WO2019066437A1 - Soluble modified respiratory syncytial virus (rsv) f protein antigen - Google Patents

Abstract

The present invention relates to a soluble respiratory syncytial virus (RSV) F protein and comprises immunogenic compositions such as vaccines for treatment and/or prevention of RSV infection.

Description

Soluble strain Respiratory syncytial virus (RSV) F Protein antigen

This application claims the benefit of Korean Application No. 10-2017-0128003, filed on September 29, 2017, and Korean Application No. 10-2018-0113291, filed on September 20, 2018, The specification and drawings are incorporated herein by reference in its entirety. The present invention relates to RSV protein antigens and / or RSV immunostimulatory compositions for the treatment and / or prevention of respiratory syncytial virus (RSV) infections. More particularly, the present invention relates to a soluble RSV F protein antigen, an RSV immunogenic composition comprising the same, a RSV vaccine, and a method for inducing RSV immunity using the same.

Respiratory syncytial virus (RSV) is a worldwide prevalent virus that causes respiratory illnesses and is a major cause of serious respiratory infections in infants and young children. Nearly all infants (> 95%) are infected within 2 years of age. In the United States, infections cause infections close to 100% in 2-3 year-old infants, and there are hundreds of direct signs / indirect signs associated with RSV, Was reportedly dead. It is also known that infants are the main infections, but infect respiratory diseases of patients with weakened immune system and elderly people and cause fatal respiratory diseases. It is the second most common cause of respiratory illness after influenza, but the annual death rate from RSV per 100,000 children under 1 year of age is 1.3 to 2.5 times higher than that of influenza. In 2002, WHO reported that 64,000 people were infected with RSV every year, of which 160,000 died.

In particular, RSV is resistant to external environments such as telephone and door knockers, which survive for six hours on a hard surface, which makes it possible to continuously infect and spread rapidly, which is very likely to outbreak in hospitals and community settings . Symptoms develop from about 4-6 days after exposure to RSV virus. Symptoms such as fever, cough, and wheezing develop from about 3 days after the onset of the first symptom, starting with runny nose and anorexia. Clinical manifestations range from relatively mild symptoms to otitis media, apnea of premature infants, asthma, pneumonia, and bronchiolitis.

RSV belongs to the order of Mononegavirales, Orthopneumovirus (genus) of Pneumoviridae (family). Viruses similar to RSV include measles virus, mumps virus causing parotitis, and parainfluenza types 1, 2, and 3. RSV is a medium-sized virus of the order of 120-200 nm, and has a genome of linear, negative strand RNA consisting of about 15,000 nucleotides. RSV is divided into serotypes A and B by G proteins. The RSV genome encodes 10 proteins and is composed of non-structural proteins and structural proteins. Nonstructural proteins are nonstructural (NS) 1, nonstructural (NS) 2, nucleocapsid (N), phosphoprotein (P) and viral polymerase (L). Among the structural proteins, matrix (M) protein, fusion (F) protein and glyco (G) protein form the envelope of the virus. Among them, F protein and G protein form a spike.

RSV F protein is an important component that fuses with cell membrane at the early stage of virus entry and is known to be a major target of vaccine and antiviral drug due to its high antigenicity. The F protein of RSV changes its structure from "pre-fusion" type to "post-fusion" type during entry into cells during the infection process. The F protein consists of about 574 amino acids and can be divided into F2 (about 20 kDa) and F1 (about 50 kDa) subunits, where the total F protein is F0. Subunit Furin cleavage between F2 and F1 is cleaved by F2 and F1 by furin protease. furin cleavage recognizes the amino acid sequences of the two sites "RARR" and "KKRKRR", and Arginine (R) acts as an important core amino acid. The F1 subunit is called the HRA (fusion domain) at the N-terminal side and the trans-membrane domain (HRB) at the C-terminal side. Two or more heptad in the HRA and HRB regions, It is confirmed that it forms. Fusion of the F protein and cell-virus fusion occurs when the hinge portion of the curved HRA is expanded. It has also been found that trimer formation is induced by purine cleavage with removal of the p27 portion.

As mentioned earlier, RSV is widely prevalent in many parts of the world and is a particularly dangerous virus for infants and young children, but there is no vaccine available. Thus, it is believed that the soluble RSV F protein obtainable with the present invention can provide the immunogen that is required for this requirement.

The present invention is directed to providing recombinant soluble respiratory cell fusion virus (RSV) fusion (F) polypeptides. The soluble RSV F polypeptides of the present invention comprise at least one epitope specific for the RSV F protein. In certain embodiments, the polypeptide is a modified form of the fusion peptide region. In certain embodiments, the polypeptide comprises one or two or more amino acid modifications.

The present invention also relates to the use of a soluble RSV F polypeptide, a nucleic acid molecule and / or a composition for inducing immunogenicity, and its use in inducing an animal and human immunological response to a soluble RSV F protein, particularly its use as a vaccine . The present invention also relates to a nucleic acid molecule encoding the RSV F polypeptide and / or a vector comprising the nucleic acid molecule and / or a polypeptide transcribed from the nucleic acid molecule in a specific context for use as a vaccine, ≪ / RTI > In particular aspects, the present invention relates to a method of inducing a neutralizing anti-respiratory cell fusion virus (RSV) F protein antibody in a subject, comprising the step of administering to the subject a nucleic acid molecule encoding the RSV F polypeptide, and / A vector comprising a nucleic acid molecule, and / or a polypeptide transcribed from a nucleic acid molecule.

Definition of common terms

The term " protein " or " polypeptide " used herein refers to a collection of amino acids that are encoded and generated in a particular nucleic acid. Here, the aggregate means a unit consisting of two, three, four, or more amino acids. Herein, " protein " or " polypeptide " may be understood as meaning "antigen" or "immunogen".

The term " antigen ", as used herein, refers to any substance that induces immunogenicity. The term " antigen " includes macromolecular molecules composed of proteins or proteins. A large protein refers to a large number of protein sequences and protein aggregates, including polymer antigens and virus-like particles (VLPs). Here, immunogenicity induction refers to both cellular immunity and humoral immunity. The term " antigen " includes, for example, all and some foreign material that has penetrated into the body, and induction of cellular immunity by the term " antigen " may mean generation of antigen-specific antibodies. Antigen-specific antibodies include neutralizing antibodies that neutralize re-infiltrated foreign material.

The term "mutated", "mutated", "modified", "mutated" or "modified" as used herein refers to any nucleic acid and / or polypeptide that forms a modified nucleic acid or polypeptide Lt; / RTI > Mutations may include, for example, point mutations, deletions or insertions of single or multiple residues in a polynucleotide, and include changes in the protein-coding region of the gene as well as changes in the protein, such as but not limited to control or promoter sequences - Contains changes in the outer region of the coding sequence. The genetic change may be any form of mutation. For example, a mutation can constitute a point mutation, a frame-shifting mutation, an insertion or deletion of all or part of a gene. In some embodiments, the mutation occurs naturally. In other embodiments, the mutation is the result of an artificial mutation pressure.

As used herein, the term " vaccine " refers to a preparation of an induced antigenic determinant used to induce the formation of antibodies or immunity against dead or weakened pathogen or pathogen. Vaccines are given to provide immunity against diseases caused by various kinds of viruses, for example, influenza and the like. The term " vaccine " also refers to a suspension or solution of an immunogen (e. G., A modified or mutated RSV F protein) administered to a vertebrate animal to cause protective immunity, i. E. it means. The present invention provides vaccine compositions that are immunogenic and capable of providing protection against diseases associated with infection.

In yet another embodiment, mutations in RSV F proteins are the result of genetic engineering.

The term " including ", as used herein, means that the content and content are together. The term " comprising " may also mean addition or content of the content and contents. It should be borne in mind, however, that the term " comprising " means that the contents and contents may be used independently or singly.

&Quot; about " includes all values that provide the same effect and result as a reference value. However, " about " gives meaning only to the reference value. Also, the scope encompassed by the term " about " may vary depending on the scope or characteristics of the content (reference value) in which the term is included. Thus, depending on the context, " about " means, for example, ± 15%, ± 10%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, or less than 1% .

All numerical ranges recited herein include all numerical values falling within numerical range values. For example, it should be understood that the numerical range of " 1000-3000 " In addition, all numerical values recited in this specification may be considered to be independent numerical ranges, and may also be accepted as a combination of numerical values - multiple ranges.

Specific details of the present invention

In order to solve the above-mentioned problems, the present invention provides recombinant RSV F proteins containing the amino acid sequence that has been modified compared to the wild-type RSV F protein. The inventors of the present invention found that the modified RSV F proteins of the present invention increase expression of RSV F proteins and improve immunogenicity compared to wild type RSV F proteins. In certain embodiments, the RSV F proteins are human RSV F proteins.

In one embodiment of the present invention, the recombinant respiratory syncytial virus (RSV) F protein antigen provides a RSV F protein antigen wherein the 525-574 position of the amino acid sequence of SEQ ID NO: 1 is deleted.

The RSV F protein antigen comprises SEQ ID NO: 2.

The RSV F protein antigen may further comprise at least one amino acid substitution at the fusion peptide position of the amino acid sequence of SEQ ID NO: 2.

In the RSV F protein antigen, at least one hydrophobic amino acid may be substituted with a hydrophilic amino acid at amino acid positions 137-145 of SEQ ID NO: 2, and preferably the hydrophobic amino acid may be substituted with an amino acid " A ". That is, the RSV F protein antigen may be a RSV F protein antigen modified with AAGAAAGAG amino acid sequence FLGFLLGVG at amino acid sequence 137-145 of SEQ ID NO: 2.

