CN117460527A

CN117460527A - Stabilized pre-fusion RSV F B Antigens

Info

Publication number: CN117460527A
Application number: CN202280013181.4A
Authority: CN
Inventors: J·P·M·朗格戴克; T·里切尔; M·J·G·巴克斯
Original assignee: Janssen Vaccines and Prevention BV
Current assignee: Janssen Vaccines and Prevention BV
Priority date: 2021-02-19
Filing date: 2022-02-18
Publication date: 2024-01-26

Abstract

The invention provides stable pre-fusionRespiratory Syncytial Virus (RSV) F _B Proteins, nucleic acid molecules and vectors encoding such proteins, compositions comprising said proteins, nucleic acid molecules and/or vectors and their use for preventing and/or treating RSV infection.

Description

Stabilized pre-fusion RSV F B Antigens

The present invention relates to the field of medicine. In particular, the present invention relates to recombinant pre-fusion RSV F _B Proteins and fragments thereof and encoding RSV F _B Nucleic acid molecules of proteins and fragments thereof, and to uses thereof, for example in vaccines.

Background

Respiratory Syncytial Virus (RSV) was soon discovered in the 50 s of the 20 th century to be a recognized pathogen associated with lower and upper respiratory tract infections in humans. Worldwide, it is estimated that 6400 thousands of RSV infections occur annually, leading to 160.000 deaths (WHO acute respiratory tract infections, updated 9 months in 2009). The most severe diseases occur especially in premature infants, the elderly and immunocompromised individuals. RSV is the most common respiratory pathogen in children less than 2 years of age, accounting for about 50% of hospitalizations for respiratory infections, with peak hospitalizations occurring between 2 and 4 months old. Almost all children are reported to be infected with RSV at the age of two. Repeated infections throughout life are due to ineffective innate immunity. In the elderly, the RSV disease burden is similar to that caused by non-pandemic influenza a infection.

RSV is a paramyxovirus belonging to the subfamily pneumoviridae. The genome encodes a variety of proteins, including membrane proteins known as RSV glycoprotein (G) and RSV fusion (F) proteins, which are the primary antigen targets for neutralizing antibodies. Antibodies against the F protein can prevent the virus from entering the cell and thus have a neutralizing effect.

RSV F fuses viral and host-cell membranes from an unstable pre-fusion conformation to a stable post-fusion conformation by irreversible protein refolding. The structure of both conformations of RSV F has been determined (McLellan JS et al, (2010, 2013, 2013); swanson KA et al, (2011)), as well as fusion proteins from related paramyxoviruses, provide insight into the complex mechanisms that such fusion proteins undergo. Like other class I fusion proteins, inactive precursor RSV F ₀ It is required to be cleaved by furin-like protease during intracellular maturation. RSV F comprises two furinsEnzyme cleavage sites, which result in three proteins: f2, p27 and F1. The p27 fragment is not part of the mature F protein and F2 and F1 are linked by two disulfide bridges, the latter containing a hydrophobic Fusion Peptide (FP) at their N-terminus. In order to refold from the pre-fusion conformation to the post-fusion conformation, refolding region 1 (RR 1), including FP and heptad repeat region a (HRA), between residues 137 and 216 must be transformed from a set of helices, loops and chains to a long continuous helix. The FP located at the N-terminal segment of RR1 can then extend away from the viral membrane and insert into the proximal membrane of the target cell. Refolding region 2 (RR 2) then forms a C-terminal stem in the pre-fusion F spike and includes a heptad repeat region B (HRB), relocates to the other side of the RSV F head, and binds the HRA coiled-coil trimer to the HRB domain to form a six-helix bundle. Formation of the RR1 coiled coil and repositioning of RR2 to complete the six-helix bundle is the most significant structural change that occurs during the refolding process.

There is currently no vaccine to prevent RSV infection, but due to the high disease burden, this vaccine is highly desirable. RSV fusion glycoprotein (RSV F) is an attractive vaccine antigen because it is the primary target for neutralizing antibodies in human serum. Most neutralizing antibodies in human serum are directed against the pre-fusion conformation, but due to its instability, the pre-fusion conformation has a tendency to refold prematurely into the post-fusion conformation, both in solution and on the virosome surface. As described above, the crystal structure has shown a large conformational change between the pre-fusion state and the post-fusion state. The magnitude of the rearrangement suggests that only a portion of the antibodies directed against the post-fusion conformation of RSV-F will be able to cross-react with the native conformation of the pre-fusion spike on the viral surface. Thus, efforts to produce anti-RSV vaccines have focused on developing vaccines comprising pre-fusion forms of the RSV F protein (see, e.g., WO20101149745, WO2010/1149743, WO2009/1079796, WO 2012/158613). So far, these efforts have focused on RSV F protein derived from RSV a strain, and until today no vaccine is available.

Human RSV (HRSV) is divided into two major subtypes; HRSV a and HRSV B are typically distinguished based on sequence differences in G proteins. Although A (F _A ) And B (F) _B ) F protein of strainShows a high degree of sequence identity (about 95% in the mature extracellular domain), but it is not clear whether the cross-reactivity of anti-F antibodies is sufficiently broad.

There remains a need for effective vaccines against RSV. The present invention aims to provide stabilized pre-fusion RSV F for use in vaccines against RSV ^B A method of protein.

Disclosure of Invention

The present invention provides recombinant stabilized pre-fusion RSV fusion (F) proteins comprising F1 and F2 domains, the F1 and F2 domains comprising the F protein of the RSV B strain (RSV F _B Protein) F1 and F2 domains.

The invention also provides a coding pre-fusion RSV F _B Nucleic acid molecules of proteins or fragments thereof, and vectors, such as adeno-associated vectors, comprising such nucleic acid molecules.

The invention also provides a composition, preferably an immunogenic composition or vaccine, comprising RSV F as described herein _B Proteins, nucleic acid molecules and/or vectors, and their use in inducing an immune response against RSV F protein, in particular their use as vaccines against RSV.

The invention also provides a method of inducing an immune response against Respiratory Syncytial Virus (RSV) in a subject comprising administering to the subject an effective amount of pre-fusion RSV F _B Protein, coding said RSV F _B A nucleic acid molecule of a protein and/or a vector comprising said nucleic acid molecule, as described herein. Preferably, the induced immune response is characterized by the induction of neutralizing antibodies and/or cellular responses against RSV and/or protective immunity against RSV infection.

The invention provides, inter alia, methods of vaccinating a subject against RSV comprising administering to the subject a composition or vaccine as described herein.

The invention also provides a method of preventing RSV infection and/or replication in a subject, the method comprising administering to the subject a composition or vaccine as described herein.

Drawings

Fig. 1: schematic of RSV F protein. F0 is enzymatically processed by furin-like protease into F1 subunit and F2 subunit at two positions, resulting in release of the p27 peptide in the mature processed protein. F1 and F2 are linked together by disulfide bonds (not shown).

Fig. 2: analysis of post-transfection cell culture supernatants measured by biolayer interferometry. The RSV F concentration and stability in the supernatant of the unstabilized and stabilized F variants as described in example 2 are shown. Total polypeptide content and pre-fusion content were measured by CR9506 and CR9501 binding, respectively. Post-fusion polypeptide content was measured by ADI-15644 (Gilman et al 2016) binding. Non-stabilized F protein RSV181177 (SEQ ID NO: 3) was compared to the stabilized variants on the day of harvest and after 7 days (A). RSV F with RSV type a (RSV 180816 and RSV 180913) or RSV type B (RSV 180910 and RSV 180907) signal peptide _B The variant RSV F expression levels, with or without a C-tag on the day of harvest (B). Untagged RSV F with several stable amino acid substitutions _B Pre-fusion F expression level of variants: RSV180913 (i152 m+k226m+d486n+s215 p+l21i+p101q, SEQ ID NO: 14) and an additional stabilizing mutation D489Y (RSV 190417;SEQ ID NO:15), wild-type amino acid residue (K226) at position 226 (RSV 190414, SEQ ID NO: 16) and drift mutation L172q+s173L (RSV 190420;SEQ ID NO:17) on day 0 and day 30 post harvest (20 μg postrsv F protein incorporated into supernatant of mock transfected cells as positive control for post F evaluation) (C). Figures 2A and 2B show the mean and error bars of two independent transfections. The data in fig. 2C are based on one transfection.

Fig. 3: SEC curve of the last purification step of the selected protein variant as described in example 3. The protein fraction is collected between the two vertical dashed lines.

Fig. 4: SDS-PAGE analysis. The combined portions of RSV180915 (SEQ ID NO: 6), RSV180916 (SEQ ID NO: 8) and RSV180917 (SEQ ID NO: 9) were protein imprinted (A) under non-reducing and reducing conditions. In B and C, the gel was stained with Coomassie. A sample of the RSV190913 (SEQ ID NO: 14) protein contained a combined peak (B) in the SEC chromatogram under non-reducing and reducing conditions. SDS-PAGE (C) of crude harvest (1) and purified F protein (2) under non-reducing (right panel) and reducing (left panel) conditions of RSV190414 (SEQ ID NO: 16), RSV190420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18).

Fig. 5: analytical SEC analysis of purified F protein. Aggregates and trimers are denoted by a and T, respectively. Proteins have been evaluated by HPLC or UPLC with trimer retention times of about 6.5 minutes or 4.5 minutes, respectively.

Fig. 6: analytical SEC analysis of purified RSV preF B-type protein (n=2) after 35 days of storage at 37 ℃. Aggregates and trimers are denoted by a and T, respectively. Proteins have been evaluated using HPLC or UPLC with trimer retention times of about 6.5 minutes or 4.5 minutes, respectively.

Fig. 7: low temperature stability of purified RSV preF B-type polypeptides of RSV 180913 (SEQ ID NO: 14), RSV19420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18). Residual pre-fusion trimer percentage after slow freezing process in different formulation buffers as measured by analytical SEC. The trimer content of the control sample stored at 4℃was set to 100%. Mean data ± SD.

Fig. 8: full length RSV-B F protein in FACS. The polypeptide was transiently expressed in expiHEK293F cells for 2 days, followed by heat stress at 37 ℃ or 55 ℃ for 10 minutes. Surface expression of PreF protein was measured with monoclonal antibody CR9501 specific for the pre-fusion conformation of RSV F.

Fig. 9: preF (preF) _B Immunogenicity of RSV190420 (SEQ ID NO: 17) in mice and cotton rats. On day 0 and day 28, RSV preF _B The proteins were administered as intramuscular immunity in mice and cotton rats. Virus neutralizing antibody titers against the indicated RSV strains were determined by a firefly luciferase-based assay (a), plaque reduction neutralization assay (B) or micro-neutralization assay (C) 2 weeks (mice) or 3 weeks (cotton rats) after the last immunization. The symbols represent the neutralization titers of the individual animals, while the average titers are indicated by horizontal lines. The lower limit of detection or identification is indicated by a dashed line. And (B): buffer is prepared.

Fig. 10: immunogenicity and protective efficacy of preF-B RSV200125 (SEQ ID NO: 18) in cotton rats. RSV preF-B protein was administered as intramuscular immunization in cotton mice on day 0 and day 28 and animals were challenged intranasally with RSV A2 on day 49 or RSV B detergent on day 50. Pulmonary and nasal viral loads (a) were determined by plaque assay in tissue homogenates isolated 5 days after challenge. Neutralizing antibodies against RSV strains in serum samples prior to challenge were analyzed by firefly luciferase-based assay (B) or plaque reduction neutralization test (C). The symbols represent viral load or neutralization titer of individual animals, while average titer is indicated by horizontal lines. The lower limit of detection or identification is indicated by a dashed line. And (B): buffer is prepared.

Fig. 11: immunogenicity of Ad26 encoding processed or single stranded variants of preF-B (SEQ ID NOS: 32 and 34) in mice. Mice were immunized with varying dose levels of ad26.Rsv. Pref-B processing or single chain. Virus neutralizing antibody titers against the indicated RSV strains were determined 6 weeks after immunization by a firefly luciferase-based assay (a) or plaque reduction neutralization test (B). RSV F-directed cellular immune responses were determined by IFN-gamma ELISPOT assay in splenocytes isolated 6 weeks post-immunization. The symbols represent responses of individual animals, while the average response is indicated by horizontal lines. The lower limit of detection or identification is indicated by a dashed line. And (B): buffer is prepared.

Fig. 12: immunogenicity and protective efficacy of the preF-B proteins RSV190414 (SEQ ID NO: 16), RSV190420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18) in cotton rats. RSV preF-B protein (50 μg) was administered as intramuscular immunization in cotton rats on day 0 and day 28, and animals were challenged intranasally with RSV B17-058221 on day 49. Pulmonary and nasal viral loads (a) were determined by plaque assay in tissue homogenates isolated 5 days after challenge. Neutralizing antibodies against RSV strains in the serum samples prior to challenge were analyzed by firefly luciferase-based assay (B) or micro-neutralization assay (C). The symbols represent viral load or neutralization titer of individual animals, while average titer is indicated by horizontal lines. The lower limit of detection or identification is indicated by a dashed line.

Fig. 13: immunogenicity and protective efficacy of Ad26 encoding processed preF-B (SEQ ID NO: 32) in cotton rats. Ad26.rsv-b.pref was administered as intramuscular immunization in cotton rats on day 0 and animals were challenged intranasally with either RSV A2 or RSV B17-058221 on day 49. Pulmonary and nasal viral loads (a) were determined by plaque assay in tissue homogenates isolated 5 days after challenge. Pre-challenge serum samples (B) were analyzed for neutralizing antibodies against RSV strains as indicated by the micro-neutralization assay. The symbols represent viral load or neutralization titer of individual animals, while average titer is indicated by horizontal lines. The lower limit of detection or identification is indicated by a dashed line.

