQ ID NO: 98: RSV210999
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGEGGSNSTGGGNSTGGEGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPS SEQ ID NO: 99: RSV211000
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGRGGSNSTGGGNSTGGRGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSDDYHFEVFNFVPCSICGNNQLCKSICKTIPS
SEQ ID NO: 100: RSV211001
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGDGGSNSTGGGNSTGGDGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSNDFHFEVFNFVPCSICSNNPTCWAICKRIPN
SEQ ID NO: 101: RSV211002
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGEGGSNSTGGGNSTGGEGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSNDFHFEVFNFVPCSICSNNPTCWAICKRIPN SEQ ID NO: 102: RSV211003
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID
AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGRGGSNSTGGGNSTGGRGGSDIDA VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSNDFHFEVFNFVPCSICSNNPTCWAICKRIPN
SEQ ID NO: 103: RSV211004
MPMGSLQPLATLYLLGMLVASVLAQIYEGKLTAEGLRFGIVASRFNHALVDRL VEGAID AIVRHGGREEDITLVRVPGSWEIPVAAGELARKEGGSGGSNSTGGGNSTGGSGGSDIDA
VIAIGVLIRGATPHFDYIASEVSKGLAQLSLELRKPITFGVITADTLEQAIERAGTKHGNK
GWEAALSAIEMANLFKSLRGGSNDFHFEVFNFVPCSICSNNPTCWAICKRIPNGGSGGSD
DYHFEVFNFVPCSICGNNQLCKSICKTIPSGGSGGSGGSEPEA

Claims

Claims
1. An immunogen comprising at least one recombinant RS V G protein ectodomain or fragment thereof linked to a protein nanoparticle subunit.
2. Immunogen according to claim 1, wherein the protein nanoparticle subunit is lumazine synthase.
3. Immunogen according to claim 1 or 2, wherein the lumazine synthase is an Aquifex aeolicus lumazine synthase (AaLS).
4. Immunogen according to claim 2 or 3, wherein the lumazine synthase comprises at least one introduced N-linked glycan.
5. Immunogen according to any one of claims 1-4, wherein the at least one N-linked glycan is introduced between the amino acid residues 70 and 71 of a lumazine synthase.
6. Immunogen according to any one of claims 1-5, wherein the lumazine synthase comprises at least two introduced N-linked glycans.
7. Immunogen according to any one of claims 1-6, wherein the at least two N-linked glycans are introduced between the amino acid residues 70 and 71 of a lumazine synthase.
8. Immunogen according to any one of the claims 2-5, wherein the lumazine synthase comprises an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
9. Immunogen according to any one of the preceding claims, wherein the RSV G protein ectodomain or fragment thereof is from a human RSV G protein.
10. Immunogen according to any one of the preceding claims 1-8, wherein the RSV G protein ectodomain or fragment thereof is from a bovine RSV G protein.
11. Immunogen according to anyone of the preceding claims, wherein the RSV G protein ectodomain fragment is an RSV G CCD peptide.
12. Immunogen according to any one of the preceding claims, wherein the RSV G protein ectodomain is an RSV Ga or RSV Gb protein ectodomain.
13. Immunogen according to any one of the preceding claims, wherein the at least one RSV G protein ectodomain or fragment thereof is fused to the N-terminal side of the lumazine synthase.
14. Immunogen according to any one of the preceding claims 1-12, wherein the at least one RSV G protein ectodomain or fragment thereof is fused to the C-terminal side of the lumazine synthase.
15. Immunogen according to any one of the preceding claims 1-12, wherein the at least one RSV G protein ectodomain or fragment thereof is fused to the lumazine synthase at an internal fusion site.
16. Immunogen according to any one of the preceding claims, comprising at least two RSV G proteins ectodomains or fragments thereof.
17. Immunogen according to claim 12, wherein the two RSV G protein ectodomains or fragments thereof are different RSV G proteins or fragments thereof.
18. Immunogen according to any one of the preceding claims, comprising a purification tag.
19. Immunogen according to any one of the preceding claims, wherein the immunogen comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-14, 16- 63, 67-69, and 72-103.
20. A nucleic acid molecule encoding the immunogen according to of any one of claims 1-19.
21. Nucleic acid molecule according to claim 20, codon optimized for expression in a human, bacterial and/or yeast cell.
22. Nucleic acid molecule according to claim 20 or 21, operably linked to a promoter.
23. A vector comprising the nucleic acid molecule of claim 22.
24. Vector according to claim 23, wherein the vector is a viral vector.
25. An isolated host cell comprising the vector according to claim 23 or 24.
26. An immunogenic composition comprising an effective amount of the immunogen according to any one of the claims 1-19, a nucleic acid molecule according to claim 20, 21 or 22, and/or vector according to claim 23 or 24, and a pharmaceutically acceptable carrier.
27. The immunogenic composition according to claim 26, further comprising an adjuvant.
28. A method for generating an immune response to RSV G in a subject, comprising administering to the subject an effective amount of the immunogenic composition according to claim 26 or 27 to generate the immune response.
29. A method for treating or preventing a RSV infection in a subject, comprising administering to the subject a therapeutically effective amount of the immunogenic composition according to claim 26 or 27, thereby treating or preventing RSV infection in the subject.
30. A method for detecting or isolating an RSV G binding antibody in a subject, comprising: providing an effective amount of the immunogen according to any one of the claims 1-19; contacting a biological sample from the subject with the immunogen under conditions sufficient to form an immune complex between the immunogen and the RSV G binding antibody; and detecting the immune complex, thereby detecting or isolating the RSV G binding antibody in the subject.
31. Use of the immunogenic composition according to claim 26 or 27 for inhibiting or preventing
RSV infection in a subject.
32. Use of the immunogenic composition according to claim 26 or 27 for inducing an immune response to RSV G protein in a subject.
PCT/EP2023/069010 2022-07-18 2023-07-10 Rsv immunogens WO2024017682A1 (en)

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