WO2024093554A1

WO2024093554A1 - Recombinant subunit vaccine against rsv and use thereof

Info

Publication number: WO2024093554A1
Application number: PCT/CN2023/119638
Authority: WO
Inventors: 蒋敦泉; 刘永江; 张士彬; 黄培鑫; 陈君娜; 阴雅洁; 刘玉莹; 张欢; 江萍; 银飞; 姜绪林; 张尧; 沈迩萃; 杨秀芬; 王艳; 高俊
Original assignee: 北京康乐卫士生物技术股份有限公司
Priority date: 2022-11-04
Filing date: 2023-09-19
Publication date: 2024-05-10
Also published as: CN117986382A

Abstract

The present invention relates to the field of biomedicine, in particular to a recombinant subunit vaccine against respiratory syncytial virus (RSV) and a use thereof, and more in particular to a recombinant fusion protein containing the Head only (RHF) domain of an RSV envelope fusion protein F and a multimerization domain such as an immunoglobulin Fc fragment, an expression construct containing the recombinant fusion protein, a preparation method for the recombinant fusion protein, and an immunogenic composition containing the recombinant fusion protein, such as a vaccine.

Description

Recombinant subunit vaccine against RSV and its application

Technical Field

The present invention belongs to the field of biomedicine, and specifically relates to a recombinant subunit vaccine against respiratory syncytial virus (RSV) and its application, and more specifically to a recombinant fusion protein comprising the Head only (RHF) domain of RSV viral envelope fusion protein F and a multimerization domain such as an immunoglobulin Fc fragment, an expression construct comprising the recombinant fusion protein, a method for preparing the recombinant fusion protein, and an immunogenic composition comprising the recombinant fusion protein, such as a vaccine.

Background of the Invention

Populations susceptible to respiratory syncytial virus (RSV) include children, the elderly, and people with weakened immune systems, among which children are the main infected population, with more than 95% of children infected with RSV by the age of 2. Globally, RSV infection is the leading cause of death in children aged 1 month to 1 year, second only to malaria. RSV infection may cause severe respiratory symptoms such as bronchiolitis, pneumonia, tracheitis, and asthma. RSV infection in the elderly often leads to worsening of obstructive pulmonary disease and is accompanied by cardiopulmonary complications. The treatment and care costs associated with RSV infection amount to billions of dollars each year, bringing a heavy burden and challenge to the global public health system.

Clinically, Palivizumab is currently used to prevent serious diseases caused by RSV infection, especially in infants and young children who are born prematurely or have congenital heart disease, but it is expensive; Nirsevimab was recently approved by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) for the prevention of RSV-related lower respiratory tract diseases in all infants and young children; Ribavirin is approved for the treatment of bronchiolitis and pneumonia caused by RSV virus infection in infants and young children, but its efficacy is limited and its side effects are huge. In terms of vaccines, although there are some candidate vaccines in the preclinical and clinical research stages, only two elderly RSV vaccines and one maternal RSV vaccine have been approved for marketing, developed by GSK (AREXVY) and Pfizer (ABRYSVO) respectively.

RSV is an enveloped, non-segmented, negative-strand RNA virus belonging to the family Pneumoviridae and the genus Orthopneumovirus. The RSV genome encodes a total of 11 proteins, of which the G and F surface proteins play an important role in the adsorption and fusion process of RSV virus. They are the most important antigenic targets for inducing the body to produce neutralizing antibodies and are also the main targets for the development of RSV virus vaccines. The F protein of RSV virus is an abundant class I fusion glycoprotein on the viral envelope and exists on the viral surface in a trimer "prefusion" state. Once the virus begins to infect cells, the hydrophobic fusion loop of the F protein will insert into the host cell membrane and undergo extensive conformational changes to fold into an energetically favorable trimer "postfusion" state.

The results of the study showed that compared with the F protein in the post-fusion conformation, the F protein stabilized in the pre-fusion conformation can activate a stronger neutralizing antibody response in animal models. For example, it was previously shown that the recombinant RSV virus F extracellular domain trimer containing "DS-Cav1" substitutions (155C, 290C, 190F and 207L), cysteine zipper (Cysteine Zipper) and "SC-DM" substitutions (N67I, S215P) triggered a much higher neutralizing antibody response in animal models than the F protein based on the post-fusion conformation. Immune response induced by the prepared immunogens (Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461862/); A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476739/); A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569726/)). In addition, most of the neutralizing immune responses produced by natural RSV infection in humans are antibodies specific to the prefusion conformation F protein, and are usually directed against the potent antigenic epitope Ф(Zero) in the distal membrane head region of the prefusion conformation F protein trimer (Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286924/)).

At present, the RSV vaccines under clinical development and approved for marketing mainly use the full-length F protein in the pre-fusion conformation as the antigen target, and adopt technical routes such as recombinant protein, mRNA and viral vector vaccines. Recombinant RSV vaccines based on the full-length F protein need to be freeze-dried because the antigen may be converted to the post-fusion conformation. mRNA vaccines usually need to be stored and transported at -20℃ or even below -70℃. Viral vector vaccines have defects such as complex production process, low yield and poor immunogenicity. Therefore, this field still needs to develop safe, effective and stable quality RSV vaccines, and more ideally, RSV vaccines that can focus on the potent antigen epitope Ф(Zero).

Brief description of the invention

In one aspect, the present invention provides a recombinant fusion protein comprising the Head only (RHF) domain and the multimerization domain of the respiratory syncytial virus (RSV) envelope fusion protein F.

In some embodiments, the RHF domain of the RSV F protein contains the RSV virus potent antigenic epitope Ф(Zero).

In some embodiments, the RSV potent antigenic epitope Φ(Zero) of the recombinant fusion protein is maintained in a pre-fusion conformational state.

In some embodiments, the respiratory syncytial virus (RSV) envelope fusion protein F is selected from subtype A human RSV virus F protein, subtype B human RSV virus F protein or bovine RSV virus F protein. Preferably, the respiratory syncytial virus (RSV) envelope fusion protein F is subtype A2 strain human RSV virus F protein.