Detailed Description

Human RSV (HRSV) is a common contributor to respiratory tract infections, causing bronchitis, pneumonia, and chronic obstructive pulmonary infections in humans of all ages. Fusion proteins (F proteins) of Respiratory Syncytial Virus (RSV) are involved in fusion of the viral membrane with the host cell membrane, which is required for infection. RSV F mRNA is translated into a 574 amino acid precursor protein designated F0 that contains a signal peptide sequence (e.g., amino acid residues 1-25 of SEQ ID NO: 1) at the N-terminus that is removed by a signal peptidase in the endoplasmic reticulum. F0 is cleaved by cellular proteases, especially furin or furin-like proteases, at two furin cleavage sites (between amino acid residues 109/110 and 136/137), removing the short glycosylation intervening sequence (also called the p27 region, comprising amino acid residues 110 to 136), and generating two domains (or subunits), designated F1 and F2 (fig. 1).

The F1 domain (amino acid residues 137-574) contains a hydrophobic fusion peptide at its N-terminus and a transmembrane region (TM) (amino acid residues 530-550) and a cytoplasmic region (amino acid residues 551-574) at its C-terminus. The F2 domain (amino acid residues 26-109) is covalently linked to F1 through two disulfide bridges. F1 heterodimer-F2 heterodimers assemble as homotrimers in virions. According to the present invention, "processed RSV F protein" refers to RSV F protein after cleavage at the furin cleavage site, i.e. without signal peptide and p27 region.

As mentioned above, no vaccine against RSV infection is currently available. One potential method of producing vaccines is to provide subunit vaccines based on purified RSV F protein. However, for this approach, it is desirable that the purified RSV F protein have a conformation similar to the pre-fusion state of the RSV F protein and are stable over time. Thus, efforts have focused on RSV F proteins that have been stabilized in a pre-fusion conformation.

Human RSV is divided into two major groups of strain antigens, subtypes a and B, primarily defined by genetic variation of the G glycoprotein. These subtypes show an irregular, alternating pattern of popularity, with subtype a having a higher cumulative prevalence than subtype B. Protein F is highly conserved between RSV a and B and induces neutralizing antibodies across both groups. However, although the F proteins of strain a and strain B show a high degree of sequence identity (about 95% in the mature extracellular domain), it is not clear whether the cross-reactivity of anti-F antibodies is sufficiently broad, and based on RSV F _A Whether the vaccine of the protein can prevent infection of the RSV B strain.

The present invention provides novel stabilized recombinant pre-fusion RSV fusion (F) proteins comprising F1 and F2 domains, the F1 and F2 domains comprising the amino acid sequences of the F1 and F2 domains of the F protein of the RSV B strain, wherein the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 215 is P, and the amino acid residue at position 486 is N. Thus, the present invention provides a stabilized recombinant pre-fusion F protein of RSV FB subgroup (RSV FB) protein or fragment thereof. The numbering of amino acid residue positions is identical to that of SEQ ID NO. 1. According to the invention, the presence of specific amino acids at the indicated positions has been shown to increase the stability of the protein in the pre-fusion conformation. According to the invention, a specific amino acid may already be present in RSV F _B In the amino acid sequence of a protein, or may be introduced into a specific amino acid residue according to the invention by substitution (mutation) of the naturally occurring amino acid residue at said position. According to the invention, e.g. with wild type RSV F _B Amino acid sequences of proteins the amino acid sequences of these proteins may thus comprise one or more mutations compared to the amino acid sequences of the proteins.

According to the invention, the term "stabilized pre-fusion protein" refers to a protein that is stabilized in a pre-fusion conformation, i.e. comprises at least one epitope specific for the pre-fusion conformation of the RSV F protein, e.g. as determined by the specific binding of antibodies specific for the pre-fusion conformation to the protein, and can be produced (expressed) in sufficient quantity.

In certain embodiments, the amino acid residue at position 203 is I. The presence of I at location 203 is shown to also increase RSV F in accordance with the invention _B Protein stability, especially in soluble RSV F _B In proteins.

Alternatively, or in addition, the amino acid residue at position 489 is Y. According to the invention, it is shown that the stability of the polypeptide is improved by the presence of this amino acid residue at the indicated position.

In certain embodiments, the amino acid residue at position 226 is M. Amino acid M at position 226 increases protein stability and expression.

In a preferred embodiment, the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 203 is I, the amino acid residue at position 215 is P, the amino acid residue at position 486 is N, the amino acid at position 357 is not R, and/or the amino acid residue at position 371 is not Y.

In a preferred embodiment, the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 203 is I, the amino acid residue at position 215 is P, the amino acid residue at position 486 is N, and the amino acid residue at position 489 is Y.

In a preferred embodiment, the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 203 is I, the amino acid residue at position 215 is P, the amino acid residue at position 486 is N, the amino acid residue at position 489 is Y, the amino acid at position 357 is not R, and/or the amino acid residue at position 371 is not Y.

The drift mutations L172Q and S173L that occur in the 2015-2016 epidemic virus population have been reported by Chen et al (Sci Rep.8 (1): 4491, 2018). In addition, lu et al (Sci Rep.9 (1): 3898, 2019) have described L172Q for strain 2015-2018&S173L is fixed, andK191R、I206M&Q209R has already occurred. Based on the sequence of the most recently circulated strain (2018-2019) deposited in ViPR and GISAID, it appears that all five positions are now fixed. Thus, in certain embodiments, the amino acid residue at position 172 is Q and the amino acid residue at position 173 is l. Alternatively, or in addition, the amino acid residue at position 191 is R, the amino acid residue at position 206 is M and the amino acid residue at position 209 is R. Thus, RSV F _B The protein is more similar to the RSV F protein of the circulating RSV B strain.

In a preferred embodiment, the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 172 is Q, the amino acid residue at position 173 is L, the amino acid residue at position 215 is P, the amino acid residue at position 486 is N, and the amino acid residue at position 489 is Y, and optionally the amino acid residue at position 203 is I.

In another preferred embodiment, the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 172 is Q, the amino acid residue at position 173 is L, the amino acid residue at position 191 is R, the amino acid residue at position 206 is M, the amino acid residue at position 209 is R, the amino acid residue at position 215 is P, the amino acid residue at position 486 is N, and optionally the amino acid residue at position 203 is I and/or the amino acid residue at position 489 is Y.

In certain embodiments, the invention provides a recombinant pre-fusion F protein as described herein, wherein the amino acid residue at position 357 is not R and the amino acid residue at position 372 is not Y.

RSV F in accordance with the invention _B The protein may comprise a naturally occurring furin cleavage site. In certain embodiments, the furin cleavage site may have been deleted. The deletion of the furin cleavage site may comprise a deletion of the p27 peptide. In these embodimentsIn this case, the F protein will remain "single chain" protein, i.e., will not be processed into F1 and F2 by furin. In certain embodiments, the furin cleavage site has been deleted by deletion of the p27 peptide, including deletion of amino acids 109-135, and replacement of the deleted p27 peptide with a linker (or a linker sequence, e.g., gsgsgsg) that connects the F1 domain and the F2 domain, optionally in combination with mutation of amino acid R at position 106 to Q (R106Q) and mutation of amino acid F at position 137 to S (F137S).

In certain embodiments, the protein comprises a truncated F1 domain. Thus, to obtain soluble RSV F _B The protein, transmembrane region (TM) and cytoplasmic region may be deleted to produce a soluble secreted F protein (sF protein). As used herein, a "truncated" F1 domain refers to an F1 domain that is not a full-length F1 domain, i.e., wherein one or more amino acid residues are deleted at the N-terminus or C-terminus. According to the invention, at least the transmembrane region and cytoplasmic tail are deleted to allow expression as a soluble extracellular domain. In certain embodiments, the F1 domain has been truncated following deletion of the amino acid at position 513, i.e., the amino acids at positions 514 through 574.

In certain embodiments, the heterotrimeric domain has been linked to the C-terminus of the truncated F1 domain, either directly or through the use of a linker (e.g., the linker sequence SAIG). Because the TM region is responsible for membrane anchoring and increased stability, the anchor-free soluble F protein is significantly less stable than the full-length protein and will even refold more easily into a post-fusion final state. Thus, in order to obtain a stabilized soluble F showing a high expression level of the pre-fusion conformation _B Proteins, heterotrimeric domains can be fused to RSV F directly or through the use of a linker (e.g., the linker sequence SAIG) _B The C-terminus of the truncated F1 domain of the protein. For example, for trimerization of soluble RSV F protein, the fibrin-based trimerization domain may be fused to the C-terminus of the extracellular domain (McLellan et al, (2010, 2013)). This fibrin domain or "folder" is derived from T4 fibrin and was previously described as a heterotrimeric domain (Letarov et al, (1993); S-Guche et al (2004).

In a preferred embodiment, the heterotrimeric domain is a folding subdomain comprising the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 2).

In certain embodiments, the protein comprises RSV F _A Signal peptide of protein to increase expression of soluble protein. The skilled artisan will appreciate that processed RSV F _B The protein does not comprise a signal peptide.

Again, it will be appreciated that the numbering of amino acid residue positions according to the invention is identical to the numbering of amino acids in SEQ ID NO. 1.

According to the invention, the presence of specific stabilizing amino acids at the indicated positions has been shown to increase the stability of the protein in the pre-fusion conformation. According to the invention, the specific amino acid may already be present in the amino acid sequence, or may be introduced into the specific amino acid according to the invention by amino acid substitution (mutation) at said position.

Thus, the present invention provides novel recombinant stabilized pre-fusion RSV F _B Protein, i.e. stabilized RSV F in the pre-fusion conformation _B Proteins, and/or fragments thereof. The stable pre-fusion RSV F proteins or fragments thereof of the invention are in a pre-fusion conformation, i.e., they comprise (display) at least one epitope specific for the pre-fusion conformation F protein. Epitopes specific for the pre-fusion conformation F protein are epitopes that are not present in the post-fusion conformation. Without wishing to be bound by any particular theory, it is believed that RSV F _B The pre-fusion conformation of the protein contains RSV F expressed on natural RSV virions _B The same epitope of the protein and thus may provide advantages for eliciting protective neutralizing antibodies.

In certain embodiments, the pre-fusion RSV F of the invention _B The protein or fragment thereof comprises at least one epitope recognized by a pre-fusion specific monoclonal antibody (e.g., CR 9501). CR9501 comprises the heavy and light chain variable regions of antibody 58C5, and thus comprises binding specificity, which has previously been shown to be a pre-fusion specific monoclonal antibody, i.e. an antibody that binds the RSV F protein in its pre-fusion conformation and not the post-fusion conformation (see WO 2012/006596).

As described above, sheets of pre-fusion RSV F proteinSegments are also encompassed by the present invention. The fragment may result from either or both of an amino-terminal deletion (e.g., by excision of the signal sequence) and a carboxy-terminal deletion (e.g., by deletion of the transmembrane region and/or cytoplasmic tail). Fragments comprising an immunologically active fragment of protein F, i.e., the portion that elicits an immune response in a subject, may be selected. This can be readily determined using computer methods, in vitro methods, and/or in vivo methods, all of which are conventional to the skilled artisan. Thus, as used herein, the term "fragment" refers to a protein having amino-and/or carboxy-terminal and/or internal deletions, but wherein the remaining amino acid sequence is identical to RSV F _B Protein sequences, e.g. RSV F _B The corresponding positions in the full-length sequence of the protein are identical. It will be appreciated that the protein need not be full length nor need to have all of its wild type function in order to induce an immune response and typically for vaccination purposes, and that fragments of the protein (i.e. without signal peptide) are equally useful.

In certain embodiments, a protein or fragment thereof encoded according to the invention comprises a signal sequence, also referred to as a leader sequence or signal peptide, corresponding to amino acids 1-25 of SEQ ID NO. 1. The signal sequence is typically a short (e.g., 5 amino acids to 30 amino acids long) amino acid sequence, present at the N-terminus of most newly synthesized proteins, directed toward the secretory pathway, and is typically cleaved by a signal peptidase to produce a free signal peptide and a mature protein.

The signal sequence may be RSV F _A Or RSV F _B Signal sequence of protein. In certain embodiments, the protein or fragment thereof according to the invention does not comprise a signal sequence.

In certain embodiments, e.g., wild-type RSV F which is not stabilized _B Protein (i.e., without stabilizing amino acids), pre-fusion RSV F of the invention _B The expression level of the protein is increased.

In certain embodiments, e.g., F with no such stable substitution _B Protein, as compared to the pre-fusion content (defined as F bound to pre-fusion specific CR9501 antibody, 7 days after harvesting of the protein after storage at 4℃ _B Part of the protein) is significantly higher. In certain embodimentsIn, e.g. F with no said stable substitution _B The pre-fusion content was significantly higher than 30 days after harvesting the protein after storage at 4 ℃. Thus, in certain embodiments, purified pre-fusion RSV F according to the invention as compared to RSV F protein having no stabilizing amino acid residues at defined positions _B The protein has increased stability when stored at 4 ℃. By "stability upon storage" is meant that the protein still displays at least one epitope specific for a particular antibody (e.g., CR 9501) prior to fusion after storage of the protein in solution (e.g., medium) at 4 ℃ for a period of time. In certain embodiments, the pre-fusion RSV F protein exhibits at least one pre-fusion specific epitope for at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, preferably at least 1 year after storage at 4 ℃.