The RSV F protein antigen may be substituted with a non-polar amino acid at one or more polar amino acids at amino acid positions 137-145 of SEQ ID NO: 2.

The RSV F protein antigen may be an RSV F protein antigen modified from the amino acid sequence FLGFLLGVG of amino acid sequence 137-145 of SEQ ID NO: 2 by QNGQNNGSG.

The RSV F protein antigen may be a RSV F protein antigen modified from the amino acid sequence FLGFLLGVG at position 137-145 of SEQ ID NO: 2 by NSGNSSGGG.

The RSV F protein antigen may be a RSV F protein antigen modified with the amino acid sequence FLGFLLGVG of amino acid sequence 137-145 of SEQ ID NO: 2 by TLSKKRKRR.

The present invention provides recombinant soluble respiratory syncytial virus (RSV) fusion (F) polypeptides comprising a mutated or modified amino acid sequence compared to the wild type RSV F protein. The present invention specifically provides the use of a novel recombinant RSV F protein obtained as a RSV immunogen, obtained through mutation or modification of one or more amino acid sequences of the wild-type RSV F protein. The present invention provides an immunogen for new forms of RSV infection.

The inventors of the present invention have confirmed that the amino acid sequence of a specific region of the F protein among the RSV structural proteins can be modified to provide excellent immunogenicity, thus completing the present invention.

In one embodiment of the present invention, the RSV F protein in which a part of the fusion peptide (F protein) of the wild-type respiratory syncytial virus (RSV) comprising SEQ ID NO: 1 is deleted can be represented by SEQ ID NO: 2, The RSV F protein of SEQ ID NO: 2 may be provided as an RSV immunogen in the body or as an antigen for RSV immunization.

Preferably, the RSV F protein of SEQ ID NO: 2 comprises a deletion of amino acid sequence positions 525-574 of the wild-type RSV F protein of SEQ ID NO: 1. The amino acid sequence position 525-574 of SEQ ID NO: 2 can be understood as a site including a transmembrane domain and a cytosol region of the F protein.

RSV immunogens of the present invention are those in which the RSV F protein of SEQ ID NO: 2, in which the amino acid sequence positions 525-574 of the wild type RSV F protein of SEQ ID NO: 1 is deleted, undergoes one or more sequence variations (amino acids or nucleic acids) , Excellent immunogenicity (i.e., high antibody affinity), and can provide body stability.

In one embodiment of the invention, the RSV immunogen is capable of providing a soluble F protein when the amino acid sequence positions 525-574 of the wild-type RSV F protein of SEQ ID NO: 1 are deleted.

In one embodiment of the invention, the RSV immunogen comprises an RSV F protein of SEQ ID NO: 2 in which the amino acid sequence positions 525-574 of the wild-type RSV F protein of SEQ ID NO: 1 are deleted is one or more sequence variants (amino acids or nucleic acids) When present, soluble F protein can be provided.

Preferably, the position at which the RSV F protein of SEQ ID NO: 2 undergoes one or more sequence variations can be understood as the fusion peptide region of the RSV F protein. Mutations in the fusion peptide region have confirmed that the recombinant RSV F protein can work with excellent immunogenicity. Preferably, the fusion peptide moiety is at position 137-145 of SEQ ID NO: 2, more preferably at position 137-144 of SEQ ID NO: 2.

The inventors of the present invention confirmed that the deletion at positions 137-144 of SEQ ID NO: 2 may contribute to the stabilization of the RSV F protein, but the synergistic effect of the antibody may be insignificant. In addition, when the amino acid of the fusion peptide was replaced with another amino acid (substitution), there was a difference in the immune response, and it was confirmed that it could act as an immunity of excellent effect.

The inventors of the present invention have developed a recombinant RSV F protein immunogen that can provide excellent antibody titer through one or more sequence variations of the fusion peptide of the RSV F protein through long term studies.

In one embodiment of the present invention, the RSV F protein immunogen can be used for prevention and treatment of RSV infection, and can be preferably provided as a vaccine for prevention of RSV infection.

Specifically, one embodiment of the present invention provides a RSV immunogen that mutates or modifies a partial sequence of a fusion peptide of RSV F protein. One embodiment of the present invention is a method for the production of a RSV F protein of SEQ ID NO: 2 wherein the RSV F protein comprises one or more mutations in the amino acid sequence positions 137-145 and / or the RSV F protein of SEQ ID NO: 2 corresponding to the amino acid sequence at positions 137-145 , A recombinant soluble RSV F protein immunogen comprising one or more mutations in the nucleotide sequence of SEQ ID NO: 24. More specifically, it is intended to provide a recombinant soluble respiratory cell fusion virus (RSV) fusion (F) polypeptide. The soluble RSV F polypeptides of one embodiment of the invention comprise at least one epitope specific for the RSV F protein. In certain embodiments, the polypeptide is a modified form of the fusion peptide region. In certain embodiments, the polypeptide comprises one or two or more amino acid modifications.

The present invention also relates to the use of a soluble RSV F polypeptide, a nucleic acid molecule and / or a composition for inducing immunogenicity, and its use in inducing an animal and human immunological response to a soluble RSV F protein, particularly its use as a vaccine . The present invention also relates to a nucleic acid molecule encoding the RSV F polypeptide and / or a vector comprising the nucleic acid molecule and / or a polypeptide transcribed from the nucleic acid molecule in a specific context for use as a vaccine, ≪ / RTI > In particular aspects, the present invention relates to a method of inducing a neutralizing anti-respiratory cell fusion virus (RSV) F protein antibody in a subject, comprising the step of administering to the subject a nucleic acid molecule encoding the RSV F polypeptide, and / A vector comprising a nucleic acid molecule, and / or a polypeptide transcribed from a nucleic acid molecule.

In one embodiment, the variant in which the amino acid sequence positions 525-574 of the wild-type RSV F protein is deleted can be understood as an RSV F protein consisting of SEQ ID NO: 2.

The inventors of the present invention have confirmed that a fusion peptide of the RSV F protein of SEQ ID NO: 2, specifically a mutation at an amino acid position of 137-145 can increase antibody titer. In addition, repeated infections can be reduced and the number of inoculations can be reduced.

The inventors of the present invention have found that a mutation or mutation induced at the RSV F protein antigen and the specific amino acid position of the antigen of SEQ ID NO: 2 increases the expression of RSV F protein in the host cell and significantly increases immunogenicity Respectively.

In one embodiment, the RSV F protein antigen may comprise a fusion peptide of SEQ ID NO: 2, more preferably at least one amino acid substituted at amino acid positions 137-145 of SEQ ID NO: 2. The amino acid sequence 137-145 of SEQ ID NO: 2 can be understood as a fusion peptide portion of the RSV F protein. In one embodiment, the amino acid substitution at amino acid positions 137-145 of SEQ ID NO: 2 may include one or more mutations or mutations of the amino acids corresponding to the position. In one embodiment, the RSV F proteins may comprise three or more modifications or mutations in the amino acids corresponding to that position.

Such substitution can be understood as meaning a modification or mutation of an amino acid. That is, the substitution may be understood to include all cases in which a part of the amino acid sequence of SEQ ID NO: 2 is changed. For example, all cases in which one or more amino acid sequences of the amino acid sequence of SEQ ID NO: 2 are altered.

In one embodiment, at least one hydrophobic amino acid at amino acid positions 137-145 of SEQ ID NO: 2 may be substituted with a hydrophilic amino acid. More specifically, the RSV F protein of SEQ ID NO: 2 is a variation of one or more hydrophobic portions of the fusion peptide portion into hydrophilic portions. More specifically, the invention comprises a RSV F protein antigen (SEQ ID NO: 3, FP1) modified with AAGAAAGAG by substituting A with the hydrophobic portion of the FLGFLLGVG sequence at positions 137-145 of SEQ ID NO:

In one embodiment, at least one of the polar amino acids at amino acid positions 137-145 of SEQ ID NO: 2 may be substituted with a non-polar amino acid. More specifically, the RSV F protein of SEQ ID NO: 2 is a mutation in which the polar portion of the fusion peptide portion is transformed into a nonpolar side chain. More specifically, the present invention encompasses a RSV F protein antigen (SEQ ID NO: 4, FP3) that has been modified from the FLGFLLGVG sequence at positions 137-145 of SEQ ID NO: 2 to QNGQNNGSG. In another embodiment, the RSV F protein antigen (SEQ ID NO: 5, FP4) is obtained by modifying the FLGFLLGVG sequence of amino acid sequence 137-145 of SEQ ID NO: 2 with NSGNSSGGG.

The inventors of the present invention have found that the RSV F protein mutated at amino acid positions 137-145 of SEQ ID No. 2, preferably FP1 (SEQ ID No. 3), FP3 (SEQ ID No. 4) and FP4 (SEQ ID No. 5) Can serve as an excellent immunogen for the purpose of Mutations may occur in the amino acid sequence at positions 137-145 of SEQ ID NO: 2, but as known to those of ordinary skill in the art, substitution, deletion, and insertion of amino acids constituting the protein may result in completely different functions and effects of the protein .

The present invention provides an optimized recombinant RSV F protein that is expected to have the best immunogenicity among the mutations induced particularly at positions 137-145 of SEQ ID NO: 2.

The RSV F protein antigen according to an embodiment of the present invention is a soluble protein, and in one embodiment of the present invention, provides a soluble RSV F protein antigen and / or an immunogenic composition comprising the RSV F protein antigen.