In some embodiments, the RHF domain comprises a first fragment and a second fragment of the RSV F protein, wherein the first fragment comprises an amino acid sequence from about position 50 to about position 108 of the RSV F protein, and the second fragment comprises an amino acid sequence from about position 145 to about position 308 of the RSV F protein, and the amino acid positions refer to SEQ ID NO:1.

In some embodiments, the RHF domain comprises a first segment and a second segment of the RSV F protein,

1) The first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 308 of the RSV F protein, and the amino acid positions refer to SEQ ID NO: 1;

2) the first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second The fragment comprises the amino acid sequence of positions 145 to 305 of the RSV F protein, wherein the amino acid positions refer to SEQ ID NO: 1;

3) The first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 306 of the RSV F protein, and the amino acid positions refer to SEQ ID NO: 1;

4) the first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 307 of the RSV F protein, and the amino acid positions refer to SEQ ID NO: 1;

5) The first fragment contains the amino acid sequence from position 51 to position 104 of RSV F protein, and the second fragment contains the amino acid sequence from position 145 to position 306 of RSV F protein, and the amino acid positions refer to SEQ ID NO: 1.

In some embodiments, the first fragment comprises the amino acid sequence shown in SEQ ID NO:4, and the second fragment comprises the amino acid sequence shown in SEQ ID NO:5; or the first fragment comprises the amino acid sequence shown in SEQ ID NO:6, and the second fragment comprises the amino acid sequence shown in SEQ ID NO:7.

In some embodiments, the first fragment and the second fragment are connected by a linker.

In some embodiments, the linker is a peptide linker of about 3 to about 21 amino acids in length.

In some embodiments, the peptide linker is G(SG)n or G(PG)n or G(AG)n, wherein n is an integer between 1-10, for example n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In some embodiments, the peptide linker is selected from GSG, GSGSG, GGSGSGSSG, GPGPG, GAGAG.

In some embodiments, the RHF domain comprises:

i) F at position 190 (190F) and L at position 207 (207L); and/or

ii) C at position 155 (155C) and C at position 290 (290C) that can form a disulfide bond;

Thus, the RSV potent antigenic epitope Ф(Zero) is maintained in a pre-fusion conformational state, wherein the amino acid position refers to SEQ ID NO:1.

In some embodiments, the RHF domain comprises an amino acid substitution relative to the wild-type sequence

i) S190F and V207L; and/or

ii) S155C and S290C;

In some embodiments, the RHF domain further includes an I at position 67 (67I) and/or a P at position 215 (215P), and the amino acid positions refer to SEQ ID NO:1.

In some embodiments, the RHF domain further comprises amino acid substitutions N67I and/or S215P relative to the wild-type sequence, and the amino acid positions refer to SEQ ID NO:1.

In some embodiments, the RHF domain comprises an amino acid sequence as set forth in one of SEQ ID NOs: 8-10 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% similarity to one of SEQ ID NOs: 8-10. Sequence identity to amino acid sequences.

In some embodiments, the multimerization domain is linked to the C-terminus of the RHF domain, either directly or through a linker.

In some embodiments, the multimerization domain is selected from the group consisting of an Fc fragment of an immunoglobulin, a Foldon sequence of T4 phage, and a ferritin subunit.

In some embodiments, the polymerization sequence is the Foldon sequence of T4 phage, for example, the Foldon sequence of T4 phage comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the multimerization domain is an Fc fragment of an immunoglobulin.

In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of an immunoglobulin from human, mouse, rabbit, cow, goat, pig, rabbit, hamster, rat, or guinea pig; or

The immunoglobulin Fc fragment is an Fc fragment of IgG, IgA, IgD, IgE or IgM; or

The immunoglobulin Fc fragment is an Fc fragment of IgG1, IgG2, IgG3 or IgG4;

Preferably, the immunoglobulin Fc fragment is the Fc fragment of human IgG.

In some embodiments, the immunoglobulin Fc fragment comprises the amino acid sequence shown in SEQ ID NO:12 or 13.

In some embodiments, the recombinant fusion protein comprises the amino acid sequence shown in one of SEQ ID NO:14-22 or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with one of SEQ ID NO:14-22.

In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the recombinant fusion protein of the present invention.

In some embodiments, the nucleotide sequence encoding the recombinant fusion protein of the present invention is codon-optimized for the host cell used for expression.

In some embodiments, the nucleotide sequence encoding the recombinant fusion protein of the present invention is operably linked to an expression regulatory sequence.

In another aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the present invention.

In some embodiments, the expression vector is a viral vector, such as an adenoviral vector, an alphavirus, a paramyxovirus, a vaccinia virus, a herpes virus, or a retroviral vector.

In another aspect, the present invention provides a host cell transformed with the isolated nucleic acid molecule of the present invention or the expression vector of the present invention.

In another aspect, the present invention provides a method for producing the recombinant fusion protein of the present invention, comprising:

(i) culturing the host cell of the present invention under conditions suitable for expression of the nucleic acid molecule or expression vector, and

(ii) isolating and purifying the recombinant fusion protein expressed by the host cell.

In another aspect, the present invention provides an immunogenic composition comprising the recombinant fusion protein of the present invention, and/or the isolated nucleic acid molecule of the present invention and/or the expression vector of the present invention, and a pharmaceutically acceptable carrier or excipient.

In some embodiments, the immunogenic composition is a vaccine and comprises one or more adjuvants.

In some embodiments, the adjuvant is selected from AS01, AS02, MF59, AlPO ₄ or Al(OH) ₃ , preferably AS01, AS02 or MF59, more preferably MF59.

On the other hand, the present invention provides the use of the recombinant fusion protein of the present invention, and/or the isolated nucleic acid molecule of the present invention and/or the expression vector of the present invention, and/or the immunogenic composition of the present invention in the preparation of a medicament for inducing an immune response against RSV F protein in a subject or for preventing and/or treating RSV infection or for preventing and/or treating a disease caused by RSV.