Pre-fusion RSV F according to the invention _B Proteins are stabilized in the pre-fusion conformation by the presence of one or more of the stabilizing amino acids (either already present or introduced by mutation), i.e., are not readily altered to the post-fusion conformation upon processing of the protein, such as purification, freeze-thawing cycles, and/or storage.

In certain embodiments, the purified pre-fusion RSV F proteins according to the invention have increased stability upon storage at 37 ℃, as compared to RSV F proteins having no stabilizing amino acid residues at defined positions.

In certain embodiments, as described in example 4, pre-fusion RSV F according to the invention as determined by measuring melting temperature, as compared to RSV F protein having a different (e.g., wild-type) amino acid residue at a defined position _B Proteins have increased thermostability.

In certain embodiments, the protein exhibits a higher trimer content after being subjected to freeze-thawing conditions in a suitable formulation buffer, as compared to RSV F proteins having a different (e.g., wild-type) amino acid residue at a defined position.

In certain preferred embodiments, RSV F _B The protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 14, 16, 17, 18, 29, 30, 32 and 34. It should be understood thatAfter expression and processing, the protein will no longer contain signal peptide and p27 peptide. Thus, in certain preferred embodiments, RSV F _B The protein comprises the amino acid sequence: the amino acid sequence comprises: an F2 domain comprising amino acids 26-109 of SEQ ID NO. 14 and an F1 domain comprising amino acids 137-513 of SEQ ID NO. 14; an F2 domain comprising amino acids 26-109 of SEQ ID NO. 16 and an F1 domain comprising amino acids 137-513 of SEQ ID NO. 16; an F2 domain comprising amino acids 26-109 of SEQ ID NO. 17 and an F1 domain comprising amino acids 137-513 of SEQ ID NO. 17; an F2 domain comprising amino acids 26-109 of SEQ ID NO. 18 and an F1 domain comprising amino acids 137-513 of SEQ ID NO. 18; or the F2 domain comprising amino acids 26-109 of SEQ ID NO. 29 and the F1 domain comprising amino acids 137-574 of SEQ ID NO. 29, or the F2 domain comprising amino acids 26-109 of SEQ ID NO. 32 and the F1 domain comprising amino acids 137-574 of SEQ ID NO. 32. Note that the proteins of SEQ ID NOS 30 and 34 will not be processed and will remain single chain proteins comprising amino acids 26-574 of SEQ ID NOS 30 or 34.

In certain embodiments, the protein comprises a HIS tag, strep tag, or c tag. His tag or polyhistidine tag is an amino acid motif in proteins, consisting of at least five histidine (H) residues; the strep tag is an amino acid sequence consisting of 8 residues (WSHPQFEK (SEQ ID NO: 27); the C tag is an amino acid motif consisting of 4 residues (EPEA; SEQ ID NO: 28); the tag is usually located at the N-or C-terminus of the protein and is usually used for purification purposes.

As described above, RSV is known to exist as a single serotype, with two antigen subgroups: a and B. The amino acid sequences of the extracellular domains of the two sets of mature process F proteins are about 95% identical. As used throughout this application, amino acid positions are given with reference to the consensus sequence (SEQ ID NO: 1) of protein F of a clinical isolate of subgroup B. As used herein, the phrase "amino acid residue of RSV F protein at position" x "thus means an amino acid corresponding to an amino acid at position" x "in the RSV F protein of SEQ ID No. 1. Note that in the numbering system used throughout this application, 1 refers to the N-terminal amino acid of the immature F0 protein consensus sequence (SEQ ID NO: 1). When using the F protein of another RSV B strain, the amino acid position of the F protein will be numbered with reference to the F protein number of SEQ ID NO. 1, by aligning the sequence of the other RSV B strain with the F protein of SEQ ID NO. 1, and inserting gaps as required. Sequence alignment may be performed using methods well known in the art, for example by CLUSTALW, bioedit or CLC bench.

As used throughout this application, nucleotide sequences are provided in the 5 'to 3' direction and amino acid sequences are provided from the N-terminus to the C-terminus as is customary in the art.

The amino acid according to the invention may be any of the twenty naturally occurring (or "standard" amino acids). Standard amino acids can be grouped based on their identity. 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 cysteines, which can form covalent disulfide bonds (or disulfide bridges) with other cysteine residues; proline, inducing torsion of the protein backbone; and glycine, more flexible than other amino acids. Table 1 shows abbreviations and properties of standard amino acids.

It will be appreciated by those skilled in the art that the protein may be mutated by conventional molecular biological procedures. Such as RSV F which does not contain these mutations _B In contrast to the proteins, the mutations according to the invention preferably lead to pre-fusion RSV F _B Increased expression levels and/or increased stability of the protein.

The invention also provides a method for encoding RSV F according to the invention _B Nucleic acid molecules of proteins. The term "nucleic acid molecule" as used herein refers to polymeric forms of nucleotides (i.e., polynucleotides), including DNA (e.g., cDNA, genomic DNA) and RNA, as well as synthetic forms and mixed polymers of the foregoing. It will be appreciated that due to the degeneracy of the genetic code, many different nucleic acid molecules may encode the same protein. It will also be appreciated that the skilled artisan can use conventional techniques to generate nucleotide substitutions that do not affect the sequence of the protein encoded by the described polynucleotide to reflect codon usage of any particular host organism in which the protein is to be expressed.Thus, unless otherwise indicated, 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 encoding proteins and RNAs may include introns. The sequences herein are provided in the 5 'to 3' direction as is conventional in the art.

In a preferred embodiment, the nucleic acid molecule encoding a protein according to the invention is codon optimized for expression in mammalian cells, preferably human cells or insect cells. Methods of codon optimisation are known and have been described previously (e.g. WO 96/09378 is described for mammalian cells). A sequence is considered codon optimized if at least one non-preferred codon is replaced with a more preferred codon compared to the wild-type sequence. In this context, a non-preferred codon is a codon that is used in an organism less frequently than another codon encoding the same amino acid, and a more preferred codon is a codon that is used in an organism more frequently than the non-preferred codon. The codon usage frequency for a particular organism can be found in a codon frequency table, such as in http:// www.kazusa.or.jp/codon. Preferably, more than one non-preferred codon, preferably most or all of the non-preferred codons are replaced by more preferred codons. Preferably, codons most frequently used in an organism are used in the codon optimized sequence. Preferably substitution of codons generally results in higher expression.

The nucleic acid sequences may be cloned using conventional molecular biology techniques or generated by DNA synthesis from the head, which may be performed by service companies (e.g., geneArt, genScripts, invitrogen, eurofins) having business in the field of DNA synthesis and/or molecular cloning using conventional procedures.

In certain preferred embodiments, the nucleic acid encodes an RSV F comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 14, 16, 17, 18, 29, 30, 32 and 34 _B And (3) protein.

In certain preferred embodiments, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs 31 and 33.

The invention also provides vectors comprising the nucleic acid molecules as described above. Thus in certain embodiments, the nucleic acid molecule according to the invention is part of a vector. Such vectors can be readily manipulated by methods well known to those skilled in the art and may, for example, be designed to replicate in prokaryotic and/or eukaryotic cells. The vector used may be any vector suitable for cloning DNA and which can be used for expressing a nucleic acid molecule of interest. Suitable vectors according to the invention are, for example, adenovectors, alphaviruses, paramyxoviruses, vaccinia viruses, herpesviruses, retroviral vectors and the like.

In certain embodiments of the invention, the vector is an adenovirus vector. Adenoviruses according to the invention belong to the family adenoviridae, and preferably belong to the family adenoviridae of the genus mammalian adenoviruses. It may be a human adenovirus, but may also be an adenovirus that infects other species, including but not limited to bovine adenovirus (e.g., bovine adenovirus 3, badv 3), canine adenovirus (e.g., CAdV 2), porcine adenovirus (e.g., PAdV3 or 5), or simian adenovirus (including simian adenovirus and simian adenovirus, such as chimpanzee adenovirus or gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV, or AdHu), or a simian adenovirus, such as a chimpanzee or gorilla adenovirus (ChAd, adCh, or SAdV), or a rhesus adenovirus (RhAd). In the present invention, if a human adenovirus is referred to as Ad without indicating a species, it means a human adenovirus, for example, the shorthand symbol "Ad26" means the same as HAdV26, which is a human adenovirus serotype 26. Furthermore, as used herein, the symbol "rAd" means a recombinant adenovirus, e.g., "rAd26" means a recombinant human adenovirus 26.

Most advanced studies have been performed using human adenoviruses, and according to certain aspects of the invention, human adenoviruses are preferred. In certain preferred embodiments, the recombinant adenoviruses according to the invention are based on human adenoviruses. In preferred embodiments, the recombinant adenovirus is based on human adenovirus serotypes 5, 11, 26, 34, 35, 48, 49, 50, 52, and the like. According to a particularly preferred embodiment of the invention, the adenovirus is a human adenovirus of serotype 26. Advantages of these serotypes include low seropositive rates and/or low pre-existing neutralizing antibody titers in the human population, as well as experience for human subjects in clinical trials.

Simian adenoviruses also typically have low seropositive rates and/or low pre-existing neutralizing antibody titers in humans, and extensive work with chimpanzee adenovirus vectors has been reported (e.g., U.S. Pat. No. 4, 6083716; WO 2005/071093; WO 2010/086189;WO 2010085984;Farina et al, 2001,J Virol 75:11603-13; cohen et al, 2002,J Gen Virol 83:151-55; kobinger et al, 2006,Virology 346:394-401; tatsis et al, 2007,Molecular Therapy 15:608-17; see also reviews by Bangari and Mittal, 2006,Vaccine 24:849-62; and Lasaro and Ertl, 2009,Mol Ther 17:1333-39). Thus, in other embodiments, the recombinant adenoviruses according to the invention are based on simian adenoviruses, such as chimpanzee adenoviruses. In certain embodiments, the recombinant adenovirus is based on simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50, or SA 7P. In certain embodiments, the recombinant adenovirus is based on a chimpanzee adenovirus, such as ChAdOx 1 (see, e.g., WO 2012/172277) or ChAdOx 2 (see, e.g., WO 2018/215766). In certain embodiments, the recombinant adenovirus is based on a chimpanzee adenovirus, such as BZ28 (see, e.g., WO 2019/086466). In certain embodiments, the recombinant adenovirus is based on gorilla adenovirus, e.g., BLY6 (see, e.g., WO 2019/086456) or BZ1 (see, e.g., WO 2019/086466).

In a preferred embodiment of the invention, the adenovirus vector comprises capsid proteins from rare serotypes, including Ad26, for example. In typical embodiments, the vector is a rAd26 virus. By "adenovirus capsid protein" is meant a protein on the capsid of an adenovirus (e.g., ad26, ad35, rAd48, rAd5HVR48 vector) that is involved in determining the serotype and/or tropism of a particular adenovirus. Adenovirus capsid proteins typically include fibrin, penton protein and/or hexon protein. As used herein, a "capsid protein" of a particular adenovirus, such as "Ad26 capsid protein" may be, for example, a chimeric capsid protein comprising at least a portion of an Ad26 capsid protein. In certain embodiments, the capsid protein is the complete capsid protein of Ad26. In certain embodiments, the hexon, penton, and fiber are Ad26.

One of ordinary skill in the art will recognize that elements derived from multiple serotypes may be combined in a single recombinant adenovirus vector. Thus, chimeric adenoviruses can be produced that combine desired properties from different serotypes. Thus, in some embodiments, chimeric adenoviruses of the invention may combine preexisting immunity lacking the first serotype with properties such as temperature stability, assembly, anchoring, yield, redirected or improved infection, stability of DNA in target cells, and the like. See, e.g., WO 2006/040330, chimeric adenoviruses Ad5HVR48, which include an Ad5 backbone with a partial capsid from Ad48, and also e.g., WO 2019/086461, chimeric adenoviruses Ad26HVRPtr1, ad26HVRPtr12, and Ad26HVRPtr13, which include an Ad26 viral backbone with partial capsid proteins of Ptr1, ptr12, and Ptr13, respectively.

In certain preferred embodiments, the recombinant adenovirus vectors used in the invention are derived predominantly or entirely from Ad26 (i.e., the vector is rAd 26). In some embodiments, the adenovirus is replication defective, e.g., because it contains a deletion in the E1 region of the genome. For adenoviruses derived from non-group C adenoviruses, such as Ad26 or Ad35, the E4-orf6 coding sequence of the adenovirus is typically exchanged with a human subgroup C adenovirus, such as E4-orf6 of Ad 5. This allows such adenoviruses to propagate in well-known complementary cell lines expressing the E1 gene of Ad5, such as, for example, 293 cells, PER.C6 cells, etc. (see, for example, havenga et al 2006,J Gen Virol 87:2135-43; WO 03/104467). However, such adenoviruses will not replicate in non-complementing cells that do not express the E1 gene of Ad 5.

The preparation of recombinant adenovirus vectors is well known in the art. Preparation of rAD26 vectors is described, for example, in WO 2007/104792 and Abbink et al, (2007) Virol 81 (9): 4654-63. Exemplary genomic sequences for Ad26 are found in GenBank accession EF 153474 and SEQ ID NO 1 of WO 2007/104792. Examples of carriers useful in the present invention include, for example, those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety.