In one embodiment of the present invention, the RSV F protein antigen is selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: Or more of the above protein antigens. In one embodiment, the RSV F protein antigen may comprise at least one protein antigen selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: More preferably, in one embodiment, the RSV F protein antigen may comprise at least one protein antigen selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

In one embodiment of the present invention, the RSV F protein antigen is selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: Or a protein antigen encoded by the above base sequence. Preferably, the RSV F protein antigen may be a protein antigen encoded by any one or more of the nucleotide sequences selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: More preferably, the RSV F protein antigen may be a protein antigen encoded by at least one base sequence selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

In one embodiment of the invention, additional mutations that maintain the stabilization of the F1 portion of the wild RSV F protein. Additional mutations associated with stabilization further include at least one additional mutation selected from the group consisting of (a) to (g) based on SEQ ID NO: 2.

(a) a mutation of the amino acid residue at the 140th position;

(b) a mutation of the amino acid residue at position 163;

(c) a mutation of the amino acid residue at position 179;

(d) a mutation of the amino acid residue at position 188;

(e) a mutation of the amino acid residue at position 189;

(f) a mutation of an amino acid residue at position 487; And

(g) Mutation of amino acid residues at position 505.

More specifically, the RSV F protein antigens of the present invention are based on SEQ ID NO: 2

(a) a mutation of W at the amino acid residue F at position 140 (SEQ ID NO: 7);

(b) a mutation of Q at amino acid residue E at position 163 (SEQ ID NO: 8);

(c) a mutation of L at amino acid residue V at position 179 (SEQ ID NO: 9);

(d) a mutation of Q at the amino acid residue L at position 188 (SEQ ID NO: 10);

(e) a mutation in the amino acid residue T at position 189 (SEQ ID NO: 11);

(f) a mutation of the amino acid residue E at position 487 (SEQ ID NO: 12); And

(g) a mutation at position 505 of the amino acid residue F to W (SEQ ID NO: 13).

In another embodiment, the RSV F protein antigen is

An amino acid corresponding to position 140 of SEQ ID NO: 2 is substituted with W, an amino acid corresponding to 163 is substituted with Q (SEQ ID NO: 14);

The amino acid corresponding to position 140 of SEQ ID NO: 2 is substituted with W, the amino acid corresponding to 163 is substituted with Q, the amino acid corresponding to 188 is substituted with Q, the amino acid corresponding to 189 is substituted with L No. 15);

An amino acid corresponding to position 488 of SEQ ID NO: 2 is substituted with W, an amino acid corresponding to 163 is substituted with Q (SEQ ID NO: 16);

An amino acid corresponding to position 488 of SEQ ID NO: 2 is substituted with W, an amino acid corresponding to 179 is substituted with L (SEQ ID NO: 17);

An amino acid corresponding to position 140 of SEQ ID NO: 2 is substituted with W, an amino acid corresponding to 505 is substituted with W (SEQ ID NO: 18);

An amino acid corresponding to position 487 of SEQ ID NO: 2 is substituted with L, an amino acid corresponding to 505 is substituted with W (SEQ ID NO: 19);

The amino acid corresponding to position 163 of SEQ ID NO: 2 is substituted with Q, the amino acid corresponding to 505 is replaced with W (SEQ ID NO: 20);

An amino acid corresponding to position 188 of SEQ ID NO: 2 is substituted with Q, an amino acid corresponding to 189 is substituted with L, and an amino acid corresponding to 505 is substituted with W (SEQ ID NO: 21);

The amino acid corresponding to position 163 of SEQ ID NO: 2 may be substituted with Q and the amino acid corresponding to 487 may be substituted with L (SEQ ID NO: 22).

The present invention provides a RSV immunogenic composition and / or vaccine comprising said RSV F protein.

Further included within the scope of the present invention are RSV F proteins other than the human RSV F protein (SEQ ID NO: 1), including variants corresponding to those described above. Such RSV F proteins may include, but are not limited to, A strains of human RSV, B strains of human RSV, strains of bovine RSV, and RSV F proteins from strains of avian RSV.

In some embodiments, this includes using known methods of protein processing and recombinant DNA technology to enhance or modify the properties of the mutated RSV F proteins mentioned above. Genes that encode the proteins are modified by codonification, and modifications of the nucleotides are known to those skilled in the art. Various forms of mutagenesis can be used to produce and / or isolate variant nucleic acids encoding the protein molecules and / or further modify / mutate the proteins of the invention. These include, but are not limited to, site-specific mutagenesis, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing template, oligonucleotide-induced mutagenesis, phosphorothioate-modified DNA mutagenesis, DNA (gapped-duplex DNA), and the like. Other suitable methods include point mismatch repair, repair-deficient host strains, restriction-selection and restriction-mutagenesis using purification, deletion mutagenesis, total gene synthesis, double-strand breakage Recovery, and the like. For example, mutagenesis involving a chimeric structure is also encompassed by the present invention. In one embodiment, mutagenesis can be guided by known information, such as sequences, sequence comparisons, physical properties, crystal structures, etc., of molecules that have been generated pre-existing or of naturally occurring molecules with acquired or mutated.

In another embodiment, the present invention provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the vaccine formulations of the present invention.

In another embodiment, the invention provides a vaccine or antigenic composition that induces an immunity to an infection or at least one disease symptom thereof in a mammal, comprising the step of adding an effective dose of a modified or mutated RSV F protein to the agent Thereby providing a method of preparing the composition. In one preferred embodiment, the infection is a RSV infection.

The modified or mutated RSV F protein of the present invention is useful for producing compositions that stimulate an immune response that provides immunity or substantial immunity to an infectious agent. Thus, in one embodiment, the invention provides a method of inducing immunity against a subject's infection or at least one disease symptom thereof, comprising administering at least one effective dose of a modified or mutated RSV F protein.

In another aspect, the invention provides a method of inducing substantial immunity to a subject's RSV viral infection or at least one disease symptom, comprising administering at least one effective dose of a modified or mutated RSV F protein.

The compositions of the present invention can induce substantial immunity in a vertebrate animal (e.g., a human) when administered to a vertebrate animal. Thus, in one embodiment, the invention provides a method of inducing a substantial immunity to a subject ' s RSV viral infection or at least one disease symptom, comprising administering at least one effective dose of a modified or mutated RSV F protein do.

RSV  F-gene recombinant baculovirus  production

(A) preparing a recombinant viral vector by introducing at least one RSV F protein coding gene selected from the group consisting of SEQ ID NO: 25 to SEQ ID NO: 44, and (b) And culturing the host cell to obtain a culture in which the RSV F protein antigen is expressed, thereby providing a method for producing the RSV F protein antigen.

The method may further comprise purifying the expressed RSV F protein or fragment thereof.

The recombinant viral vector may be, for example, a phage, a plasmid, a virus or a retroviral vector, and preferably a viral vector may be used.

In one embodiment, the vector is a recombinant baculovirus vector. The construct encoding the gene and / or the vector should be operably linked to a suitable promoter such as the AcMNPV polyhedrin promoter (or other baculovirus), the Pigilambada PL promoter, the E. coli lac, the phoA and the tac promoter, Lt; RTI ID = 0.0 > polyhedrin < / RTI > promoter.

The expression constructs will further include a ribosome binding site for translation, at the site for transcription initiation, for termination and at the transcribed region. The coding portion of the transcripts expressed by the constructs will preferably initially contain a translation initiation codon and a termination codon appropriately located at the end of the polypeptide to be translated. Preferably, the expression vectors comprise at least one selectable marker. These markers include neomycin resistance genes for dihydrofolate reductase, G418 or eukaryotic cell cultures, and tetracycline, kanamycin, or resistance genes for E. coli and other bacterial cultures . Among the vectors are Baculoviridae (for example, Virus-Autographa californica nucleopolyhedrovirus), Adenoviridae (e.g., canine adenovirus-canine adenovirus), Hepadnaviridae (E.g., Hepadnaviridae, e.g., aviepadnavirus), Vacciniaviridae (e.g., modified vaccinia Ankara virus), and Parvoviridae (e. G. And a virus vector selected from the group consisting of a virus, a virus, an autonomous parvovirus, and the like. Wherein the baculovirus is selected from the group consisting of Autographa californica nucleoside varicella virus or a modified virus strain; Or a Bombyx mori nucleoside viral virus strain or a modified viral strain thereof. Bacterial vectors may also be used. Exemplary bacterial vectors include pQE70, pQE60 and pQE-9, p BlueScript vector, phage script vector, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Of the eukaryotic vectors, pFastBacl pWINEO, pSV2CAT, pOG44, pXTl, pSG, pSVK3, pBPV, pMSG, and pSVL are preferred.

Such recombinant vectors as described above may be used for transfection, infection or transformation and may express proteins in eukaryotic and / or prokaryotic cells. Eukaryotic host cells may include yeast, insect, avian, plant, small nematode (or nematode), and mammalian host cells. Non-limiting examples of insect cells are, for example, Trichoplusiani cells such as Spodoptera frugiperda (Sf) cells such as Sf9, Sf21, and High Five cells, and Drosophila S2 cells. Examples of fungal (including yeast) host cells include S. cerevisiae, Kluyveromyces lactis (K. lactis), C. albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica Candida species. Examples of mammalian cells include human embryonic kidney lineage, Chinese hamster ovary cell lineage, Vero cell line (African green monkey lineage), MRC cell line (human lung fibroblast cell lineage), and MDCK cells (Madin-darby canine kidney cell lineage). Other cells of the African claw frog (Xenopus laevis oocyte) or amphibians can also be used. Prokaryotic host cells include, for example, bacterial cells such as E. coli, B. subtilis and mycobacteria.