On the other hand, the present invention provides a method for inducing an immune response against RSV F protein in a subject or for preventing and/or treating RSV infection or for preventing and/or treating a disease caused by RSV, the method comprising administering to the subject an effective amount of the recombinant fusion protein of the present invention, and/or the isolated nucleic acid molecule of the present invention and/or the expression vector of the present invention, and/or the immunogenic composition of the present invention.

In some embodiments, the subject is an elderly person (e.g., ≥50 years, ≥60 years, and preferably ≥65 years), a very young person (e.g., ≤5 years, ≤1 year), a hospitalized subject, and a subject who has been treated with an antiviral compound but has shown an inadequate antiviral response.

DETAILED DESCRIPTION OF THE INVENTION

I. Definition

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following documents: Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook").

As used herein, the term "and/or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and/or B" encompasses "A," "A and B," and "B." For example, "A, B, and/or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

As used herein, "polypeptide" refers to two or more amino acids covalently linked. The terms "polypeptide" and "protein" are used interchangeably herein.

An "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system different from the cells from which the polypeptide naturally originates would be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, i.e., using protein purification techniques well known in the art.

In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. In general, those skilled in the art recognize that amino acids in non-essential regions of a polypeptide may be modified to produce a more conservative amino acid. The amino acid substitutions do not substantially alter the biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub.co., p. 224).

As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), usually linked together by a phosphodiester bond.

As used herein, an isolated nucleic acid molecule is a nucleic acid molecule isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. "Isolated" nucleic acid molecules such as cDNA molecules can be substantially free of other cellular materials or culture media when prepared by recombinant technology, or substantially free of chemical precursors or other chemical compositions when chemosynthesis. The exemplary isolated nucleic acid molecules provided herein include the isolated nucleic acid molecules of the recombinant fusion protein provided by encoding.

When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of the present application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered at this time, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

Sequence "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to technicians (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without changing the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub.co., p. 224).

As used herein, "operably linked" with respect to a nucleic acid sequence, region, element or domain means that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide so that the promoter regulates or mediates transcription of the nucleic acid.

As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell, including but not limited to mammalian cells, human cells, Fungal cells, bacterial cells, insect cells, etc. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

"Codon optimization" refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp/codon/ , and these tables can be adapted in different ways. See, Nakamura et al., 2001. Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28: 292 (2000).

As used herein, "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of nucleic acids or for expression/display of polypeptides encoded by nucleic acids. Vectors are usually kept free, but can be designed to integrate genes or parts thereof into chromosomes of the genome. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.

As used herein, vectors also include “viral vectors” or “viral vectors.” Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as a vehicle or shuttle) the exogenous genes into cells.

As used herein, "expression vector" includes a vector capable of expressing DNA, and the DNA is operably connected to a regulatory sequence such as a promoter region that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate host cells, result in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and/or prokaryotic cells and expression vectors that remain free or are integrated into the host cell genome. However, expression vectors may also encompass RNA molecules that can translate proteins in cells, such as mRNA molecules.

As used herein, "amino acid position reference SEQ ID NO: x" (SEQ ID NO: x is a specific sequence listed herein) means that the position number of the specific amino acid described is the position number of the amino acid corresponding to the amino acid in SEQ ID NO: x. The correspondence of amino acids in different sequences can be determined according to sequence alignment methods known in the art. For example, amino acid correspondence can be determined using the online alignment tool of EMBL-EBI. (https://www.ebi.ac.uk/Tools/psa/), where two sequences can be aligned using the Needleman-Wunsch algorithm with default parameters.

As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment and/or cure. Prevention refers to preventing potential diseases and/or preventing symptoms from worsening or disease progression. Treatment also includes any pharmaceutical use of any recombinant fusion protein provided and the compositions provided herein.

As used herein, "therapeutic effect" refers to the effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.

As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or disorder.

As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom. A complete prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

As used herein, the term "subject" refers to a mammal, such as a human.

2. Recombinant Fusion Protein

RSV virus potent antigenic epitope Ф(Zero) is an antigenic epitope specific to the prefusion conformation RSV F protein, located in the far-membrane head region of the prefusion conformation F protein trimer, including amino acids 62-69 and 196-209 of the F protein (see Neutralizing Epitopes on the Respiratory Syncytial Virus Fusion Glycoprotein (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456247/)). Studies have shown that the monoclonal antibody 5C4 targeting the antigenic epitope Ф is 50 times more potent than palivizumab (Structure of RSV Fusion Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing Antibody (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459498/)).

In some embodiments, the RSV virus potent antigenic epitope Ф(Zero) comprises amino acids 62-69 and 196-209 of the F protein, and the amino acid positions refer to SEQ ID NO:1.

The "respiratory syncytial virus (RSV)" described herein can be human RSV or animal RSV such as bovine RSV, preferably human RSV. The RSV can be any subtype RSV, such as subtype A or subtype B. In some preferred embodiments, The RSV is a subtype A2 strain human RSV virus.

In some embodiments, the respiratory syncytial virus (RSV) envelope fusion protein F is selected from subtype A human RSV F protein, subtype B human RSV F protein or bovine RSV F protein. In some preferred embodiments, the respiratory syncytial virus (RSV) envelope fusion protein F is subtype A2 strain human RSV virus F protein.

An exemplary A2 strain human RSV virus F protein comprises the amino acid sequence described in SEQ ID NO:1 or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:1.

An exemplary subtype B human RSV F protein comprises the amino acid sequence described in SEQ ID NO:2 or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:2.

An exemplary bovine RSV F protein comprises the amino acid sequence described in SEQ ID NO:3 or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:3.