Typically, the vectors used in the present invention are generated using nucleic acids (e.g., plasmids, cosmids, or baculovirus vectors) that contain the entire recombinant adenovirus genome. Thus, the invention also provides isolated nucleic acid molecules encoding the adenoviral vectors of the invention. The nucleic acid molecules of the invention may be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded.

The adenovirus vectors used in the present invention are typically replication defective. In these embodiments, the virus exhibits replication defects by deleting or inactivating regions critical for viral replication, such as the E1 region. The region may be substantially deleted or inactivated by, for example, inserting a gene of interest within the region, such as a gene encoding the RSV F protein (typically linked to a promoter), or a gene encoding the RSV F protein (typically linked to a promoter). In some embodiments, the vectors of the invention may contain deletions in other regions, such as the E2 region, the E3 region, or the E4 region, or insertions of heterologous genes linked to the promoter within one or more of these regions. For E2-mutated adenoviruses and/or E4-mutated adenoviruses, E2-complementing cell lines and/or E4-complementing cell lines are typically used to generate recombinant adenoviruses. Mutations in the adenovirus E3 region do not require supplementation of the cell line, as E3 is not required for replication.

Packaging cell lines are typically used to produce sufficient amounts of the adenovirus vectors for use in the invention. Packaging cells are cells that contain those genes that have been deleted or inactivated in replication defective vectors, thus allowing the virus to replicate in the cell. Suitable packaging cell lines for adenovirus deleted in the E1 region include, for example, PER.C6, 911, 293 and E1A 549.

In a preferred embodiment of the invention, the vector is an adenovirus vector, and more preferably a rAd26 vector, most preferably a rAd26 vector having at least a deletion in the E1 region of the adenovirus genome, such as, for example, abink, J Virol,2007, 81 (9): the vectors described in pages 4654-4663, which are incorporated herein by reference. Typically, the nucleic acid sequence encoding the RSV F protein is cloned into the E1 and/or E3 region of the adenovirus genome.

Comprising encoding pre-fusion RSV F _B Host cells for nucleic acid molecules of proteins also form part of the invention. The pre-fusion RSV F proteins can be produced by recombinant DNA techniques that include expression of these molecules in host cells, such as Chinese Hamster Ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines (such as HEK293 cells), per.c6 cells, or yeast, fungal, insect cells, and the like, or transgenic animals or plants. In certain embodiments, the cells are derived from multicellular organisms, and in certain embodiments, they are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cell is a human cell. Generally, production of a recombinant protein, such as the pre-fusion RSV F protein of the invention, in a host cell comprises introducing into the host cell a heterologous nucleic acid molecule encoding the RSV F protein in an expressible form, culturing the cell under conditions conducive to expression of the nucleic acid molecule, and allowing expression of the protein in the cell. The nucleic acid molecule encoding the RSV F protein in an expressible form may be in the form of an expression cassette and typically requires sequences capable of causing expression of the nucleic acid, such as enhancers, promoters, polyadenylation signals, and the like. Those skilled in the art know that various promoters may be used to obtain expression of a gene in a host cell. Promoters may be constitutive or regulated, and may be obtained from a variety of sources, including viral, prokaryotic, or eukaryotic sources, or may be designed artificially.

Cell culture media can be obtained from different suppliers, and suitable media can be routinely selected for host cells to express the protein of interest, here the pre-fusion RSV F protein. Suitable media may or may not contain serum.

A "heterologous nucleic acid molecule" (also referred to herein as a "transgene") is a nucleic acid molecule that does not naturally occur in a host cell. For example, it is introduced into the vector by standard molecular biology techniques. The transgene is typically operably linked to an expression control sequence. This can be accomplished, for example, by placing the nucleic acid encoding the transgene under the control of a promoter. Other regulatory sequences may be added. Many promoters are available for expression of transgenes and are known to the skilled artisan, for example, these promoters may include viral promoters, mammalian promoters, synthetic promoters, and the like. Non-limiting examples of suitable promoters for obtaining expression in eukaryotic cells are the CMV promoter (US 5,385,839), such as the CMV immediate early promoter, e.g. comprising nucleotides-735 to +95 from the CMV immediate early gene enhancer/promoter. Polyadenylation signals, such as bovine growth hormone poly a signal (US 5,122,458), may be present after the transgene. Alternatively, several widely used expression vectors are available in the art and are available from commercial sources, such as the pcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc., which vectors can be used to recombinantly express the protein of interest, or to obtain suitable promoter and/or transcription terminator sequences, poly a sequences, etc.

The cell culture may be any type of cell culture, including adherent cell cultures, such as adherent to the surface of a culture vessel or microcarriers, as well as suspension cultures. Most large scale suspension cultures operate as batch or fed-batch processes because they operate most directly and scale up. Continuous processes based on the principle of perfusion are becoming more common and also suitable today. Suitable media are also well known to those skilled in the art and are generally available in large quantities from commercial sources or are custom made according to standard protocols. The cultivation may be carried out in a petri dish, roller bottle or bioreactor, for example, using batch, fed-batch, continuous systems, etc. Suitable conditions for culturing cells are known (see, e.g., tissue Culture, academic Press, kruse and Paterson editions (1973), and R.I. Freshney, culture of animal cells: A manual of basic technique, fourth edition (Wiley-List Inc.,2000, ISBN 0-471-34889-9)).

The invention also provides compositions comprising nucleic acid molecules, proteins, fragments thereof and/or vectors according to the invention. In certain embodiments, the invention provides a panel comprising pre-fusion RSV F protein and/or fragments thereof A compound, the pre-fusion RSV F protein exhibiting an epitope that is present in the pre-fusion conformation of the RSV F protein but not in the post-fusion conformation. The invention also provides methods for encoding such pre-fusion RSV F _B A nucleic acid molecule of a protein and/or a vector thereof and/or a vector composition. In a preferred embodiment, the composition comprises RSV F _B Proteins and/or fragments, and vectors according to the invention for simultaneous administration. For administration to humans, the present invention may employ pharmaceutical compositions comprising nucleic acids, proteins and/or carriers, and pharmaceutically acceptable carriers or excipients. In the context of the present invention, the term "pharmaceutically acceptable" means that the carrier or excipient does not have any undesired or detrimental effect on the subject to whom it is administered at the dosage and concentration used. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington, pharmaceutical Sciences, 18 th edition, a.r. gennaro, ed., mack Publishing Company [ 1990)]The method comprises the steps of carrying out a first treatment on the surface of the Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L.Hovgaard editions, taylor&Francis[2000]The method comprises the steps of carrying out a first treatment on the surface of the And Handbook of Pharmaceutical Excipients, 3 rd edition, A.Kibbe edit, pharmaceutical Press [2000 ] ]). The purified nucleic acid, protein and/or carrier is preferably formulated and administered as a sterile solution, although lyophilized formulations may also be utilized. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solution is then lyophilized or filled into a drug dosage container. The pH of the solution is typically in the range of pH 3.0 to 9.5, preferably in the range of pH 5.0 to 7.5. The nucleic acid, protein and/or carrier is typically in solution with a suitable pharmaceutically acceptable buffer, and the solution may also contain salts. Optionally, stabilizers, such as albumin, may be present. In certain embodiments, a detergent is added. In certain embodiments, the nucleic acid, protein, and/or vector may be formulated as an injectable formulation. These formulations contain an effective amount of the nucleic acid, protein and/or carrier, are sterile liquid solutions, liquid suspensions or lyophilized versions, and optionally contain stabilizers or excipients.

For example, adenovirus may be stored in a buffer which is also used in adenovirus world standards (Hoganson et al, development of stable adenovirus vector formulations, bioprocessing, month 3 2002, pages 43-48): 20mM Tris pH 8, 25mM NaCl,2.5% glycerol. Another formulation buffer suitable for administration to humans is 20mM Tris, 2mM MgCl2, 25mM NaCl, 10% w/v sucrose, 0.02% w/v polysorbate-80. Obviously, many other buffers can be used, and several examples of suitable formulations for storage and pharmaceutical administration of purified (adeno) virus preparations can be found, for example, in European patent 0853660, U.S. patent 6,225,289 and International patent applications WO 99/41416, WO 99/12568, WO 00/29024, WO 01/66137, WO 03/049763, WO 03/078592, WO 03/061708.

In certain embodiments, the composition may further comprise one or more adjuvants. Adjuvants known in the art may also increase the immune response to the antigenic determinant used, and pharmaceutical compositions comprising adenovirus and a suitable adjuvant are for example disclosed in WO 2007/110409, which is incorporated herein by reference. The terms "adjuvant" and "immunostimulant" are used interchangeably and are defined as one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response to the adenoviral vectors of the invention. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; an oil-emulsion composition (or oil-in-water composition) comprising a squalene-water emulsion, such as MF59 (see, e.g., WO 90/14837); saponin formulations such as QS21 and immunostimulatory complexes (ISCOMS) (see, e.g., US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); examples of bacterial or microbial derivatives are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3 dMPL), oligonucleotides containing CpG motifs, ADP-ribosylated bacterial toxins or mutants thereof, such as E.coli heat labile enterotoxin LT, cholera toxin CT, etc. Vector-encoded adjuvants may also be used, for example by using a heterologous nucleic acid encoding an oligomerization domain of a C4 binding protein (C4 bp) fused to the antigen of interest (e.g., solabomi et al, 2008,Infect Immun 76:3817-23). In certain embodiments, the compositions of the present invention comprise aluminum as an adjuvant, for example in the form of aluminum hydroxide, aluminum phosphate, potassium aluminum phosphate, or a combination thereof, at a concentration of 0.05mg to 5mg, for example 0.075mg to 1.0mg aluminum content per dose.

In other embodiments, these compositions do not comprise an adjuvant.

The invention also provides a vaccine against RSV comprising a composition as described herein.

The invention also provides the use of a stabilized pre-fusion RSV FB protein, fragments thereof, nucleic acid molecules and/or vectors according to the invention for inducing an immune response against an RSV F protein in a subject.

Also provided are methods for inducing antibodies against RSV F protein, particularly RSV F, in a subject _B Comprising administering to a subject a pre-fusion RSV F according to the invention _B Proteins, and/or nucleic acid molecules, and/or vectors. Also provided is pre-fusion RSV F in accordance with the present invention _B Proteins, nucleic acid molecules and/or vectors for inducing antibodies in a subject against RSV F proteins, in particular RSV F _B Is a human immune response. Also provided is pre-fusion RSV F in accordance with the present invention _B Protein, and/or nucleic acid molecules, and/or vector preparation for inducing antibodies in a subject against RSV F proteins, particularly RSV F _B For use in a medicament for immune response.

Also provided are methods of vaccinating a subject against RSV, particularly against an RSV B strain, comprising administering to the subject a composition or vaccine as described herein.

The invention also provides methods of preventing RSV infection and/or replication in a subject, particularly against an RSV B strain, comprising administering to the subject a composition or vaccine as described herein.

Pre-fusion RSV F of the invention _B The protein, fragment, nucleic acid molecule or vector may be used to prevent (prevent) and/or treat RSV infection, in particular RSV infection caused by a strain of RSV B. In certain embodiments, the prevention and/or treatment may be grouped for patients susceptible to RSV infection. Such patient groupings include, but are not limited to, for example, the elderly (e.g., 50 years, 60 years, and preferably 65 years), young (e.g., 5 years)Age 1), pregnant women (for maternal immunity), hospitalized patients, and patients who have been treated with antiviral compounds but have shown inadequate antiviral responses.

Pre-fusion RSV F according to the invention _B The proteins, fragments, nucleic acid molecules and/or vectors may be used, for example, to treat and/or prevent diseases or conditions caused by RSV alone or in combination with other prophylactic and/or therapeutic treatments such as vaccines (existing or future), antiviral agents and/or monoclonal antibodies.

The invention also provides the use of pre-fusion RSV F in accordance with the invention _B A method of preventing and/or treating RSV infection, particularly RSV infection caused by a strain of RSV B, in a subject in need thereof.

In a specific embodiment, the method for preventing and/or treating RSV infection, particularly RSV infection caused by RSV B strain, in a subject comprises administering to a subject in need thereof an effective amount of pre-fusion RSV F as described herein _B Proteins, fragments, nucleic acid molecules and/or vectors. A therapeutically effective amount refers to an amount of a protein, nucleic acid molecule, or vector effective to prevent, ameliorate and/or treat a disease or condition caused by RSV infection. Preventing 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 RSV infection. As used herein, improvement may refer to a reduction in the visible or perceptible symptoms of disease, viremia, or any other measurable manifestation of influenza infection.

In certain embodiments, the methods result in preventing reverse transcription polymerase chain reaction (RT PCR) demonstrated RSV-mediated Lower Respiratory Tract Disease (LRTD). In certain embodiments, the method results in a reduction in RSV-mediated Lower Respiratory Tract Disease (LRTD) as evidenced by reverse transcription polymerase chain reaction (RT PCR) as compared to a subject who has not been administered a combination vaccine.

Additionally, or alternatively, the method is characterized by the absence or reduction of RSV viral load in the nasal passages and/or lungs of the subject upon exposure to RSV.

In addition, or alternatively, the method is characterized by the absence or reduction of clinical symptoms of RSV in the subject upon exposure to RSV.

In addition, or alternatively, the method is characterized by the presence of neutralizing antibodies against RSV and/or protective immunity against RSV, especially RSV B strain.

In certain preferred embodiments, the method has acceptable safety features.