Pharmaceutical or vaccine preparation and administration

An embodiment of the present invention is a method for producing a protein having the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, RSV comprising at least one protein selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: Provide a preventive vaccine. Preferably, one embodiment of the present invention provides a RSV preventive vaccine comprising any one or more proteins selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO:

One embodiment of the present invention is directed to an isolated nucleic acid molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, , SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: Protein-containing RSV vaccine. Preferably, one embodiment of the present invention provides a RSV preventive vaccine comprising a protein encoded by any one or more nucleic acids selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: More preferably, an embodiment of the present invention provides a RSV preventive vaccine comprising a protein encoded by any one or more nucleic acids selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

The vaccine may further comprise adjuvants or immunostimulants. For example, an aluminum adjuvant.

The immunogenic compositions of the present invention include any pharmaceutical material that does not itself induce a harmful immune response to the vertebrate to which the composition is administered and may be any suitable diluent that can be administered with the RSV F polypeptide Or a pharmaceutically acceptable carrier comprising an excipient. As used herein, the term " pharmaceutically acceptable " refers to those listed in the United States Pharmacopoeia, European Pharmacopoeia, or other commonly recognized pharmacopoeia for use in vertebrates and more specifically in humans. The RSV F immunogen of the invention is administered in an effective amount or amount (as defined above) sufficient to stimulate an immune response against one or more strains of the RSV virus. Such compositions can be used as vaccine and / or immunogenic compositions for inducing a protective immune response in vertebrates. The composition may contain other RSV F proteins or fragments thereof.

In one non-limiting embodiment, the concentration of the immunogen is at least about 10 μg / mL, about 20 μg / mL, about 30 μg / mL, about 40 μg / mL, about 50 μg / mL, About 100 μg / mL, about 200 μg / mL, or about 500 μg / mL. In certain embodiments, the concentration of the immunogen is from about 10 μg / mL to about 1 mg / mL, or from about 20 μg / mL to about 500 μg / mL, or from about 30 μg / mL to about 100 μg / mL, To about 50 [mu] g / mL. In another embodiment, the concentration of the immunogen may be comprised between 10 μg / mL and 200 μg / mL.

In one embodiment, the pharmaceutical formulations disclosed herein comprise a RSV F protein, predominantly a spike protein; And a pharmaceutically acceptable carrier or excipient.

In another embodiment, the pharmaceutical agent comprises a purified, high affinity antibody produced in an animal to which the immunogen is administered. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer and combinations thereof. Pharmaceutically acceptable carriers, diluents and other excipients are provided in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. current edition). The formulation should be appropriate for the mode of administration. In a preferred embodiment, the formulation is suitable for administration to humans, preferably sterile, not particulate and / or pyrogenic. If desired, the composition may contain minor amounts of wetting or emulsifying agents or pH buffering agents. The composition may be in the form of a solid, liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release preparation or powder, such as a lyophilized powder suitable for recombination. Oral preparations may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the immunogenic vaccine formulations. In a preferred embodiment, the kit comprises two containers, one comprising an RSV F immunogen and the other comprising an antigen adjuvant. Announcements in the form prescribed by governmental bodies governing the manufacture, use or sale of medicinal or biological products may be incorporated into such container (s), and such announcements may be made by the institution of manufacture, use or sale for human administration Indicates approval. The agent may be packaged in a sealed container such as an ampoule or sachette indicating the amount of the composition. In one embodiment, the composition is supplied as a liquid, and in another embodiment, is supplied as a dry sterile lyophilized powder or water-removing concentrate in a sealed container, for administration to a subject, for example, with water or saline It can be reconstituted to an appropriate concentration. Preferably, the composition will preferably contain about 1 μg, about 5 μg, about 10 μg, about 20 μg, about 25 μg, about 30 μg, about 50 μg, about 100 μg, about 125 μg, about 150 μg, As a dry sterile lyophilized powder in an airtight container at a unit dose of about 200 μg. Alternatively, the unit dose of the composition may be about 1 microgram (e.g., about 0.08 microgram, about 0.04 microgram, about 0.2 microgram, about 0.4 microgram, about 0.8 microgram, about 0.5 microgram, about 0.25 microgram, ) Or greater than about 125 μg (eg, greater than about 150 μg, greater than about 250 μg, or greater than about 500 μg). Such doses can be measured as [mu] g of total RSV F protein (e.g., spike protein or fragment thereof). The immunogen of the present invention should be administered within about 12 hours, preferably within about 6 hours, within about 5 hours, within about 3 hours, or within about 1 hour after reconstitution with lyophilized powder. In another embodiment, the RSV F protein immunogenic composition is supplied in liquid form in a sealed container representing the amount and concentration of the RSV F protein composition. Preferably, the liquid form of the immunogenic composition of the invention is at least about 50 μg / ml, more preferably at least about 100 μg / ml, at least about 200 μg / ml, at least 500 μg / ml, or at least 1 mg / ml And is supplied to the sealed container.

The vaccine or immunogenic composition of the invention may be administered to an animal to induce an immune response against RSV. In one embodiment, the animal is vulnerable to RSV infection. In one embodiment, the animal is a human. Preferably, administration of the immunogen induces substantial immunity against at least one RSV strain, isolate, clade and / or species. In one embodiment, administration of an immunogen induces substantial immunity against at least two RSV strains, isolates, clades and / or species. Typically, the dosage can be adjusted within this range based on, for example, age, physical condition, body weight, age, food, time of administration, and other clinical factors. Accordingly, the invention includes a method of formulating a vaccine or immunogenic composition that results in substantial immunity to an infection of the subject or at least one symptom thereof, including the step of adding an effective amount of an immunogen to the formulation. Stimulation of substantial immunity by a single dose is preferred, but additional doses may be administered via the same or different routes to achieve the desired effect. In neonates and infants, for example, multiple administrations may be necessary to induce a sufficient level of immunity. Administration can be continued at intervals over the childhood period if necessary to maintain adequate protection against infection. Similarly, adults who are particularly susceptible to repeated or severe influenza infections, such as health workers, daycare teachers, family members of children, the elderly, and individuals with impaired cardiopulmonary function, may develop a protective immune response and / Or may require multiple immunizations to maintain. The level of induced immunity can be observed, for example, by measuring the amount of neutralizing gland and serum antibody and the adjusted dose or repeated vaccination, as is necessary to induce and maintain the desired level of protection. Thus, in one embodiment, a method of inducing substantial immunity against a viral infection or at least one symptom thereof in a subject comprises administering at least one effective amount of an RSV F protein, or fragment or aggregate thereof. Methods of administering the vaccine and / or immunogenic agent include parenteral administration (e.g., endothelium, intramuscular, intravenous and subcutaneous), epidural and mucosal (e.g., nasal and oral or pulmonary routes or suppositories) But is not limited thereto. In certain embodiments, the composition is administered intramuscularly, intravenously, subcutaneously, orally or intradermally. The composition may be administered orally or parenterally by absorption through, for example, injection or transient injection, epithelium or mucosal (e.g., oral mucosa, colon, conjunctiva, nasopharyngeal, pharyngeal, vaginal, urinary, bladder, May be administered by any convenient route and may be administered with other biologically active substances. In some embodiments, administration via the nasal passage or other mucosal route may result in a substantially higher antibody or other immune response than other routes of administration. In other embodiments, the nasal or other mucosal pathways of administration of the immunogenic composition and / or vaccine may induce antibodies or other immune responses that will induce cross protection against other strains of the virus. Administration may be systemic or local. The prophylactic vaccine preparation is administered systemically by subcutaneous or intramuscular injection using a needle and injection or by a needle-free injection device. Alternatively, the vaccine preparation is administered into the nasal cavity by drops, large particle aerosols (greater than about 10 microns), or by injection into the conduit. Any of the above pathways of delivery may cause an immune response while nasal administration provides an increased effect of inducing mucosal immunity at the site of penetration of the virus. In another embodiment, the vaccine and / or immunogenic agent is administered in a manner that targets mucosal tissue to cause an immune response to the site of immunization. For example, mucosal tissues such as gut associated lymphoid tissue (GALT) may be targets for immunization by using oral administration of a composition containing an immunogen adjuvant with specific targeting properties. Other mucosal tissues such as nasopharyngeal lymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT) may also be targets. The vaccine and / or immunogenic agent may be administered according to a dosing schedule such as administration of the initial vaccine composition followed by intensification of the administration. In certain embodiments, the second dose of the composition is administered at any time between two weeks to one year after the initial administration, preferably about 1, about 2, about 3, about 4, about 5 to about 6 months. The third dose may also be administered after the second dose and after about 3 months to about 2 years, preferably about 4, about 5, or about 6 months, or about 7 months to about 1 year after the first dose. The third dose may be administered orally when there is no specific immunoglobulin in the subject's serum and / or urine or mucosal secretions after a second dose, or when a small amount of specific immunoglobulin is detected. In a preferred embodiment, the second dose is administered about one month after the first dose and the third dose is administered about six months after the first dose. In another embodiment, the second dose is administered about 6 months after the first dose. In another embodiment, an immunogen comprising a RSV F protein can be administered as part of a combination therapy. For example, the RSV F protein or fragment thereof or a collection thereof may be formulated with other immunogenic compositions and / or antiviral agents. Dosages of the pharmaceutical preparations can be determined, for example, by first determining the effective dose to induce a prophylactic or therapeutic immune response by measuring the serum titer of a virus-specific immunoglobulin or by measuring the inhibition rate of antibodies in serum samples or urine samples or mucosal secretions Can be easily determined by those skilled in the art. In addition, human clinical studies can be performed by those skilled in the art to determine a desirable effective amount for humans. These clinical studies are routine and well known in the art. The exact dose to be used will depend on the route of administration. An effective amount can be estimated from a dose-response curve derived from an in vitro or animal testing system. As is well known in the art, the immunity of certain compositions can be improved by using nonspecific stimulators of the immune response, known as antigen-adjuvants. Antigen adjuvants have been used to experimentally improve the general increase in immunity against unknown immunogens (e. G., U.S. Patent No. 4,877,611). Immunization protocols have used antigenic adjuvants to stimulate responses for many years, and antigenic adjuvants are well known to those skilled in the art. Some antagonists affect the way antigens are present. For example, an immune response increases when protein antigens are immersed in alum. Emulsification of the antigen prolongs the antigen delivery period. Antigen adjuvants may be included. Suitable antigenic adjuvants include those described in Vogel et al., &Quot; A Compendium of Vaccine Adjuvants and Excipients (2nd Edition), which are incorporated by reference in their entirety for all purposes. Other exemplary antigenic adjuvants include the complete Freund ' (Non-specific irritant of immune response containing dead Mycobacterium tuberculosis), incomplete Freund's adjuvant and aluminum hydroxide adjuvant. Other antagonists include GMCSP, BCG, aluminum hydroxide, thur-MDP and nor- (MDP), CGP (MTP-PE), lipid A, montanide ISA 206 and monophosphoryl lipid A (MPL). Bacteria, MPL, trehalulose dimycolate (TDM) and (CWS) RIBI is considered to contain three components extracted from the cell wall skeleton (CWS) in a 2% squalene / tween 80 emulsion. MF-59, Novasom®, and MHC antigens may also be used. Well, an adjuvant is a pouch sila melanoma lipid vesicles (paucilamellar lipid vesicle) substantially with a two to ten bilayers arranged in a rectangular cover type separated by aqueous layers surrounding a large amorphous central cavity of the lipid bilayer is removed.