In some embodiments, the RHF domain comprises a first fragment and a second fragment of the RSV F protein, wherein the first fragment comprises an amino acid sequence from about position 50 (e.g., position 50 or position 51) to about position 108 (e.g., position 104, position 105, position 106, position 107, or position 108) of the RSV F protein, and the second fragment comprises an amino acid sequence from about position 145 to about position 308 (e.g., position 305, position 306, position 307, or position 308) of the RSV F protein, and the amino acid positions are referenced to SEQ ID NO: 1.

In some embodiments, the RHF domain comprises a first fragment and a second fragment of the RSV F protein, the first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 308 of the RSV F protein, and the amino acid positions refer to SEQ ID NO:1.

In some embodiments, the RHF domain comprises a first fragment and a second fragment of the RSV F protein, the first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 305 of the RSV F protein, and the amino acid positions refer to SEQ ID NO:1.

In some embodiments, the RHF domain comprises a first fragment and a second fragment of the RSV F protein, the first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 306 of the RSV F protein, and the amino acid positions refer to SEQ ID NO:1.

In some embodiments, the RHF domain comprises a first fragment and a second fragment of the RSV F protein, the first fragment comprising the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprising the amino acid sequence of positions 145 to 307 of the RSV F protein, and the amino acid positions refer to SEQ ID NO:1.

In some preferred embodiments, the RHF domain comprises a first fragment and a second fragment of the RSV F protein, the first fragment comprises the amino acid sequence of positions 51 to 104 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 306 of the RSV F protein, and the amino acid positions refer to SEQ ID NO:1.

In some specific embodiments, the first fragment comprises the amino acid sequence shown in SEQ ID NO: 4, and the second fragment comprises the amino acid sequence shown in SEQ ID NO: 5. In some specific embodiments, the first fragment comprises the amino acid sequence shown in SEQ ID NO: 6, and the second fragment comprises the amino acid sequence shown in SEQ ID NO: 7.

In some embodiments, the first segment and the second segment are connected by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is about 3 to about 21 amino acids in length. In some embodiments, the peptide linker is a glycine-serine peptide linker, a glycine-proline peptide linker, or a glycine-alanine peptide linker.

In some embodiments, the peptide linker is G(SG)n or G(PG)n or G(AG)n, wherein n is an integer between 1 and 10, for example, n = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably n = 2. In some specific embodiments, the peptide linker includes but is not limited to GSG, GSGSG, GGSGSGSSG, GPGPG, GAGAG.

In some embodiments, the RHF domain comprises:

i) F at position 190 (190F) and L at position 207 (207L); and/or

i) S190F and V207L; and/or

ii) S155C and S290C;

In this article, the amino acid substitution S190F should be understood as the substitution of S at position 190 of the wild-type RSV virus F protein sequence by F, and the amino acid position is referenced to SEQ ID NO: 1. Other similar expressions should also be understood in the same way.

In some specific embodiments, the RHF domain comprises an amino acid sequence shown in one of SEQ ID NO:8-10 or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with one of SEQ ID NO:8-10.

As used herein, the multimerization domain is a domain that enables the recombinant fusion protein to form a multimer. In some preferred embodiments, the multimerization domain is a dimerization domain that enables the recombinant fusion protein to form a dimer.

In some embodiments, the multimerization domain is directly or via a linker connected to the RHF domain. In some embodiments, the multimerization domain is directly or via a linker connected to the C-terminus of the RHF domain.

Suitable multimerization domains include, but are not limited to, the Fc fragment of immunoglobulin, the Foldon sequence of T4 phage, ferritin subunits, and the like.

In some embodiments, the polymerizing sequence is a Foldon sequence of T4 bacteriophage. An exemplary Foldon sequence of T4 bacteriophage comprises the amino acid sequence shown in SEQ ID NO:11.

In some preferred embodiments, the multimerization domain is an Fc fragment of an immunoglobulin.

The immunoglobulin Fc fragment suitable for the present invention can be human, mouse, rabbit, cow, goat, pig, rabbit, barn The Fc fragment of an immunoglobulin of a mouse, rat, or guinea pig. The immunoglobulin Fc fragment may be an Fc fragment of IgG, IgA, IgD, IgE, or IgM. Preferably, the immunoglobulin Fc fragment is an Fc fragment of IgG1, IgG2, IgG3, or IgG4. Particularly preferably, the immunoglobulin Fc fragment is an Fc fragment of human IgG. In some preferred embodiments, the immunoglobulin Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 12. In some preferred embodiments, the immunoglobulin Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 13.

In some specific embodiments, the recombinant fusion protein comprises the amino acid sequence shown in one of SEQ ID NO:14-22 or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with one of SEQ ID NO:14-22.

3. Nucleic Acids, Vectors and Methods for Producing Recombinant Fusion Proteins

In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a recombinant fusion protein of the present invention or a precursor protein of a recombinant fusion protein of the present invention, wherein the precursor protein comprises a signal peptide located at the N-terminus of the recombinant fusion protein.

Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acid molecules can encode the same protein. It should also be understood that the technician can use conventional techniques to make nucleotide substitutions that do not affect the protein sequence encoded by the nucleic acid molecule to reflect the codon usage of any specific host organism to be expressed protein. Therefore, unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may or may not include introns.

In some embodiments, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:23.

In another aspect, the present invention also provides an expression vector comprising the nucleic acid molecule of the present invention. In some embodiments, the expression vector is a viral vector, such as an adenovirus vector, an alphavirus, a paramyxovirus, a vaccinia virus, a herpes virus, a retrovirus vector, and the like.

In another aspect, the present invention also provides a host cell transformed with the nucleic acid molecule or expression vector of the present invention.

The present invention also relates to the recombinant fusion protein obtained by the above method of the present invention.

4. Composition and Application

In another aspect, the present invention also provides an immunogenic composition comprising the recombinant fusion protein of the present invention, and/or the nucleic acid molecule of the present invention, and/or the expression vector of the present invention.