In certain embodiments, the present invention provides methods for preparing a vaccine against Respiratory Syncytial Virus (RSV), particularly RSV B, comprising providing RSV F according to the invention _B Proteins, fragments, nucleic acids or vectors, and formulating them into pharmaceutically acceptable compositions.

According to the present invention, the term "vaccine" refers to an agent or composition comprising an active ingredient effective to induce a degree of immunity against a pathogen or disease in a subject that will at least cause a reduction in the severity, duration or other manifestation of symptoms associated with the pathogen infection or disease (until completely absent). In the present invention, the vaccine comprises an effective amount of pre-fusion RSV F which elicits an immune response against the F protein of RSV _B Protein, fragment, encoding pre-fusion RSV F _B A nucleic acid molecule of a protein and/or a vector comprising said nucleic acid molecule. This provides a method of preventing severe lower respiratory disease resulting in hospitalization and reducing the frequency of complications in subjects due to RSV infection and replication, such as pneumonia and bronchiolitis. The term "vaccine" according to the invention means that it is a pharmaceutical composition and thus generally comprises a pharmaceutically acceptable diluent, carrier or excipient. Which may or may not contain additional active ingredients. In certain embodiments, it may be a combination vaccine that also comprises other components that induce an immune response, such as an immune response against other proteins of RSV and/or against other sources of infection. The administration of the further active ingredient may be carried out, for example, by separate administration or by administration of a combination product of the vaccine according to the invention and the further active ingredient.

The composition may be administered to a subject, such as a human subject. RSV F in compositions for single administration _B The total dose of protein may be, for example, from 0.01 μg to about 10mg, such as from 1 μg to 1mg, such as from 10 μg to 100 μg. In a composition for single administration, the total dose of (adeno) vector comprising DNA encoding RSV F protein may be, for example, about 0.1 x 10 ¹⁰ VP/ml and 2X 10 ¹¹ VP/ml, preferably about 1X 10 ¹⁰ VP/ml to 2X 10 ¹¹ Between VP/ml, preferably 5X 10 ¹⁰ VP/ml to 1X 10 ¹¹ VP/ml.

The composition according to the invention may be administered using standard routes of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal or mucosal administration, e.g., intranasal, oral, and the like. In one embodiment, the composition is administered by intramuscular injection. The skilled person is aware of the various possibilities of administering compositions, such as vaccines, in order to induce an immune response against antigens in the vaccine.

As used herein, a subject is preferably a mammal, such as a rodent (e.g., mouse, cotton mouse), or a non-human primate, or a human. Preferably, the subject is a human subject.

Proteins, nucleic acid molecules, vectors and/or compositions may also be administered as a prime or boost in a homologous or heterologous prime-boost regimen. If booster vaccination is performed, typically such booster vaccination will be administered to the same subject for a period of one week to one year, preferably between two weeks and four months, after the first administration of the composition to the subject (in such cases referred to as "priming vaccination"). In certain embodiments, the administration comprises a primary and at least one booster administration.

In addition, the proteins of the invention can be used as diagnostic tools, for example to test the immune status of individuals by determining whether antibodies capable of binding to the proteins of the invention are present in the serum of such individuals. Accordingly, the present invention also relates to an in vitro diagnostic method for detecting the presence of an ongoing or past RSV infection in a subject, the method comprising the steps of: a) Contacting a biological sample obtained from the patient with a protein according to the invention; and b) detecting the presence of the antibody-protein complex.

Examples

Example 1: design of soluble trimeric proteins by introducing C-terminal foldlons and stable point mutations。

Several pre-fusion RSV F protein variants were generated. The soluble candidate is truncated at amino acid position 513 of the RSV B F1 domain and fused to a four amino acid linker (SAIG) to form a fibrin trimerisation domain (folder) (GYIPEAPRDGQAYVRKDGEWVLLSTFLSEQ ID NO: 2). The RSV B signal peptide (SEQ ID NO: 24) or RSV A signal peptide (SEQ ID NO: 23) of the fusion protein was used for protein expression. Some of the designs have a C-tag, C-terminal, linker and a folding subsequence to allow affinity purification (e.g., SEQ ID NO 3).

To stabilize the pre-fusion conformation of the protein, several combinations of point mutations were introduced, such as one or more of the mutations P101Q, I152M, K226M, D486N, S215P, L I and/or D489Y.

Example 2: expression and stability of RSV B F variants following transient transfection in HEK293F cells。

RSV F non-stabilized protein (SEQ ID NO: 3) based on truncated consensus sequence of subgroup B (SEQ ID NO: 1) and comprising RSV F of SEQ ID NO:1 for use as expression and stability control _B An extracellular domain of a protein comprising a C-terminal fusion via a linker to a folding subdomain (SEQ ID NO: 2) and an N-terminal signal peptide based on RSV F A-type (SEQ ID NO: 23). To allow affinity purification, a C-tag was introduced for the selected design.

A DNA fragment encoding the protein of the invention (Genscript) was synthesized and cloned into a pcDNA2004 expression vector (modified pcDNA3 plasmid with enhanced CMV promoter). The expression platform used was 293Freestyle cells in 24-deep well plates (Life Technologies). Cells were transiently transfected with 293Fectin (Life Technologies) and at 37℃and 10% CO according to the manufacturer's instructions ₂ Culturing for 5 days. For RSV180910 (SEQ ID NO: 7), RSV180916 (SEQ ID NO: 8), RSV180907 (SEQ ID NO: 13), RSV180913 (SEQ ID NO: 14), RSV190417 (SEQ ID NO: 15 RSV190414 (SEQ ID NO: 16) and RSV190420 (SEQ ID NO: 17), cells were co-transfected with RSV F plasmid and furin plasmid at a 9:1 ratio to increase furin cleavage efficiency. The culture supernatant was harvested and centrifuged at 300g for 5 minutes to remove cells and cell debris. The centrifuged supernatant was then sterile filtered using a 0.22 μm vacuum filter and stored at 4 ℃ until use.

Quantitative Octet (biological layer interferometry) was used to measure protein concentration in the supernatant on the day of harvest and after storage at 4 ℃ for 7 or 30 days. CR9501 (an antibody that specifically recognizes pre-fusion RSV F protein, which comprises the variable region of antibody 58C5 as described in WO 2012/006596) and CR9506 (a antibody that recognizes pre-fusion and post-fusion RSV F protein, and comprises a heavy chain variable region comprising SEQ ID NO:21 and a light chain variable region comprising SEQ ID NO: 22) are biotinylated and immobilized on a streptavidin biosensor (ForteBio, portsmouth, UK) by standard protocols. For the specific antibody ADI-15644 (Gilman et al 2016) after fusion, an anti-human Fc sensor was used to immobilize the antibody on the biosensor.

The coated biosensor was then enclosed in the mock cell culture supernatant. The quantitative experiments were performed as follows: the temperature was 30℃and the shaking speed was 1000rpm, and the measurement time was 300 seconds. The concentration of protein was calculated using a standard curve. Use of pre-fusion RSV F diluted in mimetic medium _A The protein was used to prepare a standard curve (SEQ ID NO:19; previously described in WO 17174568) for each of the coated antibodies CR9501 and CR 9506. For ADI-15644, equilibrium binding (2A) or initial binding rate per second (2C) is defined. In FIG. 2C, a positive control was performed against RSV postF binding, thus 20. Mu.g of RSV postF protein (SEQ ID NO: 20) was incorporated into the supernatant of mock transfected cells and measured. Data analysis was done using ForteBio data analysis 10.0.1.6 software (ForteBio).

Results and discussion：

All variants showed F expression as measured by CR9506 binding (fig. 2). Non-stabilised RSV type F B (RSV 181177, SEQ ID NO: 3) showed very low expression of the pre-fusion protein on the day of harvest, as measured by Mab CR9501 binding, and the pre-fusion F protein was also highly unstable, based on loss of binding to CR9501 after storage of the supernatant at 4℃for 7 days (FIG. 2A). In addition, for unstabilized F, a relatively high amount of post-fusion F protein was detected with Mab ADI-15644. The total amount of polypeptide and the amount of pre-fusion polypeptide in the supernatant can be increased by stabilizing mutations I152M and K226M (RSV 181178 (SEQ ID NO: 4)) (FIG. 2A). The stability in the supernatant was further improved by stabilizing mutations D486N (RSV 181179;SEQ ID NO:5) and S215P (RSV 180915;SEQ ID NO:6) for 7 days. The subsequent addition of stabilizing mutations L203I and P101Q further increased the expression level and reduced the amount of the polypeptide after fusion to a nearly undetectable level (RSV 180916; SEQ ID NO: 8) (FIG. 2A). The introduction of the stabilizing mutations D489Y, T357R and N371Y (RSV 180917 and SEQ ID NO: 9) reduced the expression level. When P101Q (RSV 181180 (SEQ ID NO: 10) and D489Y (RSV 181181 (SEQ ID NO: 11) and T357R+N371Y (RSV 181182 (SEQ ID NO: 12)) were subsequently added to the stabilized F variants with I152M, K226M, D486N and S215PF variants (RSV 180915), NO increase in expression level was observed, but increased stability was observed, since NO post-fusion F was detected after 7 days of storage at 4 ℃.

Next, the effect of the signal peptide on RSV F expression was evaluated by comparing the RSV F A-type signal peptide (SEQ ID NO: 23) with the RSV F B-type signal peptide (SEQ ID NO: 24). RSV F with or without a C-terminal C-tag _B In variants, the expression level (CR 9506 binding) and pre-fusion content (CR 9501 binding) were higher when expressed using RSV F A-type signal peptide (as in SEQ ID NO:8 and 14) (FIG. 2B).

The variant depicted in fig. 2C does not have a tag. Variant RSV1800913 (SEQ ID NO: 14) with stabilizing mutations I152M, K226M, D486N, S215P, L I and P101Q

High binding to pre-fusion specific Mab CR9501 was shown and no post-fusion F trace was detected on the day of harvest. After 30 days of storage at 4 ℃, the pre-fusion level did not decrease.

D489Y (RSV 190417;SEQ ID NO:15) is still similar to RSV 180913. Subsequent back-mutations to consensus K226 (RSV 190414: SEQ ID NO: 16) showed a slight decrease in expression in the supernatant. These variants were further studied after purification (example 4). Drift mutations L172Q and S173L (RSV 190420 (SEQ ID NO: 17) did not affect expression and pre-fusion content, and this variant was further studied after purification (example 4.) after storage of the supernatant at 4℃for 30 days, NO measurable postF binding was present.

Example 3: production and purification of selected variants

A DNA fragment encoding the polypeptide of the invention (Genscript) was synthesized and cloned into a pcDNA2004 expression vector (pcDNA 3 plasmid with internal modification to enhance the CMV promoter).

HEK293 cells were used as expression platforms for RSV180915 (SEQ ID NO: 6), RSV180916 (SEQ ID NO: 8), RSV180917 (SEQ ID NO: 9), RSV181180 (SEQ ID NO: 10), RSV181181 (SEQ ID NO: 11), RSV181182 (SEQ ID NO: 12), RSV190414 (SEQ ID NO: 16), RSV190420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18).

RSV190414 (SEQ ID No. 16), RSV190420 (SEQ ID No. 17) and RSV200125 (SEQ ID No. 18) were co-transfected with 10% furin encoding plasmids to increase the incomplete processing previously observed.

Cells were transiently transfected using 293Fectin (Life Technologies) and cultured at 37 ℃ and 10% co2 for 5 days according to manufacturer's instructions. The culture supernatant was harvested and centrifuged at 300g for 5 minutes to remove cells and cell debris. The centrifuged supernatant was then sterile filtered using a 0.22 μm vacuum filter and stored at 4 ℃ until use.

Proteins were purified using a two-step purification protocol involving CaptureSelectTM C tag affinity columns (HiTrap Capto SPImpRes column; GE Life Sciences, pittsburgh, pa.) for the C-tagged polypeptides RSV180915 (SEQ ID NO: 6), RSV180916 (SEQ ID NO: 8), RSV180917 (SEQ ID NO: 9), RSV181180 (SEQ ID NO: 10), RSV181181 (SEQ ID NO: 11) and RSV181182 (SEQ ID NO: 12) or for the non-tagged proteins RSV190414 (SEQ ID NO: 16), RSV190420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18) by cation exchange at pH 5.0. All proteins were further purified by size exclusion chromatography using a Superdex 200 column (GE Life Sciences, pittsburgh, PA, USA).

HEK293E 253 cells were used as a large scale expression platform for RSV180913 (SEQ ID NO: 14). After 6 days, the protein-containing medium was harvested by low-speed centrifugation (10 min, 1000 g) followed by high-speed centrifugation (10 min, 4000 g). Conditioned medium was concentrated using a 30kDa Quixstand hollow fiber cartridge. Next, the concentrated medium was diafiltered with 1L PBS and 1L 20mM NaOAc,100mM NaCl,pH 5.0. Aggregates were removed by centrifugation and concentrated diafiltration medium was diluted 1:1 with buffer 20mM NaOAc,pH 5.0. Next, the pre-fusion F protein was further purified using cation exchange (10 ml Capto SP-Impres (GE Life Sciences, pittsburgh, pa., USA) in XK16 column, followed by anion exchange chromatography using Resource-Q column (GE Life Sciences, pittsburgh, pa., USA) at pH 8. Finally, the protein was further purified by gel filtration using Superdex 200/600 column (GE Life Sciences, pittsburgh, pa., USA).