In one embodiment, the adjuvant effect is achieved by the use of a material such as alum, and is used as a 0.05 to about 0.1% solution in phosphate buffered saline. Alternatively, the immunogen may be prepared with a linear mixture of a synthetic polymer of sugars (Carbopol®) used in about 0.25% solution. Certain organic molecules from some antagonists, such as bacteria, act on the host rather than the antigen. An example is the muramyldipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterial peptidoglycan. In other embodiments, hemocyanin and hemoeryritin can be used. Hemostatic from the keyhole limpet (KLH) is preferred in certain embodiments, although mollusks and arthropod hemoshihenian and hemoeryritin can be used. A variety of polysaccharide adjuvants may be used. For example, the use of pneumococcal polysaccharide antigen adjuvants for antibody responses in mice has been disclosed (Yin et al, 1989). Doses that do not produce an optimal response or inhibition should be used as directed (Yin et al, 1989). Polyamine modifications of the polysaccharides are particularly like chitin and chitosan, including chitin and deacetylated chitin. In another embodiment, the muramyldipeptide lipophilic daiskaride-tripeptide derivatives disclosed for use in artificial liposomes have been formed from phosphatidyl choline and phosphatidyl glycerol. Other suitable adjuvants include bipolar surface active agents, such as saponin and derivatives such as QS21 (Cambridge Biotech). Saponin-based antigen adjuvants include those which contain substrate A and substrate C alone and in combination. Nonionic block copolymer surfactants (Rabinovich et al, 1994) can be used. Oligonucleotides are another useful group of adjuvants (Yamamoto et al, 1988). Another group of antigen adjuvants are the decrypted endotoxins such as the purified decoded endotoxin of U.S. Patent No. 4,866,034. These purified endotoxins are effective in causing an adjuvant response in vertebrates. Of course, the detoxified endotoxin can bind other antagonists to produce a multivalent-antigen adjuvant preparation. For example, the binding of the decoded endotoxin to trehalose dimethicolate is disclosed in U.S. Pat. 4,435, < RTI ID = 0.0 > 386. < / RTI > The binding of the decoded endotoxin to the trehalose dimethicolate and endotoxin glycolipids is also considered (U.S. Patent No. 4,505,899) and the binding of the decoded endotoxin to the cell wall skeleton (CWS) or CWS and trehalose dimacholate is disclosed in U.S. 4,436,727, 4,436,728, and 4,505,900. Without decoded endotoxin, the binding of CWS to trehalose dimycolyte is also disclosed in U.S. Pat. It is believed to be effective as disclosed in U.S. Pat. No. 4,520,019. Alkyl lysophospholipids (ALP); BCG; And other types of antigen-adjuvant that can be conjugated to a vaccine comprising biotin (including biotinylated derivatives). One particular antigen-reinforcing agent that specifically contemplates use is teichoic acid derived from gram-cells. This includes lipoteichoic acid (LTA), ribitol teico acids (RTA) and glycerol teico acids (GTA). Active forms of such synthetic counterparts can also be used (Takada et al, 1995).

A variety of antigen adjuvants that are not conventionally used in humans can still be used in other vertebrates, for example, when it is desired to generate antibodies or subsequently to obtain active T cells. The toxicity or other adverse effects that may arise from cells such as those that can occur using an antigen reinforcement or, for example, non-irradiated tumor cells, are independent of this environment. Other methods of inducing an immune response can be accomplished by formulating the immunogen of the invention with an " immunostimulant ". These are the body's own chemical messengers (cytokines) to increase the response of the immune system. Immunostimulants are immunosuppressive, immune-enhancing and inflammatory cytokines, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL- Cain, lymphokine and chemokine; Other immune stimuli such as growth factors (e.g., granulocyte-macrophage (GM) -colony stimulating factor (CSF)) and macrophage inflammatory factors, Flt3 ligand, B7.1, B7.2, The immunostimulatory molecules may be administered to the same formulation as an immunogen or may be administered separately. [0064] Expression vectors encoding proteins or proteins may be administered to produce an immunostimulatory effect. Can be in the range of the following lower limits: about 0.2 μg, about 0.4 μg, about 0.6 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg , About 7 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, About 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, or about 150 μg. Approximately 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 Mu] g, about 90 [mu] g, about 100 mu g, about 110 mu g, about 120 mu g, about 130 mu g, about 140 mu g, about 150 mu g, The saponin-based antigen adjuvant may be present in a range with the following lower limits: about 0.2 μg, about 0.4 μg, about 0.6 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg Saponin-based antigen-adjuvant is administered at a dosage of at least about 1 mg / kg, preferably at least about 10 mg / kg, about 4 mg / kg, about 5 mg / About 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg About 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, or about 200 μg. In certain embodiments, the saponin-based adjuvant is from about 5 μg to about 20 μg or from about 1 μg to about 10 μg. Such doses are particularly suitable in mice and can be adjusted for human use based on a typical mouse weight of 20 g versus a human body weight of about 60 kg.

In one embodiment of the invention, the RSV F protein, fragment or aggregate thereof, more preferably SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID NO: 11, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: , And SEQ ID NO: 23, or a vaccine comprising the RSV F immunoconjugate composition, wherein the RSV F immunogenic composition comprises at least one protein selected from the group consisting of SEQ ID NO: 23 and SEQ ID NO: 23.

Preferably, in one embodiment of the present invention, the method comprises administering a RSV F immunogenic composition comprising a protein selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 or a vaccine comprising the same , A method of inducing preventive immunity against RSV F infection.

In one embodiment of the present invention, there is provided a method of inducing an immune response to RSV comprising administering to a subject a composition comprising the RSV F protein antigen.

The method can reduce or prevent RSV infection.

The present invention provides immunogenic compositions for the prevention and / or treatment of RSV infection.

The present invention is able to achieve a more improved RSV F protein expression in the host cell than the wild-type RSV F protein through mutation of the RSV F protein.

The present invention can achieve high immunogenicity of the RSV F protein. That is, an antigen prepared through the sequence variation of the RSV F protein of the present invention (and / or the RSV F protein expression base sequence variation) can provide excellent neutralizing antibody titer and achieve excellent immunogenicity.

The present invention can provide a stabilized and soluble RSV F protein.

The present invention is applicable to a variety of cell lines and can provide a safe RSV immunogenic composition, more preferably a vaccine.

The RSV immunogen completed through the variation of the present invention can achieve a long duration of immune maintenance in the human body. The RSV immunogen completed through the variation of the present invention is less likely to cause repeated infections.

The RSV immunogen, which is completed through the mutation of the present invention, can induce excellent immunogenicity due to low interference with blood antibodies in the body.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing the variation of the RSV F protein mainly performed in the present invention. FIG.

FIG. 2 is an experiment for confirming the expression amount of the RSV F protein mutation performed in the present invention. This experiment measured the reactivity with palivizumab binding to the site II epitope. It was confirmed that the mutations tried in the present invention are superior to the mutation forms known in the art.

Fig. 3 shows results of western blotting and coomassie staining of the proteins expressed and purified through the mutations performed in the present invention. 3-a is the result of confirming F0 and F1 using βME. 3-b is the result of confirming FP1 and FP4 among the final purified mutant proteins.

FIG. 4 is a transmission electron microscopy photograph of RSV F protein prepared according to the present invention.