The present invention also provides the use of the recombinant fusion protein of the present invention, and/or the nucleic acid molecule of the present invention, and/or the expression vector of the present invention for inducing an immune response against RSV F protein in a subject or for preventing and/or treating RSV infection or for preventing and/or treating diseases caused by RSV.

The present invention also provides a method for inducing an immune response against RSV F protein in a subject or for preventing and/or treating RSV infection or for preventing and/or treating a disease caused by RSV, the method comprising administering to the subject an effective amount of the recombinant fusion protein of the present invention, and/or the nucleic acid molecule of the present invention, and/or the expression vector of the present invention.

The present invention also provides the use of the recombinant fusion protein of the present invention, and/or the nucleic acid molecule of the present invention, and/or the expression vector of the present invention in the preparation of a medicament for inducing an immune response against RSV F protein in a subject or for preventing and/or treating RSV infection or for preventing and/or treating a disease caused by RSV.

In some embodiments, prevention and/or treatment can target a subject group susceptible to RSV infection. Such subject groups include, but are not limited to, for example, elderly persons (e.g., >=50 years old, >=60 years old, and preferably >=65 years old), young persons (e.g., <=5 years old, <=1 year old), hospitalized subjects, and subjects that have been treated with antiviral compounds but have shown insufficient antiviral responses.

The recombinant fusion proteins, nucleic acid molecules and/or vectors according to the present invention can be used independently for the treatment and/or prevention of diseases or disorders caused by RSV, for example, or in combination with other preventive and/or therapeutic treatments (such as (existing or future) vaccines, antiviral agents and/or monoclonal antibodies).

In some embodiments, the immunogenic composition of the present invention comprises a recombinant fusion protein, nucleic acid molecule and/or vector of the present invention and a pharmaceutically acceptable carrier or excipient. In this context, the term "pharmaceutically acceptable" means that the carrier or excipient does not cause any unnecessary or adverse effects in the subject to which they are administered at the dosage and concentration employed. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients [Handbook of Pharmaceutical Excipients], 3rd edition, edited by A. Kibbe, Pharmaceutical Press [British Pharmaceutical Publishing House] [2000]. The recombinant fusion protein, or nucleic acid molecule or vector is preferably formulated and administered as a sterile solution, although lyophilized preparations may also be used. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. These solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution is typically in the range of pH 3.0 to 9.5, for example pH 5.0 to 7.5. The recombinant fusion protein is typically in a solution with a suitable pharmaceutically acceptable buffer, and the composition may also contain salts. Optionally, a stabilizer (such as albumin) may be present. In certain embodiments, a detergent is added. In certain embodiments, the recombinant fusion protein may be formulated as an injectable preparation.

In some embodiments, the immunogenic composition is a vaccine. In some embodiments, the immunogenic composition (vaccine) according to the present invention further comprises one or more adjuvants. Adjuvants are known in the art to further enhance the immune response to the applied antigenic determinants. The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that cause immune system stimulation. In this context, adjuvants are used to enhance the immune response to the recombinant fusion protein of the present invention. Examples of suitable adjuvants include, but are not limited to, aluminum salts, such as Aluminum hydroxide and/or aluminum phosphate; oil emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see, e.g., WO 90/14837); saponin formulations, such as, e.g., QS21 and immunostimulatory complexes (ISCOMS) (see, e.g., US 5,057,540, WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), oligonucleotides containing CpG motifs, ADP-ribosylating bacterial toxins or mutants thereof (such as Escherichia coli heat-labile enterotoxin LT, cholera toxin CT, etc.); eukaryotic proteins (such as antibodies or fragments thereof (such as against the antigen itself or CD1a, CD3, CD7, CD80) and receptor ligands (such as CD40L, GMCSF, GCSF, etc.), which stimulate immune responses when interacting with receptor cells. In some embodiments, the adjuvant is AS01, AS02, MF59, AlPO ₄ , Al(OH) ₃ .

In some preferred embodiments, the adjuvant is AS01. AS01 is a liposome adjuvant comprising 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and saponin QS-21, which can be obtained from supplier Beijing Huanuotai Biopharmaceutical Technology Co., Ltd. under product number HA201.

In some preferred embodiments, the adjuvant is AS02. AS02 is an oil-in-water adjuvant containing monophosphoryl lipid A (3-O-desacyl-4'-monophosphoryl lipid A, MPL) and saponin QS-21, which can be obtained from the supplier Beijing Huanuotai Biopharmaceutical Technology Co., Ltd. under the product number HA208.

In other more preferred embodiments, the adjuvant is MF59. MF59 is an oil-in-water adjuvant comprising squalene, Tween 80 and Span 85, available, for example, from supplier InvivoGen under the trade name AddaVax ^™ .

The compositions of the present invention can be administered to a subject, such as a human subject. The total dose of the recombinant fusion protein of the present invention in a composition for separate administration can be, for example, about 0.01 μg to about 10 mg, such as 1 μg-1 mg, such as 10 μg-100 μg. Determining the recommended dosage will be performed experimentally and is routine to those skilled in the art.

The administration of the composition according to the present invention can be performed using standard routes of administration. Non-limiting examples of the route of administration include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, or mucosal administration, such as intranasal, oral, etc. In some embodiments, the composition is administered by intramuscular injection. It is known to those skilled in the art that the composition (e.g., vaccine) is administered to induce different possibilities of an immune response to one or more antigens in the vaccine.

The subject described herein is preferably a mammal, such as a rodent, such as a mouse, a rat, or a non-human primate or a human. Preferably, the subject is a human subject.

Protein, nucleic acid molecule, carrier and/or composition can also be used as primary immunization in homologous or heterologous primary immunity-boosting scheme or as booster immunization.If booster vaccination is performed, typically, this booster vaccination will be administered to the subject for the first time (referred to as "primary vaccination" in such cases) between one week and one year, preferably at a time between two weeks and four months, such as 4 weeks, to the same subject.In some embodiments, the administration includes the primary administration and at least one booster administration.