Results and discussion：

Several stabilized variants of RSV fusion F B (RSV F _B Protein) by ion exchange followed by purification by SEC (fig. 3). Major peaks of RSV180915, 180916 and 180917 at about 11.5ml to 13ml volumes, and major peaks of RSV190420, 180913 and 200125 at 65ml volumes correspond to pre-trimeric RSV fusion F _B And (3) protein.

Example 4: characterization and stability of preF B-type trimer

SDS-PAGE analysis and Western blotting

Coomassie staining was performed under reducing and non-reducing conditions, and the selected representative purified proteins of example 3 were analyzed on 4% -12% (w/v) Bis-Tris nu page gel, 1x MOPS (Life Technologies). For the F variants transfected without furin co-transfection (RSV 180915, RSV180916 and RSV 180917), western blot analysis was performed as follows: semi-dry blotting was performed according to manufacturer's recommendations. 5% blot-level blocking agent (BGB)) in TBS tween, primary antibody (CR 9506 1:10.000 in 5% BGB) blocked for one hour, incubated overnight, secondary antibody (a-human IgG CW 800 (Rockland Immunochemicals, inc., limerick, PA, US) 1:5000 in 5%) incubated for 1 hour. All incubations were performed on a roller platform at room temperature. After the first and second antibodies, the blots were washed 3 times with 10ml TBS/0.05% Tween 20 for 5 minutes each, followed by a final wash with 10ml PBS. Using both 700CW and 800CW channels, the blot was visualized by scanning on an Odyssey scanner. The scan intensity for the 700CW and 800CW channels was set to 5. The scan quality is set to medium.

Results and discussion：

In fig. 4A, incomplete processing into F1 and F2 was detected for proteins produced in 293HEK cells without furin co-transfection. In fig. 4A, the corresponding band is indicated with f1+p27. Purified proteins obtained after co-transfection with furin showed a single band at the expected height of the F1 and f1+f2 extracellular domains of the reduced and non-reduced gels, respectively (fig. 4B and 4C).

RSV preF B protein trimer content

Purified RSV pre-fusion FB protein was analyzed on analytical SEC to confirm purity and trimer properties.

For RSV180915 (SEQ ID NO: 6), RSV180916 (SEQ ID NO: 8), RSV180917 (SEQ ID NO: 9), RSV181180 (SEQ ID NO: 10), RSV181181 (SEQ ID NO: 11) and RSV181182 (SEQ ID NO: 12), analysis was performed using High Performance Liquid Chromatography (HPLC) affinity 1260 series set up (Agilent). 40 μg (1 ml/min) of each purified protein was run on a TSK gel G3000SWxl column (Sigma-Aldrich). Elution was monitored by UV detector (Thermo Fisher Scientific), dawn Light Scattering (LS) detector (Wyatt Technologies), μt-rEx Refractive Index (RI) detector (Wyatt Technologies) and nanostar Dynamic Light Scattering (DLS) detector (Wyatt Technologies). The trimeric protein has a retention time of about 6.5 minutes. SEC curves were analyzed by the archra 7.3.2.19 software package (Wyatt Technology) (fig. 5).

For RSV180913 (SEQ ID NO: 14), RSV190414 (SEQ ID NO: 16), RSV190420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18), the Vanquick system (ThermoFisher Scientific) with Sepax Unix-C SEC-300 4.6X150mm 1.8 μm column (Sepax (231300-4615), injection volume 20. Mu.L, flow rate 0.3 ml/min) was used for analysis using Ultra High Performance Liquid Chromatography (UHPLC). Elution was monitored by UV detector (Thermo Fisher Scientific), dawn Light Scattering (LS) detector (Wyatt Technologies), μt-rEx Refractive Index (RI) detector (Wyatt Technologies) and nanostar Dynamic Light Scattering (DLS) detector (Wyatt Technologies). The trimeric protein has a retention time of about 4.5 minutes. SEC curves were analyzed with an archra 7.3.2.19 software package (Wyatt Technology) and chromatograms were drawn using GraphPad Prism (8 th edition) (fig. 5).

Results and discussion：

Pre-fusion F of all RSV _B Variants show high trimer content. For RSV181181 (SEQ ID NO: 11) and RSV181182 (SEQ ID NO: 12), small amounts of aggregates were observed (FIG. 5).

Stability and trimer content of FB protein before RSV fusion at 37℃for 35 days

RSV F _B The trimer content of proteins after 35 days of storage at 37 ℃ was assessed by analytical SEC to assess the stable contribution of the different mutations.

For analysis using High Performance Liquid Chromatography (HPLC) for RSV180915 (SEQ ID NO: 6), RSV180916 (SEQ ID NO: 8) and RSV181917 (SEQ ID NO: 9), see above method section for details.

For RSV181180 (SEQ ID NO: 10), RSV181181 (SEQ ID NO: 11) and RSV181182 (SEQ ID NO: 12), analysis was performed using Ultra High Performance Liquid Chromatography (UHPLC), see methods section above for details.

Results and discussion：

Pre-fusion F of purified RSV _B After 35 days of storage of the protein at 37℃as assessed by analytical SEC, variants of RSV180915 (SEQ ID NO: 6), RSV180916 (SEQ ID NO: 8), RSV180917 (SEQ ID NO: 9) and RSV181182 (SEQ ID NO: 12) remained stable (FIG. 6). RSV181180 (SEQ ID NO: 10) and RSV181181 (SEQ ID NO: 11) contain increased amounts of aggregates (fig. 5) compared to non-stressed materials, indicating that the L203I mutation is important for long term stability. D489Y (RSV 181181 (SEQ ID NO: 11)) and T357R and N371Y (RSV 181182 (SEQ ID NO: 11)NO: 12) is shown by a reduction in aggregate compared to RSV181180 (SEQ ID NO: 10).

Temperature stability of RSV F polypeptides based on melting temperature

Melting temperatures of purified polypeptides RSV180915 (SEQ ID NO: 6), RSV180916 (SEQ ID NO: 8), RSV180917 (SEQ ID NO: 9), RSV181180 (SEQ ID NO: 10), RSV181181 (SEQ ID NO: 11), RSV181182 (SEQ ID NO: 12), RSV190913 (SEQ ID NO: 14), RSV190414 (SEQ ID NO: 16), RSV190420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18) were determined by Differential Scanning Fluorometry (DSF). Purified pre-fusion F protein was mixed with a SYPRO orange fluorescent dye (Life Technologies S6650) in 96 Kong Guangxue qPCR plates. The optimal dye and protein concentrations were determined experimentally (data not shown). Protein dilutions were performed in PBS and a negative control sample containing only dye was used as reference subtraction. The following parameters were used for measurements in qPCR instrument (Applied Biosystems ViiA): the temperature is raised from 25℃to 95℃at a rate of 0.015℃per second. Data are collected continuously. Melting curves were drawn using GraphPad PRISM Software (8 th edition), tm was calculated by Spotfire suite (Tibco Software inc.) ₅₀ Values. Melting temperature was calculated at 50% of the fluorescence maximum using a nonlinear EC50 shift equation.

Results and discussion：

Pre-fusion F of RSV with multiple sets of stable mutations _B The melting temperature (Tm 50) of the variants ranged from 60 ℃ to 71 ℃, with dual or single melting events, see table 2.

TABLE 2 temperature stability of purified RSVpre-F B type polypeptides

Na=unavailable

In general, a defined high single melting event is preferred. RSV190414 (SEQ ID NO: 16) shows a single melting event with the highest stability. Addition of drift mutations at positions 172, 173 (RSV 190420 (SEQ ID NO: 17) and 191, 206 and 209 (RSV 200125 (SEQ ID NO: 18)) did not decrease stability.

Low temperature stability of RSV preF B-type trimer

RSVF of RSV180913 (SEQ ID NO: 14), RSV190420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18) were used _B Trimer was dialyzed to formulation buffer 1 or formulation buffer 2 and each formulation was diluted to 0.3mg/ml RSV protein. Formulation buffers 1 and 2 were TRIS-based or phosphate-based, respectively. 0.75ml of each formulation was filled in a glass injection vial with rubber stopper and sealed with an aluminum cap. The vials were slowly frozen to-70 ℃ over 24 hours. The samples were then thawed to room temperature and analyzed by analytical Size Exclusion Chromatography (SEC). SEC analysis was performed using a Vanquish system (ThermoFisher Scientific) using Ultra High Performance Liquid Chromatography (UHPLC), see previous segment trimer content for details.

Results and discussion：

In formulation buffers 1 and 2, the polypeptide remained trimeric after freezing (fig. 7). RSV190420 and RSV200125 are very stable in both buffers. Addition of the stabilizing mutation D489Y to RSV190420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18) reduced trimer loss after freezing in formulation buffer 2.

Example 5: antigenicity of preferred polypeptides

Binding of antibodies to polypeptides RSV190913 (SEQ ID NO: 14), RSV190414 (SEQ ID NO: 16), RSV190420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18) was measured by enzyme-linked immunosorbent assay (ELISA). First, 96-well half-zone HB plates (Perkin Elmer, cat. No. 6002290) were coated with different antibodies (1 g/mL) in Phosphate Buffered Saline (PBS), 50. Mu.L/well, and the plates incubated overnight at 4 ℃. Using pre-fusion specific antibodies: CR9501, ADI-18933, ADI-18882, ADI-18930, ADI-18928, ADI-15594, ADI-18889, ADI-18913 and ADI-15617 (Gilman et al 2016), RSD5-GL (Jones et al 2019) and hRSV90 (Mousa et al 2017). Pre-fusion and post-fusion binding antibodies were used: CR9506 comprising a heavy chain variable region comprising SEQ ID NO. 21 and a light chain variable region comprising SEQ ID NO. 22. Post-fusion antibodies were used: ADI-15644 (Gilman et al 2016). After overnight incubation, the plates were washed 3 times with 100 μl wash buffer (pbs+0.05% tween 20). To each well 100 μl of blocking buffer (2% Bovine Serum Albumin (BSA), 0.05% tween 20 in PBS) was added and the plate incubated with shaking for 1 hour at room temperature. Next, the plate was washed 3 times with 100. Mu.L of wash buffer (PBS+0.05% Tween 20). For sample preparation, the protein samples were first diluted to 4 μg/mL in assay buffer (1% bsa,0.05% tween 20 in PBS). The 4. Mu.g/mL sample was further diluted 4-fold by adding 250. Mu.g of diluent to 750. Mu.g assay buffer. Plates were incubated with shaking for 1 hour at room temperature. After incubation, the plates were washed 3 times with 300 μg wash buffer. To each well 50 μg of pre-and post-fusion binding antibody CR9506 with horseradish peroxidase (HRP) label was added at a concentration of 0.05 μg/mL. Plates were incubated with shaking for 1 hour at room temperature. After incubation, the plates were washed 3 times with 300 μl wash buffer and 20 μl POD substrate was added to each well. Plates were measured within 5-15 minutes after substrate addition (EnSight Multi-mode plate reader, HH34000000, read luminescence). ELISA curves (protein dilution vs RLU) were plotted in GraphPad Prism and IC50 values were calculated using GraphPad Prism (Table 3).

TABLE 3 RSV IC50 value of F variant

n-number of experiments; SD-standard deviation; NA-unavailable; NB-unbound; * Site zero binders described in literature

Results and discussion：

Pre-fusion F without one purified RSV _B The trimer showed binding to the specific monoclonal antibody ADI-15644 after fusion, whereas the post-fusion protein RSV150043 (SEQ ID NO: 20) did bind. For CR9501, CR9506, ADI-18882, ADI-18930, ADI-18928, ADI-15594, ADI-18889, ADI-15617 and RSD5-GL, the RSV fusion F protein RSV190913 (SEQ ID NO: 14), RSV190414 (SEQ ID NO: 14)IC50 values for ID No. 16), RSV190420 (SEQ ID No. 17), RSV200125 (SEQ ID No. 18) and RSV150042 (SSEQ ID No. 19) are comparable.

For pre-fusion specific antibodies ADI-18933 and hRSV90, NO binding was observed to RSV190420 (SEQ ID NO: 17) and RSV200125 (SEQ ID NO: 18), which may be explained by the difference in the antibody footprints of these proteins compared to RSV180913 (SEQ ID NO: 14) and RSV190414 (SEQ ID NO: 16). Furthermore, NO binding of RSV200125 (SEQ ID NO: 18) to the pre-fusion specific monoclonal antibody ADI-18913 was observed, which can be explained by the difference in the surface of the antibody footprint of this protein compared to RSV180913 (SEQ ID NO: 14), RSV190414 (SEQ ID NO: 16) and RSV190240 (SEQ ID NO: 17).

Example 6: expression of full-length, membrane-bound RSV B F variants following transient transfection in expiHEK293F cells And stability。

Full length RSV-F in processed (SEQ ID NO: 25) or single strand (SEQ ID NO: 26) form _B The non-stabilized protein served as a control for expression and stability. These sequences are based on the consensus sequence of subgroup B (SEQ ID NO: 1), which contains an N-terminal signal peptide based on RSV type F A (SEQ ID NO: 23). The C-terminal lysine residue of SEQ ID No. 1 was changed to asparagine, the corresponding residue in the RSV F A protein, to prevent cleavage of the C-terminal lysine. The stabilized full-length variants of these RSV FB-type polypeptides contain 6 stable amino acid substitutions of the processed variants (i.e., P101Q, I152M, L203I, S215P, D486N and D489Y); (SEQ ID NO: 29) and 5 stable amino acid substitutions of the single chain variant (i.e., P101Q, I152M, L203I, S215P and D486N) (SEQ ID NO: 30).