FIGS. 5A and 5B are graphs showing the results of measurement of total antibody titers to examine the induction of the mouse immunogenicity of the RSV F protein prepared according to the present invention. The total antibody titers are the values determined by ELISA analysis. Where GMT is the Geometric Mean Titer. Here, " 1ug + Adjuvant ", " 10ug + Adjuvant ", and " 30ug + Adjuvant " are substances adsorbed to 1ug, 10ug and 30ug of RSV F protein, respectively, in aluminum adjuvant. The PBS treatment time was 1.

FIG. 6 is a schematic diagram of an experimental result for confirming the concentration of a specific antibody capable of competing with palivizumab binding to a site II epitope in mouse serum in which immunity was induced by the RSV F protein prepared in the present invention.

FIGS. 7A and 7B are graphs showing experimental results for confirming the concentration of a specific antibody capable of competing with palivizumab binding to a site II epitope in a mouse serum immunized with the RSV F protein prepared according to the present invention. FIG. The result value means a GMT value which suppresses 50% of palivizumab.

FIGS. 8A and 8B are experiments to confirm the inhibition of the infection of the wild-type RSV virus in the mouse serum in which immunity was induced by the RSV F protein prepared according to the present invention. The wild-type virus used here is RSV A2 (ATCC. VR-1540). Through the neutralizing immunity test, it was confirmed that the RSV F protein produced by the present invention inhibited the infection of the wild-type RSV A2.

FIG. 9 shows the screening results of soluble F protein of FP1, 3, 4, and 6.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  One. RSV  F gene mutation

In the present invention, several kinds of amino acid sequences were mutated to obtain a stable and soluble RSV F protein. It is well known to those skilled in the art that the mutation applies to all RSV subtypes including wild RSV-A2 and RSV-B, as well as RSV-A long.

First, mutations were made to wild RSV-B (SEQ ID No. 1). The mutation removed the transmembrane domain. The amino acid sequence of the removed transmembrane domain is the amino acid at position 525-574 of SEQ ID NO: 2 (SEQ ID No. 2).

Variations in the position of the fusion peptide of the present invention (positions 137-145 of SEQ ID NO: 2), more preferably amino acids mutated to FP1, FP3, FP4, and FP6, may serve as excellent RSV immunogen, Respectively.

In one embodiment, the mutation mutated the fusion peptide portion (amino acid sequence 137-145). In one embodiment, specifically, the RSV F protein is a mutation that transforms the hydrophobic portion of the fusion peptide portion into a hydrophilic portion. Specifically, the hydrophobic part of the sequence FLGFLLGVG of the fusion peptide was substituted with A and transformed into AAGAAAGAG (SEQ ID NO: 3). For the purpose of the present invention, the optimum effect can be obtained as a result of substitution with A among hydrophilic amino acids.

In one embodiment, the polar portion of FLGFLLGVG in the fusion peptide portion was modified to a nonpolar side chain, and the modified fusion peptide portion was modified to QNGQNNGSG and NSGNSSGGG (SEQ ID NO: 4 and SEQ ID NO: 5, respectively). In another embodiment, the FLGFLLGVG portion of the fusion peptide portion was modified to TLSKKRKRR (SEQ ID NO: 6).

In one embodiment of the invention, additional mutations that maintain the stabilization of the F1 portion of the wild RSV F protein. Additional mutations associated with stabilization further include at least one additional mutation selected from the group consisting of (a) to (g) based on SEQ ID NO: 2.

(a) a mutation of the amino acid residue at the 140th position;

(b) a mutation of the amino acid residue at position 163;

(c) a mutation of the amino acid residue at position 179;

(d) a mutation of the amino acid residue at position 188;

(e) a mutation of the amino acid residue at position 189;

(f) a mutation of an amino acid residue at position 487;

(g) Mutation of amino acid residues at position 505.

More specifically, the RSV F protein antigens of the present invention are based on SEQ ID NO: 2

(a) a mutation of W at the amino acid residue F at position 140 (SEQ ID NO: 7);

(b) a mutation of Q at amino acid residue E at position 163 (SEQ ID NO: 8);

(c) a mutation of L at amino acid residue V at position 179 (SEQ ID NO: 9);

(d) a mutation of Q at the amino acid residue L at position 188 (SEQ ID NO: 10);

(e) a mutation in the amino acid residue T at position 189 (SEQ ID NO: 11);

(f) a mutation of the amino acid residue E at position 487 (SEQ ID NO: 12); And

(g) a mutation at position 505 of the amino acid residue F to W (SEQ ID NO: 13).

In another embodiment, the RSV F protein antigen is

An amino acid corresponding to position 140 of SEQ ID NO: 2 is substituted with W, an amino acid corresponding to 163 is substituted with Q (SEQ ID NO: 14);

The amino acid corresponding to position 140 of SEQ ID NO: 2 is substituted with W, the amino acid corresponding to 163 is substituted with Q, the amino acid corresponding to 188 is substituted with Q, the amino acid corresponding to 189 is substituted with L No. 15);

An amino acid corresponding to position 488 of SEQ ID NO: 2 is substituted with W, an amino acid corresponding to 163 is substituted with Q (SEQ ID NO: 16);

An amino acid corresponding to position 488 of SEQ ID NO: 2 is substituted with W, an amino acid corresponding to 179 is substituted with L (SEQ ID NO: 17);

An amino acid corresponding to position 140 of SEQ ID NO: 2 is substituted with W, an amino acid corresponding to 505 is substituted with W (SEQ ID NO: 18);

An amino acid corresponding to position 487 of SEQ ID NO: 2 is substituted with L, an amino acid corresponding to 505 is substituted with W (SEQ ID NO: 19);

The amino acid corresponding to position 163 of SEQ ID NO: 2 is substituted with Q, the amino acid corresponding to 505 is replaced with W (SEQ ID NO: 20);

An amino acid corresponding to position 188 of SEQ ID NO: 2 is substituted with Q, an amino acid corresponding to 189 is substituted with L, and an amino acid corresponding to 505 is substituted with W (SEQ ID NO: 21);

The amino acid corresponding to position 163 of SEQ ID NO: 2 may be substituted with Q and the amino acid corresponding to 487 may be substituted with L (SEQ ID NO: 22).

In some embodiments, this includes using known methods of protein processing and recombinant DNA technology to enhance or modify the properties of the mutated RSV F proteins mentioned above. Genes that encode the proteins are modified by codonification, and modifications of the nucleotides are known to those skilled in the art. Various forms of mutagenesis can be used to produce and / or isolate variant nucleic acids encoding the protein molecules and / or further modify / mutate the proteins of the invention. These include, but are not limited to, site-specific mutagenesis, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing template, oligonucleotide-induced mutagenesis, phosphorothioate-modified DNA mutagenesis, DNA (gapped-duplex DNA), and the like. Other suitable methods include point mismatch repair, repair-deficient host strains, restriction-selection and restriction-mutagenesis using purification, deletion mutagenesis, total gene synthesis, double-strand breakage Recovery, and the like. For example, mutagenesis involving a chimeric structure is also encompassed by the present invention. In one embodiment, mutagenesis can be guided by known information, such as sequences, sequence comparisons, physical properties, crystal structures, etc., of molecules that have been generated pre-existing or of naturally occurring molecules with acquired or mutated.

Example  2. RSV  F gene Expression proteins for mutations  Screening

The gene mutation for the F protein described in the previous example was confirmed by expressing the protein using 293FT cells. The candidate substance plasmid to which each mutation was applied was inserted into a vector containing a eukaryotic promoter (promoter) and the degree of expression was confirmed. The vector used in one embodiment is pcDNA3.1. It will be appreciated by those skilled in the art that not only pcDNA3.1 but also other eukaryotic expression vectors can be used as the vector. ELISA was used for the evaluation of the expression level. The antibody used was palivizumab, which binds to RSV site II. As can be seen in FIG. 2, the screening results of the expressed proteins for the applied mutations showed a higher level of expression than the mutations of the known patents.

Soluble F protein expression screening results of FP1, 3, 4 and 6

Five days after transfection, samples were harvested. The samples were harvested in two ways: lysis (Medium & Pellet) and lysis (Medium). In the absence of lysis, only the supernatant was harvested from transfected wells. For lysis, 10 μl of 10% Triton-X 100 was added to a 96-well culture (approximately 100 μl), rocked at 4 ° C for 30 minutes, and spun down for 1 minute to settle the debris. Respectively.

After adding 50 μl of PBS to the nickel-coated plate, 50 μl of the lysed sample and the non-lysed sample were added and incubated at room temperature for one hour. The primary antibody used was Sinagris sp. The secondary antibody was polyclonal rabbit anti-human IgG / HRP. TMB acts as an electron donor to reduce hydrogen peroxide and the like by peroxidase such as horseradish peroxidase (HRP). When the HRP enzyme is activated, the TMB will show blue light at 650 nm wavelength and the stop reagent will turn yellow at 450 nm when sulfuric acid is added. The difference between the two wavelengths (450nm - 650nm) was measured and the constructs that were well expressed were selected.

STDEV: standard deviation

% CV: the percent coefficient of Variation

As shown in Table 1 and FIG. 9, it was confirmed that the expression ratios of FP1, FP3, FP4 and FP6 of the present invention were excellent, and it could be used as an antigen.

Example  3. RSV  F-gene recombinant baculovirus  production

RSV-B F (SEQ ID NO: 1) The codons of the proteins were optimized and the antigens obtained through the gene mutation described above were prepared. The described RSV F gene was cloned into RSV F protein recombinant baculovirus (AcMNPV) and then inoculated into insect cells, Sf9.