Example

The following examples and drawings of the present application only illustrate specific implementation schemes for implementing the present invention. These schemes and drawings should not be understood as limiting the present invention. Any changes made without departing from the principles and essence of the present invention fall within the scope of protection of the present invention. The experimental techniques and experimental methods used in the examples of the present application are as follows: Unless otherwise specified, all methods are conventional. The materials, reagents, etc. used in the examples of this application can be obtained through regular commercial channels unless otherwise specified.

Example 1. Design and preparation of recombinant fusion protein

1.1 Vector construction

Referring to Boyington JC, Joyce MG, Sastry M, et al. (2016). Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus. PLoS One. 2016; 11(7): e0159709., two RSV F protein Head-only Domains (hereinafter referred to as “RHF”) were constructed based on the A subtype A2 strain human RSV virus F protein and the B subtype human RSV virus F protein:

RSV-A2 RHF amino acid sequence (aa 51-104+GSGSG+146-306):

RSV-B RHF amino acid sequence (aa 51-104+GSGSG+146-306):

The peptide linker between the first and second segments of the RSV F protein is underlined.

On this basis, different constructs shown in Table 1 were further obtained.

Table 1 Summary of construct designs used for RHF immunogenicity testing

A gene synthesis company was commissioned to directly synthesize the coding sequence of the above construct and clone it into the pcDNA3.1 expression vector; after the plasmid was digested, it was verified by agarose gel electrophoresis.

After confirming that the sequence information of the above constructs was correct, a third-party protein service company was commissioned to carry out protein expression and purification of constructs RSV-A2 RHF-Foldon, RSV Post-F, RSV Pre-F (DS-Cav1) and RHF i-693; the protein expression and purification of the remaining constructs were completed by the inventors, and the corresponding processes are briefly described as follows.

1.2 Transient transfection of cells

293 cells were inoculated at a cell density of 2.0×10 ⁶ cells/mL, and the cell density could reach about 4.0×10 ⁶ cells/mL after one day of culture. After cell counting on the second day, the cell viability was >95%, and the viable cell density was ≥4.0×10 ⁶ cells/mL. According to the optimized transient transfection process, a mixture of plasmid DNA and PEI of the constructs RSV-A2 RHF-hFc, RSV-B RHF-hFc, RSV-A2RHF-mFc, RSV-A2 RHF-hFc (DS-Cav1 mutation), RSV-A2 RHF-hFc (G(SG)n), RSV-A2RHF-hFc (GPGPG) and RSV-A2 RHF-hFc (GAGAG) was prepared respectively. The mixture was added to the cell culture medium for culture. After 18 hours of culture, 2 mM valproic acid (VPA) and 5% of the initial culture volume + 0.5% DN feed 2 + DN feed B2 were added. On the 7th day of culture, the cell supernatant was collected for protein purification.

1.3 Protein purification

Chromatography column: AT Protein A Diamond affinity chromatography medium, column volume CV: 5 ml, flow rate: 5 ml/min, pressure limit: ≤0.3 MPa.

Pretreatment: Rinse with purified water for at least 5 CV.

Equilibration: Elute with elution buffer for 1 CV, then equilibrate with binding buffer for at least 10 CV until the UV baseline is flat.

Sample loading: Cell culture supernatant, filtered through a 0.45 μm membrane before loading.

Elution: After loading, elute with binding buffer for at least 6 CV until the UV baseline is flat.

Elution: Elute with elution buffer, collect the eluate at the UV peak, add an appropriate amount of neutralization buffer into the collection tube according to the collection volume and mix well.

1.4 Electrophoresis detection

Take each sample collected during the purification process and prepare each sample to be electrophoresed (boiling water bath for 5 minutes) according to the ratio of 100μl sample + 25μl loading dye. Take an appropriate amount of sample for electrophoresis. The electrophoresis conditions are: gel concentration 10%, constant voltage 100V The electrophoresis was performed for 15 min at a constant voltage of 200 V until the dye front moved to the bottom of the colloid. After the electrophoresis was completed, the gel was stained with Coomassie Brilliant Blue to analyze the electrophoresis results. The results confirmed the expression of the target protein.

1.5 Protein quantification by BCA method

Dilute the test sample with injection water at different times so that the sample concentration after dilution is within the standard concentration range, and record the dilution multiple. Add the diluted reference solution and test solution to the microplate at 20ul/well, and load each sample in parallel to 3 replicate wells. Prepare an appropriate amount of alkaline copper test solution with solution A (2,2'-biquinoline-4,4' dicarboxylate sodium solution): solution B (copper sulfate) at a ratio of 50:1, mix well, add 200μl/well to the microplate, and incubate at 37℃ for 30min. Measure the absorbance value at 570nm with an enzyme reader. Make a standard curve based on the corresponding concentration of the reference substance and calculate the protein concentration of the test substance.

Example 2: Mouse immunization and neutralizing antibody detection

Female BALB/c mice aged 6-8 weeks were randomly divided into groups, with 10 mice in each group. The different recombinant proteins obtained in Example 1 were mixed with MF59 adjuvant and immunized with mice by intramuscular injection. Each mouse was immunized with 10 μg of recombinant protein. After four weeks, booster immunization was performed once. Blood was collected from mice every two weeks, and the supernatant was centrifuged and tested for neutralizing antibody titer.

The experimental method for neutralizing antibody titer detection is as follows:

1. Virus toxicity titration (TCID50):

The Hep-2 cells in the cell culture flask were washed three times with PBS, then digested with trypsin-containing digestion solution, added with an appropriate amount of nutrient solution, and transferred to a 96-well cell culture plate, 50ul per well; the harvested RSV Long strain was diluted 10 times in series with the maintenance solution, and 8 wells were inoculated for each dilution, 50ul per well. The cell lesions were observed under a microscope every day until the 5th day, and the TCID50 was calculated.

2. Microneutralization test to determine serum neutralization activity:

(1) Dilute the titrated RSV Long strain to 100 TCID50 and inoculate 50 ul per well.