Synthetically encoded full-length RSV F _B DNA fragments (Genscript) of the proteins (SEQ ID NOS: 25, 26, 29, 30, 32 and 34) were cloned into a pcDNA2004 expression vector (modified pcDNA3 plasmid with enhanced CMV promoter). The expression platform used was an expi293Freestyle cell in a 100ml shake flask (Life Technologies). Cells were transiently transfected using Expifectamine (Life Technologies) according to manufacturer's instructions and cultured at 37 ℃ and 10% co2 for 2 days. Cells were harvested by centrifugation at 300g for 5 min and resuspended in PBS. To measure preF eggs White stability cells were subjected to heat stress at 37 ℃ or 55 ℃ for 10 minutes.

Fluorescence Activated Cell Sorting (FACS) for measuring post-heat stress pre-fusion RSV F _B Expression of proteins on plasma membranes. CR9501 was fluorescently labeled with Alexa488 by standard protocol. Cells were stained at 2.5ug/ml CR9501 for 30 min, washed and analyzed on FACS Canto II. Data analysis was performed using FlowJo version 10.6.2 software.

Results and discussion：

Processed and Single Strand wild type RSV-F _B (PR_wild type and SC_wild type, respectively) showed pre-fusion F after incubation at 37 ℃respectively _B Protein expression on the cell surface, as determined by binding to Mab Cr9501 (fig. 8). Binding of Mab CR9501 to pre-fusion F was reduced after incubation at 55℃indicating two versions of wild-type RSV-F _B The proteins were all unstable (fig. 8). Processed and single stranded RSV-B F proteins containing the stabilizing mutations of the invention (PR_stabilized (SEQ ID NO: 32) and SC_stabilized (SEQ ID NO: 34), respectively) showed increased expression of the pre-fusion F protein on the cell surface after incubation at 37℃as compared to the wild-type protein. Furthermore, there was no decrease in binding after incubation at 55 ℃, confirming stabilized RSV F _B Both versions of the protein do have increased stability.

Example 7: preF B (RSV 190420, SEQ ID NO: 17) is immunogenic in mice and cotton rats and and dose-dependently induces antibodies capable of neutralizing RSVA and RSVB strains

On days 0 and 28, balb/c mice were immunized intramuscularly with 15ug, 5ug or 1.5ug of unadjuvanted RSV190420 protein (n=6 per group). On days 0 and 28, cotton rats were immunized intramuscularly with 50ug or 5ug of unadjuvanted RSV190420 or with 5ug of adjuvanted RSV190420 and AdjuPhos (n=10 per group). Control groups were subjected to muscle immunization with Formulation Buffer (FB) (mice n=3, cotton mice n=7). Virus neutralizing antibody responses were measured on day 42 on mice or on day 49 on cotton mice.

Responses were measured using a firefly luciferase (FFL) -based assay for strain RSV a CL57 or RSV B1, a Plaque Reduction Neutralization Test (PRNT) for strain RSV A2 or RSV B detergents, or a trace neutralization (MN) assay for clinical isolates RSV B11-052099 and RSV B17-058221.

Results and discussion：

Intramuscular immunization of mice or cotton mice with preF-B protein RSV190420 resulted in dose-dependent induction of antibodies that were able to neutralize different strains of RSV a and RSV B when assayed using various types of virus neutralization assays (fig. 9). These results demonstrate that the preF-B protein is immunogenic in rodents and induces cross-neutralizing antibodies.

Example 8: preF B (RSV 200125, SEQ ID NO: 18) is immunogenic in cotton rats and induces a target Protection against RSV A2 or RSV B detergent attack

On day 0 and day 28, cotton rats were immunized intramuscularly with 50ug, 5ug, or 1ug of unadjuvanted RSV200125 (each group each challenge virus n=7). Control groups were subjected to muscle immunization with Formulation Buffer (FB) (n=7). Animals were challenged intranasally with RSV A2 on day 49 or RSV B detergent on day 50. 5 days after challenge, lung and nasal tissues were isolated and viral loads in lung and nasal homogenates were determined by plaque assay. Pre-challenge serum was isolated on day 49 or day 50 and virus neutralizing antibody responses were measured on strain RSV a CL57 or RSV B1 (sample on day 49 only) using a firefly luciferase-based assay (FFL) or on strain RSV A2 or RSV B detergent (samples combining day 49 and 50) using a Plaque Reduction Neutralization Test (PRNT).

Results and discussion：

Most cotton mice immunized intramuscularly with any dose level of preF-B protein RSV200125 have no detectable viral load in the lungs after challenge with either RSV A2 or RSV B detergent. In contrast, limited protection against RSV A2 was observed in the nose, while dose-dependent partial protection against RSV B detergent challenge was observed in the nose (fig. 10A). When analyzed using various FFL-based virus neutralization assays (fig. 10B) or PRNT (fig. 10C), RSV antibodies were detected in the pre-challenge serum, which were able to neutralize the different RSV a and RSV B strains. These results demonstrate that the preF-B protein is immunogenic and induces protection in the cotton mouse RSV A2 and RSV B detergent challenge model.

Example 9: adenovirus vector encoded single strand and processed rsvp pref-B protein induces cells and bodies in mice Liquid immune response

With 10 ⁸ 、10 ⁹ Or 10 ¹⁰ Intramuscular immunization of Balb/c mice with Ad26.RSV.preF-B Single-chain Virus particles (vp) (stabilized Pre-fusion RSV F encoding SEQ ID NO:34 or Ad26.RSV.preF-B processed variants) _B Protein (stabilized RSV F encoding SEQ ID NO: 32) _B Protein) on day 0 (n=5 per group). Control groups were subjected to muscle immunization (n=3) with Formulation Buffer (FB). Serum isolated 6 weeks after immunization was assayed for virus neutralization antibody response using a firefly luciferase (FFL) -based assay for strain RSV A2, RSV a CL57 or RSV B1, and for strain RSV A2 or clinical isolate RSV B18-006171 using a Plaque Reduction Neutralization Test (PRNT). The swimming spleen isolated 6 weeks after immunization with the indicated vector or formulation buffer was stimulated with a pool of peptides covering the F sequence of RSV A2, and the RSV-F directed IFN- γ response was measured by ELISPOT.

Results and discussion：

Intramuscular immunization of mice with adenovirus vectors encoding the preF-B protein (processed or single chain variants) resulted in dose-dependent induction of virus neutralizing antibodies. Although a relatively low response to RSV a strain was observed, the apparent dose-dependent response to the various RSV B strains was readily detectable (fig. 11A and 11B). After a single immunization with both vectors, a high RSV F-directed cellular response was induced (fig. 11C).

Example 10: the preF-B proteins RSV190414 (SEQ ID NO: 16), RSV190420 (SEQ ID NO: 17) and immunogenicity and protection of RSV200125 (SEQ ID NO: 18) in cotton rats

By intramuscular on day 0 and day 28Immunization RSV preF was administered at a dose of 50 μg in cotton rats _B Protein (n=10 per group). Control group received intramuscular immunization with Formulation Buffer (FB) (n=7), and animals were challenged intranasally with RSV B17-058221, a recent clinical isolate of RSV B strain, on day 49. Pulmonary and nasal viral loads were determined by plaque assay in tissue homogenates isolated 5 days after challenge (see fig. 12A). The serum samples were analyzed for neutralizing antibodies against RSV strains prior to challenge by firefly luciferase-based assays (fig. 12B) or micro-neutralization assays (fig. 12C). The symbols represent viral load or neutralization titer of individual animals, while average titer is indicated by horizontal lines. The lower limit of detection or identification is indicated by a dashed line.

Results and discussion：

After challenge with RSV B17-058221, use any of the different prefs _B Protein intramuscular immunized cotton mice have no detectable viral load in the lungs. Although complete protection of the nose was observed in animals immunized with RSV2000125, several animals with breakthrough nose infections were observed in groups immunized with RSV190420 or RSV190414 (fig. 12A). When analyzed using FFL-based virus neutralization assays (fig. 12B) or mini-neutralization assays (fig. 12C), RSV antibodies were detected in the pre-challenge serum, which were able to neutralize the different RSV a and RSV B strains. These results demonstrate that the different preF-B proteins are immunogenic and induce protection in the cotton rat RSV B17-058221 challenge model.

Example 11: immunogenicity and preservation of Ad26 encoding processed preF-B (SEQ ID NO: 32) in cotton rats Protection device。

Ad26.rsv-b.pref was administered at dose levels indicated by cotton mouse muscle immunization on day 0 (n=6 or n=7 per group). Control subjects were subjected to muscle immunization with formulation buffer (n=7). Animals were challenged intranasally with either RSV A2 or RSV B17-058221 (a recently clinical isolate, strain RSV B) on day 49. Pulmonary and nasal viral loads were determined by plaque assay in tissue homogenates isolated 5 days after challenge (see fig. 13A). Pre-challenge serum samples were analyzed for neutralizing antibodies against RSV strains as indicated by the micro-neutralization assay (fig. 13B). The symbols represent viral load or neutralization titer of individual animals, while average titer is indicated by horizontal lines. The lower limit of detection or identification is indicated by a dashed line.

Results and discussion：

The cotton mice immunized intramuscularly with ad26.rsv-b.pref had no detectable viral load in the lungs after challenge with either RSV A2 or RSV B17-058221, with only a few cases of breakthrough lung infection in animals immunized with the lowest ad26.rsv-b.pref dose. Further, at 10 ⁷ At vp and higher vaccine doses, complete protection of the nose of RSV B17-058221 infection was observed. In contrast, ad26.rsv-b.pref did not provide complete nasal protection after RSV A2 challenge, although vaccine dose-dependent reduction in nasal viral load was observed (fig. 13A). When assayed using a mini-neutralization assay, RSV antibodies were detected in the pre-challenge serum and were able to neutralize both RSV a and RSV B strains (fig. 13B). These results demonstrate that ad26.RSV-b.pref is immunogenic and induces protection in the cotton rat RSV A2 and RSV B17-058221 challenge models.

TABLE 1 Standard amino acids, abbreviations and Properties

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Sequence(s)

RSVFB commoningFull length (SEQ ID NO: 1)

MELLIHRSSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK

SEQ ID NO. 2 (fibrin)

GYIPEAPRDGQAYVRKDGEWVLLSTFL

3RSV181177RSV F B consensus soluble polypeptide with RSV F _A Signal peptide and underlined foldlons; p27 is underlined in bold; an italic linker; bold c label.

SEQ ID NO:4 RSV181178

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGMAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO:5 RSV181179

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGMAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO:6RSV180915

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGMAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRIPNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO. 7RSV180910 (underlined RSV F) _B A signal

MELLIHRSSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGMAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQILPIVNQQSCRIPNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO:8RSV180916

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQILPIVNQQSCRIPNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYML

TNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL

PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQ

ADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSS

VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT

LYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFDASISQVNEKINQSLAFI

RRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO:9 RSV180917

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQILPIVNQQSCRIPNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYML

TNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL

PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQ

ADRCKVQSNRVFCDTMYSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSS

VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT

LYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFI

RRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO:10 RSV181180

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQLLPIVNQQSCRIPNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYML

TNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL

PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQ

ADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSS

VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT

LYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFDASISQVNEKINQSLAFI

RRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO:11 RSV181181

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQLLPIVNQQSCRIPNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYML

TNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL

PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQ

ADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSS

VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT

LYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFI

RRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO:12 RSV181182

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQLLPIVNQQSCRIPNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYML

TNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL

PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQ

ADRCKVQSNRVFCDTMYSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSS

VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT

LYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFI

RRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO. 13RSV180907 (Label free)

MELLIHRSSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYLSALRTGW

YTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQA

ANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQILPIVNQQSCRIPNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYML

TNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL

PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQ

ADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSS

VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT

LYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFDASISQVNEKINQSLAFI

RRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO. 14RSV180913 (Label free)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQILPIVNQQSCRIPNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYML

TNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL

PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQ

ADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSS

VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT

LYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFDASISQVNEKINQSLAFI

RRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO. 15RSV190417 (Label free)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQILPIVNQQSCRIPNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYML

TNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL

PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQ

ADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSS

VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT

LYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFI

RRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO. 16RSV190414 (Label free)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQILPIVNQQSCRIPNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLT

NSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPI

YGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQA

DTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSV

ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTL

YYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFIR

RSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO. 17RSV190420 (Label free)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQILPIVNQQSCRIPNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLT

NSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPI

YGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQA

DTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSV

ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTL

YYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFIR

RSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO. 18RSV200125 (Label free)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTSRVLDLKNYIN

NQILPMVNRQSCRIPNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYML

TNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL

PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQ

ADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSS

VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT

LYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFI

RRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO:19RSV150042(PRPM)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTG

WYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTP

ATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIAS

GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYI

DKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYM

LTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQ

LPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFF

PQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDV

SSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVG

NTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLA

FIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO. 20RSV150043 (post-fusion)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTG

WYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTP

ATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRAIASGVAVSKVLH

LEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNK

QSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLIN

DMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTP

CWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ

SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGA

IVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNK

QEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

SEQ ID NO. 21CR9506 heavy chain

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSRSLITWVRQAPGQGLEWM

GEISLVFGSAKNAQKFQGRVTITADESTSTAHMEMISLKHEDTAVYYCAA

HQYGSGTHNNFWDESELRFDLWGQGTLVTVSSASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI

EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE

SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK

SEQ ID NO. 22CR9506 light chain

DIVMTQSPSSLSASVGDRVTIACRASQSIGTYLNWYQQKPGKAPKLLIYA

ASSLQSGVPSRFSGSGSGTHFTLAISSLQAEDFATYSCQQSYTIPYTFGQGT

KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

SEQ ID NO. 23RSV fusion protein signal peptide

MELLILKANAITTILTAVTFCFASG

24RSV B fusion protein signal peptide of SEQ ID NO

MELLIHRSSAIFLTLAINALYLTSS

25PR wild type (full length RSV B fusion protein with RSV-A signal peptide as processed variant)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTP

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

IAVSKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTSKVLDLKNYINN

QLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLT

NSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPI

YGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQA

DTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSV

ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTL

YYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIR

RSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLS

KDQLSGINNIAFSN

26 SC_wild type (full length RSV B fusion protein with RSV-A signal peptide as single chain variant)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTP

AANNQARGSGSGRSLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALQL

TNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQ

QKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMS

SNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIK

EGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLP

SEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKN

RGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYY

DPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAII

IVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSN

29 PR_stabilized (full length RSV B fusion protein with RSV-A signal peptide as processing variant)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASG

MAVSKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTSKVLDLKNYIN

NQLLPIVNQQSCRIPNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYML

TNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL

PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQ

ADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSS

VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT

LYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFI

RRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTL

SKDQLSGINNIAFSN

30 SC-stabilized (full-length RSV B fusion protein with RSV-A signal peptide as single-chain variant) SEQ ID NO. 30

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTG

WYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQ

AANNQARGSGSGRSLGFLLGVGSAIASGMAVSKVLHLEGEVNKIKNALQ

LTNKAVVSLSNGVSVLTSKVLDLKNYINNQILPIVNQQSCRIPNIETVIEFQ

QKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMS

SNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIK

EGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLP

SEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKN

RGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYY

DPLVFPSNEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAII

IVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSN

SEQ ID NO:31 Ad26RSV019

ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACAATCCTGAC

CGCCGTGACCTTTTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATT

CTACCAGAGCACCTGTAGCGCCGTGTCCAGAGGATATCTGTCTGCCCT

GAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACA

TCAAAGAAACAAAGTGCAACGGCACCGACACCAAAGTGAAGCTGATC

AAGCAAGAGCTGGACAAGTACAAGAATGCCGTGACCGAACTGCAGCT

GCTGATGCAGAATACCCAGGCCGCCAACAACCGGGCCAGAAGAGAA

GCCCCTCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAA

CGTGTCCATCAGCAAGAAGCGGAAGCGGAGATTCCTGGGCTTTCTGCT

CGGAGTGGGATCTGCCATTGCCTCTGGAATGGCCGTGTCTAAGGTGCT

GCATCTGGAAGGCGAAGTGAACAAGATCAAGAACGCCCTGCAGCTGA

CCAACAAGGCCGTGGTGTCTCTGTCTAATGGCGTGTCCGTGCTGACCA

GCAGAGTGCTGGACCTGAAGAACTACATCAACAACCAGCTGCTGCCC

ATGGTCAACCGGCAGAGCTGCAGAATCCCCAACATCGAGACAGTGAT

CGAGTTCCAGCAGAAGAACAGCAGGCTGCTGGAAATCACCCGCGAGT

TTTCTGTGAATGCCGGCGTGACAACCCCTCTGAGCACCTACATGCTGA

CCAATAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAAC

GACCAGAAAAAGCTGATGAGCAGCAACGTGCAGATCGTGCGGCAGCA

GAGCTACAGCATCATGAGCATTATCAAAGAAGAGGTGCTGGCCTACG

TGGTGCAGCTGCCTATCTACGGCGTGATCGATACCCCTTGCTGGAAGC

TGCACACAAGCCCACTGTGCACCACCAATATCAAAGAGGGCAGCAAC

ATCTGCCTGACCAGAACCGATAGAGGCTGGTACTGCGATAATGCCGG

CAGCGTCAGCTTCTTCCCACAAGCCGATACCTGCAAGGTGCAGAGCA

ACAGAGTGTTCTGCGACACCATGAACAGCCTGACACTGCCTAGCGAG

GTGTCCCTGTGCAACACCGACATCTTCAACTCTAAGTACGACTGCAAG

ATCATGACCTCCAAGACCGACATCAGCTCCTCCGTGATCACATCTCTG

GGCGCCATCGTGTCCTGCTACGGCAAGACAAAGTGTACCGCCAGCAA

CAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACG

TGTCCAACAAAGGCGTGGACACCGTGTCTGTGGGCAACACCCTGTACT

ACGTGAACAAGCTGGAAGGCAAGAATCTGTACGTGAAGGGCGAGCCC

ATCATCAACTACTACGACCCTCTGGTGTTCCCCAGCAACGAGTTCTAC

GCCAGCATCAGCCAAGTGAACGAGAAGATCAACCAGAGCCTGGCCTT

CATCCGCAGATCCGATGAGCTGCTGCACAACGTGAACACCGGCAAGA

GCACCACAAACATCATGATCACCGCCATCATCATCGTGATCATCGTCG

TGCTGCTGTCCCTGATCGCCATCGGACTGCTGCTGTACTGCAAGGCCA

AGAACACCCCTGTGACACTGAGCAAGGATCAGCTGAGCGGCATCAAC

AATATCGCCTTCTCCAAC

SEQ ID NO:32

Transgenic amino acid sequence (RSV-B preF protein, processed): MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGMAVSKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTSRVLDLKNYINNQLLPMVNRQSCRIPNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSN

SEQ ID NO:33：Ad26RSV020

ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACAATCCTGAC

CGCCGTGACCTTTTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATT

CTACCAGAGCACCTGTAGCGCCGTGTCCAGAGGATATCTGTCTGCCCT

GAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACA

TCAAAGAAACAAAGTGCAACGGCACCGACACCAAAGTGAAGCTGATC

AAGCAAGAGCTGGACAAGTACAAGAATGCCGTGACCGAACTGCAGCT

GCTGATGCAGAATACCCAGGCCGCCAACAATCAGGCCAGAGGCTCTG

GATCTGGCAGAAGCCTGGGATTTCTGCTCGGCGTGGGATCTGCCATTG

CCTCTGGAATGGCCGTGTCTAAGGTGCTGCATCTGGAAGGCGAAGTG

AACAAGATCAAGAACGCCCTGCAGCTGACCAACAAGGCCGTGGTGTC

TCTGTCTAATGGCGTGTCCGTGCTGACCAGCAGAGTGCTGGACCTGAA

GAACTACATCAACAACCAGCTGCTGCCCATGGTCAACCGGCAGAGCT

GCAGAATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAAC

AGCAGGCTGCTGGAAATCACCCGCGAGTTTTCTGTGAATGCCGGCGTG

ACAACCCCTCTGAGCACCTACATGCTGACCAATAGCGAGCTGCTGAG

CCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGA

GCAGCAACGTGCAGATCGTGCGGCAGCAGAGCTACAGCATCATGAGC

ATTATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCTATCTAC

GGCGTGATCGATACCCCTTGCTGGAAGCTGCACACAAGCCCACTGTGC

ACCACCAATATCAAAGAGGGCAGCAACATCTGCCTGACCAGAACCGA

TAGAGGCTGGTACTGCGATAATGCCGGCAGCGTCAGCTTCTTCCCACA

AGCCGATACCTGCAAGGTGCAGAGCAACAGAGTGTTCTGCGACACCA

TGAACAGCCTGACACTGCCTAGCGAGGTGTCCCTGTGCAACACCGAC

ATCTTCAACTCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGAC

ATCAGCTCCTCCGTGATCACATCTCTGGGCGCCATCGTGTCCTGCTAC

GGCAAGACAAAGTGTACCGCCAGCAACAAGAACCGGGGCATCATCAA

GACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAAGGCGTGGACA

CCGTGTCTGTGGGCAACACCCTGTACTACGTGAACAAGCTGGAAGGC

AAGAACCTGTACGTGAAGGGCGAGCCCATCATCAACTACTACGACCC

TCTGGTGTTCCCCAGCAACGAGTTCGATGCCAGCATCAGCCAAGTGAA

CGAGAAGATCAACCAGAGCCTGGCCTTCATCAGACGCTCCGATGAGC

TGCTGCACAACGTGAACACCGGCAAGAGCACCACAAACATCATGATC

ACCGCCATCATCATCGTGATCATCGTCGTGCTGCTGTCCCTGATCGCCATCGGACTGCTGCTGTACTGCAAGGCCAAGAACACCCCTGTGACACTGAGCAAGGATCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAAC

SEQ ID NO. 34 protein (Single Strand) encoded by Ad26RSV020

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQAANNQARGSGSGRSLGFLLGVGSAIASGMAVSKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTSRVLDLKNYINNQLLPMVNRQSCRIPNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSNEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSN

Reference to the literature

Gilman et al Sci immunol.1 (6): eaaj1879 (2016)

Jones et al, plos Pathogens:https://doi.org/10.1371/journal.ppat.1007944(2019, 7, 15)

Krarup et al Nature Comm.6:8143 (2015)

Kumaria et al Journal of Virology,8, 372, (2011)

Letarov et al Moscow Biochemistry 64:817-823 (1993)

McClellan et al Science 342, 592-598 (2013)

McClellan et al Nat Struct Mol Biol, 248-250 (2010)

McClellan et al Science 340, 1113-1117 (2013)

Mousa et al, nat Microbiol: 16271.Doi:10.1038/nmicrobiol.2016.271 (2017)

S-Guche et al, J.mol. Biol.337:905-915, (2004)

Swanson et al (2011) Proc Natl Acad Sci U S A,2011, 6, 7, 108 (23): 9619-24.

Claims

1. A stabilized pre-fusion RSV fusion (F) protein comprising F1 and F2 domains, said F1 and F2 domains comprising the amino acid sequences of said F1 and F2 domains of F protein of RSV B strain, wherein the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 215 is P, and the amino acid residue at position 486 is N.

2. The protein of claim 1, wherein the amino acid residue at position 489 is Y.

3. The protein of claim 1 or 2, wherein the amino acid residue at position 203 is I.

4. A protein according to claim 1, 2 or 3, wherein the amino acid residue at position 226 is M.

5. The protein of claim 1, 2 or 3, wherein the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 203 is I, the amino acid residue at position 215 is P, the amino acid residue at position 226 is M, and the amino acid residue at position 486 is N.

6. The protein of claim 3, wherein the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 203 is I, the amino acid residue at position 215 is P, the amino acid residue at position 486 is N, and the amino acid residue at position 489 is Y.

7. The protein of any one of the preceding claims, wherein the amino acid residue at position 172 is Q and the amino acid residue at position 172 is L.

8. The protein of any one of the preceding claims, wherein the amino acid residue at position 191 is R, the amino acid residue at position 206 is M, and the amino acid residue at position 209 is R.

9. The protein of any one of the preceding claims, wherein the furin cleavage site has been deleted.

10. The protein of any one of the preceding claims, comprising a truncated F1 domain.

11. The protein of claim 10, wherein the transmembrane domain and cytoplasmic domain have been deleted, the transmembrane domain and cytoplasmic domain comprising amino acids 514 through 574.

12. The protein of claim 10 or 11, wherein a heterotrimeric domain has been linked to the truncated F1 domain.

13. The protein of claim 12, wherein the heterotrimeric domain is a folding subdomain comprising the amino acid sequence of SEQ ID No. 2.

14. The protein according to any of the preceding claims, comprising an amino acid sequence selected from the group consisting of SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 32 and SEQ ID No. 34 or fragments thereof.

15. The protein of any one of the preceding claims, wherein the protein does not comprise a signal peptide, a p27 peptide, or a tag sequence.

16. A nucleic acid molecule encoding a protein according to any one of the preceding claims 1 to 15.

17. The nucleic acid of claim 16, wherein the nucleic acid molecule is DNA or RNA.

18. The nucleic acid according to claim 16 or 17, which encodes an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 32 and SEQ ID No. 34 or a fragment thereof.

19. A vector comprising the nucleic acid of claim 16, 17 or 18.

20. The vector of claim 19, wherein the vector is a human recombinant adenovirus vector.

21. The vector of claim 20, wherein the adenovirus vector is a replication-incompetent Ad26 adenovirus vector with deletions of the E1 and E3 regions.

22. A composition comprising a protein according to any one of claims 1 to 15, a nucleic acid according to claim 16, 17 or 18 and/or a vector according to claim 19, 20 or 21.

23. A composition comprising a protein according to any one of claims 1 to 15 and a carrier according to claim 19, 20 or 21.

24. A vaccine against RSV comprising the composition of claim 22 or 23.

25. A method for vaccinating a subject against RSV, the method comprising administering the vaccine of claim 24 to the subject.

26. A method for preventing RSV infection and/or replication in a subject, the method comprising administering to the subject the vaccine of claim 24.

27. The method of claim 26, wherein the prevented RSV infection and/or replication is characterized by preventing or reducing reverse transcription polymerase chain reaction (RT PCR) confirmed RSV-mediated Lower Respiratory Tract Disease (LRTD).

28. The method of claim 26, wherein the prevented RSV infection and/or replication is characterized by an absence or reduced RSV viral load in the nasal passages and/or lungs of the subject.

29. The method of claim 26, wherein the prevented RSV infection and/or replication is characterized by the subject being absent or reducing clinical symptoms of RSV upon exposure to RSV.

30. An isolated host cell comprising the nucleic acid of claim 16, 17 or 18.

31. An isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising the nucleic acid of claim 16, 17 or 18.