The normal cell concentration of Sf9 insect cells should not exceed 3.00E6 / ml. Sf9 insect cells are cultured in sterile 125ml / 250ml / 500ml / 1L spinner flask or 5L / 50L / 100L bioreactor. In this example, serum-free serum-insect cell-specific medium containing no serum was used for the culture. Here, the serum-free insect cell-specific medium may include Insect Express (Lonza). In this example, the RSV F recombinant baculovirus for RSV F protein production is inoculated between 0.01-0.8 MOI (multiplicity of infection), wherein the concentration of Sf9 insect cells is in the range of 5.00E5-1.50E6, the initial stage of the exponential phase Lt; / RTI > The RSV F recombinant baculovirus used in this example harvests at 60-90 hours after inoculation, with viability between 75-98%. In this example, the reason why low-level viruses are inoculated to low-growth cells and harvested at relatively high viability is to minimize the target protein cleavage by proteases exiting the cells in the apoptosis stage and to reduce the amount of virus inoculum used It is to minimize.

Example  4. RSV  F antigen expression, purification, and analysis

First, the Sf9 insect cells obtained after the inoculation of the recombinant baculovirus of Example 3 are centrifuged at 3000-8000xg for 1-50 minutes to separate the cell sedimentation layer and the media supernatant. Remove the separated cell sedimentation layer and secure media supernatant. Infection of the recombinant baculovirus can occur effectively when the cells are in the early-log phase of growth and are in the 1.0E5-7.0E6 cell / ml concentration, preferably 5.00E5-2.50E6 cells / ml concentration.

Additional purification of RSV F can be accomplished by affinity chromatography, affinity chromatography, as known in the art. In one embodiment, the crude extract was further purified by passing it through anion exchange chromatography, lentile lectin affinity and cation exchange chromatography.

Additional purification of RSV F can be accomplished by affinity chromatography, affinity chromatography, as known in the art. In one example, the crude extract was further purified by passing it through anion exchange chromatography, lentile lectin affinity and cation exchange chromatography

In one embodiment, a sample of the same volume of each purification step was diluted with 2xSDS sample buffer containing βME (beta-mercaptoethanol), filled with 15-20 μl of each SDS-gel, electrophoresed, and stained with Coomassie stain Lt; / RTI > to stain whole proteins. In addition, SDS-gel was transferred to the membrane and further analyzed by WB (Western blot) using anti-single or multiple RSV F-specific antibodies. WB incubated for 4-24 hours at 4 ° C in buffer solution (PBS) containing 5% skim milk (Sigma), washed with buffer solution and then diluted 1: 2000 with RSV F-specific antibody Lt; RTI ID = 0.0 > 4 C < / RTI > for 4-24 hours. After incubation, wash three times with buffer containing 0.1% tween20, add the secondary antibody with mustard-free peroxidase (HRP) diluted 1: 5000 and incubate at room temperature for 1-2 hours. The incubated membrane is washed three times with buffer containing 0.1% tween 20, developed with ECL detection reagent (Amersham), and developed with a film. (Fig. 3)

RSV  Animal administration with F antigen / induction of immunity

Animal experiments using mouse as the antigen obtained in this example were carried out. The mouse used in the experiment can be used as an antigen used in the animal experiment itself and can also be used in combination with an immunostimulant such as an aluminum adjuvant. The adjuvant may include, for example, aluminum or calcium salts, in particular inorganic salts such as hydroxide, phosphoric acid, calcium phosphate and the like.

Aluminum or Alum-based adjuvants are currently the most commonly used adjuvants in human vaccines. Therefore, adjuvants, which are proven to be stable and effective enough to be used as a standard for developing and evaluating new adjuvants, to be. Among the alum adjuvants, aluminum hydroxide (Al (OH) ₃ ) and aluminum phosphate (AlPO4) are typical examples. These two alum adjuvants exhibit different physical and adjuvant properties. Especially, aluminum hydroxide is most widely used in aluminum adsorption vaccine. In this example, aluminum hydroxide was used as an immunity enhancer, and aluminum hydroxide was used at a level of 180 ug / once administration.

The concentration of RSV F antigen administered in this example was administered at different concentrations of 1 ug / ml, 10 ug / ml and 30 ug / ml, and the administration frequency was administered twice. The dosing schedules in this example were administered 2 times after 2 weeks of the first administration, and blood sampling was performed at 2 weeks and 4 weeks after the first administration.

RSV  Identification of immunogenicity of animals administered F antigen

The immunogenicity inducing effect of the RSV F antigen obtained in this example was confirmed. Identification of the immunogenicity induced in this example was performed by total antibody measurement, PCA measurement, and neutralizing antibody measurement.

In this example, total antibody titers to identify antigen-specific IgG antibody titers were determined using ELISA. 100 ng of RSV F antigen was coated on a 96-well plate and incubated overnight at 4 ° C. The incubated plate is washed three times with buffer containing 0.05% tween 20, then incubated with buffer solution containing 5% skim milk for 2 hours and washed again with buffer solution containing 0.05% tween 20. The obtained mouse blood is centrifuged to obtain a serum sample, the prepared serum sample is diluted to 1/20 of the original concentration, and then serial dilution is performed four times to make the final concentration 1/327680 times. Diluted serum samples are plated on 100 μl / well plates, incubated at room temperature for 2 hours, and washed three times with buffer containing 0.05% tween 20. 100 μl of goat anti-mouse IgG antibody (Invitrogen, US) combined with 1: 5000 diluted HRP-conjugated goat anti-mouse IgG was added to the washed plate, incubated at room temperature for 1 hour, Wash three times with the solution. 3,3A, 5,5A-tetramethylbenzidine supra (TMB substrate, KPL) was added to each well of the washed plate for 10 minutes, and TMB stop solution (KPL) was added to stop the reaction The absorbance 450 nm values for each well were measured using a Microtiter plate reader (Molecular Devices) and the results measured in this example show that the expressed / purified RSV F immunogen induces high antibody titers in mice to be. (Fig. 5)

In this example, a competitive ELISA was performed using the palivizumab antigen that binds to the site II epitope to confirm that the expressed / purified RSV F immunogen induces a neutralizing antibody corresponding to the site II epitope of the RSV F protein . 200 ng of RSV F antigen was coated on a 96-well plate and incubated overnight at 4 ° C. The incubated plate is washed three times with buffer containing 0.05% tween20, incubated with buffer solution containing 5% skim milk for 2 hours, and washed again with buffer solution containing 0.05% tween20. The obtained mouse blood is centrifuged to obtain a serum sample, the prepared serum sample is diluted to 1/10 of the original concentration, and then serial dilution is performed twice to make the final concentration 1/1280 times. Biotin-conjugated paribizumab is diluted to 50 ng per well and serial dilutions are performed in duplicate to achieve a final concentration of 390 pg per well. Diluted serum samples and biotin-conjugated paribisuram are incubated for 2 hours and washed three times with buffer containing 0.05% tween20. Incubate the washed plate with 1: 8000 diluted mustard radish peroxidase (HRP) conjugated streptavidin for 1 hour and wash 3 times with buffer containing 0.05% tween20. 3,3A, 5,5A-tetramethylbenzidine supra (TMB substrate, KPL) was added to each well of the washed plate, and HRP was allowed to react for 2 minutes. Then, TMB stop solution (KPL) was added to stop the reaction . The absorbance 450 nm for each well was measured using a Microtiter plate reader (Molecular Devices). It was confirmed that the palivizumab-responsive antibody measured in this example induced an antibody corresponding to site II, a neutralizing epitope of the expressed / purified RSV F antigen. (Figs. 6 and 7)

In this example, measurements of antibody titers causing a neutralizing immune response were performed. Hep2 cells were seeded in a 24-well plate at a rate of 3.5E5 per well and cultured at 37 ° C in 5% CO ₂ for one day. Serum samples from animal studies are diluted to 1/20 of the original concentration and serial dilutions are performed in triplicate to make the final concentration 1/4860. 50 μl of the diluted serum sample and 50 μl of RSV virus (40 pfu) are mixed and incubated at room temperature for 1 hour, then inoculated onto Hep2 cells of the plate seeded the day before. After 5 days of inoculation, the cells are cultured at 37 ° C and 5% CO ₂ . After incubation, the plates are stained with neutral red and neutralized. The neutralization value was calculated to provide a value (ND50) that protects 50% of the virus. (Fig. 8)

The present invention provides a soluble RSV. The soluble RSV may be provided as a vaccine composition capable of preventing RSV infection.

Amino acid sequence  List

One. SK- Seq  1 aa sequence (JN032120)

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK.