(2) Dilute the serum in multiples (2-fold, 5-fold when the titer is high), 4 wells per dilution, 50 μl per well, and incubate at 37°C _CO2 for 2 hours.

(3) After the Hep-2 cells in the cell culture flask were washed three times in a monolayer, they were digested with a digestion solution containing trypsin, and then an appropriate amount of nutrient solution was added. The cells were transferred to a 96-well cell culture plate with serum added, with 100 ul per well (approximately 3*10 ⁵ cells/well).

(4) Set up virus control wells, with 50ul virus dilution solution + 50ul MEM maintenance solution + 100ul cell suspension in each well.

Set up cell control wells, each well containing 100ul MEM maintenance medium + 100ul cell suspension.

(5) Observe the cytopathic effect under a microscope every day until the fifth day, and calculate the effective half inhibitory concentration (IC50) of each serum antibody.

IC50 (meaning: diluting the serum antibody to a concentration of IC50 can inhibit 50% of cytopathic effect) was calculated by the Reed-Muech method.

The immunogenicity of different recombinant fusion proteins designed and obtained in Example 1 is shown in Table 2 below.

Table 2 Immunogenicity of different recombinant fusion protein constructs

The results showed that RSV-A2 RHF-hFc and RSV-A2 RHF-hFc (DS-Cav1 mutation) showed the strongest immunogenicity.

Therefore, a dose response experiment of RSV-A2 RHF-hFc construct was further conducted. The results are shown in Table 3.

Table 3 Results of dose response experiments of RSV-A2 RHF-hFc constructs

The results showed that RSV-A2 RHF-hFc fusion protein had a dose effect in inducing immune response as an antigen.

In addition, the immune enhancement effects of four different adjuvants were evaluated based on RSV-A2 RHF-hFc fusion protein: AS01, AS02, MF59, AlPO ₄ , and Al(OH) ₃ . The results are shown in Table 4 below.

Table 4 Enhancement of the immune response induced by RSV-A2 RHF-hFc construct by different adjuvants

The results showed that adjuvants AS01, AS02 and MF59 had the best effects.

The inventors also tested the effects of different linkers on the immunogenicity of RSV-A2 RHF-hFc constructs. The results are shown in Table 5 below.

Table 5 Effect of different linkers on the immunogenicity of RSV-A2 RHF-hFc constructs

Example 3: Storage stability

The recombinant fusion protein RSV-A2 RHF-hFc was placed in a 37°C incubator for 15 days (equivalent to 4°C for two years), and mice were immunized according to the method of Example 2, and the serum neutralizing antibody titer was detected. The results showed that the RSV-A2 RHF-hFc recombinant fusion protein placed in a 37°C incubator for 15 days can still induce the body to produce high-titer neutralizing antibodies (results are shown in Table 6 below). This shows that the RSV-A2 RHF-hFc recombinant protein has good stability and is easy to store and transport.

Table 6

Sequence Listing

Claims

A recombinant fusion protein comprising the Head only (RHF) domain and the multimerization domain of the respiratory syncytial virus (RSV) envelope fusion protein F.
The recombinant fusion protein of claim 1, wherein the RHF domain of the RSV F protein contains the RSV virus potent antigenic epitope Ф(Zero).
The recombinant fusion protein of claim 2, wherein the RSV potent antigenic epitope Φ(Zero) of the recombinant fusion protein is maintained in a pre-fusion conformational state.
The recombinant fusion protein of any one of claims 1 to 3, wherein the respiratory syncytial virus (RSV) envelope fusion protein F is selected from subtype A human RSV F protein, subtype B human RSV F protein or bovine RSV F protein, preferably, the respiratory syncytial virus (RSV) envelope fusion protein F is subtype A2 strain human RSV virus F protein.
The recombinant fusion protein of any one of claims 1-4, wherein the RHF domain comprises a first fragment and a second fragment of the RSV F protein, the first fragment comprises an amino acid sequence from about position 50 to about position 108 of the RSV F protein, and the second fragment comprises an amino acid sequence from about position 145 to about position 308 of the RSV F protein, and the amino acid positions refer to SEQ ID NO: 1.
The recombinant fusion protein of any one of claims 1 to 5, wherein the RHF domain comprises the first fragment and the second fragment of the RSV F protein,

1) The first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 308 of the RSV F protein, and the amino acid positions refer to SEQ ID NO: 1;

2) the first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 305 of the RSV F protein, and the amino acid positions refer to SEQ ID NO: 1;

3) The first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 306 of the RSV F protein, and the amino acid positions refer to SEQ ID NO: 1;

4) the first fragment comprises the amino acid sequence of positions 50 to 108 of the RSV F protein, and the second fragment comprises the amino acid sequence of positions 145 to 307 of the RSV F protein, and the amino acid positions refer to SEQ ID NO: 1; or

5) The first fragment contains the amino acid sequence from position 51 to position 104 of RSV F protein, and the second fragment contains the amino acid sequence from position 145 to position 306 of RSV F protein, and the amino acid positions refer to SEQ ID NO: 1.
The recombinant fusion protein of claim 6, wherein the first fragment comprises the amino acid sequence shown in SEQ ID NO:4, and the second fragment comprises the amino acid sequence shown in SEQ ID NO:5; or the first fragment comprises the amino acid sequence shown in SEQ ID NO:6, and the second fragment comprises the amino acid sequence shown in SEQ ID NO:7.
The recombinant fusion protein according to any one of claims 1 to 7, wherein the first fragment and the second fragment are connected by a linker. connected.
The recombinant fusion protein of claim 8, wherein the linker is a peptide linker having a length of about 3 to about 21 amino acids.
The recombinant fusion protein of claim 9, wherein the peptide linker is G(SG)n or G(PG)n or G(AG)n, wherein n is an integer between 1-10, for example n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
The recombinant fusion protein of claim 9 or 10, wherein the peptide linker is selected from the group consisting of GSG, GSGSG, GGSGSGSSG, GPGPG, and GAGAG.
The recombinant fusion protein of any one of claims 1 to 11, wherein the RHF domain comprises:

i) F at position 190 (190F) and L at position 207 (207L); and/or

ii) C at position 155 (155C) and C at position 290 (290C) that can form a disulfide bond;