2. SK- Seq  One_ dTM  (transmembrane deletion_1-524 aa sequence )

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

3. SK-FP1 aa

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRAAGAAAGAGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

4. SK-FP3 aa

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRQNGQNNGSGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

5. SK-FP4 aa

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRNSGNSSGGGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

6. SK-FP6 aa

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRR1SAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

7. SK- Seq  One_ dTM _ F140W

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGWLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

8. SK- Seq  One_ dTM _ E163Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

9. SK- Seq  One_ dTM _ V179L

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVLSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

10. SK- Seq  One_ dTM _ L188Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVQTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

11. SK- Seq  One_ dTM _ T189L

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLLSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

12. SK- Seq  One_ dTM _ E487L

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDLFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

13. SK- Seq  One_ dTM _ F505W

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAWIRRSDELLHNVNTGKSTTN

14. SK- Seq  One_ dTM _ F140W _ E163Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGWLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

15. SK- Seq  One_ dTM _ F140W _ E163Q _ L188Q _ T189L

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGWLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVQLSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

16. SK- Seq  One_ dTM _ F488W _ E163Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEWDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

17. SK- Seq  One_ dTM _ F488W _ V179L

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVLSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEWDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

18. SK- Seq  One_ dTM _ F505W _ F140W

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGWLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAWIRRSDELLHNVNTGKSTTN

19. SK- Seq  One_ dTM _ E487L _ F505W

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDLFDASISQVNEKINQSLAWIRRSDELLHNVNTGKSTTN

20. SK- Seq  One_ dTM _ F505W _ E163Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAWIRRSDELLHNVNTGKSTTN

21. SK- Seq  One_ dTM _ L188Q _ T189L _ F505W

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVQLSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAWIRRSDELLHNVNTGKSTTN

22. SK- Seq  One_ dTM _ E487L _ E163Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDLFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

Base sequence listing

23. Wild type nt sequence (JN032120)

GGTACTGTGATAATGCAGGATCAGTATCCTTCTTTCCACAGGCTGACACTTGTAAAGTACAGTCCAATCGAGTATTTTGTGACACCATGAACAGTTTGACATTACCAAGTGAAGTCAGCCTTTGTAACACTGACATATTCAATTCCAAGTATGACTGCAAAATTATGACATCAAAAACAGACATAAGCAGCTCAGTAATTACTTCTCTAGGAGCTATAGTGTCATGCTATGGTAAAACTAAATGCACTGCATCCAACAAAAATCGTGGAATTATAAAGACATTTTCTAATGGTTGTGATTATGTGTCAAACAAAGGAGTAGATACTGTATCAGTGGGCAACACTTTATACTATGTCAACAAGCTGGAAGGCAAAAACCTTTATGTAAAAGGGGAACCTATAATAAATTACTATGACCCTCTAGTGTTTCCTTCTGATGAGTTTGATGCATCAATATCTCAAGTCAATGAAAAAATTAATCAAAGTTTAGCTTTTATTCGTAGATCCGATGAATTATTACATAATGTAAATACTGGAAAATCTACTACAAATATTATGATAACTGCAATTATTATAGTAATCATTGTAGTATTGTTATCATTAATAGCTATTGGTTTACTGTTGTATTGCAAAGCCAAAAACACACCAGTTACACTAAGCAAAGACCAACTAAGTGGAATCAATAATATTGCATTCAGCAAATAG

24. SK- Seq  One_ dTM  (transmembrane deletion_1- 524aa nt sequence )

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

25. SK-FP1 nt

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

26. SK-FP3 nt

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

27. SK-FP4 nt

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

28. SK-FP6 nt

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

29. SK- Seq  One_ dTM _ F140W

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

30. SK- Seq  One_ dTM _ E163Q

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

31. SK- Seq  One_ dTM _ V179L

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

32. SK- Seq  One_ dTM _ L188Q

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

33. SK- Seq  One_ dTM _ T189L

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

34. SK- Seq  One_ dTM _ E487L

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATCTGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

35. SK- Seq  One_ dTM _ F505W

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTGGATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

36. SK- Seq  One_ dTM _ F140W _ E163Q

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

37. SK- Seq  One_ dTM _ F140W _ E163Q _ L188Q _ T189L

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

38. SK- Seq  One_ dTM _ F488W _ E163Q

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTGGGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

39. SK- Seq  One_ dTM _ F488W _ V179L

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTGGGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

40. SK- Seq  One_ dTM _ F505W _ F140W

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTGGATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

41. SK- Seq  One_ dTM _ E487L _ F505W

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATCTGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTGGATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

42. SK- Seq  One_ dTM _ F505W _ E163Q

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTGGATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

43. SK- Seq  One_ dTM _ L188Q _ T189L _ F505W

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTGGATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

44. SK- Seq  One_ dTM _ E487L _ E163Q

GGTACTGTGACAATGCCGGTTCCGTCTCCTTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGCAAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGACCCCCTGGTGTTTCCCTCTGATCTGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

Claims (20)

1. Recombinant respiratory syncytial virus (RSV) F protein antigen,

   Wherein the protein antigen is a RSV F protein antigen, wherein the 525-574 position of the amino acid sequence of SEQ ID NO: 1 is deleted.
2. The RSV F protein antigen according to claim 1, wherein the RSV F protein antigen is SEQ ID NO: 2.
3. 3. The RSV F protein antigen according to claim 2, wherein the RSV F protein antigen further comprises at least one amino acid substitution at the fusion peptide position of the amino acid sequence of SEQ ID NO: 2.
4. 4. The RSV F protein antigen according to claim 3, wherein the RSV F protein antigen is characterized in that at least one hydrophobic amino acid is substituted with a hydrophilic amino acid at amino acid positions 137-145 of SEQ ID NO: 2.
5. 4. The RSV F protein antigen according to claim 3, wherein the RSV F protein antigen is characterized in that at least one polar amino acid is substituted with a nonpolar amino acid at amino acid positions 137-145 of SEQ ID NO: 2.
6. 5. The RSV F protein antigen according to claim 4, wherein the RSV F protein antigen has the amino acid sequence of SEQ ID NO: 3.
7. The RSV F protein antigen according to claim 5, wherein the RSV F protein antigen has the amino acid sequence of SEQ ID NO: 4.
8. The RSV F protein antigen according to claim 5, wherein the RSV F protein antigen has the amino acid sequence of SEQ ID NO: 5.
9. 3. The RSV F protein antigen according to claim 2, wherein the RSV F protein antigen is RSV F protein antigen having the amino acid sequence of SEQ ID NO: 6 by modifying the amino acid sequence FLGFLLGVG at amino acid sequence 137-145 of SEQ ID NO: 2 with TLSKKRKRR. F protein antigen.
10. 3. The RSV F protein antigen according to claim 2, wherein the RSV F protein antigen further comprises at least one additional mutation selected from the group consisting of (a) to (g) according to SEQ ID NO: 2.

    (a) a mutation of the amino acid residue at the 140th position;

    (b) a mutation of the amino acid residue at position 163;

    (c) a mutation of the amino acid residue at position 179;

    (d) a mutation of the amino acid residue at position 188;

    (e) a mutation of the amino acid residue at position 189;

    (f) a mutation of an amino acid residue at position 487; And

    (g) Mutation of amino acid residues at position 505.
11. 11. The RSV F protein antigen according to claim 10, wherein the RSV F protein antigen further comprises at least one additional mutation selected from the group consisting of (a) to (g)

    (a) a mutation of W at the amino acid residue F at the 140th position;

    (b) a mutation of Q at the amino acid residue E at position 163;

    (c) mutation of L at amino acid residue V at position 179;

    (d) a mutation of Q at amino acid residue L in the 188th position;

    (e) a mutation of L at amino acid residue T at position 189;

    (f) a mutation of L at amino acid residue E at position 487; And

    (g) Mutation of W at amino acid residue F at position 505.
12. 3. The method of claim 2, wherein the RSV F protein antigen is selected from the group consisting of

    An amino acid corresponding to position 140 of SEQ ID NO: 2 is substituted with W, and an amino acid corresponding to 163 is substituted with Q;

    An amino acid corresponding to position 140 of SEQ ID NO: 2 is substituted with W, an amino acid corresponding to 163 is substituted with Q, an amino acid corresponding to 188 is substituted with Q, and an amino acid corresponding to 189 is substituted with L;

    An amino acid corresponding to position 488 of SEQ ID NO: 2 is substituted with W, and an amino acid corresponding to 163 is substituted with Q;

    An amino acid corresponding to position 488 of SEQ ID NO: 2 is substituted with W, and an amino acid corresponding to 179 is substituted with L;

    An amino acid corresponding to position 140 of SEQ ID NO: 2 is substituted with W, and an amino acid corresponding to 505 is substituted with W;

    An amino acid corresponding to position 487 of SEQ ID NO: 2 is substituted with L, and an amino acid corresponding to 505 is replaced with W;

    An amino acid corresponding to position 140 of SEQ ID NO: 2 is substituted with W, and an amino acid corresponding to 505 is substituted with W;

    An amino acid corresponding to position 163 of SEQ ID NO: 2 is substituted with Q, and an amino acid corresponding to 505 is replaced with W;

    An amino acid corresponding to position 188 of SEQ ID NO: 2 is substituted with Q, an amino acid corresponding to 189 is substituted with L, and an amino acid corresponding to 505 is substituted with W;

    Wherein the amino acid corresponding to position 163 of SEQ ID NO: 2 is substituted with Q, and the amino acid corresponding to 487 is substituted with L.
13. The method of claim 1, wherein the RSV F protein antigen is selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: RSV F protein antigen characterized by being encoded by a nucleic acid.
14. 14. The RSV F protein antigen according to any one of claims 1 to 13, wherein the RSV F protein is a soluble protein.
15. 14. An immunogenic composition comprising the RSV F protein antigen of any one of claims 1 to 13.
16. 14. A pharmaceutically acceptable vaccine composition comprising a RSV F protein antigen according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier, wherein said RSV F protein is a pharmaceutical composition capable of eliciting an immune response in a host Acceptable vaccine composition.
17. 17. The vaccine composition of claim 16, further comprising an antigen adjuvant.
18. 18. The vaccine composition of claim 17, wherein the antigen adjuvant is aluminum.
19. 16. A method of inducing an immune response to RSV comprising administering to an individual an immunogenic composition comprising an RSV F protein antigen of claim 15.
20. 20. The method of claim 19, wherein said method reduces or prevents RSV infection.

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