Thus, the RSV potent antigenic epitope Ф(Zero) is maintained in a pre-fusion conformational state, wherein the amino acid position refers to SEQ ID NO:1.
The recombinant fusion protein of claim 12, wherein the RHF domain comprises an amino acid substitution relative to the wild-type sequence

i) S190F and V207L; and/or

ii) S155C and S290C;

Thus, the RSV potent antigenic epitope Ф(Zero) is maintained in a pre-fusion conformational state, wherein the amino acid position refers to SEQ ID NO:1.
The recombinant fusion protein of any one of claims 1-13, wherein the RHF domain further comprises an I at position 67 (67I) and/or a P at position 215 (215P), and the amino acid positions refer to SEQ ID NO:1.
The recombinant fusion protein of claim 14, wherein the RHF domain further comprises amino acid substitutions N67I and/or S215P relative to the wild-type sequence, and the amino acid positions refer to SEQ ID NO:1.
The recombinant fusion protein of any one of claims 1-15, wherein the RHF domain comprises the amino acid sequence shown in one of SEQ ID NO:8-10 or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% sequence identity with one of SEQ ID NO:8-10.
The recombinant fusion protein of any one of claims 1 to 16, wherein the multimerization domain is linked to the C-terminus of the RHF domain directly or through a linker.
The recombinant fusion protein of any one of claims 1 to 17, wherein the multimerization domain is selected from the Fc fragment of an immunoglobulin, the Foldon sequence of T4 phage, and a ferritin subunit.
The recombinant fusion protein of any one of claims 1-18, wherein the multimerization sequence is the Foldon sequence of T4 phage, for example, the Foldon sequence of T4 phage comprises the amino acid sequence shown in SEQ ID NO:11.
The recombinant fusion protein of any one of claims 1 to 18, wherein the multimerization domain is an Fc fragment of an immunoglobulin.
The recombinant fusion protein of claim 20, wherein the Fc fragment of the immunoglobulin is an Fc fragment of an immunoglobulin of human, mouse, rabbit, cow, goat, pig, rabbit, hamster, rat, or guinea pig; or

The immunoglobulin Fc fragment is an Fc fragment of IgG, IgA, IgD, IgE or IgM; or

The immunoglobulin Fc fragment is an Fc fragment of IgG1, IgG2, IgG3 or IgG4;

Preferably, the immunoglobulin Fc fragment is the Fc fragment of human IgG.
The recombinant fusion protein of claim 20 or 21, wherein the immunoglobulin Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 12 or 13.
The recombinant fusion protein of any one of claims 1-22, wherein the recombinant fusion protein comprises the amino acid sequence shown in one of SEQ ID NO:14-22 or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% sequence identity with one of SEQ ID NO:14-22.
An isolated nucleic acid molecule comprising a nucleotide sequence encoding the recombinant fusion protein according to any one of claims 1 to 23.
The isolated nucleic acid molecule of claim 24, wherein the nucleotide sequence encoding the recombinant fusion protein of any one of claims 1-23 is codon-optimized for the host cell used for expression.
The isolated nucleic acid molecule of claim 24 or 25, wherein the nucleotide sequence encoding the recombinant fusion protein of any one of claims 1 to 23 is operably linked to an expression control sequence.
An expression vector comprising the nucleic acid molecule of any one of claims 24-26.
The expression vector of claim 27, wherein the expression vector is a viral vector, such as an adenovirus vector, an alphavirus, a paramyxovirus, a vaccinia virus, a herpes virus or a retrovirus vector.
A host cell transformed with the isolated nucleic acid molecule of any one of claims 24 to 26 or the expression vector of claim 27 or 28.
A method for producing the recombinant fusion protein of any one of claims 1 to 23, comprising:

(i) cultivating the host cell of claim 29 under conditions suitable for expression of the nucleic acid molecule or expression vector, and

(ii) isolating and purifying the recombinant fusion protein expressed by the host cell.
An immunogenic composition comprising the recombinant fusion protein of any one of claims 1 to 23, and/or the isolated nucleic acid molecule of any one of claims 24 to 26, and/or the expression vector of claim 27 or 28, and a pharmaceutically acceptable carrier or excipient.
The immunogenic composition of claim 31 which is a vaccine and comprises one or more adjuvants.
The immunogenic composition of claim 32, wherein the adjuvant is selected from AS01, AS02, MF59, AlPO 4 or Al(OH) 3 , preferably AS01, AS02 or MF59, more preferably MF59.
Use of the recombinant fusion protein of any one of claims 1-23, and/or the isolated nucleic acid molecule of any one of claims 24-26, and/or the expression vector of claim 27 or 28, and/or the immunogenic composition of any one of claims 31-33 in the preparation of a medicament for inducing an immune response against RSV F protein in a subject or for preventing and/or treating RSV infection or for preventing and/or treating diseases caused by RSV.
A method for inducing an immune response to RSV F protein in a subject or for preventing and/or treating RSV infection or for preventing and/or treating a disease caused by RSV, the method comprising administering to the subject an effective amount of a recombinant fusion protein of any one of claims 1 to 23, and/or an isolated nucleic acid molecule of any one of claims 24 to 26, and/or an expression vector of claim 27 or 28, and/or an immunogenic composition of any one of claims 31 to 33.
The use of claim 34 or the method of claim 35, wherein the subject is an elderly person (e.g., ≥50 years, ≥60 years, and preferably ≥65 years), a young person (e.g., ≤5 years, ≤1 year), a hospitalized subject, and a subject who has been treated with an antiviral compound but has shown an inadequate antiviral response.